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Role of in silico tools and text mining in the risk assessment of selected alkaloids (#57)
L. Sousselier1, G. Raitano2, M. Petoumenou2, E. Benfenati2, N. Nguyen3, S. Ananiadou3, Q. T. Do4, E. Olivier5, S. Michel5, P. Rat5
1 UNITIS, Paris, France
Objective: Plant extracts are widely used, especially in food supplements and cosmetics. Their complexity and, for cosmetics, that animal tests are no longer possible, warrants an innovative approach based on natural molecules to allow for their risk assessment. This study outlines new methodologies developed in the frame of the NCSTOX project to undertake safety assessments of alkaloids based on in silico tools and text mining.
Methods: Instead of assessing each extract, information was gathered on all plant constituents.Focusing on alkaloids, which are present in numerous plants and known to exhibit safety concerns, we validated the methodology.
An innovative multi-step text mining approach, using the integrative ARGO workbench, was combined with the use of various in silico models, including those of VEGA platform, to predict safe levels of use based on 3 key endpoints: genotoxicity, systemic toxicity and sensitization. Critical compounds were identified as those associated with genotoxicity or classified as regulated allergens. Other molecules were classified according to a Threshold of Toxicological Concern (TTC) approach.
Results: Information on 3500 alkaloids was compiled. Out of them, 300 underwent a full safety assessment and the others were screened in silico. Read across was further performed among the 24 different molecular groups.
Around 30% are classified as genotoxic, specially acridones. Non-genotoxic molecules were almost exclusively belonging to Cramer class III.
On a third endpoint: sensitization, half of the molecules are classified as skin sensitizers.
Conclusions: The safety assessment of selected alkaloids validated a new methodology to establish the toxicological profile of plant constituents. Using text mining methods, we curated a novel database to provide the scientific community with animal-free, safe levels of use for plant constituents.
Keywords: Alkaloids, genotoxicity, systemic toxicity, skin sensitization, TTC
In vitro alternatives for assessing human safety of botanical mixtures (#59)
C. Mahony1, K. Vandermolen3, A. Otto-Bruc4, J. Naciff2, K. Kennedy5, G. Daston2
1 Procter & Gamble, Product Safety, Egham, United Kingdom
As complex mixtures, botanicals present unique challenges when assessing safe use, particularly when endpoint gaps exist that cannot be fully resolved by existing toxicological literature. Obtaining data for developmental and reproductive toxicity can be particularly difficult, and so a weight of evidence strategy for these endpoints will be illustrated which utilizes new in vitro approaches. Both receptor binding, enzyme activity assays and gene expression studies have been explored as tools to inform on modes of action. Several extracts of both botanicals suspected to have reproductive effects and herbs with a significant history of use were tested against a suite of receptors and enzyme activity assays at biologically relevant doses to probe developmental and reproductive activity at a molecular level. Additionally, gene expression changes in a number of different cell types were analysed using the connectivity mapping approach to identify major modes of action through a functional read across approach. Together, these two data streams have been shown to increase confidence in predictions of botanical biological mode of action, and allow for assessment of relative potency in the decision making process.
Keywords: Botanicals, human safety, high throughput, high content, in vitro
Genes associated with Parkinson’s disease respond to increasing polychlorinated biphenyl levels in the blood of healthy females (#66)
S. Bohler1, A. Espín-Pérez1, S. Gebel2, I. Bergdahl3, D. Palli4, P. Rantakokko5, H. Kiviranta5, S. Kyrtopoulos6, R. Balling2, J. Kleinjans1
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
Polychlorinated biphenyls (PCBs) are widely spread in the environment, and commonly found in human blood. PCBs have been suggested to be linked to Parkinson’s disease (PD), and non-coplanar PCBs 138, 153, and 180 were shown to have accumulated to a higher level in the brains of female PD patients . To elucidate the association between PCBs and PD risk we applied whole genome gene expression analysis in blood cells from PCB-exposed subjects.
The relationship between gene expression in blood cells and exposure levels of PCBs 118, 138, 153, 170, and 180, was studied in healthy individuals from two European cohorts (369 females, 280 males) using linear mixed model analysis. Significantly expressed gender-specific genes were selected for the individual PCBs, and compared to gene expression data available from the SN of deceased PD patients, to determine similarities.
In particular among females, blood levels of non-coplanar PCBs 153, 170 and 180 were associated with genes specifically related to PD, while none were detected among males. Among these females, 39 genes were discovered which were previously shown to display similar changes in expression levels in the SN of deceased PD patients. Especially among the down-regulated genes, transcripts of genes involved in neurotransmitter vesicle-related functions were predominant.
These investigations support the potential of PCB exposure as a PD-risk factor, and suggest an interesting overlap between the response of brain and blood cells.
 JM. Hatcher-Martin, M. Gearing, K. Steenland, AI. Levey, GW. Miller, KD. Pennell, NeuroToxicology. 33, (2012)
Keywords: Environment, MicroArray, Parkinson, Polychlorinated biphenyls, Transcriptomics, Linear Mixed Model
Ecotoxicological and toxicological assessment of substances in wastewater: making an informed decision (#110)
E. Lovsin Barle1, E. Burri1, F. Hermann1, C. Niederer2, D. Trudel2, C. Arnold1
1 Lonza AG, Basel, Switzerland
Toxicological evaluation of chemicals has been in place for many decades. A number of health based exposure limits (HBELs) such as Occupational Exposure Limits (OELs) for workers or Permitted Daily Exposure (PDE) limits for patients have been used to safely manage worker, patient, and general population health hazards. Conversely, ecotoxicological evaluation of chemicals is currently gaining momentum, especially when it comes to pharmaceuticals in the environment, for which, in the context of Marketing Authorization Applications (MAA), Environmental Risk Assessments (ERA) have to be performed. In contrast to the limits calculated for humans, the outcome of the ERA is a risk calculation based on a Predicted No Effect Concentration (PNEC) for aquatic species from three trophic levels but not considering the potential use of impacted water for drinking water production. There is a large data gap with respect to PNECs for the non-pharmaceuticals (e.g. chemicals), while OELs and even PDEs might be available.
We have evaluated data gaps for ecotoxicological and toxicological hazards of chemicals produced and used at a multipurpose manufacturing site in Switzerland. We are proposing a decision tree to identify priority substances based on existing toxicological and ecotoxicological data with the aim of reducing emissions and filling knowledge gaps for possible technical measures to reduce their emissions in wastewater in line with Swiss regulatory requirements. Additionally, we are presenting a case study for one of the large volume chemicals.
Keywords: wastewater, ecotoxicology, HBEL, PDE, OEL
Subchronic Inhaling Toxicity Study of Diphenylcyanarsine in SD Rats (#129)
L. Li1, R. Zhang1, Y. Yin1, T. Shi1, C. Wang1, X. Chen1, J. Xu1
1 /, State Key Laboratory of NBC Protection for Civilian, Beijing, China
Objective Diphenylcyanarsine(DC) is a irritating agent. Subchronic inhaling toxicity study of DC in SD rats had been done and provided the basis for establishing the environmental protection standard. Methods The groups of 20 SD rats of each sex were inhaled exposure with 0, 0.00036, 0.0011 and 0.0031μg/L of DC for 6h/d, 5d/w for 13 weeks by dynamic inhaling exposure device. The general physical conditions of rats had been observed everyday. The body weight of rats were measured and the mean food intakes per 100g BW were calculated every week. The eyes and the main breath indexes of rats had been examinated in the end of exposure. Hematological and blood biochemical indexes were analyzed and histopathology observation were evaluated in one quarter of rats every month. Results The results indicated that a few rats emerged irritating effects, such as scratching mouth, washing face, shedding tears and blood trace in eyelids side after 15-40 days of exposure in 0.0031μg/L group. The individual animals emerged blood trace in eyelids side after 60 days of exposure in 0.0011μg/L group. The results of the eyes check indicated that 12/19 rats emerged median conjunctival congestion in 0.0031μg/L group and 5/20 rats emerged slight conjunctival congestion in 0.0011μg/L group. The tidal volume of rats were significantly declined in 0.0031μg/L group compared with that of the control group. The average red blood cell volume, hemoglobin content and the thrombocytocrit were significantly increased in 0.0011 and 0.0031μg/L group. The concentrations of K, TP, ALP and the activity of LDH of serum were increased in high and median dose group, the concentration of glucose was significantly declined and the concentration of BUN was significantly increased in high dose group after 90 days of exposure. The female showed profound toxic effects compared with the male. The damages of throat, windpipe, lung, liver, kidney were more obvious in the rats exposured to high dose DC for more than 30d, while the damages of these organs were obvious in the rats exposured to the median dose of DC for 90d. These damages of organs were reversible. Conclusions The main target organs of toxic effects of DC on the rats (ih) were throat, windpipe, lung, liver, kidney. The estimated dose of NOAEL for DC was 0.00036μg/L and LOAEL was 0.0011μg/L in SD rats inhaled exposure for 13 weeks.
Keywords: Diphenylcyanarsine, Subchronic toxicity, NOAEL, LOAEL
An integrated understanding of metabolic processes relevant to mammalian and environmental toxicology in the context of REACH (#147)
Z. Mehmood1, P. Fisk1, R. Wildey1
1 PFA Brussels, Brussels, Belgium
An integrated approach to the understanding of effects, including the significance of metabolic processes, is highly desirable in a regulatory context such as REACH, for reasons explained in this poster. The integration of health and environmental regulatory endpoints can help to maximise the scientific credibility of chemical safety assessment. It is a common problem that data used for regulatory purposes have not been integrated across the endpoints.
This poster shows how there can be substantial overlap in the scientific issues affecting human and environmental toxicology; at an overview level, the example of alkanes and aliphatic alcohols is presented, alongside wider discussion. They are considered together due to the common metabolic issues: alkanes and alcohols metabolise via aliphatic acids. The metabolites are of significance biosynthetically.
The pathways themselves are shown, taken from standard literature sources. These pathways are highly conserved in mammals and micro-organisms alike. Bacterial degradation is critical for the understanding of fate of the substances in waste water treatment, surface water and soil.
Where hazard exists, the REACH Regulation calls for the setting of a derived no-effect level (DNEL) for comparison with human exposure levels. A predicted no-effect concentration (PNEC) to protect ecosystems is derived from available laboratory studies. The toxic effects of alkanes and alcohols in mammals are (with specific exceptional compounds) limited to local effects and are reviewed briefly. For aquatic organisms, hazards are found in laboratory studies. The significance of these observations, given the essentiality of the substances, is reviewed.
Given the essentiality of alcohols and acids it is highly questionable as to whether the standard ‘assessment factor’ approach is relevant to substances such as alkanes or alcohols. An alternative approach applicable to all aspects of (eco)toxicology, is suggested. In summary, this requires the assessor to consider the significance of the hazard and to limit how low a DNEL or PNEC can be, based on the natural exposure concentrations in the bodies of organisms.
Keywords: metabolic processes, REACH, ECHA, chemical safety assessment, environmental toxicology, alkanes, aliphatic alcohols, DNEL, PNEC, assessment factor, metabolism
Levels of rare earth elements in hair from a group of young Spanish adults (aged 20-24 years) (#197)
A. Peña Fernández2, S. Angulo3, M. Lobo Bedmar1
1 IMIDRA, Agroenvironmental Research, Alcalá de Henares (Madrid), Spain
Rapid agricultural, medical and industrial development is occurring on a global scale and bringing with it emerging environmental threats for humans. Contamination by rare earth elements (REE) has emerged as a public health concern due to their numerous applications in the modern industry. However, little is known about their toxicological effects despite their accumulation in different organs including brain and bone. To determine exposure to these contaminants in a young Spanish population, scalp hair samples were collected in 37 young adults (20 to 24 years-old; 28 female and 9 male) from different towns in the Community of Madrid (Spain). Despite being controversial, human hair could be an appropriate tool to determine environmental exposure to inorganic metal contaminants and to estimate the chemical burden in the individual. Lanthanum (La), cerium (Ce), praseodymium (Pr), erbium (Er) and gadolinium (Gd) were analysed in these samples by ICP-MS following appropriate methodologies. The limits of detection were (in ng/g): La (1.87), Ce (4.29), Pr (0.47), Er (0.06) and Gd (0.24 ng/g). Gd was detected in only one of the monitored samples (2.66 ng/g). The concentrations were as follows (median and interquartile range are provided in ng/g): La 5.30 (4.22, 7.13), Ce 11.18 (8.97, 15.45), Pr 1.28 (1.04, 1.72) and Er 0.19 (0.14, 0.28). In general, the presence of these metals in the Spanish group’s hair samples were lower than those reported in environmentally exposed groups, which may indicate that the studied group would have a low exposure to REE. None of these elements showed influence due to sex, although slightly higher levels were observed for La (5.57 vs. 5.17 ng/g), Pr (1.40 vs. 1.27 ng/g), Nd (2.48 vs. 2.29 ng/g) and Er (0.21 vs. 0.19 ng/g) in men’s hair and in women’s hair for Ce (11.58 vs 10.30 ng/g). Our results would be in agreement with those studies that have suggested that men may be more sensitive to REE than women.
Acknowledgement: This work was supported by Project FP-16-RESIDUA (IMIDRA, Comunidad de Madrid).
Keywords: Rare earth metals, human hair, monitoring, Spanish young adults.
Trends on PM2.5 research, 1997–2016: a bibliometric study (#208)
G. Liang1, J. Sui1, W. Wu1, T. Liu1, S. Xu1, L. Yin1, Y. Pu1
1 Southeast University, School of Public Healeh, Nanjing, China
Fine particulate matter (PM2.5) has been paid much attention in recent years. This study aims to analyze the scientific output and investigate the trend of PM2.5 research. PM2.5 related publications from 2007 to 2016 were retrieved from the Web of Science Core Collection database. Microsoft Excel 2016 and CiteSpace V software analyzed publication trend, countries/territories, journals, institutions, research areas, authors, citation counts and research frontiers. As a result, a total of 13681 papers on PM2.5 research were published by with a time limit of December 1, 2017, and the quantity of publications on PM2.5 continued to grow. The USA had the most publication (5941), citation (217,009) and H-index (172), followed by China, considered as the leading countries in this field. The journal Atmospheric Environment had the highest number of publications. Querol X has published the most papers (187), and Pope CA had the highest co-citation counts (5019) in this field. Furthermore, “climate-change,” “cardiovascular mortality,” and “long-term exposure” are becoming popular and should be closely observed in this field. In conclusion, PM2.5 is closely related to people's long-term health and we should understand it better, study it deeper, and try our best to reduce it.
Keywords: bibliometric analysis; PM2.5; citespace; trend; research frontiers
The use of a THP-1/3D reconstructed human epidermis (RHE) coculture system to assess the sensitizing potential of chemicals (#210)
M. T. Schellenberger1, U. Bock1, J. Hennen1, F. Groeber-Becker2, H. Walles2, B. Blömeke1
1 Trier University, Department of Environmental Toxicology, Trier, Rhineland-Palatinate, Germany
A key event of the adverse outcome pathway (AOP) for skin sensitization is the activation of dendritic cells. In an attempt to integrate the impact of the skin environment created by stratified keratinocyte layers on the activation of dendritic cells, we investigated whether available 3D reconstructed human epidermis (RHE) models can be cocultured together with THP-1 cells, as surrogate dendritic cells, to assess chemical-induced dendritic cell activation in vitro. For this purpose, THP-1 cells were placed underneath the RHE, which was topically exposed to increasing concentrations of chemicals with variable physicochemical properties and with different reaction mechanisms and sensitizing potential (eugenol, coumarin, ascaridole, cumine hydroperoxide and dibutylanaline). Cell surface expression of the costimulatory molecule CD86 and the adhesion molecule CD54 on THP-1 cells were analyzed by flow cytometry. Preliminary results are promising and we conclude that the coculture THP-1/RHE may allow the investigation of compounds with varying physicochemical properties and exposure at the air liquid interface.
Keywords: reconstructed human epidermis, THP-1, coculture, RHE, sensitizing potential
Next generation safety assessment strategy based on combined use of in silico tools (#227)
A. Granitzny1, S. Heinz1, T. Kawamoto2, A. Fuchs1, R. Fautz1
1 Kao Germany GmbH, Safety & Toxicology, Darmstadt, Hesse, Germany
In silico approaches and read-across are recognized as preliminary concept to perform a safety assessment (SA) without animal testing. To facilitate read-across, various in silico tools were developed in recent years, providing the basis for a next generation SA strategy. In addition, several authorities (ECHA, OECD and ECETOC) published related guidance documents. However, there is limited consensus in how to select and use each tool. Taking the first step towards clarification of this issue, several case studies were performed using raw materials such as hydrogenated polyisobutene and 2-ethylhexyl 12-hydroxystearate as well as a complex mixture of fatty acids. The aim was to establish a feasible SA strategy based on a combined use of available in silico tools. The general toxicity of compounds was assessed by means of the three tools AMBIT, COSMOS and ToxRead in order to select similar chemicals based on their chemical structure. The tools use different chemical descriptors and algorithms to calculate the pairwise similarity of compounds. Physico-chemical properties were evaluated by EPI-SUITE. The sensitization potential was predicted via TIMES-SS, followed by a quantitative risk assessment (QRA) using literature-based EC3 values. Although there is no clear definition for the threshold used to define an acceptable pairwise similarity of chemicals, retrospective analysis of published read-across case studies revealed that a similarity of 70% seems to be acceptable. AMBIT, ToxRead and COSMOS proposed differential but also overlapping analogues for the different compounds. The most conservative toxicological profile (e.g. lowest NOAEL) was selected among the identified analogues to perform the SA. TIMES-SS evaluation of the complex mixture identified nine of twelve fatty acids as non-sensitizer, while the remaining three fatty acids were predicted to have a weak to strong sensitizing potential. QRA based on the EC3 values of these three fatty acids indicated that no sensitization risk must be considered under the expected consumer exposure level. In summary, our case studies showed that a combined use of different in silico tools can result in a sufficient conservative SA. Moreover, several known chemical spaces can be covered complementarily by a combination of tools.
Keywords: In silico Tools, Read-Across, Alternative Risk Assessment, Case Study, Analogue Approach
Principles for the safety evaluation of cosmetic powders (#235)
W. Steiling2, J. F. Almeida3, H. Assaf Vandecasteele4, S. Gilpin5, T. Kawamoto6, L. O'Keeffe7, G. Pappa1, K. Rettinger8, H. Rothe9, A. Bowden10
1 Beiersdorf AG, Hamburg, Germany
Consumer exposure to cosmetic (personal care) products is mostly by dermal contact. However, additional considerations with regards to potential inhalation exposure from some cosmetics, such as sprays and powders, may be needed for a robust and reliable safety assessment.
To get a deeper understanding of the exposure to airborne particles from loose or pressed powders during product application, sufficient data on their diameter and typical doses are key. Our work describes the principles for inhalation exposure to such solids, and the derivation of safe exposure levels for cosmetic powder products. The general principles described herein are the same as for spray products as published earlier (Steiling et al. 2014). However, in contrast to liquids, where droplet maturation is important when the product is sprayed, airborne powders do not typically exhibit maturation, although aggregation and/or agglomeration of particles may occur. Moreover, certain characteristics of the powder like its dustiness also need to be considered. We have developed a decision tree with the basic elements of a tiered inhalation safety assessment approach for the different kinds of powder formulations. Furthermore, an overview of existing available inhalation in silico prediction models and real-time measurement approaches is given, as well as typical habits and practices data which are needed for the risk assessment of powder formulations following the tiered approach.
Keywords: Personal care products, spray, airborne particles, inhalation, safety assessment, inhalation exposure, dust
Development of an in chemico/in vitro testing strategy to assess the respiratory toxicity of inhalable substances from chemical-, consumer goods- and pharmaceutical industry (#246)
B. Blömeke1, J. Hennen1, U. Bock1, M. Hittinger2, H. Groß2, C. - M. Lehr2, B. Birk3, L. Ma-Hock3, R. Landsiedel3, T. Kirsch4, G. F. Gerberick4
1 Trier University, Environmental Toxicology, Trier, Rhineland-Palatinate, Germany
The research project aims at the development of an animal-free testing strategy for the prediction of respiratory toxicity in vitro (funded by German Federal Ministry of Education and Research; Acronym: AeroSafe, 031L0128A-D, 2017-2020). Overall goal is to reduce the number of animal experiments needed for safety assessment by establishing a standardized in chemico/in vitro test strategy. Based on available in vivo data of the test materials and mechanistic knowledge, AeroSafe focuses on inflammatory effects, modulation of epithelial barrier function and activation of the innate immune system. AeroSafe pursues to establish the simplest and most convenient but appropriate test strategy. We include approaches for analyzing protein reactivity and effects on target cells, imitating the complex in vivo situation. Starting from the first line of defense in the alveolar region, AeroSafe is focusing on macrophages followed by coculture of macrophages with alveolar epithelial cells, and consequently further target cells, both using cell lines and primary cells. Add on, delivery of test materials via exposure chambers is included. The Research strategy is clearly structured in a bottom-up approach – from in chemico and/or single cell cultures to complex cocultures. In doing so, we will select the number of models required to assess respiratory toxicity.
In sum, AeroSafe includes testing of representative structures of three different classes with in total 30 different materials consisting of nanomaterials, pharmaceuticals and low molecular weight respiratory allergens. Obtained in chemico/in vitro data will be used for comparison with available in vivo findings. Consequently, single or multiple test strategies will be deduced. The outcome of AeroSafe is expected to considerably contribute to significantly reduce the currently high number of so far indispensable animal experiments.
Keywords: respiratory toxicity, alveolar type I cell, macrophages
Climatic factors and aflatoxins occurrence in the feed and raw milk of dairy cows in Aguascalientes, Mexico (#278)
A. G. Valdivia1, C. R. Cruz-Vázquez2, T. Quezada-Tristán1, E. J. Rangel-Muñoz1, M. C. de-Luna-López1, E. Hernández-Valdivia1, R. Ortiz-Martínez1
1 Universidad Autonoma de Aguascalientes, Agricultural Sciences Centre, Aguascalientes, Aguascalientes, Mexico
Aflatoxins (AF) are toxic, immunosuppressive and carcinogenic compound; AF often contaminate agricultural commodities intended for human and animal consumption. AF are produced as secondary metabolites of Aspergillus spp., which can be influenced by water-temperature stress. The objective of study was to evaluate the association between climatic factors and the occurrence of AF contamination in the dairy feed and their residues in milk. Three dairy farms (DF) located in Aguascalientes, Mexico (21°48’N, 102°03’ W; 2000 mamsl; semi-temperate climate) were selected by non-probabilistic convenience method. DF were monitored monthly for two years (2015-2016). AF were tested by HPLC and ELISA in 266 samples of total mixed feed and 156 samples of raw milk; samples were obtained directly from each feeder and from the milk collection tank for each batch of dairy cows. The climate information of the near (<15 km) Huizache Weather Station (Num. 48291) of the National Network of Stations of the INIFAP was accessed; data were analyzed by one-way ANOVA, logistic regression and correlation using statistical software. AF contamination were detected in 99% of feed samples and 27% of milk samples; one sixth of them (17.3 and 16.7%) with AF concentrations above the maximum permissible limit (MPL, 20 µg/kg and 50 ng/kg). The AF concentration in the cows diet were positively associated (P<0.05) with the daily average temperature (DAT) and precipitation (DAP; P<0.01, R-square 37.6%); DAT and DAP, in turn, were associated with relative humidity and potential evapotranspiration. Other participating factors (cold hours, radiation, wind, and annual or DF-associated effects) did not show statistical significance. Compared to autumn and winter, the summer season recorded a higher DAT (18.2-18.7a, 15.4-15.9b and 13.9-14.5c °C, IC 95%, Tukey´s HSD test); but due to lower rainfall, spring had the highest DAT (19.6-20.2°C, P<0.05). Consequently, the incidence of milk and feed samples with AF above MPL were higher in the summer than in autumn and winter (Relative Risk: 1.4, 12.9 and 1.6, 5.4, respectively), but not that spring (0.9 and 1.0). These results suggest that the AF occurrence in the dairy food chain is related to DAT-DAP combination, so it is expected that the risk of exposure and damage may increase in predictable global warming scenarios.
Keywords: climate change, aflatoxins, dairy food chain, dairy industry, risk of exposure
The Threshold of Toxicological Concern (TTC) approach can be applied to organosilicon compounds (#279)
B. G. Schmitt1, E. Jensen2, M. C. Laufersweiler3, J. L. Rose4
1 Dow Silicones Belgium SPRL, Toxicology and Environment Research & Consulting, Seneffe, Belgium
The TTC is a pragmatic risk assessment screening tool to establish a safe exposure level for substances without an adequate toxicological data set. This approach has found acceptance among regulators globally. However, since the Munro database for non-cancer endpoints, on which the TTCs for non-genotoxic compounds were based, did not contain any organosilicon compounds, some regulators exclude this substance class from the TTC approach.
We evaluated all public available robust study summaries of oral repeated dose and reproductive/developmental toxicity studies of organosilicon compounds to confirm whether the TTC approach can also be applied to this chemistry. Only studies that were found to be adequate for the use as stand-alone studies for human health risk assessment were taken into consideration. If more than one study was available per substance, the one providing the most human relevant point of departure for risk assessment was chosen as the key study for this project.
Reliable and relevant high-quality key studies for 46 different organosilicon compounds could be identified, representing the chemical space of low molecular weight siloxanes, silanes and silanols. For repeat dose studies uncertainty factors were used to extrapolate to chronic exposure.
All identified NOAELs, with appropriate uncertainty factors, were compared to the Munro Cramer Class III TTC threshold, the default substance class for silicon-containing substances. Based on this comparison, in all cases, the derived human risk values for silicon-containing substances were greater than the Cramer Class III threshold.
Keywords: Threshold of Toxicological Concern, organosilicon compounds, siloxanes, silanes, risk assessment
Updates and overview of derivation of subacute guidance values for contaminants in drinking water in Japan (#281)
M. Matsumoto1, T. Kawamura1, K. Inoue1, T. Yamada1, N. Kobayashi2, A. Hirose1
1 National Institute of Health Sciences, Division of Risk Assessment, Kanagawa, Japan
The health-based values for lifetime exposure of contaminants in drinking water have been established as “Drinking Water Quality Standards (DWQS)” and notified as target values for “Complimentary Items” of the DWQS under the Japanese Water Supply Act. In addition to these two criteria lists, chemicals that can be detected in natural water sources but not yet managed are listed as “Items for Further Study” because of provisional risk assessment. We previously derived Subacute Reference Dose (saRfD) for 35 chemicals from the above three criteria lists to propose the subacute guidance values. We present the latest updates for 8 chemicals listed as “Items for Further Study” and an overview of our work. To derive the saRfD, the NOAEL or BMDL10 of a one to three-month study was basically used, and a total uncertainty factors of 100 was generally applied. Three out of 8 chemicals were genotoxic carcinogens. Therefore, 10 times of the Virtual Safe Dose at 10-5 risk was defined as the saRfD for these chemicals. The saRfD for perchloric acid was evaluated as same as a TDI, because the TDI was based on the human short-term effect. By using the saRfDs, subacute guidance values were culculated. The most of subacute guidance values for 43 chemicals became three to several dozen times as high as the corresponding health-based values for lifetime exposure. Especially, as for genotoxic chemicals or reproductive toxic chemicals, the subacute guidance values are less than ten times. Sources of water supply can be transiently influenced by local weather or chemical accidents. In such cases, the subacute reference values we proposed are considered to be useful for risk management. ACKNOWLEGMENT: This study was supported by a Health and Labour Sciences Research Grant (H28-Kenki-Ippan-005) from the Ministry of Health, Labour and Welfare, Japan.
Keywords: drinking water quality standard, subacute reference dose, subacute guidance value
Pro-inflammatory responses to PM0.25 from airport and urban traffic emissions (#303)
1 RIVM, Bilthoven, Netherlands
Due to the rapid development of the aviation industry and high demand for air transportation, the concomitant airport pollution has attracted increasing attention in recent years. Airport particulate matter (PM) emissions are the known source of air pollution in the proximity of an airport. Often large airports are located near metropolises, and airport emissions may have a potentially considerable impact on public health in the surrounding urban areas. However, little is known about the sources that are relevant to air quality and health in the vicinity of airports. Therefore, the effect of the chemical composition of airport-related particulate matter (PM) on adverse health risks was investigated in comparison to urban traffic emissions.
PM0.25 were collected at the Los Angeles International Airport (LAX) and at a central Los Angeles site (University of Southern California, USC campus), along with PM2.5 collected directly from turbine and diesel engines. The chemical composition, oxidative potential (OP) (measured by means of ascorbic acid (AA), and electron spin resonance (ESR)) of particles as well as the reactive oxygen species (ROS) activity, inflammatory potential (interleukin (IL) 6 and 8 and tumor necrosis factor (TNF) –α release) and cytotoxicity on human bronchial epithelial (16HBE) cells were assessed. From the results we obtained, chemical composition measurements confirmed that aircraft emissions were the major source to LAX PM0.25, while the sources of USC samples were more complex, including traffic emissions, suspended road and soil dust, and secondary sulfate. The traffic-related transition metals (Fe and Cu) in LAX and USC samples mainly affected OP values of particles, while multiple factors such as compositions, size distribution and internalized amount of particles contributed to the promotion of ROS generation in 16HBE cells during 4 h exposure to 10 μg/mL particles. Internalized particles in cells might also play an important role in activating inflammatory responses during cell recovery period, with LAX particles being more potent.
Our results demonstrate considerable toxicity of airport-related particles, even at low exposure concentrations, which suggests that airport emission as source of PM0.25 may also contribute to the adverse effects on public health attributable to PM. The potency of such particles is in the same range as those collected at a site in urban area with dense traffic.
Keywords: PM0.25, Aviation emission, Traffic emission, Oxidative potential, Pro-inflammation
Spatiotemporal Variability of N-Nitrosamines Exposure in Drinking Water of A High Incidence Area of Esophageal Cancer (#327)
L. YIN1, 2, C. Zhao1, 2, Y. Feng1, 2, Y. Miao1, 2, R. Liu1, 2, Y. Pu1, 2, L. Yin1, 2
1 Southeast University, School of Public Health, Nanjing, China
As a group of emerging unregulated disinfection by-products (DBPs) in drinking water, N-nitrosamines are perceived to pose a greater health risk due to their strong carcinogenicity and mutagenicity. In this study, the presence of nine nitrosamines in source water and finished water from eleven drinking water treatment plants in Nanjing with a low incidence and Huaian with a high incidence of esophageal cancer was evaluated by a solid phase extraction-gas chromatograph-mass spectrometry (SPE-GC-MS) method in March, October 2017 and January 2018. Overall, N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitrosopyrrolidine (NPyr) topped the first three commonly detected nitrosamines list and the detection rate was 71.0, 48.4 and 37.1%. The highest concentrations of them in source water were up to 119.8, 57.3, 22.9 ng/L, and in finished water were 58.3, 51.0 and 21.0 ng/L, respectively. The occurrence of N-nitrosodimethylamine (NDMA) was in 19.3% of water samples and the average and maximum value were 2.2 and 49.4 ng/L. Meanwhile, the average exposure level of total nitrosamines in source water was 2.9 times as high as that in finished water. Seasonal distribution of total nitrosamines showed that samples in October had the highest level, followed by January and March. It was obviously high to compare detection rate and concentrations of NDPA, NDBA, N-nitrosodiethylamine (NDEA) and total nitrosamines in water samples from Huaian with these of water samples from Nanjing. The risk level indicated that the average carcinogenic risk of total nitrosamines in finished water from Huaian district was 4.8 times that of Nanjing. Among them, NDPA, NDBA and NDEA were the main carcinogenic risk contributor for waters from Huaian, and NDMA was the major risk contributor for waters from Nanjing. These findings showed that N-nitrosamines were popular in drinking water and may be a potential health risk factor for population and the local water environment. It is very important for regulators to reinforce evaluation of the health risk with long-term exposure to N-nitrosamines in drinking water. (Supported by National Natural Science Foundation of China, 81573191).
Keywords: Disinfection by-products, N-nitrosamines, drinking water, GC-MS
Assessing Acute Inhalation Exposure to Hydrogen Fluoride from Thermal Runaway of Lithium-Ion Batteries (#336)
J. Hercegovac1, J. Frangos2
1 Golder Associates, Melbourne, Victoria, Australia
Lithium-ion battery storage is becoming more frequent as the use of this technology increases due to their high energy and power densities. Exceeding the thermal stability limits of a lithium-ion cell can result in thermal runaway and such failures can generate intense heat, smoke and toxic gases. A human health and toxicological risk assessment of hydrogen fluoride (HF) was undertaken for a lithium-ion battery testing facility to assess potential HF exposures to staff if thermal runaway of lithium-ion battery occurred during testing. While instances of thermal runaway occurring are rare, assessment of risk to worker health was still recommended. The project required consideration of acute exposure duration (seconds) under a defined workplace scenario. The key exposure pathway assessed was inhalation as HF is readily absorbed in the upper respiratory tract which presents the most sensitive target of acute toxicity.
The assessment required estimation of emission rates for HF and subsequent conversion to HF indoor air concentrations. Emission rates of HF depended on the state of charge of the batteries and this was also incorporated in modelled scenarios adding to the complexity and range of results. Due to the acute exposure durations assessed (5 to 15 seconds) obtaining appropriate short-term acute exposure standards or toxicological guidelines was a challenge. Occupational exposure limits and emergency guidelines were considered and Haber’s Law was used for time-scale downward extrapolations of the selected acute guidelines.
The results showed that the modelled indoor air concentrations exceeded the selected guideline and the potential health consequences required further analysis. A toxicological assessment of the concentration-time response was undertaken to characterise potential risks to workers. Combining risk assessment principles with toxicological understating of HF behaviour allowed a more detailed assessment of the actual (likely) risks. Consideration of mitigation measures was then undertaken to provide an aid in reducing the risks inferred by the study. The project illustrated the challenges and complexities in assessing rare emergency exposure scenarios that may not fall within the routine guidance and methodologies employed in environmental contaminants risk assessment.
Keywords: lithium ion, hydrogen fluoride, inhalation exposure
Safety evaluation of the migration in oily cosmetic products in contact with plastic packagings (#338)
P. MURAT2, 3, V. SIMON3, M. GALONNIER1, F. Pierre-Jacques1, S. COSLEDAN2
1 Pierre Fabre Dermo Cosmetique, Safety and Cosmetovigilance Department, Toulouse, France
The concern about container-content interactions has really increased this last decade, especially with the growing preoccupation about endocrine disruptors. In fact, lots of plasticizers are endocrine disruptors and are able to migrate from packagings into products. In cosmetic industry, these studies are complicated since no protocol exists in actual regulations. Moreover, there are no acceptance thresholds available for potential contaminants. In this context, a strategy of risk evaluation were developed, combining migration tests and safety evaluation for container-content interactions. Due to the complexity of cosmetic matrices, the study were made on simulants, as for studies in food industry. In a previous work, aqueous simulants such as water or ethanol were used to develop the strategy. Positive results led to extend this work to oily simulants that represent an important part of beauty products. Unlike aqueous simulants, oily simulants cannot be directly injected into the analytical system. An extraction step is necessary. In order to obtain the best results possible, two extraction methods were compared: liquid-liquid extraction and solid phase micro-extraction (SPME). Liquid paraffin and glycerin were chosen as oily simulants, and put in contact with eleven selected plastic packagings. As for aqueous simulants, 12 potential contaminants were screened: 10 phthalates, bisphenol A and distearyl thiodipropionate (European Pharmacopoeia plastic additive n°17). After one month at 50°C and extraction steps, the oily simulants were analyzed by GC-MS and obtained results were evaluated using a safety evaluation developed specifically for this study. This work complete the container-content interactions evaluation realized on cosmetic simulants and corroborate the strategy developed.
Keywords: Container-content interactions, Cosmetics, Safety assessment
Chemical analysis of metallic trace elements of toxicological concern in Lebanese pita and risk characterization for the consumers (#352)
N. Lebbos1, E. Bou-Maroun2, C. Daou3, R. Ouaini3, H. Chebib3, C. Keller4, M. Afram1, P. Curmi5, M. - C. Chagnon6
1 Lebanese Agricultural Research Institute LARI, Heavy metals Department, Beirut, Lebanon
Among metallic trace elements that can be present in food, some are known as toxic and must be monitored. Regarding food safety, it is relevant to study the case of the pita since it represents a large part of the daily diet of the Lebanese population. The objective of this study was primarily to quantify As, Cd, Co, Cr, Hg, Ni and Pb in Lebanese pita. Then, to evaluate the consumer exposure to metal contaminants via white pita consumption, a survey was carried out in supermarkets of 5 different Lebanese regions. Thousand individuals (half men, half women) were grouped as follows: 800 adults (15 years and above) and 200 children divided in two categories (6-9 and 10-14 years). The exclusion criteria were chronic or severe pathology patients and adults who had stayed in Lebanon for less than 15 years. Different bread brands, sizes and numbers of pita consumed per day were taken into consideration.
Pitas were sampled according to EU regulation No 836/2011. Three bags of bread were collected from 3 bakeries (B1, B2 and B3), and 3 pitas out of 8 were taken from each bag. The mineralization of samples was carried out in nitric acid and hydrogen peroxide using microwave digestion. As, Cd, Co, Cr, Ni and Pb were analyzed by atomic absorption spectrometry and Hg was analyzed by a direct mercury detector. To ensure the methods reliability, a certified reference material (BCR-191) was tested at the same time as samples.
The metal contents were different depending on the selected bakeries. The highest quantities of Ni and Co were found in bread of B1, As, Pb and Cd in bread of B2, and Cr and Hg in bread of B3. Maximum limits in Lebanese bread are only defined for Pb and Cd. The content of these elements in breads respect the Lebanese standard.
The human dietary exposures of the population categories were compared to toxicological reference values, when available, to characterize the risk for the Lebanese consumer. The population between 6 and 9-years-old was the most exposed to As, Pb and Co. Concerning these elements, the highest exposures (95th percentile) were above toxicological reference values or with a margin of exposure too low.
Keywords: Survey, Metallic trace elements analysis, Lebanese pita, Human exposure, risk
Silicones – do they all behave the same way in the environment regarding biodegradability / degradability? (#360)
T. CREUSOT1, G. LOUVET2, A. BELLEMAIN3, B. COMBOURIEU3, S. CATOIRE1, H. E. FICHEUX1
1 THOR Personal Care Department, Toxicology and Risk assessment, Compiegne, France
Many molecules are found under the generic name of silicones. Applications are numerous and silicones are found in industrial, pharmaceutical, medical device and personal care products.
Revisiting the concept of biodegradation and degradation in such a complex family is challenging. For the purpose of this study fluid silicones with various viscosity levels were used, such as those found in the cosmetic industry.
Biodegradability was investigated using in silico tools (Epiwin and QSAR toolbox) together with in vitro OECD approaches.
In parallel, a non-conventional approach was used with the aim of studying potential relevant effects such as a solar simulator to study UV effects combined with abiotic degradation in wet and dry soils.
Combining the results of each approach lead to the following observations:
A low level of polymerisation leads to easily hydrolysed silicones by enzymatic and non-enzymatic reactions of the grafted carbon chain. A higher level of polymerisation provides greater protection against degradation.
The ultimate stage, represented by the siloxane backbone degradation, may be obtained by the combined effect of bacteria and UV light leading to formation of silicates.
Physico-chemical properties, in part driven by the polymerisation level, play a crucial role as they influence the physical state: solid, liquid, volatile, gel. Functionalisation of alkyl dimethicone compounds is also a key parameter for biodegradability.
Consequently (bio)degradability should be approached on a case-by-case basis.
Even if (bio)degradation, with respect to the OECD definition for very high molecular weight silicones, is not totally achieved, a degradation of lower molecular weight silicones, as used in the personal care industry, is achieved by other means such as UV and abiotic transformation.
Keywords: Silicones, Biodegradability, Degradability, In Silico
Outdoor and indoor air pollution exposure: a cross-sectional study in Dakar city (Senegal) (#362)
A. NDONG1, 2, A. VERDIN1, J. THOMAS4, F. CAZIER6, M. CABRAL2, A. GARAT4, D. ALLORGE4, G. GARCON3, J. - M. LOGUIDICE3, A. DIOUF2, D. COURCOT1, M. GUALTIERI1, 5, M. FALL2
1 Université du Littoral Côte d'Opale, Unité de Chimie Environnementale et Interactions sur le Vivant, Dunkerque, France
The harmful effects of indoor and outdoor air pollution on health are of great concern in several regions of the world. Several epidemiological studies have established a correlation between exposure to atmospheric pollutants and the onset of respiratory pathologies. Dakar, the capital and biggest city of Senegal, is characterized by a vehicle fleet characterized by poorly efficient combustion technologies that largely contributes to the deterioration of air quality. The aim of this study was to assess individual exposure level to air pollution and its potential health impact on professionals (bus drivers and traders) and housewives in Dakar city center, where road traffic is particularly dense and in a rural area with minor traffic related emissions.
BTEX analysis revealed that toluene was the volatile pollutant with higher concentrations, ranging from 800 to 1950 µg/m3. Benzene and xylenes concentrations were from 150 to 650 µg/m3 while ethylbenzene was always around about 50 µg/m3. Benzene concentration was significantly higher for the housewives group (649 and 568 µg/m3 respectively for urban and rural sites) than for professionals (192 and 425 μg/m3 respectively for bus drivers and traders). The high concentrations found for housewives is probably due to the cooking habits (coal used in kitchens as combustible), local practices (important incense burning) and use of cleaning products that are important emitters of volatile organic compounds. Different urinary metabolites were selected to assess human exposure. t-t-MA was not detected (below the detection limit) in urine samples, while S-PMA values were higher for housewives (1.3 and 1.2 µg/g creatinine for urban and rural sites respectively) when compared to professionals (1.1 and 0.8 µg/g creatinine for traders and drivers respectively). The level of 1-HOP, used to assess a pyrene exposure, was slightly higher in drivers (0.4 µg/g creatinine) compared to others groups (0.2, 0.3 and 0.2 µg/g creatinine respectively for traders, city housewives and district semirural housewives). Statistical analysis revealed that urinary 1-HOP levels were significantly higher for urban site housewives compared to those at the semirural district. These results showed that indoor air pollution seems to be greater than outdoor air pollution, confirming previous observations on the BTEX exposure level and therefore indicating a non-negligible risk also for indoor exposure to benzene.
Keywords: Air pollution, Risk assesment, toxicology, biomarkers, Urine metabolites
Effect-directed monitoring tools as a toxicological fingerprint for ecological and human risk assessment of water bodies. (#368)
V. De Gussem1, 2, L. Schuijt3, P. van den Brink3, J. van Dijk1, M. Jonker2, N. Kramer2, A. van Wezel1, 4
1 Utrecht University, Copernicus Institute of sustainable development, Utrecht, Netherlands
Classical analytical methods to monitor water quality have their limitations: i) not all chemicals of emerging concern can be measured sensitively enough in water, ii) they do not assess the presence and levels of all unknown chemicals (e.g. transformation products), and iii) they do not assess chemical mixture effects. For these reasons, the use of bioassays to assess the health risks associated with chemical contaminants in water has been promoted these past few years. We propose a battery of sensitive, 3R-based and high-throughput bioassays, covering ecologically- and human-relevant toxicity pathways. These effect-directed monitoring (EDM) tools will assess the ecological and human health risks of chemicals in different types of water bodies taking into account the whole complex mixture of compounds (known and unknown). To do so, we linked the protection goals for humans and ecosystems described in (supra)national guidelines and a list of priority chemicals with related adverse outcome pathway (AOP). The most critical and sensitive adverse outcomes (AO) were selected, from which we compiled a set of key events (KE) to test in 3R bioassays. We chose the most suitable bioassays to measure these KEs, predicting critical ecological or human endpoints. This way, a toxicological “fingerprint” of a water sample can be obtained by testing the sample in the proposed 3R test battery. Thanks to the integrative information provided by these EDM tools, the risks assessment of water bodies should be more relevant for water quality monitoring.
Keywords: Adverse Outcome Pathways, bioassays, water pollution, ecological health, human health
A homeopathic ophthalmic ointment containing Echinacea purpurea, Euphrasia officinalis and Calendula officinalis extracts: a PDE case report. (#384)
E. GILLIO TOS1, M. D. RODDA1, A. IAVELLO1, L. BRUNASSO CATTARELLO1, A. CONTO1
1 CHEMSAFE SRL, PHARMA BUSINNESS UNIT, COLLERETTO GIACOSA (TO), Italy
In 2014, a guideline of the EMA was published introducing the concept of Permitted Daily Exposure (PDE) values. The approach for setting PDE limits is the same outlined in ICH Q3C consensus guideline on residual solvents and in ICH Q3D consensus guideline on elemental impurities. The background for that was the request of the pharmaceutical industry to be able to decide itself on a risk-based approach whether a product can be produced in a multipurpose facility, or not.
Medicinal plants are an integral part of traditional medicine since ancient era. Homeopathy is a natural form of medicine used by over 200 million people worldwide to treat both acute and chronic conditions. It is based on the principle of ‘like cures like’. In other words, a substance taken in small amounts will cure the same symptoms it causes if taken in large amounts.
The homeopathic ophthalmic ointment containing Echinacea purpurea (3%), Euphrasia officinalis (5%) and Calendula officinalis (5%) extracts is used for treatment of conjunctivitis and other inflammatory disorders of the eye and its annexes. A thin line of the ointment (corresponding to around 18 mg) has to be placed along the inside of the lower eyelid.
A common approach for the selection of the Point of Departure (POD) and Adjustment Factors (AFs) can be applied to the three extracts. The three extracts’ toxicological and pharmacological profile is poorly characterized and there are few useful quali-quantitative data to be used to allow a NOAEL or LOAEL determination, so the PDE for the three extracts is based on the lowest ocular recommended dose of 1.62 mg/day (Echinacea) or 2.7 mg/day (Euphrasia and Calendula) in adults.
Regarding selection of the AFs have to be considered: intra-species and inter-species variability, duration of therapy, adverse effects, therapeutic dose and bioavailability adjustment.
The resulting calculated ocular PDE values for each extract are the following: 162 µg/day for Echinacea purpurea extract; 270 µg/day for Euphrasia officinalis and Calendula officinalis extracts.
Keywords: PDE, ophthalmic homeopathic ointment, Echinacea purpurea, Euphrasia officinalis, Calendula officinalis
Derivation of a Point of Departure for Alkyl-Substituted Urea Compounds using a Category-Based Read-Across Approach (#385)
B. J. Lampe1
1 NSF International, Toxicology Services, Ann Arbor, Michigan, United States of America
The goal of this research is to apply a category-based read-across approach toward identifying a point of departure (POD) for the alkyl-substituted urea compounds (ASUCs) with no relevant toxicological data. The ASUCs are commonly used as reaction intermediates in the production of both polymers and pesticide products, and nearly 20 ASUCs were identified without adequate data to derive a chemical-specific POD.
Boundaries for the chemical category were established based on the presence or absence of specific structural attributes, and in order to identify ASUCs with relevant toxicological data, 13 ASUCs previously detected as drinking water contaminants by NSF International were profiled in the OECD QSAR Toolbox software (version 4.1). In this manner, 15 additional ASUCs with relevant data from OECD guideline-compliant studies were identified, which were analyzed to determine systemic and reproductive/developmental NOAELs. Additionally, this software tool was utilized to address mutagenicity and clastogenicity data gaps for ASUCs with no relevant data for these endpoints.
The available OECD guideline-compliant mutagenicity and clastogenicity data on the evaluated ASUCs, combined with data gap filling using read-across, suggests low genotoxic concern. The most conservative point of departure (POD) identified was 30 mg/kg-day, a NOAEL derived from a developmental toxicity study on 1,3-dimethylurea, which is supported by 50 mg/kg-day NOAELs from 28-day and 90-day repeated dose studies on 1,3-dimethylurea and diethyldiphenylurea, respectively. The critical effects associated with this POD included reduced maternal food intake (21%), reduced maternal body weight gain (66%), and increased incidence of fetuses with hydroureter. As the POD derived from 1,3-dimethylurea is based most sensitive adverse effect for the entire category, it is therefore adequately health-protective for read-across to other ASUCs for which no chemical-specific POD can be derived.
Keywords: Read-across, Risk Assessment, Urea Compounds, Point of Departure
Effects of Heavy Metals on Nile Tilapia (Oreochromis niloticus L.) obtained from Meycauayan River, Philippines: Studies on Health Risks, Bioaccumulation and Oxidative Stress (#396)
R. I. Guaring1, E. Espiritu1
1 Ateneo de Manila University, Department of Environmental Sciences, Quezon City, Metro Manila, Philippines
The heavy metals coming from the effluents of industrial facilities, predominant of which are leather tanning and jewelry factories, render the Meycauayan River in Bulacan, Philippines polluted. Through bioaccumulation, the heavy metals in water are eventually retained in the tissues of fishes cultured in fishponds downstream of the river such as Nile tilapia (Oreochromis niloticus L.).
This study assessed the potential risks to human health that may result from consuming Nile tilapia exposed to heavy metal contaminated waters from four (4) fishpond sites located downstream of the Meycauayan River. Tilapia samples were collected in March 2017 and were analyzed for the presence of cadmium (Cd), lead (Pb), nickel (Ni), chromium (Cr) and manganese (Mn) using atomic absorption spectrometry; and for mercury (Hg) using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Target Hazard Quotient (THQ) was used to estimate the associated health risks on long-term consumption. Biomarkers were also measured to evaluate the level of oxidative stress affecting the Nile tilapia samples.
The heavy metals chromium, nickel, lead, and manganese were found to be bioaccumulated in the edible tissues of Nile tilapia samples wherein nickel and lead concentrations exceeded international quality standards for fish and fish products. Calculated THQs show that lead (Pb) contributes the greatest risk to human health from all the pollutants that were analyzed in the fish tissues. The activity of enzyme biomarkers, catalase and superoxide dismutase and the lipid peroxidation level in tilapia were also found to be significantly elevated.
From a public health perspective, long-term consumption of tilapia obtained from Meycauayan River is not recommended due to adverse effects which may be contributed by the highly elevated level of lead.
Keywords: Bioaccumulation, Oxidative stress, heavy metals, health risk
Occupational risk assessment of pesticides with different EU dermal absorption and Ko-POEM in Korea (#408)
A. S. You1, N. Lee1, S. Park1, Y. Jo1, J. A. Oh1, Y. - K. Park1
1 National Institute of Agricultural Sciences, Agro-Food Safety and Crop Protection, Jellabukdo, Republic of Korea
The default of dermal absorption is fixed as 10% in Korean guidance unlikely to EFSA guidance (2017) for operator risk assessment of plant protection products. In case of application of EFSA guidance, the risk was considered to increase because 10% dermal absorption is minimum default. Default of dermal absorption was classified for 169 products according to EFSA guidance. 24 products were classified as 25% dermal absorption in mixing and 75% in application, 107 products as 10% in mixing and 50% in application. It was investigated to change the operator risk through assessment by different default of dermal absorption with Ko-POEM in 223 cases of 169 products by crops. 33% of cases without PPE and 19% of cases with PPE were indicated the risk was high in Korean guidance. 61% of cases without PPE and 52% of cases with PPE were indicated the risk was high in EFSA guidance. The cases of using oral absorption were 40 and 5 cases among these were indicated that the risk was low. As a result, the high risk assessed by the different default of dermal absorption was increased 2.6 times compared with only 10% default
Keywords: dermal absorption, exposure, operator, assessment
Life-course exposure and physical activity impact on genomic biomarkers of a group of older adults (#414)
A. Teixeira-Gomes1, 2, B. Lage1, 2, S. Silva2, F. C. Esteves1, 2, J. Carvalho3, V. Valdiglesias1, 4, J. P. Teixeira1, 2, S. Costa1, 2
1 University of Porto, EPIUnit – Instituto de Saúde Pública da Universidade do Porto, Porto, Portugal
The world’s population is ageing rapidly. Elderly are a well-recognised susceptible population due to the decline of immune defences and the burden of multiple chronic diseases. As a susceptible population, the burden of environmentally induced disease and lifestyle factors in the elderly is an increasing concern. The major public health goal for older adults is to prevent disability and to enhance active life expectancy. Several studies established a link between genomic instability and age and age-related disorders. Physical activity, on the other hand, has been demonstrated to be effective in the prevention and treatment of different chronic conditions, including age-related disorders. Thus, it is hypothesised that physical exercise programs should impact some biological endpoints. The present study sought to investigate the impact of regular exercise in the evaluation of genomic biomarkers in the elderly. Lifestyle factors were also addressed to account for its possible influence in the results obtained. A group of around 70 older adults (≥65 years old) was engaged in this longitudinal study. A physical exercise programme of 9 months was ministered to the participants. Blood samples were collected before and after the physical exercise programme for genomic biomarkers evaluation. Primary DNA damage and oxidative DNA damage were measured through comet assay and H2AX phosphorylation was measured through gH2AX assay. A life-course exposure questionnaire was also included to characterise and identify lifestyle factors and key exposures. Data here obtained addresses the differences between the levels of primary DNA damage, oxidative DNA damage and H2AX phosphorylation before and after the physical exercise programme. The influence of key exposures and life-style factors, such as the consume of home-produced vegetables, living near traffic zones, smoking habits, etc, was also observed. The ageing phenomenon offers great challenges for all societies, being important to ensure that the increase in longevity is accompanied by the preservation of health and good quality of life. Obtaining other conclusions on the biological effect of regular exercise may help to clarify its role in prevention of age-related diseases. Acknowledgments:Armanda Gomes and Solange Costa are supported by FCT under the grants SFRH/BD/121802/2016 and SFRH/BPD/100948/2014, respectively. Vanessa Valdiglesias is supported by Xunta de Galicia postdoctoral fellowship (ref. ED481B 2016/190-0).
Keywords: Biomarkers, Older adults, Life-course exposure, Physical exercise
Exposure reducing remedies in cytotoxic drug preparation. (#416)
M. D. Karakoç1, B. Y. Taşköylü1
1 Denizli Public Hospital, Zafer Goksin Oncology Centre, Denizli, Turkey
Purpose: Among anticancer agents, cytotoxic drugs display nonspecific activity towards both tumor and the normal cells which is responsible for their numerous and severe side effects. The toxicity of these agents depends on several parameters like their intrinsic carcinogenic character, concentration in tissues and their length of contact with the same tissues. Then, in case of fortuitous contamination of healthy people, cytotoxic agents can cause acute and/or delayed toxicity. Consequently, preparation of cytotoxic drugs by healthcare workers exposes them to a professional contamination risk. The aim of the study is reducing the healthcare workers duration of exposure to the cytotoxic agents without increasing the unused dose wasting during preparation by avoiding the use of small size vials of five different drugs that frequently used in oncology centers.
Methods: All antineoplastic agents are prepared in negative pressurized clean rooms with using closed system transfer devices throughout the study. Doxorubicin (10 and 50 mg), epirubicin (10 and 50 mg), cisplatin (10, 50 and 100 mg), oxaliplatin (50, 100 and 200 mg) and carboplatin (50, 150 and 450 mg) wasted doses and preparation time (potential exposure time) were measured for six months. The usage of small dose vials which causes prolongation in preparation time has been avoided and larger dosage forms were preferred as much as possible in last trimester. Wasted drug dose amounts and preparation periods were recorded. Subsequently, the data compared between the first and the last trimesters.
Results: The total preparation period of the selected antineoplastic agents with different size vials was 1407 minutes. In the second trimester, which avoided the small size vial usage as much as possible it is also prepared in 1058 minutes. There was no significant changes between the first and the last trimester of the study in terms of patients numbers that receiving chemotherapy ( 504 and 505 patients respectively) and the rate of unused drug doses extermination (totally 840 mg and 782 mg respectively) (p>0.05). On the other hand, drug preparation time, in other words the health employee's exposure period to cytotoxic drugs was reduced 24.81% (349 minutes).
In the study, it has been shown that by making rational choices about drug usage can reduce the healthcare workers duration of exposure to the cytotoxic agents without increasing the unused dose wasting during preparation.
Keywords: Cytotoxic, antineoplastic, exposure, toxicity, healthcare worker
An Evaluation of U.S. Forest Service Worksheets and their usefulness in modern risk assessment for exposure to pesticides. (#425)
K. Phang1, D. Bonnar1
1 Blankinship & Associates, Davis, California, United States of America
The U.S. Forest Service Risk Assessment Worksheet Tool (FSWT) is a highly sophisticated Microsoft Suite based application developed to perform risk evaluations based on toxicity values, chemical properties, water and off-site soil contamination rates, and built-in exposure assumptions. The FSWT is a set of interlinked word documents, excel spreadsheets, and database that prompt and guide a user through the risk assessment process. Risk assessors are presented with the challenge of evaluating risk in a variety of situations. Computational tools such as the FSWT can expedite the risk evaluation process and allow comparisons to be made under multiple scenarios.
This talk compares the FSWT to other current risk assessment methodologies. The FSWT has unique features that provide both advantages and limitations. These advantages and limitations will be presented along with proposed enhancements to improve the FSWT.
Keywords: database, computational, risk assessment, pesticides
Degradation of cyclophosphamide using plasma activated water and UV/H2O2 for the abatement of persistent pharmaceuticals in wastewater (#459)
M. H. Graumans1, W. F. Hoeben2, F. G. Russel3, P. H. Leenders4, P. T. Scheepers1
1 Radboud Institute for Health Sciences, Radboudumc, Nijmegen, Netherlands
Background: With the increase of unwanted pollutants in the environment, this study focuses on the abatement and release of pharmaceuticals in the wastewater stream by using the new plasma-activated-water (PAW) technology. PAW is a promising technique for the pre-treatment of contaminated waters, Electric gas discharges in air over water produces reactive hydroxyl and nitrogen species with oxidative degradation capacities.
Purpose: To evaluate the treatment effect of PAW and UV-C light combined with hydrogen peroxide (UV/H2O2) for the oxidative degradation of cyclophosphamide (CP).
Methods: Different CP concentrations were spiked in tap water and subsequently treated with the advanced oxidation processes (AOPs) PAW and UV/H2O2. Plasma activation of aqueous CP solution was applied at 50, 100 and 150 W electric power input. The UV/H2O2 oxidation process was carried out by adding approximately 10 mg/L 30% (v/v) H2O2 to the samples and by placing an UV-C lamp 20 cm above the solution for immediate radiation. Both AOPs were tested for 120 min. and analysed by LC-MS/MS to determine the removal rate and possible formation of degradation products.
Results and discussion: PAW removes CP at a rate of 94.6 % at 100 W and 100 % at 150 W within 120 minutes. With UV/H2O2 treatment, within 60 min. CP was almost completely degraded (> 0%). Although this rapid degradation of CP indicates that a photochemistry process is more efficient, it is demonstrated that multiple oxidative reaction by-products are formed compared to PAW treatment. UV/H2O2treatment produce compounds such as 4-keto-CP, 4-hydroxyperoxy-CP and carboxyphosphamide, whereas only 4-keto-CP was seen with PAW oxidation. Identification of by-products and the reaction kinetics indicate that two studied approaches results in distinct patterns of oxidative degradation. UV/H2O2 oxidation can be explained as a pseudo-first order reaction where PAW oxidation shows a bi-model degradation of the pseudo-first order reaction.
Conclusion: The oxidative degradation of CP in spiked tap water shows efficient removal rates indicating that PAW is a promising alternative methodology for the elimination of CP from wastewater. Further research should reveal whether PAW may be suitable for the removal of other persistent pharmaceuticals from wastewater.
Keywords: oxidative degradation, medicine residues
Human induced pluripotent stem cell-derived neurospheres develop into electrically active neuronal networks which suit as an alternative method to study neurotoxicity in vitro (#472)
L. Nimtz1, M. Hofrichter1, Y. Kabiri1, S. Theiss2, J. Adjaye2, E. Fritsche1
1 IUF - Leibniz research institute of environmental medicine, Modern risk assessment and sphere biology, Duesseldorf, North Rhine-Westphalia, Germany
For improving hazard and risk assessment processes, a paradigm switch in toxicity assessment from apical endpoint evaluation in rodents towards a mode-of-action (MoA)-based testing in human-relevant in vitro methods was proposed. This switch was inspired by the high attrition rates in new drug development when moving from the preclinical phase of animal experimentation into clinical trials with humans. Therefore, it is thought that in vitro methods based on human cells might increase predictivity of compound testing for humans due to the elimination of species differences. One field with amongst the highest attrition rates concerns CNS diseases. Thus, such human-relevant alternative methods are needed that have the ability to predict adverse effects on the nervous system. We are using human induced pluripotent stem cells (hiPSC) for neural induction into 3D neurospheres, which consist of neural progenitor cells (hiNPC). Upon plating on laminin-coated single- or multi-well microelectrode arrays (MEAs), hiNPC differentiate into neurons and astrocytes and thereby form electrically active neuronal networks. In contrast to neuronal networks derived from rat NPC, hiNPC-derived networks exhibit less electrical activity (spikes and bursts) and synchronicity over differentiation time. To improve hiNPC network activity and maturation we established a differentiation medium with maturation-supporting supplements (CINDA medium). HiNPC differentiated with CINDA medium need shorter times for first network activities, display higher spike as well as burst frequencies and show network synchronicity. Challenging of networks with pharmacological compounds indicates presence and functionality of synapses and inhibitory and excitatory receptors. On the molecular level, CINDA medium induces gene expression of NSE2, GFAP, as well as synapse-, receptor- and neuronal subtype-specific markers compared to medium without CINDA supplements. These data suggest that hiNPC-derived neuronal networks formed in presence of CINDA medium are a promising system for setting up test methods for neurotoxicity evaluation.
Keywords: in vitro, brain development, stem cell, MEA, neurotoxicity testing
USAGE PATTERNS OF COSMETIC PRODUCTS BY PREGNANT WOMEN (#490)
T. Nguyen1, M. - P. Berrada-Gomez1, A. Rielland2, M. Bellec2, S. Gil3, D. De Javel2, P. - J. Ferret1
1 Pierre Fabre Dermo-Cosmetique, Safety Assessment Department, Toulouse, France
Purpose: Nowadays, personal care products are widely consumed by all categories of persons. The European Regulation (EC) N°1223/2009 concerning cosmetic products defines pregnant women as a vulnerable population. Thus, a specific risk assessment with accurate exposure data is required. However, exposure values from SCCS guidelines do not consider pregnant women and little information is available in the literature. The aim of this study was to obtain frequencies of use and to identify potential disparities in cosmetic habits during and outside pregnancy period.
Methods: 111 adult pregnant women (mean age = 32 y/o) from 7 different regions of France answered a web-based or paper form survey distributed by healthcare professionals (gynecologists, midwives, pharmacists and physiotherapists). The questionnaire was in-house developed, including items about socio-demographic and pregnancy characteristics and focusing on frequencies of use of 37 cosmetic products, selection criteria, purchasing places and safety perception of personal care products during and outside the pregnancy.
Results: The percentage of women expecting a baby who modified their frequency of use was obtain for each product. Some products were less frequently used during the pregnancy: nail polish remover (46%), nail polish (44%), perfume (32%), foundation (27%), face mask (26%), lipstick (25%), mascara (23%), hair dye product (23%) and eye pencil (22%). On the contrary, pregnant women increased their frequency of use of stretch-marks care (68%), hydrating body cream (40%), bust care (26%) and massage oil (23%) indicating these products are commonly used during this specific period. In addition, pregnancy appears to have an impact on selection criteria of cosmetic products. The most reported criterion of choice outside pregnancy is the price while during pregnancy it is the presence of natural ingredients. This study provides frequencies of use of cosmetic products and highlights the influence of pregnancy on usage patterns which could be useful for the risk assessment of products dedicated to pregnant women.
Keywords: pregnancy, cosmetic product, usage pattern, exposure, risk assessment
Investigation of the effects of alternative flame retardants on embryonal and fetal neurodevelopmental processes using 3D human in vitro models (#498)
J. Klose1, F. Bendt1, K. Dach1, U. Hübenthal1, B. Kühne1, L. Nimtz1, M. Schmuck1, E. Fritsche1
1 IUF-Leibniz Research Institute for Environmental Medicine, Modern risk assessment and sphere biology, Düsseldorf, North Rhine-Westphalia, Germany
Flame retardants (FRs) inhibit or delay the spread of fire by suppressing the chemical reactions in the flame or by forming a protective layer on the surface of a material. In the past decades, FRs like brominated diphenyl ether-99 (BDE-99) have been identified as threats to human health, especially to the developing brain. Less persistent alternative flame retardants (aFRs) have been released onto the market, however, toxicities of these compounds have not been investigated yet. Therefore, it is essential to assess the developmental neurotoxicity (DNT) potential of these aFRs.
We have developed two in vitro 3D neurosphere models, i.e. human second trimester neural stem/progenitor cells (NPCs) and human induced pluripotent stem cell (hiPSC)-derived NPCs. Both represent distinct processes of brain development mimicking specific neurodevelopmental key events (KE), e.g. proliferation, migration, differentiation into neural effector cells (astrocytes, neurons and oligodendrocytes), interference with thyroid hormone-dependent oligodendrocyte maturation and the formation of functional neuronal networks. The two FR Tetrabrombisphenyl A (TBBPA) and BDE-99 interfere with some of these neurodevelopmental KE within the testing battery. Currently, the adverse effects of selected aFRs (Tris(chloroethyl)phosphate (TCEP), Triphenylphosphate (TPHP), Tris(2-butoxyethyl)phosphate (TBOEP) and Bis-(2-butoxyethyl)phosphate (BBOEP)) are studied.
Hence, our assays represent an array of neurodevelopmental KE that can be used for the DNT assessment of aFRs. In addition, they are under discussion to be a part of an in vitro DNT testing battery for compound screening at the OECD level.
Keywords: in vitro, Developmental Neurotoxicity, 3D in vitro model, alternative flame retardant, induced pluripotent stem cells, human progenitor cells
Cosmetic Europe’s Long Range Science Strategy – A Non-Animal Safety Assessment Case Study for Phenoxyethanol, a Cosmetic Ingredient (#500)
M. Nepelska3, M. Cronin2, R. Cubberley3, M. Dent3, J. Firman2, J. Fisher4, G. Kenna7, C. Mahony5, B. Nicol3, S. Piechota3, K. Przybylak3, A. Schepky6, S. Tozer5, J. Troutman4, B. Desprez1
1 Cosmetics Europe, Science and Research Department, Brussels, Belgium
Cosmetics Europe’s Long Range Science Strategy (LRSS) focusses on developing approaches allowing non-animal safety assessments for cosmetic ingredients. Case study safety assessments are conducted to evaluate and gain experience of using new approaches when making safety decisions. We present an example of an exposure-based ab initio case study for a cosmetic ingredient associated with significant systemic exposure, with the purpose of replacing repeat dose toxicity animal testing.
The Scientific Committee on Consumer Safety (SCCS) assessed phenoxyethanol as safe in cosmetics at its current use level (up to 1% in rinse-off and leave-on cosmetics). This case study aims to deem if this conclusion can be drawn using only data from non-animal approaches.
We used the tiered SEURAT-1 ab initio workflow, and each tier can allow safety decisions. Information used includes traditional safety assessment approaches such as toxicological threshold of concern (TTC) and read across, and then moves into in silico predictions (Tier 0), internal TTC based on bioavailability and formation of mode of action hypotheses using available mechanistic data such as ToxCast and transcriptomics (Tier 1). Tier 2 focusses on targeted non-animal testing, biokinetic refinement and calculating points of departure.
In Tier 0 we considered the exposure scenario and available non-animal data. We determined that the level of exposure could not be supported using TTC but were able to perform a read across risk assessment for some health effects e.g. skin sensitisation, but not for systemic effects due to lack of data on analogues.
In Tier 1 we used an aggregate exposure input and predictions from physiologically-based biokinetic modelling to make predictions of systemic exposure. We considered which modes of action may be relevant to the case study ingredient by performing in silico predictions and using or generating high throughput & high content information.
Tier 2 involves assessing if modes of action are likely to be relevant at the systemic exposures experienced following the use of cosmetic products and determining whether further testing is needed to reach a final conclusion on the safety assessment.
We believe that this ab initio workflow is useful in guiding non-animal mechanistic and exposure-based safety assessment. Moreover, transparently documenting the logic of the risk assessment can improve confidence in decision making.
Keywords: Ab initio; safety assessment; non-animal methods; (Q)IVIVE; TTC; read across
Human exposure to disinfection by-products in indoor swimming pools - looking at the genotoxic effects (#504)
F. C. Esteves1, 2, A. Amaro1, 2, S. Silva1, C. Costa1, 2, S. Costa1, 2, J. P. Teixeira1, 2
1 National Institute of Health, Environmental Health Department, Air and Occupational Health Unit, Porto, Portugal
Disinfection of pool water is an extremely important process that ensures the safety of its use avoiding water-borne infections caused by microbial pathogens. However, the chlorine used as disinfectant, in the presence of organic matter, may generate disinfection by-products (DBPs), including trihalomethanes (THMs), a class that includes genotoxic compounds, which are inhaled and absorbed by the skin. Previous studies have shown that exposure to these DBPs are associated with adverse health outcomes.
The aim of this study was to assess DNA damage in swimming pool users exposed to THMs. To evaluate DNA damage, blood samples were collected from a group of approximately 150 swimmers in different indoor chlorinated pools located in north of Portugal. Levels of DNA strand breaks and oxidized purines (formamidopyrimidine DNA glycolase) sites were determined in lymphocytes using the comet assay. Individual exposure to THMs compounds was analysed using a predictive model; analysis included not only total THMs but also chloroform, dibromochloromethane, bromochloromethane and bromoform separately.
Data obtained will increase knowledge on the potential genotoxic risks associated to THMs exposure in indoor swimming pools. Furthermore, results may be useful to perform a human health risk assessment model essential for development of risk management strategies.
This work was supported by Project NORTE-01-0145-FEDER-000010 - Health, Comfort and Energy in the Built Environment (HEBE), cofinanced by Programa Operacional Regional do Norte (NORTE2020), through Fundo Europeu de Desenvolvimento Regional (FEDER)
Keywords: DNA damage, comet assay, exposure model, trihalomethanes, swimming pools
Genetic damage in young swimmers exposed to pool disinfection by-products (#505)
R. D. N. Amaro1, 2, F. Esteves1, 2, S. Silva1, C. Costa1, 2, S. Costa1, 2, J. P. Teixeira1, 2
1 National Institute of Health, Environmental Health Department, Air and Occupational Health Unit, Porto, Portugal
Swimming is seen as an activity with innumerous wellbeing and health benefits. For the maintenance of the quality of swimming pools’ water, and consequently for the safety of its users, there is a need to add disinfection products. The interaction between these products and organic matter, naturally introduced in the water by users, leads to the formation of disinfection by-products (DBPs), many of them with mutagenic and carcinogenic characteristics. Despite the relevance of this issue in public health, there is limited research on the subject.
The aim of this study was to evaluate genetic damage levels in young federated swimmers exposed to pool DBPs. A sample of urine, buccal mucosa and blood was collected from 12 young federated swimmers at the end of season competition period. Samples of a control group of 12 healthy individuals, matched for age and gender were also collected. Genetic damage was analyzed using CBMN assay in peripheral blood lymphocytes and micronucleus assay in buccal and urothelial cells. Alkaline and enzyme (FPG) versions of comet assay were also performed in order to measure the primary and oxidative DNA damage in lymphocytes.
Younger individuals constitute a susceptible group and their exposure to genotoxic compounds may lead to health problems later in life. Despite some limitations, results here obtained contribute to the understanding of the possible genotoxic effects of DBPs in children providing support for possible implementation of coherent measures for disease prevention in early stages of life.
This work was supported by Project NORTE-01-0145-FEDER-000010 - Health, Comfort and Energy in the Built Environment (HEBE), cofinanced by Programa Operacional Regional do Norte (NORTE2020), through Fundo Europeu de Desenvolvimento Regional (FEDER).
Keywords: Swimming pools, disinfection by-products, genotoxicity, young swimmers
Guidance for the Risk Assessment of Enzyme-Containing Consumer Products (#508)
F. H. Kruszewski1, B. Concoby2, M. Simonsen3, R. Bookstaff4, M. Baccam4, J. Paschal5
1 American Cleaning Institute, Washington, D.C., United States of America
The American Cleaning Institute’s (ACI) “Guidance for the Risk Assessment of Enzyme-Containing Consumer Products” provides valuable information on the potential health hazards of enzymes used in consumer products and provides a framework for manufacturers of these products to conduct risk assessments to help ensure the safety of new products containing enzymes. Enzymes like many other proteins can act as allergens and induce the production of enzyme-specific IgE antibody upon repeated inhalation or exposure to mucous membranes that may lead to allergy symptoms, including asthma. The primary challenge associated with enzyme use is preventing the generation of enzyme-specific antibody and the development of symptoms of respiratory Type 1 hypersensitivity. This hazard is the primary focus for risk assessment of enzymes, and it must be managed carefully. Another hazard that should be addressed is primary irritation of the eye and skin which can be caused by enzymes belonging to the class of proteases. Only enzymes of the class of protease will have this irritating effect.
It is recommended that companies using enzymes in consumer products responsibly consider how they are managing enzyme safety, including the conduct of appropriate risk assessments and risk management programs. Such programs will include measures to manage exposures to enzymes. Program designs should be developed on a case-by-case basis to address parameters specific to the type of product and its applications. Experience in the cleaning products industry demonstrates that the potential risk of adverse effects can be successfully managed by identifying the hazards, carefully assessing exposure, characterizing the risk and then applying appropriate risk management. An updated ACI guidance document provides risk assessment approaches and risk management strategies that have been used successfully by the cleaning products industry and other industries.
Keywords: enzymes, risk assessment, consumer products, cleaning products, respiratory sensitizatiion
Ad hoc risk assessments by Front Office Food and Product Safety (#520)
S. Jeurissen1, R. de Jonge1, G. Schuur1, P. Keizers1, R. Hoogenboom2
1 National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands
In case of urgent food and consumer product safety questions, it is important that the relevant authorities and ministries receive an independent and unambiguous assessment of the possible health risks within a short time frame. On the basis of these urgent risk assessments, authorities can take preventive and protective risk management measures.
In the Netherlands, the Front Office Food and Product Safety is operated by the National Institute of Public Health and the Environment (RIVM) and RIKILT – Wageningen University and performs ad hoc (rapid) risk assessments commissioned by the Office for Risk Assessment and Research (BuRO) of the Netherlands Food and Consumer Product Safety Authority (NVWA). These assessments are the basis of the advices of BuRO for the ministries or the Inspector General of the NVWA.
The Front Office has been established to answer ad hoc questions on the chemical and microbiological safety of food and feed, and on genetically modified organisms. In addition, questions regarding chemical substances in consumer products such as toys and cosmetics are answered. The Front Office also conducts chemical analysis and risk assessment of food supplements.
Approximately 30 ad hoc risk assessments are commissioned each year. In general, these requests result from specific cases or occasions. Examples are results of analyses by NVWA and/or RIKILT, questions from the ministries, information from industry and topics in the media.
The risk assessments usually need to be performed within a short time frame and are based on available analytical results, data on maximum (use) levels in food, feed and consumer products, legal criteria, (existing or newly derived) health-based guidance values and information on exposure. In some cases, a risk assessment has to be based on very limited information. Where applicable, limitations of the assessment and their impact on the outcome are specified.
Recent topics for ad hoc risk assessments include fipronil in eggs, mineral oils in cheese biscuits, recreational use of nitrous oxide (laughing gas) and the possible extension of shelf life for perishable products (eggs). More information on previous assessments can be found at rivm.nl.
Keywords: Rapid risk assessment, food safety, consumer product safety, microbiology
Monitoring of pesticides in Sais groundwater of Morocco for human health and ecotoxicological risk assessment (#528)
I. Berni1, I. El Ghazi1, A. Menouni1, R. C. Duca2, L. Godderris2, 3, S. El Jaafari1
1 Moulay Ismail University, Cluster of Competency “Environment and Health”, Meknes, Morocco
Pesticides in Morocco are widely used in agriculture. Their usage may pose risk to human health and environment. The aim of this study was to monitor pesticides levels in groundwater from Sais plain in Morocco during summer 2017 and winter 2018. For this purpose, passive samplers were deployed between 14 to 20 days in 21 traditional wells from the study area. Twelve pesticide compounds were analyzed including fungicides, insecticides, herbicides and their metabolites. Moreover, based on the pesticide monitoring results we performed a human health risk assessment for both adults and children using the model described by Hu et al. (2011), as well as an ecotoxicological risk assessment using Risk Quotient (RQ) method (Vryzas et al., 2009). RQ was predicted based on the ratio between the measured environmental concentration and the predicted no effect concentration. In the study area, seasonal variations of pesticides concentrations were observed in all wells. The highest concentrations were recorded during summer campaign, right after pesticide application, especially chlorpyrifos methyl (0.1 μg/L) and chlorpyrifos ethyl (0.015 μg/L). Based on the human health risk assessment, pesticides in groundwater of Sais plain posed low potential non carcinogenic risk on human through drinking water as exposure route, except for chlorpyrifos methyl in the case of children. In this case, the ratio of the potential exposure dose to the chloprpyrifos methyl and the level at which no adverse effect are expected was greater than one (1.6), due to this compound adverse health effect were possible. In addition, simazine showed unacceptable carcinogenic risk estimated 4.51 x 10-6. High ecotoxicological risk for two pesticides was observed according to the RQ approach, namely chlorpyrifos methyl and chlorpyrifos ethyl (i.e. 462.6 and 1.12 respectively). The coupling of environmental monitoring data to probabilistic human and ecotoxicological risk estimates showed that cancer risk is unlikely due to drinking water but there is a high ecotoxicological risk.
Hu, Y., Qi, S., Zhang, J., Tan,L., Zhang, J., Wang, Y., Yuan, D., 2011. Assessment of organochlorine pesticides contamination in underground rivers in Chongqing, Southwest China. J. Geochem. 111, 47–55.
Vryzas, Z., Vassiliou, G., Alexoudis, C., Papadopoulou-Mourkidou, E., 2009. Spatial and temporal distribution of pesticide residues in surface waters in north- eastern Greece. Water Res. 43 (1), 1–10.
Keywords: Human health assessment, ecotoxicological assessment, pesticide, groundwater, passive samplers.
Exploring the presence of ESBL-producing bacteria in different aquatic ecosystems in England (#546)
U. Anjum1, R. Reid1, H. Hoosen1, M. C. Lobo-Bedmar2, A. Peña-Fernández1
1 De Montfort University, School of Allied Health Sciences, Leicester, United Kingdom
Recent evidence has shown an increasing presence and distribution of extended-spectrum beta-lactamases (ESBLs) in the environment, which could represent a threat to public health. However, their presence in open water systems in England remains unknown despite the significant role of water in disseminating contamination and as a reservoir for biological hazards. Therefore, three sets of 30 water samples each were collected from different open water environments in Leicestershire (UK) including Leicester city in three periods of time (summer, autumn and winter) in 2017. Water samples were collected in the same locations each season using a portable water pump connected to a foam filter module according to the 1623 method developed by the US Environmental Protection Agency. The environments monitored were: the course of the River Soar and the Grand Union Canal (a canalised section of the River Soar) throughout the city including the River Biam; lakes highly frequented for fishing or leisure (e.g. John Merricks' Lake, Kings Lear’s Lake; Bennion Pools Fishing Lake); and a marina. The River Soar is rich in wildlife and attracts large numbers of users. Water samples were concentrated using the IDEXX® Filta Max system according to manufacturer's instructions and 1623 method. The DNA was extracted from each concentrated water sample with a Fast DNA® Kit. Genotypic prevalence of ESBL-producing bacteria was investigated by means of multiplex PCR. The assay was performed to detect the ESBLs: blaTEM, blaSHV, blaOXA and blaCTX-M using a CTX-M-15 producing strain of Escherichia coli as a positive control. All 90 samples assessed for ESBLs were negative. However these results should be considered inconclusive, as different factors such as dilution in the surface water could have affected them, as we have previously detected the presence of ESBLs (specifically CTX-M-15) in animal faecal samples (6 waterfowls, 3 dogs and 2 foxes) collected in different parks in Leicester city highly frequented by the public in 2016. Further studies will be needed to determine the presence and distribution of ESBLs in the open water systems in England as the World Health Organisation has highlighted ESBLs as a priority to be controlled to response to the bacterial resistance phenomenon. Information about the presence and distribution of human-pathogenic bacteria harbouring ESBL enzymes is needed to develop national intervention strategies to protect the public.
Keywords: ESBLs, CTX-M, open water systems, Leicestershire, E. coli antibiotic resistance
Developing an adverse outcome pathway framework for carcinogenicity from alerts in an expert rule-based system (#760)
R. Foster1, A. Cayley1, L. Fisk1, S. Guesne1, S. Canipa1, S. Kane1
1 Lhasa Limited, Leeds, United Kingdom
Derek Nexus is an in silico, expert rule-based system capable of predicting for multiple toxicity endpoints. Accompanying the predictions are informative descriptions highlighting known toxicants and a plausible biological mechanism of action. Structural alerts are currently organised and presented to the user according to a hierarchy of toxicity endpoints, an approach which has proved useful for numerous use cases.
An alternative approach to organising this knowledge is the use of adverse outcome pathways (AOPs). An AOP is a mechanistic representation linking a molecular initiating event (MIE) to an adverse outcome (AO) through sequential key events (KEs). KEs are described at a defined level of biological organisation and linked together according to known biological mechanisms which are supported by empirical evidence. Similar knowledge is contained in Derek Nexus, having been collated by the alert writer.
In this work we sought to rearrange the knowledge already contained in Derek Nexus relating to the AO of carcinogenicity, into a format more compatible with an AOP framework. Accompanying information relating to the mechanism of action for structural alerts from endpoints related to carcinogenicity (carcinogenicity, chromosome damage, mutagenicity) was analysed and MIEs were assigned to the alerts where possible. MIEs linking a compound class to carcinogenicity were assigned to 268 alerts in this way. The alerts and their relationship with MIEs were then used to profile a carcinogenicity data set to establish which MIEs are most prevalently associated with the data and which may be the most predictive of the AO.
Organising knowledge in this way means that it can be reused and combined with other information sources such as experimental assay data and pharmacokinetic studies more effectively. The grouping of hazards according to MIE may also be more appropriate in current regulatory use cases. In the future we expect the reformatted alerts will ultimately support development of a network of MIEs and KEs that also relate to other toxicological endpoints and increase the application of Derek as a tool for risk assessment across multiple endpoints of concern.
Keywords: Adverse outcome pathway (AOP), Alternative approach, Carcinogenesis, in silico, Risk assessment
Evaluation Of The Dose-response And Fate In The Lung And Pleura Of Chrysotile Containing Brake Dust Compared To Chrysotile Or Crocidolite Asbestos In A 28 Day Inhalation Toxicology Study (#559)
B. Toth2, D. M. Bernstein1, R. A. Rogers3, P. Kunzendorf4, H. Ernst5
1 Consultant in Toxicology, Geneva, Switzerland
This study was designed to provide an understanding of the biokinetics and potential toxicology in the lung and pleura following inhalation of brake dust (from brakes manufactured with chrysotile) in a 28-day repeated multi-dose inhalation toxicology study (6 h/d, 5 d/wk. 4 wk) followed by 28-days of exposure free recovery and served as a range finding study for a subsequent ongoing 90-day repeated dose inhalation toxicology study with lifetime recovery. Comparative fiber control groups were included of a similar grade commercial chrysotile as used in the brakes and a commercial crocidolite asbestos sample. The aerosol fiber distribution of the chrysotile (chry) and crocidolite (croc) asbestos were similar (Fibers L>20 μm/cm3: Chry-LD 42, Chry-HD 62; Croc-LD 36, Croc-HD 55; WHO fibers/cm3 Chry-LD 192, Chry-HD 219; Croc-LD 211, Croc-HD 255). The total number of particles in the aerosol of the brake dust groups was similar to that in the chrysotile groups (Part/cm3: B-D 710 - 1065; Chry 532 - 1442). A macrophage dose-response to the brake dust groups was observed with no fiber related effects. In the fiber control groups, the study differentiated between similar exposures to chrysotile and crocidolite asbestos. The chrysotile exposure resulted in a dose-dependent particle laden macrophage response characterised as Wagner Grade 1 to 3. In contrast, following crocidolite exposure there was a dose-response accumulation of fiber laden macrophages and interstitial fibrosis during exposure (Wagner score Grades 3 to 4) which increased in incidence following exposure (Wagner score Grade 4). Confocal microscopy images (determined following deep freezing of the chest wall at sacrifice) revealed no difference between the air control, brake dust and chrysotile high-dose exposure groups and no difference in the visceral or parietal pleura thickness at 28 or 56 days. The crocidolite exposure resulted in more than double the thickness of the visceral pleura and parietal pleura, with associated extensive inflammatory response and collagen development observed including adhesions between the visceral and parietal surfaces. These results provided a basis for the design of the 90 day study. (Study funded by Honeywell International Inc).
Keywords: respiratory toxicology, inhalation toxicology, lung, pulmonary, respiratory system, Brake dust, Chrysotile, Crocidolite
Effect of the herbicide paraquat in larval phase of the Bullfrog (#779)
A. C. Razo Estrada1, B. G. Jiménez-García2, N. - A. Romero Pérez1, R. D. C. Guzmán-Ibarra1
1 Instituto Politécnico Nacional, Mexico City, Mexico
Paraquat is a non-selective herbicide commonly used in the world to eliminate weeds in agricultural fields. Its use has benefits, however, it has been described that exposure to paraquat generates adverse consequences depending on the concentration and time of exposure. Acute exposure has reportedly caused the death of workers and the subacute exposure causes damage to the respiratory and central nervous system in humans and animals. It has not been fully described what the mechanisms of toxicity of paraquat are, but it has been shown that the herbicide can increase the production of ion superoxide (O2.-), which has resulted in an increase in the generation of reactive oxygen species capable of oxidizing molecules necessary (NADPH and reduced glutathione) for proper cell functioning. In addition, it has been described that paraquat is capable of generating nitric oxide (NO) that can interact with the superoxide ion, generating peroxynitrite (ONOO-) causing cellular damage. The application of herbicides contributes to an increase of productivity in the agricultural fields, however, the inappropriate use has negative consequences in cultivation sites and nearby bodies of water where many species live. The American Bullfrog (Lithobates catesbeianus) is an amphibian that develops both in aquatic and terrestrial environments so it can be affected by herbicides. The acute toxicity of paraquat was determined at 48 hours of exposure, as well as the antioxidant response that includes the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and Lipoperoxidation (TBARS) in a concentration non-lethal of 1.13 mg / L for 6, 12, 24 and 48 hours of exposure in bullfrog tadpoles. The median lethal concentration of paraquat during 48 hours of exposure was 11. 30 mg/L. To assess enzymatic activity it was observed that SOD activity increased after 12 hours and remains so until the end of the time of exposure; CAT activity increased only at 6 hours, GPx showed no changes; regarding the TBARS, an increase was observed from 6 hours until the end of the exposure time. It can be concluded that at the sublethal concentration of 1.13 mg / L of paraquat, oxidative stress is generated in bullfrog tadpoles.
Keywords: Parquat, Larval Phase, Oxidative Stress, Amphibian, Enzymatic Activity
The usefulness of BALB/c strain mice in the LLNA: BrdU-ELISA (#585)
D. Krakowian1, A. Drzewiecka1, D. Gądarowska1, M. Paleczny1, J. Faron1, I. Mrzyk1, K. Gruszka1, R. Sornat1, M. Wołany1, A. Daniel-Wójcik1
1 Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Toxicological Studies, Pszczyna, Poland
The Local Lymph Node Assay (LLNA): BrdU-ELISA utilises 5-bromo-2-deoxyuridine (BrdU) is an ELISA-based (Enzyme-Linked Immunosorbent Assay) test system to measure lymphocyte proliferation. The LLNA: BrdU-ELISA has been validated and is recommended as useful method for identifying skin sensitizing and non-sensitizing test substances (at the last point of the AOP strategy). The current OECD Test Guideline no. 442B (2010) for the conduct of the LLNA recommends the use of only CBA/J strain mice for the assessment of skin sensitization potential for chemicals. However, other strains can also be used when sufficient data are generated to demonstrate that significant strain-specific differences response do not exist. Thus, this study was conducted to determine whether BALB/c strain mice can also be used in the LLNA:BrdU-ELISA.
In this study, two chemicals with known sensitizing properties (hexyl cinnamic aldehyde and eugenol; both in acetone: olive oil (4:1, v/v)) were examined at two concentrations (25 % and 10 %). Female CBA/J (Jackson Laboratory) and BALB/c (Medical University of Białystok) mice were randomly allocated to groups (four mice per group). Mice were topically treated with chemicals or vehicle on both ears for 3 days. Following intraperitoneal injection of BrdU, bilateral lymph nodes were isolated separately and undergone ELISA (Cell Proliferation ELISA, BrdU kit; Roche) to determine the level of BrdU incorporated to lymph node cells. The Stimulation Index (SI) was calculated and statistical analysis was performed.
The results revealed no significant difference between CBA/J and BALB/c at all concentrations. The average SI values were similar for both stains for hexyl cinnamic aldehyde (2.3, 1.7 for CBA/J mice and 2.8, 1.7 for BALB/c mice at the following concentrations: 25 %, 10 %, respectively) and for eugenol (2.3, 1.5 for CBA/J mice and 2.7, 1.6 for BALB/c mice at the concentrations 25 % and 10 %, respectively). The differences were not statistically significant for each concentration (p>0.05).
In conclusion, this study indicated that female BALB/c strain mice can be used as an alternative to CBA/J strain mice in the LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.
Keywords: LLNA, BrdU-ELISA, CBA/J, BALB/c, mice
Environmental risk assessment and spatiotemporal variability of atmospheric Volatile Organic Compounds and Nitrogen Dioxide using passive sampler in Meknes City (Morocco) (#634)
I. EL GHAZI1, I. BERNI1, A. MENOUNI1, M. - P. KESTEMONT2, M. AMANE1, S. EL JAAFARI1
1 Université Moulay Ismail, Cluster of Competence “Environment & Health, Biologie, Meknes, Morocco
Epidemiological evidence links nitrogen dioxide and volatile organic compounds (VOC) trends to a wide range of health effects. In this context, a key issue is the ability of the monitoring to serve as an indicator of personal exposure to these atmospheric pollutants. In Morocco, only few regions have air quality monitoring stations. In Meknes where no air quality monitoring station exists, the use of diffusive sampling tubes for monitoring and mapping of air quality is relevant.
The purpose of this study was to evaluate the air quality in Meknes by measuring levels of VOC and NO2 in relation with road traffic during summer 2014 and winter 2015. Thus, passive diffusion tubes were deployed in 16 near-road and residential sites for 14 to 30 days to measure BTX and NO2. In parallel to the winter campaign, road traffic counting sessions were conducted at the main roads of the city. Collected samples were analyzed and pollutants concentrations were determined using spectrophotometry and gas chromatography techniques. Our study results show that the average concentrations of BTX and NO2 are highest in the city center, from where we noticed a decreasing gradient of pollutant levels. No season impact was noticed. The average concentration of benzene in Meknes during the two measurement campaigns is equal to 2.065 ug / m3 which exceeds the quality objective set by the European Union (2 ug/m3) but still lower than the annual limit value set in Morocco (10 ug / m3). Concerning the average of NO2 concentration in all sites and measurement campaigns, it is approximately 30.652 ug /m3, which lowers than the admissible limit value set by the European Union (40 ug/m3) . Sites with high levels of benzene have also shown high levels of nitrogen dioxide (R2= 0.8714). VOC and NO2 present in the atmosphere of our study area are mainly generated by road traffic. This study shows that population is exposed to atmospheric pollutants which may lead to emerging health effects. Our results also invite to examine the various lines of setted limit values in different countries and to assess the collective evidence.
Keywords: passive sampler, air quality, BTX, NO2, Meknes
Use of read-across in EU approved biocidal active substances (focus on human health) (#640)
P. PAPADAKI1, A. Airaksinen1, D. Antal1, M. Damsten1, C. Estevan Martinez1, G. Muller1, K. Myohanen1, M. U. Naeem1, L. Ruggeri1
1 European Chemicals Agency, Directorate Risk Management , Biocides Unit, HELSINKI, Finland
Under Biocidal Products Regulation (BPR) 528/2012, read-across is a possible adaptation to the standard testing and is listed in Annex IV of BPR. Read-across entails the use of results from tests on other (analogue) substances to predict properties for the active substance under evaluation. The use of read-across can mean that applicants do not need to carry out one-by-one active substance testing of each toxicological property to fulfil their data requirements and is therefore a way to avoid unnecessary animal testing. However, it also has to be ensured that such prediction of a property based on read-across is reliable, can be used for risk assessment and classification and labelling, and complies in general with the provisions in BPR for the source study proposed.
The current poster presents the use of read-across in the assessment of biocidal active substances approved under BPR and Biocidal Products Directive (BPD) 1998/8/EC. Specific characteristics of the read-across are presented such as the basis of read-across justification / hypothesis and the toxicological endpoint(s) for which read-across is applied. The analysis of data shows that read-across was used in one third of the assessments of the approved active substances, which makes read-across an extensively used adaptation to data requirements of BPR and BPD. Further guidance on the use and applicability of read-across in biocidal active substance approval is needed.
Keywords: Biocidal Products Regulation, read across, approved biocidal active substances
Risk assessment of formaldehyde present in food and drinking water. (#646)
E. E. Nielsen1, M. Egebjerg1, G. A. Pedersen1, A. Sharma1, P. Olesen1, M. Hansen1
1 Technical University of Denmark, National Food Institute, Kgs. Lyngby, Denmark
Formaldehyde occurs naturally in the environment and is ubiquitous in the environment. Formaldehyde occurs in very low levels in water as it is rapidly hydrated and therefore, predominantly is found as methylene glycol. Formaldehyde occurs naturally in food. Reported background levels vary considerable. Formaldehyde can also occur in food if released from melamine resin food contact materials. Being very reactive, formaldehyde is essentially present in food bound reversibly and irreversibly to different constituents. In humans, as in other animals, formaldehyde is an essential metabolic intermediate in the physiological one-carbon pool (central to many biological processes).
The general population is exposed to formaldehyde from many sources. The European Food Safety Authority has estimated that the contribution of formaldehyde from food does not exceed 100 mg/person/day. Drinking water is only a minor source of exposure.
The critical effects of formaldehyde following repeated oral exposure are considered to be the non-neoplastic histopathological changes observed in the forestomach and stomach (erosion, ulceration, inflammation and hyperplasia, most likely due to the irritative potential of formaldehyde) in experimental animals (long term studies drinking water). A NOAEL of 260 mg/l is considered.
Formaldehyde is genotoxic, with effects observed in vivo in cells from first site of contact tissues (i.e. nasal tissue); however, there is no evidence of genotoxicity locally in the gastro-intestinal tract. The weight of evidence indicates that formaldehyde is not carcinogenic by the oral route.
A tolerable concentration of formaldehyde in drinking water is estimated to 30 mg/l (rounded value) based on the NOAEL of 260 mg/l and assessment factors of 2.5 and 3.2 for interspecies and inter-individual variability, respectively, in toxicodynamics. Based on this tolerable concentration and an estimated (worst-case) concentration of formaldehyde in drinking water of 30 µg/l, no risk for adverse effects from intake of formaldehyde in drinking water is identified.
Based on the available data, a tolerable concentration of formaldehyde in food cannot be estimated. Therefore, the risk for adverse effects from intake of formaldehyde in beverages and foods could not be evaluated.
Keywords: formaldehyde, risk assessment, food, drinking water, food contact materials
Behavioral effects changes in rats exposed to real-life chemical mixture at doses around Toxicological Reference Values (#649)
A. O. Docea1, D. Calina2, E. Gofita1, A. L. Arsene4, O. Zlatian4, A. Crenguța Nicolae5, C. M. Drăgoi5, A. Nosyrev6, B. Izotov7, A. M. Buga8, A. Tsatsakis9
1 University of Medicine and Pharmacy, Department of Toxicology, Craiova, Romania
Consumers in real life are not exposed to single chemicals but to real-life chemical mixture at doses around toxicological reference values. The aim of this study was to assess the chronic toxicity of a real life mixture of pesticides with food additives and common consumer products chemicals using doses that simulate the real life exposure scenario. Four groups of 10 Sprague Dawley rats (5 males and 5 females) were treated with a mixture of dimethoate, glyphosate, triadimefon, methyl parathion, carbaryl, aspartame, benzoic acid, calcium disodium ethylene diamine tetra-acetate (EDTA), ethylparaben, butylparaben, bisphenol and acacia gum in doses of 0, 0.25 x acceptable daily intake (ADI), ADI and 5xADI doses for 12 months. The behavioural changes were assessed every 3 months using open field exploratory test and elevated plus-maze test. After 12 months of exposure there was an increase in locomotor activity, quantified as number of crossing over external squares in low dose group (46.8 ±11.57) compared with control (13.70±6.15) (p=0.038). The spatial orientation activity, quantified as number of rearings, was significantly increased in the low dose exposed rats (22.20 ±4.77) compared with the control rats (6.90 ±1.61) (p=0.015). In medium and high dose exposed rats the locomotor activity and special orientation activity decreased compared with the low dose group almost till the control levels in high dose exposed group. Mixture exposure determine a two-phase dose-dependent effect, the chronic use of a small dose has a stimulating effect on the organism while the higher doses have some additional negative effects that eliminate/mask the stimulating effect of the low dose.
Keywords: pesticides, food additives, chronic exposure, neurological changes
EVALUATION OF HUMAN HEALTH RISKS ASSOCIATED WITH TOXIC METALS PRESENT IN COASTAL ECOSYSTEM: A STUDY ON TROPHIC LEVEL TRANSFER OF METALS IN BAY OF BENGAL, INDIA. (#671)
1 CSIR-IMMT, Environment and sustainability, Bhubaneswar, Orissa, India
Introduction: The rapid industrial growth coupled with the development of modern technologies has resulted in the introduction of diverse toxic substances, including toxic metals into the environment. Toxic metal ions are imperishable and have lethal effects on living beings when their quantity surpasses a certain threshold level. The presence of the metals like Cr, Cd, Pb, Mn and Ni above the threshold limits exhibit carcinogenic and genotoxic effects on humans. The industrial or harbor zone is typically more populated which is responsible for creating anthropogenic pressure on the coastal environment.
Material and method:
The toxic metal content of marine water and sediment and fishes from the five selected sites of the east coast of Bay of Bengal have been collected on a seasonal basis during Pre-monsoon, Monsoon and Post-monsoon during (2014-2016).
The enrichment level of metals in the different trophic level of marine ecosystem (Water/sediment → Phytoplankton→ zooplankton → benthic organisms and fishes) of Bay of Bengal has been quantified. Prediction of environmental risks, as well as risks associated with humans using different pollution indices from the available sources for toxicity information, has been calculated.
The outcome of this study clearly indicates that the bioaccumulation was lowest in phytoplankton compared to organisms of higher trophic levels. There is a clear evidence of the anthropogenic origin of all toxic metals. It was found that the selected study sites of the coastal Bay of Bengal are mostly polluted by Chromium followed by Arsenic and lead. Human health risk assessment indicated consumption of seafood could increase cancer risks. Among all the heavy metals analyzed, Cr is the most critical and possess highest carcinogenic and non-carcinogenic health threat for seafood consumers. The risk assessment of coastal sediments indicated, there are probabilities of developing cancer and other health-related issues in the coastal population after long-term exposure. The biomagnifications were observed only for Cr, Ni, and Pb in tissues and not in As, Hg, and Cd. THQCr possesses highest non-carcinogenic risks, which contributes about 50% to HI. Likewise, the contribution of carcinogenic risk (CR Cr) to TCR is about 60%. The non-carcinogenic risk was found to be beyond the safety limit in children, below 15 years, than adults in the study site (HI child >1). The sediment was profoundly polluted with Arsenic.
Keywords: Toxic metals, Human health risk assessment, Ecological risks, Bay of Bengal coast, Food chain
Toxicological impacts of the fungicide isoprothiolane on common carp, Cyprinus carpio: a multi-biomarker assessment (#309)
J. H. Hur1, S. Manoharan1, K. J. Hur1, 2, J. Kim1
1 Kangwon National University, Biological Environment, Chuncheon, Republic of Korea
Pesticides used in agriculture have led to widespread occurrence in the aquatic environment that causes detrimental effects on the aquatic organisms even at very low concentrations. In this context, the present study was aimed to examine the impact of lower levels of isoprothiolane (2.7 and 27 µg/L; reported concentration in Yung San River, Korea) on the common carp Cyprinus carpio under 96 h exposure using various biomarkers (hematological, ionoregulatory, biochemical, and enzymological parameters). The concentrations used in this study significantly (p<0.05) decreased the hemoglobin (Hb), hematocrit (Hct), red blood cell (RBC), serum glucose, cholesterol, triglycerides, aspartate aminotransferase, and gill Na+/K+-ATPase levels in fish exposed to isoprothiolane. In contrast, the white blood cell (WBC), serum sodium (Na+), chloride (Cl-), and albumin levels were increased (p<0.05) when compared to that of their control groups. Further, there were slight changes in mean cellular volume (MCV), mean cellular hemoglobin (MCH), mean cellular hemoglobin concentration (MCHC), serum potassium (K+), protein, globulin, and alkaline phosphatase levels. Taken together, the present study suggests that alterations of hematological, ionoregulatory, biochemical, and enzymological parameters can be used as efficient biomarkers in monitoring the toxicity of pesticides in aquatic organisms. Further, the long-term effects of isoprothiolane need to be investigated in our future studies with more endpoints.
Keywords: Isoprothiolane, Toxicity, Cyprinus carpio, Blood, and Gills
Primary risk assessment done after detection of fipronil in eggs in Belgium (#810)
S. Fierens1, T. Lernout2, C. Vleminckx1
1 Sciensano, Scientific Direction Chemical and Physical Health Risks, Brussels, Belgium
In Belgium the assessment of risk to public health in the framework of the International Health Regulations is coordinated by Sciensano (former Scientific Institute for Public Health). In July 2017, the Federal Agency for the Safety of the Food Chain communicated the finding of fipronil in eggs through a notification via the EU Rapid Alert System for Food and Feed (RASFF). Because of the high attention in the media and divergent interpretations on the risk for public health, the Belgian Risk Assessment Group (RAG) was requested by the Federal Public Service Health, Food chain Safety and Environment, to perform a risk assessment.
Keywords: fipronil, risk assessment
NFIL-3 regulates dendritic cells functions following nickel and cobalt exposure (#131)
M. Pallardy1, R. Bechara1, M. Nabhan1, M. Hullo1
1 Inflammation Chimiokines et Immunopathologie, INSERM, Fac. de pharmacie-Univ. Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France
Dendritic cells (DC) play a major role in the regulation of immune responses to a variety of antigens. Indeed, metallic haptens such as nickel and cobalt are able to induce DC maturation through TLR4 activation leading to a T cell-mediated inflammatory skin disease called allergic contact dermatitis. We recently showed that nickel but not cobalt induced the production of IL-12p40 and IL-23 in human monocyte-derived dendritic cells (MoDCs) leading to Th17 cell polarization. Although both metals are considered to be TLR4 agonists, we wanted to better understand the particular mechanism behind metals-induced IL-12p40 production and we were interested in the NFIL-3 transcription factor, a recently described regulator of IL-12p40. Using an in vitro model, we showed that both nickel and cobalt were able to induce NFIL-3 expression, in human MoDCs. Pretreatment of MoDC with a TLR4 blocking antibody followed by metals stimulation did not modify NFIL-3 expression suggesting that NFIL-3 induction by nickel and cobalt was independent of the TLR4 pathway. However, this expression of NFIL-3 was dependent on the Jak-STAT pathway and oxidative stress since the pretreatment of MoDC with Jak Inhibitor I or N-acetylcysteine followed by metals stimulation induced a decrease in NFIL-3 expression. Moreover, we showed that cobalt is a strong inducer of reactive species, measured using dichlorofluorescin by flow cytometry, which may explain why cobalt is a strong inducer of the NFIL-3 pathway, compared to nickel, and thus its inability to induce IL-12p40 production. In summary, our data suggested that the NFIL-3 signaling pathway plays an important role in regulating the expression of IL-12 cytokine family in metals-exposed DC that may be implicated in ACD.
Keywords: dendritic cells, allergy, IL-12 cytokine family, NFIL-3
Does altered gut barrier function facilitate food allergy development? (#145)
E. K. Drønen1, 2, E. Namork2, H. Dirven2, K. Hylland1, U. C. Nygaard2
1 University of Oslo, Department of Biosciences, Oslo, Norway
The integrity and function of the mucosal epithelial barrier plays an important role in maintaining the gut homeostasis. Research indicate a link between altered barrier function and the development of food allergy. Barrier disruptors, like cholera toxin (CT), house dust mite (HDM), and the mycotoxin deoxynivalenol (DON) have been shown to facilitate allergy development. We hypothesized that this is due to 1) damage to intestinal epithelial cells, leading to impaired barrier integrity and increased uptake of allergens, and/or 2) the production of cytokine ‘alarmins’ by intestinal epithelial cells. The aims of this project were a) to detect early markers of a disrupted gut barrier, and b) to investigate if the tested barrier disruptors could promote development of food allergy.
This study was based on a food allergy model in mice, being composed of two short-term experiments for gut barrier assessment and one allergy model experiment. In the short-term experiments, C3H/HeOuJ mice were exposed intragastrically to one of the compounds DON, HDM or CT, and the food allergen peanut (PE). In the second experiment, we excluded PE, and included the pesticide glyphosate. 4, 24 and 48 hours after exposure, intestine and draining lymph nodes and/or Peyer’s patches were excised, and serum was collected. The intestines were homogenized and cells from lymphatic tissue were stimulated ex vivo for determination of cytokine secretion. ELISA and flow cytometry were used to detect markers of disrupted barrier and allergy.
Preliminary results indicate that the barrier disruptors elicit early effects. In DON-treated groups, the levels of the pro-inflammatory cytokines TNF-α and IL-6 in the draining lymph nodes were higher than in the other groups at 4 hours in both experiments. The levels of IL-33 in ileum and TSLP in duodenum were higher at 4 hours in the first experiment, when PE was given together with DON. This trend was not detected in the second experiment when PE was excluded, suggesting a role of PE.
Keywords: gut barrier, food allergy, deoxynivalenol, peanut
Identification of Potential Tattoo Allergens by Combining In Vitro Assays and Chemical Analysis (#171)
I. Schreiver1, M. Kühn1, S. Yildirim3, L. - M. Eschner1, K. Hutton-Carlson2, J. Serup2, A. Luch1
1 Federal Institute For Risk Assessment, Chemical and Product Safety, Berlin, Berlin, Germany
Tattoo allergies are severe side effects related to the permanent deposition of pigments in the dermal layer of the skin. Even though most type IV delayed hypersensitivity reactions are observed in the red color spectrum, the true allergen is rarely identified. So far, just a few metallic allergens and one organic pigment were reliably linked to allergic tattoo reactions. Organic pigments are only thought to act as pro- or pre-haptens since they lack water solubility and therefore hardly interact with biomolecules in physiological media. Based on this, degradation products became the main suspects in focus.
Based on the analysis of more than 100 skin samples of patients with red tattoo allergies, we identified the most common organic tattoo pigments correlating with these reactions. Possible degradation products of these pigments were deduced from substances found after laser irradiation, sunlight exposure and pyrolysis. In addition, we identified UV degradation products using untargeted quadrupole time-of-flight tandem mass spectrometry (qTOF-MS/MS). We selected known sensitizers, irritants, carcinogens and other suspected substances for further testing. The Direct Peptide Binding Assay (DPRA) was applied to identify substances that are able to bind to peptides, which is a key event in sensitization. Additionally, the activation of immune cells was analyzed using the Human Cell Line Activation Test (hClat).
We found that the majority of organic pigments in the tattoo allergy skin samples belong to the azo pigment class. These pigments are known to be easily degraded upon sun light or laser irradiation. Also, cleavage of the azo bond by reductases in vivo may occur. Most common degradation products are primary aromatic amines cleaved from either azo or amide bonds present in the molecule. Among other substances, 2-aminophenol showed positive test results in the DPRA. This substance is as yet only classified as “suspected of causing genetic defects” by the harmonized classification of the European Chemicals Agency (ECHA). However, ECHA refers to data from manufacturers supporting that 2-aminophenol may cause skin sensitization.
The results derived from this study should lead to the development of a skin patch test series for identification of sensitizing substances in tattoo allergies. In the long run, tattoo pigments likely to release these substances should be banned for use in tattoo inks.
Keywords: tattoo, allergy, chemical analysis
In vitro assessment of medical device extracts potential to produce skin sensitization. (#189)
F. Cotterez2, C. Pellevoisin1, K. Coleman3, H. Groux2
1 EPISKIN Academy, Lyon, France
The ISO norm 10993 part 10 describes the procedure for the assessment of medical devices and their constituent materials with regard to their potential to produce irritation and skin sensitization. The success of the recent Round Robin Study to assess in vitro skin irritation of medical devices extracts, with Reconstructed Human Epidermis (RhE) models, opened the way to in vitro approaches for this endpoint. For skin sensitization of medical devices, animal methods are still requested. The goal of this study was to evaluate the capacity of the SENS-IS assay to predict skin sensitization of medical devices extracts.
Based on a comprehensive analysis of skin sensitization, the SENS-IS assay uses the quantitative analysis of multiple specific biomarkers, expressed in EPISKIN model, a 3D reconstructed human epidermis. ImmunoSearch, developed this assay after analyzing genes modulated during the sensitization process either on mice (LLNA) or human (blisters). The panel of sensitization biomarkers defined includes genes already identified as markers, such as the ARE family, and others not yet associated with the sensitization process (called SENS-IS gene subset).
Solvents used for extraction of medical devices, polar (physiological saline) and nonpolar
(sesame oil), were spiked with different concentrations of sensitizer and successfully detected with SENS-IS assay in the Episkin model and the SkinEthic RHE model. Then, samples of polymers used in medical device industry, MED-2000 silicone, were loaded with 6 known sensitizers (10% final concentration): 1-phenyl-1,2 propanedione, 1-Chroro-2,4-dinitrobenzene, Diethyl maleate, p-Benzoquinone, Propyl gallate and Phenyl Benzoate and extracted in polar and nonpolar solvents in accordance with ISO 10993-12:2012.
After optimization of the original protocol used for neat chemicals, the SENS-IS method was able to correctly classify the 6 polymers.
These preliminary results show that the EpiSkin model and the SkinEthicTM-RHE model could be used in the SENS-IS assay to assess the sensitizing potential of medical devices extracts. Further studies are engaged with a more comprehensive set of molecules. If confirmed, these results would allow toxicologists, familiar with either the EpiSkin or the SkinEthic-RHE model to assess skin sensitization with the SENS-IS assay.
Keywords: skin sensitization, medical device, RHE, alternative method
Evaluation of an Integrated Strategy for Skin Allergy Risk Assessment using six ingredients and two cosmetic product types (#233)
E. Vandenbossche1, M. Baltazar1, J. Butcher1, R. Cubberley1, N. Gilmour1, C. MacKay1, R. Pendlington1, J. Reynolds1, G. Maxwell1
1 Unilever, Safety and Environmental Assurance Center, Sharnbrook, UK, United Kingdom
Our aim is to apply mechanistic and clinical understanding to develop a risk assessment approach for skin allergy that does not require new animal test data and addresses novel exposure scenarios, while better characterising uncertainty.
Our Integrated Strategy for Skin Allergy Risk Assessment (SARA) is a tiered model-based approach that predicts the probability of human skin sensitisation to an ingredient occurring following a given product exposure, with explicit uncertainty. The first tier is a probabilistic, weight of evidence (WoE) human potency model designed to use historical in vivo (HRIPT), in vitro (DPRA, Keratinosens, hCLAT, SENS-IS and U-SENS) and in silico (DEREK-NEXUS) hazard information to inform an initial prediction of human skin allergy risk which can be combined with an applied dose model. A skin toxicokinetic (TK) model may also be utilised as an additional tier that can further reduce uncertainty in the risk prediction from the clinical potency model by extrapolating from the HRIPT exposure to a market scenario.The Integrated Strategy for SARA has been evaluated using six case study ingredients included at 0.2% in two products types, face cream and shampoo. The results from the first-tier analysis are given as the probability of human skin sensitisation occurring following a given product exposure. The output from the Integrated Strategy for SARA allows a decision to be made using probability of skin sensitisation induction as a risk metric without the need for new animal data.
Keywords: skin sensitisation, risk assessment, Bayesian model
Implication of TGF-β in the immunosuppressive functions of mesotheliomagenic macrophages (#259)
H. Kiyambu1, C. Al Hatem1, M. Palmai-Pallag1, M. Orsi1, D. Lison1, F. Huaux1
1 UCL ( Université Catholique de Louvain), LTAP, Bruxelles, Belgium
Introduction: Malignant Mesothelioma (MM) is a cancer of peritoneal and pleural serous membranes induced by asbestos and carbon nanotubes (CNT). MM remains a contemporary health problem, the World Health Organisation (WHO) is expecting a peak of incidence in 2020. We suggested that carcinogenic fibers possess the intrinsic capacity to induce a preferential, rapid and sustained accumulation of macrophages that promote early immunosuppressive microenvironment, tumor cell evasion and mesothelioma development.
Aims: The goal of the present study is to determine by which mechanisms mesotheliomagenic macrophages are immunosuppressive and specifically suppress T lymphocyte functions after carcinogenic fiber exposure.
Methods and results: We analyzed the entire transcriptome profile (Illumina array) of peritoneal macrophages collected from Wistar rats treated with mesotheliomagenic (asbestos or CNT-7, i.p. injection) and non-mesotheliomagenic fibers (tangled carbon nanotubes or CNT-T) and identified specific sets of genes which may explain the immunosuppressive properties of mesotheliomagenic macrophages. We found that pathways related to the TGF-β superfamily could be used for distinguishing macrophages from peritoneal mesothelioma. In particular, Syndecan-1, a transmembrane heparan sulfate proteoglycan implicated in TGF-β signaling, is specifically expressed by peritoneal macrophages from CNT-7-treated rats. Mesotheliomagenic macrophages also express integrin β3 recently implicated in the activation of TGF-β. Transglutaminase-2, an extracellular matrix enzyme that plays a role in the interaction between latent TGF-β and extracellular matrix, represents an additional TGF-β activation signature. Moreover, we also noticed an increase of TGF-β levels in peritoneal fluid from rats exposed to CNT-7 comparatively to CNT-T.
Conclusions: Our data support an important role of TGF-β activation in immunosuppressive functions of macrophages during the development of experimental mesothelioma.
Keywords: mesothelioma, nanotubes, immunosuppression, macrophages, TGF-β
Microbiome composition of air samples from livestock farms and their effect on innate immune receptors and cells (#262)
D. Liu1, 2, R. Mariman1, M. E. Gerlofs-Nijland1, J. F. Boere1, G. Folkerts3, F. R. Cassee1, 2, E. Pinelli1
1 National institution for public health and environment, Centre for environment health, Bilthoven, Netherlands
Patients with respiratory diseases in rural areas have been reported to have enhanced responsiveness to ambient particulate matter (PM) (Brunekreef et al 2002, Pope et al 2009). In addition to the physical and chemical components, ambient PM can contain microorganisms or parts thereof which is referred here as BioPM. The aim of this study is to characterize the microbial composition of BioPM originating from livestock, and to investigate whether these BioPM can trigger the activation of innate receptors and cells.
Coarse (2.5-10 μm) and fine (< 2.5 μm) indoor BioPM were collected from various animal farms using the versatile aerosol concentration enrichment system (VACES). The fungal and bacterial community was assessed with an amplicon based approach using Next Generation Sequencing (NGS). In parallel, HEK-Blue cells expressing different pattern recognition receptors (Toll like receptors (TLR) 2,3,4,5,7,8,9 and NOD 1,2) and a human monocytic cell line (MM6) were exposed to increasing concentrations of BioPM from these sites. The levels of secreted embryonic alkaline phosphatase (SEAP) and cytokine (Interleukin-6, IL-6) were measured indicating TLR and MM6 activation respectively. TLR antagonists or blocking antibodies were used to examine the role of specific TLR on MM6 activation.
Results indicate distinct airborne microbiota profiles associated with the corresponding animal farm. Moreover, the various BioPM contained mainly ligands for TLR2 and TLR4 resulting in a dose-dependent production of IL-6 by MM6 cells. In addition to TLR2 and TLR4 only the pig-derived BioPM induced TLR5 activation. Blocking experiments indicate that only blocking TLR4 interfered with the IL-6 production by the MM6 cells upon stimulation with these BioPM.
These findings indicate that BioPM from livestock can activate innate cells and therefore, trigger inflammatory responses. Knowledge on the microbial composition of BioPM derived from different farms and understanding what type of inflammatory responses they induce is crucial for future studies on the effect of that exposure to BioPM may have on respiratory symptoms. Such as asthma, which is expected to increase in the next two decennia, resulting in expenditures up to 900 million euros per year in the Netherlands.
This work was supported by the Dutch Ministry for Public Health and the Environment under grant S/121012/01.
Keywords: Particulate matter, livestock, microbiota, innate immune
Effects of exposure of T cells to fine particulate matter and the influence of age - a prospective pilot study (#376)
M. Al Zallouha1, S. Billet1, Y. Landkocz1, M. Borgie1, S. Poddighe1, R. Delepée2, F. Ledoux1, F. Cazier5, J. J. Vitagliano3, F. Visade4, A. Verdin1, P. Martin1, P. Gosset1, 6, D. Courcot1
1 Université du Littoral Côte d’Opale, EA4492 UCEIV, Dunkerque, France
Air pollution is known to be associated with increase morbidity and mortality. Atmospheric fine particulate matter (FP) is able to enter the lungs where some compounds can reach the bloodstream and enter in contact with immune cells. Ageing is accompanied by many changes in the body: immunosenescence involves changes in the organization and the functions of the immune system making elderly more vulnerable. The project aims to identify the effects of FP on human T lymphocytes while attempting to firstly determine biomarkers related to exposure and secondly to evaluate the variation of the cellular response as a function of age. T cells were isolated from blood samples of 90 healthy volunteers belonging to three age groups (20-30, 45-55, 70-85 years) then exposed ex vivo for 72h to 45μg/μL of FP collected in Dunkirk. Analyses of the exposed T cells were then performed using transcriptomics, metabolomics, DNA adductomics and cytokines quantification. We have demonstrated that T cells exposure to FP caused an induction of the genes CYP1A1, CYP2S1 and NQO1 coding for the enzymes involved in the metabolic activation of organic compounds such as PAHs identified in the PF sample. We could then detect DNA adducts of organic metabolites, BaP Diol Epoxyde (BPDE) and formaldehyde, and of oxidative stress markers, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde. The implication of oxidative stress in this toxicity was confirmed by the detection of a decrease of cystein level during the metabolomics analysis performed on T cells supernatants. Characterization of the T cells profile made it possible to propose a mixed Th1/Th2 profile caused by the exposure. The transcriptomic study, using Affymetrix array then confirmed by qPCR, finally revealed an overexpression of several miRNAs such as miR-124-3p involved in the regulation of several functions in the immune system and miR-1290 involved in several types of cancer. Concerning the influence of age, overexpression of the genes coding for the antioxidant enzymes (NQO1 and HMOX1), an increase in the concentration of cytokines (IL-4 and IL-13) as well as a modification of the expression profile of some miRNAs were noted in the elderly. These results showed that exposure of T cells to FP causes effects at the transcriptomics, metabolomics and DNA adductomics levels.
Keywords: Fine particulate matter air pollution, T lymphocyte, age, metabolic activation, miRNA
Effect of kynurenic acid and metformin on mercury-exposed SH-SY5Y cells (#401)
A. B. Engin1, D. Karakisa1, E. D. Engin2
1 Gazi University, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey
Environmental mercury exposure is an inevitable threat to human health. It has been proposed that the reduction in adenosine monophosphate activated protein kinase (AMPK), a regulator of cellular energy homeostasis, makes cells highly susceptible to methyl mercury toxicity. Glutamate activates AMPK through N-methyl-D-aspartate (NMDA) receptor, while metformin promotes phosphorylation of AMPK. Methyl mercury easily crosses the blood‑brain barrier, however it is not known whether the NMDA receptor inhibition by kynurenic acid (KynA) or stimulation of AMPK by metformin might reduce the mercury-induced damage in dopaminergic neurons. This study evaluated the effects of KynA and metformin on mercury-induced toxicity in SH-SY5Y cells. SH-SY5Y neuroblastoma cells were exposed to various concentrations of methyl mercury at different time periods. Mercury exposed cells were incubated with KynA or metformin. Total mitochondrial metabolic activity and oxidative stress intensity coefficient were determined, and nitric oxide levels were measured spectrophotometrically. Mercury compound caused a remarkable increase in the mitochondrial oxidative stress of SH-SY5Y cells in glutamine-containing medium. By increasing dose and duration of mercury exposure, metformin exacerbated the neuronal oxidative stress and decreased mitochondrial metabolic activity. KynA supplementation presented a significant protective effect against methyl mercury toxicity with glutamate transmission. Surprisingly, despite being an AMPK activator, metformin strongly enhanced the toxic effect of 5µM methyl mercury on SH-SY5Y cells. Further studies should be made considering the association between AMPK and methyl mercury toxicity.
This study was partially supported by The Scientific and Technological Research Council of Turkey, 214S112.
Keywords: Methyl mercury, metformin, SH-SY5Y human neuroblastoma cells, mitochondrial oxidative stress, kynurenic acid
IMMUNOPROTECTIVE EFFECTS OF SELENIUM ON HEPATORENAL TOXICITY INDUCED BY SCORPION VENOM (#405)
A. Megdad-Lamraoui1, S. Adi-Bessalem1, F. Laraba-Djebari1
1 University of Sciences and Technology Houari Boumediene, Cellular and Molecular Biology, ALGIERS, Algeria
Scorpion venom compounds are able to induce alteration and dysfunction of the host organs such as liver and kidneys. These effects can be attributed to the activation of an inflammatory response. Trace elements such as selenium are known to exert multiple beneficial effects including anti-inflammatory and antioxidant action related to various diseases (inflammatory bowel diseases, arthritis…). However, studies related to the protective effects of trace elements against the induced inflammatory response by scorpion venom are scarce. The aim of this study is to investigate the potential immunoprotective effects of selenium in the induced hepatorenal toxicity by Androctonus australis hector venom.
Selenium was therefore, administered to mice orally during 2 weeks prior to the injection of a sublethal dose of scorpion venom. The hepatorenal inflammation response was assessed twenty four hours after the envenomation of mice by the measurement of vascular permeability changes, edema formation, oxidative/nitrosative stress markers levels and also by histological examination.
The results showed that the venom induced inflammatory disorders characterized by an increase of levels of reactive oxygen/nitrogen species, lipid peroxidation, and a decreased antioxidant defense. Moreover, significant alterations of the hepatic tissue such as hemorrhages and cell degeneration were also observed. The administration of selenium prevented the induced hepatorenal toxicity by the venom, as evidenced by a decreased edema formation, leukocyte infiltration, nitric oxide and thiobarbituric acid-reactive substances levels in the liver and the kidneys, as well as a reduced incidence of hepatorenal tissue alteration.
These results indicate that the selenium is able to exhibit potent protective effects against the induced inflammation response and oxidative/nitrosative stress by the venom, in hepatic and renal tissues, probably by inhibiting oxidative damage and increasing antioxidant defense. However, more researches are needed to better understand the exact mechanism by which selenium prevents the induced hepatorenal toxicity by scorpion venom.
Keywords: Scorpion envenomation, selenium, hepatorenal inflammation response, oxidative stress.
Up-regulation of bisphenol A-mediated BAFF expression in Raw264.7 murine macrophages (#426)
S. H. Lee1, S. S. Yoon1, E. Y. MOON1
1 Sejong University, Bioscience and Biotechnology, Seoul, Republic of Korea
Rheumatoid arthritis (RA) is a long-term autoimmune disorder that is pathologically occurred by immune responses of many cell types, including T cells, B cells, and macrophages. B-cell activating factor (BAFF) plays a role in the maturation and maintenance of B cells, which is associated with RA. Fibroblast-like synoviocytes (FLS) also play a key role in producing inflammatory cytokines that contribute to cartilage destruction. Rheumatoid FLS develop a unique aggressive phenotype that increases invasiveness into the extracellular matrix and further exacerbates joint damage. We previously reported that BAFF expression was increased in MH7A synovial cells transfected with the SV40 T antigen. In addition, FLS could activate macrophages to increase BAFF expression. Bisphenol A (BPA), an organic synthetic compound, is widely used to make certain plastics such as water bottles, sports equipment, DVDs and CDs. Epoxy resins containing BPA are also used to line water pipes, as coatings on the inside of many food and beverage cans and in making thermal paper such as that used in sales receipts. BPA is a xenoestrogen, exhibiting estrogen mimicking, hormone-like properties. Here, we investigated whether BPA affect BAFF expression in macrophages to influence the damage of synovial tissues. We are also studying the polarization into M1- or M2-type macrophages by BPA. Taken together, these data demonstrate that macrophages were activated by the incubation with BPA, which up-regulated BAFF expression. This suggests that individuals at high risk for autoimmune disease should avoid long-term exposure to BPA and its analogues.
Keywords: Bisphenol A, B cell activating factor, Macrophage, Rheumatic arthritis
Evaluation of cytotoxicity and in silico epitope prediction of a PEGylated snake venom serine protease: a step closer to therapeutic applications. (#454)
E. L. Pinheiro-Junior1, J. Boldrini-França1, T. R. Costa1, A. A. S. Takeda2, S. V. Sampaio1, M. R. M. Fontes2, E. C. Arantes1
1 School of Pharmaceutical Sciences of Ribeirão Preto - University of São Paulo, Ribeirão Preto, São Paulo, Brazil
Introduction and purpose: rCollinein-1 is a recombinant snake venom serine protease (SVSP) derived from Crotalus durissus collilineatus venom. This SVSP has a thrombin-like effect and interferes in blood coagulation, being considered a promising molecule targeting the development of novel biopharmaceuticals to treat hemostatic disorders. However, heterologous proteins may be immunogenic. In this regard, PEGylating proteins emerges as a strategy targeting the suppression of the immune system response. Thus, this work proposed the comparison of the cytotoxicity in peripheral blood mononuclear cells (PBMC) of rCollinein-1 and its PEGylated form and the in silico epitope prediction within the structure of rCollinein-1. Methods: rCollinein-1 was expressed in Pichia pastoris system and purified employing immobilized metal affinity (IMAC) and ion exchange chromatographies. The protein was PEGylated using a 5 kDa PEG-derived polymer. Protein structure was evaluated using circular dichroism (CD). The degradation of a chromogenic substrate was employed to evaluate their enzymatic parameters. Cytotoxicity was investigated in human PBMC in five concentrations. In silico epitope prediction was performed using ABCpred server (imtech.res.in/raghava/abcpred/). SWISS-MODEL (swissmodel.expasy.org) was employed to build the molecular model. Results: PEGylated rCollinein-1 maintained its enzymatic properties and CD revealed the secondary structure contents were similar between rCollinein-1 and its PEGylated form. These results indicate the preservation of protein conformation after PEGylation. Both enzymes showed to be non-toxic to PBMC in all tested concentrations, with cell viability higher than 85%. Regarding epitopes prediction, four peptides with 16 amino acid residues presented a high score to be immunogenic (>0.85). They are located in the surface of the protein structure, near to possible sites of PEGylation. Conclusions: PEGylating rCollinein-1 did not change its structure and has no influence on its toxicity. The epitopes with highest score to be immunogenic lie close to sites of PEGylation. Consequently, PEGylating rCollinein-1 may be an interesting modification targeting the reduction of immunogenicity, allowing its therapeutic applications.
Keywords: snake venom serine protease, PEGylation, immune system response
Irritant-induced asthma to hypochlorite in mice due to impairment of the airway barrier (#481)
J. Vanoirbeek1, S. Van Den Broucke1, L. Pollaris1, G. Vande Velde2, A. - C. Jonckheere3, T. Decaesteker4, E. Verbeken5, B. Nemery1, P. Hoet1
1 KU Leuven, Centre for Environment and Health, Leuven, Belgium
Inhalation of commonly present irritants, such as chlorine and chlorine derivatives, can cause adverse respiratory effects, including irritant-induced asthma (IIA). We hypothesize that due to airway barrier impairment, exposure to hypochlorite (ClO-) can result in airway hypersensitivity.
C57Bl/6 mice received an intra-peritoneal (i.p.) injection of the airway damaging agent naphthalene (NA, 200 mg/kg body weight) or vehicle (mineral oil, MO). In vivo micro-computed tomography (CT) images of the lungs were acquired before and at regular time points after the i.p. treatment. After a recovery period of 14 days an intranasal (i.n.) challenge with 0.003% active chlorine (in ClO-) or vehicle (distilled water, H2O) was given, followed by assessment of the breathing frequency. One day later, pulmonary function, along with pulmonary inflammation was determined. Lung permeability was assessed by means of total broncho-alveolar lavage (BAL) protein content and plasma surfactant protein (SP)-D levels. Dendritic cells (DC) and innate lymphoid cells (ILC) of the lungs are measured using FACS.
In vivo micro-CT imaging revealed enlargement of the lungs and airways early after NA treatment, with a return to normal at day 14. When challenged i.n. with ClO-, NA-pretreated mice immediately responded with a sensory irritant response. Twenty-four hours later, NA/ClO- mice showed airway hyperreactivity (AHR), accompanied by a neutrophilic and eosinophilic inflammation. NA administration followed by ClO- induced airway barrier impairment, as shown by increased broncho-alveolar lavage (BAL) protein and plasma surfactant protein (SP)-D concentrations; histology revealed epithelial denudation.
These data proves that NA-induced lung impairment renders the lungs of mice more sensitive to an airway challenge with ClO-, confirming the hypothesis that incomplete barrier repair, followed by irritant exposure results in airway hypersensitivity.
Keywords: irritant-induced asthma, innate immunology, lung barrier, mice
GMO SAFETY ASSESSMENT IN THE RUSSIAN FEDERATION: THE IMMUNOTOXICOLOGICAL STUDIES (#495)
M. D. Trebukh1, N. V. Tyshko1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The Russian system of genetically modified organisms (GMO) safety assessment within the framework of their state registration includes the study of toxic and allergenic properties of the new product in in vivo experiments. The study of immunotoxic and sensitizing features is carried out in experiments on mice of CВА and C57B1/6 lines in four tests: (1) the effect on the humoral immunity is evaluated with hemagglutination test; (2) the effect on the cellular immunity is evaluated with delayed-type hypersensitivity reaction; (3) the sensitizing effect is evaluated with histamine sensitivity test; (4) the resistance is evaluated on a model of Salmonella typhimurium (strain 415) intraperitoneal infection. The study of GMO allergenic potential is studied on a model of rats’ systemic anaphylaxis, induced by ovalbumin. The method includes the assessment (1) of systemic anaphylaxis severity and (2) of circulating antibodies (subclasses IgG1 + IgG4) level.
The advent of new genetic engineering technologies and new GM plants requires the existing safety assessment system improvement, not excepting the methodology of the immunotoxicity and allergenicity research.
As the most promising parameters that can describe the immune status, are being considered the main cytokines: pro - and anti-inflammatory (IL-1,IL-6, IL-8,TNF-α,IFN-γ;IL-4,IL-10,IL-13), cell regulators (IL-1, IL-2, IL-10,IL-12,IFN-γ) and humoral immune response regulators (IL-4, IL-5, IL-6, IL-10, IL-13, IFN-γ), the basic immunoregulatory chemokines, the adhesion molecules, and immunoglobulins (IgG, IgE) in serum, and the cell population structure (CD) markers on the cell surface.
Taking into account the wide range of indicators that can be determined with modern research methods, one of the main tasks of GMO safety assessment system improving is the formation of an optimal list of parameters that can be used in immunotoxicological studies.
This work was carried out within the state assignment of FASO Russia (theme No. 0529-2014-0047).
Keywords: GMO safety assessment, immunotoxicological studies, immune status
1,2-Diacetylbenzene with inflammatory related pathway associated neurotoxicity (#544)
M. - S. Kim1, S. - W. Kang1
1 Sunchon National University, Pharmacy, Suncheon, Republic of Korea
The older people are more susceptible by environmental factors. Organic solvents are used widely in industry and in everyday life; they are found in paints, cosmetics, drugs, and gasoline. We’ve been reported environmental neurotic metabolite, 1,2-diacetylbenzene (DAB), DAB causes central and peripheral neuropathies that lead to motor neuronal deficits. DAB increases oxidative stress and protein adducts formation along with impaired hippocampal neurogenesis in mice.
To explore the link between neurodegenerative disease and exposure to DAB, we treated the DAB (3.0 mg/kg) in young (6 months) and old (21 months) Sprague Dawley rats for 1 week. RNA was isolated and applied RNAseq technique. The transcriptome analysis of mRNA reveals that DAB has the different effects on young and old rats. Young rats showed increased the genes which are related to metabolism and excretion. However, old rats showed increased response in inflammatory and immune responses. The inflammatory responses in old rats by DAB was very similar with gene expression pattern which is regulated by the bacterial endotoxin lipopolysaccharide (LPS). Especially in old rat group showed increased prolactin level. Prolactin plays an essential role in metabolism, regulation of immune response. We also confirmed up-regulated genes that are related to inflammation-related pathway by real time PCR. These results might be helpful to elucidate the underlying mechanism of neurotoxicity by DAB especially in older people.
Keywords: 1, 2-diacetylbenzene, neuroinflammation, Organic solvent metabolite, RNAseq
Dose-dependent sensitization effects of transcutaneously exposed acid-hydrolyzed wheat protein (#548)
Y. - M. Cho1, J. - I. Akagi1, Y. Mizuta1, T. Toyoda1, N. Tamehiro2, Y. Kimura2, R. Adachi2, K. Ogawa1
1 National Institute of Health Sciences, Division of Pathology, Kawasaki, Japan
Novel mouse skin sensitization model with ovalbumin (OVA) as antigen has been developed which is sufficient to detect key immune pathway necessary for immediate hypersensitivity reaction. But more alternative and widely exposed antigen than OVA is needed to consider in this model since it is rear to be transcutaneously sensitized with OVA. We investigated immunological and histopathological changes of mice transcutaneously sensitized with acid-hydrolyzed wheat protein (HWP), which was reported to induce immediate hypersensitivity to human.
HWP at a dose of 0.22, 1.1, 5.4, 27 and 135 µg was applied in skin patches to the left flanks of 8-week old BALB/c female mice 3 days a week for 4 weeks, and immune responses were evoked with 270 µg of HWP given via intraperitoneal route. Rectal temperatures, scores on anaphylactic responses, plasma histamine levels and histopathological changes were evaluated. In over 5.4 µg HWP groups evocated via intraperitoneal injection, increased IgG1 and IgE levels after sensitization and with augmented anaphylaxis scores after evocation were noted as compared with vehicle control. In over 27 µg HWP groups and 135 µg HWP group, decreased rectal temperature and increased plasma histamine levels were observed as compared with vehicle control, respectively. Ki67-positive germinal center in the regional axillary lymph node was observed more frequently in over 5.4 µg HWP groups.
Thus when mice were sensitized transcutaneously to HWP, sufficient sensitization potential in skin sensitization model was showed at low dose. These results suggest that HWP is promising effective antigen at comparable low dose to OVA (2 µg).
Keywords: Acid-hydrolyzed wheat protein, Mouse, Skin sensitization model, Transcutaneous exposure
ADVANCES IN PRECLINICAL HUMAN IMMUNE RESPONSE STUDIES – REDEFINING IMMUNOGENICITY (#605)
M. Alam1, R. Choudhury1, R. - J. Lamers1
1 Immundnz Ltd, Macclesfield, United Kingdom
We raise the important issue of understanding drug immunogenicity. There is much ignorance on the term and the current perception of the ‘immunogenicity’ of a drug is rather misguided and limited to its ‘antigenicity’ property only, hence the scope of understanding and testing the actual immunogenicity of a drug remains very much ignored and misunderstood. The immunology of a drug and its immunologic safety are critical matters in the development of a drug. One third of all drugs in development fail prior to reaching the market due to drug-induced organ toxicity of which immunotoxicity or immunogenicity could be a contributing factor. This poses a significant financial burden to drug-developing companies. One major shortcoming in the preclinical study is that the testing of ADAs is a measure of the antigenicity of a drug only rather than the full scale immunogenicity. The immune response against drug-induced damaged tissue is ignored and drugs with the greater risk of causing secondary pathology in vital organs such as lungs or nervous system pass onto clinical phase and even the market. Drugs can also injure non-target tissue generating serious pathology, such as lung fibrosis by amiodarone. Another shortcoming is the viability of preclinical studies due to the dependence on animal models with translation from animals to human being a serious risk. This leaves the possibility of injury or fatality in clinical trials. The FDA and EMA require better understanding of drug immuno-toxicology that can be achieved through integrated assays that analyse novel markers of damage and immune response. We are running a series of in vitro human cell based assays to test drug induced tissue damage, biomarkers and the immune response against damaged tissue. Our aim is to develop an in vitro platform that can model immunogenicity of drugs and robustly predict the human immune response to a greater extent. Compared to downstream analysis in liver and kidney in conventional toxicology such studies are very upstream in the drug pathway. This will bring a whole meaning to immunogenicity and make drug development more translational and safer. This platform applies to both small and large molecules and to all therapeutic areas including oncology. By generating more critical information on drug immune safety at preclinical stage we can avoid TeGenero-like scenarios and be better prepared rather than waiting for dangerous outcomes to happen in the clinical phase.
Keywords: immunogenicity, in vitro, human, immune modelling, preclinical
Reduced risk of allergy induction for hairdressers? An occupational risk assessment for the new oxidative hair dye 2-methoxymethyl-p-phenylenediamine (#674)
C. Goebel1, E. M. Gargano1, A. A. Gaspari2, B. Blömeke3
1 Coty, Global Product Stewardship, Darmstadt, Germany
Introduction of a methoxymethyl side chain into PPD yielded the oxidative hair dye precursor 2-methoxymethyl-p-phenylenediamine (ME-PPD) with excellent hair coloring performance, and a significantly reduced sensitizing potency compared to the strong sensitizers p-phenylenediamine (PPD) and p-toluenediamine (PTD). Occupational exposure of hairdressers to PPD and PTD has been associated with the development of allergic contact dermatitis involving the hands. The exposure of hairdressers’ hands to ME-PPD was assessed following typical working conditions. Samples were obtained after hair dyeing treatments using a hand rinse method and HPLC analysis. Based on the exposure data a quantitative risk assessment (QRA) for the induction of contact sensitization was conducted. Daily hand exposure concentrations were derived by accounting for wet work, uneven exposure and inter-individual variability for professionals. Daily hand exposure was compared with the sensitization induction potency of ME-PPD defined as the No Expected Sensitization Induction Levels (NESIL).
First estimations for ME-PPD indicate that the hairdresser hand exposure level is approximately two orders of magnitude below its NESIL. Correspondingly, the risk of contact allergy induction for hairdressers to ME-PPD is significantly reduced compared to PPD and PTD, with hand exposure levels only 2.7 and 5.9 fold below the NESIL, respectively. In conclusion, the QRA indicates that induction risk for contact sensitization to ME-PPD is very low for hairdressers, because their occupational daily hand exposure to ME-PPD is approximately two orders of magnitude below the NESIL.
Keywords: skin sensitization, quantitative risk assessment, hair dye, 2-methoxymethyl-p-phenylenediamine (ME-PPD), hairdresser
Development Of Cell Assays To Predict Cytokine Storm Responses Of Novel Biological Therapeutics (#695)
J. R. Munday1
1 Covance, Immunology & Immunotoxicology, Harrogate, North Yorkshire, United Kingdom
There are many examples of immunomodulatory compounds inducing a significant safety risk associated with cytokine storm responses. Many investigators have assessed in-vitro cellular assays for evaluation of this response but these do not always accurately reflect the true biologic mechanisms taking place. Importantly, as many new therapeutics are being developed that are human specific there is an increasing need for human cell based assays that help predict the safey risks associated with cytokine storm. This poster reviews some of the assay formats that have been tested in the field and reviews the disadvantages and advantages of the specific approaches. The reproducibility and predictive nature of these approaches will be discussed with the aim of showing how these approaches can be used to accurately predict the potential for cytokine storm for novel biologics.
Keywords: cytokine storm, cytokine release assay, in-vitro cellular assay, immunomodulatory compound, immunotoxicology
Zinc oxide nano- and bulk-materials interfere with immunometabolism (#718)
N. Herlin2, K. Becker3, M. Paparella1, P. Fagundes dos Santos1, 3, H. Schennach4, D. Fuchs3, J. M. Gostner1
1 Medical University of Innsbruck, Medical Biochemistry, Innsbruck, Austria
Immunoregulatory pathways tip the scale for either activation of inflammatory responses or the mediation of suppressive effects. Upon chemical exposure, type and duration of the immunological responses decide on the direction of outcomes. For this, metabolic reprogramming is critical. Importantly, both physical and chemical characteristics of a chemical may influence the reaction cascades.
The interferon-gamma-induced metabolic pathways of tryptophan breakdown to kynurenine via indoleamine 2,3-dioxygenase (IDO-1), and neopterin formation via GTP-cyclohydrolase in human peripheral blood mononuclear cells (PBMC) can be used as a reliable readout to investigate immunomodulatory effects of compounds and materials.
Zinc oxide (ZnO) nanomaterials are used in technical and chemical industry, in pharmaceutical, food and cosmetic applications are frequent, due to the relatively low toxicity and biodegradability. Exposure of the healthy skin to ZnO and nanomaterials is generally considered as safe, but there are concerns regarding ZnO containing aerosols. In real life, the possibility for interferences with biological processes cannot be excluded.
ZnO bulk material and nanoparticles were analysed in this in vitro setting in a concentration range from 2.3 to 37.5 µg/ml. Nanoparticles showed higher cytotoxicity compared to the bulk material. The kynurenine to tryptophan ratio, a measure of IDO-1 activity, decreased with all materials dose-dependently in mitogen-stimulated cells. However, nanomaterial treatment of unstimulated cells increased IDO-1 activity, while this effect was less prominent with the bulk treatment. Neopterin concentrations showed the same trend.
Data indicate that within a certain concentration range, ZnO nanoparticles may have some activating effect on unstimulated PBMC, while in stimulated cells and with higher concentrations immunosuppressive effects prevail. A closer elucidation of these effects is clearly warranted.
Keywords: immunometabolism, tryptophan, IDO-1, neopterin, interferon gamma, ZnO, nanoparticles, PBMC
Olive-derived hydroxytyrosol shows anti-inflammatory effect without immunotoxicity (#732)
Y. Yonezawa1, 2, H. Nejishima1, Y. Takeda3, K. Imai3, H. Ogawa2, 3
1 Kaken Pharmaceutical Co., Ltd., Pharmacokinetics and Safety Department, Drug Research Center, Fujieda, Shizuoka, Japan
The control of inflammation, which arises from complex biological responses to harmful stimuli, is an important determinant of both clinical outcomes and patient comfort. However, the side effects of many current therapies such as non-steroidal anti-inflammatory drugs mean that new safe treatments are required. We previously reported that 12.5 mg/ml hydroxytyrosol (HT) suppressed gene expression of the inducible nitric oxide (NO) synthase (iNOS) isoform, and NO production, in mouse peritoneal macrophages treated with lipopolysaccharide (LPS), where nuclear factor-κB (NF-kB) gene expression was not altered. The present study evaluated the anti-inflammatory effects of various concentrations of HT in LPS-induced RAW264.7 mouse macrophage cells. HT suppressed NF-kB signaling and downregulated LPS-mediated expression of iNOS, cyclooxygenase-2, and interleukin-1b at concentrations ≥ 12.5 mg/ml, resulting in reduced production of NO and prostaglandin E2. Additionally, we aimed to determine whether HT suppresses COX-2-induced inflammation in a carrageenan-induced rat paw edema model. Additionally, we compared its activity with those of the selective COX-2 inhibitor, celecoxib, and a representative nonsteroidal anti-inflammatory drug (NSAID), indomethacin. HT, celecoxib, and indomethacin significantly suppressed swelling in carrageenan-injected rat paws. Although HT was less effective than celecoxib and indomethacin, it had a delayed onset of action, suggesting that the combined use of HT and celecoxib or indomethacin might yield a sustained anti-inflammatory effect. Moreover, we evaluated whether HT aggravates gastric damage, which is a typical adverse effect associated with NSAIDs, in an aspirin-induced gastric damage rat model. Unlike celecoxib and indomethacin, HT did not cause gastric damage when co-administered with aspirin. Our results indicate that HT displays potential as a new anti-inflammatory drug without gastric toxicity.
Keywords: hydroxytyrosol, anti-inflammatory, celecoxib, indomethacin, gastric damage, macrophage, lipopolysaccharide
Differently functionalized CuO nanoparticles: efficient antimicrobial against Escherichia coli or the poison to human macrophages? (#737)
A. - L. Kubo1, 2, H. Vija1, A. Kahru1, 3, O. Bondarenko1
1 National Institute of Chemical Physics and Biophysics, Laboratory of Environmental Toxicology, Tallinn, Estonia
CuO nanoparticles (CuO NPs, 1-100 nm) are used in industrial and consumer products due to their antibacterial properties. The macrophage-NP interaction is highly important for the distribution, fate and toxicological profile of NPs.
Here we hypothesized that antibacterial metal-based NPs, including CuO NPs, that are already used in consumer products can be harmful to human cells. Since toxicity of CuO towards various cells may depend on their surface functionalization, four CuO NPs with primary sizes about 30 nm and different surface functionalizations (coatings) were studied: uncoated CuO, CuO functionalized with amino group (CuO-NH2), CuO functionalized with carboxyl group (CuO-COOH) and CuO functionalized with polyethylene glycol (CuO-PEG). Escherichia coli MG1655 was selected to test antibacterial effects of differently functionalized CuO. Macrophages differentiated from THP-1 cells in vitro, were selected for immunotoxicity testing. Differently from all the previous studies, antibacterial and immunotoxicity tests were performed in the same conditions: incubation at 37◦ C in 10%-serum supplemented culture medium. Alamar blue assay was used as the toxicity endpoint in all cases. In parallel, we measured dissolution of CuO NPs in these test conditions and the toxicity of ionic Cu (as CuSO4) as a control for solubility. Toxicity was expressed as EC50 values adjusted to the Cu content.
All tested CuO NPs and ionic control CuSO4 were toxic to bacterial cells. The antibacterial toxicity (24 h EC50) varied from 19 mg Cu/l (CuO-PEG) to 42 mg Cu/l (uncoated CuO) and the EC50 values increased in the order CuO-PEG < CuO-NH2< CuO-COOH < CuSO4 50) varied from 15 mg Cu/l (CuO-NH2) to >30 mg Cu/l (CuO-PEG) and the EC50 values increased in the order CuO-NH2< uncoated CuO < CuSO4 < CuO-COOH < CuO-PEG. Thus, CuO-PEG could be the most suitable antibacterials as they were the most toxic to E. coli cells and not toxic to human immune cells in vitro. In contrast, CuO-NH2 was significantly more toxic to THP-1 cells then to E. coli cells and is the least favorable as the antibacterial. This knowledge can be used for the synthesis of more efficient and safe antimicrobials. This work was supported by Estonian Research Council grants IUT23-5 and PUT1015.
Keywords: immunotoxicology, CuO nanoparticles, biocompatible surface coatings, antimicrobials, nanotoxicology
Quercetin attenuates ergotamine induced toxicity in rat liver hepatocytes (#34)
J. Khalili Fard1, 2, M. Sattari3, H. Hamzeiy3, M. A. Eghbal3, S. Jafari4
1 Lorestan University of Medical Sciences, Faculty of Pharmacy, Pharmacology and Toxicology Department, Khorramabad, Iran (Islamic Republic of)
Background: Mycotoxins are secondary metabolites and capable of causing life-threatening effects. Ergot alkaloids are mycotoxins which formed by Claviceps species. Among these alkaloids, ergotamine has been commonly prescribed for migraine headaches. Its mechanisms of toxicity nevertheless has not yet been completely understood. Among polyphenols, quercetin is mostly distributed in different fruits and vegetables.
Materials and Methods: In this study hepatocyte lysis and ROS formation have been investigated using accelerated cytotoxicity mechanism screening (ACMS).
Results: On the base of our results ergotamine induces ROS formation and lipid peroxidation in freshly isolated rat hepatocytes in dose dependent manner and these toxicity markers have been significantly prevented by quercetin.
Conclusion: It is concluded that the ROS formation play a key role in the ergotamine induced toxicity and quercetin could be considered as a supplement for who suffers from migraine.
Keywords: ergotamine, migraine, quercetin, toxicity
CHARACTERIZATION OF CYTOCHROME P450 INDUCTION BY THE TYPE II PYRETHROID LAMBDA-CYHALOTHRIN (#75)
A. Anadón1, I. Ares1, J. - L. Rodríguez1, M. Martínez1, B. Lopez-Torres1, D. Roura-Martínez1, M. - R. Martínez-Larrañaga1, M. - A. Martínez1
1 Universidad Complutense de Madrid, Madrid, Spain
Many pesticides including pyrethroid are subjected to metabolic biotransformation by hepatic cytochrome P450 (CYP) enzymes. CYP enzyme induction alters balance between detoxification and activation and in cases this may lead to enhanced toxicity. There are numerous examples where CYP enzymes catalyze metabolic activation of chemically inert agents to electrophiles. This work showed induction of CYP catalytic activities and mRNA levels by the pyrethroid lambda-cyhalothrin. Testosterone oxidative metabolism was determined in hepatic microsomes from control and treated rats. There are evidences that CYP3A1/2, CYP2A1, CYP2C11 and CYP2B1 isozymes catalyze testosterone oxidation at the 6β-, 7α-, 16α- and 16β-positions. Microsomal 6β-, 7α-, 16α- and 16β-testosterone hydroxylase activities were evaluated by conversion of testosterone to 6β-, 7α-, 16α- and 16β-hydroxytestosterone metabolites by HPLC. Quantitative real-time PCR assays for rat CYP1A1, CYP1A2, CYP2A1, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1 mRNA were also performed to analyze mRNA gene expressions. Oral exposure of rats to lambda-cyhalothrin resulted in a dose-dependent significant increase of hepatic microsomal testosterone 6β-, 7α- and 16β-hydroxylase activities. Lambda-cyhalothrin (8 mg/kg bw 6 days) produced significant increases in testosterone 6β-hydroxylase (68%, reflecting CYP3A1/2 activity), testosterone 7α-hydroxylase (52%, reflecting CYP2A1 activity) and testosterone 16β-hydroxylase (412%, reflecting CYP2B1 activity). CYP2E1 and CYP3A1/2 mRNA levels increased significantly (at 4 and 8 mg/kg bw 6 days). The major significant increase of mRNA levels was observed in CYP2B1 (1463% and 961%) and CYP2B2 (604% and 501%) in both treatment groups. It is concluded that lambda-cyhalothrin induces CYP enzymes which may enhanced the toxicity of these compounds. Work supported Projects Ref. S2013/ABI-2728, Comunidad de Madrid, and Ref. RTA2015-00010-C03-03, Ministerio de Economía, Industria y Competitividad, Spain.
Keywords: Pyrethroids, CYP enzymes, rat liver
Reactive Oxygen Species: the hidden face of biodegradable Fe-based alloys (#79)
E. Scarcello1, M. Tomatis2, F. Turci2, A. Thomas3, P. J. Jacques3, D. Lison1
1 Université catholique de Louvain, Louvain Centre for Toxicology and Applied Pharmacology, LTAP, Brussels, Belgium
Fe-based materials are currently considered for manufacturing biodegradable coronary stents. Here we document, for the first time, the generation of hydroxyl radicals (OH•) during the corrosion of Fe-based materials and their deleterious impacts on endothelial cells. The generation of OH• was documented by two independent acellular assays, terephtalic acid hydroxylation (fluorescence) and formation of spin trapping adducts (electron paramagnetic resonance spectroscopy). All tested Fe-based materials exhibited a strong potential to generate OH•, directly from the incomplete reduction of dissolved oxygen and/or via Fenton chemistry mediated by Fe ions. The reduction of these signals in presence of D-mannitol confirmed their specificity for OH•. Cellular responses were assessed in vitro on two human endothelial primary cells (HUVECs and HAOECs) using colorimetric and luminescence cytotoxicity assays. Cells were exposed directly to Fe powder, or to corrosion extracts. Only direct contact with Fe materials affected cell viability, consistent with the cytotoxic activity of short-lived OH• produced in direct contact with cells. To confirm the cellular impact of OH•, mRNA expression of oxidative stress response genes (heme oxygenase-1 (HO-1) and glutamate cysteine ligase modifier subunit (hGCLM) was also assessed. The expression of oxidative stress genes was dose-dependently increased 4h after direct exposure to the particles, not to extracts.
The demonstration of OH• production during corrosion and consequent oxidative stress on endothelial cells provides a new perspective on the biocompatibility of biodegradable Fe-based alloys, the future design of Fe-based vascular implants, and the protocols for assessing their biocompatibility in vitro.
Keywords: iron alloys, biocorrosion, oxidative stress, reactive oxygen species, stent
Gene Profiles expressed in Hippocampus of Aluminum-treated Rats and the Significance for Predicting Al-induced Neurotoxicity (#82)
1 Shanxi Medical University, School of Public Health, Taiyuan, China
Objective: Aluminum is a widely exposed neurotoxicant. However, the molecular mechanism underlying Aluminum toxicity remains to elucidate. In this study, expression profiling was performed using Illumina next generation sequencing to show the interfered hippocampus transcription induced by aluminum exposure. The ultimate goal was to find the key transcription changes in Aluminum-induced neurotoxicity.
Materials and Methods: Sub-chronic intraperitoneal injection of Aluminum-maltolate complex[AL(mal)3] was performed for 3 months. cDNA libraries were constructed from six rats’ hippocampus (3 in control group and the other 3 in aluminum-exposed group). DNA-seq analysis of the two groups was performed using the Illumina HiSeq platform. After transcripts were assembled, expression profiling was performed. Differentially expressed genes (DEGs) were analyzed using DEseq2 method. Gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was employed to analyze the function of DEGs.
Results: 96 up-regulated and 652 down-regulated DEGs were identified, comparing to the control group. GO analysis results showed that multiple functional genes are most significantly affected by aluminum exposure, including gial cell differentiation, neural transmission and vesicle trafficking. Furthermore, KEGG pathway analysis results revealed that these DEGs were clustered in several signal pathway, including ECM-receptor interaction; PI3K-Akt signaling pathway; Focal adhesion and cAMP signaling pathway.
Conclusion: Expression profiling identified that the genes involved in gial cell differentiation, cell adhesion and vesicle trafficking were candidate genes to be used in the research on aluminum induced neural toxicity.
(This work is supported by NSFC 81430078, 81372968)
Keywords: Genome wide transcriptome, high throughput sequence, neurotoxicity, Aluminum, Rat
In vivo comparison between two nephrotoxic agents, sodium fluoride and uranyl nitrate: phenotypic aspects and molecular mechanisms involved (#83)
A. BONTEMPS1, L. Conquet1, C. Elie1, V. Magneron1, C. Gloaguen1, D. Kereselidze1, K. Tack1, O. Barbier2, Y. Guéguen1
1 IRSN, PSE-SANTE/SESANE/LRTOX, Fontenay-Aux-Roses, France
Humans can be exposed at low concentrations to nephrotoxic agents with anthropogenic and natural origins such as uranium and fluoride. Nevertheless, there is a lack of knowledge of their mechanisms of nephrotoxicity. This study aims to compare these mechanisms in mouse to identify the cellular and molecular pathways of toxicity.
C57Bl6 mice are exposed by intraperitoneal injection of uranyl nitrate (UN) (0, 2, 4, 5 mg/kg) or sodium fluoride (NaF) (0, 2, 5, 7.5, 10 mg/kg) and euthanized 48h and 72h later. Renal phenotypic aspects and biological mechanisms are evaluated by urinary biochemistry, gene and protein expressions, enzyme activity, and histological analyses. Exposure to UN and NaF induces nephrotoxicity in a dose-dependent manner. 5 mg/kg of UN induces mild histopathological alterations and respectively 44 and 6-fold increase in gene expressions of nephrotoxicity markers KIM1 and osteopontin. In comparison, 10 mg/kg of NaF induces high nephrotoxicity with histopathological alterations scored as severe and late appearing parameters of toxicity whereas 7.5 mg/kg induces mild histopathological scoring and gene expressions of KIM1 and clusterin enhanced respectively by 70 and 4-fold compared to control. No signs of nephrotoxicity are observed below 5 mg/kg of NaF. Clusterin is respectively increased by 2.4 and 4.4-fold in urines after 7.5 mg/kg and 10 mg/kg injections of NaF. Apoptosis is evaluated through caspases 3/7 activity which is increased by 210% after UN treatment (5mg/kg) whereas NaF does not induce apoptosis. Inflammation is implied in UN and NaF acute nephrotoxicity as shown by gene and in situ overexpressions of ICAM and VCAM.
UN and NaF acute exposures resulted in a dose and time-dependent nephrotoxicity with a higher nephrotoxicity after 72h. Inflammation and apoptosis are both involved in UN or NaF toxicity. These observations allow us to identify the mechanisms that will be studied in a low-dose exposure protocol after a chronic exposure.
Keywords: Uranium, Fluoride, Nephrotoxicity
Expression of Aryl Hydrocarbon Receptor in the Developing Mouse Brain (#85)
E. Kimura1, 2, F. Maekawa1, C. Tohyama3, 4
1 National Institute for Environmental Studies, Center for Health and Environmental Risk Research, Ibaraki, Japan
Epidemiological and experimental studies have shown that perinatal exposure to dioxin induces impaired cognitive function and behavioral abnormalities in humans and laboratory animals. Disruption of neuromorphology and expression of neurotransmitter receptor genes was observed in the brain of rodent offspring in utero and lactationally exposed to dioxin, suggesting impaired neural circuit structure and function by dioxin exposure. The aryl hydrocarbon receptor (AhR) that avidly binds dioxin is a ligand-activated transcription factor, and ligand-AhR complex induces various toxicities through activation of downstream signaling pathway. In addition, AhR plays important roles in the developing nervous system of invertebrates and vertebrates. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study was performed to examine AhR expression in the brain of C57BL/6J mice using western blot and in situ hybridization techniques. AhR protein was detected in the brain of 3-day-old mice although its expression level in the brain was significantly lower than that in the liver, lung, kidney, thymus, and spleen. Next, we examined AhR mRNA expression in the brain of embryonic and postnatal mice. On embryonic day 12.5, AhR mRNA was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebellum, and olfactory bulb of 3- and 14-day-old mice. In the hippocampus, mRNA expression in the CA1 and CA3 pyramidal cell layers was higher than stratum radiaum and oriens, suggesting that AhR is expressed in a subregion-specific manner. These results reveal temporal and spatial patterns of AhR expression in the mouse brain, which may contribute to understanding mechanisms of developmental neurotoxicity of AhR ligands, such as dioxin.
Keywords: Aryl hydrocarbon receptor, Brain, Mouse
Aerosol trapping , optimization and characterization for comparative in vitro assessment of combustible and heat-non burn tobacco platforms . (#87)
D. P. Goedertier1, C. Pak1, A. Kondylis1, G. Vuillaume1, J. Hoeng1, C. Mathis1, S. Frentzel1
1 Philip Morris Products S.A, PMI R&D, Neuchâtel, Neuchâtel, Switzerland
There are more than 6,000 chemical constituents in cigarette smoke (CS), many of them known toxicants or carcinogens. In vitro models, commonly used in toxicology, are often based on submerged cell cultures that cannot be exposed to smoke or aerosol directly. It is thus essential to develop adequate protocols to quantitatively trap smoke/aerosols in bio compatible solvent to allow testing them in submerged cell cultures.
With a rigorous Design of Experiment approach, we aimed to optimize the process of collecting the smoke from the 3R4F reference cigarette and the aerosol generated from the Tobacco Heating System (THS) 2.2 in phosphate-buffered saline (PBS) or in ethanol. In this study, the protocols to generate two different fractions, total particular matter (TPM) and aqueous extract (AE), were optimized for these two tobacco products to improve trapping efficiency, A detailed characterization of the chemical composition of each produced fraction was subsequently performed.
Trapping efficiencies for TPM and AE were determined by measuring nicotine concentration using a gas chromatography with a flame ionisation detector and the levels of eight carbonyls quantified by liquid chromatography with electrospray ionization mass spectrometry respectively . When comparing the extraction methods (shaking or syringe) from the Cambridge filter pad, nicotine concentration for THS 2.2 TPM fraction was slightly higher when shaking, while for 3R4F TPM, no difference was observed between the two extraction methods. In order to obtain the highest nicotine concentration per test item on one filter pad, we found that the smoke from six 3R4F cigarettes and the aerosol from four THS 2.2 test items were needed. We also found that for the AE preparation, collecting the aerosol before the puff volume-generating pump in an impinger filled with ice-cold PBS was important to obtain the highest carbonyl concentration per test item. Finally, the use of an impinger that contained glass pearls also improved the trapping efficiency for 3R4F AE smoke fraction.
In summary, we present optimized protocols for the collection of TPM and AE fractions from 3R4F and THS 2.2 tobacco products that may be leveraged in systems toxicology based in-vitro assessments.
Keywords: aerrosol trapping, cigarette smoke, smoke fraction
Unravelling mechanisms of toxicity induced by classical PPAR-gamma agonists through transcriptomic analysis of hiPSC-derived kidney organoids (#112)
S. Busch1, J. Sagemark1, A. Jonebring2, A. - K. Sjögren1, R. Hicks2, M. Persson1, J. Hornberg1
1 AstraZeneca, Discovery Safety, Drug Safety and Metabolism, IMED Biotech Unit, Mölndal, Sweden
The use of peroxisome proliferator-activated receptor-gamma agonists as anti-diabetic drugs has been limited due to serious adverse effects. PPAR-gamma targeting drugs such thiazolidinediones (TZDs) have been implicated with heart failure which has been linked to increased fluid retention. However, the exact underlying molecular mechanisms of PPAR-gamma mediated water resorption in the kidney are largely unkown. As part of an exploratory discovery safety strategy we seek to analyze whole transcriptome profiles upon drug exposure as a tool to decipher PPAR-gamma associated kidney-specific toxicity. Human induced pluripotent stem cell (hiPSC)-derived kidney organoids more closely recapitulate the complexity of kidney tissue in vitro than cell line models with regard to cellular composition and organization. As TZD-induced adverse outcome in vivo is manifested upon long-term treatment, an additional benefit of the 3D organoid model is that it allows for long-term culture and repeated dosing. Therefore, kidney organoids were the model of choice representing a meaningful model system to study TZD-mediated effects in vitro.
In an effort to cover acute and prolonged drug respone, TZD-treated kidney organoids are harvested after several exposure durations and processed for generation of sequencing libraries from mRNA, to obtain information from the coding transcriptome. Acquired data from high throughput whole transcriptome sequencing are subjected to bioinformatic preprocessing prior to integrated analysis.
Validation of kidney organoid model demonstrates suitable detection of drug-induced gene expression changes associated with kidney toxicity, water resorption and ion transport function. Quantification of gene expression data upon TZD exposure indicate the induction of known PPAR-gamma target genes as well as distinct transcriptional programs over time. We believe that in conjunction with future applications using samples of long-term treated animals, transcriptome analysis is a promising platform to discover novel mechanisms of toxicity and biomarkers. Ultimately, identification of safety biomarkers with regard to TZD-induced transcriptional changes will be crucial in order to overcome the lack of reliable in vitro methods to de-risk PPAR-gamma during early drug discovery.
Keywords: PPAR-gamma, RNA sequencing, kidney organoids, discovery safety
Involvment of ATM/Chk2-p53 signaling pathway in B[a]P-induced neural cell apoptosis (#135)
J. Nie1, 2, Z. YAN1, L. DUAN1, X. WANG1, D. TANG2, 1, Q. NIU1
1 Shanxi mediacal University, Occuptional Health, Taiyuan, /, China
A growing body of evidence suggests that air pollution can harm the brain, accelerating cognitive aging, and may even increase risk of Alzheimer’s disease and other forms of dementia. Neuronal apoptosis plays a crucial role in neurodegenerative diseases such as Alzheimer’s disease. Benzo[a]pyrene (B[a]P), a typical component of air pollution , can lead to neural cell apoptosis. However, little is known about the molecular mechanisms in B[a]P-induced neural cell apoptosis. In this study, Male SD rats were used to establish models of B[a]P-induced apoptosis, rats were randomly divided into five groups: the blank control group received no treatment and the others were received intraperitoneal injection of B[a]P (0, 1.0, 2.5, 6.25mg/kg.bw) for 1, 2 and 3 months respectively. The results of immunohistochemistry showed that B[a]P increased neural cell apoptosis and BPDE-DNA adducts in rat cortex . In the three dose groups, after 3 months treatment, B[a]P upregulated expressions of ATM, Chk2, Chk2thr68, p53, p53ser20 , PUMA and cleaved caspase3 proteins in a dose- and time-dependent manners in rat cortex . The findings suggested that B[a]P-induced cortex neurons apoptosis through DNA damage, in which ATM/Chk2/p53 pathway play an important role.
Keywords: Benzo[a]pyrene，neural cell apoptosis，ATM，Chk2，p53
Respiratory hazard of Li-ion battery components: elective toxicity of lithium cobalt oxide (LiCoO2) particles via IL-1β and HIF-1α (#172)
V. Sironval1, L. Reylandt2, S. Ibouraadaten1, M. Palmai-Pallag1, Y. Yakoub1, M. Tomatis3, B. Ucakar4, R. Vanbever4, E. Marbaix5, D. Lison1, S. van den Brule1
1 Université catholique de Louvain, Louvain centre for Toxicology and Applied Pharmacology, Brussels, Belgium
Background: Rechargeable Li-ion batteries (LIB) are increasingly used worldwide. They contain micrometric and low solubility particles consisting of toxicologically relevant elements. Their production and use has increased sharply in recent years, implying a potential for inhalation exposure in occupational settings. New proposed applications such as printable or spray-paintable batteries might also expose consumers. The health hazard of these materials is not documented. We performed the first study on the respiratory hazard of 3 leading LIB components (LiFePO4 or LFP, Li4Ti5O12 or LTO, and LiCoO2 or LCO) and investigated their mechanisms of action.
Methods: Particles were characterized physico-chemically (composition, morphology, size distribution) and elemental bioaccessibility was documented at neutral and acidic pH. Lung inflammation, oxidative stress, fibrotic responses, and particle persistence were assessed in C57BL/6 and interleukin (IL) -1β deficient mice after oro-pharyngeal aspiration of LIB particles (0.5 or 2 mg) or crystalline silica (2 mg) used as reference. The implication of hypoxia-inducible factor (HIF)-1α in lung toxicity was assessed in vivo by using chetomin, an inhibitor of HIF-α activity.
Results: LIB compounds contain a significant fraction of respirable particles and their metallic elements are differently bioaccessible. Acute inflammatory lung responses (including IL-1β) and oxidative stress were recorded with the 3 LIB particles and silica, LCO being the most potent. Inflammation persisted 2 m after LFP, LCO and silica, in association with fibrosis in LCO and silica lungs. LIB particles persisted in the lung after 2 m and formation of ferruginous bodies was detected in LCO lungs. In vivo, only LCO and silica stabilized intracellular HIF-1α, a marker of Co ion activity, known to be implicated in inflammation and pro-fibrotic events. IL-1β deficiency or inhibiting HIF-1α activity significantly reduced in vivo lung inflammation induced by LCO.
Conclusions: We report here the varying respiratory toxicity of particles used in LIB, and conclude that they represent a respiratory hazard. LCO was at least as potent as crystalline silica to induce inflammatory and fibrotic responses. IL-1β and HIF-1α might represent key mediators of the elective lung toxicity of LCO particles.
Keywords: Lung, inflammation, fibrosis, silica, oxidative stress
Neurotoxicity assessment of four synthetic cathinones in neuroblastoma cells at in vivo relevant concentrations (#176)
J. Parra1, A. R. T. S. Araujo1, 2, F. Carvalho3, H. Carmo3, J. P. Silva3
1 School of Health Sciences, Polytechnic Institute of Guarda, Guarda, Portugal
Synthetic cathinones (SC) comprise a broad group of new psychoactive substances structurally related to cathinone, a naturally occurring stimulant found in the khat plant (Catha Edulis). The rapidly increasing use of SC, the second largest group of monitored new drugs, represents a major public health concern in view of several reports of intoxications and deaths. However, their toxicological data remains scarce. Pregnant and women of childbearing potential comprise particular risk groups, due to the potential onset of neurodevelopment disorders in the offspring.
This work aimed at evaluating the in vitro neurotoxicity of four commonly reported SC, as a first assessment of their effects on neuronal differentiation.
Briefly, different toxicological parameters (i.e. cell viability, mitochondrial integrity, energy metabolism) were evaluated in NG108-15 neuroblastoma cells exposed to increasing concentrations (1nM – 500μM) of Ethcathinone (Eth-Cat), 4-Chloroethcathinone (4-CEC), 4-chloro-alpha-pyrrolidinopropiophenone (4-Cl-α-PPP) or 4-fluoro-Pyrrolidinohexanophenone (4F-PHP). Neurite outgrowth was measured as the number of newly formed neurites per total number of cells, following differentiation with forskolin and retinoic acid, in the presence or absence of SC (single addition at day 0) at in vivo relevant concentrations (< 10μM).
4F-PHP showed the highest neurotoxicity, significantly decreasing metabolic activity (MTT reduction) above 1nM and was the only one inducing membrane integrity loss (LDH release) at 500μM. 4-Cl-α-PPP only decreased metabolic activity at the highest concentration tested, while neither Eth-Cat nor 4-CEC affected cell viability up to 500μM. Interestingly, only Eth-Cat induced the hyperpolarization of the mitochondrial membrane following exposure to 1 and 10μM and none of the SC significantly altered intracellular ATP levels up to 100μM. Moreover, none of the SC tested affected neurite outgrowth up to 10μM.
Overall, the tested SC showed different neurotoxicity profiles, suggesting the involvement of distinct toxicity mechanisms, which do not seem to affect neuronal differentiation at in vivo relevant concentrations.
Keywords: new psychoactive substances, β-keto-amphetamines, neurodevelopment, cell viability
Prenatal high fat diet alters DNA methylation and gene expression of metabolic genes in mouse offspring. (#205)
S. Rouschop1, T. Karl2, L. Maas1, A. Risch2, A. Opperhuizen1, 3, F. J. van Schooten1, R. Godschalk1
1 Maastricht University, Pharmacology & Toxicology, Maastricht, Netherlands
Background: A parental prenatal high fat diet leads to an impaired metabolic phenotype in mouse offspring. The underlying mechanisms, however, are not yet fully understood. Therefore, this study investigated whether the impaired metabolic phenotype could have been programmed through altered gene expression and DNA methylation in promoter regions.
Methods: Both parent mice received either a high fat or a low fat diet starting from 6 weeks before gestation, continuing throughout pregnancy and lactation. At weaning, all offspring mice were switched to a high fat diet. Analyses were done on liver samples from male offspring at 12 and 28 weeks of age. Gene expression was assessed using microarray. Several differentially expressed genes (Cd163, Hmgcr, Aacs, Lpin1, Saa1, Orm2 and Il1r1) were subsequently selected for bisulfite pyrosequencing of the promotor region.
Results: Gene set enrichment analysis of microarray data revealed that metabolic pathways were indeed enriched as a result of the prenatal high fat diet. Bisulfite pyrosequencing of the promotor regions of Cd163, Hmgcr, Aacs, Lpin1, Saa1, Orm2 and Il1r1 indicated that DNA methylation levels were altered at several transcription factor binding sites.
Conclusion: These results suggest that an impaired metabolic phenotype in offspring mice due to prenatal high fat diet may be mediated by gene expression and DNA methylation.
Keywords: Prenatal programming, Epigenetics, High fat diet
Revisiting the paradigm of silica pathogenicity: silanols, not crystallinity, as key determinant (#293)
C. Pavan1, F. Turci2, 3, M. Tomatis2, 3, R. Leinardi2, 3, L. Pastero3, 4, M. Fabbiani2, 5, G. Martra2, 5, B. Fubini2, 3, D. Lison1
1 Université catholique de Louvain, Louvain Centre for Toxicology and Applied Pharmacology (LTAP), Institute of Experimental and Clinical Research (IREC), Brussels, Belgium
Exposure to respirable silica dusts is associated to severe lung diseases, including silicosis and lung cancer, and is still a current source of concern for the heath of many workers and those using silica in nanotechnologies. Crystallinity has always been considered as the main feature of hazardous silica dusts. However, crystalline silicas are not all equally pathogenic, and some still unidentified physico-chemical differences between particles account for the variability of the silica hazard.
We recently synthesized a set of quartz crystals of respirable size, exposing as-grown, intact crystal faces. These particles showed a very low biological activity in a series of cellular tests relevant for the pathogenicity of quartz, indicating that the activity of quartz dust is not necessarily contingent to crystallinity. Mechanical grinding of these quartz crystals markedly increased their activity. By combining this finding with studies on a large set of both amorphous and crystalline silica particles, the surface spatial/energetic configuration of silanols emerged as a relevant characteristic accounting for damage to cellular membranes. We also correlated the ability of quartz particles to cause membranolysis with their capacity to activate the enzymatic machinery inflammasome and trigger a pro-inflammatory response, a step at the origin of silicosis and lung cancer. Our current hypothesis is that characterizing silanols through physico-chemical and in vitro analyses allows identifying the pathogenic activity of silica materials.
We are currently validating our hypothesis in vivo and developing a set of assays to identify and predict the respiratory hazard of silica particles based on the analysis of their surface silanol distribution. The results obtained on a panel of model quartz samples prepared ad hoc (i.e. particles with defined and designed physico-chemical properties) showed significant differences in their membranolytic and cytotoxic activity consistent with the hypothesis, confirming the relevance of silanols. Preliminary analyses via diffuse reflectance infrared spectroscopy (DRIFTS) indicated that this technique is promising to clearly reveal the silanol distribution of the silica particles.
Keywords: silica, quartz, silanol, membranolysis, inflammasome
Role of Hif-1a and its target gene PTP4A3 in regulating cell proliferation during benzene exposure (#331)
Y. Pu1, 2, R. Sun1, 2, F. Sun1, 2, J. Zhang1, 2, L. Yin1, 2, Z. Man1, 2, Y. Pu1, 2
1 Southeast University, Nanjing, China
Benzene is a widespread environmental and occupational pollutant. The major adverse health effect of benzene exposure is hematotoxicity. Hif-1a regulates the expression of genes encoding molecules that participate in cell proliferation. In the present study, we aimed to investigate the effects and molecular regulation of hif-1a on cell proliferation during benzene exposure. Male C57BL/6 mice were administered by subcutaneously injection with or without benzene for 2 weeks, hematotoxicity, cell proliferation and hif-1a protein level were evaluated. PTP4A3, a proliferation-related gene, was found by ChIP-Seq screening as a hif-1a target gene. Next, the hif-1a high expression mouse model (C57BL/6- hif-1a+) was constructed, the protein level of hif-1a and PTP4A3 were measured by western blot in WT and C57BL/6- hif-1a+ mouse. The bone marrow (BM) cell proliferation was also analyzed by BD FITC BrdU Flow Kit. Then the PTP4A3 inhibition K562 cell line was established by transfecting with the specific shRNA. Cells were exposed to 1,4-Benzoquinone (1,4-BQ) for 24 hours. The cell viability, proliferation, cell cycle and key protein levels in PI3K-ATK pathway were estimated. The results showed that Benzene exposure caused significant reduction in numbers of leukocytes, red blood cells, platelets and decreased hif-1a level in benzene exposed mice. Meanwhile, a significant decrease in the percentage of hematopoietic stem cells (HSCs) and an increase in the proliferation were observed in benzene exposed mice. Then we found that augmentation of hif-1a in C57BL/6 mouse significantly elevated the proliferation of BM cells and increased PTP4A3. In vitro, after treated with 1,4-BQ, PTP4A3 was significantly reduced in PTP4A3 inhibition and control cells. Compared with control cells at the same 1,4-BQ concentration, the cell viability and cell proliferation were significantly lower in PTP4A3 inhibition cells. Protein levels of PI3K and ATK were also dramatically decreased in cells with inhibited PTP4A3. In addition, G2/M phase found in the cells with inhibited PTP4A3 exposed to 1,4-BQ than those in control cells. In conclusion, the hif-1a regulates cell proliferation through PTP4A3 and PI3K-ATK pathway during benzene exposure. (Supported by National Natural Science Foundation of China 81573189, 81730087 and 81703265).
Keywords: Benzene, Hif-1a, Proliferation, PTP4A3
Mechanism of chronic ethanol consumption-induced oxidative stress in mice renal cortex: involvement of iNOS (#369)
C. B. P. Silva1, 2, N. A. Gonzaga2, C. S. Ceron3, J. D. Santos4, M. M. Castro4, C. R. Tirapelli2
1 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Sao Paulo, Brazil
Introduction: Ethanol consumption is described to induce the expression of the inducible isoform of the enzyme nitric oxide synthase (iNOS). Purpose: Evaluate the role of iNOS in the renal toxicity induced by chronic ethanol consumption. Methods: Male C57BL/6J wild-type and iNOS-deficient (iNOS-/-) mice were randomized into 4 groups: Control (WT); Ethanol (E-WT); iNOS (iNOS-/-) and Ethanol iNOS-/- (E-iNOS-/-) (Ethics committee 14.1.847.53.0). The animals were treated with water or ethanol 20% (v/v) ad libitum for 10 weeks, and the temporal assessment of body weight and liquid and solid intake was determined. Systolic blood pressure (SBP) was measured. To evaluate the renal function, serum levels of urea, creatinine, K+ and Na+ ions were determined. The renal cortex was collected for the assessment of levels of: superoxide anion (O2˙-), hydrogen peroxide (H2O2), nitrate/nitrite (NOx), thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), oxidized glutathione (GSSG) and cytokines. Catalase (CAT), superoxide dismutase (SOD), metalloproteinases (MMP) and myeloperoxidase (MPO) activities, besides the Nox1, Nox2, Nox4, eNOS and SOD2 protein expression were evaluated. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s test (p<0.05). Results: Long-term ethanol consumption induced change in nutritional status and increased SBP, creatinine serum, IL-1β and GSH levels in WT and iNOS-/- mice. Ethanol-treated animals showed high O2˙- levels and MPO activity, however these responses were lower in E-iNOS-/-. High Nox4 expression, TNF-α and TBARS levels was noticed in E-WT mice. NOx levels were not modulated by ethanol, but the kidney of iNOS-/- animals showed lower concentrations than WT. CAT, SOD, MMP-2 and MMP-9 activities, Nox1, Nox2, eNOS and SOD2 protein expression, GSSG, H2O2, IL-6, IL-10, urea, K+ e Na+ ion levels did not differ among the groups. Conclusion: Chronic ethanol consumption induces rise of O2˙-, TBARS and TNF-α levels, Nox4 protein expression and inflammatory cell infiltration in renal cortex, and these responses are mediated by iNOS. These results suggest a role for iNOS in renal toxicity induced by ethanol consumption. Financial support: FAPESP/CAPES
Keywords: Chronic ethanol consumption, iNOS, Renal cortex
Singlet Molecular Oxygen Generated by Cholesterol, Oleic, Linoleic Hydroperoxides with Nitronium Ion (#373)
P. Di Mascio1, A. C. Scalfo1, S. Miyamoto1, F. M. Prado1, M. H. G. Medeiros1
1 University of São Paulo, Chemistry Institute, Department of Biochemistry, São Paulo, São Paulo, Brazil
Evidence has been accumulated during the last three decades on the strong implication of several oxidants including hydroxyl radical, one-electron oxidants and singlet molecular oxygen [1O2] in the generation of hydroperoxides from several nucleobases, amino acids, and unsaturated lipid components. 1O2 is one of the major species responsible for the cytotoxic effects of photodynamic treatment and is being implicated in diseases such as porphyria and cataracts. In addition, 1O2 can trigger ultraviolet A-induced biological effects through activation of gene expression. Cell membrane contains polyunsaturated fatty acids, which are vulnerable to attack from many reactive species. The outcome of this process is lipid peroxidation and its primary products are lipid hydroperoxides. In addition, hydroperoxides can react with reactive nitrogen species leading to nitro or nitrosyl products. The aim of present work is to study the generation of 1O2 through the reaction of lipid hydroperoxides with nitronium tetrafluoroborate (NO2BF4). Ultraweak chemiluminescence arising from biomolecules oxidation has been attributed to the radiative deactivation of 1O2 and electronically excited triplet carbonyl products involving dioxetane intermediates. The approach used to unequivocally demonstrate the generation of 1O2 in these reactions is the use of the direct spectroscopic detection and characterization of 1O2 light emission. Characteristic light emission, corresponding to the singlet delta state monomolecular decay was observed.Chemiluminescence measurement of the monomol light emission in the near infrared region (λ= 1270 nm) has indicated that the reaction of organic and lipid hydroperoxides (cumene, t-butyl, oleic, linoleic and cholesterol hydroperoxides) with NO2BF4 generate 1O2.The use of sodium azide as a physical quencher of 1O2, associated with chemiluminescence measurements, contributed to identifying the generation of 1O2. Besides Russell mechanism, other possible mechanisms for 1O2 generation are under investigation.1O2 might be generated as a byproduct of lipid peroxidation, in conditions where reactive nitrogen species interact with lipid hydroperoxides. This might contribute to a better understanding of this complex event and physiological or physiopathological implications. Acknowledgments: FAPESP 2012/12663-1, CEPID 2013/07937-8, CNPq 301307/2013-0, CAPES NAP Redoxoma 2011.1.9352.1.8 and John Simon Guggenheim Memorial Foundation (P.D.M. Fellowship).
Keywords: singlet oxygen, lipid, hydroperoxides, chemiluminescence, nitronium ion
DNA adducts mapping as possible urban pollution biomarker (#380)
M. H. G. Medeiros1, A. B. Sanchez1, P. Di Mascio1
1 University of São Paulo, Chemistry Institute, Department of Biochemistry, São Paulo, São Paulo, Brazil
Globally, pollution problems are more severe in industrialized regions or in high density populated regions. The São Paulo Metropolitan Region is an example of a megalopolis with a unique set of emissions, solar flux and meteorological conditions that determinates its higher atmospheric pollution. Previously, we have shown the accurate quantification of 1,N2-propanodGuo in human urinary samples collected from residents of a polluted city (SP) and an unpolluted region showed significantly higher 1,N2-propanodGuo levels in the samples from SP donors than in samples from donors of the unpolluted region (Garcia et al. Chem. Res. Toxicol. 26, 1602-1604, 2013). This result provided evidence that elevated levels of 1,N2-propanodGuo in urinary samples may be correlated with urban air pollution. Here, we described a highly sensitive method involving HPLC/ESI-MS detection in SRM mode and employing isotope-labeled internal standards for the analysis of several DNA adducts (adductome) with aldehydes such as formaldehyde (1,N2-hydroxy-methyl-deoxyguanosine), acetaldehyde (1,N2-propano-2’-deoxyguanosine), crotonaldehyde, acrolein 1,N2-(α-Hydroxy) propano-2’-deoxyguanosine and 2,4-decadienal. The methodology was used to quantify the levels several DNA-aldehyde adducts in calf thymus DNA exposed to tobacco and cannabis smoke. In control DNA, 8-oxo-7,8-dihydro-2’-deoxyguanosine and 1,N2-hydroxy-methyl-2’-deoxyguanosine were detected. In the groups exposed to tobacco and cannabis smoke, all the analyzed adducts were detected. With the exception of the acetaldehyde adduct 1,N2-propano-2’-deoxyguanosine adduct, the groups exposed to tobacco smoke present higher levels of DNA adducts in relation to the group exposed to cannabis smoke. The method described herein can be used to study exocyclic DNA adducts through the quantification of different adducts in humans and experimental animals after air pollution exposure. Supported by CEPID-Redoxoma (FAPESP: Proc. 2013/07937-8), NAP-Redoxoma (PRPUSP: Proc. 2011.1.9352.1.8), CNPq (301404/2016-0 and 301307/2013-0).
Keywords: DNA adducts, air pollution, mass spectrometry, biomarkers
The synthetic psychostimulant 4-fluoromethamphetamine induces in vitro hepatotoxic effects, via CYP2E1 metabolism (#393)
R. R. D. S. Bravo1, P. Moreira2, F. Carvalho1, M. D. L. Bastos1, H. Carmo1, D. Dias da Silva1
1 UCIBIO/REQUIMTE, Laboratory of Toxicology, Biological Sciences Department, Faculty of Pharmacy, University of Porto, Porto, Portugal
4-Fluoromethamphetamine (4-FMA) is a stimulant and entactogenic amphetamine, with alleged nootropic effects. Many drug users describe its subjective effects as similar to those of other amphetamines, but the potential health effects of recreational use of 4-FMA is hitherto unknown.
Because the liver plays a pivotal role in the metabolism and toxicity of amphetamines, we set out to evaluate the cytotoxic effects of 4-FMA using three complementary in vitro hepatocyte models.
Viability of human immortalised HepG2 and HepaRG cells, and primary rat hepatocytes (PRH) was evaluated by MTT reduction, after exposing cells for 24h to a concentration range of the drug that enable obtaining complete concentration vs. effect curves. 4-FMA was shown to induced concentration-dependent toxicity, primary rat hepatocytes being the most sensitive in vitro model (LC50 2.21 mM), followed by HepaRG and HepG2 (LC50 5.59 mM and 9.57 mM, respectively). Also, 4-FMA significantly impaired the glutathione and energetic storages in PRH, as well as disrupted redox homeostasis. Finally, 4-FMA activated the apoptosis common pathway, following activation of caspase-8 and caspase-9. The co-incubation of PRH with 4-FMA and cytochrome P450 specific inhibitors suggested that its toxicity is metabolism-dependent. CYP2D6 inhibition increased 4-FMA-induced cytotoxicity, while CYP2E1 inhibition decreased the cytotoxicity. These data suggest that 4-FMA may be metabolically activated by CYP2E1, while metabolism through CYP2D6 results in detoxification. CYP3A4 inhibition had no impact in the toxic effects of 4-FMA.
Our results show that 4-FMA is potentially hepatotoxic to its users. Since the metabolism of the drug is likely to greatly affect its toxicity, interindividual variability in susceptibility and potential for toxicologically relevant drug interactions are of concern.
Keywords: 4-Fluoromethamphetamine (4-FMA), Hepatotoxicity, In vitro metabolism, Oxidative stress, Mitochondrial dysfunction
Autophagy plays a protective role in synthetic cathinones-induced nephrotoxicity (#397)
I. Vaz1, A. Castro1, A. M. Araújo2, M. J. Valente3, P. Guedes de Pinho2, M. D. L. Bastos2, M. Carvalho1, 2
1 UFP Energy, Environment and Health Research Unit (FP-ENAS), University Fernando Pessoa, Porto, Portugal
Synthetic cathinones (β-Keto amphetamines; β-KA) abuse has been associated with renal injury. However, the exact mechanism behind the nephrotoxic effects of β-KA is not yet understood. This study aimed to investigate the role of autophagy in β-KA-induced nephrotoxicity in human kidney (HK-2) cells. To this purpose, HK-2 cells were exposed to two different concentrations of 3,4-dimethylmethcatinone (3,4-DMMC; 0.25 or 0.5 mM) and 3,4-methylenedioxymethcathinone (methylone; 2 or 4 mM) for 24 h. Both drugs prompted the formation of acidic vesicular organelles (evaluated by fluorescence microscopy of acridine orange-stained cells) and autophagosomes (evaluated using a CytoID Autophagy Detection Kit) in a concentration-dependent manner. Moreover, the autophagy inhibitors 3-methyladenine and wortmannin significantly potentiated cell death (evaluated by the reduction of the tetrazolium salt MTT), indicating that autophagy serves as a cell survival mechanism that protects β-KA exposed cells. We further examined the role of oxidative stress in autophagy induction and found that antioxidant treatment with N-acetylcysteine (NAC) or ascorbic acid significantly potentiated cell death induced by these cathinone derivatives, most likely due to inhibition of ROS-mediated autophagy and potentiation of apoptosis induced by β-KA. Further studies are ongoing to verify this theory.
Acknowledgements: This work was supported by national funds through FCT – Fundação para a Ciência e a Tecnologia, in the scope of FCT Project UID/Multi/04546/2013. A.M.A. thanks Fundação para a Ciência e Tecnologia (FCT), Portugal, for her PhD grant (SFRH/BD/107708/2015).
Keywords: β-Keto amphetamines, Nephrotoxicity, Autophagy, Antioxidants, Autophagy inhibitors
Relative potency ranking of azoles altering cranio-facial morphogenesis in rat: an in vitro data modelling approach (#409)
M. Battistoni1, F. Di Renzo1, F. Metruccio2, A. Moretto2, 3, E. Menegola1
1 Università degli Studi di Milano, Department of Environmental Science and Policy, Milano, Italy
Oral clefts in any form (cleft lip and/or palate alone or associated with other head skeletal defects) represents one of the most frequent abnormality in humans (1:700 live births) showing increasing interest also because of their demonstrated multifactorial origin, involving both genetic and environmental risk factors. Environmental agents inducing cranio-facial malformations in experimental models include some pesticides, especially azole fungicides. The proposed common mode of action for azoles teratogenic effects is the inhibition of CYP26 enzymes, involved in the retinoic acid (RA) catabolism, with a consequent perturbation in endogenous RA levels in specific embryo segments. Risk assessment now focuses on combined exposures to molecules and, for those sharing the same mode of action, the use of relative potency factor (RPF) approach has been suggested. The aim of the work is to define RA-relative potency of four antifungal azoles by fitting postimplantation rat whole embryo culture (WEC) experimental data by using PROAST software analysis. The chemicals selected are all-trans retinoic acid (RA 0.025-1µM), cyproconazole (CYPRO 7.8-250µM), triadimefon (FON 6.25-125µM), flusilazole (FLUSI 1.56-125µM) and prochloraz (PCZ 6.25-50µM). Dose-related specific defects of the cranio-facial structures (reduced or fused branchial arches), were detected in rat embryos exposed in vitro to increasing concentration of RA or of the selected azoles. Collected dicotomic data were then modelled using PROAST 62.3 software in order to characterize the single dose-response curves. Curve parameter comparison showed that for all the used teratogenic agents steepness values were not dissimilar, so parallelism hypothesis and, by consequence, the hypothesis of a common mode of action shared between RA and the different tested azoles could not be rejected. Due to the azole RPFs versus RA, the potency ranking of the tested molecules resulting by the present work is RA>FLUSI>PCZ>CYPRO>FON. The obtained data suggest that WEC results could be a simple but predictive alternative method applicable to the novel hazard evaluation based on relative RPFs.
Keywords: teratogenesis, PROAST, embryo, azoles, ranking
Unravelling essential key events in mitochondrial adverse outcome pathways (#424)
W. van der Stel1, S. Darici1, B. van de Water1, E. Danen1
1 Leiden University, Drug Discovery and Safety, Leiden, Netherlands
The study focuses on key events relating compound-induced mitochondrial perturbation to hepatotoxicity. The obtained data serve as input for the establishment of quantitative adverse outcome pathways (qAOPs) specific for mitochondrial toxicity. Ultimately, these qAOPs could contribute to the improvement of toxicity prediction and risk assessment of new and existing chemicals.
Analytical tools for assessment of mitochondrial functioning in HepG2 cells, including membrane potential, ROS formation, GSH expression and ATP production, were optimised for use in an automated real time confocal microscopy platform. The parameters were monitored along with measurements of cell functioning, including viability. In addition, gene and protein expression are studied using TempoSeq for transcriptomics and using BAC-GFP reporter technology for single cell real time analysis of candidate protein expression.
Time and concentration-resolved high content analysis of mitochondrial functionality and cell fate outcome were applied in a single exposure setting for mitochondrial toxicants including complex I, II and III inhibitors. Cluster analysis based on all mitochondrial functioning parameters demonstrated a clear separation of the different complex inhibitors. CI and CIII inhibitors reduced membrane potential with different potencies. CI inhibitors induced ROS formation and diminished ATP production at lower concentrations compared to CIII inhibitors. No effect was observed after CII inhibition in the applied concentration range. Cell viability after CI and CIII inhibition was reduced only when cells were switched from glucose- to galactose-containing medium, indicating that a switch to glycolysis represented an adaptive response to CI/III inhibition.
Besides compound characterisation, the data was used to develop a qAOP. A temporal relationship between parameters supported the AOP paradigm: for concentrations above the threshold value a reduction in membrane potential was observed along with a subsequent induction of an unfolded protein response and thereafter a loss of cell viability.
Ongoing experiments address the impact of repeat dose exposures and generate information on global transcriptional changes. The obtained data provides further insight into the chain of events triggered by mitochondrial toxicants. Altogether, this will support optimisation of the testing platform for assessment of mitochondrial toxicity.
Funded by EU Horizon2020 research grant agreement No 681002
Keywords: Mitochondria, AOP, liver, confocal imaging
2,4-Dinitrophenol (2,4-DNP) induces toxicity in primary hepatocytes through oxidative stress and mitochondrial dysregulation (#435)
P. Guedes de Pinho1, D. Sousa1, F. Carvalho1, H. Carmo1, M. D. L. Bastos1, D. Dias da Silva1
1 Porto University, Biological Sciences, Porto, Portugal
The desire of boasting a healthy and fitbody leads many individuals to use ‘fat burners’ as a quick way to reduce body weight, under a misperception that such products endure a low health risk. An example of such substances is 2,4-dinitrophenol (2,4-DNP), well-known for its weight loss enhancer properties, but also for the occurrence of several related fatal cases among users. Although liver damage has been associated to the toxicity of 2,4-DNP, the underlying mechanisms remain to be established.
The aim of the present study was to evaluate the toxicity of 2,4-DNP in primary rat hepatocytes. In vitro assays were performed in primary cultures of rat isolated hepatocytes, obtained with a two-step collagenase perfusion. Our data showed that 2,4-DNP induced concentration-dependent toxicity in primary hepatocytes, at concentrations ranging from 0.55μM to 1 mM, at 37° C for 24 h, as assessed by three viability assays [neutral red uptake assay: EC50 98.05 μM; MTT reduction assay: EC50 96.82 μM; and lactate dehydrogenase leakage assay: EC50 174.83 μM]. A significant concentration-dependent increase of intracellular levels of reactive oxygen species (p<0.01, at ≥50 μM; Dunnett’s test) concurrent to a concentration-dependent decline in antioxidant defenses [decreased reduced glutathione (redGSH; p<0.01, at ≥100 μM; Dunnett’s test); decreased total glutathione (p<0.01, at ≥100 μM; Dunnett’s test); increased oxidized glutathione (GSSG; p<0.05, at ≥1 μM; Dunnett’s test); and decreased redGSH/GSSG (p<0.001, at ≥1 μM; Dunnett’s test)] were observed. Significant increase of mitochondrial membrane potential was observed at 10 μM and 50 μM (p<0.05; Holm-Sidak’s test), in accordance with the exacerbated synthesis of adenosine triphosphate (ATP). Energy-dependent activation of apoptosis was also confirmed by activation of pro-caspase-8 at all tested concentrations (p<0.05; Dunnett’s test) and by the increased activities of caspase-3 and -9, at concentrations higher than 10 μM (p<0.05; Dunnett’s test).
Keywords: Dinitrophenol, toxicity, fat burners
High throughput adaptive stress response pathway activation to support read across of valproic acid analogue-induced liver steatosis (#439)
N. Vrijenhoek1, R. Graepel1, S. Wink1, S. Escher2, B. van de Water1
1 Leiden University, Drug Discovery and Safety, Leiden, Netherlands
Hepatic steatosis is a common liver disease that can lead to hepatotoxicity. Many drugs, including the anticonvulsant valproic acid (VPA), can induce hepatic steatosis as a side effect. Besides human clinical data, VPA and some of its structural analogues have shown to induce steatosis in rodents. Transcriptomics data derived from yeast cells suggest that VPA activates several stress response pathways, which are related to hepatotoxicity. This study aims to investigate stress response pathway activation by VPA and its structural analogues by using HepG2 BAC-GFP reporter cell lines in a high throughput screening platform. Activation of different stress responses were visualized by GFP tagged to target proteins SRXN1 (Nrf2-dependent oxidative stress response), P21 (p53-dependent DNA damage), BiP (unfolded protein response) and A20 (NFκB-dependent inflammatory signaling). Cells were treated with VPA and 18 structural analogous to generate dynamic concentration and time response datasets on pathway activation using high content confocal imaging. SRXN1 activation could discriminate between stimulation of VPA and structural in vivo steatosis positive analogues and in vivo negative analogues. P21 and A20 expression was also changed upon VPA exposure, where BiP did not show a response. Additionally, we observed no cytotoxic effects in our dose range. However, cells treated with valproic analogues that cause steatosis and co-stimulated with TNFα showed an increase in apoptosis, further supporting activation of common signaling pathways by the various toxic valproic analogues that drive onset of cytotoxicity. In conclusion, our current data support the application of a high content imaging stress response reporter platform to provide biological weight-of-evidence support for read across-based risk assessment strategies by providing mechanistic mode-of-action information.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002.
Keywords: Stress response pathways, valproic acid, high content imaging platform
Inhalation exposure to cigarette smoke exacerbates vascular fibrosis and causes endothelial nitric oxide synthase uncoupling in streptozotocin-induced diabetic rats (#465)
Q. V. Do1, K. - H. Park1, Y. - S. Seo1, M. - Y. Lee1
1 Dongguk University, College of Pharmacy, Goyang-si, Gyeonggi-do, Republic of Korea
Smoking is a well-established risk factor for vascular diseases and vascular complication is a major outcome of diabetes. This study was performed to test the effect of cigarette smoke exposure on the progress of vascular impairment in diabetic condition. Seven-week old male Sprague-Dawley rats were divided into four groups consisting of each 10 rats; control group (C), cigarette smoke-exposed group (CS), diabetic group (D), and diabetic, cigarette smoke-exposed group (DCS). Diabetes was induced by a single, intraperitoneal injection of 60 mg/kg streptozotocin, which was confirmed by blood glucose level higher than 300 mg/dL. After 4 weeks, CS and DCS was exposed to cigarette smoke generated from 3R4F reference cigarettes at total particulate matter concentration of 200 microgram/L, 4 h/day, 5 day/week for 4 weeks. Filtered air was supplied instead of cigarette smoke for C and D. After treatment, thoracic aorta and blood plasma were analyzed for pathological changes by cigarette smoke. Medial area and thickness were decreased in aorta of diabetic rats, whereas internal diameter was not changed. However, cigarette smoke exposure had minimal effect on the structure of aorta. Collagen content was increased in diabetic aorta and such increase was further augmented by cigarette smoke exposure, although cigarette smoke did not affect collagen content in aorta of control rats. Cigarette smoke exposure caused endothelial nitric oxide synthase (eNOS) uncoupling only in diabetic aorta which was evidenced by an increase in monomer/dimer ratio. Malondialdehyde was increased and glutathione was decreased in the plasma of diabetic rats. However, such alterations of oxidative stress markers were not further changed by cigarette smoke exposure. Expression of NADPH oxidase (NOX) 2 and NOX4, representative reactive oxygen species-generating enzymes in aorta, were enhanced by cigarette smoke exposure in diabetic rats, not in control rats. The results of this study indicate that cigarette smoke aggravates vascular fibrosis and eNOS uncoupling in diabetes, which may accelerate the progress of diabetic macrovascular impairments such as loss of arterial elasticity and endothelial dysfunction.
Keywords: smoking, cigarette, diabetes, vascular fibrosis, endothelial nitric oxide synthase uncoupling
The Mechanism for Potentiation of the Combined Effects of 2,4-D and Methylmercury in Warm-Blooded Organisms (#502)
V. Rakitskii1, T. Sinitskaya1
1 FBES «FSCH Named after F.F. Erisman» of the Rospotrebnadzor, Mytishchi, Russian Federation
2,4-D is a derivative of the chlorophenoxyacetic acid, it is a part of pesticides with herbicidal activity. In most cases, mercury is contained in environmental objects in the form of methylmercury; therefore, the heavy metal under investigation was used in the experiment in the form of methylmercury.
The purpose of the present studies was to establish the pathogenetic mechanism for potentiation of the combined effects of global environmental pollutants 2,4-D and methylmercury on warm-blooded organisms. The experiments were performed on albino rats in acute and subchronic experiments. Literature data on the toxicity of the derivatives of chlorophenoxyacetic acid and mercury were referred to when choosing research methods to determine the specific features of toxicodynamics of the studied substances.
Keywords: Chlorophenoxyacetic acid, Methylmercury, Oxidative stress, Antioxidant enzyme, Lipid peroxidation
ROS generation and oxidative stress induced by hexachlorobenzene in human intestinal caco-2 cells (#506)
H. Chalouati1, 2, D. Boussoufa1, L. Payrastre2
1 University of Tunis Al Manar, Faculty of Sciences of Tunis, Tunis, Tunisia
Purpose: Many organochlorine compounds have been classified by the International Agency for Research on Cancer (1979) as carcinogenic in animals and also suspected to be carcinogenic in humans, hexachlorobenzene (HCB), is a typical member of this family. HCB has been found in feed in several regions of the world. In our study, we performed an in vitro evaluation of ROS generation and oxidative stress induced by HCB on the human intestinal Caco-2 cell line as a target cell type regarding to oral exposure.
Methods: Caco-2 cells were cultured in an atmosphere of 5% carbon dioxine and 95% air at 37°C. HCB was dissolved in absolute ethanol, for a stock solution of 2mM, and the same final concentration of the vehicle (0.1%) was used in the corresponding control. After 15 hours of seeding, cells were washed with serum free medium and then incubated with 4% FBS and treated with HCB (0.04 to 2000 nM). Treatments were repeated every 48 hours in all experimental designs for 14 days. Intracellular production of ROS was analyzed using the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (H2-DCFDA) which characterized H2O2 production. Antioxidant enzymes activities like catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were determined in the cytosolic fraction.
Results: Our results showed that exposure of caco-2 cells to HCB elicited a dose-dependent induction of the intracellular production of H2O2 and the highest intensity of fluorescence being observed upon a 3 hours-period of incubation. Indeed, treatment of caco-2 cells with HCB induced an oxidative stress demonstrated by the increased activity of antioxidant enzymes including SOD, CAT and GPx. This induction was significantly observable at dose 4 nM and was more marked at the highest dose tested (2µM).
Normal cellular functions depend on the balance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms in the cell. In our study the cellular enzymatic antioxidants are unable to control the production of ROS, and an oxidative stress is occurs, leading to damaged cell functions.
Keywords: ROS, Antioxidant, HCB
Chronic organophosphate exposure triggers ROS-mediated injuries at organismal and sub-organismal levels of non-target Drosophila melanogaster (#519)
1 The University of Burdwan, Zoology, Burdwan, West Bengal, India
The study demonstrates ROS-mediated organismal and sub-organismal injuries in Drosophila melanogaster following chronic organophosphate (acephate) exposure. Drosophila was reared on food supplemented with sub-lethal concentrations (1-6 µg/mL) of acephate (LC50 8.71 µg/mL). The treated adults demonstrated reduced longevity (~to half at 6 µg/mL exposure) along with reduced neuromuscular coordination and physical activities. Conspicuous developmental defects in compound eyes were confirmed through detection of apoptotic lesions in larval eye imaginal discs. Larval gut manifested tissue damage at various sites. Neural and fat cell viability was reduced by ~1.89 and ~3.38 fold at 6 µg/mL acephate treatment. Significant reduction in hemocyte viability confirmed the immunotoxic potential of acephate. Nearly 1-3fold increase in expression of various oxidative stress-markers (MDA, protein carbonyl contents, SOD, Catalase and HSP70) in treated larvae served as evidence of reactive oxygen species (ROS) production. Post-treatment increases in CYP450 and GST activities reflects ‘switch-on’ states of phase-I and phase-II detoxification mechanism. Genotoxic potential of Acephate was confirmed through alkaline single cell gel electrophoresis. Thus, findings of present study validate the fact that, besides traditional cholinesterase inhibition, chronic sub-lethal exposure to acephate potentially induces ROS-mediated toxic responses in non-target model organism Drosophila.
Keywords: Acephate, Drosophila melanogaster, oxidative stress, ROS
A human relevance investigation of PPARα-mediated key events in the hepatocarcinogenic mode of action of propaquizafop in rats (#521)
P. Singh1, C. Strupp2, W. H. Bomann3, F. Spézia1, F. Gervais1, R. Forster1, L. Richert4, 5
1 Citoxlab France, Evreux, France
Propaquizafop is an herbicide with a long history of use. Chronic dietary exposure of rats led to liver tumors without genotoxicity, subchronic studies demonstrated dose-dependent increases in liver weights with hepatocellular hypertrophy, and two weeks administration induced liver CYP4A and peroxisomal enzymes. A rodent-specific mode of action (MOA) via activation of the peroxisome proliferator-activated receptor α (PPARα), increased liver peroxisomal activity, hypertrophy, liver enlargement, subsequent cell proliferation and finally adenoma formation was postulated by others based on the alignment of these findings with an established MOA for fibrates. Experience with PPARα-inducing pharmaceuticals indicates a lack of human relevance. In this study, the results of a MOA investigation following two weeks of dietary feeding in wildtype (WT) and PPARα-knockout (KO) rats are presented to confirm the dependency of key events following propaquizafop administration on PPARα. In WT animals dose-dependent liver weight increases (65-84%), liver CYP4A (20 fold) and acyl-CoA oxidase (10-15 fold) induction, hepatocellular hypertrophy and proliferation were observed, while in KO rats only a marginal increase in liver weight (24%) was observed without any effect on the other parameters confirming PPARα-dependency and sequelae comparable to a well-understood hepatocarcinogenic MOA in rodents. Based on an evaluation of the data according to the MOA-Human Relevance Framework, we conclude that liver tumors observed in rodents after long-term dietary administration of propaquizafop do not pose a relevant health risk to humans.
Keywords: Propaquizafop, carcinogenicity, hepatocarcinogenic, PPARα, liver, peroxisome proliferators, mode of action, human relevance framework
Protective action of caffeine against acetaminophen-induced liver injury in male CD-1 mice (#534)
Y. Masubuchi1, M. Ebato1
1 Chiba Institute of Science, Faculty of Pharmaceutical Sciences, Choshi, Chiba, Japan
Caffeine is often used as an analgesic adjuvant combined with acetaminophen (APAP) in several prescription drugs and over-the-counter drugs. Apart from the therapeutic benefit of APAP-caffeine combination, possible risk of caffeine to potentiate APAP-induced liver injury (AILI) is of clinical concern. In animal experiments, it has been shown that caffeine both increase and decrease AILI, which might depend on the animal species used and the relative contents of hepatic cytochrome P450 enzymes (CYP). In the present study, we evaluated the effects of caffeine on AILI in male CD-1 mice, which are susceptible to AILI, in relation to changes in CYP isoforms contents and inflammation mediators. Overnight-fasted male CD-1 mice were given APAP intraperitoneally. Some mice were pretreated with phenobarbital or acetone to induce hepatic CYP2B/3A or CYP2E1, respectively. Caffeine was administered at various time points before or after the APAP injection. AILI was assessed by serum leakage of alanine aminotransferase (ALT). Glutathione (GSH) contents were determined in the liver homogenates. Hepatic TNF-α mRNA expression was assayed by real-time RT-PCR. Serum ALT leakage in male CD-1 mice treated with APAP (300 mg/kg) was attenuated by coadministration of caffeine. Caffeine administration 1 hour before APAP was also effective against AILI, but that 16 hour before or 1 hour after APAP did not. Phenobarbital and acetone potentiated AILI. The protective effect of caffeine against AILI was reduced in the mice pretreated with phenobarbital, whereas it was pronounced in acetone-pretreated mice, suggesting the importance of CYP2E1 in the protective effects of caffeine. Early depletion of hepatic GSH, which has been observed prior to initiation of AILI and an index of the rate of APAP activation into N-acetyl-p-benzoquinone imine, was slightly attenuated by caffeine, but the hepatic GSH levels in the mice treated with both APAP and caffeine were still much lower than control mice. On the other hand, caffeine suppressed basal TNF-α expression and early TNF-α induction by APAP. These results suggest that caffeine attenuates AILI via suppression of TNF-α, which has been implicated in the pathogenesis of AILI as an inflammatory mediator, in addition to the inhibition of CYP2E1.
Keywords: drug-induced liver nijury, acetaminophen, glutathione, TNF-α, caffeine
Sodium molybdate attenuates lipid accumulation in the livers of mice fed a diet deficient in methionine and choline (#540)
D. - Y. R. Ryu1, S. Lee1
1 Seoul National University, College of Veterinary Medicine, Seoul, Republic of Korea
Lipid accumulation and oxidative stress are major pathologic contributors to the development of hepatic steatosis. Treatment with molybdate reduces hepatic levels of lipids in diabetic rats. Potential activities of molybdate as an antioxidant have also been demonstrated in various animal models. In the present study, we evaluated the effects of sodium molybdate dihydrate (SM) on hepatic steatosis and associated disturbances in a widely used mouse model of the metabolic disease. Male C57Bl/6 mice at 10 weeks of age were fed a diet deficient in methionine and choline (MCD) and bottled water containing SM for four weeks. The SM treatment markedly attenuated MCD-induced accumulation of lipids, mainly triglycerides, in the liver. Lipid catabolic autophagic pathways were activated by SM in the MCD-fed mouse livers, as evidenced by a decreased level of p62 expression. MCD-induced oxidative damage, such as lipid and protein oxidation, was also alleviated by SM in the liver. However, the level of MCD-induced hepatocellular damage was not affected by SM. Taken together, these findings suggest that molybdate can be used in the treatment and prevention of hepatic steatosis without inducing adverse effects in the liver. To the best of our knowledge, this is the first experimental study to investigate the effects of molybdate in non-alcoholic fatty liver disease, and also the first that demonstrates molybdate-induced autophagy.
Keywords: steatosis, molybdate, liver, autophagy
Hepatic and thyroid effects of phenobarbital in the minipig (#564)
J. Boje Nilsen1, J. Decorde2, C. - A. Erratico2, C. Parmentier3, M. Untrau3, C. Bansard2, P. Ancian2, M. Fonsi2, L. Richert3, R. Forster2, P. Singh2
1 Citoxlab Denmark, Lille Skensved, Denmark
Rodent models are often used to study biochemical mechanisms of toxicity and to establish a mode of action (MOA) for adverse, chemical-induced effects. Phenobarbital (PB) has been shown to induce differential gene expression and CYP2B, CYP3A and UGT enzymes in the rodent liver leading to increased thyroid hormone clearance and subsequent stimulation of cell proliferation in the thyroid that eventually produces thyroid tumors after long-term exposures. This pathway has not been completely investigated in nonrodents. In this study, male Gottingen minipigs were orally administered 15 mg/kg/day PB for 6 days followed by investigation of phase I and II liver enzymes (mRNA expression and enzyme activities), circulating thyroid hormone levels and differential gene expression in the liver, in addition to standard toxicological evaluations. PB-administered minipigs had 46% increased absolute and 42% increased relative liver weights with mild, diffuse hepatocellular hypertrophy versus the control group. PB reduced plasma concentrations of T3 (by about 27% and 47%) and T4 (by about 15% and 20%) versus predose values at 24 hours and after 6 days of administration, respectively. PB induced a 2-fold increase in CYP3A429 and CYP4A24, 5-fold increase in CYP1A2 and around 11-fold increase in CYP2B22 compared to the control group, with increases in mRNA expression of these genes. A 1.6-fold increase in T4-specific UGT phase II enzyme activity was observed in the PB-administered group, and CYP2B22, CYP2A19, CYP2C42, CYP3A39 and CYP3A46 were among the top 20 upregulated genes. This study indicates that several of the early biochemical key events in the PB-induced (rodent) MOA are present in minipigs. It is concluded that the minipig is an interesting model for further study of the effects of xenobiotics on the liver-thyroid axis.
Keywords: Mechanisms of Toxicity, Alternative Animal Models, Target Organ Toxicity
Cytotoxicity and lipid peroxidation of beauvericin and ochratoxin A individually and combined in HepG2 cells (#573)
A. Juan-García1, S. Carbone2, M. Fernández-Franzón1, M. - J. Ruiz1
1 University of Valencia, Preventive Medicine, Burjassot, Valencia, Spain
This work is focused on two mycotoxins: ochratoxin-A (OTA), produced by the fungi os the genus Aspergillus and Penicillium, and beauvericina (BEA) mainly produced by Fusarium. The cytotoxic effect in HepG2 cells exposed to OTA, BEA and OTA+BEA was performed by the MTT method at different concentration ranges (OTA 0.35-100 μM, BEA 0.003-25 μM, OTA+BEA 10:1 ratio) and at three exposure times (24, 48 and 72h); while lipid peroxidation (LPO) through the malondyaldehyde (MDA) production assay. The IC50 values of individual mycotoxins were for BEA 12.5 ± 0.047 µM, 7 ± 0.055 μM and 5.5 ± 0.071 μM at 24, 48 and 72h, respectively; while for OTA 75±0.047 μM, 52.62±0.067 μM and 36±0.09 μM at 24, 48 and 72h, respectively. The IC50 values decreased over the time; so increasing potential of toxicity; however, a greater toxic potential of BEA vs OTA is observed at all times tested in HepG2 cells. Combination OTA+BEA (10:1 ratio) at 24h no IC50 was reached; while at 48h and 72h IC50 values were 3.4±0.045 μM and 2.9±3.034 μM, respectively. The isobologram method in combination showed antagonistic effect at 24h, synergism effect at 48h and 72h at CI25, CI50 and CI75; while additive effect was observed at CI90. The results obtained in LPO assay showed that BEA and OTA increased the production of MDA from 2.1- to 3.6 fold and from 2- to 6.2-fold respect to control, respectively; while the combination of OTA+BEA (10:1 ratio) reached the highest increases of MDA production, which ranged from 3- to 4.6-fold respect to the control. In conclusion, simultaneous presence of mycotoxins in food and feed could increase toxic risk to human and animal health.
Acknowledgments: Spanish Ministry of Economy and Competitiveness (AGL2016-77610R).
Keywords: mycotoxins, cytotoxicity, interaction, lipid peroxidation
Altered microRNA expression patterns related to polyhexamethyleneguanidine phosphate-induced lung toxicity in human alveolar adenocarcinoma (A549) cells (#596)
E. H. Jang1, D. M. Kim1, S. H. Yoo1, Y. B. Han1, K. H. Chung1
1 Sungkyunkwan university, School of pharmacy, Suwon, Gyeonggi-do, Republic of Korea
Polyhexamethyleneguanidine phosphate (PHMG-phosphate), an active component of humidifier disinfectant, was elucidated as a major cause of idiopathic pulmonary fibrosis that resulted in deaths of pregnant women and infants in South Korea. Fibrosis is triggered by recurrent damage in epithelial cells and is largely affected by epigenetic effects including microRNAs (miRNAs). In our previous study, epithelial-mesenchymal transition(EMT), one of the mechanisms in lung fibrogenesis, was observed, and mRNA-miRNA regulatory networks of PHMG-phosphate were investigated. The aim of this study was to elucidate the mechanism underlying PHMG-phosphate-induced EMT through miRNA. Microarray of miRNA profiling was conducted using Affymetrix GeneChip miRNA 4.0 array and was demonstrated by real-time qPCR. To show the specificity of PHMG-phosphate, miRNA expressions were compared with paraquat and transforming growth factor- β(TGF-β). In vivo study rats were repeatedly exposed to PHMG-phosphate 0.1 mg/kg 6 times for 4 weeks. As a result in A549 cells exposed to PHMG-phosphate 0.75 and 3 μg/ml for 24 and 48 hours, expression of miR-33b-3p was signicantly decreased and that of miR-6126 was increased. Moreover the specificity of miR-6126 up-regulation by PHMG-phosphate exposure was showed by comparison with paraquat and transforming growth factor- β(TGF-β). Otherwise miR-33b expressions were decreased similarly by exposure to all sorts of chemicals. Also expression of miR-6126 was markedly increased in lung of rats exposed to PHMG-phosphate repeatedly. To study the functions of miR-6126 at EMT caused by PHMG-phosphate, several proteins involved in EMT were assessed in A549 cells transfected with miR-6126 mimic or negative control mimic using western blot analysis. In conclusion, EMT was induced by miR-6126 up-regulation, so miR-6126 expression can be one of the markers for lung injury by PHMG-phosphate. For further understanding of the effects by miR-6126, more research on the targets of miR-6126 is needed.
Keywords: PHMG-phosphate, miR-6126, lung fibrosis
Multiple targets/molecular initiating events, for OP neurotoxicity may be related with interactions of cholinesterase and phenylvalerate activities in chicken, the model of delayed neurotoxicity (#597)
J. Estévez1, M. Benavent1, V. Selva1, I. Mangas1, E. del Río1, M. A. Sogorb1, E. VILANOVA1
1 University Miguel Hernández, Institute of Bioengineering, Elche (Alicante), Spain
Organophosphorus compounds (OPs) caused several effects and not all can be explained by the classical recognized target: acetylcholinesterase (AChE). A fraction of the membrane proteins with phenylvalerate esterase activity (PVase) is called or neuropathy target esterase (NTE) and is involved in the key initiating molecular event in the OP-induced delayed neuropathy (OPIDN) for which adult chickens is the model animal for testing. We showed that several PVase enzymatic components could be discriminated by their inhibitory kinetic properties in the soluble fraction in chicken brain (Eα, Eβ, and Eγ) and four ChE activity components (C1, C2, C3, and C4). We demonstrated that the PVase activity of the Eα component showed cholinesterase (ChE) activity and it is a butyrylcholinesterase protein, while the other components have no ChE activity. C1 was identified with the Eα activity. We suggested that it might be related to the potentiation/promotion phenomenon of the OPIDN. Human butyrylcholinesterase (hBuChE) also shows PVase activity. Evidences are showed that both activities are associated to the same active center: (1) Both substrates (acethylthiocholine and phenyl valerate) compete in their activities. (2) Mipafox, iso-OMPA and PMSF inhibited with similar kinetic behavior. Further kinetic and structural research is under study. In the membrane brain fractions, four components (EPα, EPβ, EPγ and EPδ) of PVase have been discriminated by their inhibitory kinetic properties using irreversible inhibitors (paraoxon, mipafox, PMSF). Only EPα PVase activity were inhibited by acetylthiocholine and its cholinesterase activities by phenylvalerate. Four ChE activity enzymatic components (CP1, CP2, CP3 and CP4) were discriminated in membrane fraction, on the basis of inhibitory kinetic with irreversible inhibitors (mipafox, paraoxon, PMSF and iso-OMPA). The ChE components CP1 and CP2 could be related to the PVase activity component EPα; This is resistant to PMSF and is highly sensitives to mipafox and paraoxon but is spontaneously reactivated when inhibited by paraoxon. The relation of PVases with ChEs need to be included in the molecular adverse pathways of OP delayed neurodegenerative toxicity under approaches with multi-target or multi-initiating molecular events.
Keywords: cholinesterase, phenylvalerate esterase, NTE, mipafox, paraoxon, iso-OMPA, PMSF
The effect of PI3K/Akt and Notch signaling pathway in polyhexamethylene guanidine-phosphate induced epithelial-mesenchymal transition in human alveolar epithelial A549 cells. (#611)
E. H. Jang1, M. H. Jeong1
1 Sungkyunkwan University, School of pharmacy, Suwon, Gyeonggi-do, Republic of Korea
Polyhexamethylene guanidine-phosphate (PHMG-phosphate) was widely used as a biocidal disinfectant to inhibit microbial growth in household humidifiers, which caused severe lung injury including idiopathic pulmonary fibrosis in hundreds of victims in South Korea. One of the mechanisms in lung fibrogenesis is epithelial-mesenchymal transition (EMT), a process in which epithelial cells gain a mesenchymal phenotype. Our previous study showed that PHMG-phosphate exposure caused EMT in human alveolar epithelial A549 cells in vitro. PI3K/Akt and Notch signaling have been shown to induce EMT after repressing transcription of the E-cadherin gene. To elucidate the mechanism of EMT induced by PHMG-phosphate, the activation of PI3K/Akt and Notch pathway by PHMG-phosphate was examined. The expressions of representative markers of PI3K/Akt and Notch pathway, p-Akt and NICD, after exposure to PHMG-phosphate in A549 cells were increased dose-dependently in Western blotting. The effect of PI3K/Akt and Notch pathway in EMT was confirmed using PI3K inhibitor, LY294002 and Notch inhibitor, DAPT. Moreover, the transcription factor expressions of down-stream targets of PI3K/Akt pathway, ZEB and β-catenin, and Notch pathway, Hes-1, were increased in A549 cells exposed to PHMG-phosphate. Our results demonstrate that PHMG-phosphate can trigger the activation of PI3K/Akt and Notch signaling pathway, which mediates EMT induced by PHMG-phosphate. To provide further understanding of the molecular effects induced by PHMG-phosphate, the mechanisms of Akt and Notch activation are required to be studied.
Keywords: PHMG-phosphate, lung fibrosis
Sterigmatocystin induces oxidative stress in male Wistar rats (#630)
D. Rašić1, D. Želježić2, V. Micek3, D. Kifer4, D. Jakšić5, M. Peraica1, N. Kopjar2, M. Šegvić Klarić5
1 Institute for Medical Research and Occupational Health, Toxicology Unit, Zagreb, Croatia
Sterigmatocystin (STC) is a polyketide mycotoxin with a bifuran ring structurally similar to aflatoxin. It is a precursor in aflatoxin biosynthesis in some Aspergillus species from the section Flavi and the final product in a number of Aspergilli from the section Versicolores. STC has been detected in various foodstuffs as well as indoor environments such as damp, moldy dwellings, carpets, and grain dust. In acute oral exposure it targets the liver and the kidneys. Its oral LD50 in male rats is about 160 mg kg-1 b.w. Similar to aflatoxin, STC's mechanism of action has been linked to the formation of exo-epoxide, which readily forms DNA adducts. Considering that aflatoxin toxicity has also been associated with oxidative stress, the aim of this study was to check whether the same is true for STC toxicity. With that in mind, we treated male Wistar rats with single oral STC doses of 1/4, 1/8, and 1/16 of LD50 (40, 20, and 10 mg kg-1 b.w., respectively) and measured superoxide dismutase (SOD) activity in plasma on a plate reader using a commercial kit. We also measured glutathione (GSH) and malondialdehyde (MDA) in plasma, kidney, and liver tissue using a spectrophotometer and HPLC, respectively. To assess oxidative DNA damage in the liver and kidneys we used comet tail intensity (i. e. DNA % in tail) obtained with the hOGG1-modified comet assay. STC did not affect GSH concentrations. SOD activity in plasma dropped significantly only in rats treated with 1/8 of LD50 (9.26±0.26 vs. 10.08±0.82 U mL-1 in control). All doses significantly increased MDA concentrations in plasma and kidney, but not in the liver. Oxidative DNA damage showed up in the liver and kidney cells, and was more pronounced in the kidneys, particularly at 1/8 of LD50. Our findings suggest that acute oral exposure to STC causes greater oxidative stress in the kidneys than the liver. This work has been fully supported by the Croatian Science Foundation under the project MycotoxA (HRZZ-IP-09-2014-5982).
Keywords: DNA damage, glutathione, malondialdehyde, sterigmatocystin, superoxide dismutase
A comprehensive comparison of in vitro assays utilised to detect mitochondrial toxicity (#636)
R. A. Maclennan1, J. Eakins1, C. Bauch1, B. Park1, P. Walker1
1 Cyprotex Discovery, Toxicology, Alderley Park, Macclesfield, United Kingdom
Mitochondrial dysfunction has been implicated in numerous drug induced adverse events, such as liver failure and cardiac toxicity. The potential of drugs to be mitochondrial toxicants can be determined by either comparing the increase in cytotoxicity of compounds in media containing galactose compared to glucose (Glu/Gal assay). In galactose conditions, cells are more reliant on oxidative phosphorylation and therefore more sensitive to mitochondrial disruption. Alternatively mitochondrial toxicants can be determined using a mitochondrial respiration assay which measures cellular oxygen consumption rate (OCR), reserve capacity (RC) and extracellular acidification rate (ECAR). A third approach utilises fluorescence dyes to measure changes in mitochondrial membrane potential (MMP) using high content imaging and compared to ATP depletion and cell loss. Alteration of MMP is strongly associated with mitochondrial toxicity. These approaches were analysed for the predictivity of compounds known to result in mitochondrial toxicity by different mechanisms of action.
Seventy compounds (known mitochondrial toxicants with varying mechanism of action and compounds with no mitochondrial effect) were screened through the Glu/Gal assay, the MMP and cytotoxicity assay, and the mitochondrial respiration assay, in HepG2 cells. Comparing the ECAR and OCR data with Glu/Gal data showed there were a number of toxins such as rosiglitazone, amiodarone and perhexiline which were not detected in the Glu/Gal assay, however were flagged as being positive for mitochondrial toxicity based upon the mitochondrial respiration assay. The MMP and cytotoxicity assay is higher throughput in comparison to the mitochondrial respiration assay with the benefit of better specificity (e.g. positive response to rosiglitazone) than the Glu/Gal assay and can provide some additional mechanistic information.
In summary, determining cellular OCR, reserve capacity and ECAR provides a more predictive, and sensitive measure of mitochondrial toxicity and in addition gives an understanding of potential mechanisms of action when compared to the Glu/Gal assay. The MMP and cytotoxicity assay provides a more sensitive alternative to the Glu/Gal assay where high throughput is required in early drug discovery.
Keywords: in vitro, mitochondrial toxicity, Seahorse, MMP, Glu/Gal, Cyprotex, Evotec
Caffeic acid boosted the efficiency of benzyl isothiocyanate to induce the death of human breast adenocarcinoma (#651)
M. H. A Rahaman1, W. B. Wan Omar2, N. H. Abd Kadir1
1 Universiti Malaysia Terengganu, School of Fundamental Science, Kuala Terengganu, Terengganu, Malaysia
Background: Combining potential drug originated from edible plant such as benzyl isothiocyanate (BITC) with antioxidant like caffeic acid (CA) could be more compatible to human body and produce synergistic effect to kill cancer. Our recent study demonstrates the trends and mechanisms of cytotoxicity effects of BITC, CA and the novel combination of BITC and CA to enhance the effectiveness of the cancer treatment.
Methods: Cytotoxicity effects of BITC, CA and their combination toward human breast adenocarcinoma (MCF-7) cells were evaluated by using MTT assay after 24 and 48 hours of treatments. We explored mechanisms of cytotoxicity effects by examining the protein expression of glutathione-S-transferase (GST)-pi and GST-alpha, p38, ERK 1/2, Nrf-2 and Bcl-2 by Western blot technique. Glutathione (GSH), reactive oxygen species (ROS) and caspase 3/7 level were measured fluorometrically.
Results: High dose of CA needed to cause the death of MCF-7 cells. Interestingly, we found that the combination of BITC and CA created the synergistic effect to kill MCF-7 cells in time and dose-dependent. BITC increased the ROS level and exploit it to induce MCF-7 cell death whereas the combination of CA and BITC decrease ROS level but effectively reduced MCF-7 cells viability. Coordination of p38 MAPK, ERK1/2, NRF-2, Bcl-2, caspase 3/7 and phase II of xenobiotic metabolism by the up-regulation of GS- pi and GST-alpha revealed the intracellular signaling to cause MCF-7 cells death.
Conclusion: Conclusively, CA boosted the efficiency of BITC to kill the cancer cells. The elevation of GST catalyzed the conjugation of GSH with BITC, CA and their combination, thus lowered the amount of GSH to create the imbalance of redox homeostasis and cause the death of cancer cells.
Acknowledgment: Ministry of Higher Education Malaysia and UMT for funding provided (Vot. 53131).
Keywords: cell death, chemopreventive, xenobiotic metabolism, oxidative stress, synergistic effect
COMBINED ASSAYS ON ISOLATED HEART MITOCHONDRIA AND COR.4U hiPS-DERIVED CARDIOMYOCYTES TO PREDICT DRUG-INDUCED CARDIOTOXICITY (#670)
C. Martel1, C. Pertuiset1, B. Aubry1, M. Porceddu1, N. Buron1, R. Kettenhofen2, A. Fouassier2, A. Borgne-Sanchez1
1 Mitologics, Paris, France
Drug-induced mitochondrial toxicity is a major issue for patients because such toxicity can trigger acute or chronic injury involving different organs or tissues including liver, heart, muscle, kidney and adipose tissue. We developed a medium-throughput in vitro screening platform to detect loss of mitochondrial function and integrity on isolated liver mitochondria  and HepaRG differentiated cells  investigating acute, indirect and chronic mitochondrial alterations for DILI prediction. For early detection of cardiotoxicity potential of lead compounds, we set up multiparametric assays on isolated rat heart mitochondria and terminally differentiated human iPSC-derived cardiomyocytes, Cor.4U cells. Both models were characterized in terms of mitochondrial (OxPHOS) activity and Cor.4U cells were shown to present highly functional mitochondria suitable for compound testing. Four reference drugs selected on their clinical-determined likelihood to induce (amiodarone, sulindac, tamoxifen) or not (erlotinib) mitochondrial damages in human heart were tested on both models. On isolated mitochondria their ability to induce swelling, transmembrane potential loss, inhibition of O2 consumption either driven by complex I, complex II or fatty-acids beta-oxidation (FAO) was challenged. Amiodarone, sulindac and tamoxifen were found to directly inhibit mitochondrial respiratory chain while erlotinib did not alter rat heart mitochondria. In addition, Cor-4U cells were treated for 3 hours with compounds and subjected to O2 consumption driven by Complex I or Complex II, mitochondrial transmembrane potential and mtROS production measurements. Results showed that amiodarone and tamoxifen presented strong mitochondrial toxicity in Cor.4U cells, while sulindac did not induce strong mitochondrial toxicity (only slight inhibition of CII-driven O2 consumption). Surprisingly, erlotinib was shown to have an uncoupling-like effect that was not measured on isolated mitochondria. Thus, using a combination of both rat mitochondria and human cardiomyocytes models allows a good understanding of compounds mechanism of action. Overall, such assays help to early investigate the potential mitochondrial toxicity of new compounds, in addition to classical preclinical studies, to select safer drug candidates.
 Porceddu M et al (2012) Toxicol Sci. 129: 332-45.
 Porceddu et al (2018) Methods in Pharmacology and Toxicology, Eds Chen, M & Will Y.
Keywords: Mitochondria, cardiotoxicity, iPS-derived cardiomyocytes, Respiratory chain, oxidative stress
Dependence of Developmental Toxicity for Polyaromatic Hydrocarbons on Aryl Hydrocarbon Receptor: Range-finding with Benzo(a)pyrene (#676)
H. Ketelslegers1, C. North2, F. Van Otterdijk3, D. Adenuga2
1 European Petroleum Refiners Association, Concawe division, Brussels, Belgium
Previous assessment of heavy and poorly refined petroleum substances demonstrate a strong association of developmental toxicity with levels and types of polyaromatic hydrocarbon (PAH) content. While suggestive evidence exists indicating a role for the aryl hydrocarbon receptor (AHR) in this toxicity, no direct experimental evidence exists. To assess the role of AHR in developmental toxicity of PAHs, a positive control PAH was sought. Although benzo[a]pyrene (BaP) is coal-derived, as it is only present at low ppb levels in most petroleum products, BaP is among the best characterized PAHs thus was identified as a reasonable candidate. Groups of five or six pregnant wild-type (WT) and AHR-/- rats (Sprague-Dawley background) were exposed to 0 or 300 mg BaP/kg/d by single daily oral gavage from gestation day (GD) 6 through 20. Maternal body weight gain significantly decreased in BaP exposed WT and AHR-/- dams from GD9 and GD12, respectively. Reduced weight gain resulted in final body weights 11% lower in BaP exposed WT dams and 18% lower in AHR-/- dams. BaP exposure significantly increased total number of fetal resorptions, particularly early resorptions, in WT dams (56.4% of fetuses [55.0% early and 1.4% late] versus 3.7% in WT control), but did not significantly affect fetal resorptions in AHR-/- dams (4.0% of fetuses versus 0.0% in AHR-/- control). Pre-implantation losses were also increased in BaP exposed WT rats (16.7% compared to 2.4% in WT control, 0.0% in AHR-/- control, and 1.7% in BaP exposed AHR-/- rats). Consistent with increased resorptions, WT BaP exposure significantly decreased number of viable foetuses (43.6% versus 96.3% in control) and post-implantation loss (56.4% versus 3.7% in control), but not AHR-/- dams. The results suggest 300 mg BaP/kg/d is an acceptable dose as a positive control for developmental toxicity in WT rats. Overall, this dose range finding experiment provides a first indication for a role of the AHR in PAH related developmental toxicity.
Keywords: mode of action, Ah Receptor, AhR, knock-out rats, benzo(a)pyrene, PAH toxicity, developmental toxicity, reprotox
RISK ASSESSMENT AND MECHANISM OF TOXICITY OF MYCOSYNTHESISED SILVER NANOPARTICLES TO PROKARYOTIC AND EUKARYOTIC MODEL (#690)
I. R. GUPTA1, 2, M. K. Rai2
1 GOVERNMENT INSTITUTE OF SCIENCE, DEPARTMENT OF BIOTECHNOLOGY, AURANGABAD, MAHARASHTRA, India
Among various nanomaterial silver nanoparticles (AgNPs) has been used widely for many application. However, with the rise in their applications the concerns of their safety are also increasing at alarming rate. Therefore, it is need of the hour to study their nanotoxicological aspect. Therefore, the present study was designed to evaluate the toxicity of mycosynthesised AgNPs. They were synthesised by using three species of Fusarium and a species of Phoma. Later on, they were characterised by UV-Vis Spectrophotometry to confirm their synthesis, Fourier Transform Infra-Red spectroscopy (FTIR) to confirm the protein capping on synthesised AgNPs, Zeta potential to know their stability, Nanosight LM20 to know their siez in working condition, X-ray diffraction (XRD) to confirm the silver as elemental composition and Transmission Electron Microscopy (TEM) to know their size and shape. The AgNPs, at different concentration, were then exposed to a specially designed Psueodomonas putida biosensor having lux gene. For eukaryotic studies, they were exposed to various cell lines including Caco-2, a colon cancer cell line, A549, a human alveolar cell line and IEC6, a rat intestinal epithelial cell line.
In result we found stable AgNPs to reduce the culturability of P. putida at the concentration range of 0.4 to 0.8 μg/ml. However, with the help of MTT assay, the AgNPs within size range of 5 to 25 nm were found to show the IC50 of 8 μg/ml against cell lines. The effect of AgNPs on interaction with cell membrane, formation of reactive oxygen species and fragmentation of DNA was tested by using LDH leakage assay, ROS assay and DNA fragmentation assay. The study revealed that AgNPs damages the cell membrane, promoting the accumulation of reactive oxygen species and damaging the nuclear material. Overall these mechanisms contribute to the toxicity of mycosynthesised AgNPs. This study is probably unique in performing the risk assessment and the mechanism of toxicity of mycosynthesised AgNPs on prokaryotic and eukaryotic model.
Keywords: MYCOSYNTHESIS, SILVER NANOPARTICLES, IN VITRO TOXICITY, MECHANISM OF TOXICITY, RISK ASSESSMENT
Microsomal metabolism and mutagenicity of cis- and trans-anethole (#691)
J. A. Fuhlbrueck1, E. C. Schwind1, A. Lencioni1, M. J. Carlsson1, A. T. Cartus1
1 Technische Universitaet Kaiserslautern, Food Chemistry and Toxicology, Kaiserslautern, Rhineland-Palatinate, Germany
Anethole is a natural constituent in many herbs and spices such as anise, star anise and fennel and belongs to the group of phenylpropenes. In natural products, anethole is usually present as a mixture of its trans- (1) and cis-configured (2) stereoisomers. Anethole or anethole containing plant components are commonly used as flavoring substances for foods, cosmetics and drugs, e.g. tea, alcoholic beverages like ouzo, or mouthwashes. Although 1 has been given GRAS status (generally recognized as safe) by FEMA (Flavor and Extract Manufacturers Association), recent data showed the DNA adduct formation of trans-anethole in fetal turkey livers. To get more insights in the toxicological properties of both, trans- and cis-anethole, we investigated the microsomal metabolism, mutagenicity and DNA reactivity of 1 and 2 and their main metabolites trans- (3) and cis-anethole epoxide (4).
The microsomal metabolism of 1 and 2 was investigated using liver microsomes of different species. Metabolites were characterized using HPLC-UV/Vis and HPLC-MS/MS. The mutagenicity of 1–4 was examined using the Ames fluctuation test with different Salmonella typhimurium strains. Nucleoside adducts were synthesized by the reaction of 3 or 4 with 2′-deoxyadenosine (dA) and 2′-deoxyguanosine (dG).
We were able to identify eleven metabolites of 1 and 2 from microsomal incubations. The threo- and erythro-configured dihydroxydihydrodiols, likely formed from 3 and 4 as precursors, as well as 3′-hydroxylated metabolites were identified as the main metabolites of 1 and 2. The Ames test showed no mutagenic effect for 1 and 2 in the tested concentrations (up to 1 mM), whereas 3 and 4 were found to be mutagenic at concentrations at or above 100 µM. The reaction of 3 and 4 with dA and dG resulted in the formation of adducts with a m/z of 416 and 432, respectively.
Our results show that both anethole isomers, 1 and 2, are metabolized mainly via epoxidation of the side-chain. The intermediate formed reactive epoxides 3 and 4 may be responsible for the formation of DNA adducts detected in recent studies, since both epoxides react readily with dA and dG to form nucleoside adducts of the expected mass. Investigations to elucidate if trans- or cis-anethole forms these DNA adducts also in vitro and in vivo are currently ongoing in our group.
Keywords: DNA adducts, HPLC-MS/MS, mutagenicity, microsomal metabolism, phenylpropenes
The driving forces of stem cell plasticity under chemical stress: a central role for TSGs and the stem cell niche (#708)
A. Wouters1, A. Van Roten1, A. Barakat2, 3, T. Tran4, A. - S. Stevens1, J. - P. Ploem1, N. Leynen1, L. Gentile2, 4, K. Smeets1
1 Hasselt University, Centre for Environmental Sciences, Diepenbeek, Belgium
Tissues are a complex entity of different cells. Tissue heterogeneity increases variability in toxic responses, and makes it difficult to interpret adverse outcomes. A different intracellular signature, differentiation state and surrounding niche result in cell type-specific coping strategies. Within the complexity of multicellular tissues, stem cells often have a lower sensitivity to toxic compounds. Stem cells are characterized by a low metabolic state, a preference to maintain low oxygen levels and a well functioning repair system. Their presence in developing and carcinogenic tissues makes them an even more relevant toxicological target.
In this study, we focused on the coping strategies of pluripotent stem cells to chemically-induced damage in an in vivo situation, i.e. in the planarian Schmidtea mediterranea. Planarians are long known for their regenerative ability, which hinges on pluripotency. This renders the opportunity to study pluripotent stem cell responses while stem cells reside in their original niche. Cellular damage was induced via different toxicants: MMS, Cd and AgNPs. Pluripotent stem cells were able to recover from the induced cellular and DNA damage and successfully regenerated an entire organism. Depending on the microenvironment and needs of the organism, stem cell responses toggled between apoptosis and DNA repair, with p53 as a probable regulator of the stem cell’s self-renewal switch. Stem cell repair responses were further investigated by combining two top-down approaches, an in silico screening and a proteomic screening after chemical exposure. Underlying stem cell plasticity, we observed the involvement of several TSGs, either known or novel, who’s loss-of-function, in combination with chemical exposure, destabilized the stem cell compartment, triggering tumor formation. A knock down of matrix metalloproteinase B induced different types of protrusions, suggesting a control exerted by the ECM on the stem cell compartment. Although a further effort is needed to unravel how the integrated network of TSGs prevents stem cell transformation and tumorigenesis, our work pinpoints that planarian tumors originate from the ectopic, uncontrolled proliferation of the ordinary stem cells, and that TSGs and the stem cell niche have a central role in their transformation.
Keywords: stem cell, pluripotency, alternative model, stem cell toxicology
Exploring the usefulness of morphological profiling of cells to study toxicity mechanisms (#716)
P. Georgieva1, W. Schaal1, O. Spjuth1
1 Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden
Although there have been major technological advances in how we study biological processes and systems in the last years, studying toxicity pathways still remains difficult, time-consuming and resource-intensive. High-content imaging of cells stained with multiplexed dyes, “Cell Painting”(1), has been successfully applied in drug screening(2) of chemical compounds based on morphological changes they induce. However its application in the field of toxicity is still rather unexplored and comprises an exciting opportunity to unravel toxicity mechanisms and pathways.
Our initial experiments show promising results for mechanistic classification of toxicity mechanisms and we hypothesize that the method could be used for pathway enrichment. To this end we will strive towards automating the platform, and develop robust analysis pipelines to handle data/analysis to be able to study mechanisms on larger scale
(1) Bray, Mark-Anthony et al. "Cell Painting, A High-Content Image-Based Assay For Morphological Profiling Using Multiplexed Fluorescent Dyes". Nature Protocols 11.9 (2016): 1757-1774.
(2) Simm, Jaak et al. “Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery”. Cell Chemical Biology, In press
(3) Carpenter, Anne et al. “CellProfiler: image analysis software for identifying and quantifying cell phenotypes”. Genome Biology (2006) 7:R100
Keywords: cell painting, High-content imaging, image-based morphology profiling
A System-based Comparative Proteomics Approach to Investigate Heavy metals mixtures toxicity mechanism relates to the neurodegeneration on Hippocampal cell line (#639)
V. Kumar1, V. Karri1, M. Edwin2, S. Coort3, C. Evelo3, E. Oliveira4, M. Schuhmacher1
1 Universitat Rovira i Virgili, Environmental Engineering Laboratory, Departament d'Enginyeria, Tarragona, Barcelona, Spain
Exposure to neurotoxic metals lead (Pb), arsenic (As), and methylmercury (MeHg) occurs to mixtures and not to single contaminants. Metal interactions have posed a big challenge in toxicity characterization and human health risk assessment of environmental mixtures. To characterize the impacts of metal mixtures interactions on protein and cytotoxicity responses, we established an in vitro omics approach integrating bioinformatics, and statistics, to measure mixtures interactions at protein level in hippocampal cells. Multivariate statistical analysis showed the correlation of protein response between mixtures. Gene functional and pathway analyses of proteomic data confirms alterations related to mitochondrial dysfunction, oxidative stress, m-RNA splicing, and ubiquitin system dysfunction related neurodegeneration. Bioinformatics analysis of mixtures protein data are presented in comparative manner. Our results showed that, the significant differences of affected proteins in the mixtures. This study provides a novel proteomic approach for characterization neurotoxicity of metal mixtures in hippocampal cells.
Keywords: Metal mixtures, Cytotoxicity, Proteomics analysis, Systems biology
Carbon nanotubes influence surface properties of the phospholipid membrane (#124)
D. Kondej1, T. R. Sosnowski2
1 Central Institute for Labour Protection – National Research Institute, Department of Chemical, Aerosol and Biological Hazards, Warsaw, Poland
Carbon nanotubes due to their structure and specific chemical, mechanical, thermal, optical and electrical properties have a great potential for applications and constitute an important group of nanomaterials. The aim of this study was to investigate the effect of selected carbon nanotubes on the surface properties of a phospholipid monolayer as the simplest model of biological membranes. Carbon nanotubes of various types, dimensions and modifications were chosen. Identification involved microscopic analysis using scanning electron microscopy (SEM), measurement of specific surface area (BET) and analysis of particle size distribution in liquid dispersion (DLS). The research was carried out using Langmuir-Wilhelmy balance (KSV, Finland). The phospholipid monolayer was prepared by applying 1 mg/ml solution of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Sigma-Aldrich, USA) on the surface of the aqueous subphase and evaporation of the solvent. The suspensions of carbon nanotubes at surface concentrations ranging from 10.3 ng/cm2 to 205.1 ng/cm2 were instilling on DPPC monolayer. The DPPC monolayer was isothermally compressed (37 °C) with constant velocity (1.25 cm2/s). It was found that carbon nanotubes cause (a) the shift of compression isotherms and compressibility curves towards higher values of surface pressure and surface area per one DPPC molecule, (b) the increase of maximum compressibility of DPPC monolayer and the corresponding surface pressure, (c) the reorganization of DPPC molecules at the air-liquid interface in gaseous phase (G), liquid-expanded phase (LE), liquid-condensed - liquid-expanded coexistence (I) and liquid-condensed phase (LC). The changes in surface properties of DPPC monolayer indicates a disruption of elasticity of the phospholipid membrane in the presence of the carbon nanotubes tested.
This paper has been based on the results of research task No. II.N.10 carried out within the scope of the fourth stage of the National Programme “Improvement of safety and working conditions” partly supported in 2017–2019 — within the scope of research and development — by the Ministry of Science and Higher Education/National Centre for Research and Development. The Central Institute for Labour Protection – National Research Institute is the Programme’s main co-ordinator.
Keywords: carbon nanotubes, surface properties, phospholipid, biological membranes
The applicability of zebra fish animal models for acute toxicity testing of silver nanoparticle (#141)
Y. - J. Wang1, Y. - H. Lee2
1 National Cheng Kung University, Department of Environmental and Occupational Health, Tainan, Taiwan
Nanoparticle-induced toxicological mechanisms have become one of the most studied topics in toxicology during the last few years. Because of their excellent antimicrobial activity, silver nanoparticles (AgNPs) are recognized as a promising nanomaterial with widespread applicability. However, the impact of AgNPs on biological systems, regarding their possible effects and fate in living cells are still limited. In order to assess AgNPs hazard and risk, reliable and reproducible screening approaches are needed to test the basic materials as well as nanoenabled products. In recent years, zebrafish was recommended as the nanomaterials toxicity screening animal model. The purpose of current study is to investigate the toxic effects and mechanism of AgNPs on zebrafish embryos. The results indicated zebrafish embryos exposed to AgNPs led to reduced survival ratio in a size-, dose-, and time dependent manner. Abnormal phenotypes (abnormal jaw, axis flexure, pericardial edema, and yolk sac edema) and developmental retardation can also be found. In addition, oxidative stress, ER stress and apoptosis are involved in the underlying toxic mechanisms. These results demonstrated that different size of the AgNPs can trigger different toxicity, in which the larger size cysteamine-modified AgNPs was more toxic than smaller size AgNPs.
Keywords: silver nanoparticle, zebra fish toxicity, particle size
Potential cytotoxic risk and chronical inflammation after exposure to graphene platelets (#158)
T. Svadlakova1, A. Malkova2, P. Kulich3, J. Masek3, F. Hubatka3, P. Turanek Knotigova3, Z. Fiala2, L. Borska4, J. Turanek3
1 Charles University, Faculty of Medicine, Institute of Clinical Immunology and Allergology, Hradec Kralove, Czech Republic
Graphene represent two-dimensional (2D) nanomaterial with unique physical and chemical properties. Unlike graphene oxide less is known about biological effects of other derivatives such as graphene platelets and sheets (GPs) which workers are exposed during manufacturing. These non-biodegradable materials could pose a risk especially for respiratory system after exposure by inhalation. It has been proved that GPs of a certain size can be delivered beyond the ciliated airways and deposit in alveoli. There are internalized mostly by alveoli macrophages or simply persists in intercellular spaces, where can physically interact with other cells. This may lead to inflammation, disruption off cell metabolism and tissue damage. This study represents preliminary data of possible interactions between two different commercially available GPs and selected monocyte/macrophage cell lines.
Dynamic light scattering method and Transmission Electron Microscopy (TEM) were used for characterization of GPs. THP-1 cells, THP-1 null cells, RAW BLUE cells and primary monocytes were used for experiments. The viability of cells was evaluated via WST-1 and LDH assays after 24, 48 and 72 hours of exposition. Localization of GPs was detected by TEM and confocal microscopy. Potential immune response was detected by activation of inflammasome NLRP3 test.
TEM confirmed presence of both GPs in cytoplasm after 24 hours, but no particles were located in nucleus. First results have shown that both GPs caused neither acute cytotoxicity nor activation of inflammasome NLRP3 after 24 as well as 48 hours of exposition. However, levels of LDH which is released through damaged cell membrane increased with GPs concentration after 48 and 72 hours exposition of RAW BLUE cells. Interestingly, there was no increase of LDH after exposition of activated THP-1 cells and monocytes.
Supported by program PROGRES Q-09 and Q-10, ERDF/ESF projects NANOBIO CZ CZ.02.1.01/0.0/0.0/17_048/0007421 and FIT CZ.02.1.01/0.0/0.0/15_003/0000495, project CENATOX GAP503/12/G147.
Keywords: Graphene platelets, Cytotoxicity, Inflammasome, Cell cycle
Behavioral alterations in the short and long term after chronic exposure of silver nanoparticles to young rats (#160)
J. Orzelska-Gorka1, L. Struzynska2, S. Talarek1, E. Kedzierska1, G. Biala1
1 Medical University of Lublin, Chair and Department of of Pharmacology and Pharmacodynamics, Lublin, Poland
The purpose: Silver nanoparticles (nanoAg) are used in a wide array of industry and biomedicine. The increasing applications of nanoAg may lead to necessitate a general health and environmental risk assessment of them. The toxicity of nanoparticles depends on their unusual features including growing reactivity and particle-to-cell contact (due to a large number of particles). Despite a few toxicological reports, in vitro and in vivo, neurotoxic effects of nanoAg are poorly known. The aim of the present study was to investigate the possible behavioural changes of rats after prolonged neonatal exposure (21 days) to small (10 nm) citrate-stabilized nanoAg at a dose of 0.2 mg/kg b.w. using a wide range of behavioural assessments.
Methods: Rat pups (14 days old) were administered intragastrically with nanoAg (0.2 mg/kg) or silver citrate (Ag+) for 21 days. Behavioural tests were performed twice – one day and one month after the discontinuation of substance administration. The measurement of locomotor activity (in actimeters), motor coordination (rota-rod test), memory performance (novel object recognition test), depression (forced swimming test) and anxiety-like behavior (elevated plus maze test), generally accepted in investigations of the central activity of new compounds, were made.
Results and conclusion: NanoAg exposure did not significantly influence locomotor activity and motor coordination of rats, both in the short and long term. Pro-depressive, anti-anxiety and pro-cognitive effects were observed after prolonged administration of nanoAg, but only one day after the discontinuation. These results were not shown one month after the discontinuation. Our data may suggest that nanoAg exerts a significant central action, together with low neurotoxicity as measured in behavioural animal tests.
The study was supported by the Statutory Activity provided by the Polish Ministry of Science and Higher Education for Medical University of Lublin (DS 23/2016).
Keywords: silver nanoparticles, behaviour, rats, central activity, neurotoxicity
Potassium octatitanate fibers are possibly carcinogenic in male Fischer 344 rats (#185)
M. A. M. Abdelgied1, 3, A. El-Gazzar1, 5, D. Alexander1, W. Alexander1, T. Numano1, M. Iigo1, A. Naiki2, M. Abdelhamid4, 6, H. Takase7, A. Hirose8, Y. Taquahashi9, J. Kanno10, S. Takahashi2, H. Tsuda1
1 Nagoya City University, Nano toxicology Project, Nagoya, Japan
Potassium octatitanate (POT) fibers are commonly used as alternative to asbestos. Their shape and biopersistence suggest that they are possibly carcinogenic. However, previous studies showed that inhaled POT fibers had little carcinogenic potential. Therefore, we conducted a short-term study in which we administered well-dispersed POT fibers to rats using transtracheal intrapulmonary spraying (TIPS). We found that POT fibers had greater biopersistence in the lung and pleural cavity and caused stronger tissue response than either anatase titanium dioxide nanoparticles (a-nTiO2) or rutile titanium dioxide nanoparticles (r-nTiO2), which have been evaluated as group 2B carcinogens. Consequently, we are conducting a 2-year study of POT fibers to investigate their carcinogenicity using MWCNT-7, a known carcinogen in rats, and inert r-nTiO2 as reference materials. Male F344 rats were administered 0.25 and 0.5 mg POT fibers and r-nTiO2 and 0.5 mg MWCNT every other day over a period of 15 days by TIPS. Rats were killed 3 weeks and 52 weeks from the start of experiment. In the lung, POT and MWCNT fibers induced significant increases in alveolar macrophage number, granuloma formation, reactive hyperplasia and thickening of the alveolar wall, pulmonary collagen deposition, and pulmonary 8-OHdG levels. In contrast, for both of the r-nTiO2 treated groups, the tested parameters were not different from the controls. Both visceral and parietal pleura showed increase in thickness, PCNA labeling indices, and few areas of visceral pleural hyperplastic proliferation in the 0.5 mg POT and the MWCNT treated groups. The pleural cavity lavage showed significant increases in total protein levels in the 0.5 mg POT fiber group, approximately 4 fold higher than in MWCNT treated group. Also, numerous POT fibers but few MWCNT fibers were detected in the pleural cavity lavage cell pellet. In conclusion, POT fibers were persistent inside the lung and pleura and induced a similar or greater tissue response compared to MWCNT-7, indicating the possibility that inhaled POT fibers are carcinogenic in rats.
This work was supported by Health and Labour Sciences Research Grants of Japan, the 5th Term Long-Range Research Initiative (2017) by Japan Chemical Industry Association, and Egyptian cultural affairs and missions sector.
Keywords: Potassium octatitanate fibers, MWCNT, Transtracheal intrapulmonary spray, Male Fischer 344 rats
The PLATOX project: Combining in vitro and in vivo investigations to generate valid toxicity data for risk assessment of graphene nanoplatelets (#223)
O. H. Creutzenberg1, C. Ziemann2, D. Schaudien3, H. Oliveira4, L. Farcal5
1 Fraunhofer ITEM, Inhalation Toxicology, Hannover, Lower Saxony, Germany
Carbon-based nanoplatelets (CNP) represent a new class of 2-D nanostructures in multiple variants and with interesting functional properties (e.g. material enforcement and electrical conductivity). The hazard characterization of CNP is still incomplete. Commercial CNP candidates (ACS Material, USA) were selected, covering single layer (SLG) or multi layer graphene (MLG), carboxyl graphene, single layer graphene oxide, and graphite oxide. Technical soot (Printex 90) served as non-platelet reference. Sterility and endotoxin content of the CNP were analysed and the morphology (SEM pictures) and the specific surface area (BET method) were re-evaluated. As in vitro screening models both, primary rat alveolar macrophages (AM) and MRC-5 human lung fibroblast cells were analysed on membrane damage (LDH release) and metabolic activity (AlamarBlue test). Interestingly, the two SLG induced marked concentration-dependent membrane damage in AM after 24h of incubation, with a BMD30 of 3.2 and 2.5 µg/cm2, whereas no such effect was observed for MRC-5 cells. Some LDH release was also shown for single layer graphite oxide (BMD30: 39.3 µg/cm2). The other materials were nearly inactive. Significant effects on metabolic activity were not observed. In AM, SLG additionally induced direct DNA damage and release of PGE2 and TXB2. In conclusion, SLG showed a (geno)toxic potential in vitro in AM, but not in lung fibroblasts. Based on the in vitro screening data and for validation, an SLG (highest) and an MLG (lowest toxic potential) were selected for in vivo investigations. In a dose range finding (DRF) test, with dosing by intratracheal instillation (0.02 and 0.2 mg/rat, each) SLG was confirmed as the stronger inflammogenic sample (bronchoalveolar lavage fluid - BALF analysis) inducing a significant recruitment of neutrophils and - surprisingly - of eosinophils. In a subsequent 4-week nose-only inhalation study with the same total doses (predicted by MPPD model) the pro-inflammatory effect of SLG was weaker and no eosinophils were detected in BALF. Again, SLG but not MLG exhibited some inflammogenic potential. The histopathological examination mirrored the results of the BALF analysis. Granulocyte infiltration and giant cells were observed only in the SLG high dose group. – Funding: FP7- ERA-NET SIINN - BMBF-PtJ
Keywords: graphene, inhalation, risk assessment, in vitro screening, nanomaterials
(Eco)Toxicity Assessment of Commercial Engineered Nanomaterials for Plastic Industry in Zebrafish (#226)
M. Passos1, I. Pinheiro1, A. Vieira1, 2, J. C. Martins3, A. Campos3, B. Espiña1
1 INL- International Iberian Nanotechnology Laboratory, Braga, Portugal
International awareness towards safe and sustainable nanostructured materials have conducted to the development of pioneering and fangled green and smart nanosolutions, and have boosted a remarkable expansion potential for multiple industry sectors. Yet, current uncertainty in the regulatory framework for the commercialization of these nano-sized structures, the barely available literature on their potential effects towards human health and environmental safety, and the scarce information on their specific properties have limited their use in the industry.
Our research group has screened the in vivo nanotoxicity of calcium carbonate, antimony tin oxide and titanium dioxide nanoparticles. All these engineered nano-sized structures are already commercially incorporated in materials developed in the industrial sector of plastics improving their barrier, mechanical and conductive properties. The above-mentioned nanoparticles were characterized for their biocompatibility via in vivo zebrafish embryo toxicity (ZET) protocol and the results were contrasted with the ones from zebrafish acute toxicity testing in order to assess the ability to substitute for the acute fish test in nano EHS (environmental health and safety) investigation .
Fertilized zebrafish eggs were monitored via continuous waterborne exposure to the different nanoparticles at the nominal concentrations of 0.01-100 mg.mL-1 for 80 hours postfertilization, permitting a fast-track screening of the embryonic lethality, developmental delay signals, phenotypical malformations, spontaneous movements and free-swimming patterns, heart rate and hatching. F0 parental zebrafish (i.e. adult fish) were further waterborne exposed to the same nanoparticles and at the same nominal concentrations range for 1 week.
Adult zebrafish acute toxicity testing results were accurately extended by in vivo ZET data that allowed for a sensitive and early warning signal of nanotoxicity. Inversely to titanium dioxide nanoparticles, calcium carbonate and antimony tin oxide nanoparticles did not cause significant (sub-)lethality to zebrafish. Yet, all nanoparticles tested induced-developmental cardiotoxicity.
Keywords: commercial engineered nanostructured materials, acute fish toxicity, ZET, nanosafety profile
THE MEMBRANE TOXICOLOGY OF RODS AND SPHERIC ZINC OXIDE NANOPARTICLES (#257)
Y. Harmaza1, A. Tamashevski1, D. Jevdokimovs2, R. Viter3, E. Slobozhanina1
1 Institute of biophysics and cell engineering of National academy of sciences of Belarus, Laboratory of medical biophysics, Minsk, Belarus
It is assumed that the key role in the cytotoxicity of nano-sized ZnO compared to other metallic nanoparticles is played exactly Zn2+ due to ion-shedding effect, which is capable in a high concentration to trigger an apoptosis. On the other hand, the shape of the nanomaterial can also contribute to these processes under interaction with the components of cell membrane.
The aim of work – to conduct a comparative analysis of the membrane effects of nano-sized ZnO in different shapes: rods (ZnO NRs) and spherical particles (ZnO NPs), after their interaction with human lymphocytes.
Materials and methods: Lymphocytes were isolated from heparinized peripheral blood of healthy donors. The morphology of the nanostructured ZnO was studied by scanning electron microscopy (SEM). PI test is used for cells viability assessment. Fluorescent labeling with TMA-DPH, laurdan, N-(1-pyrenyl)-maleimide, fluorescamine allow examining the conformation of cell membrane components.
Results and discussion. It was shown that incubation of isolated lymphocytes with 1– 100 μg/ml ZnO NRs and ZnO NPs during 20 and 40 h leads to a dose-dependent increase of count cells in a late apoptotic phase but the highest cytotoxity was revealed for ZnO NPs. Study of physical state of membrane lipids after exposure of lymphocytes to ZnO NRs revealed a decrease of lipid microviscosity of the hydrophilic membrane area but increase it in hydrophobic area. The effects of ZnO NPs on membrane lipid components were not significant. Investigations of the membrane proteins conformation state revealed rise of protein NH2-groups level on the membrane surface only after lymphocytes exposure to ZnO NRs. At that time, level of the membrane protein SH-groups after action of ZnO NPs was increased, otherwise interaction of ZnO NRs with cells lead to oxidation of thiol groups. According to the results of SEM, the geometric size of the spherical ZnO NPs did not exceed 50 nm, the diameter of the ZnO NRs was 50-80 nm and the length did not exceed 500 nm. So, the obtained results of toxicological tests can be partly explained a possibility of spherical ZnO NPs to enter into cell while the more probable mechanisms for interaction with cell of NRs are electrostatic interaction or membrane puncturing.
The work is supported by the BRFFR Grant B17-128 and the EU Horizon 2020 programme under grant № 778157.
Keywords: ZnO nanoparticles, ZnO nanorods, human lymphocytes, membrane components, viability
INFLUENCE OF DISPERSION METHOD ON SILICA NANOPARTICLE SIZE DISTRIBUTION/AGGREGATION AND THEIR CHRONIC TOXICITY IN-VITRO (#296)
S. MURUGADOSS1, S. VAN DEN BRULE2, N. SEBAIHI5, F. BRASSINNE4, J. MAST4, L. GODDERIS3, D. LISON2, P. HOET1
1 Katholieke Universiteit Leuven, Unit of Environment and Health, Leuven, Belgium
Increasing number of in-vitro acute toxicity studies suggest that amorphous silica nanoparticles (SiNPs) could induce adverse effects. However, different dispersion methods are being used to disperse SiNPs and their effect on size distribution/Aggregation (not agglomeration) were not properly addressed. We found that the acute toxicity of SiNPs was strongly influenced when dispersed using different methods. The main objective of this study was to investigate the adverse effects of repeated low dose exposure (estimated from OELs) in-vitro to SiNPs prepared by different dispersion methods. NM-200 (precipitated silica) was used in this study and two stock dispersions were prepared by ultra-sonication and vortexing respectively. Characterization of these suspensions by Transmission Electron Microscopy (TEM) and Dynamic Light scattering (DLS) indicated that only nanofractions were present in the sonicated suspension while a mixture of nano and micro (aggregated) particles were found in the vortexed suspension. Human bronchial epithelial cell line (16HBE14o-) and Human monocytic cell line (THP-1) were repeatedly exposed to low dose (0.76 µg/cm2) of these suspensions, three times a week for three weeks and the cell viability, cell number, total glutathione, IL-8, IL-6 and genotoxicity were evaluated at the end of third week. Sonicated suspensions strongly reduced the cell number/cell growth rate, induced strong increase in IL-8 and IL-6 only in HBE cells. No significant effect was noticed in THP-1 exposed to any of these suspensions. Our results suggest that the dispersion method strongly influenced the size distribution of SiNPs and therefore, acute and chronic in-vitro toxicity of SiNPs.
Keywords: Silica nanoparticle, Dispersion method, Size distribution, Aggregation, Toxicity
Clastogenic and aneugenic effects of short-time and long-time exposure to nanoparticles in a human population (#310)
A. Rossnerova1, D. Pelclova2, V. Zdimal3, F. Elzeinova1, K. Vrbova1, J. Schwarz3, J. Ondracek3, M. Kostejn3, M. Komarc4, 5, S. Vlckova2, Z. Fenclova2, S. Dvorackova6, P. Rossner Jr.1
1 The Czech Academy of Sciences, Institute of Experimental Medicine, Department of Genetic Toxicology and Nanotoxicology, Prague 4, Czech Republic
The application of nanomaterials has been rapidly increasing during the last years. Current studies show, that inhalational exposure to nanoparticles (NP) probably brings about negative toxic effects, but there is a critical lack of human studies, especially related to possible DNA alterations. In this study we analyzed twice (pre-shift and post-shift) a group of 20 persons, long-time working (17.8±10.0 years) in nanocomposites research, and 21 matched controls. Detail aerosol exposure monitoring during working shift in a sampling day (including processes as welding, smelting, machining) to assess the differences in exposure to a wide range of particulate matter (PM) including fractions <25 – 100 nm, and chemical analysis of nano-sized fraction was completed. The micronucleus assay combined with fluorescence in situ hybridization using Human Pan Centromeric Chromosome Paint was applied to recognize not only the frequency of total micronuclei (MN) in binucleated cells (BNC), but also other types of chromosomal damage (losses and breaks), including the centromere positive (CEN+) and centromere negative (CEN-) micronuclei. The monitoring data showed differences in the risk of exposure to NP related to individual working processes as well as differences in chemical composition of nano-fraction. Cytogenetic results demonstrated possible adaptation to long-time (years) exposure of NP (related to total frequency of MN), although this exposure may be responsible for DNA damage pattern changes (increase of chromosomal breaks from 36% to 48% – clastogenic effect). However, short-time (daily) exposure could be a reason for the increase of chromosomal losses (aneugenic effect) as well as breaks (clastogenic effect) in the exposed group, depending on the particular type of exposure. Supported by the Grant Agency of the Czech Republic #18-02079S and the Ministry of Education Youth and Sports Czech Republic #LO1508.
Keywords: DNA damage, Micronuclei, Nanoparticles, Occupational exposure, Pan-centromeric FISH
Whole-genome expression analysis in THP-1 macrophage-like cells exposed to diverse metal-containing nanomaterials (#311)
P. Rossner1, T. Brzicova1, H. Libalova1, K. Vrbova1, J. Sikorova1, V. Philimonenko2, J. Klema3, J. Topinka1
1 Institute of Experimental Medicine, Department of Genetic Toxicology and Nanotoxicology, Prague, Czech Republic
From the perspective of the immune system, nanomaterials (NMs) represent invading agents. Macrophages are immune cells residing in all organs and tissues as the first line of defense. Interactions of macrophages with NMs can determine the fate of NMs as well as their potential toxic effects. In the present study, we compared toxicity of four different types of NMs [NM-100 (TiO2, 110 nm), NM-110 (ZnO, 20 nm), NM-200 (SiO2, 150 nm) and NM-300K (Ag, 20 nm)], in THP-1 macrophage-like cells. Cells were incubated with non-cytotoxic concentrations (1-25 µg/ml) of NMs for 24 hours and microarray technology was used to analyze changes in whole-genome expression. Gene expression profiling revealed a substantially different molecular response following exposure to diverse NMs. While NM-100 did not exert any significant effect on gene expression profile, all other NMs triggered a pro-inflammatory response characterized by an activation of NF-κB transcription factor and induced expression of numerous chemokines and cytokines. NM-110 and NM-300K further affected processes such as p53 signaling, oxidative and replication stress as well as modulation of cell cycle and DNA repair. NM-300K, the most toxic nanomaterial, was also characterized by enhanced expresion of genes coding protesomal complex involved in degradation of diverse proteins. We suppose that genotoxicity of ZnO and Ag NMs leading to DNA damage in THP-1 macrophages is probably caused by the extensive intracellular dissolution of these NPs, as confirmed by TEM imaging. Supported by the Ministry of Youth, Education and Sports of the Czech Republic (LO1508).
Keywords: nanomaterials, immune system, whole-genome gene expression
Characterization of the TiO2 E171 food additive (#366)
F. Brassinne1, S. De Vos1, E. Verleysen1, P. - J. De Temmerman1, M. Ledecq1, J. Mast1
1 Sciensano, Trace Elements and Nanomaterials, Brussels, Brussels, Belgium
E171 (Titanium dioxide) is an EC approved food additive (EC 1129/2011), authorized to be used as color in foodstuffs. It is widely used for its refractive properties (shiny coating, UV protection) in the food and pharmaceutical industries. It is intended and assumed to be present in bulk form. A nanofraction may be present.Dispersion is crucial step to characterize the particle properties of food additives as it allows to separate the primary particles. To better characterize titanium dioxide food additives (E171) their dispersion method was optimized.
A dispersion methodology based on the Guiot and Spalla approach was applied. It electrosterically stabilizes the (nano)materials, dispersed by sonication, using BSA at a pH determined by zeta potential measurement. Dispersion efficiency was examined by descriptive TEM and using a combination of TEM imaging and image analysis. The latter approach allows to assess the distribution of the particle properties (size, shape, surface structure) quantitatively. For both the pristine TiO2 food additive E171 and the JRC TiO2 representative test material zeta-potential measurement allowed to identify the conditions (pH) where a stable dispersion with a minimal level of agglomeration was observed. The stability of the dispersion was confirmed by descriptive TEM: preparing dispersions of TiO2 through a pH adjustment provides a stable dispersion of single primary particles and small aggregates and agglomerates.
Under the optimized conditions, the minimal external dimension of the primary particles could be measured more precisely and accurately by a combination of EM imaging and image analysis than in metastable conditions, such that the materials could be better classified according to the EC definition of a nanomaterial.
Evaluation of tight junction disruption induced particles on airway epithelial cells using an automatized imaging method. (#367)
E. Alfaro-Moreno1, M. Engel1, G. de Marco1, V. Munic Kos1
1 Swetox, Södertälje, Sweden
There is strong evidence indicating that some inhaled particles can induce systemic effects. Translocation of particles from the airways or the lung is one of the possible ways for particles to induce systemic effects. Some nanomaterials have been proved to induce changes in the tight junctions of airway epithelial cells. In the present study we are using an automatized imager to evaluate changes in tight junctions and comparing with the results obtained by measuring the transepithelial electric resistance (TEER). We are using human airway epithelial cell line 16HBE as a model of epithelial barrier. To standardize the conditions of our cell cultures we seeded different cellular densities of 16HBE (8,000 to 33,000 cells) inside inserts with a surface of 0.3 cm2. Once the cultures look confluent, we measured the TEER every day. After 5 to 7 days in culture, the higher density of cells did present a large increase in the TEER values, and these values remain high during 3-5 days. When exposing to NaHClO at concentrations from 0.0003 to 0.3% we observed drops in the TEER values. At the highest concentrations (0.3 and 0.03%) there was no recovery on the TEER values even after 24 h of exposure. With the lowest concentrations, after the TEER values get reduced in the first 2-5 h, there was a recovery after 24 h. Some particulate matter was used to evaluate the effect on the TEER values. TiO2, PM10 and PM2.5 were tested with transient drops on TEER values at concentrations above 10 mg/cm2. Using the same cellular densities as on inserts, we seeded 96 well plates (with a surface of 0.3 cm2/well). When we register an increase in the TEER values on the inserts, we treated the 96 well plates in the same way. To evaluate alterations in the tight junctions we did immunofluorescence staining for ZO-1 (tight junction protein) and evaluating changes in the tight junction patter using ImageXpress Micro equipment. With this method we are looking to automatize the evaluation of changes in tight junction patterns. Due to the variability on TEER values measurement, more efficient ways of evaluating changes in tight junctions is needed to evaluate and screen large amount of chemicals and particulate matter. Our preliminary results indicate that this approach is promising.
Keywords: Nanoparticles, inhaled particles, TEER, tight junctions
Silica nanoparticles impact the permeability of human intestinal barrier. (#387)
R. Cornu1, C. Chrétien1, A. Béduneau1, H. Martin1
1 Université de Bourgogne Franche-Comté, PEPITE EA4267, Besançon, France
Nanoparticles (NPs) are colloidal systems with a size below 100 nm. They are present in some innovative drugs called "nanomedicines" and in many food products. Among them, silica NPs are used as a lubricant in the production of tablets but also as an anti-caking agent in powdered foods (named additive E551). Some recent information has alarmed on a potential human health risk induced by the oral ingestion of the additive E551 composed of a high part of silica NPs. The lack of knowledges regarding their effects due to their unique physicochemical properties (size and specific surface area) requires a rigorous evaluation of the safety of these NPs, especially at the level of the intestinal barrier. Indeed, a modification of its permeability could have deleterious consequences for human health. The aim of the present work is to determine the effects of silica NPs and the corresponding food additive on the permeability of the intestinal barrier. For this purpose, silica NPs at different sizes and the additive E551 were added for 2h to Caco-2/HT29-MTX cells in co-culture. This in vitro model is recognized as a relevant model to mimic human intestinal barrier. The integrity of the intestinal barrier was evaluated through the measurement of transepithelial electrical resistance (TEER). In addition, paracellular transport was studied through the quantification of lucifer yellow, a membrane integrity marker. The level of reactive oxygen species (ROS) and different cytokines (IL-1α, IL-6, IL-8 and IL-10) was measured to evaluate the oxidative cellular stress and the inflammatory process. At non-cytotoxic concentrations, silica NPs dramatically alters the integrity of the intestinal barrier. This effect was not strictly inversely proportional of the NP size since the most deleterious effects were observed with the 30nm NPs. The enhanced paracellular transport could promote the passage into the body of potentially toxic molecules for humans. Moreover, the ROS production and the proinflammatory signaling pathway were increased after NP treatment. Taken altogether, these results suggested that the silica NPs can strongly alter the barrier function of the human intestine and interact with the epithelial cells by inducing an inflammatory response and an oxidative stress.
Keywords: intestinal permeability, silica nanoparticles, E551, Caco-2/HT29-MTX cell co-culture
Effects of double-walled carbon nanotubes on the pneumonia in respiratory syncytial virus-infected mice (#423)
W. Watanabe1, T. Akashi2, A. Hirose3, A. Miyauchi2, H. Yoshida2, M. Kurokawa2
1 Kyushu University of Health and Welfare, Department of Clinical Engineering, Nobeoka, Japan
Double-walled carbon nanotubes (DWNTs) have been expected to be one of important materials in the fields of nanotechnology, because they have not only high mechanical strength and thermal stability but also unusual electronic and optical properties. But their safety for human health is poorly known. To evaluate immunotoxicity of DWNTs, effects of DWNTs on the pneumonia in respiratory syncytial virus (RSV)-infected mice were assessed.
We used DWNT-1 (1 μm in length), DWNT-7 (7 μm in length) and DWNT-15 (15 μm in length) in this study. The RSV infection test was performed as reported previously (Hashiguchi et al., 2015). Briefly, female (6 weeks old) BALB/c mice were exposed intranasally to DWNTs (0 - 0.25 mg/kg) on days 1, 3 and 5 before RSV infection under anesthesia. These mice were intranasally infected with 3.5 × 105 PFU of RSV under anesthesia.
The levels of chemokine CCL5 (RANTES), a representative marker of pneumonia, in the bronchoalveolar lavage fluids (BALF) of RSV-infected mice were increased due to DWNT-1-exposure (0.025 mg/kg) compared with the control on day 5 post-infection. Histopathological analysis for lung tissues showed that the infiltration of mononuclear cells in alveolar wall was also increased due to DWNT-1-exposure compared with the control. Ingestion of the carbon tubes in macrophages was observed in the lung tissues of DWNT-1-exposed (0.25 mg/kg) RSV-infected mice. Effects of DWNT-7 and DWNT-15 on the pneumonia were not clear compared with DWNT-1.
Thus, exposure to DWNTs exacerbated the pneumonia in RSV-infected mice and might influence macrophages/monocytes in an early phase of RSV infection.
*Hashiguchi et al., 2015. Environ. Toxicol. Pharmacol. 39, 879-886.
Keywords: DWNTs, Pneumonia, RSV
Iron oxide nanoparticle toxicity on human cell lines, aquatic and soil organisms and interactions with metal pollutants (#448)
J. CABELLOS1, V. GONZÁLEZ-ANDRÉS1, M. DIEZ-ORTIZ1, G. JANER1
1 LEITAT TECHNOLOGICAL CENTER, HUMAN & ENVIRONMENTAL HEALTH AND SAFETY, BARCELONA, Barcelona, Spain
The use of iron oxide nanoparticles (IONPs) as an environment remediation tool is based on their ability to adsorb metals and decrease their bioavailability. Within the Reground project, a humic acid coating was introduced on IONPs (ha-IONPs) to improve their reactivity and stability in the environment and could as well affect their toxicity. Cytotoxicity assays on human cell lines (A549 and Caco-2) and acute toxicity tests on soil and aquatic organisms (enchytraeids, plants and daphnids) were used to evaluate the toxicity of these particles. In addition, possible toxicological interactions between adsorbed zinc ions (Zn, as a model pollutant) and the ha-IONPs were evaluated.
ha-IONPs caused no toxicity effects on A549 and Caco-2 cells, except at rather high concentrations (above 100 mg/mL), in which assay artefacts induced by the NPs could not be totally excluded. In the ecotoxicological studies, ha-IONPs did not show any toxic effect to soil invertebrates (enchytraeids) and plants (Lactuca sativa), but caused immobilization to Daphnia magna (EC50 > 900 mg/L).
The co-incubation of Zn ions with ha-IONPs led to slight changes in cellular toxicity when compared to the cellular response of the metals alone. Although the EC50 values did not change, the shape of the dose responses became shallower in the presence of ha-IONPs. This suggests a protective effect of the NPs at high metal concentrations and an enhanced toxicity at low concentrations. To further investigate the mechanisms leading to such increased toxicity, the mixture was also tested on 21-day differentiated Caco-2 cells, which are characterized by minimal nanoparticle uptake. In this model, Zn toxicity was much lower than in A549 cells, regardless of the presence of ha-IONPs. These results, together with additional evidence showing the precipitation of Zn salts in the assay medium, suggest that cell internalization as particles is a determinant event of metal toxicity.
In the Daphnia magna assays, co-exposure to Zn salts and ha-IONP, decreased the toxicity of the Zn salts (EC50 increased from 0.013 mg Zn/L to 0.575 mg Zn /L), showing a protective effect of the particles. This could be due to the reduced bioavailability of the Zn ions due to settling in association with ha-IONPs.
Acknowledgments. Supported by the EU H2020 Project REGROUND, ID: 641768 (2015-2018).
Keywords: Iron oxide nanoparticles, zinc, Caco-2 cells, A549 cells, Daphnia magna, mixture toxicity
In vitro toxic effects of L-glutamic acid-g-p(HEMA) polymeric nanoparticle (#468)
B. BAKAN1, 4, S. Gülcemal2, S. Akgöl3, P. H.M. Hoet4, N. U. Karabay Yavaşoğlu1
1 Ege University, Faculty of Science, Department of Biology, Bornova, Izmir, Turkey
Polymers are widely used as biomaterials due to their unique properties such as biocompatibility, easy design preparation and a variety of structures. Several methods have been developed to improve polymer design and it based conjugates provides many advantages, especially in clinical studies. There is limited number of studies on this subject. The aim of the study was characterized and determined of in vitro toxicities of newly synthesized L-glutamic acid-g-p(HEMA) nanoparticles. The characterization analyses showed that polymers have an average size of around 194.6 nm and zeta potential is a negative value which is -18 mV. SEM image was also supported these findings. The sample was scanned from 750 to 4000 cm-1 for FT-IR spectroscopy and the characteristic peaks of stretching band were observed in spectrums. It was determined that the nanoparticle (12.5-100 µg/ml) has not been showed cytotoxic effect on healthy cell line (HEK 293, ATCC-CRL-1573) with WST-1 test after 24 and 48 hours of exposure. It has not hemolytic activity on rabbit erythrocytes even at the highest concentration (20-160 µg/ml). Also, Ames Test results showed that it was not caused genotoxic effects on Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 strains (even at 5000 µg/ml concentration). So, it has shown promising results for the future works.
Keywords: L-glutamic acid-g-p(HEMA), characterization, in vitro toxicity
Relationship between developmental toxicity of multi-wall carbon nanotubes and lung inflammation in pregnant mice after repeated intratracheal instillation (#470)
M. Hojo1, N. Kobayashi2, Y. Hasegawa1, Y. Sakamoto1, S. Murakami1, Y. Yamamoto1, Y. Tada1, A. Maeno1, Y. Kubo1, H. Ando1, M. Shimizu1, Y. Taquahashi2, T. Suzuki1, D. Nakae3, A. Hirose2
1 Tokyo Metropolitan Institute of Public Health, Tokyo, Japan
[Background] Some studies showed reproductive and developmental toxicity of multi-wall carbon nanotube (MWCNT) in mice, but the mechanism remains unknown. We preliminary found that the strength of the toxicity was varied depending on pretreatments for MWCNT. We here conducted a comparative study using the MWCNT samples with different pretreatment. [Materials and Methods] Pregnant ICR mice were intratracheally instilled with three types of MWCNT samples: bulk (B-group), heat-treated (incubation 2-hr at 250 oC; H-group) and well-dispersed by Taquann method (T-group). Animals were repeatedly administered on gestational days 6, 9, 12 and 15 at the dose of 4 mg/kg/day, and then dissected on gestational day 17 for evaluation of reproductive parameters. Pulmonary toxicity of the dams was analyzed at gestational day 15 after three times instillation. [Results and Discussion] The body weight of dams in H-group significantly decreased at the day 17 comparing to vehicle control. Number of dead fetuses was increased in H-group, although the change was not significant. Body weight of fetuses was significantly decreased in B- and H- groups, and the mean value in H-group was lower than that in B-group. Frequency of visceral malformations of the fetuses was significantly high in B- and H-groups. In contrast, these parameters were not changed in T-group. Histological analysis of the lung parenchyma of dams revealed that the level of inflammation was high in order of H- > B- > T- group. LDH and total protein levels in lavage of the lung were increased in all exposed groups, but to a lesser degree in T-group. Further, expression of inflammation-related genes in the lung, such as ccl-2 and Il-6, was markedly upregulated in H-group. These data suggest that the developmental toxicity of MWCNT could be depending on the level of inflammation in the dam’s lung. [Acknowledgement] This study was supported by a Health and Labour Sciences Research Grant (H30-Kagaku-Shitei-004) from the MHLW, Japan.
Keywords: carbon nanotube, inflammation, developmental toxicity, intratracheal instillation
Hazard assessment for a Ti-based nano-additived material: from core-shell production to final part (#486)
S. Verstraelen1, A. Van Rompay1, A. Jacobs1, K. Hollanders1, F. Boonen1, G. Geukens1, E. Frijns1, L. Geerts1, R. Weltens1
1 Vlaamse Instelling voor Technologische Onderzoek (VITO), Unit Environmental Risk and Health, Mol, Belgium
VITO started to implement the ‘Safe Innovation’ concept in the H2020 NANOTUN3D project (Grant Agreement 685952; www.nanotun3d.eu/; 2015-2019) concerning the development of nano-modified alloys for additive manufacturing. It’s a way to incorporate Health, Safety, and Environmental (HSE) aspects already at an early stage of the innovation process in order to guarantee safety at the workplace, for consumers, and the environment. Identification of exposure scenarios, exposure modelling, and on-site measurements provided information on the relevant exposure routes. The human and ecological hazard assessment was performed for these routes and these results are shown for 7 NANOTUN3D test items, i.e. silicium carbide (SiC, 50 and 500 nm), titanium dioxide (TiO2, 50 nm), SiC@TiO2 core-shell nanoparticle (50 and 500nm), and yttrium oxide (Y2O3, 40 nm, Alfa Aesar and HWNANO).
Human toxicity screening consisted of acute exposure of in vitro cultured human cell models (airways: A549 alveolar epithelial cells type II), skin: SkinEthicTM Reconstructed Human Epidermis (OECD Test Guideline (TG) 439), and eye: SkinEthicTM Human Corneal Epithelium (SkinEthicTM HCE, OECD TG 492)) and endpoint analysis of cytotoxicity and oxidative stress (A549 cells), and irritation responses (RHE, HCE). For eye irritation, the Bovine Corneal Opacity and Permeability test (OECD TG 437) was performed in a testing strategy together with SkinEthicTM HCE. Ecological hazard identification for the aquatic environment was assessed. To simulate potential emissions to the aquatic compartment, leachable fractions (LF) were prepared. The acute aquatic toxicity of the LFs was measured (Algae growth inhibition (OECD TG 201) and Daphnia acute immobility (OECD TG 202)) for all compounds. Also the chronic ecotoxicity for selected compounds SiC 50 nm and Y2O3 was measured (Algae NOEC/LOEC (OECD TG 201) and Daphnia reproduction (OECD TG 211)).
No human health hazard effects were observed for the NANOTUN3D compounds. Adverse effects were seen in the Algae growth inhibition test for TiO2 and Y2O3 (EC50 value = 73% LF (730 mgeq/l); NOEC/LOEC = 250/500 mgeq/l). No adverse acute effects were seen in the Daphnia test. SiC 50 nm also had no inhibiting effect on Daphnia (reproduction test). Y2O3 showed chronic toxicity for Daphnia (NOEC/LOEC = 500/1000 mgeq/l).
Keywords: Keywords: Safe Innovation, HSE, human health hazard, ecological hazard
PEG-Serine nanoparticles as novel nanostructure for attenuation of organophosphate Poisoning: Synthesize, Characterization, invitro and invivo studies (#575)
H. Mohammadi1, P. ebrahimnejad1, A. Davoodi1, H. Irannejad1
1 Mazandaran University of Medical Sciences, Sari, Iran, Department of Toxicology and Pharmacology, Sari, Iran (Islamic Republic of)
Introduction: Different usages of organophosphate (OP) poisons and availibility of these compounds for most peoples cause serious intentional and accidental toxicity. Hydroxyl group of serine in active site of acetylcholine estrase (AChE) enzyme inhibits by OPs and inactivat the enzyme. Treatment of OPs poisoning is not always successful. Use of synthetic polymers because of biocompatibility and biodegredetivity is very important. In this study we attempt to attach serine amino acid to the polyethylenglycol (PEG) as a novel antidote for Ops poisoning.
Methods: Serine and polymer were conjugated with reductive amination reaction. nanoparticles were purificated by ultrafilteration. Structure of conjugated compound were analyzed by 1H NMR, 13C NMR, IR and DSC. Particle size of this compound determined by dynamic light scattering method. Finally, amount of blood hemolysis and its cytotoxicity effects on SKBR3 cell line were calculated. Effectivness of NPs were evaluated in Albino poisoned mice. NPs at doses of (100, 200, 400 mg/kg) were administered (ip) 20 minutes after a single dose of diazinon (LD50 = 166 mg/kg). Atropine (20 mg/kg, ip) with Pralidoxime ( 20 mg/kg, ip) or alone as compared to the standard treatment were used. LD50 decreasing, cholinestrase reactivation enzymes in brain, RBC and serum and oxidative dammage in brain mitochondria were assessed in mice.
Results: According to NMR, IR and DSC data, conjugation of PEG to Serine was achieved succesfully. Peak 9.5 ppm in 1HNMR spectrum of PEG-aldehyde which is attributed to the aldehyde hydrogen, does not exist in PEG-Serine spectrum and also appearance of peak 179.89 ppm in 13C-NMR of PEG-serine are indications of the product correct structure. Avarage particle size of nanomicelle was determined 192.4 nm. Amount of hemolytic activity of this NP was calculated 0.867 % and IC50 was calculated 36 mg/ml. LD50 significantly decreased (25 %) by NPs when compared with only diazinon group. Cholinestrase enzymes activity, lipid peroxidation, protein carbonyl content, and mitochondrial function significantly improved by NPs when compared with diazinon group.
Conclusion: Synthetized conjugated compound is very biocompatible with none toxicity that can successfully decrease the acute toxicity of diazinon as a new combination therapy.
Keywords: PEG-Serine, nanoparticle, antidote, organophosphate, Diazinon.
Single-walled and multi-walled carbon nanotubes induce epigenetic alterations in association with the nuclear deposition in 16 HBE cells (#581)
M. Ghosh1, D. Öner1, H. Bové2, M. Moisse3, 4, B. Boeckx3, 4, R. C. Duca1, J. A. Vanoirbeek1, M. Ameloot2, B. Bekaert6, 7, D. Lambrechts3, L. Godderis1, 5, P. H. Hoet1
1 KU Leuven, Unit of Environment and Health, Department of Public Health and Primary Care, 3000, Leuven, Belgium
In order to understand the epigenetic toxicity, in particular DNA methylation alterations, of SWCNTs and short MWCNTs, we performed global/genome-wide, gene-specific DNA methylation and RNA-expression analyses after exposing human bronchial epithelial cells (16HBE14o- cell line). The presence of CNTs on/in the cell nucleus was evaluated in a label-free way using femtosecond pulsed laser microscopy. A higher number of SWCNTs, compared to MWCNTs, was deposited at both the cellular and nuclear level after exposure. No global DNA methylation alteration on 5-methylcytosine (5-mC) or LINE-1 elements were observed for both CNTs. After exposure to MWCNTs, 2398 genes were hypomethylated (at gene promoters), and after exposure to SWCNTs, 589 CpG sites (located on 501 genes) were either hypo- (N = 493 CpG sites) or hypermethylated (N = 96 CpG sites). Significant changes in sequence-specific methylation was observed also observed in at least one CpG site for DNMT1 (SWCNT), HDAC4 (MWCNT), NPAT/ATM (MWCNT and SWCNT), MAP3K10 (MWCNT), PIK3R2 (MWCNT and SWCNT) and MYO1C (SWCNT). The study did not reveal any significant alteration in the miRNA expression, associated with MWCNT and SWCNT exposure. Cells exposed to MWCNTs exhibited a better correlation between gene promoter methylation and gene expression alterations. Differentially methylated and expressed genes induced changes (MWCNTs > SWCNTs) in different cellular pathways, such as p53 signalling, DNA damage repair and cell cycle. Based on our results, CNT induced DNA methylation and expression changes could have significant impact on several critical cellular processes.
Keywords: Carbon nanotubes, DNA methylation, Gene expression, Nuclear uptake, Toxicity, Epigenetics, Epigenomics, Genotoxicity, Nanoparticles, Nanomaterials, In vitro
Effects of Silver Nanocomposite (AgI) on Allium cepa: Part I DNA damage assessment with COMET assay (#598)
M. Gunes1, B. Yalcin1, H. Ertugrul1, N. Kaya1, E. Akarsu2, B. Kaya1
1 Akdeniz University, Department of Biology, Antalya, Turkey
Nanomaterials, occurring naturally or engineered for many different purposes, have a wide range of applications due to have a small size and large surface area. Silver nanoparticles (AgNPs) are used as antimicrobial agent in various products such as textiles, cosmetics, medical tools, food packaging. This widespread use of this materials causing concern about the effects of human and environmental health. Moreover, to increase the antimicrobial activity, different groups are added to AgNPs. AgI, nano-sized a silver halide, is noticeable composite in this area. With the advances in nanotechnology, new materials are produced day by day, although the information on the potential toxicity of these materials is very limited. In this study, the effects of nano silver composite AgI (AgI NP) was investigated with the COMET assay (single cell gel electrophoresis) on Allium cepa root cells. A. cepa root cells were exposed to AgI NP during 18 hours. Five onion bulbs were used for each dose and three were randomly selected. 50 cells were counted for each dose and the genotoxic damage caused by the nanocomposite was assessed according to the tail length parameter. Distilled water and ethyl methane sulfonate were used as a negative and positive control, respectively. The statistical analysis was performed using SPSS software and One-Way ANOVA was used to evaluate the differences between the distilled water and the treated groups. The results obtained from this study showed statistically significant positive results at 0.05 mM and 0.1 mM doses of AgI NPs. The genotoxicity observed at the lowest dose of 0.01 mM and the highest dose of 0.5 mM was not statistically significant. This study revealed that AgI nanocomposite induced DNA damage on Allium cepa root cells.
Keywords: Nanocomposite, single cell gel electrophoresis, genotoxicity, DNA damage, Allium cepa
Effects of Silver Nanocomposites (AgI) on Allium cepa: Part II Cytotoxic, Genotoxic and Mutagenic Assessment (#600)
B. Yalcin1, M. Gunes1, N. Kaya1, E. Akarsu2, B. Kaya1
1 Akdeniz University, Department of Biology, Antalya, Turkey
Silver nanoparticles (AgNPs) are widely used due to their physico-chemical properties in nanotechnology, especially their antibacterial effect. Recent studies are progress to produce new silver composites to enhance the antibacterial activity of AgNPs. Since silver nano composites are new products, there is limited information about the effects on the environment and the living organisms during their production, use and disposal. Determination of the genotoxic potential of these AgNPs composites will enable for a safer use of AgNPs. In this study, micronucleus (MN) test, mitotic index (MI) and other chromosome aberrations (CA) tests were applied Allium cepa root meristematic cells in order to investigate the potential genotoxic effects of AgI NPs. The cells were exposed to four different doses (0.01, 0.05, 0.1 and 0.5 mM) of AgI NP during 18 hours. The CA test was evaluated according to different categories such as bridge, c-mitosis, polar deviation, binucleate, nuclear bud, laggard, early movement, and other deviations. The mutagenic effect of the NP tested was examined by the MN test. The cytotoxic effect was also determined by calculating the MI by the ratio between the number of dividing cells and the total of cells. In the tests, 3 repetitive 1000 cells (total 3000 cells) were evaluated.
Analysis of variance (ANOVA) and Dunnett’s t-test (p<0.05) was used for the statistical analysis of MI, MN, and CA data. Our results reveal that although the mitotic index did not statistically different from control group, chromosomal damages were significantly induced in all doses. On the other hand, incidence of MI was not statistically significant at any doses.
Keywords: Silver nanocomposite, chromosome aberrations, mitotic index, micronucleus, Allium cepa
Insights into the role of the physicochemical properties in the toxicity of SiO2 nanoparticles in rat alveolar epithelial RLE-6TN cells (#672)
M. F. P. Brandão1, 2, M. J. Bessa1, 2, C. Costa1, 2, S. Fraga1, A. Haase3, J. P. Teixeira1, 2
1 University of Porto, EPIUnit-Institute of Public Health, Porto, Portugal
Silica nanoparticles (SiO2 NPs) are easily produced in different sizes and surface modifications enabling their use in several industrial and biomedical applications, which increases the risk of human exposure to these nanoparticles. Because the toxicological profile of nanoparticles is dependent upon their physicochemical properties, the aim of this study was to investigate the influence of the physicochemical properties (size, surface capping and hydrophilicity/hydrophobicity) in the cyto- and genotoxicity of SiO2 NPs in rat alveolar epithelial cells (RLE-6TN), a primary target during inhalation exposure.
To evaluate the cytotoxic potential of SiO2 NPs, RLE-6TN cells were exposed for 24 h to different concentrations (0-56 µg/cm2) of differently capped (naked, phosphonate- and amino-modified), differently sized (7, 15 and 40 nm), and with different hydrophilicity/hydrophobicity nature variants dispersed in serum-free culture medium. Cytotoxicity of the SiO2 NPs was assessed by determining the LDH release and WST-1 metabolization in RLE-6TN cells. To evaluate the genotoxic potential of the SiO2 NPs, RLE-6TN cells were exposed to subtoxic concentrations of the different variants tested (0-28 µg/cm2) and the potential DNA damage was assessed by the alkaline comet assay.
Exposure to hydrophilic SiO2 NPs induced a concentration-dependent cytotoxicity in RLE-6TN cells, being the cytotoxic effect more marked in the cells exposed to the smallest SiO2 NPs. Amino and phosphonate surface modification mitigated the cytotoxicity of the SiO2 NPs as demonstrated by the lower LDH released levels and higher metabolization of WST-1 comparing with cells exposed to naked SiO2 NPs with the same size. Exposure to hydrophobic SiO2 NPs did not induce cytotoxicity on RLE-6TN cells. No DNA damage was observed in RLE-6TN cells after 24 h of exposure to all tested SiO2 NPs.
Our data shows that size and hydrophilic/hydrophobic nature influence the cytotoxic profile of SiO2 NPs. In turn, amino and phosphonate surface modification attenuates cytotoxicity of SiO2 NPs on RLE-6TN and might constitute a strategy to increase biocompatibility of SiO2 NPs. Nevertheless, further research such as the evaluation of other toxicity endpoints, should be conducted to support our findings.
This work is supported by Fundação para a Ciência e a Tecnologia through European project NanoToxClass (SIINN/0001/2013) and under the grants SFRH/BD/101060/2014, SFRH/BPD/96196/2013 and SFRH/BD/120646/2016.
Keywords: RLE-6TN cells, silica nanoparticles, cytotoxicity, genotoxicity, physicochemical properties
ICONS - Integrated Testing Strategy for Mechanistically Assessing the Respiratory Toxicity of Functionalized MWCNTs – Comparative In Vitro Investigations (#677)
C. Ziemann1, S. M. Reamon-Buettner1, D. Lison2, J. C. Bonner3, S. van den Brûle2, S. Simon4, O. Creutzenberg5
1 Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Genetic Toxicology & Tumor Research, Hannover, Lower Saxony, Germany
Multiwalled carbon nanotubes (MWCNTs) represent tube-like structures with nano-sized diameters, made of multiple carbon layers. MWCNTs exhibit promising properties, including electrical/thermal conductivity and high tensile strength, thus practically pointing to a wide range of applications in the electronics and composite materials sectors. However, hazard characterization of MWCNTs, highly needed for work place and consumers’ safety, is still incomplete. Besides morphology, surface functionalization of MWCNTs may have marked impact on adverse biological activity. The ERA-NET SIINN project ICONS (“International Collaboration On Nanotube Safety”) thus aimed at mechanistically evaluating and ranking the pro-fibrotic and genotoxic potential of eight differentially purified (chemically or thermally) and functionalized (-COOH and -NH2) MWCNT samples, made from one batch of industrially relevant Nanocyl NC7000 (tangled morphology). One focus of the subproject, carrried out by Fraunhofer ITEM (BMBF-funded: FKZ: 03XP0063), deals with the comparative in vitro (geno)toxicity testing of the eight samples, using primary human lung fibroblast MRC‑5 and primary human peritoneal mesothelial LP9 cells. MWCNTs were intially tested for sterility and endotoxin, followed by development of an ultrasound-based dispersion protocol. Scanning electron microscopy indicated presence of both free fibers and agglomerates, and turbidity measurements pointed to differential sedimentation and thus perhaps unequal in vitro doses. Subsequent in vitro testing revealed concentration-dependent membrane damage (LDH release), differential inhibition of proliferation (RICC, mitotic index), slight induction of micronuclei (1 µg/cm2, 24 h), and loss of chromosomes in MRC-5 cells. In LP9 cells (5 µg/cm2, 24 h), induction of LDH release, differential accumulation of MWCNTs around the cell nucleus and impairment of the cytoskeleton (α-tubulin immunofluorescence) were noted. But, no direct DNA damage (γH2A.X or 53BPI foci) was evident, and only sporadic γH2A.X panstaining occurred to account for induction of apoptosis or cellular senescence. Our results point so far to a rather mechanical than clastogenic adverse activity of the MWCNTs. Two candidate MWCNTs will be selected for a 28-day inhalation test in rats.
Keywords: nanomaterials, multiwalled carbon nanotubes, genotoxicity, surface functionalization, in vitro
Transcriptional changes underlying cerium dioxide nanoparticle modulation of allergen induced type II airway inflammation. (#678)
K. Meldrum1, 2, S. B. Robertson1, T. W. Gant1, T. D. Tetley2, R. Smith1, M. O. Leonard1
1 Public Health England, Toxicology Department, Chilton, Didcot,, United Kingdom
Cerium dioxide nanoparticles (CeO2NPs) have the potential to impact disease outcome but have not been investigated for their effect on models of asthma and inflammatory lung disease. We therefore set out to examine the impact of CeO2NPs on a house dust mite (HDM) allergen induced model of asthma in Balb/c mice. Repeated intranasal instillation of CeO2NPs in the presence of HDM resulted in a type II inflammatory response, characterised by increased pulmonary mast cell markers, plasma IgE and airway goblet cell metaplasia. This was accompanied by increases in IL-4, CCL11, mucin levels and inflammatory regulators CLCA1 and SLC26A4 within the lung. Repeated HDM instillations followed by a single exposure to CeO2NPs failed to produce changes in type II inflammation but did result in alterations in the neutrophil activation marker CD177. RNA-SEQ was used to explore early effects of CeO2NPs and HDM exposure after 24hr exposure. Changes in serum amyloid A3 expression paralleled increased neutrophil levels within BAL fluid, while no changes in eosinophil or lymphocyte levels were observed. RNA-Seq was also used to investigate transcriptional responses within the lung after repeat CeO2NP and HDM treatments. Changes in macrophage differentiation markers indicative of type II inflammation were prominent among a general inflammatory profile. Overall changes in gene expression were consistent with regulation of dendritic cells, macrophage functionality and interferon signalling as the potential key events for how CeO2NPs may guide pulmonary responses to allergen towards type II inflammation. As this type of inflammatory response is present within asthmatic endotypes our findings may have implications for how occupational or incidental exposure to CeO2NPs should be considered for those susceptible to disease.
Keywords: Transcriptomics, Asthma, Allegy, Airway disease
Toxicity of ceramic nanoparticles in human alveolar epithelial A549 cells at the air-liquid interface (#704)
S. Fraga1, 2, M. J. Bessa1, 2, F. Brandão1, 2, P. Fokkens3, J. Boere3, D. Leseeman3, A. Salmatonidis4, M. Viana4, F. Cassee3, J. P. Teixeira1, 2
1 University of Porto, EPIUnit-Institute of Public Health (ISPUP), Porto, Portugal
Several ceramic industries have already incorporated within their production processes the manufacture of different types of ceramic nanoparticles, as well as the application of those nanomaterials on conventional products, which increases the risk of human exposure to these nanoparticles, particularly in occupational settings.
The aim of this study was to investigate the in vitro toxicity of ceramic nanoparticles (ZrO2, and CeO2 and Sb2O3•SnO2 NPs) in human alveolar epithelial cells, a primary target during inhalation exposure. The cells were grown for 7 days onto Transwell inserts in RPMI 1640 medium supplemented with Glutamax, 25 mM HEPES, 10% FBS, 50 U/mL penicillin and 50 µg/mL streptomycin. Cells were exposed to aerosolized nanoparticles for 2 and 4 h at air-liquid interface using a Vitrocell® exposure system and cytotoxicity assessed by the LDH release and WST-1 metabolization assays immediately after exposure or in the recovery time (24h). DNA damage was also assessed by the alkaline comet assay at 24 h after exposure. The percentage of DNA in the tail and the olive tail moment (OTM) were used as a measure of the amount of DNA damage.
A concentration-dependent increase in LDH release was observed after 2 and 4 h of exposure to all the tested aerosolized NPs comparing with cells exposed to clean air (exposure control). However, 24 after exposure, no differences in LDH release levels were observed among the exposed cells (exposure control and NPs aerosol-exposed cells) but those were significantly higher than the incubator control. A concentration-dependent decrease in cellular metabolic activity, as assessed by the WST-1 assay, was observed 24 h after exposure to all tested NPs aerosols. Regarding the DNA damage induced by exposure to the NPs aerosols, no significant increase in % tail intensity and OTM was detected comparing with control cells.
Under our experimental conditions, exposure to the NPs aerosols affected plasma membrane integrity and the metabolic activity of A549 cells. Further research should be conducted to get further insight into the mechanisms involved in the toxicity of the selected ceramic nanoparticles.
This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the CERASAFE project (SIINN/0004/2014). MJB and FB thank FCT for their PhD scholarships (SFRH/BD/120646/2016 and SFRH/BD/101060/2014).
Keywords: A549 cells, ceramic nanoparticles, cytotoxicity, genotoxicity, Air-liquid interface exposure
MEtabolomics standaRds Initiative in Toxicology (MERIT) (#123)
H. Kamp1, R. Beger2, J. - L. Dorne3, T. Ebbels4, D. Ekman5, C. Guillou6, A. Goetz7, G. Loizou9, P. Leonards8, B. van Ravenzwaay1, S. Sperber1, M. Viant10, T. Walk11
1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany
The MEtabolomics standaRds Initiative in Toxicology (MERIT) is a European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) supported project to provide guidance on best practice, quality standards and the reporting of analytical and computational metabolomics methods used in regulatory toxicology. ECETOC relies on world-wide expert collaboration “to develop an agreed understanding on how the state of the science can be used to improve [chemical] risk assessment by developing novel tools”. Although there is strong interest in using metabolomics from industry and regulatory scientists, the lack of best practice guidelines is a concern that has hindered its use in regulation. Fourteen metabolomics scientists from academia, industry, regulatory and government agencies were recruited as the MERIT core team, with a much larger number acting as a wider consultative group. The primary objective of MERIT is to develop best practice guidelines and minimal reporting standards in the context of metabolomics in regulatory toxicology. Several case studies representing near-future applications of metabolomics - including for the discovery of molecular key events and biological read-across - are being used to ensure the relevance of the guidelines to regulatory toxicology. MERIT will publish its recommendations for best practice and reporting standards and is liaising with other groups, such as the NIH Metabolomics Quality Assurance and quality Control Consortium (mQACC) and the Metabolomics Society, to ensure harmonization of the recommendations.
Keywords: metabolomics, quality assurance, ecetoc, MERIT, guidance
DMSO-induced drastic changes in cellular processes and epigenetic landscape in vitro (#166)
M. C. Verheijen1, M. Lienhard2, Y. J. Schrooders1, O. Clayton3, R. Nudischer3, B. Timmermann2, S. Boerno2, N. Selevsek4, R. Schlapbach4, H. Gmuender5, S. Gotta5, R. Herwig2, J. Kleinjans1, F. Caiment1
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
The use of 0.1% DMSO (dimethyl sulfoxide) as solvent for in vitro assays is widespread and effects are often assumed to be negligible. Initially, DMSO was researched for medical applications although clinical trials were halted in 1965 due to adverse effects whereupon DMSO was labeled extremely toxic. Years later, upon reviewing new results, the FDA classified DMSO in the safest category. Thereafter, DMSO was used in applications such as cryopreservation or cell differentiation inducer, but mainly as a solvent. This study uses complete transcriptome and epigenome sequencing (RNA-seq & MeDIP-seq) to chart the effects of 0.1% DMSO on 3D cardiac and hepatic microtissues for the purpose of assessing whether DMSO induces bias in analyzing findings from toxicant-treated in vitro assays. RNA-seq detected over two thousand differentially expressed genes (DEGs; FDR >0.05) in both cardiac and hepatic tissue (compared to untreated microtissues). Pathway analysis of these DEGs identified hundreds significantly overrepresented pathways (FDR <0.05). Although the amount of DEGs per biological process differs between the tissues, similar processes are affected, indicating a consistent mode of action of DMSO. Since affected pathways displayed a majority of downregulated genes, processes of transcriptional regulation were investigated in detail, focusing on DNA methylation. Differentially methylated regions (FDR <0.05) found in both tissues indicate changes in the epigenetic landscape, suggesting that DMSO can interfere with regulatory systems in cardiac and hepatic tissues. While the field is evolving towards more sophisticated in vitro models, using 3D conformation and/or physiological-consistent low dose concentrations, omics technologies clearly demonstrate that the effect of DMSO on cell regulation has to be kept in mind when designing in vitro studies and interpreting the data. Consequently, the lowest possible dose of DMSO should be used as even low incubation concentrations may induce solvent-induced bias.
Keywords: DMSO, transcriptomics, microRNA, 3D microtissues, epigenetics, omics
Omics-Based Analysis of the Impact of Cigarette Smoke and Cessation in Mouse Liver and Kidney Following a 6-Month Exposure (#229)
K. Matsumura1, T. Hirata1, Y. Motohashi1, S. Ito1, T. Mine1, H. Suzuki1
1 Japan Tobacco Inc., Scienetific Product Assessment Center, Yokohama, Japan
Smoking cessation has been reported as one of the most effective approaches to reducing the effects of cigarette smoke (CS). We used integrated omics analyses to investigate the impact of smoking cessation in murine liver and kidney, the primary organs involved in xenobiotic response. Female C57BL/6J mice were exposed to filtered room air (sham) or K3R4F reference CS for 6 months. Moreover, two additional groups as smoking cessation groups, mice were exposed to CS for 5 or 13 week, followed by filtered air up to 6 months. Livers and kidneys were subjected to transcriptomic and proteomic analysis. The number of differentially expressed genes (DEGs) was higher in livers than in kidneys in all exposure groups, suggesting a greater impact on liver. Notably, the number of DEGs decreased as the duration of the cessation period increased, in particular DEGs related to cell stress, oxidative stress, and inflammatory responses. Cessation periods reduced the numbers of DEGs in both organs, approaching the numbers in the sham group. Canonical pathway analysis predicted that CS inhalation would activate the aryl hydrocarbon receptor signaling pathway in the liver, showing a good correlation between gene expression and the abundance of proteins related to aryl hydrocarbon receptor signaling. These findings suggest that CS-inducible cell stress, especially in the liver, is mediated by aryl hydrocarbon receptor signaling. Moreover, this impact can decrease with the duration of smoking cessation.
Keywords: cigarette smoke, omics analysis, liver, kidney, smoking cessation
Data-driven identification of interspecies pathway perturbations in vivo and in vitro: the case of Nrf2 (#288)
T. M. Souza1, J. C. S. Kleinjans1, D. G. J. Jennen1
1 Maastricht University, Department of Toxicogenomics, Maastricht, Netherlands
The dynamic, time- and dose-dependent perturbation of intracellular pathways is an important mechanism underlying toxic responses in several species, in vivo and in vitro; the identification of such perturbations is essential to understand the mechanisms and to design effective predictive models. Despite the large amount of gene expression data generated for this purpose, certain limitations exist when using available analytical methods, including database annotation, arbitrary thresholds for statistical testing, as well as species- and model- specific responses. In this study, we sought to overcome these limitations by applying a data-driven approach to identify perturbations to pathways/gene sets applying ordinary differential equations (ODEs) to exposed time series data. Our hypothesis is that different stimuli will lead to different dose-dependent perturbations of component nodes. For this, we focused on perturbations to (orthologue) genes from the Nrf2 pathway, which were then modelled using TG-GATEs sets (rat in vivo repeated and single dose; rat in vitro and human in vitro) exposed to compounds with different pathological outcomes after chronic exposure in vivo: acetaminophen (APAP), carbon tetrachloride (CCl4) and diazepam (DIZ). A clear separation of the perturbation clusters following single and repeated exposure regimens was observed: single dose exposures reflected compound-specific effects while repeated dose effects seemed to reflect pathology. Genes such as G6pd, Abcc4 and Slc39a7 were identified as high-ranked perturbations in APAP and CCl4; Nfe2l2 and Nqo1 were correlated with increasing doses of APAP, while Ces1f and Tgfb1 were identified as dose-dependent targets of CCl4. Comparisons to in vitro models revealed a different set of genes perturbed in vitro but confirmed perturbations to Abcc4 in both human and rat models in APAP and CCl4, Nqo1 for APAP as well as genes from theTgf family for CCl4. Perturbations in vitro common to rat and human included Cbr3 and Dnajb1 for APAP and Ptgr1 and Maff for CCl4. None of these targets showed substantial perturbation across DIZ doses. In conclusion, we provide a data-driven method to overcome some limitations inherent to gene expression data interpretation, i.e., the identification of perturbations and translatability to other species.
Keywords: translation, in vivo, in vitro, acetaminophen, ODEs
Is the prediction of nephrotoxicity and its mode of action by metabolome analysis in vitro in NRK-52e cells feasible? (#322)
B. Birk1, S. Sperber1, H. - A. Huener1, A. Verlohner1, T. Walk2, V. Haake2, M. Spitzer2, 1, H. Kamp1, B. van Ravenzwaay1
1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany
Nephrotoxicity is a relevant toxicological endpoint for new and existing chemicals (pharmaceuticals, pesticides, cosmetics or other classes of chemicals). Beyond the identification of nephron-toxicity in new compounds, identification of the mode of action (MoA) gets more and more relevant. Therefore, reliable and robust in vitro methods are the preferred methods to reduce animal testing but also to identify the toxicological modes of action. Metabolomics in vitro in kidney cells is a novel approach that could enable the early identification of nephrotoxicity and contribute to understanding the mode of action.
Therefore, a robust system based on NRK-52e cells was established (partly within the InnoSysTox Project “Risk-IT” founded by BMBF, Germany) by cultivation of the cells on Lumox dishes (Sarstedt), treatment for 48h and sensitive harvesting of the cells. Nine different nephrotoxic compounds classified in three different mode of actions (covalent protein binding, lysosomal overload and mitochondrial DNA-interaction) and caffeine acting as negative control were applied to the system. Gentamycin-sulfate, belonging to the MoA “lysosomale overload” was tested in several concentrations and in several runs.
Principle component analysis (PCA) showed a clear dose response relationship and demonstrated the high reproducibility of the data. Caffeine, which does not share any aspects of the three different MoA of nephrotoxicity induced by the other reference compounds clustered nicely with the vehicle control (DMSO 0.5%). For all 9 compounds a separation of the clusters in the PCA was shown compared to vehicle and negative control. Further evaluation on a specific fingerprint per mode of action is ongoing.
These observed differences in the in vitro metabolome in NRK-52e cells of the 9 known nephrotoxic substances with three different modes of action suggest that the metabolome analysis could be a suitable tool for analysis of the mode of action of nephrotoxicants. This method can support on one hand side the screening process but can also contribute to identify and understand the mode of action of nephrotoxicity and has thus the potential to be applied for regulatory purposes.
Keywords: nephrotoxicity, metabolome analysis in vitro, NRK-52e cells, mode of action, omics
Global proteomic analysis of major metabolic tissues from Wistar rats chronically exposed to the contaminants bisphenol A and S (#402)
L. F. Azevedo1, M. F. H. Carneiro1, C. R. P. Dechandt2, J. S. Cassoli3, L. C. Alberici2, F. Barbosa Júnior1
1 University of Sao Paulo, Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences, Ribeirao Preto, Sao Paulo, Brazil
Exposure to the endocrine disruptors bisphenol A (BPA) and bisphenol S (BPS) has been associated with development of metabolic disorders, however the associated mechanisms are still not fully understood. The present study aimed to evaluate protein expression changes by shotgun proteomics in liver and pancreas from male Wistar rats chronically exposed to BPA or BPS in order to identify possible metabolic pathways dysregulated by these compounds. Rats were distributed in 3 groups (n=6) and exposed to the contaminants through drinking water for 20 weeks as follows: (I) vehicle - 0.1% v/v ethanol, (II) BPA - approximately 50 µg/kg/day of BPA and (III) BPS - approximately 50 µg/kg/day of BPS (ethical approval n. 15.1.979.60.7). After sample preparation from tissue lysates (in duplicates), proteomic analyses were performed in a nanoUPLC tandem nanoESI-HDMSE platform by multiplexed data independent acquisitions experiments. Proteins with mean changes of 1.5-fold in normalized abundance between groups were considered differentially expressed. No statistical difference was observed in body weight gain. Hepatic proteins differentially expressed in BPA and BPS exposed groups compared to control group were mostly related to lipid metabolism and synthesis, with downregulation of enzymes involved in fatty acid β-oxidation (peroxisomal acyl-coenzyme A oxidase 2, 0.3-fold in BPA; peroxisomal bifunctional enzyme, 0.2-fold in BPS) and upregulation of triglycerides and cholesterol synthesis (glucokinase activity related sequence 1, 1.8-fold in BPA and 2-fold in BPS; HMG-CoA synthase cytoplasmic, 1.7-fold in BPS). In pancreas of BPA treated animals compared to control, effectors proteins of MAPK pathway were upregulated (Ras-related C3 botulinum toxin substrate 1, 2-fold; serine/threonine-protein kinase PAK 1, 2.1-fold; MAPK-activated protein kinase 2, 2.8-fold). Current studies are focused on assessing what proteins in pancreas are affected by BPS exposure. In addition, BPA and BPS dysregulated expression of enzymes involved with detoxification and of mitochondrial proteins in both tissues. In summary, our studies shed light on potential pathways disrupted by both BPA and BPS in tissues critically important for metabolic homeostasis raising new concerns over the safety of BPA alternatives.
Keywords: Bisphenol A, bisphenol S, metabolic disorders, shotgun proteomics
Percellome Toxicogenomics Project: Newly Designed Repeated Dose Study. (#406)
J. - Kanno1, 2, S. - Kitajima2, R. - Ono2, K. - I. - Aisaki2
1 Japan Bioassay Research Center, Japan Organization of Occupational Health and Safety, Director, Hadano, Kanagawa, Japan
The Percellome Project aims at reinforcing and replacing the “safety (uncertainty) factor”by comprehensively identifying the transcriptomic networks induced by xenobiotics. “Percellome” method was developed (BMC Genomics 7:64, 2006) to generate absolute copy numbers of mRNAs in a “per one cell” basis. Data from the Affymetrix MOE430 2.0 GeneChip are absolutized and visualized in 3-D graphs (time x dose x copy number). Datasets of mouse liver (4 time points x 4 dose levels, triplicate, 48 GeneChip data per chemical/organ) on 140 chemicals are compiled. Here, we report the progess on the newly designed repeated dose study which is insipred by a single dose study on gene knockout mice; wild type mice are repeatedly exposed to a chemical to create a “chemically-induced transgenic state”. Then, a single dose of a chemical was given and the liver was sampled at 2, 4, 8 and 24 hours thereafter. Now we have data on CCl4, tributyltin, deet, clofibrate, valproic acid, acetaminophen, phenobarbital, thalidomide, 5-fluorouracil and acephate on single exposure and 4 repeat + single exposure pairs. Repeated dose of CCl4 induced suppression of baseline expression of genes related to ER stress and attenuated the effect of chemicals given after CCl4. On the other hand, valproic acid did not show such effect. Tributyltin and deet were similar to CCl4, whereas, acetaminophen and phenobarbital were partly similar to CCL4 but repeated dose had elevated the baseline with increase in transient response as well. Thalidomide showed complex response, suppresion of 2hr response and enhancement of 4 to 8 hr response. 5-FU tends to show elevation of baseline with increased transient response. Acephate was similar to valproic acid, i.e. no effect of repeated dosing. It becomes clear that the relation between baseline and transient responses are more complicated in thoses chemicals. Basic epigenetic molecular mechanisms of chronic toxicity will be discussed.
Keywords: Percellome, toxicogenomics, gene network, mice, repeated dose, chemically-induced transgenic state
Benzo[a]pyrene induced regulation of mRNA translation and its impact on gene expression (#441)
E. Smit1, F. Caiment1, J. Piepers1, J. C. Kleinjans1, T. van den Beucken1
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
Exposure to benzo[a]pyrene (BaP) induces profound changes in cell phenotype that may contribute to an adverse outcome. These phenotypic changes are assumed to be mediated largely by changes in gene expression, which occur through various mechanisms. One of the key mechanisms is the transcriptional response driven by transcription factors like AhR and p53. Nevertheless, BaP-induced changes in gene expression may also be influenced by its effect on protein synthesis. The goal of this study was to determine the impact of regulating mRNA translation on gene expression changes induced by BaP on a genome-wide scale. Primary mouse hepatocytes were exposed to 1 and 10 µM BaP for 1 and 24 hours. At each time point mRNAs were isolated using sucrose gradients to obtain 4 fractions with increasing translation rates. Next, RNA sequencing was performed on these 4 fractions as well as on the total mRNA to determine gene-specific changes in mRNA translation. From these data we determined both transciptomics changes (mRNA abundance) and translatomics changes (translated mRNA) after BaP exposure. Surprisingly, 1h exposure to 10 µM BaP only resulted in differential gene expression for 4 genes due to changes in mRNA abundance and for 6 genes due to differential translation. While after 24h exposure to BaP, 190 genes were altered with respect to mRNA abundance (FDR<0.05) and +2000 genes were differentially translated. Based on this analysis we were able to distinguish 3 categories of genes; (i) genes with concordant changes in mRNA abundance and mRNA translation, (ii) genes with altered mRNA abundance only and (iii) genes with changes in mRNA translation only. GO term analysis showed that genes in the “mRNA translation only” category were involved in specific biological processes including: translation, apoptosis and oxidation-reduction process. Genes from the “concordant” group were mainly associated with BaP metabolism and anti-oxidant response. To test the robustness of these findings we validated 3 genes from each category by means of qPCR. All 9 genes tested revealed similar expression changes. In conclusion, our data reveals that gene expression changes in response to BaP occur more on the level of mRNA translational than on the level of mRNA abundance, which adds new mechanistic insights into the cellular response to BaP.
Keywords: mRNA translation, Benzo[a]pyrene, Primary mouse hepatocytes, gene expression
Enabling High Throughput Transcriptomics (HTTr) for Mechanistic Toxicology by Tuning Dynamic Range (#509)
H. VanSteenhouse1, J. Yeakley1, P. Shepard1, J. McComb1, B. Seligmann1
1 BioSpyder Technologies, Carlsbad, United States of America
HTTr offers a powerful and sensitive method to molecularly characterize biological responses to compound exposures in vitro or in vivo. However, expression profiling by RNA-seq can be costly and labor intensive in large-scale, statistically powered dose-response studies. One limitation has been that highly expressed genes dominate sequencing output, increasing costs and limiting detection of low abundance RNAs that may obscure classifiers for response mechanisms. Attempts to overcome this issue typically add cost and effort. A better method would be to tune the dynamic range to decrease the impactof highly abundant transcripts predictably and quantitatively, thereby not influencing results and permitting measurement of relative abundance of each gene and maximizing sensitivity to changes in rare transcripts.
TempO-Seq® is a targeted expression profiling assay with NGS readout, measuring content up to the whole transcriptome. Well suited for HTTr, it does not require purification or cDNA synthesis and runs as a simple add-only assay. To attenuate the impact of abundant transcripts, non-assay oligos that compete for hybridization of TempO-Seq Detector Oligos (DOs) were added to the assay at known ratios. Assay sensitivity for measuring rare transcripts was assessed across a range of attenuation from 0.5%-10% of probes (29%-79% of total reads). Signal attenuation across this range was linear, independent between probes, and could be accurately back-calculated to unattenuated equivalents, whereas differential expression fold changes and average percent CVs were unaffected. Sequencing space was better utilized, resulting in 3-fold greater read counts for each of the unattenuated transcripts at the highest level of attenuation. Thus, when 40% of total reads were attenuated to half the maximum residual level, there was a saving of 42% of sequencing space, equivalent to ~72% more samples being included in the same run. Selectively limiting the dynamic range of TempO-Seq through attenuation of abundant transcripts increased its sensitivity for rare transcripts and offers substantial benefit for HTTr, enabling toxicologists (or other researchers, for example, in drug discovery) to perform statistically powered studies.
Keywords: transcriptomic, alternatives, high throughput
Gene-specific 5’-UTR methylation vs. promoter methylation in leukocytes from workers exposed to different levels of VOCs (#516)
O. Jiménez-Garza1, A. Dávalos-Pérez1, J. Alegría-Torres2, L. Ruiz-García1
1 University of Guanajuato, Health Sciences Division, Leon, Guanajuato, Mexico
As a result of exposure to toxic substances, different human and animals cells have displayed either global DNA hypomethylation or gene-specific hypermethylation. Specifically in occupational volatile organic compound (VOC) exposure, we previously found hypermethylation in TOP2A, SOD1 and TNF-a promoter area in leukocytes from people exposed to a complex VOC mixture, while participants exposed to a single VOC did not show any differences compared to controls. The aim of this study was to analyze the methylation pattern in the 5’-UTR region, considered for some authors as an “internal/alternative promoter area” in people occupationally exposed to a single VOC compared to VOC mixtures exposure as well as a reference, non-exposed group. Methods: PCR-pyrosequencing was performed in order to analyze methylation status in CpG’s located at the 5’-UTR region of the TOP2A, SOD1, IL6, TNF-a and CYP2E1 genes in leukocytes from Mexican workers laboring at three different scenarios with VOC exposure compared to a reference group. Results: 5’-UTR region from IL6 and TOP2A genes showed hypomethylation for two populations exposed to a VOC mixture, while TNF-a showed hypermethylation; methylation in the SOD1 gene did not show differences between groups. DNA obtained from the reference group showed hypermethylation in the 5’-UTR region from the CYP2E1 gene compared to all exposed subgroups. Conclusions: 5’-UTR region in certain genes is more prone to undergo epigenetic modifications compared to promoter area as a result of VOC exposure. 5’-UTR methylation status must be considered in a near future as an early biomarker of effect in human chemical toxicity
Keywords: VOC's mixtures, epigenetics, promoter area, 5'-UTR area, gene-specific DNA methylation
In vitro toxicogenomic and proteomic assessment of e-cigarette aerosol compared to cigarette smoke (#621)
I. Verrastro1, L. Haswell1, S. Corke1, A. Baxter1, D. Thorne1, D. Breheny1, E. Minet1, M. Gaca1
1 British American Tobacco, Southampton, United Kingdom
E-cigarette use has increased globally and could potentially offer a lower risk alternative to cigarette smoking. Differential gene and protein expression studies may offer perspectives for assessing the reduced risk potential of e-cigarettes. In this study, we compared the transcriptional and proteomic profile of primary 3D human airway tissues (MucilAirTM) exposed to e-cigarette aerosols or smoke from a scientific reference cigarette (3R4F), using an acute exposure regimen. Transcriptomic analysis was conducted using next-generation sequencing, and the functional profile of differentially expressed RNAs was assessed using gene set enrichment analysis. Using both a targeted and “shotgun” LC-MS proteomic approach, we quantified proteins in the air-surface liquid (ASL) collected from exposed MucilAirTM tissues relative to air control. Using pFalse Discovery Rate (FDR) <0.01 and fold change (FC)>2, 873 and 205 differentially expressed RNAs were identified at 24hrs and 48hrs post- 3R4F exposure, respectively. Using a looser threshold of pFDR<0.05 and FC>2, only 3 differentially expressed RNAs were identified with e-cigarette aerosol. Gene set enrichment analysis revealed a clear response from lung cancer, inflammation and fibrosis-associated genes upon 3R4F exposure, and a low-confidence response from metabolic/biosynthetic, extracellular membrane, apoptosis and hypoxia genes upon e-cigarette exposure, suggesting a reduced impact of e-cigarette acute exposure on gene expression. Using a [FC]>1.5 and p-value<0.05 threshold, for a selected panel of 21 ASL proteins, significant changes were observed 24hrs post- 3R4F exposure for 1 protein involved in angiogenesis (ANGL3), and at 48hrs post- 3R4F exposure for 2 proteins involved in adaptive immune response at mucosal surfaces (PIGR and MUC1), while no response was observed for e-cigarette aerosol. Using a FC >1.5 and p-value<0.05 threshold, shotgun proteomic analysis of ASL proteins revealed a strong change in protein expression at both 24hrs and 48hrs post-3R4F exposure, while minimal changes were observed for e-cigarette aerosol, relative to air controls. In conclusion, e-cigarette aerosol induced minimal protein and gene expression compared to a conventional cigarette after acute exposure in vitro.
Keywords: omics, e-cigarette, proteomics, transcriptomics, tobacco
FDA SEND Process streamlining and implementation - CT-Compatible Simulated Study - (#29)
T. Anzai1, 2, R. Aerni2, M. Wasko2, F. Mura2, S. - I. Horikawa3, S. - I. Sato3, Y. Murase3, H. Hatakeyama3
1 Showa University School of Medicine / PDS Life Science, Hamamatsu-shi, Japan
The Standard for the Exchange of Nonclinical Data (SEND), adopted by the US FDA, is part of a set of regulations and guidances requiring the submission of standardized electronic study data for nonclinical and clinical data submissions. Thus, non-US countries must consider and develop approaches to SEND that meet their needs.
Since becoming the first Japanese CRO to make a successful trial submission to the FDA in 2015, Ina Research (INA) has been pro-active in educating the Japanese community regarding the steps that led to this success, as well as the processes and precautions that should be considered for those planning to incorporate SEND into their research planning. During these sessions, INA has continued to stress the importance of preparing for SEND in the early research stages - preparing a study protocol in accordance with the most recent guidelines and employing data collection methods that will allow effective QC.
In light of the gradual standardization of SEND Controlled Terminology (“CT”), INA has more recently conducted a simulated CT-compliant study. In order to increase the rate at which terminology used in the final report matched CT in a standard toxicology study, INA investigated :
1) matters that needed consideration during the planning stages
2) system issues that became obvious only when collecting mock data in the same manner as a real study
3) methods allowing quicker, more efficient SEND conversion. Following this, INA also considered the visualization of SEND-converted data, and how it may be better used in study evaluation.
Keywords: SEND, FDA, Controlled Terminology (CT), non-US countries, CDISC, PhUSE
European Food Safety Authority (EFSA) protocol for the re-assessment of the safety for consumers of Bisphenol A (BPA) (#88)
C. Croera1, A. F. Castoldi1, C. Putzu1, F. Barizzone2, J. Cara Carmona1, U. Gundert-Remy3
1 European Food Safety Authority (EFSA), Food Ingredients and Packaging (FIP) Unit, Parma, Italy
To ensure a methodologically rigorous re-assessment of the European temporary Tolerable Daily Intake (TDI) for BPA of 4 µg/kg bw per day set in 2015, EFSA has developed a protocol detailing a priori the approach and methodology for performing the BPA hazard identification and characterisation. The protocol defines upfront the methods and criteria to be used for data collection, study inclusion, evidence appraisal (internal and external validity) and evidence integration (e.g. weight of evidence, confidence in the body of evidence). Pre-defined criteria for evidence inclusion (e.g. publication date after December 2012, any exposure route, any toxicological endpoint, etc.) have been set. A full systematic review approach will be applied to human and animal evidence potentially suitable for deriving a TDI, whereas other types of evidence, e.g. cross-sectional and toxicokinetic studies, will be dealt with narratively. An appraisal tool adapted from the 2015 NTP-OHAT Approach for Systematic Review and Evidence Integration will be used to rate the internal validity of individual human or animal studies, according to study design and endpoint. For the external validity of animal studies (i.e. the relevance for humans of measuring a given endpoint in a given animal) criteria taken from the 2014 Science in Risk Assessment and Policy (SciRAP) tool will be applied. Confidence ratings in the body of evidence for each endpoint will be translated into levels of evidence for a certain health or no health effect, separately for humans and animals. These will then be integrated into likelihoods of a health or no health effect. For hazard characterisation, studies supporting likely effects of BPA will undergo a BMD analysis to identify a suitable point of departure for setting the TDI.
Keywords: Bisphenol A, Systematic Review, Protocol, Hazard assessment, Study reliability, Study validity
Stereotactic Surgeries and MRI-guided Convection-Enhanced Delivery (CED) into the Striatum in Cynomolgus Macaques (#107)
J. Luft1, A. von Keutz1, F. Runge1, T. V0ß1, S. Grote-Wessels1, S. Korte1
1 Covance Preclinical Services, Münster, North Rhine-Westphalia, Germany
Gene therapy is a promising area of drug development for a number of diseases including neurological disorders. NHPs (M. fascicularis) show high nucleotide sequence homology with humans are used when test article cross-reactivity occurs. An IACUC-approved toxicity study required bilateral administration of a gadolinium labeled viral vector to 4 locations to the Striatum (Caudate nucleus+putamen) with temporarily implanted catheters. MRI was used to calculate the trajectory and for surveillance of test article delivery during convection-enhanced delivery.
24 NHPs were prepared for anesthesia: analgesia, induction with ketamine/medetomidine, intubation, clipping, disinfection and placement in a stereotactic frame. Isoflurane inhalation anesthesia for 6-8 h. Animals were transferred to the MRI for calculation of position, angle and catheter depth, assuring that trajectory will not cross blood vessels or ventricles.
Following transfer to the surgical suite, a stereotactic frame was used to ensure correct placement of catheters based on the coordinates determined by MRI. The skin was reclined from the skull and holes were carefully drilled. Catheters were cut to specific length and inserted along calculated trajectories to administer the viral vector to 4 different locations into the Striatum. Catheters were connected to infusion lines containing the gadolinium labeled test article. Infusion (0.3 mL/h) was monitored in the MRI for 1.5 h to verify correct administration into the Striatum. Animals were transferred back to surgical suite and catheters were explanted. Animals recovered from anesthesia within 15–30 min and were treated with analgesics and antibiotics. 2 animals presented laryngeal swelling were treated with corticosteroids successfully. No animal showed neurological abnormalities.
Keywords: NHP, Stereotactic Surgery, Convection-Enhanced Delivery, MRI, Striatum Putamen
Application of a new biochip array platform to the simultaneous screening of drugs of abuse in urine and oral fluid in under 20 minutes (#143)
V. Anderson1, S. Cardwell1, D. Doone1, J. Dicks1, A. Speers1, J. Darragh1, P. Vance1, O. Dyttus1, M. L. Rodríguez1, M. E. Benchikh1, R. I. McConnell1, S. P. FitzGerald1
1 Randox Toxicology Ltd, Crumlin, United Kingdom
Background. Biochip array technology enables multi-analytical screening of drugs of abuse from a single sample in under 20 minutes with the new biochip analyser Evidence MultiSTAT. This study reports the analytical evaluation of this application to the simultaneous screening of drugs of abuse in urine (creatinine included) and oral fluid.
Methods. Simultaneous competitive chemiluminescent immunoassays on a biochip surface were applied to the fully automated Evidence MultiSTAT analyser, which processes a self-contained cartridge containing all the components required for the assays. Sampling against a cut-off sample, the results are qualitative.
Results. Drugs detected and cut-offs in urine and oral fluid respectively: AB-PINACA [2.5ng/mL (urine)], alpha-PVP (5ng/mL, 2ng/mL), amphetamine (200ng/mL, 50ng/mL), barbiturates (200ng/ml, 50ng/mL), benzodiazepines (150ng/mL, 10ng/mL), benzoylecgonine/cocaine (150ng/mL, 20ng/mL), buprenorphine [1ng/mL (urine and oral fluid)], ETG [750ng/mL (urine)], fentanyl (2ng/mL, 1ng//mL), ketamine [50ng/mL (oral fluid)], JWH-018 (20ng/mL, 5ng/mL), LSD [1ng/mL (oral fluid)], 6-MAM (10ng/mL, 2ng/mL), methadone (300ng/mL, 4ng/mL), PCP [5ng/mL (oral fluid)], methamphetamine (200ng/ml, 50ng/mL), opiate (200ng/mL, 10ng/mL), oxycodone (50ng/mL, 8ng/mL), THC (20ng/mL, 2ng/mL), tramadol (5ng/mL, 4ng/mL), UR-144 [10ng/mL (urine and oral fluid)] and creatinine [20mg/dL (urine)].
For both matrices, precision (+50% and -50% cut-off samples analyzed across 2 analyzers) and accuracy evaluation showed percentage agreement values≥90%. Percentage agreement with LC/MS (urine): 100% (6-MAM, oxycodone), 97% (benzodiazepines, methadone, opiates), 93% (amphetamine, buprenorphine, methamphetamine, THC), 80% (benzoylecgonine/cocaine). All samples screened positive for creatinine indicating that no sample dilution had occurred. Percentage agreement with LC/MS (oral fluid): 100% (10 analytes), 96% (methamphetamine, opiates) and 93% (benzoylecgonine/cocaine). No positive samples found for alpha-PVP, barbiturates, fentanyl, JWH-018, tramadol or UR-144.
Conclusion. Data indicate optimal analytical performance and applicability of the Evidence MultiSTAT system to the simultaneous screening of drugs of abuse in <20 minutes in urine and oral fluid.
Keywords: Biochip Array, Drugs of Abuse, Urine, Oral Fluid, Rapid Screening
Toxicology Out of The Box - an overview of the Lush Prize (#159)
1 The Lush Prize, Manchester, United Kingdom
The Lush Prize is pleased to attend and present at EUROTOX 2018. This year's theme of 'Toxicology out of the Box' aligns with the mission of the Prize, which since its launch in 2012 has awarded over € 2 million in bursaries for outstanding projects and achievements in the research, development and promotion of innovative approaches in human relevant toxicology.
Current regulatory requirements for chemical safety testing in the pharmaceutical, industrial and consumer product industries are resource-intensive in terms of money, time and animal use. The results are also of great concern with regard to their ability to predict human safety and disease, resulting in both scientific and ethical challenges. However, exciting advances in in vitro, in chemico and in silico technologies have achieved considerable success to date and provide irrefutable evidence for a future based on high calibre, human relevant research.
Each year, Lush Prize funding of up to € 400,000 is available across five categories; Science, Young Researchers, Training, Lobbying, and Public Awareness. There is also a special sixth category- the Black Box Prize, which may award in any one year a further prize of €285,000 for breakthrough achievements in human-relevant approaches. The first Black Box Prize was awarded in 2015 to several organisations for their work on human in vitro methods linked to the Adverse Outcome Pathway (AOP) for skin sensitisation.
The prize continues to fund innovative new research across, Europe, Asia, USA and the rest of the world. It was established to address an urgently needed shift towards more 'fit for purpose' human-relevant toxicity testing methods, in order to meet the increasing demands of high throughput, chemical safety assessment. The presentation will provide an overview of the Lush Prize and showcase the success of over 90 researchers and organisations funded to date. EUROTOX 2018 delegates are invited to attend and also find out how their research could be eligible for prize nomination in 2019 and beyond.
Keywords: Lush Prize, human relevant, in silico, in vitro, funding, 3Rs, 1R, replacement, alternatives, non-animal, award, bursary
Retrospective histopathology evaluation of respiratory tract tissues from two rat 90-day OECD 413 inhalation studies: Tobacco Heating System 2.2 versus Mentholated Tobacco Heating System 2.2 compared with conventional and mentholated reference cigarettes (#248)
A. Oviedo1, P. F. Neves3, E. T. Wong2, C. Foong2, G. Vuillaume1, D. Sciuscio1, M. Peitsch1, P. Vanscheeuwijck1
1 Philip Morris Products S.A., Neuchatel, Neuchâtel, Switzerland
For this cross-study assessment, a single pathologist performed a retrospective histopathological evaluation of respiratory tract tissues collected from two sub-chronic 90-day OECD inhalation studies in Sprague Dawley rats following Test Guideline 413, and compared results obtained from two versions of the Tobacco Heating System (THS 2.2): THS 2.2 Regular and THS 2.2 Menthol (THS 2.2M). Results obtained from effects of Reference Cigarettes (with or without menthol) tested in the 2 studies were also compared.
Rats were exposed to: a) filtered air (sham), THS 2.2 Regular (at 15, 23, and 50 µg nicotine/L) the 3R4F reference cigarette (at 8, 15, and 23 µg nicotine/L) in one study; and b) sham, THS 2.2M (at 15, 23, and 50 µg nicotine/L ), 3R4F and two mentholated reference cigarettes (MRC), MRC low and MRC high menthol (at 23 µg nicotine/L), in the other study. Respiratory tract tissues from each group were evaluated microscopically using a severity grade score and compared with each other.
Exposure to the smoke from reference products (MRCs and 3R4F) resulted in treatment-related adaptive and degenerative findings in the nasal cavity, larynx, trachea, and lungs of rats. However, the severity of histopathological changes observed in the respiratory tract of THS 2.2 and THS 2.2M aerosol-exposed animals was significantly lower when compared with those observed from exposure to reference cigarettes’ smoke (all concentrations). Findings observed in THS 2.2-exposed groups (regular and mentholated) included basal cell hyperplasia, squamous cell metaplasia, goblet cell hyperplasia, decreased/loss of goblet cells, necrosis, degeneration, increased pigmented macrophage, and goblet cell metaplasia.
Similar test item-related findings were observed in the respiratory tract of THS 2.2 compared with THS 2.2M groups but showed few significant high-severity observations in THS 2.2M-exposed animals, mainly at high concentration (50 µg nicotine/L). This suggests increased toxicity of the test item when menthol was added only at high concentration of nicotine.
Comparison of 3R4F-exposed groups showed consistent results across studies. Similar pathological findings were observed when comparing 3R4F to MRCs.
Keywords: Histopathology, Inhalation Toxicology, Menthol, THS2.2
Di (2-ethylhexyl) Adipate (DEHA): Systemic Toxicity in Rats Following 28-Day Intravenous Exposure (#308)
H. Xu1, B. Musi2, Z. Wang3, T. Zhou3, Q. Huang4, J. Liu1, T. Li1, E. Koo5
1 Baxter Healthcare Corporation, PreClinical, Suzhou, China
Background: Di (2-ethylhexyl) adipate (DEHA) is a plasticizer (softner) and a potential alternative for di-2-ethylhexyl phthalate (DEHP). Toxicity of DEHA has been studied mostly via oral exposure. DEHA is used in various medical devices including equipment for intravenous administration, but the toxicity of DEHA has not been assessed after repeated intravenous exposure. The present study shows the results of a systemic toxicity study in male and female rats with intravenous administration of DEHA once daily for 28 consecutive days. The reversibility, persistence, or delayed occurrence of toxic effects following a 14-day recovery period were also assessed.
Methods: The study was done according to ISO10993 Part 11 guidelines-under GLP conditions. Four groups of rats (15/sex/group) each received either vehicle (10% lipid emulsion) or DEHA in vehicle (100, 200, or 450 mg/kg/day). Criteria for evaluation included viability (morbidity/mortality), clinical observations, body weight, food consumption, clinical pathology (hematology, serum chemistry, coagulation, urinalyses), gross (necropsy) evaluation, organ weight and histopathological evaluation.
Results: There were no DEHA-related changes in all the endpoints evaluated at 100 or 200 mg/kg/day. At the 450 mg/kg/day dose, DEHA-related findings included clinical observations (prostration, salivation, abnormal gait, decreased activities, atonia post bolus dose), increased liver weight in females associated with minimal hepatocellular hypertrophy, and decreased thymus weight in males and females without histopathology findings. There were no test article-related changes in hematology, coagulation, serum chemistry, urinalysis, or gross findings at necropsy at 450 mg/kg/day.
Conclusion: The 200 mg/kg/day dose is considered to be the No-Observed-Effect Level (NOEL) based on the results from this study.
Keywords: DEHA, intravenous exposure, toxicity, rat
EXPOSURE DATA FOR BABY WIPE PRODUCTS (#314)
A. NALIN1, C. HERBIN1
1 EUROFINS EVIC France, Safety and Regulatory Affairs - Cosmectic Product Testing, Aix-en-Provence Cedex 3, /, France
Wipes are widely used cosmetic products. Among all available choices, Baby Wipes is one of the most popular. In order to assess the safety of use it is important to determine the quantity that will remain on the skin after a single use. This is considered an estimate of the exposure to the product. But, managing the exposure of baby wipes is challenging, as there are no guidelines to provide support. And multiple sources of daily amount applied data are available!
It is the responsibility of the safety assessor to choose the best exposure value, that will guarantee the health of consumers, taking into account the inter variability, and normal or foreseeable conditions of use, while remaining realistic. Available studies showed disparate exposure values. This review tries to address this issue by referencing the different data sources and offers a choice encompassing maximum value from real life context. This will help to a better harmonization between the safety assessors in choosing the most relevant value.
Keywords: Cosmetics; Risk assessment; baby wipes; exposure
Updates to OECD Guidance Document 23: Aquatic toxicity testing of difficult substances and mixtures (#341)
G. Stoddart1, W. Hunter2, M. Halder4, E. Salinas3, C. Faßbender1
1 PETA International Science Consortium Ltd., London, United Kingdom
The Organisation for Economic Cooperation and Development (OECD) Guidance Document (GD) on Aquatic Toxicity Testing of Difficult Substances and Mixtures (GD 23) provides critical guidance that supplements OECD Test Guidelines (TG) for studies conducted for regulatory purposes. OECD GDs undergo periodic review and updates due to scientific progress, changing regulatory needs, and animal welfare considerations. The GD 23 was first published in 2000 and recently updated to provide state-of-the-science approaches for aquatic toxicity tests involving difficult-to-test chemicals. This poster presentation provides an overview of the updated GD 23, which is currently awaiting approval by the OECD, including expansion of the guidance on testing of poorly water-soluble test chemicals and substances of unknown or variable composition, complex reaction products, and biological materials (UVCBs). As part of the updates, particular attention was paid to updating exposure methods that do not employ a solvent in order to eliminate solvent effects and reduce the number of animals used in aquatic toxicity tests (i.e. via eliminating the need for a solvent control). The updated GD 23 is a useful tool to regulators, industry, and contract research organisations to aid in conducting (or reviewing) valid and reliable aquatic toxicity studies, while minimising both the number of animals used and the need to repeat studies.
The views, conclusions and recommendations expressed in this presentation are those of the authors and do not necessarily represent the policies or positions of the United States Food and Drug Administration, the PETA International Science Consortium Ltd., the International Council on Animal Protection in OECD programmes, the European Commission or the OECD.
Keywords: Aquatic toxicity, animal welfare, guidance document, OECD
FOLLOW-UP ON WORTHINESS OF LiCl USE IN THE RATS’ DIET (#381)
E. O. Sadykova1, S. I. Shestakova1, A. N. Timonin1, M. D. Trebukh1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
This publication presents the results of research of Wistar rats’ morphofunctional parameters, which were exposed to diet (AIN-93) with Lithium chloride (LiCl) in its composition (control group) and LiCl free diet (test group). For the assessment of rats’ growth and development the integral parameters (body weight, internal organs weight, white fat weight), hematological and biochemical parameters were investigated.
The results of comparative assessment of LiCl influence on rats revealed a number of statistically significant differences of some parameters. Analysis of the differences allows us, with high level of assurance, to link them to particular physiological and biochemical processes: for instance decrease in spleen weight of experimental group rats might’ve been caused by the decrease in content of blood formed elements, mainly of erythrocytes, while concentration of erythrocytes in blood of test group rats was significantly higher than of control animals. The consequence of increase in concentration of erythrocytes in blood, as well as increase in haematocrit, may be higher the oxygen consumption per body weight unit, provided that O2-binding ability of erythrocytes and O2-availability of tissues were equal. The assumption regarding higher oxygen consumption per body weight unit of test group rats was indirectly confirmed by the increase in concentration of serum iron, which is the central atom of many mono- and dioxygenases as well as of mitochondrial cytochrome enzymes. High oxygen consumption, in its turn, proves the high intensity of metabolic processes.
It was established that adding of LiCl into standard diet significantly decreased metabolism intensity without the change of structure of the processes themselves.
To sum up, the change of LiCl concentration in diet composition can be viewed as alimentary regulator of metabolic processes intensity, and therefore, the necessity of its application should be differentiated depending on experiment goals. For instance, adding of LiCl into a diet appears to be justified in model experiments for obesity study, metabolic syndrome study, dislipidemy study, and is not justified in toxicological or reprotoxicological research, alimentary factors impact study.
This work was supported by Russian Science Foundation grant No. 16-16-00124.
Keywords: laboratory animals, metabolism intensity, lithium chloride, LiCl
Comparison of organ weight (absolute and relative to brain) in wistar han rats at different ages (#395)
S. M. McPherson1
1 WuxiApptecc, Toxicology, Suzhou, China
Organ weights are routinely assessed during the conduct of toxicology studies. This study was conducted to see if there were any differences in organ weight both relative and absolute in Wistar Han rats at three different ages: 11-12, 20-21 and 33-34 weeks. Rats were sourced from BioLasco Taiwan. The data sets were analyzed using graphs of organ weight to body weight and organ weight to brain weights. The data was further analyzed looking at the Y-intercepts, slopes, correlation coefficient (r) and coefficient of determination (r^2). The results of the analysis showed:. Organs with Highest Relative Growth Rates in Absolute Weights in Males and/or Females: Thyroid > Liver > Heart > Kidneys > Lungs > Spleen > Pituitary. Growth rates were similar in both males and females for the heart, kidneys, lungs and spleens but greater in the males for the thyroid, liver and pituitary. Organs with the highest correlation to body weight: Liver > Heart > Kidneys > Lung > Thyroid > Spleen. Correlations to brain weight were generally low and in the order of: Lung > Heart >Liver > Kidney > Spleen > Thyroid > Pituitary. Organs with Decreases in Relative Growth Rates in Absolute Weights in Males and/or Females: Thymus > Adrenal Glands. Correlations to body weight were low and in the same order. There was practically no correlation to brain weight. Males Organs with Highest Relative Growth Rates in Absolute Weights: Prostate > Epididymus > Testes. Male organs with the highest correlation to body weight and brain weight: Epididymus > Prostate > Testes. Female Organs with Highest Relative Growth Rates in Absolute Weights: Uterus > Ovaries. The uterus and ovaries were poorly correlated to body weight and brain weight.
These background data collected from this study can serve as a tool to help the toxicologist evaluate study data and put potential findings in context when compared to both the concurrent controls and these data sets.
Keywords: organweights, background data
My Family and Other Animals: Comparative Requirements for Human and Animal Metabolism Studies (#412)
1 Fera Science Limited, Chemical Safety, York, United Kingdom
There is an ever increasing requirement to provide high quality metabolism data to meet the regulatory challenges in both pharmaceutical (pre-clinical and clinical) and environmental fate studies (ADME, e-fate and dietary). The gold standard study uses radiolabelled test compound, but the samples taken for analysis often contain very low levels of radioactivity.
Preliminary data on the nature of metabolites are often obtained during the discovery phase of compound development. The data obtained is often semi-quantitative in nature, relying on mass spectrometer (MS) response factors. For the data generated during the critical phases of development to be useful, accurate methods of metabolite quantification are required. Structural analysis can be performed using MS or in some cases NMR. By linking the structural data to the chromatographic profiles, a metabolism pathway can be elucidated on a quantitative level.
In pharmaceutical development the key matrix for structural elucidation and quantification of metabolites is the plasma, whilst for agrochemicals and veterinary products it is the tissues.
The talk will focus on the similarities and differences between human and livestock ADME studies, look at the study objectives and discuss typical detection limits encountered. There will also be a review on recent developments that improve the limit of quantification for High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). Measurements that can provide added value to the experiment.
The talk will provide examples from ADME, dietary and e-fate studies. The techniques discussed will include tissue distribution, 2D-TLC, HPLC with online detection and multiwell microplate scintillation counting. Advantages and disadvantages of the different methods will also be included.
Keywords: ADME, Metabolism, Radioactivity, Quantification
Reviewing the use of two species in toxicology studies supporting drug development (#429)
H. Prior1, N. Gellatly1, F. Sewell1, I. Kimber2
1 NC3Rs, London, United Kingdom
The NC3Rs and the Association of the British Pharmaceutical Industry (ABPI) are collaborating to review the utility of two species in regulatory toxicology studies. The purpose is to explore circumstances when data from a single species could be sufficient to enable safe progression in humans, for a broader range of molecule types than currently accepted. An international working group of 37 representatives from pharmaceutical/biotechnology companies, contract research organisations, consultants, academia and regulatory bodies is reviewing information gathered via a data sharing exercise completed in 2017, to examine if there are options to reduce the number of species used for toxicology studies at different stages of development.
Blinded data from 18 organisations were collected, for 92 small molecules, 46 monoclonal antibodies (mAbs), 15 recombinant proteins, 13 synthetic peptides and 6 antibody-drug conjugates (ADCs); 114 compounds were in active development whilst 58 had stopped. Compounds covered a wide range of therapy areas (including oncology: 45; central nervous system: 27; immunomodulatory (anti-inflammatory): 27) and had progressed to pre-First-in-Human (FIH) studies (32), FIH package (93) or post-FIH longer-term studies (47).
Two species were used by 89 small molecules, 14 mAbs, 12 recombinant proteins, 13 synthetic peptides and 5 ADCs. If used, the non-rodent was dog (59, 0, 2, 7 and 0 compounds respectively), non-human primate (29, 14, 10, 6 and 5 compounds respectively) or minipig (1 small molecule). Only 2 ADCs and 5 mAbs (all following ICHS6 guidelines) and 1 small molecule (following ICHM3 guidelines) reduced to a single species during the package. All other compounds retained use of two species: 94 followed ICHM3 or ICHS9 guidelines whilst 31 followed ICHS6 guidelines (including 11 compounds at post-FIH stage).
Further analysis is ongoing to investigate the value of data generated in two species, particularly for the post-FIH compounds. Understanding the differences or similarities in toxicities between species may highlight reasons for the low adoption of existing opportunities (single species chronic studies) by biologicals and may provide evidence to expand these principles to a wider range of molecule types (e.g. small molecules) or therapeutic areas (e.g. oncology).
Keywords: drug development, non-rodent, rodent, safety assessment, toxicology
An overview of popular (Q)SAR packages for regulatory purposes: an agrobusiness point of view (#432)
B. E. S. Calçada1, P. V. B. Dias1, C. S. R. Sousa1
1 Ascenza Agro SA, Physical-Chemical Department, Setúbal, Portugal
(Q)SAR - (Quantitative) Structure-Activity Relationship - models are an important tool for the qualitative or quantitative prediction of physicochemical, toxicological, and ecotoxicological properties of compounds based on the knowledge of their chemical structure.1 These models are being developed and improved in a regular basis and are specially relevant for agrochemical compounds1 since they could be used not only for screening purposes, but also, if sufficient and based in a weight of evidence (WoE) approach, as substituents of in vitro and/or in vivo studies, thus contributing to an higher implementation of the 3Rs policy (Refinement, Reduction and Replacement).
Herein, we present a comparative study of the predictive capability of popular (Q)SAR packages in the context of agrobusiness. Two computational software were used: the expert-based software Derek Nexus®2 and the statistical software OECD (Q)SAR Toolbox.3 For a collection of relevant compounds, (eco)toxicity was predicted recurring to all the models available in the software for a variable group of general endpoints (e. g. Carcinogenicity, Mutagenicity and Aquatic toxicity). A comparison with their respective official classification and evaluation by authorities (e. g. EFSA and ECHA), thus assessing the predictive power of the software, will be presented and discussed. We will also present relevant outliers, trying to rationalize the reasons that led the software to give an erroneous prediction.
1 European Chemical Agency, Practical guide – How to use and report (Q)SARs, version 3.1, July 2016
2 Carol A. Marchant, Katharine A. Briggs, Anthony Long, In silico tools for sharing data and knowledge on toxicity and metabolism: derek for windows, meteor and vitic. Toxicol. Mech. Methods, 2008; 18(2-3):177-87. doi: 10.1080/15376510701857320
3 Application manual of OECD QSAR Toolbox v.4.1, www.qsartoolbox.org
Keywords: (eco)toxicity, assessment, software, classification, regulatory
Use of recovery animals for human safety assessment across the pharmaceutical development package (#440)
F. Sewell1, N. Gellatly1, H. Prior1
1 National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs), Gibbs Building, London, United Kingdom
As part of the NC3Rs/ABPI (Association of the British Pharmaceutical Industry) initiative to review the use of two species in regulatory toxicology packages, an international expert group (representing 37 pharmaceutical/biotechnology companies, contract research organisations and regulatory bodies) has analysed the dataset to examine current approaches to the inclusion of recovery animal groups. It is a regulatory requirement to assess recovery at some point during drug development, to assess whether effects observed persist or reverse once treatment ends. However, it is not stipulated how, where or when recovery animals should be included (if at all). In 2014, it was recommended that inclusion later in development be considered, once more information on toxicity is available1.
Data was available for 62 small molecules and 37 monoclonal antibodies (mAbs) with studies conducted to support First-in-Human (FIH) and later phase clinical trials. For FIH packages, 41/62 small molecules included recovery in at least one study, usually in two species. However, 21 compounds did not include recovery in any FIH study. This is a higher proportion than in the 2014 dataset (34 v. 23%). For mAbs, 29/37 included recovery in studies to support FIH, usually in one non-rodent species. 8 compounds did not include recovery in any study (22 v. 11% in 2014). When recovery was included, reduced study designs used fewer animals via fewer dose groups, with cases of high dose only (no control) and/or assessment in a single sex.
For a subset of compounds (22 small molecules and 13 mAbs) there was information on additional studies to support post-FIH packages. Variable approaches were adopted for small molecules: most included recovery in almost all studies, for both FIH and post-FIH studies (9 compounds). However, some included recovery in FIH (6 compounds) or post-FIH studies only (4 compounds), and 3 compounds did not include recovery in any study. For the 13 mAbs, recovery animals were always included: 10 compounds included recovery in both FIH and post-FIH studies, whilst others included recovery in FIH studies (2 compounds) and post-FIH studies only (1 compound).
Industry is moving towards a more case-by-case approach, however there remain opportunities to expand uptake of the previous recommendations, and reduce the use of recovery animals across the wider drug development pathway without impacting on human safety.
1Sewell et al. (2014) Reg Tox Pharmacol 70: 413-429.
Keywords: drug development, recovery, first-in-human (FIH), safety assessment, regulatory toxicology
Probabilistic risk of decreased levels of triiodothyronine following chronic exposure to PFOS and PFHxS (#450)
M. Öberg1, 2, A. Da Silva1, J. Ringblom1, K. Scott4, C. Lindh4, K. Jakobsson3
1 Karolinska Institutet, Unit for Toxicology Sciences (Swetox), Södertälje, Sweden
High concentrations of perfluoroalkyls (PFASs) have been found in drinking water in areas with extensive use of firefighting foams. We performed an integrated probabilistic risk assessment analysis correlating the serum levels measured in humans from a municipality with drinking water PFAS contamination with data from a subchronic study in monkeys exposed to PFOS. The critical effect size was set to a 10% decrease in triiodothyronine (T3) levels.
The study populations was participants from a biomonitoring study in Ronneby, Sweden, a municipality where 1/3 of the households for decades had municipal drinking water with very high levels of perfluorooctanesulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS) and to a lesser degree perfluorooctanoic acid (PFOA). All participants aged 3 and above, who were living in the exposed water district in Dec 2013 when clean water was provided, were included (n=1845).
Serum levels (5th and 95th percentiles) were between 63 – 830 ng/mL for PFOS, and 45 – 790 ng/mL for PFHxS. A benchmark dose analysis was employed to describe the dose-effect relationship for decreasing total T3 hormone levels in Cynomolgus monkeys. Extrapolation distributions were employed to estimate the human benchmark dose for the same effect, and to divide it with the human internal exposure data. By using uncertainty distributions recommended by the WHO for the different extrapolation factors, we estimated a median probability of critical exposure (PoCE) of 2.11% (i.e. 2,100 per 100,000) for a 10% T3 decrease following a combined exposure to PFOS and PFHxS. For PFOS exposure only, the estimated PoCE was 0.95 % (i.e. 950 per 100,000). Among the sources of uncertainty, extrapolation from subchronic to chronic exposure and intraspecies toxicodynamics variability were the greatest contributors.
To our knowledge, this is the first risk assessment analysis combining benchmark dose-modelling, uncertainty distributions and potential T3 decrease following PFASs exposure in drinking water. This study exemplifies the use of probabilistic assessment as a tool for risk assessment and risk management support in relation to exposure of PFASs in the general population. Ongoing biomonitoring studies in a larger population from the area, exploring free T3, free T4 and TSH responses in relation to exposure, will allow for comparisons.
Keywords: Health risk assessment, Probabilistic risk assessment, PFOS, PFHxS, drinking water, triiodothyronine
Toxicity evaluation of flavored e-CIG condensed aerosols on in vitro models of the lung (#453)
R. D. Bengalli1, Y. Piunno1, E. Ferri2, P. Mantecca1
1 University of Milano-Bicocca, POLARIS Research Centre, Dept. of Earth and Environmental Sciences, Milano, Italy
Since 2016, EU countries transposed directive 40/2014/UE and Tobacco Product Directive (TPD) became effective for the first time about vaping products. Through TPD, member states ask the manufacturers/importers to notify vaping products and declare them as safe for human consumption. Therefore, market demands for fast and efficient toxicity screening systems, able to test copious references and to discriminate the effects according to the different ingredients. The present study aims to contribute in this field by testing the effects of condensed aerosols (CAs) from different main e-liquid categories (tobacco, fruity, creamy, mentholate) and investigating the mechanisms of toxicity. The commercial e-liquids tested had the same PG:VG composition (50:40) and varied only in term of concentrated flavored blends (4-6%). A vaping machine equipped with an aerosol condensing trap was connected to a flask containing 25mL of cell culture medium. Puff regime was set to 55mL puff volume, 3s draw, 60s puff interval and 200 puffs. A549 lung cells were used to test cell viability and interleukin-8 (IL-8) release after 24h exposure to CAs at increasing doses. Data were compared to conventional cigarette smoke extracts. The mode of action of the different CAs on lung cells was also investigated through Hoechst/PI staining, ROS detection, cell morphology and cell cycle analysis. Furthermore, the effects were evaluated on an in vitro model of the air-blood barrier (ABB) composed by a 3D co-culture of epithelial and endothelial cells, where barrier integrity, cell viability and IL-8 release were assessed. Data show that only few flavors affected cell viability at the highest doses, while most of the flavors induced a dose-dependent decrease of IL-8 release. Specific flavors with a cytotoxic profile induced ROS production, cell cycle alteration and necrotic cell death. Data from the ABB model evidenced that in a complex system the effects are significantly mitigated. These results suggest that more efforts should be dedicated to the study of e-CIG mechanisms of toxicity, but at the same time point out that multiple assays with different in vitro models are able to discriminate the acute inhalation toxicity of CAs from different flavored e-liquids, providing useful tools for the preliminary screening of marketable products.
Acknowledgements: Authors wish to thanks FlavourArt Srl for supporting the study.
Keywords: e-cigarettes, condensed aerosols, in vitro toxicity, air-blood barrier
Rotational Thromboelastometry for Ex Vivo Assessment of Drug Effects on Platelet Function (#501)
S. Authier1, 2, F. Silva Negro1, C. Saint-Jean2, C. Li1, R. Forster3, O. Foulon3
1 Citoxlab North America, Laval, Québec, Canada
High sensitivity assays to identify pro or antithrombotic effects are critical in preclinical safety testing considering the life-threatening consequences of drug-induced thrombosis or hemorrhage. This study evaluated the sensitivity of a global coagulation assessment platform, to detect alterations in platelet response, clot formation and thrombus lysis ex vivo. Blood was obtained from Cynomolgus monkey and human normal volunteers. Rising concentrations or inhibitors, such as heparin and acetylsalicylic acid, and platelet activator, such as collagen, were added to the blood samples, ex vivo. Viscoelastic properties of the clot formation were assessed by thromboelastometry (ROTEM(®, for 1.5 hours after recalcification of citrated whole blood with calcium chloride (Star-tem reagent). Platelet response in whole blood decreases abruptly after 2 hours post collection. In Cynomolgus monkeys, mean coagulation time (CT) increased by 17%, two hours post collection while clot formation time (CFT) increased by 51% and maximum clot firmness (MCF) decreased by 9% in the same interval. The results after 4 hours were similar to those at 2 hours. Because of the inter-individual variations observed, the results were presented as the % of change from an untreated baseline, which was the value obtained without any agonist or inhibitor. With this approach, inter-individual variations were significantly reduced. With rising collagen concentrations (0ug/mL, 1ug/mL, 2ug/mL, 5ug/mL), the CT was shortened by 23%, 31% and 36% with collagen concentrations of 1, 2 and 5 ug/mL, respectively. Signs of saturation were observed at higher collagen concentrations. Collagen had no significant impact on the CFT and the MCF. As expected, heparin at pharmacologically relevant concentrations completely inhibited clot formation at concentrations of 1 units/mL or above. However, at a lower concentration of 0.1 units/mL, CT was extended by 148%, CFT was prolonged by 454% while MCF was reduced by 32%. As previously reported, acetylsalicylic acid (0.1 mg/mL, 1 mg/mL, 10 mg/mL) did not significantly affect rotational thromboelastometry. This study reports ex vivo platelet response characterization using rotational thromboelastometry as a strategy for pharmacological evaluations of drug effects using non-human primates and human blood.
Keywords: thromboelastometry, drug effect, platelet function, ex vivo assessment, antothrombotic effect, coagulation, non-human primates
Can we do better with paracetamol poisoning in rural Asia? - A model based approach to reduce cost of management of paracetamol poisoning in Sri Lanka (#532)
V. M. Pathiraja1, I. B. Gawarammana1, 2, N. A. Buckley1, 3, F. Mohamed1, 4, S. F. Jayamanna1, 5, A. H. Dawson1, 6
1 South Asian Clinical Toxicology Research Collaboration, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka
There has been a significant increase in paracetamol poisoning in Sri Lanka. Cost of management of paracetamol poisoning exceeds all other poisonings. There is a variable adherence to ideal management prescribed by National Management Guidelines (NMG) leading to unnecessary and costly transfers to larger hospitals. The objective of the study was to compare actual costs incurred with that of the ideal cost that should have been spent in the management of paracetamol poisoning in rural Sri Lanka.
We prepared models of actual costs incurred and the ideal cost that should have been spent. Data was extracted from two observational studies in two large districts, Kurunegala and Matara of Sri Lanka in 2011 and 2017. The actual model was developed using the following costs: cost for a hospital bed day; cost for health care personals; cost of transfers to tertiary care hospitals and cost of antidotes. We then developed an ideal model where all patients were considered to have been managed according to the NMG. We factored in the impact of changes of antidotes prices to the models to decide if changes of pricing have an impact on the model.
There were 2670 and 459 paracetamol poisoning admissions in Kurunegala and Matara districts respectively. Actual per patient cost was $134 (in 2011) and $123 (in 2017) while ideal cost was $47 (in 2011) and $50 (in 2017). Had the NMG been followed a total of $181,816 could have been saved in 2011. Despite the reduction of prices of NAC in 2017, the savings would still have been $151,535. The main reason for the increased actual cost was hospital bed cost associated with intravenous N- acetylcysteine (NAC) administration which remained constant throughout. Additional costs were spent on unnecessary transfers to larger hospitals for intravenous NAC administration.
These costs could be reduced by regulating the delivery of care in the rural hospitals. Adherence to NMG would increase the use of shorter duration oral methionine instead of the longer duration intravenous NAC and thereby reduce hospital bed costs. Regulation to encourage antidote stocking in rural hospitals will further reduce costs associated with transfers.
Keywords: Paracetamol, self-poisoning, Management Guidelines, cost, antidote
DNA microarray analysis on characteristics of hepatocyte-like cells derived from human iPS cells for the application to the cell based drug safety tests (#533)
S. Ishida1, S. Horiuchi1, Y. kuroda1, R. Fujii1, S. - R. Kim1, Y. Kanda1
1 National Institute of Health Sciences, Division of Pharmacology, Kawasaki, Kanagawa, Japan
[Purpose] In in vitro test for safety evaluation of drug candidates at the early stage of drug development, the human primary / cryo-preserved hepatocytes are mainly used. However, there are drawbacks to overcome, such as the differences between lots based on the individual donor variations or limited supply of each lot. To circumvent these points, hepatocyte-like cells derived from human iPS cells (hiPSC-hep) are expected as an alternative cell source. In this study, liver-like characteristics of hiPSC-hep were compared with cryo-preserved hepatocytes (cryo-hep), as well as human liver cancer cells (HepG2) and hepatocytes derived from liver-humanized mice (PXB-cells), to evaluate their potential of application to cell based drug safety tests.
[Methods] Genopal focused DNA microarray (Mitsubishi Chemical. Co., Ltd., Tokyo, Japan ), which is customized to measure 183 liver-related gene expressions, was used for analysis of liver-like characteristics of hiPSC-hep. Liver-related gene expressions were measured in hiPSC-hep obtained from three vendors (Vendor A, B and C), cryo-hep, PXB-cells, and HepG2 by Genopal. Principal Component Analysis (PCA) and hierarchical clustering were performed using gene expression data. Additionally, functional characteristics enriched in each gene cluster were analyzed.
[Results] When the gene expression patterns of hiPSC-hep, cryo-hep, HepG2, and PXB-cells were compared by PCA, the group of cryo-hep and PXB-cells and the group of hiPSC-hep and HepG2 were separated by principal component 1. In addition, the group of cryo-hep and PXB-cells and the group of hiPSC-hep and HepG2 were separated in hierarchical clustering. Genes related to phase Ι drug metabolism enzymes and drug transporters were significantly enriched in the cluster of genes whose expressions were lower in hiPSC-hep than in cryo-hep and PXB-cells. These indicated the immature characteristics of hiPSC-hep. However, genes related to phase II drug-metabolizing enzymes and those related to metabolism of lipid, carbohydrate and amino acid were significantly enriched in the cluster of genes whose expressions were high in hiPSC-hep from vendor C. These results suggest that a part of hiPSC-hep functions is close to those required for cell based drug safety tests.
Keywords: human iPS cell, hepatocyte, DNA microarray, drug safety test, drug matabolism
Implementation of in vitro methods in the safety evaluation of the skin sensitization potential of chemicals under REACH regulation (#547)
C. Rovida1, M. Locatelli2, E. Corsini3
1 Konstanz University, CAAT-Europe, Konstanz, Germany
REACH Regulation (EC 1907/2006) asks for a complete registration dossier for all substances marketed in the EU in quantity above 1 t/y. This registration dossier contains toxicological information with increasing complexity in relation to the yearly tonnage band of the registered substance. Since 2007, EU Companies have registered more than 18,000 substances to ECHA, the European Chemical Agency.
Skin sensitization endpoint is considered fundamental to guarantee safety of both workers and consumers and the assessment is mandatory for all substances, excluding only intermediates used in low quantities and under strictly controlled conditions.
Annex VII of REACH contains provisions about how to assess skin sensitisation in the registration dossier. When the regulation was published, the suggested test was the in vivo method LLNA (Local Lymph Node Assay) according to OECD TG 429 or 442a-b. In the following years, some in vitro methods were successfully validated and in 2016 the REACH Regulation was amended and now Annex VII asks to test in vitro first, following the Adverse Outcome Pathway described in OECD 168a-b with correspondent OECD TGs. The ECHA endpoint specific guidance on Information Requirements (R7a) contains detailed instruction on how to apply the new strategy.
In spite of the many available guidance documents, REACH registrants have to cope with the uncertainty of using a new approach and CROs (Contract Research Laboratories) are overloaded of requests on methods for which they still have little experience.
Through the activity of a consultancy company, there was the possibility to assess many test reports about skin sensitisation evaluation for REACH purposes and understanding the difficulties of the companies in applying in vitro methodologies. The analysis of the final results allowed the definition of some statistics about how to use the guidance defined approach, the costs, the quality of different CROs and the examples when the in vitro strategy was not applicable. In few cases, non standard methods were also successfully applied in a weight of evidence approach, according to the provisions of REACH Annex XI and including epidemiological data.
Acknowledgment of the hurdles in applying new approach methodologies is fundamental in improving acceptability and implementation of in vitro methods.
Keywords: REACH, in vitro methods, skin sensitisation, IATA
Selective evaluation of potentially carcinogenic polycyclic aromatics in mineral oil by the DMSO based IP346 method (#577)
J. C. Carrillo1, P. J. Boogaard1
1 Shell International, den Haag, Netherlands
Mineral oils are produced by vacuum distillation of petroleum at temperatures of ~300⁰C – ~600⁰C. Refining processes to eliminate the carcinogenic potential of mineral oils (by extraction and/or hydrotreatment) are based on the principle of removing substances associated with carcinogenic activity; i.e. PAC (polycyclic aromatic compounds), which include PAH and N or S heterocycles. Traditionally, the refined product was tested in the mouse skin painting carcinogenicity assay. This bioassay is considered the golden standard to study the process of carcinogenesis and its relevance to humans since it uses the most sensitive species and route of exposure. Mouse skin painting studies have also been important in understanding the toxicity of two types of aromatic compounds which are in recent literature lumped together as ‘mineral oil aromatic hydrocarbons’ or MOAH. The first type includes the 3-7 ring PAC associated with potential carcinogenic effects that may be found in the 340-535⁰C boiling range. The second type, includes highly alkylated aromatic compounds (predominantly 1-2 rings) which are not bioactivated and non-carcinogenic. Because mouse skin painting tests take too long, an alternative method was developed which is based on several common features of PAC’s. This method, known as IP346 is capable to distinguish the two types of MOAH, and is validated against a large data base of mouse skin painting studies. It is the only existing analytical method with biological significance and currently the EU legal regulatory standard to assess carcinogenicity of mineral oil. These chemical and biological features of PAC structures assessed by IP346 are presented and discussed in detail, especially the crucial role of the DMSO extraction step which allows to discriminate between the two types of MOAH. This selectivity is based on boiling point, molecular weight, carbon chain length, content of heterocycles; all of which are dictated by refining conditions. Thus, the DMSO based IP346 is a clear reflection of refinement efficacy by linking manufacturing conditions and biological activity of an oil. In conclusion, for the accurate interpretation of MOAH, the basics of mineral oil refining, the toxicological data base and the historical developments that led to the establishment of IP346 must be properly understood.
Keywords: mineral oil, MOAH, PAC, IP346, carcinogenicity
Guidance for synthesising epidemiology evidence for the UK Committee on Toxicity (COT) and on Carcinogenicity (COC) of chemicals in foods, consumer products and the environment. (#578)
D. Gott1, A. Hansell1, 2, C. Potter1
1 Food Standards Agency, London, United Kingdom
The UK Committee on Toxicity (COT) and on Carcinogenicity (COC) regularly review epidemiological evidence in their risk assessments. However, there was a need for guidance on the approaches used, in part to ensure public transparency. To that end, the committees established the Synthesising Epidemiology Evidence Subgroup (SEES) to review and document current practice, and make recommendations for COT/COC guidance.
Systematic review and meta-analysis are gold standard methods for combining epidemiological studies, but may not always be feasible for COT/COC, because of time and resource constraints.
Epidemiological reviews by COT/COC in the last 10 years were identified and reviewed. They covered a wide range of topics and used a variety of methods of review. This review was invaluable in developing guidance that would meet the needs of the committees.
For systematic reviews and meta-analysis, SEES recommended use of the MOOSE and PRISMA guidance. Quality assessment of studies was considered integral to review. SEES considered that if a numerical scoring tool is used, it should (i) aid narrative assessment, and (ii) help direct sensitivity analyses. Specific issues related to quantitative risk assessment and meta-analysis were identified, particularly study heterogeneity. Documentation of uncertainty and potential conflict of interests was considered important.
SEES prepared a report/guidance, which has been accepted by the COT and COC. It will be trialled by the COT for 2 years under review, along with a COT/COC checklist for meta-analyses and systematic reviews, based on MOOSE.
All authors contributed equally to the work
Acknowledgements: D Bodey, A Boobis, J Cade, D Lovell, N Pearce, J Peto, L Rushton, H Walton, D Benford, L Williams, B Gadeberg and F Pollitt
Keywords: epidemiology, toxicology, carcinogenicity
Safety of Hopoxia-cultured Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells : subchronic toxicity, tumorigenicity (#591)
E. Kwon1, J. You1, J. Yoon1, B. Kang1, 2
1 Seoul National University Hospital, Department of Experimental Animal Research, Biomedical Research Institute, Seoul, Republic of Korea
Hopoxia-cultured Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells (HC-hUCB-MSCs) was known to enrich the phosphorylation in the anti-apoptosis pathway and the secretion of several angiogenic proteins from cells and leading to alleviate the ischemic injury of hindlimb of mice. Recently, the more interest in stem cell therapy has increased, the more its safety is emphasized. The aim of this study was to evaluate the safety of HC-hUCB-MSCs for the subchronic toxicity and tumorigenicity. We injected the HC-hUCB-MSCs at the dosage of 2×105, 1×106, 5×106 cells/mouse by single-dose intramuscular administration. In the 13-week subchronic study, no significant HC-hUCB-MSCs-related changes (body weight, food/water consumption, clinical symptom, urinalysis, hematology, clinical chemistry, organ weight, and histopathology) were observed. In a 26-week tumorigenicity study, no mice developed tumor related to HC-hUCB-MSCs transplantation up to 5x106 cells/mouse. There was no systemic toxicity or neoplastic finding either. These results suggest that Hypoxia-cultured hUC-MSCs have great potential for future clinical treatment of various disorder.
Keywords: Hopoxia-cultured, Umbilical Cord Blood-Derived Mesenchymal Stem Cells, subchronic toxicity, tumorigenicity
Introducing WikiPathways to link molecular pathways to Adverse Outcome Pathways to support regulatory risk assessment (#645)
M. Martens1, E. Willighagen1, P. Nymark3, 4, R. Grafström3, 4, L. Burgoon7, H. Aladjov5, F. Torres Andón6, C. Evelo1, 2
1 Maastricht University, Bioinformatics - BiGCaT, Maastricht, Netherlands
In the last decade, omics-based approaches such as transcriptomics, proteomics and metabolomics have become valuable tools in toxicological research, and are finding their way into regulatory toxicity. A promising framework to bridge the gap between the molecular-level measurements and risk assessment is the concept of Adverse Outcome Pathways (AOPs). These pathways comprise mechanistic knowledge and connect biological events from a molecular level towards an adverse effect after exposure to a chemical or nanomaterial. However, the implementation of omics-based approaches in the AOPs and acceptance by the risk assessment community is still a challenge. Therefore, tools are required for omics-based data analysis and visualization, and to link the data to the traditional AOPs.
Here we show how WikiPathways, an open science pathway database, can serve as a viable tool for this purpose. Therefore, an AOP Portal (aop.wikipathways.org)has been created with a rapidly growing collection of molecular-level AOPs on which omics datasets can be mapped an analyzed, currently consisting of 15 pathways by 14 authors that are structured in various ways. Besides that, we are making WikiPathways more interoperable with aopwiki.org, the main knowledge-base that collects and stores AOPs. The open and collaborative nature makes WikiPathways a fast growing platform that is applicable in a wide range of biomedical research fields in which omics-based approaches are used. Also, its use of ontologies, OpenAPI documentation and FAIR (Findable, Accessible, Interoperable, Reusable) approaches makes WikiPathways interoperable with many other data sources. By introducing AOPs in WikiPathways and linking these with the AOPs in aopwiki.org, we aimed to make WikiPathways a useful tool for the regulatory toxicity community and for toxicological research in general. Eventually this could lead to implementation of WikiPathways as a data-source for decision-making in REACH (Registration, Evaluation, Authorization, and restriction of Chemicals) dossiers for risk assessment of chemicals. This project has received funding from the European Union’s Horizon 2020 research and innovation programme project EU-ToxRisk under grant agreement No. 681002 and EINFRA-22-2016 programme project OpenRiskNet under grant agreement No. 731075.
Keywords: Adverse Outcome Pathways, Regulatory risk assessment, WikiPathways, Omics, Interoperability
Study Rating – is Reliability enough? An appeal for revisiting Relevance and Adequacy, particularly in environmental risk assessment (ERA) (#680)
R. A. Wess1
1 Innovative Environmental Services (IES) Ltd, Witterswil, Solothurn, Switzerland
The challenge of decision making between several and unequivocal experimental data occurred in regulatory with the systematic hazard and risk evaluation of existing chemicals, namely the OECD High Production Volume program and eventually the EU REACh legislation. The working experience resulted in the recommendation of a systematic approach (Klimisch et al. 1997), considering not only Reliability but also Relevance and Adequacy of data. The regulatory implementation in the REACh legislation and the IUCLID database tool for data submission were anyhow restricted to the assignment of a Reliability score, thus rating only the intrinsic quality of the available study information. Due to endpoint specific refinement of the criteria for Reliability evaluation, also the publication of Küster et al. (2009) became a standard in regulatory, specifically for the environmental risk assessment (ERA) of pharmaceuticals. Finally the Reliability evaluation methodology has been further developed to gain increased reproducibility of the ratings (Kase et al. 2016). In result, a state-of-the-art conclusion on the Reliability of a study is a work and documentation intensive task.
As the Reliability of an irrelevant and/or inadequate study is completely meaningless, these points may be worth to be checked and documented before undertaking investigations on the Reliability. Studies correctly rateable as “reliable without restriction”, can still be unsuitable for assessment. This holds typically true in cases of test artefacts, i.e. if the experimental protocol is unsuitable for a substance. An example is the aquatic toxicity of test items releasing metal cations in case the test medium contains chelating agents. Other reasons to rate a study irrelevant may be the use of the wrong test item, e.g. a dissociating pharmaceutical formulated as an organic salt with a readily biodegradable and toxicologically almost irrelevant organic cation or anion, should not be used in the biodegradation screening-test as it will be present as two substances, one of them feeding the test microorganisms. Also a radiolabel in a carboxylic acid group, being subject to readily decarboxylation would question the relevance and/or adequacy of a study qualifying as reliable due to the proper study documentation.
Keywords: Reliability, Relevance, Adequacy, Environmental Risk Assessment (ERA), Pharmaceuticals
Critical Evaluation of Thresholds for Respiratory Effects of Toluene Diisocyanate (#701)
R. L. Prueitt1, H. N. Lynch2, I. Mohar1, J. E. Goodman2
1 Gradient, Seattle, Washington, United States of America
In 2016, the American Conference of Governmental Industrial Hygienists lowered the 8-hour Threshold Limit Value - time-weighted average (TLV-TWA) for toluene diisocyanate (TDI) from 5 ppb to 1 ppb, and the 15-minute short-term exposure limit (STEL) from 20 ppb to 5 ppb, to protect against respiratory effects. We critically evaluated the available human and animal evidence upon which these occupational exposure limits (OELs) were based. We found that the human evidence indicates that maintenance of 8-hour average TLV-TWA concentrations less than or equal to 5 ppb and peaks less than 20 ppb (i.e., the previous 8-hour TLV-TWA and 15-minute STEL) is protective of occupational asthma (OA) in most workers, and is also protective of lung function decrements and other respiratory effects. Some of the available studies suggest that occupational asthma cases at TWA concentrations less than 5 ppb were likely affected by very high peak exposures, well above 20 ppb. Advances in industrial hygiene measures have reduced peak exposures and the incidence of upset conditions such as spills and accidents, so these high peak exposures are unlikely to occur in modern TDI manufacturing facilities. The animal literature supports the human evidence and indicates that TDI-induced asthma is a threshold phenomenon. We conclude that the evidence does not indicate that the lower TDI TLVs will result in a lower incidence of respiratory effects, including OA. Our evaluation is applicable to the setting of OELs for TDI by other regulatory agencies worldwide.
Keywords: Toluene diisocyanate, Occupational exposure limits, Occupational asthma, Lung function
Antibody-Drug Conjugates: strategies, experiences and challenges from the non-clinical development to clinical development in cancer treatment in France (#361)
M. G. Burbank1, C. Menoret1, 2, S. Lopes1, V. Gazin1, L. Boudali1
1 French National Agency for Medicines and Health Products Safety (ANSM), Oncology division, Saint Denis, France
Antibody-drug conjugates or ADCs are an emerging class of cancer treatment agents that combines the selectivity of targeted treatment with the cytotoxic potency of chemotherapy drugs. Antibody conjugation to cytotoxic drugs appears to be a promising way to reduce the systemic toxicity of drugs by targeting them specifically to tumor tissue.
Recent efforts to improve the therapeutic index of ADCs for the next generation require the optimization of each component in combination with the others, resulting in a multitude of new technologies that could have an impact on the safety and pharmacokinetics of ADCs.
Since the first approval of MYLOTARG (Gemtuzumab ozogamicin) in 2000, ADCs market has evolved considerably and now comprises around 20% of the clinical pipeline of antibodies for cancer, but only 11% of all antibodies in clinical development (Reichert, 2016). To date, 3 ADCs have received market approval in Europe while there are approximately 200 ADCs in clinical/preclinical stages of development showing that the field is currently going through a gradual transition (Antibody Drug Conjugates Market; 4th Edition).
These molecules are taking a growing place within the oncology division of the ANSM and will represent in the future a major focus. We present here an inventory of the development (main indications, different stages of development, summary of assessment methods, …) of the various conjugated antibodies in France over the last years within applications for clinical trial authorization for the French territory submitted to the ANSM.
A synthesis of their main toxicities (ADCs and/or payloads) showing the different causes of rejection at the non-clinical level is presented and compared to clinical toxicity (hematology toxicity, immunogenicity, anti-drug antibodies,...). Current challenges include a careful selection of targets, a better understanding of ADCs mechanism of action, the management and understanding of ADC off-target toxicities, as well as the selection of appropriate clinical settings (patient selection, dosing regimen) where these molecules can bring highest clinical benefit.
One of the main issue for the French Medicines Agency as well as for the European Medicines Agency (EMA) would be the elaboration of new guidelines on these ADCs allowing a more precise development of these molecules located between biological and chemical entities in the treatment of both hematologic malignancies and solid tumors.
Keywords: Antibody-Drug Conjugates, oncology, ANSM, Non-clinical toxicities, immunogenicity, payload, ADA
The Validation of GARDskin (#659)
P. Sandberg1, M. Agemark1, A. Johansson1, R. Gradin1, A. Forreryd1, O. Larne1, A. Edwards2, V. Hoepflinger3, F. Burleson2, H. Gehrke3, E. Roggen1, H. Johansson1
1 SenzaGen, Lund, Sweden
The development and welfare of our society is dependent on chemicals, but exposure to certain chemicals, sensitizers, may induce allergic contact dermatitis (ACD). The disease has a prevalence of >20% in the western world and is associated with poor quality of life and subsequent socioeconomic effects. For safer products and occupations, regulatory authorities require chemicals to be safety tested. Traditional sensitization tests comprise animals, but recent regulations ban or advice non-animal testing. The Genomic Allergen Rapid Detection skin – GARDskin – is a state of the art in vitro assay for assessment of chemical sensitizers. To demonstrate the validity of the assay, we here present the results of the GARDskin ring trial (OECD TGP 4.106).
The initiation of skin sensitization in vivo includes activation of dendritic cells (DCs). The GARDskin assay relies on in vitro stimulation of SenzaCells; a myeloid cell line resembling human DCs. Following exposure of the cells to a test substance, chemicals are classified as either sensitizers or non-sensitizers based on the readout of a genomic biomarker signature comprising 200 genes involved in immunological pathways associated with skin sensitization.
For validation of GARDskin, three independent laboratories were selected: two external CROs and the in-house development laboratory. The structure of the study was designed to cover training and transferability of GARDskin to the two external test laboratories. The final study included assessment of reproducibility within and between the three laboratories (WLR and BLR respectively), as well as assessment of predictive performance.
In the transfer study, the two external test laboratories tested eleven chemicals by GARDskin in three consecutive experiments. All substances (11/11) were correct classified (100% accuracy), confirming the assay transferability. In the final validation study 28 coded chemicals were analyzed by all three laboratories. The WLR was calculated to 82-89% and the BLR to 92% (range 92-100%). Moreover, the test performance was excellent: accuracy 94%, sensitivity 93% and specificity 96%. The overall conclusions from the study was that GARD is easy to transfer to naïve laboratories, highly reproducible, and a powerful tool for the assessment of skin sensitizers.
Keywords: skin sensitization, GARD, in vitro, validation
Characterisation of uncertainties in chemical safety assessment: available guidance related to Integrated Approaches to Testing and Assessment (IATA) (#76)
A. Richarz1, S. K. Bopp1, R. Corvi1, A. P. Worth1
1 European Commission Joint Research Centre, Directorate for Health, Consumers and Reference Materials, Ispra, Italy
The characterisation and transparent documentation of any uncertainties encountered in risk assessment are essential for informed decisions and risk management measures. A scoping exercise was undertaken, anchored to the framework of Integrated Approaches to Testing and Assessment (IATA), to identify existing guidance and evaluate the inclusion of uncertainty assessment. IATA are composed of building blocks such as Test Guideline and non-standard studies, high throughput in vitro assays, omics and in silico data. The multiple data sources are integrated in a weight of evidence (WoE) to conclude on the chemical hazard/risk. Guidance documents were searched for and hierarchically grouped at different levels, following the knowledge pyramid, from cross-cutting issues concerning data and methodological quality to the different IATA components and WoE integration. The analysis included documents from regulatory sources to peer-reviewed literature, assessing in particular how uncertainty was considered and whether user-friendly templates were included. More guidance was available on basic aspects related to the input data and methods than for the integration. Basic steps towards standardisation and increasing of data quality facilitate the WoE and reduce overall uncertainties. They are therefore considered an important integral part of the process to improve confidence in safety assessment results. Another finding was that available guidance is fragmented and duplicated across sectors, scientific areas, countries and pieces of legislation. The provision of overarching advice on the availability and use of different guidance would be beneficial. As a first step, the creation of a one-stop inventory for reference will raise awareness of all existing guidance and aspects possibly having an impact, in order to facilitate and streamline safety assessment at a global level. This work is also contributing to a project under the OECD's Working Party on Hazard Assessment (WPHA).
Keywords: uncertainty assessment, safety assessment, guidance, Integrated Approaches to Testing and Assessment (IATA), data quality
Anticancer Effect of Arsenic Compounds on Apoptosis in Oral Cavity Cancer Cells (#140)
B. - M. HUANG1
1 National Cheng Kung University, Cell Biology and Anatomy, Tainan, Taiwan
Arsenic is a well-documented environmental toxicant. Epidemiological survey implicates that exposure to arsenic will induce neurotoxicity and peripheral vascular disease (known as blackfoot disease). However, arsenic trioxide (ATO) has also been used for medicinal purposes, originally to treat acute promyelocytic leukemia (APL), showing ability for anticancer treatment. Oral cancer has been in top 10 common cancers for decades in Taiwan, and the incidence rate still increases year after year. Around 75 percent of oral cancers are linked to modifiable behaviors, such as tobacco use and excessive alcohol consumption. Also, betel chewing in some certain areas, especially in Southeast Asia, is known to be a strong risk factor for developing oral cancers. Due to the high rate of occurrence and mortality, three oral squamous carcinoma cells (Fadu, OEC-M1, and OC3) treated by sodium arsenite (NaAsO2) and dimethylarsenic acid (DMA) were investigated to determine whether the arsenic compounds could be the anti-cancer agents. Results show that cells appeared rounded up and became membrane blebbing after treatments with NaAsO2 (1 μM ) and DMA (1 mM) for 24 hr in OEC-M1 and OC3 cell lines, and NaAsO2 (10 μM ) and DMA (5 mM) for 24 hr in Fadu cell line, respectively. These morphological changes revealed characteristics of apoptosis. In cell viability test, the surviving percentage of all three cell lines significantly decreased as the dosage of arsenic compounds increased (10 to 100 μM NaAsO2 and 1 to 100 mM DMA). The impact of arsenic compounds on cell cycle regulation was detected by flow cytometry. Results showed that the percentage of subG1 and G2/M phase cells among three cell lines increased in both NaAsO2 and DMA treatments. In addition, activation of the caspases, such as caspase-8, -9, and -3, and cleavage of poly ADP-ribose polymerase (PARP) were examined by western blot, and results showed that NaAsO2 and DMA activated caspase-8, -9, and -3 cleavages. Moreover, both arsenic compounds could activate JNK, ERK1/2, and p38 phosphorylation among these cell lines. Taken together, NaAsO2 and DMA could induce cell apoptosis through extrinsic and intrinsic apoptotic pathways and cause the activation of MAPK pathway in Fadu, OEC-M1, and OC3 oral cancer cell lines.
Keywords: Arsenic compounds, Anti-cancer, Apoptosis, Oral Cavity Cancer
Association between perfluoroalkyl substances in cord blood and birth weight in Belgian population. (#149)
P. Dufour1, C. Pirard1, M. - C. Seghaye2, C. Charlier1
1 Université de Liège, Service de Toxicologie clinique, médico-légale, de l'environnement et en entreprise, Liège, Belgium
Introduction: Perfluoroalkyl substances (PFAS) are man-made chemicals used in numerous industrial processes and characterized by very long half-lives in the environment. We highlighted PFAS contamination in cord blood samples collected in Belgium. In this population, we also found a negative correlation between PFNA concentration in cord blood and TSH level measured 3 days after delivery . Beside the TSH level, birth weight is also an important health parameter, since low birth weight (<2500 g) is related to several health adverse outcomes. Many studies explored the potential association between PFAS contamination in newborn and birth weight but the results are still inconsistent and the situation was never assessed in Belgium. The objective of the present work is thus to assess the potential correlation between PFAS levels measured in cord blood and birth weight in Belgium.
Materials and methods: Cord blood samples (n=281) were collected at the university hospital of Liege (Belgium) between August 2013 and March 2016. Levels of perfluoroalkyl substances were determined using LC-MS. Birth weight and additional data about newborns and their mothers (gestational age, mother age, pre-pregnancy BMI, tobacco habits, parity, and sex of the newborn) were collected using the medical records. Multivariate analyses were performed using R software to assess the association between birth weight and PFAS levels.
Results and discussion: No significant correlation was highlighted between birth weight and PFAS contamination in our population. Nevertheless, the concentration measured in our population are low (median PFOA concentration: 0.68 ng/mL and median PFOS concentration: 0.73 ng/mL) compared to populations where significant negative correlations were highlighted (e.g. in English newborns, median PFOA concentration: 3.7 ng/mL and median PFOS concentration: 19.6 ng/mL ).
Conclusion: Contrary to the results of some other studies, in our population, birth weight and PFAS levels were not significantly associated. This discrepancy may be due to the lower contamination of Belgian newborns.
 P. Dufour, C. Pirard, M.-Ch. Seghaye, C. Charlier, Environ. Pollut. 238C (2018) 389-396.
 M. Maisonet, M.L. Terrell, M.A. Mc Geehin, K.Y. Christensen, A. Holmes, A.M. Calafat, et al., Environ. Health Perspect. 120 (10) (2012) 1432–1437.
Keywords: birth weight, perfluoroalkyl substances
Aryl hydrocarbon Receptor (AhR) signaling in human primary trophoblast cells (#195)
R. El Dairi1, J. Rysä1, M. Pasanen1, P. Huuskonen1
1 University of Eastern Finland, Pharmacology and Toxicology, Kuopio, Finland
Introduction. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls a wide variety of developmental and physiological events. AhR is highly expressed in placenta and it upregulates the most important xenobiotic metabolizing enzyme of the placenta, cytochrome P450 1A1 (CYP1A1). Chemicals including pharmaceuticals and environmental contaminants binding to AhR in the placenta, may contribute to adverse effects on the fetal development, as well as fetal metabolism and reproductive system.
Methods. Human primary trophoblast cells were isolated from full term placenta after delivery. The trophoblasts were exposed to 25 µM of AhR agonist β-naphthoflavone (BNF) for 72 hours and gene expression profiling was done by using Illumina Human HT-12 expression beadchips. Differential expression of selected genes was confirmed with RT-qPCR. Ingenuity pathway analysis (IPA) was performed to identify BNF induced biological functions and downstream signaling pathways within the gene expression data.
Results. In response to BNF treatment, 68 genes were up regulated and 294 genes were down regulated as compared to control cells. The majority of the top upregulated genes were genes related to inflammatory response, polycyclic aromatic hydrocarbons (PAHs) and dioxin-related response, fatty acid, steroid and xenobiotic metabolism. Whereas, the majority of the top downregulated genes play a role in placental growth and development, regulating pregnancy-related hormones and metabolism, and modulating the activity and function of immune cells.
Conclusions. Our study indicates that AhR signaling in placenta is involved in regulation of various physiological processes beyond xenobiotic metabolism. Therefore, any disturbances of AhR signaling can have significant consequences on maintenance of pregnancy.
Keywords: AhR, BNF, placenta, DNA microarray, developmental toxicology.
Paracetamol (acetaminophen) may interfere with the human fetal ovary development in an ex vivo model. (#206)
L. L. Lecante1, S. Leverrier-Penna1, V. Lavoué2, A. D. Rolland1, S. Mazaud-Guittot1
1 Univ Rennes 1, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMR-S1085, Rennes, France
Purpose: Paracetamol (APAP i.e. acetaminophen), taken by more than half of pregnant women, is the most used over-the-counter analgesic along pregnancy. Although considered safe for long, a raising number of studies show that APAP intake may be associated with troubles in children exposed in utero and notably, a higher risk of male genital tract abnormalities (hypospadias, cryptorchidism). These data have been correlated with human studies showing that paracetamol may interfere with the male fetal hormones. On the female side, if rodent studies suggest that a fetal exposure to paracetamol may have long-term effects on the reproductive function, there is, up to now, no evidence of its effects on the human female gonad development. Provided that any disruption of the fetal ovarian development may impact woman reproductive health, we investigated whether in utero exposure to APAP interferes with the human fetal ovary development.
Methods: Human fetal ovaries collected from medical terminations of pregnancies aged from 7 to 12 developmental week (DW) were cultivated during 7 days with APAP (10-8 to 10-3 M). For each individual, a negative control was cultivated with the drug vehicle and compared with its treated counterpart(s).
Results: After 7 days of exposure, APAP reduced the total cell number in a non-monotonic manner only in 10-12 DW samples, but not in 7-9 DW ones. We therefore addressed the question whether it impacted apoptosis and/or cell proliferation. While APAP had no specific effect on the percentage of cell death by using flow cytometry, we showed by immunostaining of cleaved caspase 3, a trend to increase apoptosis in 7-9 DW samples. However, APAP induced no major alteration in the percentage of the subpopulation of M2A-positive germ cells, whatever the concentration and the studied age group. Altogether, our data indicate that APAP may impact the human fetal ovarian development during the first trimester by increasing apoptosis without decreasing specifically the M2A-positive germ cell subpopulation ratio. Nevertheless, crucial events for the ovarian pool establishment occurring after, further study is needed to assess its potential effects later in pregnancy.
Funded by Agence nationale de la recherche (# ANR-15-CE34-0001-01) and Agence nationale de sécurité du médicament (# 2014S032)
Keywords: human, fetal ovary, development, paracetamol
A new screening approach for the evaluation of retinoic acid receptor alpha (RARα) interaction of chemicals (#315)
B. Birk1, P. Demuth1, N. Roth1, K. Sessler1, H. - A. Hüner1, A. Verlohner1, B. Flick1, B. van Ravenzwaay1, R. Landsiedel1
1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Rhineland-Palatinate, Germany
Early and robust identification of reproductive toxicity properties of chemicals and their mode of action is crucial during the development of new products. It is known that inappropriate regulation of the retinoic acid receptor alpha (RARα) can lead to a range of adverse effects including teratogenicity and testicular toxicity and is a relevant mode of action (MoA) in the context of reproductive toxicity. The RARα is a nuclear receptor present in the cellular nucleus and bound to the retinoic acid response elements in front of target genes. A key step in the ligand induced transcription of those controlled genes is the recruitment of cofactors by RARα.
Two different methods (a cell based system and a micro array system) were tested to determine their strength and limitations to identify RARα-interaction (agonism, antagonism and non-response) of xenobiotics. Therefore, the cofactor binding patterns (tested by MARCoNI, PamStation®) for several known agonists (e.g. ATRA, AM580), antagonists (e.g. BMS195614, BMS493) and non-responder (e.g. Ketoconazole) were investigated and compared with results from reporter cell line test (HEK293), expressing RARα, to evaluate the applicability for toxicological assessment of xenobiotics.
The dose response curves (EC50) of peptides and cofactors, known as relevant for the RARα interaction, corresponded nicely to the dose response curve of the HEK293 cell line. In detail: after ATRA treatment 16 selected cofactors/45 peptide motifs revealed EC50 values between 6.6∙10-9 M and 1.0∙10-8 M and was comparable to EC50 value of 1.9⋅10-9M for ATRA in HEK293 cells.
All results nicely show that RARα interaction of xenobiotics can be demonstrated by above mentioned methods. While the HEK293 cell line is suitable to determine the agonistic and antagonistic property of a chemical in an easy and economical way, the experiments in the MARCoNI-system revealed the molecular initiating event by influencing corepressor or coactivator-binding. Both methods can compensate the limitations of each other and therefore the combination of both methods increases the value of each individual method. A smart testing strategy was implemented to investigate the RARα interaction including mechanistical understanding of chemicals in the screening phase but also for specific regulatory questions.
Keywords: RAR, MARCoNI, HEK293- cells, reprotoxicity, Mode of action
New approach for assessment of Wistar Hannover male rats reproductive toxicity (#337)
Y. Kolianchuk1, M. Prodanchuk1, N. Nedopytanska1, I. Rashkivska1, V. Bubalo1, T. Usenko1
1 L I. MEDVED’S RESEARCH CENTER OF PREVENTIVE TOXICOLOGY, FOOD AND CHEMICAL SAFETY OF THE MINISTRY OF HEALTH OF UKRAINE, Institute of Experimental Toxicology and Bio-medical Research, Kiev, Ukraine
Nowadays there are many schemes for pesticides reproductive toxicity assessment based on investigations after treatment phase of experiment. We provide standard reprotox research procedure of Lambda-cyhalothrin (LC), which is studied insufficiently, which was supplemented with extended studies after recovery phase. 40 Wistar Han males were treated by gavage for 11 weeks with generic LC 98.06 % in doses 0; 0.3; 3 and 10 mg/kg/bw/day. After exposure period males were recovered for one full cycle of spermatogenesis (70 days). Reproductive parameters (RP): testicular, epididymis weight, testes and epididymis coefficients of relative weight, total amount of sperm, sperm motility, percentage of abnormal forms of germ cells were studied. RP of intact females, who mated with experimental males were taken into account. Hematological and cytochemical parameters (SDH, APH), hemogram with morphological disturbances of blood cells were evaluated. All abovementioned indicators were determined after exposure (E) and recovery (R) period. Significant decrease of sperm quality parameters after E was noted. This changes had been saved and intensified to R. Tendencies to decrease of HGB and RBC were observed. SDH activity significantly decline in directly proportional dependence to gonadotoxicity signs after E and normalize after R in all dose, despite of 10 mg/kg, where it was compensatory increased. Based on the results we conclude that LC cause adverse effects on reproductive male function. Hematological alterations were noted. Determined WBC cytochemical status corelate with morpho-cellular functions of gonads and can predict the probable pathological changes in reproductive system of Wistar Han males.
Keywords: cytochemical, peripheral blood, gonadotoxicity, reproductive, pesticides
MINERAL MIX DIET COMPOSITION FOR REPROTOXICOLOGICAL EXPERIMENTS IN VIVO: LITHIUM SALT (#349)
N. V. Tyshko1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The optimized formula of semisynthetic casein diet (SCD), similar to AIN-93 in the composition of vitamin-mineral complex, was used in a numerous reprotoxicological experiments on rats. The experience of the SCD usage revealed that reproductive function of rats showed the decrease in mating efficiency from 75-85% to 55-65%. As a result of conducted comparative analysis of vitamin-mineral mix the hypothesis about the leading role of lithium salts for formation of detected changes was put forward.
Lithium salts are characterized by a wide range of physiological action, particularly, lithium is known to be sedative medication. Taking into consideration the properties of lithium salts, necessity of their inclusion into the laboratory animals diet under conditions of long-term experiments seems doubtful.
The 120-days experiment was performed on Wistar rats with the initial body weight of 90-110 g. The animals were randomly divided into 2 groups (20 males and 20 females in each group, accordingly): the rats in control group were exposed to SCD, the rats in the test group were exposed to Lithium chloride free SCD.
Research of female fertility in control and test groups showed the absence of significant differences: out of 20 co-housed females in each group, pregnancies and deliveries were registered for 9 females in control and 11 females of test group, accordingly. Within the framework of the experiment influence of lithium salt in the diet composition on female fertility was not concerned, however, later we compared the fertility of control group rats which were used in eight different experiments; in four experiments the animals received the diets with the inclusion of lithium salt, in the other four experiments the lithium-free salt mix was used. It was revealed that under the conditions of the lithium salt intake, 194 of 264 co-housed females were impregnated (74% fertility), and the rate of potential fertility was 83% (parameter includes females co-housed with a sterile male (194+26); whereas under the conditions of Lithium-free diets 221 of 256 co-housed females were impregnated (86% fertility) and the rate of potential fertility was 90% (221+8).
The data indicate a stable trend in fertility increase of rats on Lithium-free diets.
This work was supported by Russian Science Foundation grant No. 16-16-00124.
Keywords: laboratory animals, fertility, lithium salt, mineral mix composition
Effects of silver and gold nanoparticles on ovarian granulosa cell viability and steroidogenesis (#483)
S. Scsukova1, A. Bujnakova Mlynarcikova1, A. Sirotkin2, 3
1 Biomedical Research Center Slovak Academy of Sciences, Institute of Experimental Endocrinology, Bratislava, Slovakia
Despite a great potential benefit of silver (Ag) and gold nanoparticles (Au NPs) in the areas of biomedical and industrial applications, there has been an increased interest in studying their possible deleterious effects in biological systems. Current data have suggested that NPs may pose risks to female reproductive health by inducing cytotoxic effects on ovarian cells, impairing follicle maturation, and altering sex hormone levels. Ovarian granulosa cells (GCs) play a major role in maintaining ovarian function, health, and female fertility. In the present study, we investigated in vitro dose-dependent and time-course effects of Ag and Au NPs with different size on viability and steroidogenic activity of ovarian GCs.
The immortalized GC line COV434 and primary culture of GCs isolated from porcine ovarian follicles (3–5 mm in diameter) were cultured with Ag NPs (10, 20, 100 nm; 0.1–10 µg/ml) or Au NPs (20, 100 nm; 0.3–33x1010 and 0.2–19x108 particles/ml, respectively) in the absence or presence of follicle-stimulating hormone (FSH, 100 ng/ml) for 24, 48, and 72 h. Cell viability was assessed by MTT and CytoTox-ONE Homogenous Membrane Integrity (LDH) assays. Progesterone levels in culture media were measured by radioimmunoassay commercial kits.
Treatment of COV434 cells and porcine GCs with Ag and Au NPs induced a significant concentration- and time-dependent inhibition of cell viability. The smaller NPs showed more deleterious effects. Exposure of porcine GCs to Ag and Au NPs at all sizes significantly decreased, in a dose-dependent manner, basal as well as FSH-stimulated progesterone secretion by GCs after 48 and 72 h of culture.
The obtained results indicate that disruption of gonadal cell functional state via NPs may affect steroidogenic output and thus perturb mammalian reproductive function. Possible sites of NPs action on the steroidogenic pathway should be further elucidated.
This work was supported by the Slovak Research and Development Agency under the contract No APVV-15-0296 (acronym ENDONANOSAFE) and VEGA Grant 2/0187/17.
Keywords: nanoparticles, ovary, granulosa cells, cell viability, progesterone
Coenzyme Q10 supplementation rescues infertility and reproductive outcomes following exposure to the endocrine disruptor bisphenol A in Caenorhabditis elegans (#525)
M. F. Hornos Carneiro1, 2, N. Shin1, M. P. Colaiacovo1
1 Harvard Medical School, Department of Genetics, Boston, Massachusetts, United States of America
Given the widespread prevalence of endocrine disruptors in our environment, it is critically important to understand their impact on reproductive health and to identify ways to mitigate their effects and improve fertility and early embryogenesis outcomes. Here, utilizing advantageous features of Caenorhabditis elegans as a model for germline studies and the conservation of the CoQ pathway, we show that the natural supplement CoQ10, an antioxidant, rescues the increased chromosome nondisjunction and meiotic defects resulting from exposure to the endocrine disruptor bisphenol A (BPA). CoQ10 rescues sterility (decreased brood size), embryonic lethality and larval lethality and high-resolution microscopy revealed that worms treated with CoQ10 showed a significant decrease in the levels of chromosome defects in oocytes at diakinesis (worms were exposed to different doses of BPA starting as L1 larvae and were supplemented with 100 µg/ml of CoQ10 starting as young adults when the gonads are fully formed). CoQ10 treatment also decreased the elevated levels of germ cell apoptosis and pCHK-1, a protein involved in activation of the DNA damage checkpoint, seen in BPA-only treated worms. In addition, impaired DSB repair, as revealed by elevated levels of foci for RAD-51, a protein involved in strand-exchange during DNA double-strand break (DSB) repair following BPA exposure, were significantly decreased following CoQ10 supplementation. Finally, use of a reporter strain carrying GFP expressed under the control of the glutathione-requiring prostaglandin D synthase (gst-4) promoter showed significantly less GFP signal in gonads of CoQ10-treated worms suggesting that BPA exposure generates oxidative stress in the germ cells which is neutralized by CoQ10 supplementation. Our study shows that supplementation with CoQ10 can serve as a low-risk and low-cost intervention for significant rescue of infertility and reproductive outcomes following exposure to BPA.
Keywords: Bisphenol A, Coenzyme Q10, infertility, reprotoxicity
Possible role of n-hexane as endocrine disruptor in occupationally exposed women at reproductive age (#527)
L. RUIZ-GARCÍA1, J. M. MALACARA-HERNÁNDEZ1, N. FIGUEROA-VEGA1, G. B. BARTOLOUCCI2, M. CARRIERI2, F. SALAMON2, O. JIMÉNEZ-GARZA1
1 UNIVERSIDAD DE GUANAJUATO, DIVISIÓN CIENCIAS DE LA SALUD, LEON, GUANAJUATO, Mexico
Introduction. Several studies in experimental animals and cellular lines have associated n-hexane and its metabolite 2,5-hexanodione exposure (2,5-HD), as an ovarian toxic and therefore as hazardous for fertility. In humans, the evidence with women occupationally exposed to n-hexane has been reported as subfertility:affecting time for getting pregnant and menstrual cycle length. The previous reports did not specify solvents individual exposure levels for n-hexane.
Methods. We studied a group of Mexican women laboring in a shoe Factory (n=34). Individual environmental levels for seven volatile organic compounds, included n-hexane, were measured. Also, urinary 2,5HD and FSH and anti-Müllerian hormone (AMH) serum concentrations as potential biomarkers of ovarian toxicity, in addition to a gyneco-obstetric history were obtained, reproductive history was asked. We performed all tests and questionnaires in a reference group as well (n=32).
Results. Mean exposure levels to n-hexane (49.2 ± 39.6 mg/m3) and toluene (30.8 ± 24.5 mg/m3) were the highest observed. There were no significant differences in serum FSH and AMH concentrations between groups (p >0.05). Exposed group showed prolonged menstrual cycles (p= 0.007, OR 1.26,95% CI=1.07-1.50) and longer time for getting pregnant compared with controls (p= 0.015, OR 6.25, 95% CI=1.24-31.2).Also in the exposed group, significant correlations were observed between FSH serum levels and n-hexane (r = -0.34, p= 0.028) as well as FSH and 2,5HD urinary levels(r=-0.33, p= 0.029).
Conclusions. The results of this study suggest that n-hexane contained in a mixture of solvents could behave as an endocrine disruptor and therefore interfere in the menstrual cycle and in the fertility of women occupationally exposed to this solvent.
Keywords: n-hexane, endocrine disruptor, menstrual disorders
ReproTracker, a Human Stem Cell-Based Reporter Assay for In Vitro DART Assessment (#549)
P. Racz1, I. Brandsma1, T. Zwetsloot1, G. Hendriks1
1 Toxys BV., Leiden, Netherlands
Testing for developmental and reproductive toxicology (DART) is a crucial part of the toxicological risk assessment. Today, DART mostly relies on animal testing although alternative in vitro tests such as embryonic stem cells based assays are increasingly being used. However, these in vitro assay often lack mechanistic insight and are difficult to translate to human risk due to inter-species differences.
To improve in vitro identification of developmental toxicants, we developed a human induced pluripotent stem cell (hiPSC) based reporter system. To quantitatively assess the major developmental processes, we identified 43 potential biomarkers marking different developmental stages from stem cells to mature tissues. To test whether compounds affect development, we optimised the differentiation protocols for hiPSC towards cardiomyocytes and hepatocytes and confirmed expression of selected biomarkers (OCT4, BMP4, MYH6, FOXA2, SOX17, AFP, ALB) by qPCR. During differentiation, expression of the stem cell marker OCT4 decreased, while expression increased for MYH6 in cardiomyocytes or ALB and AFP in hepatocytes. Next, we exposed the cells to two established teratogens: 5-fluorouracil and thalidomide during cardiomyocyte and hepatocyte differentiation and observed downregulation of the cardiomyocyte-specific biomarker MYH6 and hepatocyte-specific markers ALB and AFP. This results indicates the disruption of the developmental program and confirms the teratogenic potential of the compounds. We are currently validating our assay using a set of known teratogenic compounds including the ECVAM compound library.
In conclusion, following the differentiation program by reporter genes is a suitable tool for high throughput, quantitative measurement of differentiation for DART screening.
Keywords: ReproTracker, Development, Teratogens, Stem cells, Biomarkers
Prenatal administration of SMe1EC2 changed anxiety- and depression-like behavior with changes in the brain oxytocin expression of adult offspring (#734)
P. Stefanik2, L. Olexova2, Z. Dzirbikova2, E. Ujhazy1, M. Mach1
1 Centre of Experimental Medicine SAS, Institute of Experimental Pharmacology and Toxicology, Bratislava, Slovakia
SMe1EC2 is a pyridoindole derivative with low toxicity and potentially anxiolytic effect. SMe1EC2 was orally administered daily to the pregnant Wistar rats from 15thday of gestation to the 10thday of postpartum in three doses: 5mg/kg (dose1), 50mg/kg (dose2) and 250mg/kg (dose3). Adult offspring underwent behavioral tests – elevated plus maze (EPM) for assessment of anxiety-like behavior at age 55 days, and forced swim test (FST) for assessment of depression-like behaviour at age 90 days. Oxytocin (OT) mRNA levels were measured in the supraoptic (SON) and paraventricular nucleus (PVN) of hypothalamus. We found the highest effect of SMe1EC2 in dose2 group in both behavioral tests. In the EPM dose2 spent significantly more time in the open arms. In the FST dose2 group exhibited higher immobility duration and higher number of immobility episodes in comparison with all other groups. In situ hybridization revealed significantly increased OT expression in the PVN of dose1 group compared to control group and decreased OT expression in the SON of dose1 and dose2 males in comparison with any other males and females groups. Our results indicate that administration of SMe1EC2 during pregnancy and early postnatal period had effect on anxiety-like and depression-like behaviour and expression of OT in the offspring in adulthood.
Keywords: SMe1EC2, prenatal treatment, anxiety, depression, behavior, oxytocin
Electrophysiological evaluation of developmental neurotoxicity induced by the prenatal exposure to 1-bromopropane (#271)
Y. Fueta1, T. Ishidao1, S. Yoshida2, H. Hori1, D. Yamasaki3, Y. Kanda3, S. Ueno4
1 University of Occupational and Environmental Health, Department of Environmental Management and Control, Kitakyushu, Japan
Purpose: 1-Bromopropane (1-BP) is an organic solvent mainly used as a degreasing agent and spray adhesive, and has been reported to exhibit reproductive and neuronal toxicity in human and animal studies. However, the mechanism underlying developmental neurotoxicity (DNT) is not known. Recently, we reported that prenatal exposure to 1-BP induced delayed DNT in rats after growth (JOH, 2018). We aimed to investigate whether DNT caused by exposure to 1-BP can be evaluated during the juvenile period before adulthood using the electrophysiological approach. Methods: Pregnant Wistar rats were exposed to 1-BP vapour at a concentration of 400 or 700 ppm for 20 days during the gestational period (1–20 days). Fresh air was provided to the control pregnant rats. Hippocampal slices were prepared from postnatal days (PNDs) 13–15 juvenile rats belonging to the 1-BP and control groups. The integration of synaptic excitation potentials to generate an action potential, referred to as excitatory postsynaptic potential-spike coupling (E-S coupling), determines the neuronal output. We, then, analysed the stimulation/response (S/R) association of the slope of the field excitatory postsynaptic potential (fEPSP) and the amplitude of population spike (PS) evoked in the CA1 area. Data of E-S coupling obtained from each individual slice were fitted to a logistic curve using its maximal PS value. We compared two parameters, namely, the fEPSP slope value at half maximal PS (Eslope50) and the slope factor at the midpoint (Hillslope), among the experimental groups. Results and discussion: The day of eye-opening was PND 15–16 among the groups. The enhancement in the association of S/R with PS and fEPSP was observed on PND15 in the control group and on PND14 in the 1-BP groups. In addition, the maximal values of PS and the Eslope50, and the Hillslope values obtained from the 1-BP groups were significantly higher than those from the control group on PND14. There was no difference in the values of the parameters between the 400 ppm and 700 ppm 1-BP groups. Conclusions: Our results suggested that prenatal 1-BP exposure might transiently increase the neuronal activity before eye-opening, which may lead to potential DNT. Our electrophysiological analysis of E-S coupling may serve as a useful evaluation index for DNT in juvenile rats.
Keywords: developmental neurotoxicity, 1-bromopropane, E-S coupling
Six-Month Systems Toxicology Inhalation/Cessation Study in ApoE−/− Mice to Investigate Cardiovascular and Respiratory Exposure Effects of Two Reduced Risk Products Compared with Conventional Cigarettes (#150)
J. G. Szostak1, B. Phillips2, G. Emmanuel1, E. T. Wong2, B. Titz1, F. Martin1, G. Vuillaume1, P. Leroy1, A. Buettner3, P. Vanscheeuwijck1, M. Peitsch1, J. Hoeng1
1 PMI R&D, Philip Morris Products S.A.,, PMI Science and Innovation, Neuchatel, Neuchâtel, Switzerland
Cigarette smoking causes adverse health effects that may occur shortly after smoking initiation and lead to the development of cardiovascular disease, respiratory disease (chronic obstructive pulmonary disease), and various cancers. To reduce the risk of smoking-related diseases, Philip Morris International is developing Reduced Risk Products (RRP) to which adult smokers can switch instead of continuing to smoke cigarettes.
Engaging a systems toxicology approach combining physiological, histological, and omics endpoints, the effects of a six-month exposure to cigarette smoke (CS) or to aerosols from two RRPs, the Carbon Heated Tobacco Product (CHTP) and the Tobacco Heating System (THS), were investigated in ApoE-/- mice. In addition, the impact of cessation or switching to CHTP aerosol exposure after three months of CS exposure was evaluated.
Our results demonstrated that exposure to CS at a concentration of 28 µg nicotine/L causes adverse effects on the lungs, including increased lung volume, lung inflammation, aortic plaque formation, and a dysregulation of the heart and lung transcriptome. In contrast, exposure to either THS or CHTP aerosol at matched nicotine concentrations did not induce lung inflammation or enhance plaque development. Cessation or switching to CHTP aerosol exposure reversed lung inflammatory responses and halted progression of aortic plaques. Transcriptomics analysis revealed that multiple biological pathways were impacted significantly in heart and lung tissue by CS exposure but not by exposure to CHTP or THS aerosols. Both cessation and switching to CHTP aerosol reduced these perturbations to levels similar to those in sham-exposed animals.
In conclusion, in this ApoE-/- mouse study, exposure to aerosol from either THS or CHT had minimal adverse respiratory and cardiovascular effects. In addition, cessation or switching to CHTP aerosol exposure delayed the progression of CS-induced atherosclerotic and lung emphysematous changes.
Keywords: Cigarette smoking, adverse effects, transcriptomics, cardiovascular, respiratory, histopathology
Sharing in the INTERVALS platform study details and data from the assessment of the impact of aerosol from the carbon-heated tobacco product (CHTP) 1.2, a potential modified risk tobacco product, on human organotypic gingival cultures. (#230)
S. Boue1, F. Zanetti1, J. Hoeng1, M. C. Peitsch1
1 Philip Morris Products S.A., Biomedical Research, Neuchatel, Switzerland
The U.S. FDA defines modified risk tobacco products (MRTP) as any tobacco product sold or distributed to reduce harm or the risk of tobacco-related disease associated with commercially marketed tobacco products. Establishing a product’s potential as an MRTP requires scientific substantiation, including toxicity studies and measures of disease risk relative to that of cigarette smoking.
As part of a 21st-century toxicology assessment framework, human gingival epithelia organotypic cultures were exposed repeatedly to nicotine-matching concentrations of Carbon-Heated Tobacco Product (CHTP) 1.2 aerosol and 3R4F cigarette smoke (CS), as well as a non-diluted (100%) CHTP 1.2 aerosol, and subsequently characterized. The results demonstrated the absence of cytotoxicity and reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP 1.2 aerosol, as compared with 3R4F CS. To study the molecular changes in the epithelia further, mRNA and miRNA profiles were evaluated.
All relevant data files from this assessment study were deposited on INTERVALS (www.intervals.science), an online platform developed by PMI to enable independent, third-party collaboration and data analysis by sharing protocols, tools, and data from assessment studies proactively. Data files are accompanied by the relevant information to foster reproducible research and by high-level summaries of obtained results. As the platform also hosts protocols and results from in vivo inhalation studies and in vitro studies describing the chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol from this and other candidate and potential MRTPs, it can serve as an invaluable source of scientific assessment data. Strengthened by community and peer-review features, INTERVALS aims to enable the necessary dialogue between industry, independent reviewers, the public health community, and regulatory agencies that can validate the harm reduction potential of these products.
PMI is inviting other institutions producing relevant data on potential MRTPs (from aerosol characterization to molecular data) to contribute to this infrastructure to facilitate scientific exchange and data reusability in the context of tobacco harm reduction.
Keywords: Transparency, tobacco harm reduction, in vitro assessment
Towards the endocrine disruptive potential of Alternaria mycotoxins in complex mixtures (#539)
D. Marko1, L. Dellafiora2, C. Dall´Asta2
1 University of Vienna, Department of Food Chemistry and Toxicology, Vienna, Wien, Austria
Alternaria alternata are black molds reported to grow on a wide variety of food and feed items, known to produce more than 120 secondary metabolites. Early studies indicated that alternariol (AOH), one of the most studied Alternaria toxins, might possess estrogenic activity. However, due to the apparent low estrogenic potency this observation was not followed with high priority at that time. The advances in mycotoxin analysis in the last decade demonstrated that contaminated food and feed might contain not only single mycotoxins but a spectrum of secondary fungal metabolites.
Scope: In the light of the potential occurrence of Alternaria toxins in complex mixtures we addressed the questions:
Methods: The estrogenic activity of potential combinations were studied in Ishikawa cells, an endometrial carcinoma cell line, expressing both isoforms of the estrogen receptor. An estrogenic stimulus activates the expression of alkaline phosphatase. We identified AME as well as phase I metabolites of AOH as estrogenic activators in this test system. Moreover, the presence of nanomolar concentrations of AOH were sufficient to enhance the estrogenic impact of the known mycoestrogens (ZEN, α-ZEL). Over a broad range of concentrations and ratios, synergistic effects were observed. An in silico/in vitro workflow based on ligand-based virtual screening and structure-based modeling using AOH as a scaffold identified the ERs as well as 17-β-hydroxysteroid dehydrogenase (17βHSD), an enzyme involved in estrogens production. The close analysis of the binding pose in comparison to those reported by crystallographic studies of polyphenolic inhibitors revealed a strong fitting into the binding pocket.
Results: In addition to AOH, further metabolites with estrogenic potential might have to be considered. Also acting as a weak estrogenic single compound, AOH shows substantial synergistic effects in mixtures with other xenoestrogens. In addition to binding to the ER also further impact on key elements in estrogenic pathways might be of relevance.
Keywords: Alternaria alternata, alternariol, combinatory effects, mycoestrogen, zearalenone
Effect assessment of target tissue doses of cadmium and decabrominated diphenyl ether on GSH level in kidneys (#589)
M. Curcic1, A. Buha Đorđević1, V. Milovanović1, S. Janković2, S. Vučinić3, E. Antonijević1, D. Đukić-Ćosić1, Z. Bulat1, V. Matović1, B. Antonijević1
1 University of Belgrade - Faculty of Pharmacy, Department of Toxicology, Belgrade, Serbia
The objective of this study was to assess effect of target tissue doses of cadmium (Cd) and decabrominated diphenyl ether (BDE-209) on GSH kidney levels. Effects were examined on male Wistar rats, weighing 200-230 g, exposed to dose of 2.5, 7.5 or 15 mg Cd/kg/day, to doses of 1000, 2000 or 4000 BDE-209/kg/day, and all nine combinations, by gavages, during 28 days. Experimental protocol was approved by the Ethics Committee of the Military Medical Academy, No. 9667-1/2011. Concentrations of Cd in kidney were measured by atomic absorption spectrophotometry after mineralization with 8 ml of nitric acid in microwave oven. Standard Reference Material 1577c-Bovine liver, NIST, NY, SAD was used for quality control. Concentration of BDE-209 was determined in kidney by GC/ECD method after tissue homogenates preparation using QuEChERS. For quality control CIL-EDF-2524 Clean Fish (slurry) - Organic contaminants (LG standards) was used. Slopes and steepness of dose response curves were calculated using PROAST software. Concentrations of Cd in kidneys were 5, 20 and 30 mg/kg of kidneys, respectively corresponding to applied doses. Slopes of effect assessment curves for mixtures were significantly different when compared to the slope of Cd tissue dose–response curve (Cd increases GSH level). Similarly, curves derived for mixtures differed with the dose–response curve obtained for BDE-209 only. BDE-209 concentrations were 0.2, 0.5 and 0.9 mg/kg, respectively and also increased GSH level. Multiple factorial regression analysis confirmed significant positive influence of BDE-209 on Cd absorption rate in kidney and vice versa positive influence of Cd on BDE-209 kidney concentration. These findings confirm interactions between Cd and BDE-209 at the level of gastrointestinal or renal absorption but also at the level of GSH content. Concentrations of both pollutants in target tissue can be proposed as better parameter for effect assessment interpretation than doses used in experiment. [Project III 46009].
Keywords: Cd, BDE-209, mixture, target tissue dose
Preclinical safety assessment of aqueous medicinal plant extracts containing thujone (#603)
C. Turek1, P. Molnar-Pfitzner1, N. Mörbt1, M. B. Müller1, P. Vögele1, F. C. Stintzing1
1 WALA Heilmittel GmbH, Bad Boll, Germany
Thujone, a mixture of the monoterpenoid α- and β-isomers, occurs widely in plants. Amongst them, sage (Salvia officinalis L.) and wormwood (Artemisia absinthium L.) are commonly used for medicinal and alimentary purposes [1,2]. A neurotoxic potential has been ascribed to thujone interacting with GABAA receptors . Therefore, derived from a No Observed Effect Level (NOEL) for epileptic seizures, an Acceptable Daily Intake (ADI) of 7 mg/kg has been established in the food sector as an amount which can be ingested over a lifetime without an appreciable health risk. In 2012 and confirmed in 2017 [e.g. 4], the Committee on Herbal Medicinal Products of the European Medicines Agency proposed to reduce this highest acceptable amount by the possible dietary intake, estimated at up to 1 mg/day, to derive 6 mg as limit value for daily thujone exposure from herbal medicine .
To evaluate the possible thujone burden by medicines containing aqueous plant preparations, extracts produced according to pharmaceutical standards were analysed in the present study for their total thujone contents. Risk assessment of herbal medicines containing those extracts was performed by comparison with officially accepted limit values. To complete their safety profiles, whole aqueous extracts were additionally evaluated in bacterial reverse mutation assays following OECD standards .
Results show that thujone contents in aqueous plant extracts do not pose any special concern and can be well monitored in the final products. Furthermore, no evidence for genotoxic potential of the plant extracts was revealed. Therefore, a good safety profile could be derived for the herbal medicinal products evaluated. Data presented contribute to broadening the knowledge of the preclinical safety of aqueous plant extracts containing thujone and preparations thereof.
 Pelkonen, O. et al. 2013. Regul. Toxicol. Pharmacol. 65, 100-7
Keywords: in vitro, mutagenicity, herbal, aqueous extract, thujone
The in vitro biological assessment of the stability of cigarette smoke aqueous extracts (#624)
M. Taylor1, S. Bozhilova1, A. Baxter1, A. Terry1, D. Thorne1, O. Oke1, M. Gaca1
1 British American Tobacco, R&D, southampton, United Kingdom
The generation of cigarette smoke aqueous aerosol extracts (AqE) has been widely used for assessing tobacco products, particularly with in vitro cardiovascular disease models, oxidative stress and inflammation. Little is known of the long-term stability of AqE, leading researchers to favour the generation of fresh extracts which can be time consuming and result in batch-to-batch variability. This could be minimised if large batches of AqE could be generated and stored for later use.
Here we describe a study using both chemical and biological measurements to assess the stability of cigarette smoke AqE at various timepoints during a 2 month period.
AqE from a scientific reference cigarette (3R4F) was generated on a Borgwaldt-KC RM20H using the Health Canada Intense (HCI) regime, bubbling into 20mL of cell culture media. Twenty batches of AqE were generated and pooled, aliquoted and stored at -80oC.
Nicotine levels were quantified in AqE by GC/MS and optical density at OD320nm and OD260nm were utilised as indicators of tar and nicotine levels, respectively.
Biological measurement included cell viability and quantification of intracellular glutathione ratio in human lung epithelial cells (NCI-H292), and an assessment of the inhibition of human umbilical vein endothelial cell (HUVEC) migration rates using a wound healing assay.
Exposure to freshly generated (100%) AqE reduced H292 viability to 28+1%, and lowered the glutathione ratio to 2+1% of negative control. Exposure to freshly generated diluted (35%) AqE reduced HUVEC viability to 68+4% and reduced migration rate to 9+3% of negative control. No significant change in biological responses were observed between fresh 3R4F AqE and after 8 weeks of -80oC storage (unpaired t-test for glutathione ratio assay (p=0.80), cell viability assay (p=0.70), wound healing assay (p=0.99)). Neither were there any significant changes in the AqE physical measurements. Freshly generated AqE measured 8.8 +1.4µg nicotine, 0.59 OD320nm and 2.34 OD260nm. After 8-weeks of -80oC storage these values were 8.1+1.4 nicotine, 0.58 OD320nm and 2.29 OD260nm.
In conclusion, AqE generated using our protocol was stable for a period of 8 weeks for the tested endpoints; we are currently exploring longer storage times. Therefore, it may not be necessary to use freshly generated AqE for each study.
Keywords: in vitro, cigarette smoke, endothelial migration, glutathione, shelf life
Toxicity of DEP and metal oxide nanoparticles mixtures in human lung cells (#654)
A. Zerboni1, P. Mantecca1
1 University of Milano Bicocca, POLARIS Research Centre, Dept. of Earth and Environmental Sciences, Milan, Italy
Airborne ultrafine particles in urban areas and workplaces mainly derive from combustion sources (e.g. incomplete diesel fuel combustion, diesel exhaust particles, DEP), abrasion sources (non-exhaust particles, e.g. brake and tire wear particles), or from the unintentional release of engineered nanoparticles (NPs). Metal and metal oxide are among the most relevant NPs emitted by the above mentioned processes, since they are used as fuel additives, are produced by brakes and tires wearing and are among the most globally produced nanomaterials (NMs). The aim of this study was to analyze the combined in vitro effects of DEP and metal oxide NPs (ZnO, CuO) on human lung cells. The human alveolar A549 cells were exposed for 24h to mixtures of standard DEP (NIST 2975) at the subtoxic concentration of 100 μg/ml and increasing concentrations of ZnO and CuO (10, 15, 20, 25 μg/ml). In parallel cells were treated only with the NPs at the same concentrations used in the mixture. After exposure, MTT test and clonogenic assay were used to assess the cytotoxicity. MTT test showed a concentration-dependent decrease in cell viability after exposure to both DEP-ZnO and DEP-CuO mixtures, although ZnO and CuO NPs themselves resulted more cytotoxic than the respective mixtures with DEP. On the contrary, no significant effects were obtained from the clonogenic assay, in term of number of colonies formed after treatment with mixtures, in comparison with the negative control. Additional microscopic analyses are taking place to establish possible NP interference with cells and colonies at morphological level (e.g. cytoskeleton organization, adhesion and migration behavior). These preliminary data suggest that the presence of DEP may introduce new physico-chemical interactions able to reduce the cytotoxicity of ZnO and CuO NPs, likely interfering with their bio-availability and/or bio-reactivity. In conclusion, in the light of providing experimental results reflecting more strictly the environmental conditions, the possible synergistic or antagonistic effects deriving from the mixture of different hazardous airborne particles should be considered, as well as the opportunity to integrate the results with additional toxicity assays and exposure methods.
Acknowledgements: EU Horizon 2020 project PROTECT (grant agreement No 720851)
Keywords: mixture toxicity, Diesel exhaust particles, metal oxide nanoparticles, lung cells
Toxicogenomics analysis of phthalates and bisphenol A mixture: obesity and comorbidities (#675)
K. V. Baralić1, D. Jorgovanović1, V. Matović1, B. Antonijević1, Z. Bulat1, M. Ćurčić1, E. Antonijević1, D. Đukić-Ćosić1
1 University of Belgrade, Faculty of Pharmacy, Belgrade, Serbia
Phthalates and bisphenol A (BPA), to which people are simultaneously exposed as a result of their use in a variety of consumer products, are in the class of the most well-known obesogens. Among the phthalates, diethylhexyl phthalate (DEHP), and dibutyl phthalate (DBP) are considered to have the greatest impact on the development of metabolic disorders in humans.
The aim of this study was to explore the relationship between the exposure to the mixture of BPA, DEHP, and DBP and obesity, as well as its comorbid conditions, using the toxicogenomics approach.
The Comparative Toxicogenomics Database (CTD; http://ctd.mdibl.org), along with its different tools, was used to identify the genes which are affected by these substances and connected to obesity and its comorbidities. GeneMania predictive server (http://www.genemania.org) was used to explore the interaction between the identified genes common to all three substances.
The results obtained by Set Analyzer CTD tool indicate that DEHP, DBP and BPA correlate with the development of obesity - by interacting with 60, 84 and 172 genes, respectively. MyVenn CTD tool identified 43 genes affected by all three substances. Further analysis of these 43 common genes by GeneMania predictive server revealed that more than half of them (52.52%) were in co-expression. Additional Batch Query CTD analysis of 43 common genes revealed that these genes were involved in other comorbidities of obesity - neoplasms (35 genes), cardiovascular diseases (29 genes), endocrine system diseases (26 genes), diabetes mellitus (17 genes), insulin resistance (13), etc. All three substances bound to and resulted in the increased activity of the two genes involved in all investigated obesity comorbidities - PPARA, and PPARG, key factors of lipid metabolism. Furthermore, all three substances interacted with INS gene, implicated in the pathology of insulin resistance and diabetes mellitus.
These results provide a basis for further in vitro and in vivo investigation of molecular mechanisms of obesogenic properties of investigated mixture of chemicals.
Keywords: phthalates, bisphenol A, mixture, toxicogenomics, obesity and comorbidities
A comparative high-content screening-based assessment of e-cigarette liquids in primary bronchial epithelial cells (#712)
D. Marescotti1, M. Taylor2, I. Gonzalez-Suarez1, S. Acali1, P. Walker3, V. Belcastro1, F. Martin1, S. Frentzel1, D. Thorne2, M. Peitsch1, C. Proctor2, M. Gaca2, J. Hoeng1
1 Philip Morris International, Neuchatel, Neuchâtel, Switzerland
In 2009, the U.S. National Research Council’s Toxicity Texting in the 21st Century outlined approaches using human-based in vitro systems and computational and molecular biology to support chemical safety assessment. This facilitated a paradigm shift from traditional in vivo toxicology approaches towards the greater use of high-throughput in vitro screening technologies. High-content screening (HCS) approaches employ cellular systems assessing multiple biological endpoints in parallel and are used routinely for the rapid screening of drug candidates and chemical safety assessment. Recently, HCS has been used to assess novel next-generation nicotine and tobacco products. E-cigarette use has increased globally and could potentially offer a lower-risk alternative to cigarette smoking. E-liquids typically comprise a mix of propylene glycol (PG), vegetable glycerin (VG), flavorings, and nicotine. Given the constant evolution of the category and the growing number of e-liquids, new approaches need to be considered for rapid safety screening.
In this study, two independent laboratories (Philip Morris International and British American Tobacco) evaluated the use of HCS to assess e-liquids. Primary bronchial epithelial cells isolated from two different healthy, nonsmoker donors were exposed to solutions containing different concentrations of PG, VG, and nicotine to demonstrate the dynamic range of the screen. Multiparametric measurements of cellular viability were performed via real-time cellular analysis and HCS employed across 10 endpoints and over 2 timepoints. Osmolarity of the e-liquid was also assessed in parallel.
A comparable dose-dependent cytotoxicity was determined upon exposure to PG and VG mixtures at both test sites. Dose-dependent increase in osmolarity was also observed. Dose-dependent increases in osmolarity were also observed. Responses occurring at concentrations where osmolarity exceeds the normal physiological levels (300 mOsm/kg) may result from hyperosmotic shock.
These data suggest that HCS may be considered as an approach for the biological assessment of e-liquids. As part of a framework for e-liquid investigation, the current multiparametric analysis is now expanded at both sites to assess e-liquid ingredients as well as the range of commercially available e-liquids.
Keywords: E-liquids, In vitro assessment, 3R
Acute mixture toxicity of pesticide formulations and perspective of use in silico methods to complement risk assessment of plant protection products (#722)
S. Kolesnyk1, O. Riabukha1, M. Prodanchuk1, P. Zhminko1, O. Vasetska1
1 L.I. Medved's Research Center of Preventive Toxicology, Food and Chemical Safety Ministry of Health, Institute of Experimental Toxicology and Bio-Medical Research, Kyiv, Ukraine
Humans are exposed to mixtures of chemicals both through diet and in occupational environments. Currently, many plant protection products are complex mixtures of few active ingredients and other ingredients here called adjuvants. In this work, we report studies of acute mixture toxicity of eight plant protection products taking into account not only active ingredients but also other components of the formulation. As whole mixture testing is not feasible as a regular practice for risk assessment of mixtures, we explore possibilities to use in silico methods to guide further experiments and risk assessment.
We conducted studies of acute toxicity (Wistar Han rats, OECD 425) of eight pesticide formulations, contained 2 or 3 pesticides (azoxystrobin, cyproconazole, difenoconazole, flutriafol, imidacloprid, lambda-cyhalothrin, propiconazole, spiroxamine, tebuconazole, thiabendazole, thiram, triadimefon, triadimenol). Results showed that coefficient of additivity calculated on the basis of active ingredients (AI) LD50 characterize potentiation of toxicity (4 formulations); in two formulations we have observed antagonistic interaction; one formulation caused dose addition effect for males and synergistic effect for females. Given that not for all components of formulation LD50 experimental values were found in literature we used QSAR modeling using publicly available tools to derive such values. Results of calculation of additivity coefficient taking into account other ingredients of plant protection products showed the slight difference, however same direction of additivity coefficient in most cases.
To verify findings, further acute toxicity studies with mixtures of only active ingredients and subacute studies with formulations and mixtures of active ingredients were conducted and results will be reported.
To decide if active ingredients of tested plant protection products may be grouped in same cumulative assessment groups we conducted in silico toxicity modeling of AI and adjuvants using different publicly available QSAR and molecular docking models (with the accent on endocrine disruption mode of action and developmental and reproductive toxicity).
Keywords: mixtures, pesticides, acute toxicity, in silico
Perinatal exposure to secondary organic aerosol influences neurobehavioral development and inflammatory markers in rats (#65)
T. - T. Win-Shwe1, Y. Fujitani1, S. Hirano1
1 National Institute for Environmental Studies, Ceter for Health and Environmental Risk Research, Tsukuba, Japan
Background and Aims: Secondary organic aerosol (SOA) is a major component of PM 2.5 and formed in the atmosphere by oxidation of products from anthropogenic and biogenic volatile organic compounds. In this study, we aimed to examine the effects of gestational and lactational exposure to SOA on social dominance behavior and gene expression of inflammatory markers in male rats.
Methods: Sprague-Dawley pregnant rats were exposed to clean air, DEP and SOA in the whole-body exposure chamber (Sibata) for 5 h per day on 5 days of the week from gestational day 8 to postnatal day 21. The male offspring rats at PND 21 were allocated into three different groups (n = 6 per group) as follows: 1) rats exposed to clean filtered air; 2) rats exposed to DE; 3) rats exposed to SOA. Social dominance behavior was investigated at 10 week-old rats using a social dominance tube test (Muromachi Kikai Co. Ltd., Japan). Prefrontal cortex was removed under deep anesthesia to examine inflammatory biomarkers using real-time RT-PCR.
Results and Discussion: We found that SOA exposed rats were more socially dominant compared with the control and DEP-exposed rats. The mRNA expression levels of interleukin (IL) 1 b, microglia marker ionized calcium-binding adapter molecule (Iba)1 and heme oxygenase (HO)-1 in the prefrontal cortex were upregulated compared to the control rats. We suggest that, although the potential toxic substances contained in SOA have not yet been identified, they may reach the brain via placenta of pregnant mice during fetal period and via the olfactory nerve route or systemic circulation during neonatal period and induce neurotoxicity.
Conclusion: Our results indicate that perinatal exposure of SOA may affect social dominance behavior of male rats by modulating social behavioral-related genes and proinflammatory markers in the prefrontal cortex. Among the constituents of SOA, organic carbon is the potential candidate to induce neurotoxicity.
Keywords: neurotoxicity, secondary organic aerosol (SOA), social dominance, prefrontal cortex, rat
NEUROTOXICITY IN VIVO and IN VITRO BY GLYPHOSATE (#74)
M. - R. Martínez-Larrañaga1, J. - L. Rodríguez1, I. Ares1, B. Lopez-Torres1, M. Martínez1, D. Roura-Martínez1, A. Anadon1, M. - A. Martínez1
1 Universidad Complutense de Madrid, Department of Pharmacology and Toxicology, Madrid, Please Select, Spain
Glyphosate is a post-emergent, systemic and non-selective herbicide intended for use against growth of unwanted annual and perennial weeds, woody brush and trees in agricultural, industrial, forestry and residential weed control settings. Glyphosate data are constantly revaluated by regulatory agencies in a science-based process for many reasons including its volume of production and new uses. Nevertheless, questions regarding its safety are periodically raised. This work evaluates: (i) in CNS-tissues from male Wistar rats, the glyphosate effects after oral exposure (35, 75, 150 and 800 mg/kg bw, 6 days) on neurotransmitter levels [serotonin (5-HT), dopamine (DA) and metabolites 5-hydroxy-3-indolacetic acid (5-HIAA), 3,4-hydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] by HPLC and (ii) whether glyphosate affect the expression of selected target genes linked to developmental neurotoxicity in human neuroblastoma SH-SY5Y cells, such as camk2α and camk2β, gap-43, tubulin-α and tubulin-β using RNA isolation, reverse transcription and qPCR analysis. Glyphosate caused signiﬁcant changes in a brain regional- and dose-related manner in neurotransmitter levels. Analysis of 5-HT and DA contents showed a loss of these neurotransmitters, in striatum (5-HT content decreased -49%) and prefrontal cortex and hippocampus (DA content decreased -83% and -71%) after highest dose 800 mg/kg bw. Glyphosate at 800 mg/kg bw produced significant increase of 5-HIAA/5-HT turnover in striatum (88%) and of DOPAC+HVA/DA turnover in prefrontal (153%) and hippocampus (150%), critical brain regions that regulate cognitive functions. Glyphosate treated SH-SY5Y cells showed up-regulation of camk2α, camk2β, and gap-43 transcripts. In conclusion, the glyphosate neurochemical effects observed in this study are an important public health concern. Supported Projects Ref. S2013/ABI-2728, Comunidad de Madrid, and Ref. RTA2015-00010-C03-03, Ministerio de Economía, Industria y Competitividad, Spain
Keywords: Glyphosate, neurotoxicity
Impact of serum on calcium transient assays performed on human-induced pluripotent stem cell-derived cardiomyocytes (#204)
M. Petit1, 2, M. Paquet1, 2, L. Paulhac1, 2, P. Kitchener1, 2
1 Porsolt, LE GENEST-ST-ISLE, France
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as a preclinical tool for detecting cardiotoxicity. These cells express physiologically relevant cardiomyocyte channels and through integrated measurements, allow for the assessment of compounds which may simultaneously affect multiple channels or have an indirect effect on other signaling pathways. icell® Cardiomyocytes ² (Cellular Dynamics international (CDI)) were used in this work as the potential cardiotoxicity model.
CDI recommends the use of serum supplemented growth medium for calcium assays, however, serum can potentially bind molecules and affect their potency. The aim of this study was therefore to evaluate the effects of serum on the contractile activity of hiPSC-CMs after treatment with Cisapride, Quinidine and Nifedipine.
Cryopreserved icell® Cardiomyocytes ² were plated and maintained according to manufacturer’s instructions, in serum-containing media on gelatin-coated 384-well plates. Seven days after plating, hiPSC-CMs were loaded with a calcium indicator and calcium transients were monitored from spontaneously beating cells either in Tyrode, without serum, or in Maintenance Medium with serum. Basal beat frequency, in untreated hiPSC-CMs, was similar in both assay media. One-hour treatments with Cisapride, tested up to 300 nM, and Quinidine, tested up to 10 µM, resulted in dose-dependent decreases in beat frequency in serum-containing Maintenance Medium. In Tyrode (without serum), the same treatments had no effects on beat frequency. Treatment with Nifedipine resulted in a dose-dependent increase in beat frequency in both assay media, with a curve of higher slope in serum-containing Maintenance Medium than in Tyrode. In addition, beat arrest was observed with 1 µM Nifedipine in Maintenance Medium but not in Tyrode.
These results highlight the importance of carefully considering the choice of assay medium to perform toxicologically relevant calcium transient assays on icell® Cardiomyocytes ², and their potential influence on the results.
Keywords: Human-induced pluripotent stem cell-derived cardiomyocytes, serum, calcium transients, cardiotoxicity
Attenuation of livery injury with hypothermia in acute hepatotoxicity (#217)
Y. L. Tan1, 2, H. K. Ho1, 2
1 NUS Graduate School for Integrative Sciences & Engineering, Centre for Life Sciences, Singapore, Singapore
The notion of hepatotoxicity has been prevalent and distressing, especially when the liver is frequently exposed to drugs, herbs or toxicants and many may pose a risk to liver injury. Furthermore, its early asymptomatic manifestation may impede timely treatment, thereby escalating deterioration and worsens outcome. Hence, therapeutic hypothermia has been explored in this study as a potentially simple and effective method to preserve liver functions. Hitherto the concept of hypothermia for toxicity management has been contentious and we aim to debunk the conflict with a systematic investigation of hypothermic effects in an acetaminophen (APAP)-induced liver injury model.
Cytotoxic levels of APAP (1-10 mM) was used to induce liver injury of different severities in TAMH. The temporal relationship between moderate hypothermia, 32 ᵒC for 24 h, and onset of hepatotoxicity was explored for any protective (i.e. hypothermia induced before toxicity), preservative (i.e. hypothermia induced during toxicity) or recovery (i.e. hypothermia induced after removal of toxicants) effects. The extent of hypothermic effects was also investigated by rewarming cells back to 37 ᵒC for 24 h. Both the extent of cell viability and cell death was examined and we attempted to elucidate its protective mechanism with gene and protein expression of cold shock proteins (CSPs), caspase 3 activity, microarray and cell-cycle analysis.
Moderate hypothermia could promote liver preservation, with most prominent effect seen in 5 mM APAP-induced liver injury. The increased cell viability and reduced cell death continued for 24 h after rewarming. These cytoprotective effects could stem from different mechanisms as seen with differential gene expressions and distinct contrast in caspase 3 activity following temperature transition - with a predominant CSPs-induced translational regulation and mRNA stabilization during hypothermia and a complex interplay with transcriptional regulation upon rewarming, involving Tsc22 leucine zipper domains, independent of heat shock.
Moderate hypothermia may effectively alleviate liver injury in an unconventional way and allow lasting protection beyond treatment duration. This study thus unveils a prelude for an efficient drug-free approach to attenuate injury.
Keywords: Hypothermia, Toxicity Management, Liver
Toxicity of synthetic cathinones in human kidney (HK-2) cells (#275)
T. Carvalho1, I. Vaz1, A. M. Araújo2, F. Remião2, M. L. Bastos2, M. Carvalho1, 2
1 UFP Energy, Environment and Health Research Unit (FP-ENAS), University Fernando Pessoa, Porto, Portugal
Synthetic cathinones, also known as bath salts, emerged in the recreational drug market in the mid-2000s as alternatives to illicit drugs such as amphetamines and cocaine and represent nowadays a large class of new popular drugs of abuse. The use of synthetic cathinones is associated with adverse health effects, including renal injury, although the underlying mechanisms are not yet understood. The aim of this study was to evaluate the potential nephrotoxic effects of five commonly used cathinone synthetic derivatives, namely 3,4-methylenedioxypyrovalerone (MDPV), methylone, pentedrone, 3,4-dimethylmethcatinone (3,4-DMMC) and 4-methylethcatinone (4-MEC), using the human kidney (HK-2) cell line as an in vitro model. The HK-2 cells were exposed to a wide range of concentrations, specifically 0.01-3 mM for 3,4-DMMC and 0.1-10 mM for all the others synthetic cathinones, for 24 and 48 hours. Cytotoxicity was evaluated by measuring by measuring mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and by assessing lysosomal uptake of neutral red (NR). It was observed that all tested compounds induced cell death in a concentration- and time-dependent manner. 3,4-DMMC was found to be the cathinone derivative that exhibited the highest toxicity for HK-2 cells, followed by MDPV, pentedrone, methylone, and 4-MEC. To the best of our knowledge, this is the first study to demonstrate the in vitro nephrotoxic potential of synthetic cathinones.
Acknowledgements: This work was supported by national funds through FCT – Fundação para a Ciência e a Tecnologia, in the scope of FCT Project UID/Multi/04546/2013. A.M.A. thanks Fundação para a Ciência e Tecnologia (FCT), Portugal, for her PhD grant (SFRH/BD/107708/2015).
Keywords: Synthetic cathinones, Bath salts, Nephrotoxicity, HK-2 cells, Cell viability
Protective effects of SIRT1 antagonist on diabetic-induced renal fibrosis. (#283)
J. S. Kim1, K. S. Kim1, A. Kundu1, H. S. Kim1
1 Sungkyunkwan University, Collage of Pharmacy / Molecular toxicoloy, Suwon-si, Gyeonggi-do, Republic of Korea
Chronic kidney disease (CKD) is a leading cause of mortality in patients with diabetes mellitus (DM). Recent studies have shown that SIRT1 is closely related to the occurrence and development of diabetic nephrotoxicity. Silent information regulator 2 (Sir2) is a nicotinamide adenine dinucleotide- (NAD+-) dependent deacetylase. The homology of SIRT1 and Sir2 has been extensively studied. SIRT1 deacetylates target proteins using the coenzyme NAD+ and is therefore linked to cellular energy metabolism and the redox state through multiple signaling and survival pathways. In the kidneys, SIRT1 may inhibit renal cell apoptosis, inflammation, and fibrosis. Therefore its activation may also become a new therapeutic target in the patients with CKD including diabetic nephropathy. Here, we evaluated the roles of SIRT1 on the kidney fibrosis in diabetic animal model. We found that acetylation of p65 and STAT3 was increased in the kidney of high fat diet-induced ZDF rats. The expression of a-SMA, collagen I, and fibronectin levels were markedly increased in diabetic-induced ZDF rats. Furthermore, SIRT1 inhibitor attenuated the diabetic-induced kidney fibrosis. Our findings strongly support that SIRT1 inhibitor may use as a protective agent for renal fibrosis in chronic hyperglycemia condition.
Keywords: SIRT1, chronic kidney disease, renal fibrosis, diabetes mellitus
Effects on cigarette smoke extract on cell proliferation and apoptosis in mouse embryonic stem cells via reactive oxygen species-induced endoplasmic reticulum stress signaling pathways (#286)
C. - W. Kim1, R. - E. Go1, K. - C. Choi1
1 Chungbuk National University, Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Cheongju, Republic of Korea
Cigarette smoke contains thousands of chemicals, and many components have reproductive and developmental toxicity that is harmful to humans and animals. Previous studies have reported that cigarette smoke or cigarette smoke extract (CSE) have negative effects on embryo development through in vivo and in vitro studies. However, there is no mechanism study on how CSE affects the cellular signaling pathway for apoptosis and oxidative stress in embryonic cells, or how the two pathways cross-link. Therefore, we investigated the effects of CSE on apoptosis and oxidative stress in mouse embryonic stem cells (mESCs). We measured changes in cell viability by three CSEs (3R4F and two domestic cigarettes CSE1 & CSE2) using the MTT assay and the NRU assay, resulting in decreased cell viability in a concentration-dependent manner. Then, we examined the expression of cell cycle-related protein by Western blot analysis, the expression of cyclin D1 and cyclin E1 was increased and the expression of p21 and p27 was increased by each CSE in mESCs. We hypothesized that this inhibition of cell proliferation might be related to apoptosis, so conducted Terminal deoxynucleotidyl transferase (TUNEL) staining following by CSE exposure in mESCs. In the mESCs, the number of TUNEL-stained cells was increased by CSE exposure in a concentration- and time-dependent manner, and expression of Bax and Caspase-3 was increased and expression of Bcl-2 was decreased by Western blotting. Finally, we performed 2 ', 7' -dichlorofluorescin diacetate (DCF-DA) assay and ROS-Glo™ H2O2 assay to confirm the generation of reactive oxygen species (ROS) by CSE, we could found the increase of ROS by CSE. The increased ROS by CSE was associated with up-regulated translational levels of Keaf-1 and CHOP. Overall, we concluded that the CSE inhibited cell proliferation by regulating cell cycle-related protein expression and increased the oxidative stress by regulating the expression of Keap-1 and CHOP, which regulated the Bcl-2 family signaling pathway, then resulted in apoptosis in mESCs. [This research was supported by a grant (14182MFDS977) from the Ministry of Food and Drug Safety, Republic of Korea, in 2017.]
Keywords: cigarette smoke, mouse embryonic stem cells, apoptosis, oxidative stress, reactive oxygen species
An In-vitro investigation in rat L6 and mouse C2C12 cell lines into the skeletal muscle toxicity observed in the mouse of Trypanosoma Cruzi whole cell inhibitor compounds. (#386)
D. Grimsditch1, G. Brunori1, J. Armitage1, C. Taylor1, T. Dare1, M. Lennon1, E. Jimenez-Navarro2, S. Gonzalez-Del Valle2, S. Gresham1, J. Lyon1, G. Dear1
1 GlaxoSmithKline R&D, ISDM, Ware, United Kingdom
As part of an investigation into clinical signs observed in mouse efficacy studies with a chemical series being assessed for the potential treatment of Chagas disease, four compounds were evaluated for their toxicity profile in 4 day repeat dose toxicity study in the CD-1 mouse. The principal findings were a lack of tolerability at high doses and a common histopathological change in 3 out of the 4 compounds of degeneration/regeneration of myocytes in the skeletal muscle with associated increases in aldolase (ALD), aspartate aminotransferase (AST) and creatine kinase (CK). To investigate these findings further and identify a suitable in vitro platform for screening back-up molecules plus understanding if the rat would also be sensitive to these target organ effects, murine C2C12 and rat L6 myoblast cell lines were selected for in vitro investigations. Briefly, using both cell lines the 4 compounds were evaluated for their effect on mitochondrial bioenergetics using the Seahorse XFe96 bioanalyser, and the translation of biomarker release from in vivo to in vitro, evaluating AST, ALD, CK. Lactate dehydrogenase (LDH), and skeletal troponin I (sTnI) after 24h incubation. To aid the mechanistic investigations the compounds were also evaluated for their ability to directly affect mitochondrial function using the calcium loading capacity assay in freshly isolated rat liver mitochondria. Results from this in vitro investigative study suggest that all compounds tested affect mitochondrial function at different potencies in both mouse and rat cell lines. This was also associated with an increase in the level of skeletal muscle damage markers with sTnI being the most sensitive marker of damage in the absence of cytotoxicity (MTT). These investigations provided strong evidence that the skeletal muscle toxicity observed in vivo was most likely associated with the mitochondrial activity of the compounds assessed and that the rat is also likely to be sensitive to the in vivo changes and therefore there was no value in progressing an exemplar to a rat toxicity study. These data supported the termination of all 4 molecules in the lead optimisation phase and presented the discovery scientists with a platform to screen further molecules for their potential to induce skeletal muscle toxicity.
Keywords: mitochondria, skeletal muscle, in vitro, Seahorse, mouse, rat
Effects of 2,4- dichlorophenoxyacetic acid on the biochemical parameters and oxidative stress in rats: the protective role of pomegranate (punica granatum) juice. (#394)
R. MOSBAH1, 2, A. CHETTOUM3, Z. DJERROU4, A. MANTOVANI5
1 University of Boumerdes, Department of Biology, Boumerdes, Algeria
2,4-Dichlorophenoxyacetic acid (2,4-D) is a systemic herbicide used since 1941 in agriculture. Subchronic exposure to 2,4-D may lead to several negative health impacts on human and animals. Pomegranate (POM, Punica granatum) juice is a rich source of antioxidants like polyphenols which make it one of the most popular drinks in the world. So, it has wonderful nutritional values and therapeutic benefits towards a variety of ailments such as cancer, hypertensive, diabetes, stomach, cardiac, inflammatory and erectile problems.
In this study, we attempt to assess the effects of 2,4-D on the biochemical parameters and oxidative stress as well the protective role of POM juice against 2,4-D induced toxicity. Thirty six male Wistar rats were randomly allocated into four groups of 9 each as follows: control (CT), 2,4-D (150 mg/kg), POM juice (100 mg/kg) and in combination (2,4-D + POM) respectively. The treated animals had received the chemicals by gavage (5 days/weekly) for 9 weeks.
Results showed that the herbicide 2,4-D declines significantly the levels of glucose, total proteins and uric acid, meanwhile, cholesterol, urea, creatinine, transaminases (AST, ALT) and lipids (LDL) levels were elevated. Likewise, thiobarbituric reaction species (TBARS) levels in plasma, liver and kidney were significantly increased in 2,4-D treated animals. The supplementation of POM juice along with 2,4-D could ameliorate clearly the most altered biochemical parameters induced by 2,4-D and inhibiting lipid peroxidation as evident by a decrease in TBARS levels in plasma, liver and kidney.
These findings suggest that the consumption of POM fresh juice as daily beverage can prevent against the toxic effects induced by 2,4-D regarding to its antioxidant properties and capacity in scavenging free radicals and reducing oxidative stress.
Keywords: 2, 4-Dichlorophenoxyacetic acid, Pomegranate, biochemical parameters, oxidative stress, protective role
Increased susceptibility to troglitazone-induced mitochondrial permeability transition in type 2 diabetes mellitus model rat (#422)
M. Segawa1, 2, S. Sekine1, T. Sato1, K. Abe2, K. Ito1
1 Chiba University, Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba, Japan
Drug induced liver injury (DILI) is recognized as a major adverse drug reaction, one of the risk factors have been thought to depend on host characteristics such as disease and nutritional condition. Troglitazone, an antidiabetic drug, was withdrawn from the market because it caused severe hepatotoxicity. One of the mechanisms for this hepatotoxicity is thought to be mitochondrial toxicity, but little research has been conducted on their host factors in type 2 diabetes mellitus (T2DM), involved in troglitazone-induced hepatotoxic effects. To investigate the characteristics of troglitazone-induced liver toxicity in detail, we examined the toxicological effects of troglitazone on liver mitochondria using a rat model of T2DM. Liver mitochondria was prepared from male Zucker Diabetic Fatty (ZDF, fa/fa) and lean (?/+) rats. Among antidiabetic drugs, troglitazone (10-50 µmol/L), pioglitazone (10-50 µmol/L) and phenformin (30-300 µmol/L) were used. Assessment of mitochondrial toxicity was conducted by measurement of mitochondrial permeability transition (MPT), mitochondrial membrane potential and respiratory function. To examine redox status of liver mitochondria, amounts of lipid peroxidation products, reduced glutathione (GSH) and its oxidized form contents were determined. Troglitazone was found to increase MPT in the liver mitochondria of ZDF fa/fa rats to a greater extent than in ZDF lean rats, and susceptibility to MPT after treatments with pioglitazone and phenformin was slightly but significantly potentiated in ZDF fa/fa rats. However, susceptibilities of mitochondrial membrane potential and respiratory function to these antidiabetic drugs were not affected. In mitochondria of ZDF fa/fa rat, we found a decrease in GSH content and an increase in phospholipid peroxidation. Moreover, incorporation of oxidized cardiolipin, a mitochondrion-specific phospholipid, was involved in the troglitazone-induced alteration in susceptibility to MPT. These results suggested that liver mitochondria displayed disease-associated mitochondrial lipid peroxidation in T2DM, which facilitates the higher susceptibility to troglitazone-induced MPT. Thus, greater susceptibility of liver mitochondria will be a host factor leading to troglitazone-induced hepatotoxicity in T2DM.
Keywords: Diabetes, Troglitazone, Liver mitochondria, Mitochondrial permeability transition, Lipid peroxidation
A High-Content Imaging approach for AOP development of kidney proximal tubule toxicity (#463)
S. Di FIore1, F. Schmelter1, B. Ellinger2, S. Jarzina3, A. Mally3
1 Fraunhofer Institute for Molecular Biology and Applied Ecology - IME, Division Molecular Biotechnology, Aachen, Germany
The nephrotoxic effect of some drugs such as polymyxins is a major drawback that restricts the treatment of bacterial infections in humans. Nevertheless, the use of such drugs is often unavoidable, since they are regarded as a last-resort intervention against e.g. multi-resistant pathogens. To circumvent these problems the most appropriate strategy would be the development of drugs with decreased nephrotoxic potential. However, nephrotoxic effects are difficult to predict during preclinical drug development and nephrotoxicity is typically detected late in drug discovery. Within the project Risk-IT we focus on predicting nephrotoxicity based mechanism-based in vitro endpoints and in vitro to in vivo extrapolation modelling. We applied the adverse outcome pathway (AOP) concept to analyse two kidney cellular models: the rat NRK52E and the human RPTEC/hTERT1 cell line. Based on what is known about the mechanism of polymyxin nephrotoxicity, we developed an AOP for nephrotoxicity due to lysosomal overload, including key events describing morphological and functional changes that recapitulate lysosomal failure. Together with data generated for an alternative AOP we demonstrate that High Content Imaging (HCI) assays are an affordable and robust platform capable of delivering time resolved data for analysis of lysosomes in cells exposed to drugs such as polymyxins. Our data provide strong evidence that HCI is a powerful technology for characterization of key events associated with AOPs, with sensitivity and throughput sufficient to clearly discriminate differences in response between cell models of the proximal tubule.
Keywords: High Content imaging., lysosomal overload, in vitro testing, AOP
The effects of dioxin action on hr-deficient epidermal keratinocytes are associated with changes in expression of AhR-regulated genes both in vitro and in hairless mouse skin in vivo. (#515)
S. G. Rudyak1, V. V. Shinin2, L. A. Usakin2, N. N. Belushkina1, M. A. Paltsev1, A. A. Panteleyev2
1 Emanuel Institute of Biochemichal Physics Russian Academy of Sciences, Moscow, Russian Federation
Hairless mice, bearing a mutation in hr gene, have an increased skin sensitivity to the ligand of aryl-hydrocarbon receptor (AhR), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These specific effects of TCDD action are manifested in severe chloracne-like cutaneous lesions and skin carcinomas, lacking in wild type mice. Hairless gene (hr) encodes histone demethylase, which is highly expressed in epidermal compartment of the skin, but missing from most other tissues. It was suggested that hr enzyme is involved in regulation of gene silencing in keratinocytes, although its immediate targets remain unknown. We have used hairless mice and keratinocyte cultures obtained from the skin of hairless and wild-type animals to determine the range of TCDD-induced skin effects in hr/wt backgrounds. 1,6 ug of TCDD were applied to skin of hairless mice for 25 days, while hr and wt keratinocyte cell cultures were treated with 0-100 nm TCDD and its non-toxic analogue 6-Formylindolo[3,2-b]carbazole (FICZ). In hr mice, topical application of TCDD produced an increased skin inflammation, infiltration of neutrophils in the epidermis, involution of sebaceous glands, and changes in localization of keratinocyte differentiation markers. Concomitantly, treatment of hr/wt keratinocytes with TCDD/FICZ resulted in upregulation of AhR-regulated genes (Cyp1A1, etc.), with varying levels of induction between hairless and wild-type cells, and highly dependent on conditions and time of incubation. These results suggest that hr might be involved in regulation of AhR-dependent TCDD response and elucidate novel potential targets of hr- and TCDD-mediated gene regulation.
Supported by the Russian Foundation of Basic Research grant 18-04-01332 and by intramural research grant from NRC “Kurchatov institute”.
Keywords: keratinocytes, skin, dioxin, hairless
The Adverse Outcome Pathway (AOP) concept as a basis for the development of mechanism-based animal sparing approaches: Kidney toxicity due to lysosomal overload and inhibition of mtDNA polymerase-γ as case studies (#583)
S. Jarzina1, P. Reiser1, J. Scherer1, S. DiFiore2, B. Ellinger3, A. Mally1
1 University of Würzburg, Department of Toxicology, Würzburg, Germany
Adverse outcome pathways (AOP) are widely recognized as a pragmatic tool that may facilitate transition of toxicity testing from measurement of apical endpoints in animals to prediction based on mechanistic information. As a case study, we established AOPs for proximal tubule injury initiated by (1) lysosomal overload and (2) inhibition of mtDNA polymerase-γ and used these as a mechanistic basis for the development of in vitro assays reflecting key events (KE) across the AOPs. Nephrotoxicity of polybasic drugs such as polymyxin antibiotics results from receptor-mediated endocytosis (MIE), disturbance of lysosomal function (KE1), lysosomal swelling and disruption associated with release of lysosomal components (e.g. cathepsins) (KE2) that induce proximal tubule cell toxicity (KE3). Renal toxicity of antiviral drugs such as tenofovir is thought to be due to inhibition of mtDNA polymerase-γ (MIE), leading to mtDNA depletion (KE1), mitochondrial dysfunction (KE2) and proximal tubule cell toxicity (KE3). In vitro endpoints reflecting each KE were assessed in rat (NRK-52E) and human proximal tubule cells (RPTEC/TERT1) treated with model compounds. High content imaging using LysoView™ stain was used for lysosomal analysis (KE1). Some of the readouts (e.g. lysosome numbers per cell) appeared to reflect cytotoxicity (KE3) rather than specific effect on lysosomes, highlighting the need for time-resolved data to support the causal and temporal relationship between KEs. In contrast, lysosomal-associated membrane protein 2 (LAMP2) (KE1) and Cathepsin D release (KE2) were observed even at subtoxic concentrations. Importantly, significant differences in polymyxin uptake and susceptibility were observed between cell-lines, underscoring the importance of in vitro biokinetics to define an appropriate point of departure for risk assessment. Only minor effects on mitochondrial toxicity (KE2) and cytotoxicity (KE3) were observed in response to antiviral drugs. In contrast to the mechanistic hypothesis, treatment with antiviral drugs lead to an increase rather than decrease in mtDNA copy number (KE1). Possibly, longer treatment times may be required to achieve the decrease in mtDNA copy number observed in patients, highlighting some of the challenges in mimicking in vivo mechanisms in short-term in vitro systems.
Keywords: Adverse outcome pathway, in vitro toxicity, nephrotoxicity, kidney
Natural antioxidants prevent contrast-induced nephropathy by enhancing nitric oxide synthesis in an animal model (#588)
I. Fragkiadoulaki1, C. Mamoulakis2, A. Alegakis1, M. Tzatzarakis1, V. Karzi1, A. Stratidakis1, E. Renieri1, A. Vardavas1, G. Leon1, C. Tsitsimpikou3, A. Tsatsakis1
1 University of Crete, Laboratory of Toxicology, Medical School, Heraklion, Crete, Greece
Purpose: Contrast-induced nephropathy (CIN) is a reversible form of acute renal failure that begins soon after iodinated contrast media (CM) administration. CM induces renal vasoconstriction, through oxidative stress by inhibiting vasodilators such as nitric oxide (NO). Asymmetric Dimethylarginine (ADMA) and Symmetric Dimethylarginine (SDMA) are inhibitors of NO synthesis and elevated levels indicate renal damage. The aim of this study was to evaluate the protective effect of natural antioxidants against CIN in an animal model by estimating the SDMA and ADMA levels.
Materials and Methods: New Zealand white male rabbits were divided in four groups control, iopromide, iopromide plus resveratrol (RVT) and iopromide plus lycopene (Lyc). Iopromide was administered intravenously (single dose of 7.5 mg/I/kg), RVT and Lyc were administered orally (single dose of 4.28 mg/kg and 4.0 mg/kg respectively), 24h prior to iopromide exposure. Blood samples were collected immediately after iopromide exposure (0h), at 24h and at 48h after iopromide exposure. SDMA and ADMA levels were estimated by High-Performance of Liquid Chromatography (HPLC).
Results: SDMA and ADMA levels were significantly elevated in iopromide group compared to all other groups (p<0.05) at 0h and 24h after CM exposure, showing a trend to decline towards baseline levels at 48h.
Conclusion: RVT and Lyc seem to prevent iopromide-induced nephropathy by enhancing NO synthesis in the rabbits.
Keywords: contrast-induced nephropathy, resveratrol, lycopene, ADMA, SDMA, NO
The interaction between the guanidine polymers of disinfectants and cytoplasmic organelles. (#617)
S. H. Yoo1, M. H. Jeong1, E. H. Jang1, D. M. Kim1, K. H. Chung1
1 Sungkyunkwan, Pharmacy, Suwon, Gyeonggi-do, Republic of Korea
Guanidine polymer is a series of polymers bearing guanidino groups, which are mainly used as an antimicrobial agent. In 2011, many patients of lung damage were occurred, so a number of epidemiological studies were conducted and the association of humidifier disinfectant with lung injury was demonstrated.
Polyhexamethylene guanidine phosphate (PHMG-p), poly (oxyalkylene guanidine) hydrochloride (PGH) and polyhexamethylene biguanidine (PHMB) were guanidine polymer used as disinfectants for the prevention of microorganism growth in humidifiers. In vitro and in vivo studies revealed that inhaled disinfectants induced the biological responses associated with pulmonary fibrosis. However, the mode of action of these disinfectants is unknown yet. In this study, we synthesized PHMG-p-, PGH- and PHMB-FITC conjugates and assessed the uptake into lung epithelial A549 cell line. To examine cell localization, the A549 cells were treated with PHMG-p-, PGH- and PHMB-FITC conjugates, counter-stained with various cytoplasmic organelles, and observed by confocal microscope. Covalent conjugation with fluorescein isothiocyanate yielded labeled PHMG-p, PGH and PHMB by dialysis. Both PHMG-p- and PGH-FITC were detected within cytoplasmic space and co-localized in endoplasmic reticulum and lysosome, but not in mitochondria. PHMB-FITC was found in endoplasmic reticulum and lysosome only after 24 hours of exposure. In addition, PHMG-p induced the autophagy and apoptosis in A549 cells. Further studies are necessary to elucidate the relationships between endoplasmic reticulum, lysosome and mitochondria damages.
Keywords: PHMG, PGH, PHMB, FITC
A classical and an immunoaffinity-proteomic study to identify and validate drug-induced kidney injury biomarker in canine (#631)
W. Naboulsi1, 2, H. Planatscher1, 2, M. Sonee3, Y. Chen4, S. Bryant3, C. Johnson3, L. Nguyen4, B. Scott4, T. Joos1, 2, J. E. Mcduffie4, O. Poetz1, 2
1 Signatope GmbH, Reutlingen, Germany
Canines remain one of the most common non-rodent species used in pre-clinical safety studies. Drug‑induced kidney injury (DIKI) is still one of the major reasons for failure in drug development. Not many data about proteome changes in DIKI in canines is available. Here, we conducted a three phases proteomic experiment to highlight the proteome changes in DIKI and to verify some biomarkers known from rat and human studies. In a 10-day (D) low dose-study, animals received the nephrotoxic tobramycin (60 mg/kg, n=6) or vehicle (n=3). Minimal to moderate proximal tubular injury was histologically confirmed. First, a discovery proteomic study, we performed a label-free (LF) quantification on the collected kidney tissue samples. 558 proteins were significantly differentially regulated (q-value < 0.05) between tobramycin-treated canines and vehicles. Based on the function of the regulated proteins, 12 proteins were selected further for analysis. These included proteins which are suggested as DIKI-biomarker such as osteopontin, clusterin and kidney injury molecule 1 (KIM-1) and unknown proteins such as Serpin A5 and Fatty acid-binding protein. Second, we developed a peptide-centric mass spectrometry-based immunoassay panel (IP-LC/MS) to quantify the candidates in the kidney cortex and in canine’s urine. In the IP-LC/MS assay, targeted peptides are enriched by antibodies which recognize a short epitope motif. In accordance with the LF experiment, 8 proteins were found to be significantly up-regulated via the IP-LC/MS assay following the treatment. KIM-1 was mostly affected with 50‑fold higher in the treated dogs. Finally, we further validated the IP-LC/MS assay (assay linearity, digestion kinetics, intra- and inter variation) to absolutely quantify the biomarker candidates in the dog urine. Upregulation of urinary osteopontin, clusterin, retinol binding protein 4, KIM-1 and protein alpha-1-microglobulin/bikunin on D7 and D10 versus controls was demonstrated while no changes in the standard parameters such as serum creatinine and urea nitrogen were detected. This indicate the applicability of the IP-LC/MS assay to detect changes in urine-based proximal tubular injury biomarkers in canines. Utilizing our short epitope motif enrichment strategy, the developed assay can be applied in monkey, human, mouse, rat and cat studies.
Keywords: Biomarkers, Drug-induced kidney injury, Canines, Urine, mass spectrometry-based immunoassay, nephrotoxicity, Tobramycin, Kidney, IP-LC/MS
Natural antioxidants prevent contrast-induced nephropathy in an animal model: Results from a cytological study (#650)
A. M. Tsatsakis1, I. Fragkiadoulaki1, A. Kalogeraki2, I. Tsiaoussis3, F. Karkala1, K. Kalliantasi1, P. Stivaktakis1, P. Fragkiadaki1, C. Psycharakis1, C. Mamoulakis4
1 University of Crete, Laboratory of Toxicology, Medical School, Heraklion, Crete, Greece
Purpose: Contrast-induced nephropathy (CIN) is considered a reversible form of acute renal failure that begins soon after iodinated contrast media (CM) administration. Iodinated CM may exert their nephrotoxic effects in several different ways. Hemodynamic alterations resulting in renal medulla ischemia and direct toxic effects on renal tubular epithelial cells leading to apoptosis and tubular vacuolization seem to be major factors contributing to CIN. The aim of this study was to evaluate the protective effect of natural antioxidants against CIN in an animal model by estimating renal tissue damage.
Materials and Methods: New Zealand white male rabbits were divided in four groups control, iopromide, iopromide plus resveratrol (RVT) and iopromide plus lycopene (Lyc). Iopromide was administered intravenously (single dose of 7.5 mg/I/kg), RVT and Lyc were administered orally (single dose of 4.28 mg/kg and 4.0 mg/kg respectively), 24h prior to iopromide exposure. After 48h of iopromide administration rabbits were sacrificed and kidneys were collected. Renal tissue damage was estimated using the touch preparation technique.
Results: Major renal damage including tubular apoptosis, cell degeneration and tubular vacuolization was observed only in iopromide group. Renal tissues in RVT and Lyc groups tended to be similar to control group; cells appeared normal with minimal renal damage detected.
Conclusion: RVT and Lyc seem to prevent iopromide-induced nephropathy by reducing acute renal damage in the rabbits.
Keywords: contrast-induced nephropathy, antioxidants, resveratrol, lycopene, renal damage, cytological study
Natural antioxidants prevent contrast-induced nephropathy in an animal model: Results from a biochemical study (#699)
R. Zisis1, I. Fragkiadoulaki2, A. Alegakis2, F. Karkala2, E. K. Vakonaki2, E. Apalaki2, G. A. Vaki2, L. Kovatsi2, A. M. Tsatsakis2, C. Mamoulakis1
1 University General Hospital of Heraklion, Department of Urology, Heraklion, Crete, Greece
Purpose: Contrast-induced nephropathy (CIN) is considered a reversible form of acute renal failure that begins soon after iodinated contrast media (CM) administration during angiographic or other procedures. CIN is characterized by an increase in serum creatinine (Scr) levels >25% compared to baseline values that generally appears within the first 48h after CM exposure, reaching a peak within the following 5 days. Antioxidants have been found to prevent lipid peroxidation, reduce oxidative stress by scavenging free radicals and prevent renal ischaemia after CM exposure. The aim of this study was to evaluate the protective effect of natural antioxidants against CIN in an animal model by estimating Scr levels.
Materials-Methonds: New Zealand white male rabbits were divided in four groups control, iopromide, iopromide plus resveratrol (RVT) and iopromide plus lycopene (Lyc). Iopromide was administered intravenously (single dose of 7.5 mg/I/kg), RVT and Lyc were administered orally (single dose of 4.28 mg/kg and 4.0 mg/kg respectively), 24h prior to iopromide exposure. Blood samples were collected immediately after iopromide exposure (0h), at 24h and at 48h after iopromide exposure. Scr levels were measured by a biochemical analyzer (Architect c4000, Abbott, Abbott Park, Illinois, U.S.A).
Results: There was no difference in Scr levels among groups at 0h but there was an increase >25% in iopromide group compared to control, RVT and Lyc group after 24h and 48h of iopromide administration.
Conclusion: RVT and Lyc seem to prevent iopromide-induced nephropathy in the rabbits.
Keywords: contrast-induced nephropathy, resveratrol, lycopene, antioxidants, serum creatinine, prevention
Combination of energy drink and alcohol and its associated toxicity (#729)
M. D. Arbo1, M. Costa-Valle2, B. Tonietto2, L. Altknecht1, C. Duarte1, S. C. Garcia1, E. Dallegrave3, M. B. Leal2
1 Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia, Porto Alegre, Brazil
The consumption of energy drinks is high among adolescents and young adults, who 24 often associate them with alcoholic beverages, such as vodka and whisky. The aim of this study was to evaluate the acute toxicity of the association of energy drink and alcohol in male Wistar rats. Animals (n=4-5/group) were treated by oral gavage with 10 ml/kg distilled water (control); 10ml/kg energy drink (ED10); 2g/kg alcohol 20%; and 2g/kg alcohol 20% + ED10. Behavioral alterations were observed for 6h after treatment. After 24, animals were euthanized, blood and urine collected for biochemical analysis (transaminases, ureia, creatinine, microalbumin and NAG activity). Histopathological analysis and lipoperoxidation were performed in liver and kidneys. The experiments were approved by the University Ethics Committee (Number 26689) Animals presented differences in the frequency of rearing, ambulation, grooming, self-cleaning, be awake and tachypnea along time. No significant alterations in serum biochemical parameters was observed. Lipoperoxidation was significantly increased (p<0.05, ANOVA/Bonferroni) in livers of ED10 treated group compared to control. The association of ED10+alcohol induced nephrotoxicity through the significant increase of the early kidney damage biomarker NAG urinary activity (p<0.05, ANOVA/Bonferroni). Histopathological analysis showed the presence of hydropic and hyaline degenerations, and congestion in the livers of ED10+alcohol treated rats. In kidneys, hyaline degeneration was observed in ED10 and ED10+alcohol groups. Hemorrhage was present in the kidneys of all groups. The combination of energy drinks and alcohol, are not safe for their consumers. Therefore, precautionary measures should be disseminated among risk populations, specially the teenagers.
Acknowledgements: Brazilian National Council for Scientific and Technological Development - CNPq; CAPES Foundation - Brazil, and Universidade Federal do Rio Grande do Sul (UFRGS).
Keywords: alcohol, energy drink, nephrotoxicity
Subchronic toxicity of Al2O3, TiO2 and SiO2 nanoparticles in different combinations for rats and its attenuation with a complex of innocuous bioprotectors. (#35)
S. V. Klinova1, I. A. Minigalieva1, L. I. Privalova1, V. G. Panov2, O. G. Makeev3, I. E. Valamina3, E. V. Shishkina4
1 The Medical Research Center for Prophylaxis and Health Protection in Industrial Workers, Ekaterinburg, Russian Federation
For the animal experiment, stable water suspensions of nanoparticles (NPs) were obtained by laser ablation of 99,99% pure elemental aluminum, titanium or silicon under a layer of deionized water. Male outbred rats with initial body mass 290 g were injected intraperitoneally 3 times a week (up to 18 injections) with suspensions of Al2O3-NP, TiO2-NP or SiO2-NP in doses 0.25, 0.5 and 0.5 mg per rat, respectively, either separately, or in binary, or in ternary combinations of the same doses, the controls receiving injections of deionized water in the same volume. Toxic effects where estimated with a lot of functional, biochemical and morphometric indices for the organism’s status. The results obtained demonstrated that, in many respects, the Al2O3-NP was the most toxic as such and the most dangerous component of the studied combinations. Mathematical modeling with the help of the Response Surface Methodology has shown that the organism’s response to a simultaneous exposure to any two of the element oxide (ElO) NPs under study is characterized by all possible types of combined toxicity (additivity, subadditivity or superadditivity of unidirectional action and different variants of opposite effects) depending on which outcome this type is estimated for as well as on the levels of the effect and dose. With any third ElO-NP species acting in the background, the type of combined toxicity displayed by the other two ElO-NPs can change significantly. Many adverse effects produced in rats by the combined [Al2O3-NP+TiO2-NP+SiO2-NP] exposure, including the genotoxic one, were substantially attenuated by per oral administration of a complex of innocuous bioprotective substances during the entire exposure period.
Keywords: nanoparticles, subchronic effects, comparative and combined toxicity, bioprotectors
Pharmacokinetics of HuperzineA-PLGA-nanoparticlesin in mice (#114)
R. zhang1, T. shi1, C. wang1, X. chen1, L. li1
1 /, State Key Laboratory of NBC Protection for Civilian, Beijing, China
Objective:To study plasma and brain tissue pharmacokinetics of different sizes HuperzineA-PLGA-nanoparticles (HupA-PLGA-NPs) in mice. Methods: Thirty ICR mice were divided into three groups randomly. Native HupA and HupA-PLGA-NPs with mean diameter 46.4 nm and 208.5 nm were injected into mice by tail vein with dose of 0.5 mg/kg,respectively. Blood samples were collected at various time points(2min, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 12h and 24h)after intravenous injection and the mice were sacrificed. The brain tissues were excised, weighed and homogenized.The 200 μl serum or brain tissue samples were mixed and vortexed with 10 μl sodium hydroxide, then 1 ml ethyl acetate were added and vortexed to precipitate protein and centrifuge for 15min, the supernatants were collected and transfered to another tube and dried under a stream of nitrogen at room temperature. The residue samples were resuspended in 200 μl mobile phase and following centrifuged. The samples(10 μl) were analyzed by method of LC-MS/MS. Results: The distribution of different size HupA-PLGA-NPs showed remarkable changes compared with that of HupA in vivo, the particle size of nano-drugs emerged significant affect for the pharmacokinetics parameters. The Cmax values of 46.4 nm and 208.5 nm HupA-PLGA-NPs in plasma were almost equaled to the that of HupA.The T1/2 values were 1.53 times (46.4 nm) and 1.96 times (208.5 nm)compared with that of HupA, respectively. The bioavailability of 46.4 nm and 208.5 nm HupA-PLGA-NPs were 2.02 and 2.35-folds higher than that of HupA, respectively. The Cmax value of 46.4 nm HupA-PLGA-NPs was as much as that of HupA, but the Cmax value of 208.5 nm HupA-PLGA-NPs was 1.6 times compared with that of HupA in brain tissue. The Tmax values of both nanoparticles were 1.25h，which was obviously longer than that of HupA (0.5 h). Meanwhile, the T1/2 values were 1.82 times (46.4 nm HupA-PLGA-NPs) and 1.23 times(208.5 nm HupA-PLGA-NPs) compared with that of HupA (6.89 h), respectively. Besides, brain targeting indexes of 46.4 nm and 208.5 nm HupA-PLGA-NPs was 1.48 and 0.81, respectively, the former showed obvious brain targeting effect, but the latter had not the effect.Conclusion: This study may provide technical support for the application of HupA in controlled drug deliveryand in drug brain-targeting.
Keywords: HuperzineA, HupA-PLGA-NPs, Pharmacokinetics, LC-MS/MS
In Vitro to In Vivo Extrapolation of Valproic Acid hepatotoxicity: a Biokinetic and Physiologically Based Toxicokinetic Informed Approach (#247)
C. P. Fisher1, O. Hatley1, B. van Vugt4, F. Bois2, S. Escher3, I. Gardner1
1 Certara UK Limited, Simcyp Division, Sheffield, United Kingdom
Valproic acid (VPA) is used in the management of seizures, bipolar disorder, and migraines however, its use is associated with a number of adverse effects including hepatic steatosis. Whole-body physiologically based toxicokinetic (PBTK) models were developed to simulate the tissue concentrations of VPA in the rat and human. The models were parameterised using physicochemical properties and reverse translation approaches from published data. In order to recover the extended systemic exposure observed in rodent pre-clinical studies, it was necessary to incorporate the enterohepatic recirculation resultant from deconjugation of biliary cleared glucoronidated metabolites in the gut, and the subsequent reabsorption of parent compound. In vitro reporter assays showed activation of the pregnane X receptor (PXR) and peroxisome proliferator activated receptor alpha (PPARa) in response to VPA exposure; both previously identified as molecular initiating events (MIEs) in the hepatic steatosis adverse outcome pathway. Using a steady-state biokinetic model of in vitro distribution, nominal VPA treatment concentrations identified as activating PXR and PPARa in vitro were used to predict the corresponding intracellular concentrations. Using the verified rat and human PBTK models, a reverse-dosimetry approach was employed to determine the oral dose resulting in maximal hepatic concentrations corresponding to the intracellular concentration associated with activating these MIEs in vitro. Doses of 3.7mg/kg and 1.7 mg/kg in rat and human, respectively, were determined to result in hepatic concentrations associated with activation of MIEs in the steatosis AOP. As might be expected, the MIE activating dose determined in rat, was more than two orders of magnitude lower than the oral LOAEL identified in rat repeat dose toxicity studies (500 mg/kg) linked with histopathological scoring of hepatic steatosis. However, the MIE activating dose in humans, equivalent to ~125mg, is well within the therapeutic dosing range. Repeat dosing at therapeutic levels could result in sustained activation of MIEs associated with adverse hepatic outcomes. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002.
Keywords: toxicokinetics, biokinetics, AOP
Incorporation of Toxicodynamic Feedback in a Physiologically-Based Toxicokinetic Model of Acetaminophen Overdose (#249)
D. Reddyhoff1, C. Fisher1, I. Gardner1
1 Certara UK Limited, Simcyp Division, Sheffield, United Kingdom
The hepatotoxicity of acetaminophen (APAP) has been extensively studied and modelled. The putative mechanism underlying observed toxicity under overdose conditions is driven by saturation of the main sulfotransferase (SULT) mediated clearance pathways, resultant from the depletion of the 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) co-factor. The fraction of the dose metabolized through CYP450 mediated clearance pathways is subsequently increased, resulting in excessive formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). While minimal amounts of NAPQI produced at therapeutic levels can be eliminated through conjugation with reduced glutathione (GSH), overdose results in a depletion of GSH, formation of protein adducts, and oxidative stress.
Here we present a physiologically based toxicokinetic model (PBTK) model incorporating the critical co-factor dependence of SULT mediated metabolic clearance, GSH depletion, regional drug metabolism, and feedback of NRF2-mediated upregulation of basal metabolism of GSH, as well as the rate of reduction of oxidized glutathione (GSSG) to GSH. We implement a 6-compartment liver model, which includes hepatic zonation of metabolism and its effect on the clearance of drugs/metabolites. Incorporation of metabolic heterogeneity in our liver model reproduces increasing CYP and UGT mediated metabolism in the perivenal region of the liver lobule. A coupled toxicodynamic model incorporates the depletion of the PAPS co-factor, depletion of GSH, the glutathione redox cycle and NRF2-mediated protective responses to reactive oxygen species (ROS), and the subsequent feedback on toxicokinetic parameters.
Our model reproduces in vivo time-course data of APAP plasma levels, and captures the depletion of cofactors at increasing APAP doses. NRF2 reduces the depletion of GSH in response to high APAP doses, resulting in lower NAPQI concentrations across the liver. Zonation of hepatic metabolism results in a concentration gradient from the periportal to perivenal region of the liver. This is consistent with in vivo findings, where the most acute and extensive cell damage is observed in the perivenal region.
Keywords: toxicokinetics, hepatotoxicty, toxicodynamics
In vitro to in vivo extrapolation of effective concentrations in alternative assays for developmental toxicity testing: Application of generic PBK modelling. (#348)
S. Fragki1, A. H. Piersma1, 2, E. Rorije4, M. J. Zeilmaker3
1 RIVM, Centre for Health Protection, Bilthoven, Netherlands
Incorporation of kinetics to QIVIVE (quantitative in vitro to in vivo extrapolations) is an important step for the realization of a non-animal testing paradigm, in the field of regulatory toxicology. The use of Physiologically-Based Kinetic (PBK) modelling for estimating systemic doses of chemicals at the target site is accepted to be an indispensable element for such purposes. Nonetheless, PBK models are commonly constructed for a single or a group of compounds and are considered to be demanding, with respect to experimental data needed for model parameterization. Alternatively, we evaluate here the use of a more generic approach, ie. the so-called IndusChemFate model, which is based on incorporated QSAR model parametrization. The model was used to describe the in vivo kinetics of three diverse classes of developmental toxicants: triazoles, glycol ethers’ alkoxyacetic acid metabolites and phthalate primary metabolites. The model needed specific input per each class of compounds. These substances were previously tested in three alternative assays: the whole- embryo culture (WEC), the zebrafish embryo test (ZET), and the mouse embryonic stem cell test (EST). Thereafter, the PBK-simulated blood levels at toxic in vivo doses were compared to the respective in vitro effective concentrations. Comparisons pertaining to relative potency and potency ranking with integration of kinetics were similar to previously obtained comparisons. Additionally, all three in vitro systems resulted in quite comparable outcomes, and hence, a combination of alternative tests is still preferable for predicting the endpoint of developmental toxicity in vivo. This approach is put forward as biologically more plausible since plasma concentrations, rather than external administered doses, constitute the most direct in vivo dose metric.
Keywords: QIVIVE, Generic PBK-model, Triazoles, Glycol ethers, Phthalates
High-throughput platform for rapid TEER measurement of Organ-on-a-Chip endothelial and epithelial tubules. (#350)
W. Strijker1, A. Nicolas1, T. Olivier1, H. L. Lanz1, S. J. Trietsch1
1 Mimetas BV, Leiden, Netherlands
Organ-on-a-chip technology has rapidly grown in the past decade, driven by the need for better predictive in vitro models for drug efficacy and toxicity assessment. These systems enable the formation of endothelial and epithelial tubules that are used to mimic in vivo cues such as flow exposure, mixed co-culture and overall micro-environment. In conventional drug toxicity and transport studies, Trans Epithelial/Endothelial Electrical resistance (TEER) is used to determine the integrity of barrier tissues. However, current approaches to TEER measurement involve the use of chopstick electrodes, incompatible with high-throughput Organ-On-a-Chip platforms, such as the OrganoPlate®, a microfluidic platform for perfused 3D cell culture.
To this end, we developed a fully automated TEER measurement platform capable of addressing up to 96 tubules in an OrganoPlate. The developed system makes use of an electrode interface compatible with the OrganoPlate microfluidics layouts. The system is lightweight, fits in an incubator and can be used in combination with a rocker platform to provide perfusion in parallel to long-term TEER experiments. The device can read out an entire OrganoPlate with 96 perfused tubules within 60 seconds and allows programmable measurements over the entire duration of an epithelial/endothelial study.
We quantified TEER values in multiple epithelial/endothelial tubules, including widely used primary and immortalized cell lines such as Caco2, Huvec and HepG2, and developed an automated signal analysis solution, suited for high-throughput assays in the OrganoPlate. We used the platform to investigate drug toxicity and inflammatory processes on OrganoPlate grown gut tubules, which exhibit TEER values relevant to characterized caco2 models, and demonstrate the use of TEER in combination with the OrganoPlate for long term compound exposure studies.
Complementing the OrganoPlate scope of application, we developed a novel technique for on-a-Chip epithelial/endothelial tissue TEER investigation with very fast measurement times, automated signal extraction and compatible with tissue culture incubator environments. This provides a valuable tool for drug toxicity and transport studies in Organ-on-a-Chip.
Keywords: Trans Epithelial/Endothelial Electrical resistance (TEER), organ-on-a-chip
Evaluation of 6 in silico skin penetration models to predict the in vitro cutaneous distribution of 25 chemicals (#359)
M. Klaric1, R. Cubberley2, I. Sorrell2, H. Duplan3, J. Eilstein4, C. Ellison5, S. Gregoire4, N. Hewitt1, C. Jacques-Jamin3, D. Lange6, C. Lester5, H. Rothe7, S. Salhi8, A. Schepky6
1 Cosmetics Europe, Brussels, Belgium
In this study, 3 open source (EPA DermWin, Kasting CDC, University of Surrey) and 3 commercial (GastroPlusTM TCATTM, Certera Simcyp, Scientific Consilience DSkin) in silico skin penetration models were evaluated. These varied in complexity but were mainly based on physical chemistry of diffusion, with different degrees of physiological relevance built in. Simulations of the cutaneous distribution of 25 chemicals were compared with measured data from in vitro, finite dose, human skin penetration studies. Each group was provided with the same set of input parameters, including physicochemical properties and details of the skin penetration protocol. The DermWin model was included because it is used by safety assessors; however, it predicts Kp and is not suitable for the prediction of cutaneous distribution and was therefore excluded from several comparisons.
The prediction of the dermal delivery (DD, amount in epidermis + dermis + receptor fluid) of 24 chemicals applied in PBS (1 was applied in ethanol) varied between the models. The best correlation with in vitro data was observed for the TCATTM model, with only 2 outlier chemicals over- or under-predicted by more than 2-fold. The over-prediction of DD of 6-8 chemicals by 3 other models is considered to be conservative in terms of potential use of these models within human safety assessment. The ability to predict the amounts in epidermis and dermis varied between models; however, the maximum amounts measured in ex vivo human skin were only 10% and 3% of the applied dose, respectively. The predicted amounts in the receptor fluid by each model were generally within 2-fold of the measured values (which ranged up to 95% of the applied dose). None of the models adequately predicted the amount of chemical that evaporated. This was shown to be important since the prediction of DD was improved when the evaporated amount was accounted for in the simulations (estimated using the mass balance).
In conclusion, our evaluation highlighted important differences in 6 models. The 5 mechanistic in silico models could predict the DD of 24 chemicals relatively well, especially if the fraction evaporated was considered. Amounts of chemical in the epidermis and dermis were less well predicted, as was the amount evaporated. Future work will investigate how measured data can be used to improve the models further.
Keywords: in silico skin penetration model, dermal delivery
TKPlate: A web-based platform for toxicokinetic modelling in food safety (#370)
N. Quignot1, W. Wiecek2, B. Amzal2, J. L. Dorne3
1 Analytica LASER, Paris, France
The integration of toxicokinetics (TK) is a critical issue for all areas of human health risk assessment of chemicals including the food safety area. In parallel, high-throughput screening methods, in vitro to in vivo extrapolation and in silico models for predicting chemical properties are increasingly being developed and improved.
Here, in the context of an EFSA-funded project, TKPlate has been built as a web-based suite of generic TK tools and models within the R freeware as an extension of the US-EPA ‘httk’ package. TKPlate allows the prediction of TK parameters for single and multiple compounds in data poor and data rich situations. Simple TK tools and physiologically-based models were built from data collection reporting human and animal physiological data as well as meta-analyses of human TK variability for parameters reflecting both acute and chronic exposure. In addition, chemical-specific data for a number of compounds, available in the ‘httk’ package and from specific case studies, were integrated in the platform.
In food safety, data availability for TK model parameterisation range from basic in vitro TK data and compound’s physicochemical properties to a full set of in vivo TK parameters. In TKPlate, distributions describing inter-individual TK variability can be modelled using Monte Carlo methods rather than mean values. Under data poor situations, default values are also available in TKPlate for each input across the platform (i.e., both means and variability).
TK models have been applied to selected case studies. Simulations for single compounds and binary mixtures have been performed, according to realistic and EFSA relevant exposure scenarios. The flexibility of the platform allows prediction of TK parameters for subgroups of the human population (e.g., polymorphic phenotypes for the CYP2C9 and CYP2C19 metabolic pathways or age subgroups such as children, elderly), as well as magnitudes of TK interactions for binary mixtures. Overall, the results from case studies illustrate the applicability and validity of the platform. It is foreseen that TKPlate will allow the further development of predictive tools in the broad context of food safety, particularly in data poor situations where inter-species extrapolations or the use of in vitro data and read-across methods may be required.
Keywords: Food safety, Interactions, Modelling, Risk assessment, Toxicokinetics
Hierarchical Bayesian meta-analysis of human variability in CYP3A4 metabolism and CYP3A4-related uncertainty factors for human risk assessment (#446)
K. DARNEY1, E. TESTAI2, N. KRAMER3, J. - L. DORNE4, C. BECHAUX1
1 Anses, Paris, France
Historically, the default 100 fold uncertainty factor (UF) has been applied to sub-chronic toxicity data in test species for the derivation of chronic safe levels of exposure to thresholded chemicals. The default UF allows interspecies differences (10) and human variability (10) in toxicokinetics (3.16) and toxicodynamics (3.16). For human toxicokinetics, research efforts have been refined the UFs using human variability data in metabolism to derive pathway-related UFs or chemical specific adjustment factors. CYP3A4 is the major cytochrome P450 isoform metabolising more than 50% of known xenobiotics. Here, inter-individual variability in CYP3A4 metabolism for human subgroups (i.e. neonates, children, adolescents, healthy adults, elderly) was investigated for the oral and intravenous routes using parameters for acute (i.e Cmax) and chronic exposure (i.e. clearance, half-life) for 15 CYP3A4 probe substrates. All relevant human pharmacokinetic studies were extracted from a systematic review and meta-analyses, using a hierarchical Bayesian model in the R freeware, were then performed to derive parameter, route and subgroup specific-distributions for inter-individual variability in CYP3A4.
Overall, distributions for CYP3A4-inter-individual variability provided the basis to derive CYP3A4-related UF to cover 95% of the population. For chronic oral exposure, the CYP3A4-related UF in healthy adults was higher (5.0 [4.4-5.6] (median [CI90]), 10 compounds) than that for intravenous exposure (3.6 [3.3-4], 5 compounds). CYP3A4-related UFs for the elderly and neonates were respectively 5.2 [3.9-7.1] (3 compounds) and 6.3 [3.5-10.6] (1 compound). Future work includes the integration of isoform-specific variability distributions for inter-individual differences in human metabolism and in vitro data into physiologically based toxicokinetic (PBTK) models to further develop quantitative in vitro in vivo extrapolation (QIVIVE) models and refine the use of UFs in human risk assessment.
Keywords: human variability, toxicokinetics, uncertainty factor, CYP3A4-related uncertainty factor
PBPK/PD coupled HPG SB model to analyze the reproductive toxicity of EDs: case study on mixture effects of DEHP and TCDD (#582)
R. P. Sharma1, M. Schuhmacher1, V. Kumar1
1 Universitat Rovira i Virgili, Departament d'Enginyeria Quimica, Tarragona, Catalonia, Spain
In-silico modelling and simulation in pharmacokinetics and systems biology (SB) along with high throughput in vitro assays is gaining attention to assess the environmental chemicals exposure related human health effects. With the recent advances in in vitro high throughput techniques and their quantitative extrapolation based on in-vitro in-vivo extrapolation (IVIVE) approach we are able to develop high throughput physiologically based pharmacokinetics model (PBPK) predicting concentration-time profiles for large classes of chemicals even without the need of in vivo studies. Combined with SB models of cellular response at the target site will provide more realistic mechanism based predictive toxicology with a wide spectrum of important applications in the field of toxicology. The objective of this study is to analyse the reproductive toxicity of EDs based on an integrated tool that describes both the kinetics and the toxicodynamic effects via coupling PBPK to a hypothalamus–pituitary–gonadal (HPG) axis SB model, taking case study of Di (2-ethylhexyl) phthalate (DEHP) and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) mixture. Both chemicals are endocrine disruptors (ED) chemicals affecting women fertility. Ubiquitous presence of dioxins and phthalates in the environment and raised concern over their common adverse effects on ovarian cycle such as hypo-estrogenic, polycystic ovary and anovulation has catch attention towards their mixture effects. Ovarian cycle in women involves periodic single oocyte maturation and ovulation, which is controlled and regulated by hormones and their feedback loops along the HPG axis. To achieve this objective, two levels of hierarchy were used: First both a PBPK model describing the chemical-concentration time-course inside the body and a systems biology model describing time evolutions of HPG hormones, were made. Second, the two models were coupled so as to predict the ovarian toxicity considering HPG hormones as a surrogate biomarker for fertility. Developed PBPK/PD coupled HPG SB model is able to demonstrate the effects of EDs on HPG hormones, thus predicting effects on ovulations and mixture simulation. Results showed a synergistic action possibly due to their crosstalk mechanism.
Keywords: Systems Biology, PBPK/PD, EDs, Crosstalk effects, HPG axis
New approaches for parameterization and evaluation of PBK models using human in silico, in vitro, biomonitoring and volunteer data (#648)
J. Bessems1, G. Schoeters1, T. Santonen2
1 VITO, Mol, Belgium
Initially, PBK (physiologically-based kinetic) modelling was largely based on animal in vivo kinetic data and in vitro information on animal metabolic clearance parameters. In order to predict human kinetics the animal model parameters were subsequently adjusted to anatomical and physiological values of humans and the metabolic parameters were scaled (and sometimes measured using in vitro human metabolic systems). Nowadays, there is a need to construct human models straight on using only human data as to include a priori interindividual human variation and age-dependency.
This talk will illustrate the concept of bottom-up model parameterization using in silico approaches like QSAR and QPPR in combination with human and in vitro data. This bottom-up concept allows first tiers predictions of variations in human blood concentration over time (C,t)-curves as well as urinary excretion of parent chemical and/or metabolite(s) and in combination with human in vitro toxicity data also screening of chemicals for potential risks (margin of internal exposure – MOIE).
It will be shown how human biomonitoring data and data from human volunteer studies (blood concentrations or urinary excretion) can be used to evaluate the predictive capacity of these bottom-up models.
 Bessems JGM, Paini A, Gajewska M, Worth A. The margin of internal exposure (MOIE) concept for dermal risk assessment based on oral toxicity data - A case study with caffeine. Toxicology. 2017 Dec 1;392:119-129. doi: 10.1016/j.tox.2017.03.012. Epub 2017 Mar 10.
 Jongeneelen FJ, Berge WF. A generic, cross-chemical predictive PBTK model with multiple entry routes running as application in MS Excel; design of the model and comparison of predictions with experimental results. Ann Occup Hyg. 2011 Oct;55(8):841-64. doi: 10.1093/annhyg/mer075.
Keywords: PBK OR PBBK OR PBTK OR PBPK, human biomonitoring, human volunteer study, animal-free, intra-individual variation
Incorporation of public exposure models into the PLETHEM pharmacokinetic framework (#682)
S. N. Pendse1, I. Diallo1, C. I. Nicolas1, A. Y. Efremenko1, C. E. Hack1, C. Housand1, P. D. McMullen1, M. Yoon1, 2, H. J. Clewell1, 3
1 ScitoVation, Research Triangle Park, North Carolina, United States of America
An outstanding challenge in the acceptance of alternatives to animal testing is the incorporation of computational models into decision making pipelines. Fifteen years ago, the US EPA Office of Research and Development's framework for computational toxicology emphasized the need for computational methods to bridge the source-to-outcome continuum. We and others have suggested that this can be achieved by linking exposure estimation methods, physiologically based pharmacokinetic (PBPK) modeling, and computational systems biology pathway modeling tools into a standardized framework. To that end, we have developed the Population Lifecourse Exposure To Health Effects Model (PLETHEM) suite, a modular open source modeling platform that provides users the ability to create, run, share, and audit PBPK models. The platform consists of a database of chemicals, QSAR models, life-stage specific physiological and metabolism parameters needed to parameterize PBPK models, an R- based engine to perform model simulations, and an interactive user interface to define and select parameter sets for the models. PLETHEM implements easy to use interfaces for a generic PBPK model and a high-throughput IVIVE model. These model interfaces along with the included databases provide capabilities necessary for rapid analysis of chemicals using PBPK modeling. PLETHEM includes the ability to run Monte Carlo analyses to investigate population variance and a set of life-stage equations to investigate life stage-based sensitivities. The PLETHEM database also incorporates ontogeny profiles for key metabolic enzymes that can be used to calculate in vivo metabolic clearance using measured in vitro clearance. In addition, PLETHEM has an ability to link to a number of EPA and OECD exposure estimation programs, including CEM, TRA, SEEM2, and SHEDS-HT. These models, which estimate exposures in the workplace and the general populations, can be used to drive PBPK model-based estimates of resulting internal exposures to support risk assessments. PLETHEM is now freely available as an R package through the Bitbucket and Github open source repositories.
Keywords: pharmacokinetics, computational toxicology, source-to-outcome continuum, exposure modeling
Application of PBPK modelling of enterocyte exposure aids in vitro to in vivo translational risk assessment of µ-opioid-induced clinical constipation. (#724)
S. Kavanagh1, L. Rosenbrier Ribeiro1, T. Noeske2, P. Sharma3
1 AstraZeneca, Discovery Safety, Drug Safety & Metabolism, IMED Biotech Unit, Cambridge, United Kingdom
The µ-opioid receptor is often screened as part of in vitro secondary pharmacological profiling during drug discovery as agonism activity is known to lead to safety liabilities of constipation, respiratory depression and drug abuse potential. µ-opioid receptors are expressed in the gastrointestinal (GI) tract, localised to the myenteric plexus and nerve endings innervating the mucosa and enterocytes. Opioid drugs are known to act directly upon receptors locally in the GI tract (e.g. Loperamide) blocking muscle contraction and transport of mucosal fluids, leading to constipation. As many drugs are primarily optimised for oral administration, unwanted activity at the µ-opioid receptor could lead to dose limiting constipation and hinder both clinical compliance and efficacy.
Here we apply physiologically-based pharmacokinetic (PBPK) modelling of five marketed/clinical µ-opioid agonists to simulate local drug exposure at the site of action in the enterocyte of the GI tract in relation to constipation. The correlation between enterocyte concentration (Cmax), µ-opioid receptor activity (EC50) and clinical occurrence of constipation for µ-opioid receptor agonists illustrated >1x cover in vitro agonist EC50 was required to drive constipation. Enterocyte concentration was simulated for two project lead molecules at the current predicted dose to man to enable a risk assessment of their off target µ-opioid receptor agonist activity to predict the likelihood of constipation within the clinic. The data enabled compound differentiation and selection for progression. In addition the analysis provides semi-quantitative translational understanding that can be applied to risk assess future molecules that possess off-target µ-opioid receptor agonist activity.
Keywords: Opioid, MOP, PBPK, Translation
An inter-species comparison approach for triclopyr, utilizing in silico, in vitro and in vivo toxicokinetic properties, for risk assessment purpose. (#738)
M. Corvaro1, M. Bartels2
1 Dow Agrosciences, Human Health assessment, Abingdon, United Kingdom
Triclopyr is a synthetic auxin, a broad-weed herbicide, used in pasture and Rice. Hence, external human exposure is possible via dietary and non-dietary route. Herein, we present a multi-faceted approach, utilizing newly generated data from in vitro systems and existing internal exposure data from in vivo studies performed in mammalian toxicology species and human volunteers, as well as in silico predictions of systemic exposure to recapitulate oral gavage and dietary exposure profiles in various species, including humans. The oral absorption of triclopyr is complete in mammals as well as in human volunteers. In vitro comparative metabolism confirms lack of metabolism in humans, similar to other species. Additional in vitro plasma protein binding and renal clearance in primary proximal tubule cell experiments will be utilized to determine if excretion mechanisms previously investigated in an in vivo renal clearance study in dog are unique to that species, while a more rapid renal elimination in rat and human would correlate with known lower systemic levels vs. dog. Overall the results of these studies will be utilized to confirm that the triclopyr toxicokinetic profile of rats is more similar to humans, while dogs present a very specific excretion system of organic acids. These data are key to interpret toxicity features in rats. In addition, since the point of departure for triclopyr risk assessment are bases on rat studies, the similarities between these two species greatly reduce uncertainties (and, possibly, safety factors) for the use in human health risk assessment.
Keywords: Triclopyr, ADME, In vitro DMPK, exposure, kindey
Acute lead poisoning in a golden eagle from Italian Alps: an evaluation to metals exposure through blood and feathers analysis (#41)
S. Squadrone1, R. Orusa2, S. Robetto2, M. mantia1, M. Rizzi1, G. Colombero1, E. Parovel3, M. C. Abete1
1 Istituto Zooprofilattico Sperimentale, Chemistry, Turin, Italy
Lead (Pb) is a metal with high toxicity for wildlife, posing a great risk especially in long-lived predators such as golden eagle (Aquila chrysaetos), a widely distributed species of eagle in the Northern Hemisphere. The poisoning risk for raptors is manly due to the ingestion of fragments of Pb ammunition present in big- and small-game prey. A golden eagle with an acute lead poisoning (3.7 mg kg-1) was found in Aosta district and was subjected to chelation treatment in the local Center for the recovery of wildlife. The metals body burdens was determined in blood and feathers by ICP-MS five days after the onset of chelation therapy.
In blood we found with the following decreasing mean concentrations (mg/Kg): Fe (322) > Zn (6) > Cu (1.4) > Pb (0.93) > Al (0.75) > Se (0.19) Ni (0.12) > Sn (0.089) > Mn (0.056) > Cr (0.032); As, Cd and Co were under the limit of quantitation (LOQ= 0.010 mg kg-1). In feathers we found: Zn (80) > Fe (36) > Pb (32) > Al (27) > Cu (18) > Mn (1.5) > Ni (1.0) > Se (0.57) > Cr (0.34) > Sn (0.13) > As (0.11) > Co (0.055) > Cd ( <LOQ).
We report an eagle lead poisoning, with abnormal concentration of lead (0.93 mg kg-1 and 32 mg kg-1 respectively) in blood and feathers. In fact, a blood level above 0.2 mg kg-1 indicates lead exposure, while a level above 0.6 mg kg-1 could lead to lethal poisoning without chelation treatments. Metals other than lead did not show values of concern in the examined eagle, and are in the range found in birds of prey.
Moreover, avian feathers are a reliable noninvasive method that well reflected the golden eagle body burden. In fact, the concentrations of trace elements in feathers represent circulating levels in blood at the time of their formation, due to the input of blood to the feathers; and after feather maturation it still indicate metals level in the birds. We suggest that feathers could be successfully employed as a noninvasive method of choice for revealing exposure to environmental contaminants in birds.
Keywords: lead, eagle, ICP-MS, poisoning, feathers, blood
Optimization of differentiation condition for K562 cell line and rat erythroleukemia cell line (#94)
M. Otani1, K. Iwashita1, T. Utsumi1, S. Kawamura1
1 Sumitomo Chemical Co., Ltd., Environmental Health Science Laboratory, Osaka, Japan
K562 is a human cell line established from the pleural effusion of a patient with chronic myeloid leukemia in blast crisis. Rat erythroleukemia (REL) cell line is derived from transplantable tumors from 7,12 dimethylbenz (α) anthracene-induced erythroleukemia in the Long-Evans rat. Inducers such as hemin, butyric acid and hydroxyurea are known to induce differentiation of K562 cells into erythroid cells, and glycine, dimethyl sulfoxide (DMSO) and hexamethylenebisacetamide (HMBA) are known to induce differentiation of REL cells into erythroid cells. Therefore, these cells are used as in vitro model for erythroid cells. However, optimal condition for differentiation of these cells into erythroid cells was not fully examined.
We investigated several inducers and the concentration of inducers, and medium composition such as fetal bovine albumin (FBS). For K562 cells, hemin, sodium butyric acid (NaB) and DMSO were chosen for candidate inducers. For REL cells, glycine and HMBA were chosen. As a result, 1 mM sodium butyric acid and 10% FBS in Roswell Park Memorial Institute Tissue Culture Medium (RPMI) 1640 was optimal for K562 cells, and 1 mM HMBA and 40% FBS in RPMI 1640 were optimal for REL cells. Under these conditions, K562 and REL cells can stably differentiate into erythroid cells.
Keywords: K562, rat erythroleukemia, anaemia
High-throughput and non-depletive quantification of chemicals in a 3D liver microtissue in vitro assay. (#98)
A. Teixeira1, M. - Y. Lee1, B. Nicol1
1 Unilever, Safety and Enviromental Assurance Centre, Bedford, United Kingdom
In principle, quantitative in vitro to in vivo extrapolation may be performed based on nominal effect concentrations of chemicals in an in vitro assay and relating these to total plasma concentrations in vivo. However, one aspect that this approach neglects is that the actual biologically effective dose both in vivo and in vitro can be substantially different from the applied or total dose due to processes of partitioning/binding, transport, metabolism, degradation, solubility or evaporation.
In this study, a semi-automated method was applied to determine the free concentration and the depletion by metabolism of diclofenac and dextromethorphan directly from a 96-well plate format in vitro assay (3D liver microtissues), over a period of 48h. The method makes use of novel ultra-thin solid phase micro-extraction fibers that allow repetitive, non-destructive sampling from low volume (<100uL) wells/samples.
Results were comparable to those of traditional liquid sampling, whilst allowing sampling multiple time points from the same well (reducing the number of microtissues required) and the direct measurement of the free concentration in solution.
Keywords: solid phase micro-extraction, free concentration, in vitro biokinetics, metabolism
Training to prepare human health undergraduate students to respond to biological incidents (#118)
A. Peña Fernández1, F. Machado-Ferreira1, M. C. Lobo-Bedmar2
1 De Montfort University, Faculty of Health and Life Sciences, Leicester, Leicestershire, United Kingdom
Academics with experience from the field during the 2014-16 Ebola outbreak (West Africa) have created basic competences to train human health science students how to respond to chemical/biological incidents following major competences recently identified by the European Commission. Our competencies were divided into six domains: identification of the risk and risk analysis; toxicological effect of chemical/biological agents; planning and organisation of an intervention programme; environmental planning; communication and information management; safety and personal protective equipment; societal and ethical reflections. We have developed a brief educational programme composed of different training sessions (lectures and research-led workshops), which cover each of the different phases of an appropriate response to any of these incidents. The biological training was tested in different programmes at De Montfort University (UK) in 2016/17: BSc Biomedical Science (BMS; n=121) and Medical Science (BMedSci; n=24). Biomedical scientists are critical for provision of an appropriate response to these events, as early diagnosis is of paramount importance to contain the spread of hazards and for patient care. The training developed seemed successful in providing these students with the created competences. Thus, 93.1% of BMS students indicated that they acquired some knowledge of prevention and preparedness against a biological incident (63% agreed; 30.1% strongly agreed); and 94.8% reported that they learnt how to establish some public health interventions to protect humans in the aftermath of a biological incident (63.2% agreed; 31.6% strongly agreed). In relation to the BMedSci students, only 6.7% of them highlighted that they did not learn how to tailor a remedial programme and nearly 70% reported that the use of the “UK Recovery Handbook for Biological Incidents” (Public Health England, 2015) was an appropriate resource for tailoring a recovery response and aided their environmental recovery learning. To our knowledge, this is the first attempt at introducing basic training to respond to biological incidents in an undergraduate programme. Finally, this training could easily be adapted and introduced into other health science programmes to provide this relevant training to future health professionals.
Keywords: biological incidents training, emergency response, recovery and restoration, environmental decontamination, undergraduate training
Activities of the Iberoamerican Network of Toxicology and Chemical Safety (#190)
E. de la Peña1
1 Consejo Superior de Investigaciones Científica, Environmental Toxicology, Madrid, Spain
There is done an exhibition of the activities that from the Iberoamerican Network of Toxicology and Chemical Safety (RITSQ) is realized so much in the information and advertisement of congresses and meetings, since of the information of events of toxicological interest, which have realized and developed in iberoamerica, throughout the years passed from the creation of the RITSQ (2007) and of the web (2008) and the continued presentation of 100 posters of the RITSQ in Congresses and Meetings. The RITSQ has realized three meetings in IUTOX's: Congresses celebrated in Montreal 2007 (Canada), Barcelona 2010 (Spain) and Merida 2016 (Mexico). Throughout this time from March 2008 to 2018, the number of visits to the web page has increased sensitively, having a total of 60,842 users of 140 country, with 90,027 meetings and a total of 188,673 visits to pages. The RITSQ wants to be a platform of communication of all the toxicological activities be going to be realized and he compromises himself in publishing and spreading the above mentioned activities of toxicological interest.
Keywords: networks, toxicology, iberoamerican, visits
Development of a novel in vitro aerosol exposure system: the Independent Holistic Air-Liquid aerosol exposure system (InHALES) (#192)
S. Steiner1, P. Herve1, S. Majeed1, C. Pak1, S. Frentzel1, J. Hoeng1
1 PMI R&D, Philip Morris Products S.A. (part of Philip Morris International group of companies), R&D, Neuchatel, Neuchâtel, Switzerland
Simulating the functional and structural complexity of the human respiratory tract for in vitro aerosol exposure experiments poses a significant technical challenge. This is due to a need for preservation of relevant exposure parameters that occur naturally during breathing (e.g., flow velocities and patterns, gradual changes in test atmosphere properties, residence times, and test atmosphere dilution).
We developed a novel aerosol exposure system, the Independent Holistic Air-Liquid Exposure System (InHALES), that resembles the human respiratory tract structurally and functionally. The key features of the system include:
Keywords: in vitro aerosol exposure, Exposure system, Respiratory tract simulation
Evaluation of sex differences in diazepam and amphetamine conditioned place preference in Sprague Dawley rats. (#207)
S. Breche1, E. Sablé1, V. Castagné1, N. Madar-Balakirski2, H. Kedar2, C. Froger-Colléaux1
1 Porsolt S.A.S., Le Genest Saint Isle, France
The revised FDA guideline January, 2017 related to the assessment of abuse potential of drugs, include the recommendation for a New Chemical Entity to be evaluated in both male and female rodents. Nevertheless, despite evidence for an impact of sex difference in humans, the corresponding impact in rodents needs further evaluation. The aim of this study was to evaluate the impact of sex on diazepam and amphetamine -induced conditioned place preference (CPP) in rats using a two-chambered CPP apparatus and a biased procedure.
Four groups of 12 male or 12 female Sprague Dawley rats were subjected to a pre-conditioning session with free access to both sides of the CPP apparatus and were then assigned to groups based on initial preferences for a compartment. Rats were then given 2 conditioning sessions per day for 4 days, where they were allowed to explore one side of the two-chambered box after an i.p. administration of drug (diazepam or amphetamine) or vehicle (physiological saline). Drugs were paired with the least preferred compartment. 24 hours after the conditioning, the rats were evaluated for preference for the drug-paired compartment. The data are expressed as percent time spent in the drug-paired compartment test session (20 minutes) and compared to the control group using unpaired Student’s t test.
In male rats, diazepam 2.5 mg/kg i.p. did not affect the percent of time spent in the drug-paired compartment compared with vehicle controls (+7%, NS). Amphetamine 2 mg/kg i.p. significantly increased the percent of time spent in the drug-paired compartment (+27%, p < 0.01) with a similar trend at 1 mg/kg i.p. (+18%, p = 0.0675). In female rats, diazepam 2.5 mg/kg was also devoid of effects, whereas amphetamine 1 and 2 mg/kg i.p. significantly increased the percent of time spent in the drug-paired compartment (+35%, p < 0.001 and +27%, p< 0.001, respectively).
These data indicate the presence of reinforcing properties for amphetamine at 2 mg/kg i.p. in male and female rats and at 1 mg/kg i.p. in female rats, suggesting no marked sex difference in drug-induced CPP in the present experimental conditions. Studies evaluating additional drugs would be useful to support the new guideline, taking into account the 3R’s principle in preclinical research.
Keywords: Abuse Liability, Place conditioning, Rat, Sex
Ni (II) complexes with different ligands express various degree of cytotoxicity (#241)
L. V. Dyakova1, T. Zhivkova2, M. Georgieva3, G. Miloshev3, R. Kalfin1, R. Tudose6, G. Marinescu5, I. Pantcheva4, E. - M. Mosoarca6, D. - C. Culita5, L. Patron5, O. Costisor6, R. Alexandrova2
1 Bulgarian Academy of Sciences, Institute of Neurobiology, Sofia, Bulgaria
The aim of our study was to evaluate comparatively the cytotoxic activity of eight Ni(II) complexes with different ligands: mannich bases (N,N’-bis(4-antipyrylmethyl)-piperazine - BAMP or N,N’-tetra-(antipyryl-1-methyl)-1,2-diaminoethane - TAMEN), non-steroidal antiinflammatory drugs (meloxicam, piroxicam, isoxicam) and monensin, on viability and proliferation of cultured human tumor and non-tumor cells.
The following permanent cell lines were used as model systems in our investigations: MCF-7, SK-BR-3, MDA-MB-231 (breast cancer), A549 (non-small cell lung cancer), HeLa (cervical carcinoma), HT29, Caco2 (colorectal adenocarcinoma), HepG2 (hepatocellular carcinoma), 8MGBA (glioblastoma multiforme) and Lep-3 (non-tumor embryonic fibroblasts).
The investigations were performed by thiazolyl blue tetrazolium bromide (MTT) test, neutral red uptake cytotoxicity assay, crystal violet staining, double staining with acridine orange and propidium iodide, comet assay and Annexin/FITC assay in short-term experiments (24 – 72 h) with monolayer cell cultures. Long-term experiments (16-30 days) were carried out to examine the influence of the compounds on 3D cancer cell colony formation.
The obtained results reveal that the compounds investigated express various degree of cytotoxicity that is time- and concentration-dependent. Ni(II) complexes with BAMP are more cytotoxic than Ni(II) complexes with TAMEN. Both mannich bases (BAMP and TAMEN) do not decrease significantly viability and proliferation of the treated cells. Ni(II) complex of monensin has been found to be the most pronounced cytotoxic agent among the compounds investigated. Lep-3 non-tumor cells are also sensitive to the cytotoxic effect of the tested Ni(II) complexes.
Acknowledgements: This study was supported by: Operational Programme “Science and Education for Smart Growth” 2014-2020, co-financed by the European Union through the European Structural and Investment Funds, Grant BG05M2OP001-2.009-0019-С01 from 02.06.2017; Fund “Scientific Research”, Bulgarian Ministry of Education and Science, Grant № Б 02 30 from 12.12.2014; bilateral project between Bulgarian Academy of Sciences and Romanian Academy.
Keywords: mannich bases, non-steroidal antiinflammatory drugs, metal complexes, cytotoxic activity, permanent tumor cell lines
A lipase biosensor using digital camera for the determination of neurotoxic compounds (#289)
M. Pohanka1, J. Zakova1, I. Sedlacek2
1 University of Defence, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic, Czech Republic
This work is focused on the construction of a biosensor with interdigitated unique bacterial homogenate with high lipase activity and colorimetric type of assay where camera of a smartphone was chosen as a detector. The biosensor was constructed as a tool of lipase inhibitors and paraoxon served as a representative analyte inhibiting the lipase.
Psychrophilic strains of bacteria isolated from in Antarctica were tested and the best isolate P4368 having huge lipase activity was chosen. Tween assay was performed as a standard method for lipase activity determination and indoxylacetate served as a substrate of lipase measurable by smartphone camera and R, G and B color channels digital analysis. Bacteria were homogenized and immobilized on polyvinylidene difluoride membrane and indoxylacetate was immobilized in the proximity of the homogenate. Paraoxon ethyl was analyzed by the standard method and by the camera-based biosensor.
The biosensor-based assay was found to determine paraoxon with limit of detection 3.72×10-8 mol/l and IC50 value for paraoxon was 4.00×10-6 mol/l. A volume 5 µl of sample was sufficient for the assay and the sample was applied directly without any processing.
In a conclusion, simple colorimetric biosensor for the determination of venomous compounds like organophosphorus pesticides and nerve agents was constructed.
Keywords: biosensor, fatty acids, neurotoxin, paraoxon, acetylcholine, pesticide, RGB, colorimetry, analytical toxicology
Useful series of positive control compounds for colony formation cytotoxicity test (#307)
K. Isama1, N. Katsuyama1
1 Teikyo Heisei University, Faculty of Pharmaceutical Sciences, Tokyo, Japan
Purpose: Relatively strong positive control compounds, such as zinc diethyldithiocarbamate (ZDEC), and zinc dibutyldithiocarbamate (ZDBC), are commonly used to test medical devices for in vitro cytotoxicity; however, they are not suitable for testing relatively weak cytotoxic compounds. Thus, we established a new series of positive control compounds that can be used to classify a wide range of cytotoxic compounds.
Keywords: cytotoxicit, positive control, phosphite triester, phosphate triester, 4-hydroxybenzoate
Treatment of impaired wounds – Understanding the therapeutic mechanisms of umbilical cord-derived mesenchymal stem cells (#325)
S. P. Camões1, A. de la Fuente2, F. Carvalho3, M. Abal2, J. M. Santos4, J. P. Miranda1
1 Faculty of Pharmacy, University of Lisbon, iMed.ULisboa - Research Institute for Medicines, Lisbon, Portugal
Mesenchymal stem cells (MSCs) have been shown to be implicated in wound healing, in a process that involves mechanisms such as homing and cell recruitment. Our previous work showed that a specific population of human umbilical cord tissue-derived MSC (UC-MSCs) and human bone marrow-derived MSCs (BM-MSCs) present different secretomes, leading to distinct effects in wound healing. Specifically, UC-MSCs secrete granulocyte-colony stimulating factor (G-CSF), which is a key factor for the recruitment of BM-MSCs by UC-MSCs. This study aimed at elucidating the ability of UC-MSCs to home to a wound site and to recruit BM-MSCs. Additionally, the UC-MSC therapeutic effect on an impaired wound healing model was also evaluated. An in vivo transplantation model for chemoattraction was used to evaluate the UC-MSC recruiting ability, confirming that systemically administered BM-MSCs, labelled with DiD fluorescence marker (BM-MSC-DID), migrated towards UC-MSCs seeded in a scaffold previously implanted in severe combined immunodeficient (SCID) mice. For exploring the homing of UC-MSCs to wounds, an injury was inflicted 1 cm far from the scaffold containing UC-MSC-DID, which attracted them to the wound site. Moreover, shRNA silencing of G-CSF in UC-MSCs (UC-MSC shG-CSF) led to the loss of their homing ability, suggesting that MSC-specific homing properties depends on G-CSF. Finally, to evaluate if UC-MSCs were able to induce in vivo healing of impaired wounds, an excisional wound healing model was used, in which characteristics of impaired healing, such as retarded closure, were achieved by inducing diabetic state in C57BL/6 mice. Preliminary observations revealed that UC-MSCs induced healing of impaired wounds by accelerating wound closure when compared to untreated wounds. Additionally, UC-MSC shG-CSF-treated wounds tendentially presented a delayed wound resolution, when compared with UC-MSC-treated wounds, further supporting the role of G-CSF in UC-MSC regeneration capacity. Overall, this work contributes for the understanding of the therapeutic mechanisms of UC-MSCs in wound healing, also providing safety data for future clinical applications in different pathological or toxicological contexts.
Keywords: Human neonatal mesenchymal stem cells, secretome, 3D cultures, impaired wound healing, G-CSF
Prevalence, Knowledge, Beliefs, and Attitudes of Doping among Future Coaches and Athletes (#340)
S. M. Aly1, M. Abdelrazik2
1 Suez Canal University, Forensic Medicine & Clinical Toxicology, Faculty of Medicine, Ismailia, Egypt
Backgrounds: Because of tremendous numbers of new substances used in doping, anti-doping program shifted their policy from detection to prevention. So it became mandatory to understand attitudes, beliefs, and knowledge to prepare efficient educational course. No Egyptian work has addressed this issue before especially it is important to study at different culture and locality. Purpose: We aimed to identify knowledge, beliefs, attitudes, and prevalence of doping among a student sample from Physical Education Faculty -Suez Canal University. Those students will be coaches in the future and already some of them athletes in different sports. Method: An anonymous self-reported validated questionnaire was administered to a representative student sample. Results: the prevalence among the surroundings was very high in comparison to self reported personal use and whom willing to use in the future. Most students showed lack of knowledge although their anti-doping attitude with negative beliefs regarding top athletes and doping control. Conclusion: it was recommended to this faculty to change their policy by early teaching an updated extended course about doping instead of giving them light course at their last academic year. This is very important to immunize the students against the surroundings with the updated knowledge that have impacts on their beliefs, behaviors, and attitudes.
Keywords: Doping, substance abuse in sport
Interplay of four genetic high risk variants for urinary bladder cancer (#364)
S. Selinski1, K. Ickstadt2, H. Gerullis3, 4, T. Otto3, E. Roth5, F. Volkert5, D. Ovsiannikov6, 7, O. Moormann6, G. Banfi8, P. Nyirady8, S. H. Vermeulen9, M. Garcia-Closas10, J. D. Figueroa11, A. Johnson12, M. R. Karagas13, M. Kogevinas14, 15, N. Malats16, 17, M. Schwenn18, D. T. Silverman10, S. Koutros10, N. Rothman10, L. A. Kiemeney9, J. G. Hengstler1, K. Golka1
1 Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), Dortmund, Germany
Little is known whether genetic variants identified in genome-wide association studies interact to increase bladder cancer risk. Recently, we identified two- and three-variant combinations associated with a particular increase of bladder cancer risk in a urinary bladder cancer case-control series (IfADo, 1501 cases, 1565 controls).
Keywords: bladder cancer, variants, risk, smoking habits, genetics
A pilot education program in collaboration with World Health Organization to increase knowledge and awareness amongst medical students of “Environmental Preventive Medicine” (#398)
E. Todaka1, S. Shiga1, K. Poore2, P. Fenichel3, C. Mori1
1 Chiba University, Centre for Preventive Medical Sciences, Chiba-City, Japan
Environmental medicine in modern society is increasingly important: reducing environmental risks will help to prevent possible adverse health effects for future generations (1). To date, this subject has not been a major subject in medical education, therefore we aim to increase knowledge and interest of the issue amongst future doctors. A unique collaboration between Chiba University, Japan, the University of Nice Sophia Antipolis, France and World Health Organization (WHO) designed an environmental education course for medical schools.
A pilot medical education course took place at University of Nice Sophia Antipolis on 22-23 November, 2017. The course was offered to medical students from University of Nice Sophia Antipolis (year 4) and Chiba University (Graduate students). More than 100 medical students attended the course, given by 10 lecturers from France, UK, USA and Japan. WHO’s existing educational modules regarding children’s environmental health (2) were used as guides for the presentations that included information on the unique susceptibility of children, developmental origins of health and disease (DOHaD), epigenetics, the effects of a range of different environmental exposures throughout the life course and possible interventions to reduce risk. Case studies were presented. Students’ knowledge of the topics was assessed before and after the course using smartphone software and course evaluation was performed by questionnaire (questions ranked 1 (lowest) to 5 (highest)) and free comments.
Results and discussion:
Knowledge of the topics was increased by the course in all students. Students found the course highly intellectually stimulating (average score 4.7) and the course was given an overall rating of 4.4 out of 5. Suggestions from students and our own appraisal of the pilot course will allow for improvements to future deliverance of the course, for example making it available to students from any year group and other health care trainees. A promotional video created from the course will be displayed during the poster presentation.
Keywords: Environmental medicine, Medical school education, World Health Organization's Module, Endocrine Disruptors, Developmental Origins of Health and Disease
A new credit system for re-registration of toxicologists adopted in The Netherlands (#438)
P. T. Scheepers1, E. D. Kroese2, M. A. van Velthoven3, J. Louisse4, J. H. Arts5
1 Radboud Institute for Health Sciences, Radbudumc, Nijmegen, Netherlands
By January 1st 2019 the Netherlands Society of Toxicology (NVT) implemented a new credit system for re-registration in line with the EUROTOX registration guidelines for the European Registered Toxicologist (ERT) of 2016.
The registrant earns credits for activities in two categories: ‘receiving’ and ‘sending’. Receiving covers advancement of the registrant’s own knowledge, expertise and skills by continuous professional development activities. Sending relates to active contributions such as to the development of the field of toxicology (talks at conferences, teaching, publishing papers etc.) or to the role of toxicology in the society (e.g. active contributions to national and international expert committees, public appearances, etc). Since both aspects are important, a minimum of 4 credits per year should be spent on ‘receiving’, and 4 credits on ‘sending’. The remaining 2 credits may be allocated to either of the two categories. The minimum requirement is 50 credits over a five year period. A surplus of credits can be transferred to the next year within the five year registration period.
The registrant declares the time spent on toxicology as part of the job description. For special projects registrants may claim credits in a portfolio. If details cannot be disclosed for reasons of confidentiality the registrant may provide a reflection report to describe the candidate’s toxicology contribution on a meta level.
The NVT validates committee memberships and other active contributions to the society directly into an online system. Likewise, the coordinator of postgraduate education in toxicology (PET) validates courses that have been successfully completed by the registrant. Two RT members of the Registration Committee will perform an independent evaluation upon the request for re-registration as ERT. Conflicting evaluations are discussed in the next Registration Committee meeting. If the registrant does not agree with the decision of the Registration Committee, an appeal can be filed that will be considered by the independent committee of appeal. This new credit system will also be facilitated by an online system to be developed for which other toxicological societies are invited to join.
Keywords: registration; continued professional development; guideline; European Registered Toxicologist
A simplified numerical scoring system to quantify skin and eye irritation/corrosion (#443)
M. PEREA1, C. CHARMEAU-GENEVOIS1, 2, F. SAHIGARA2, P. THOMAS1, 2
1 CEHTRA, L'Isle d'Abeau, France
Before in vitro studies were developed, to replace them, the assessment of skin or eye irritation using the OECD TG 404 or 405 guidelines was based on the evaluation of lesions of skin or eyes in rabbit studies according to the Draize system. This required the translation of in vivo observations into a generic scoring index subjectivity by a technician. Prior to the global harmonization system (UN GHS, 2003), other scoring systems existed, such as the Primary Irritation Index (PII) for skin irritation and the Modified Maximum Average Scores (MMAS) for eye irritation. However, neither scores can be interpreted according to the current harmonized EU CLP/ UN GHS using the cut-off limits for classification.
The aim of this work was to determine which lesion(s) of skin or eye principally drive classification in order to create a relevant, simplified score that can be used as a QSAR model input to predict the skin and eye irritation potential.
Following validation of hundreds of studies obtained from the literature, a quantitative approach was proposed for skin irritation substance classification, calculating a Simplified Irritation Index (SIISKIN) based on erythema only. For eye irritation, the quantitative approach was based on two descriptors: corneal opacity and conjunctival redness. Depending on the cut-off limits determined respectively for the SIISKIN and SIIEYE, the substances are classified as non-irritant, potentially irritant, irritant or corrosive to skin or eyes.
The SIISKIN and SIIEYE were based on validated studies from multiple chemical groups demonstrating a:
-correlation between the SIISKIN or SIIEYE and the classification following the CLP/GHS criteria.
-proportionate relationship between the SIISKIN and the PIIs and,
-poor relationship between the SIIEYE and the MMAS for the same set of substances.
Using a conservative approach, several substances were classified as corrosive instead of irritant to the eyes using the SIIEYE and the criteria for classification in Category 1 in the CLP.
SIISKIN and SIIEYE are therefore relevant scores to simplify the results available from the huge amount of in vivo data facilitating modelling to predict skin irritation and eye irritation potential, respectively.
Keywords: skin irritation, eye irritation, EU CLP/ UN GHS classification
Anti-inflammatory activity of Portuguese thermal waters (#473)
A. C. Silva1, 7, C. V. Vaz2, A. S. Oliveira2, S. Correia2, R. Ferreira2, 3, L. Breitenfeld2, 3, J. Martinez de Oliveira2, 4, R. Palmeira de Oliveira2, 5, C. Pereira1, 6, A. Palmeira-de-Oliveira2, 5, T. Cruz1, 7
1 University of Coimbra, Center for Neuroscience and Cell Biology, Coimbra, Portugal
Introduction: Thermal therapy has been used for centuries and Portugal is one of the richest European countries in mineral medicinal waters. However, in contrast to other European thermal centers, scientific studies sustaining the beneficial effects of Portuguese thermal waters (TW) are scarce. These natural resources are frequently used to reduce or treat symptoms related to skin and respiratory disorders, which are associated to an unfavorable inflammatory milieu.
Aim: In order to fill this gap the present study was focused in the evaluation of the anti-inflammatory activity of several TW in vitro, pertaining to thermal spa’s located in the Centre Region of Portugal. The molecular mechanisms behind their bioactivity were also disclosed.
Methods: The bioactivity of TW with different chemical profiles, namely four Sulphurous/Bicarbonated/Sodic (SBS), three Sulphurous/Bicarbonated/Sodic/Fluoridated (SBSF) and one Magnesium/Ferric (MF) waters, was evaluated on the following inflammatory parameters using the in vitro model of inflammation consisting of mice macrophages (RAW 264.7) stimulated with lipopolysaccharide (LPS): i) nitric oxide (NO) production; ii) expression of the pro-inflammatory enzyme inducible nitric oxide synthase (iNOS); iii) TW’s scavenging activity. Cell metabolism was also assessed through the Alamar blue assay to monitor cell viability.
Results: MF, SBSF and one SBS TW diminished NO production triggered by LPS, which seems to occur, in general, in a pH dependent manner. Interestingly, the molecular mechanisms implicated in NO depletion are apparently different since:
- Cells exposed to the MF TW (acidic pH) did not show alterations in metabolism or in iNOS expression levels but exhibited scavenging activity;
- Cells exposed to the SBS TW (basic pH) showed a decrease in iNOS expression;
- All SBSF TW (basic pH) upregulated iNOS but reduced cellular metabolism.
Conclusions: Overall, the reduction in NO production sustains the anti-inflammatory activity of several Portuguese TW, suggesting their application in several dermatological conditions according to their specific characteristics. In addition, TW able to reduce cell metabolism can be useful as therapeutic strategies to hyperproliferative skin pathologies.
This work was financed by the European Regional Development Fund (ERDF), Centro 2020 Regional Operational Programme and Economic Enhancement Program for Endogenous Resources - 3rd phase - PROVERE: project CENTRO-04-3928-FEDER.
Keywords: Thermal Waters, Inflammation, Macrophages, iNOS
Microextraction by Packed Sorbent (MEPS) for the determination of Polybrominated Diphenyl Ether (PBDE) in eggs by Gas Chromatography-Mass Spectrometry (GC-MS) (#518)
M. C. O. Souza1, B. A. Rocha1, J. M. O. Souza1, F. Barbosa Junior1
1 University of Sao Paulo - School of Pharmaceutical Sciences of Ribeirao Preto, Department of Clinical Analysis, Toxicology and Food Science, Ribeirao Preto, Sao Paulo, Brazil
The flame retardants (FR) are classified as emerging contaminants and persistent organic pollutants, they are used to reduce the fire hazards in materials used in buildings, and an important class of FR is the Polybrominated Diphenyl Ether (PBDE). Some studies have suggested the potential for harmful effects to human health for many of these compounds. The mains of exposure routes are through foods, including eggs, fish, meat, and milk, and also through of inhalation and ingestion dust. In this present study, a novel method combining microextraction by packed sorbent (MEPS) and gas chromatography coupled to mass spectrometry (GC-MS) was developed and validated for the extraction, preconcentration, and determination of 6 congeners of PBDEs (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, and BDE-154) in eggs samples. The advantages of this method include small amount of sample required in the analyzes (20 mg of freeze-dried egg), low quantification limit (LOQ = 0.4 µg/g lipid), absence of matrix effect , high recovery, reproducibility and repeatability of the results, and use of the sample preparation technique not yet described for determination of PBDEs in food samples. The method was applied in 40 eggs commercial samples, and the BDE-47 was quantified in 30% of them. The BDE-100 and BDE-153 were quantified in 5 analyzed samples. And the BDE-99 and BDE-154 were determined in 2 and 1 samples, respectively. The mean of the sum of PBDE was 1.41 µg /g lipid. The Estimated Daily Intake (EDI) calculated was 0.135 ng de PBDE/kg-day. The results of this study provide preliminary and unpublished data regarding the concentrations of PBDEs in foods consumed in Brazil.
Keywords: Polybrominated Diphenyl Ether, Microextraction By Packed Sorbent, eggs, GC-MS, emerging contaminants, analytical toxicology
JTp and Tpe as Biomarkers of Proarrhythmic Risk in Nonclinical Models: Historical Data Evaluation by the HESI Consortium (#523)
S. Authier1, 2, M. M. Abernathy3, E. Boulay1, R. Chui4, G. S. Friedrich5, N. Gendron-Parra1, A. Greiter-Wilke6, J. - M. Guillon7, D. J. Leishman3, J. Nichols8, J. Pierson9, M. Skinner10, M. K. Pugsley11, J. - P. Valentin12, H. M. Vargas4, T. Wisialowsky13
1 University of Montreal, St-Hyacinthe, Québec, Canada
A comprehensive analysis of T-wave morphology changes has recently emerged as a testing strategy to evaluate drug-induced ion channel blockade and proarrhythmic risk using ECG data from clinical trials. This retrospective study assessed the JTpeak and Tp-e intervals as novel non-clinical proarrhythmia biomarkers. Lead II ECG telemetry data was analyzed from Beagle dogs and monkey studies by members of the HESI consortium using pattern recognition methods with various software platforms. JTpeak and Tp-e duration was also quantified in groups given known drugs. Baseline JTpeak and Tp-e were HR dependent in both species and individual rate correction methods were evaluated as JTpeak corrected (JTpc) and Tp-e corrected (Tp-ec). Drugs such as sotalol, dofetilide, thioridazine, cisapride, quinine and moxifloxacin were generally associated with an increase in JTpc similar to QTc interval prolongation. Drugs such as captopril, verapamil, nifedipine, minodixil and milrinone were generally associated with minimal effects or shortening of JTpc. The anesthetic medetomidine decreased body temperature, increased JTpc in monkeys; however, shortened the Tp-ec interval. Atenolol and pimobendan showed no notable effect on these two QT subinterval biomarkers. Both JTp-c and Tp-ec demonstrate promise as complementary biomarkers to drug-induced QT interval changes and may play a role in proarrhythmia risk assessment. However, experimental methods and factors that influence variability of these ECG parameters in non-clinical species need to be evaluated. Evaluation of a broad range of control drugs to validate these measures is required to establish their translational and discriminative value in drug safety pharmacology studies.
Keywords: proarrhythmic risk, ECG, JTp and Tpe intervals, proarrhythmia biomarkers, QTc prolongation
TOX-OER MOOC: THE LIVER AS TARGET ORGAN (#526)
C. Rocha-Pereira1, J. Soares1, A. G. Casanova2, H. Carmo1, F. Carvalho1, M. D. L. Bastos1, F. Remião1
1 UCIBIO/REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences of Faculty of Pharmacy of University of Porto, Porto, Portugal
TOX-OER (Learning Toxicology through Open Educational Resources) is an Erasmus+ Action KA2 Project, involving seven European countries, which aims to develop an international Massive Open Online Course (MOOC) on Toxicology. Its purpose is to enhance digital integration in learning, teaching, training and youth work at various levels by developing scientific, pedagogical, informative and formative materials. TOX-OER MOOC platform is already available online (https://toxoer.com/), being the MOOC in English and all partner-country languages in a construction process.
The MOOC is organized into seven modules: General Concepts; Pharmaco-Toxicokinetics; Main Groups of Xenobiotics; Environmental Pollutants; Target Organ Toxicity and Biomarkers; Environmental Toxicology; and Patents and Patent Application. They constitute a total of 31 ECTS and include an introduction to the module, video lessons, intermediate evaluation or active online learning activities, text based learning resources, a final evaluation test and bibliography.
The Target Organ Toxicity and Biomarkers module (8 ECTS) includes 5 topics, one of which is dedicated to liver toxicity (2 ECTS). Hepatic physiology and structure are described, as well as their tight relationship with hepatotoxicity. Hepatotoxicity mechanisms are presented, namely those related to intrinsic and idiosyncratic (allergic and non-allergic) xenobiotic-induced hepatotoxicity. Both direct and indirect mechanisms of toxicity, such as the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), lipid peroxidation, mitochondrial injury, cytoskeleton rupture, massive calcium entry, or binding to cell macromolecules (eg, DNA and proteins), are described. Additionally, a full unit is dedicated to the explanation of the main types of liver damage, underlying mechanisms and examples of agents inducing hepatotoxicity. Particular relevance is given to hepatocellular death, inflammation/hepatitis, steatosis, fibrosis and cirrosis, cholestasis and tumors. A section refers to clinical manifestation of hepatotoxicity and biomarkers of liver damage.
The use of this pedagogical tool focused in the liver will allow the learning of Toxicology, not only in the classroom but also in any computer of the world. This project has been funded with support from the European Commission.
Keywords: Toxicology Education, MOOC, Liver, Hepatotoxicity, Erasmus+
Requirement of Nrf2 for termination of liver regeneration caused by acute proliferative response (#531)
S. Takasu1, Y. Ishii1, A. Kijima1, K. Ogawa1, T. Umemura1, 2
1 National Institute of Health Sciences, Division of Pathology, Kawasaki-shi, Kanagawa, Japan
Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcriptional factor that regulates expression of detoxification and antioxidant enzymes in response to cellular events such as electrophilic and oxidative attacks. Recently, it has been reported that lack of Nrf2 delays the completed recovery in terms of the weight after partial hepatectomy (PH), suggesting that Nrf2 plays an important role in the process of liver regeneration. However, its detailed molecular mechanisms are not fully understood. In present study, the kinetics of proliferative hepatocytes in Nrf2-deficient mice after PH was compared with those in their wild type of mice. A PH or sham operation was performed on 10 or 11-week-old male Nrf2-deficient and Nrf2-wild-type mice. The labelling indices (LIs) of proliferating cell nuclear antigen (PCNA)-positive hepatocytes were examined at 6, 24, 48, 96 or 168 hours after PH. The LIs were elevated and reach the peak at 48 hours after PH in Nrf2-wild-type mice. Although the LIs returned to the normal level at 168 hours after PH,in Nrf2-wild-type mice, those in Nrf2 deficient mice remained at high level and reach the peak at this time. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level were significantly elevated from 6 to 48 hours and decreased from 96 to 168 hours after PH in both genotypes, indicating that there were no inter-genotype differences in the extents of hepatocyte injuries. Western blot analysis showed that the level of phosphorylated STAT3 in the liver of Nrf2-wild-type mice was higher than that in Nrf2-wild-type mice at 168 hours after PH. The overall data suggested that Nrf2 might be required for terminating liver regeneration. Considering no inter-genotype changes in liver injury, Nrf2 might directly regulate hepatocyte proliferation by inactivation of STAT3-related mitogenic responses.
Keywords: Nrf2, liver regeneration
Spontaneous Lesions Associated with Test Article-Unrelated Deaths in Repeated Dose Rat Studies (#537)
L. Pan1, S. McPherson1, T. Zhou1
1 WuXi AppTec (Suzhou) Co., Ltd., Toxicology, Suzhou, China
Distinguishing test article-related and –unrelated deaths is critical in interpreting nonclinical study data as deaths of unknown cause that may be test article-related warrant further understanding of the mechanism of the drug candidate and bring more challenges to risk assessment and ultimately regulatory submission and approval. Conventionally and scientifically, the cause of the deaths occurred in animal studies is judged by comprehensive considerations including comparison with the concurrent control, consistency of dose dependency (more applicable to small molecule chemical drugs), time and incidence of deaths, reference to background pathology lesions of the specific species, and comparison of findings with animals that survived to scheduled termination. The purpose of this investigation is to analyze the spontaneous lesions that resulted in test article-unrelated deaths in rat studies conducted at the facility and provide references as background data. A retrospective analysis was performed on approximately 100 repeated dose rat studies of 4-week duration or longer completed so far. The animals used on studies were Sprague Dawley rats sourced from BioLASCO Taiwan Co., Ltd. and Vital River Laboratory Animal Technology Co., Ltd. Beijing and Wistar Han rats sourced from Charles River Laboratories, USA. The results showed that test article unrelated deaths due to spontaneous lesions were noted in approximately 13% studies and represented approximately 1‰ of total animals used on the studies. Among these, about half of the incidences were related to spontaneous tumors including lymphosarcoma, histiocytic sarcoma, mammary adenomas, lymphocytic leukemia, and other solid tumors (masses); the remaining incidences were considered related to other lesions in various organs.
Keywords: Spontaneous Lesions, Rat, Death, Repeated Dose
Evaluating the usefulness of human-induced pluripotent stem cell-derived intestinal organoids in drug discovery research (#543)
A. Nakanishi1, D. Onozato1, H. Okumura1, T. Hashita1, T. Iwao1, T. Matsunaga1
1 Nagoya City University, Graduate School of Pharmaceutical Sciences, Clinical Pharmacy, Nagoya, Japan
The small intestine is endowed with drug transporters and drug metabolizing enzymes, and thus has a pivotal role in the absorption of orally administered drugs. Currently, the pharmacokinetics in the small intestine is mainly evaluated using animal models and Caco-2 cells. However, the results are not necessarily accurate owing to species differences or disparities in the expression levels of drug metabolizing enzymes. Therefore, novel evaluation models that imitate the human tissue need to be developed. Recently, human intestinal organoids (HIOs) were generated from human-induced pluripotent stem cells, and contain an epithelium besides the various cells which constitute the intestinal tissues. Although the HIOs are featured as a novel in vitro evaluation system, they are still immature. Hence, we generated more mature HIOs and evaluated their applicability as a useful evaluation system in drug discovery research. The mRNA expression of the intestinal markers and pharmacokinetics-related genes was markedly increased with the addition of differentiation-inducing small molecule compounds. Using immunostaining, tight junction proteins, cell proliferation markers, and drug transporters were also examined. Thus, we suggest that the small molecule compounds used are useful for promoting differentiation in HIOs. Additionally, we maintained the HIOs for two more months and noticed sustained high gene expression levels. Furthermore, the HIOs were transplanted under the kidney capsule of immunodeficient mice and observed their engraftment and expansion after eight weeks of transplantation. The usefulness of HIOs for predicting the human intestinal pharmacokinetics and drug toxicity is evident from our results. HIOs are also expected to be applied to cell sources for the treatment of inflammatory bowel disease as well as the generation of in vivo models that have a human intestine.
Keywords: Cell differentiation, Experimental models, Induced pluripotency, Intestinal organoids, Pharmacokinetics
Developmental neurotoxicity study of zeta-cypermethrin (#574)
N. Nedopytanska1, M. Prodanchuk1, N. Kornuta1, Y. Kolianchuk1, T. Usenko1, I. Rashkivska1
1 L.I. Medved's Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Laboratory of experimental toxicology and mutagenesis, Kiev, Ukraine
Influence of chemicals, in particular pesticides, on the developing organism represents a serious risk in development of neurological disorders. According to the epidemiological data, children who exposed to pesticides during prenatal or early postnatal periods suffer from different neurobehavioral disorders, which include hyperactivity, learning disabilities, autism, behavioral and emotional problems. Epidemiological observations can point out only at the relation between pesticides exposure and various neurological deficits. While experimental studies on laboratory animals can show the neurotoxic effects of pesticides on developing organism. The present study was conducted in accordance with OECD 426 and GLP requirements. Developmental neurotoxicity study includes special approaches in investigating clinical observations, physical development, behavioral ontogeny, neuropathology, learning and memory in different periods of rats development. Dams Wistar Hannover were administered by zeta-cypermethrin orally at doses 0; 5.0; 12.5; 35.0 and 70.0 mg/kg/bw from day 6 of gestation to day 21 of lactation. Young and adolescent rats were more sensitive to exposure of zeta-cypermethrin at doses 35.0 and 70.0 mg/kg (by landmarks of physical development, clinical observations and repeated behavioral reactions). In adult rats were detected changes in sexual behavior and memory testing at dose 70.0 mg/kg. In comparison between sexes males were more sensitive. Exposure of zeta-cypermethrin from day 6 of gestation to day 21 of lactation at doses 35.0 and 70.0 mg/kg/bw induce the developmental neurotoxic effects in rats. Developmental neurotoxicity study should be conducted for pesticides which show neurotoxic effects on adult rats in toxicity investigations.
Keywords: Developmental neurotoxicity study, pesticides, zeta-cypermethrin, behavioral testing.
Review of methods for detection of toxic compounds involved in animal poisoning (#587)
I. Valverde1, S. Espín1, I. M. Navas1, E. Martínez-López1, R. Mateo2, A. J. García-Fernández1
1 University of Murcia, Sociosanitary sciences, Murcia, Spain
Illegal use of poisoned baits to kill animals is a serious threat to wild and domestic animals and public health. Although European law condemns and pursues this practice, animal poisoning is still common. A suitable post-mortem examination and toxicological analysis are essential to determine poisoning cases. These cases are a challenge for Toxicology laboratories mainly due to the variety and complexity of biological matrices with different stages of decomposition and the numerous products that may be involved. The aim of this review is to collect information regarding different methods used to analyzed the main compounds involved in animal poisoning, such as anticholinesterase pesticides, rodenticides, and other products used to kill animals, including α-chloralose, strychnine or metaldehyde, in samples of animal origin, reported in the last decades, which will help to develop and optimize new techniques applicable to cases of poisonings. For this purpose, an extensive search of the literature available was conducted using keywords (forensic, poisoning, animal, analysis), plus the noun of the searched compound. Published techniques are mainly described for the analysis of anticholinesterase pesticides, rodenticides, strychnine and metaldehyde. The most commonly analyzed samples are blood and liver. In addition of these matrices, it is important to highlight the analysis of baits. The most frequently used methods for extraction of toxic compounds are based on solid phase extraction procedures (SPE and dSPE). Depending on the chemical compound and the analytical technique used, different sample amount is needed. The extraction solvent more frequently used is acetonitrile. Both, LC-MS and GC-MS, are the analytical techniques more widely used. The matrix interference could be one of the most interfering factors on the analytical results, therefore, current methods should be assessed and optimized to be used on different matrices and to detect different toxic compounds involved in animal poisoning. Acknowledegments: To SENECA Foundation (MASCA’2014 project. 19481/PI/14 and 20031/SF/16). To MICINN (RECODEP Project CGL2013-40975-R).
Keywords: rodenticides, anticholinesterases, strychnine, metaldehyde, animal poisoning
Toxicological assessment of S-(diethyl-amino) ethanol-ether of para-brom-benz-thio-hydroxime acid hydrochloride in animals (#593)
M. Prodanchuk1, P. Zhminko1, V. Kryvenchuk1, M. Zinovieva1
1 L.I.Medved's Research Center of Preventive Toxicology, Food and Chemical Safety Ministry of Health, Ukraine, Institute of Experimental Toxicology and Bio-Medical Research, Kyiv, Ukraine
Experimental toxicity assessment by adopted OECD guidelines is an important stage of selection of candidate chemical substances during the development of a new specific purpose product. Toxic properties of the synthesized substance – S-(diethyl-amino) ethanol-ether of para-brom-benz-thio-hydroxime acid hydrochloride (S-XX) having properties of reactivator of cholinesterase inhibited by organophosphorus compounds were studied. Purpose of the study was to evaluate acute and repeat dose toxicity of S-XX in laboratory animals.
Acute toxicity of intramuscularly (i.m.) administered S-XX was studied in accordance with OECD Test Guidance 425 in rats, and mice. Repeat dose toxicity of S-XX administered i.m. was studied in accordance with OECD Test Guidance 407 in rats.
S-XX exhibited low acute toxicity when administered i.m. to rats and mice. LD50 of S-XX (i.m.) for rats and mice was found to be greater than 500 mg/kg of body weight. No substantial interspecies variability of S-XX acute toxicity was observed. Daily i.m. administration of S-XX to rats at dose equivalent 1/20 LD50 during 10 consecutive days did not cause any adverse effect.
Keywords: para-brom-benz-thio-hydroxime, reactivator of cholinesterase, toxicity
Evaluation of e-cigarette liquids on labelling, packaging and technical features prior the adoption of the Tobacco Products Directive (TPD) in multiple European countries. (#619)
M. N. Tzatzarakis1, C. Girvalaki1, A. Vardavas1, P. Stivaktakis1, A. Nosyrev1, G. Leon1, A. M. Tsatsakis1, C. Vardavas1, 2
1 University of Crete, Laboratory of Toxicology, Medical School, Heraklion, Greece
Aim: This study aimed to identify, record and evaluate the compliance of e-cigarette liquids to regulations on labelling, packaging and technical design features of e-cigarettes before the implementation of Article 20 of the Tobacco Products Directive (TPD).
Methods:Within context of the Horizon2020 (EUREST-PLUS project), 122 of the most commonly sold e-liquids in 9 European Countries (France, Poland, Germany, the Netherlands, United Kingdom, Spain, Romania, Hungary and Greece) were randomly selected and purchased. Once all products were obtained and in order to minimize human error, 2 researchers independently evaluated them as for their compliance to technical features noted under the TPD on the vial, external packaging and leaflet.
Results: In general, the products evaluation showed good compliance with the most of the parameters. A low compliance of the products was recorded regarding the tamper proof seal in total (59.0%). The products with the lowest compliance scores were Romania (20.0%) and Spain (38.9%) while the remaining countries ranged from 50.0% (Poland) to 83.3% (United Kingdom). The 85.3% of the products evaluated were within the limit of ≤10 ml refill volume cut off while the countries with the lowest compliance were Romania (20.0%) and Spain (66.7%). While assessing for nicotine content ≤ 20mg/ml, 95.9% of the products were in compliance with TPD. The majority of the products (93.4%) were also in good compliance regarding the child proof seal except for Germany (66.7%) and Romania (80.0%). The presence of 5 basic components or a list of ingredients was also evaluated. In 94.3% of the products, a list of ingredients was identified. Good compliance was also noted in most of the 5 parameters evaluated (nicotine content, 97.5%; batch number, 89.3%; recommendation for child safety, 97.5%) except for descending order of the weight and nicotine delivery per dose which was not noted in any of the products purchased.
Conclusions:To our knowledge, the present study is potentially the first comprehensive evaluation of e-cigarette refill liquids across multiple EU MS, which will serve as a pre-TPD reference assessment.
Funding: This work was supported by a grant from the European Commission (Horizon2020 HCO-6-2015; EUREST-PLUS: 681109).
Keywords: e-cigarette liquids, labeling, packaging, technical features, tobacco products directive
Polyethylemine coated silver nanoparticles induce human neutrophils’ oxidative burst via NADPH oxidase, through the activation of PKC (#620)
T. Soares1, M. Gónzalez-Gómez2, J. Rivas2, P. Freitas3, F. Carvalho4, E. Fernandes1, M. Freitas1
1 LAQV, REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry, Faculty of Pharmacy, University of Porto, Porto, Portugal
Silver nanoparticles (AgNPs) have a wide spectrum of applications that reach far beyond therapeutics, being easily found in the everyday human life. Due to their tendency to aggregate, AgNPs are typically synthesized with surface coatings for stabilization. Branched polyethylenimine (PEI) is a multifunctional polymer that has been recently used in functionalized AgNPs with promising results. Neutrophils are the most abundant leukocytes in the blood and the first line of defense against xenobiotics, as nanoparticles. During phagocytosis, neutrophils increase their consumption of molecular oxygen, with the consequent production of reactive species (RS), a process name oxidative burst. The sustained overproduction of RS is associated with several health disorders.
Therefore, it was our aim to evaluate the activating effect of two different sizes of PEI-AgNPs (4 and 19 nm) on human neutrophils´ oxidative burst.
Our results indicate that PEI-AgNPs-induced the production of RS by human neutrophils. The use of diphenyleneiodonium (DPI), a NADPH oxidase inhibitor and Go6983, a protein kinase (PKC) inhibitor, prevented the production of RS. Interestingly, the effect was not size-dependent, since the two tested sizes have similar effects.
These results suggest that PEI-functionalized AgNPs-induce human neutrophils’ oxidative burst via NADPH oxidase, in a concentration, but not size–dependent manner, through the activation of PKC, which contributes to better understand the human immune response against these nanoparticles type.
This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013, and “Programa Operacional Competitividade e Internacionalização” (COMPETE) (POCI-01-0145-FEDER-029248), and under the framework of QREN (NORTE-01-0145-FEDER-000024). Tânia Soares acknowledges FCT the financial support for the PhD grant (SFRH/BD/128647/2017), in the ambit of "QREN - POPH - Tipologia 4.1 - Formação Avançada", co-sponsored by Fundo Social Europeu (FSE) and by national funds of Ministério da Ciência, Tecnologia e Ensino Superior (MCTES).
Keywords: Neutrophils, silver nanoparticles, polyethlemine, reactive species
Determination of environmental persistent organic pollutants (POPs) in hair samples from wild terrestrial mammals (#652)
E. Iatrou1, M. Tzatzarakis1, E. Vakonaki1, S. Papachristou1, E. Renieri1, K. Golokhvast2, 3, I. Seryodkin2, 3, V. Tsygankov2, R. Valerii4, A. Tsatsakis1
1 University of Crete, Laboratory of Toxicology, Medical School, Heraklion, Greece
Aim. The global contamination from the released amounts of POPs into the environment has raised great concern by the adverse impacts in humans and several species of marine and terrestrial ecosystems. The objective of the current study was to assess the burden of POPs in wild terrestrial mammals.
Materials and Methods. A total of 16 hair samples were collected from wild animals (leopard cats, musk deers, wolf, amur hedgehog and raccoon dogs) that found dead due to traffic accident or by hunting of Primorsky Krai, Russia. The analysis of hair samples was focus on the determination of HCB (hexachlorobenzene), DDTs (opDDE, ppDDE and opDDD), PCB congeners (28, 52, 101, 118, 138, 153 and 180) and PAHs (acenaphylene, fluorene, anthracene, phenathrene and pyrene). The detection of pollutant was conducted by a one-step hair extraction method, using headspace SPME technique and analyzed by a simple GC-MS.
Results: The use of HSSPME in the current study provides low detection limits for most of the POPs. The majority of wild animal hair samples were found positive. More specific, the percentages of positive hair samples for HCB, opDDE and ppDDE were 93.8%, 100.0%, 93.8 %, respectively. In contrast, opDDD was detected only in the 18.8 % of the samples. DDTs’ detection levels ranged from 1.26 to 52.06 pg/mg. The most frequently detected PCB congeners were PCB28, PCB52, PCB101 and PCB138 (from 93.8 to 100.0%) in a concentration range from 0.73 to 31.34 pg/mg. PCB118, PCB153 and PCB180 was detected in the 18.8, 68.8 and 6.3 % of the samples, respectively. Fluorene and anthracene/phenathrene were detected in the 100% of the samples in a range from 6.53 to 23.56 pg/mg, while the acenaphylene and pyrene in the 87.5% and 75.0% of the samples providing levels below the LOQ values.
Conclusion. The highest mean concentrations levels of all tested pollutants were found for musk deer compared to the other species. For HCB, DDTs and PCBs the order of higher to lower mean detected concentrations was: musk deer> leopard cat> wolf> raccoon dog> amur hedgehog. To the best of our knowledge, the present study is the first that provide results for the contamination levels in hair samples by persistent organic pollutants in five different terrestrial mammals.
This work was supported by the Russian Science Foundation (project No. 18-14-00120)
Keywords: Hair, terrestrial mammals wildlife, persistent organic pollutants, HSSPME-GC/MS
Psychotropic effects of nicotine and cotinine in mice (#668)
A. M. Vlasceanu1, C. Chirita2, D. P. Mihai2, C. D. Marineci2, M. Stan1, D. Gradinaru3, D. L. Baconi1
1 University of Medicine and Pharmacy Carol Davila Bucharest, Toxicology, Bucharest, Romania
Cigarette smoking is considered a chronic medical disorder, as nicotine has a highly addictive potential.
In the last decades, studies were focused on the pharmacological effects of nicotine and its main metabolite cotinine.
Nicotine and cotinine have been suggested to be psychoactive in humans and animals, facilitating memory, cognition, and executive function.
Epidemiological studies indicate that the incidence of cigarette smoking is very high among depressed individuals.
Experimental animal studies revealed the antidepressant effects of acute or chronic nicotine treatments. In addition, nicotine and other nicotinic agonists have been demonstrated to improve performance on attention and memory tasks. In animal models, cotinine was demonstrated to have antipsychotic, anxiolytic, and antidepressant properties.
Both nicotine and cotinine are very similar structurally, but despite this structural similarity, cotinine shows distinctive effects from nicotine.
The objective of this study was to investigate comparatively the antidepressant and anxiolytic effects of cotinine and nicotine in mice.
Nicotine 0.5 mg/kg and cotinine 5 mg/kg were administered orally to NMRI mice for 14 days. Pharmacological tests were performed after 1, 7 and 14 days. Antidepressant effect was evaluated using forced swimming test (FST) after acute administration and tail suspension test (TST) after 7 and 14 days. Plus suspended maze test was used for anxiolytic effect evaluation in the final day of the experiment. In order to eliminate false positive results, we also determined the mice motor activity at each test moment with the Ugo Basile activity cage.
Immobilisation time in TST was shorter in cotinine group than the control after 7 days (24.7%; p<0.05), close to imipramine reference group (31.85%; p<0.05). After 14 days, this effect no longer occurs, probably due to a significant reduction in motor activity for cotinine group which could influence the immobility time values in TST. Time spent in open arms of the labyrinth was longer for nicotine group than the control (129.61%; p<0.05), which supports a consistent anxiolytic effect.
Both nicotine and cotinine are psychoactive in mice. Further studies are needed to understand the mechanisms for these effects.
Keywords: nicotine, cotinine, antidepressant, anxiolytic
Low size citrate coated silver nanoparticles are potent inducers of human neutrophils’ oxidative burst (#686)
T. Soares1, M. Freitas1, F. Carvalho2, E. Fernandes1
1 LAQV, REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry, Faculty of Pharmacy, University of Porto, Porto, Portugal
Silver NPs (AgNPs) have emerged as an important class of nanomaterials, due to their unique optical, antimicrobial, electrical and physical properties, which enabled the use of these nanoparticles in various fields, including medical, food, health care, consumer, and industrial purposes. However, the widespread use of AgNPs raises unmet safety concerns, which may be related to their size and the coating agents used for its stabilization.
Neutrophils, as host defense cells, play a key role in recognizing, ingesting, digesting, and eliminating foreign agents, including nanoparticles. Despite the central role of neutrophils as key players in immune system, little is known about their interaction with AgNPs, with different coatings and sizes. In that sense, it was our aim to study the effect of three different sizes of citrate and polyvinylpyrrolidone (PVP) -AgNPs (5, 10 and 50 nm) in human neutrophils cell death and oxidative burst.
It was possible to conclude that the effect of AgNPs was size and coating-dependent.The citrate and 5 nm AgNPs were the most cytotoxic and potent inducers of oxidative burst, through the activation of NADPH oxidase, via the action of PKC.
This work brings new insights about the interaction of AgNPs and human neutrophils which is essential for the safer use of these nanoparticles.
This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013, and “Programa Operacional Competitividade e Internacionalização” (COMPETE) (PTDC/QEQ-QAN/1742/2014 – POCI-01-0145-FEDER-016530), and under the framework of QREN (NORTE-01-0145-FEDER-000024). Tânia Soares acknowledges FCT the financial support for the PhD grant (SFRH/BD/128647/2017), in the ambit of "QREN - POPH - Tipologia 4.1 - Formação Avançada", co-sponsored by Fundo Social Europeu (FSE) and by national funds of Ministério da Ciência, Tecnologia e Ensino Superior (MCTES).
Keywords: Silver nanoparticles, neutrophils, PKC, NADPH oxidase
Chemical Alternatives Assessment of Selected Low Aromatic C7–C18 Isoparaffin Solvents, A Class of UVCB Substances (#694)
M. zachary1, M. Whittaker1
1 ToxService LLC, Washington,, United States of America
Isoparaffins are branched aliphatic hydrocarbons with a carbon skeleton length ranging from approximately C7 to C20. They are mainly used as solvents in textile cleaning products (dry-clean cloth) due to their low odor, low surface tension and excellent cleaning efficiency. Many of the isoparaffins are multi-constituent mixtures with variable and often unknown composition and have been defined as substances with unknown or variable composition (UVCB). Because of the compositional complexity, the assessments of their toxicity and environmental fate is complicated. Grouping these chemicals according to their carbon chain length and the aromatic content is suggested by recognized scientific and advisory bodies to be an ideal way to assess their toxicity. To this end and as part of ToxServices’ Full Materials Disclosure (ToxFMD®) program, we used the GreenScreen® hazard assessment tool to assess and compare the hazard properties of four isoparrafins solvents with low aromatic content (< 2%) that are used as solvents for commercial textile cleaning. The substances belong to three different categories (short carbon chain (C7-9), medium chain (C9-14) and long-chain (C14-C20)). Our results show that all four investigated isoparrafins, regardless of the carbon length chain, are associated with a high hazard for aspiration toxicity, a common concern for such type of solvents, as their measured kinematic viscosity parameters are below the GHS Guidance value for Category 1. Aspiration hazard was recently incorporated in the GreenScreen® methodology under systemic toxicity single exposure endpoint. In terms of environmental fate (persistence) and toxicity, the properties vary with the carbon chain length; substances with short chain (C7-C11) have low potential to persist, but are toxic to aquatic organisms. In contrast, substances with a carbon length greater than 11 are not readily biodegradable but are not expected to be toxic to aquatic organisms. All compounds have high potential for bioaccumulation. These results can be utilized to characterize hazards for other isoparaffin solvents with similar properties (carbon length/aromatic content) and provide informed choices for selecting less hazardous isoparaffin solvent alternatives.
Keywords: GreenScreen, Hazard Identification, Isoparrafin
Determination of proinflammatory cytokines level and NF-κB activation level in the human lung cells line stimulated with essential oils for a vehicle ventilation system application (#714)
A. Janicka1, J. Molska1, M. Zawiślak1, A. Czarny2, E. Zaczyńska2
1 Wroclaw University of Science in Technology, Division of Automotive Engineering, Wrocław, Poland
The aim of the research was to choose non-toxic fragrance oils for use in an aroma-dispenser innovative device applied in a vehicle ventilation system. The levels of proinflammatory cytokines IL-6 and TNF-α and NF-κB activation level were determined in the human lung cells line A549 stimulated with selected essential oils.
Twenty commercial fragrance oils was used for the experiment in two concentrations: 25% and 12.5%. The A549 cell culture was stimulated with the studied fragrance oils according to the procedure. The A549 cell culture (ATCC CCL 185) was performed in Dulbecco's culture fluid (DMEM) supplemented with 10% inactivated calf serum, 100 μ/ml penicillin, 100 μg/ml streptomycin, and L-glutamine. The cell culture was adjusted to a density of 2x106 cells per ml and then incubated in 37°C until a single-layer culture was obtained.
The level of TNF-α and IL-6 cytokines was determined by immunoenzymatic ELISA using the R&D system kit and results were given in pg/ml. 96-well microplates were coated with anti-TNF or anti-IL-6 monoclonal antibodies, rinsed with buffer (0.05% Tween80 in PBS) and then blocked with PBS with 10% fetal bovine serum. Samples of 100 μl (supernatant of the cell culture) were applied to microplates. The reaction were stopped and the absorbance was measured at wavelength λ = 470 nm with a correction to λ = 570nm using the BioTek reader. The obtained results were referenced to the standard curve.
The diluted anti-NFκB p-65 primary antibody (Chemicon Internationatl Inc., USA), the biotinylated antibody (Novocastra Laboratories Ltd.) and the Novostain Super ABC Reagent solution (Novocastra Laboratories Ltd.) were applied to microscopic slides according to the procedure (using the cell culture). The DAB solution (0,1% DAB solution in 0,1 M Tris buffer containing 0,02% H2O2) (Novocastra Laboratories Ltd.) was used to stain microscopic slides, which allowed to determine the percentage of cells with positively stained nucleus and determine the level of NF-κB activity.
Studies have shown that only a few tested fragrance oils do not stimulate A549 cells for the production of proinflammatory cytokines such as TNF-α and IL-6 and do not affect NF-κB activation in the human lung cells line, so they are recommended for use in the innovative fragrance dispenser for vehicle interior refreshing.
Keywords: vehicle, ventilation system, fragrance oils, proinflammatory cytokines, NF-κB
The effect of thiamine on oxidative stress and apoptosis in the liver of Japanese quails treated with chlorpyrifos (#723)
D. Ćupić Miladinović1, S. Borozan2, S. Peković3, S. Dacić4, D. Đukić-Ćosić5, V. Ćupić1, S. Ivanović1
1 University of Belgrade, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Belgrade, Serbia
The aim of this study was to investigate the influence of vitamin B1 (thiamine) on biochemical changes in the liver of Japanese quail (Coturnix japonica) treated with chlorpyrifos. The following parameters of oxidative stress were examined: concentration of malondialdehyde –MDA, activity of catalase–CAT, glutathione S-transferase–GST, myeloperoxidase–MPO, but also and activity of butyrylcholinesterase–BChE, and cyclooxygenase –COX and extracellular signal–regulated kinase –ERK (apoptotic regulators).
The study was conducted on eighty male Japanese quails (2 control groups and 6 experimental groups, n= 10), 3-4 weeks old. One control group was treated only with vitamin B1, while the second one received pure corn oil. CPF dissolved in corn oil was administered to three groups of quails by gavage for 7 consecutive days at doses of 1.5 mg/kg BW, 3 mg/kg BW and 6 mg/kg BW. Another three groups were treated with 10 mg/kg BW of vitamin B1 i.m. 30 min after CPF administration (in above mentioned doses) for 7 consecutive days.
Activity of BChE, CAT GST and MPO, and concentration of MDA were measured spectrophotometrically using a Cecil CE 2021 UV/VIS spectrophotometer. For detection of COX and ERK, we used Western blot analysis.
Our studie have shown that CPF significantly inhibited butyrylcholinesterase in the liver, while vitamin B1 increased activity of this enzyme in a dose dependent way. Also CPF has led to increase in the concentration of MDA, activity of CAT, GST and MPO, but after thiamine treatment there has been a decrease of these parameters. Increase of COX and ERK expression after CPF treatment demonstrates an increase of apoptotic vulnerability of cells exposed to CPF, while vitamin B1 caused a decrease in their expression.
Overall these results confirm that CPF causes oxidative stress and apoptosis, and also are giving new insights into thiamine research as an "antistress vitamin".
Keywords: chlorpyrifos, thiamine, butyrylcholinesterase, oxidative stress, apoptosis
Toxicity assessment of Cistus ladanifer extracts: determination of citotoxicity and antioxidant activity (#725)
A. Palmeira de Oliveira1, 2, L. Ramos1, C. Vaz1, C. Gaspar1, 2, J. Rolo1, A. Oliveira1, R. Palmeira de Oliveira1, 3, J. Martinez de Oliveira1, F. Delgado4
1 CICS-UBI, FCS, Covilha, Portugal
The interest of plant extracts as phytopharmaceuticals has recently increased, due to their natural bioactivity, as antimicrobial and antioxidant effects. Consequently, the toxicity assessment of plant extracts is relevant to evaluate its potential use as ingredients for pharmaceutical products. Cistus ladanifer medicinal plant has been shown to be of interest in this field, due to the presence of phenolic compounds and compounds belonging to the terpene family in its composition. This species is traditionally used for dermal application due to putative cicatrization effect.
This study intends to assess the toxicity profile of Cistus ladanifer different extracts and their antioxidant bioactivity.
The antioxidant potential of 2 different aqueous extracts (hydrolates) from Cistus ladanifer (A and B), as well as an essential oil, were enrolled. The antioxidant effect was tested by measuring the extracts capacity to reduce the compound 2,2-diphenyl-1-picryl hydrazyl (DPPH). EC50 was calculated as the amount of sample able to reduce half of DPPH initial concentration, by comparison with a DPPH concentration calibration curve. The cytotoxicity was tested, cells of Skin fibroblasts (3T3) were treated with the hydrolates of Cistus ladanifer and the MTT assay was performed.
The hydrolates did not show relevant antioxidant activity, since a reduction of at least 50% of DPPH initial solution was not observed. On the other hand, the essential oil presented a EC50 of 12.73 ul/ml. The fibroblasts presented cell viability above 50% after treatment with up to 10% of hydrolate A and 35% of hydrolate B.
Keywords: Pharmaceutical ingredients, cosmetics, efficacy, safety
Microorganisms Reduce Indoor Air Pollution (#726)
J. Verdonck1, J. Vanoirbeeck1, K. Poels1, P. Hoet1, L. Godderis1
1 KU Leuven, Department of Public Health and Primary Care, Leuven, Belgium
Indoor air pollution plays a major role with regard to public health. There are three established strategies to reduce indoor air pollution, namely source control, ventilation and mechanical air cleaners. Despite the existence of these established strategies, indoor air pollution is still one of the world’s largest environmental problems according to World Health Organization. Consequently, new methods or strategies to reduce indoor air pollution are continuously being sought. The purpose of this study was to investigate a novel technology that aims to construct an indoor self-cleaning ecosystem by using microorganisms, namely Bacillus species. Instead of applying conventional cleaning products in indoor spaces, microorganisms are nebulized on surfaces, objects and in the indoor air. The key element of the self-cleaning ecosystem is bioremediation, which is the use of living (micro)organisms for the cleaning up of contaminated soil, water, sediment or air. The pollutants might be metabolized to less hazardous metabolites. Scientific evidence concerning the effectiveness of this novel technology to reduce indoor air pollution is still lacking. Therefore, a crossover pilot study was carried out in an urban region in Flanders and lasted 3 weeks. The microorganisms were nebulized in one office building. Of this building, two offices were included in this study. One office of a well-matched neighbouring office building was included as control. The indoor air quality was assessed by tracking particulate matter, carbon dioxide, aldehydes, polycyclic aromatic hydrocarbons, volatile organic compounds, temperature, relative humidity and ventilation rate. Despite the complexity of the real-life study concerning the large amount of variables, the data obtained in this real-life pilot study indicates that constructing a self-cleaning ecosystem might help in reducing the indoor levels of acetaldehyde, particulate matter and total amount of hydrocarbons. The percentage decreases of acetaldehyde and particulate matter concentrations were respectively up to 1.5 and 4 times larger in the offices equipped with a self-cleaning ecosystem than in the control office (P<0.05).
Keywords: Indoor air quality, microorganisms, microbial purification, cleaning, office
The accelerated removal of kidney stones by concomitant use of camel thorn distillate and Rowatinex drug: A case report (#222)
G. Arabrahmatipour1, A. Ebadollahinatanzi2
1 Medical Sciences of Tehran University, Department of Biochemistry,Farabi Hospital Laboratory, Tehran, Tehran, Iran (Islamic Republic of)
Purpose: Kidney stone is one of the most prevalent diseases in globe. It can result in obstructing urinary tract which followed by renal colic and severe pain. The treatment considered for renal colic pain relief is to use nonsteroidal anti-inflammatory drugs or opioid analgesics and/or drug combinations which cause side effects and toxicity. In traditional medicine, camel thorn distillate (Alhagi persarum) is widely used to treat urinary stones. This study was carried out to speed up the kidney stones removal through concomitant use of camel thorn distillate and Rowatinex drug.
Case: The case was a 45-year-old woman with symptoms of flanks pain and kidney stone diagnosis. The sonograms of the patient showed that, there were two stones with 3.5mm and 3mm diameters in the left and right kidney calyx, respectively. There was also seen another stone with diameter of 3.5 mm on the front right distal urethral and ureterovesical junction. In the right kidney; there were hydro-urethra and nephritic grade I which was evident based on two diagnostic imagines.
The treatment protocol on this patient was started with Rowatinex capsule (two times daily) and 150 ml of camel thorn distillate which followed by a thirty- minute walk. This treatment was continued for 20 days.
Results: The sonograms taken one month after treatment showed; there was no trace of kidney stones. The concomitant use of camel thorn and Rowatinex can efficaciously help kidney stones in small sizes to be removed more appropriately and drug toxicity will be lowered.
Keywords: Kidney stone, Rowatinex, Camel thorn
Alginate microencapsulated capsaicin suppresses ROS production and sustains human dermal fibroblasts cells viability (#415)
S. Dragomir1, A. Hudita2, B. Galateanu2, M. Costache2, R. M. Ion3, O. Ginghina4, 5, M. Stan1, D. Baconi1, C. Negrei1
1 “Carol Davila” University of Medicine and Pharmacy, Toxicology, Bucharest, Romania
Capsaicin, the principal pungent ingredient of hot chili is wide used in various topic formulations to relieve diabetic neuropathic pain. However, this natural ingredient exhibits a very high cytotoxicity and finally induces apoptosis by ROS (Reactive Oxygen Species) production. The use of a drug delivery system for capsaicin could alleviate the toxicity of free capsaicin extracts and decrease the numeral side effects that free capsaicin administration triggers. In this context, the aim of our study was to investigate in vitro the potential of novel synthetized capsaicin alginate microcapsules to generate ROS and induce apoptosis of human dermal fibroblasts.
In this view, the bare alginate microcapsules and capsaicin loaded alginate microcapsules were washed with PBS supplemented with 5% antibiotic – antimicotic and then immersed in complete culture medium. During 24h, 4 extracts were collected at predefined time points and subsequently frozen until analysis. The potential of the novel alginate microencapsulation of capsaicin to induce ROS production was investigated by measuring the level of hydrogen peroxide in CCD – 1070Sk human dermal fibroblasts cell culture after treatment with the defrosted extracts. Moreover, the apoptotic status of the cells exposed to defrosted extracts was also investigated by flow cytometry.
Our results show that the capsaicin free alginate microcapsules doesn`t induce ROS production or apoptosis. Furthermore, the capsaicin loaded alginate microcapsules treatment doesn`t determine an increase of the hydrogen peroxide levels, but caused the appearance of a very small population of apoptotic cells. However, the un encapsulated capsaicin displayed a statistically significant increase of ROS production, correlated with a dramatically considerable increase of the apoptotic cells.
In conclusion, the novel synthetized capsaicin alginate microcapsules represent a much safer approach for capsaicin therapeutic use and might be further included in different pharmacological formulations for topic administration in diabetic neuropathy.
Keywords: capsaicin, alginate, microencapsulation, drug delivery, diabetic neuropathy
Effects of zinc chloride on Zebrafish locomotion, circadian rhythm, social behavior and memory: correlation between toxicity and neurobehavior changes. (#761)
1 Chung Yuan Christian University, Biotechnology, Taoyuan, Taiwan
Zinc is a micronutrient at low level but at high concentrations leads to environmental contamination and causes health problems. The aim of this study was to evaluate the effects of Zinc Chloride (ZnCl2) exposure on cognition and locomotion behavior in adult zebrafish (Danio rerio) and correlate these findings with different tissue accumulation of Zn, overall brain AChE and ROS level, Cortisol and Metallothionine levels in the nervous system. Although low level heavy metal exposure poses a potential risk to the zebrafish larvae, little or no data about the effects of ZnCl2 exposure on zebrafish adult fishes. We therefore, exposed adult zebrafishes for 4 days (0.5,1 and 1.5mg/L) displayed decreases locomotor behavior, such as average speed and time in upper zone and an increase speed of meandering. Interestingly, adult fishes exposed to ZnCl2 for 4 days showed impaired frigid long-term memory in the passive avoidance test. Furthermore, zinc chloride treated fish showed memory deficit, increased ROS and AChE levels and decreased locomotor and swimming activities compared to control. A significantly positive correlation was observed between memory and AChE activity, as well as between locomotion and ROS production. These results indicate that acute exposure to ZnCl2 in adults leads to anxiogenic effects, impaired memory and decrease aggressive behavior that might be associated with damage caused by this metal in the CNS, particularly in the cholinergic system.
Keywords: Zebrafish, AChE activity, zinc chloride, locomotor behavior, cholinergic system