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One-month repeated cigarette smoke exposure of human organotypic bronchial epithelial cell culture. (#40)
S. Ito1, K. Ishimori1, S. Ishikawa1
1 Japan Tobacco Inc., R&D Group, Science Product Assessment Center, Yokohama, Japan
Cigarette smoke (CS) is a causative factor in inflammatory responses, and chronic exposure to CS can eventually lead to the development of chronic inflammatory diseases. Organotypic culture models enable the conduct of chronic inhalation studies in vitro because of their long shelf life. We exposed MucilAir human primary bronchial organotypic cultures to 5–50 μg/mL of CS extract for four 24-h periods, and measured the levels of seven cytokines, including interleukin (IL)-8 and growth-related oncogene (GRO), secreted into the basolateral medium. We found a clear concentration-response relationship for IL-8, even though no obvious morphological alterations were observed, suggesting that this concentration range would be suitable for repeated CS exposures. Next, we conducted a one-month repeated-exposure study to assess changes in the levels of cytokines associated with inflammation. The tissues were exposed to 1–20 μg/mL of CS extract, a range exerting no significant acute effects on tissue morphology, and we analyzed changes in the levels of inflammatory cytokines and matrix metalloproteinases secreted into the basolateral medium, as well as long-term alterations in tissue morphology. A concentration-dependent increase in IL-8 levels was observed throughout the experimental period. Moreover, IL-8 levels in tissues exposed to more than 10 μg/mL CS extract increased over time. GRO and matrix metalloproteinase levels also increased, suggesting an exacerbation of the inflammatory response. A focal morphological change was observed in tissues exposed to 20 μg/mL CS extract. Baseline levels of the inflammatory mediators decreased gradually over time, suggesting that an appropriate acclimation period is required. Overall, our results demonstrate that even at low concentrations, repeated CS exposure induces inflammatory effects. Organotypic cultures of human primary cells are useful tools in reproducing tissue-specific responses to chronic CS exposure in vitro.
Keywords: Cigarette smoke, organotypic culture, inflammation, repeated exposure
Cytotoxicity Testing of Therapeutic Nanosystems for Pulmonary Infection using an air-lifted interphase in-vitro test system (#44)
J. Knebel1, D. Ritter1, M. Niehof1, T. Hansen1, M. Strandh2, C. Falciani3, M. Flores4, Y. te Welscher5, C. van Nostrum5, R. Gracia6, M. Marradi6, I. Loinaz6
1 Fraunhofer ITEM, Preclinical Pharmacology and In vitro Toxicology, Hannover, Lower Saxony, Germany
While searching for effective antibiotics against resistant bacteria, nanocompounds are developed today, which may serve as drug carriers for the antibiotic active substances. In the case of respiratory infections, the administration of such combined antibiotic nanosystems (NS) via the inhalation route is the first choice in order to combat the bacteria directly at the site of action. Here we report about the in vitro investigation on the cytotoxic effects of 4 nanosystems and a free antimicrobial peptide (AMP) on a human lung cell line (A549) in a realistic test environment. Test substances as well as vehicle, positive (CuSO4) and negative (Lactose) controls were aerosolized. Cells, grown on membranes at the air-liquid interphase, were exposed to the airborne test and reference substances using the P.R.I.T.® ALI technology. After exposure, cells were postincubated for 24hrs before cellular viability was determined by use of the tetrazolium salt conversion assay (WST-1). The results indicated that the exposure scenario had no adverse effect on the cells as revealed by the vehicle controls. CuSO4, known as a mild human lung toxicant, led to a clear dose-effect relationship, whereas increasing concentrations of lactose showed no cytotoxic effect. The free antimicrobial peptide displayed the strongest toxicity of all test substances and revealed more toxic potency than the positive control (CuSO4). The comparison on dosage based grouping between the free AMP and the nanosystem, tested at the same relative antibiotics concentration, revealed that packing into a micelle nanosystem significantly reduced the toxicity of the AMP. When comparing the effect of the four NS at the highest dosage the results show, that NS #2 had a significantly higher impact on the cell viability than the other three nanosystems. Therefore it is concluded that the protective effects of the nanosystems were dependent on the antibiotic/nanocompound combination. EU grant agreement No 604434.
Keywords: in-vitro, air-lifted interface, antimicrobial peptide, therapeutic nanosystem
The development of in vitro embryotoxicity testing using human iPS cells (#77)
1 Kyowa Hakko Kirin Co., Ltd, R&D Division, Translational Research Unit, Shizuoka, Japan
Embryotoxicity, which affects the next-generation is a serious toxicity. To predict the clinical embryotoxic potential of drug candidates in the preclinical setting, a method of in vitro human-induced pluripotent stem cell (iPS) embryotoxicity testing (iPSET) was developed by modifying a mouse embryonic stem cell test (EST), which is one of the more promising animal-free approaches, to assess the embryotoxic potential of drug candidates. I previously reported on the human-specific embryotoxicity of thalidomide in preliminary iPSET (J Pharmacol Sci, 2014). In the present study, to validate the clinical prediction by iPSET, some other drugs were assessed.
The drugs were assessed according to the following three endpoints: the inhibition of the cardiac differentiation of iPS and the cytotoxicity on iPS and human dermal fibroblasts. The spontaneous differentiation of iPS to cardiomyocytes was considerably difficult to achieve in comparison to mouse embryonic stem cells; however, I developed a simple protocol through which the application of biological substances promotes the differentiation of iPS into cardiomyocytes. Reference drugs that are reported to have embryotoxicity, were used as positive controls (e.g., thalidomide, sodium valproate and methotrexate). For convenience, reference drugs that have not been reported to be associated with embryotoxicity (e.g., ascorbic acid and saccharin) were used as negative controls. These test drugs were classified into three classes, class-1 (no embryotoxicity), class-2 (weak embryotoxicity) and class-3 (strong embryotoxicity), according to the classification of the potential risk of embryotoxicity by the EST prediction criteria using the three endpoints.
All of the positive control drugs were classified as class-2 or -3. All of the negative control drugs were classed as class-1. These findings suggested that the iPSET would be a useful assay for evaluating the human embryotoxicity of drug candidates in the preclinical setting.
Keywords: embryotoxicity, iPS cells, in vitro toxicology
A novel model of alphavirus-induced neurotoxicity for studying gene x environment interactions (#99)
C. M. Bantle1, S. M. Roche2, R. B. Tjalkens1
1 Colorado State Univeristy, Environmental Health and Radiological Sciences, Fort Collins, Colorado, United States of America
The etiology of neurodegenerative diseases such as Parkinson’s (PD) and Alzheimer’s disease is unknown but likely linked to combinatorial interactions between genetic risk factors, environmental stressors including viruses, and environmental neurotoxins. Our understanding of the environmental links to PD and related neurodegenerative disorders remains extremely limited, emphasizing the need for better animal models in which to test interactions between environmental toxins and genetic backgrounds. Current animal models based on the use of neurotoxins and transgenic mice may show loss of neurons but often lack other key hallmarks of these diseases, such as neuroanatomical specificity, progressive neuronal loss, glial activation and protein aggregation. Notably, certain neurotropic mosquito-borne alphaviruses, such as Western equine encephalitis virus (WEEV), can target the midbrain, as well as cortical regions, with high fidelity and have been shown for many years to cause neurological symptoms resembling PD in individuals who develop encephalitic disease. Using recombinant WEEV expressing firefly luciferase in outbred CD-1 mice, we demonstrate that infection by simple intranasal inoculation results in rapid distribution throughout the cortex and basal ganglia, pronounced neurobehavioral abnormalities, marked glial activation and loss of dopaminergic neurons in the SNpc. To prevent mortality, mice were treated with polyclonal antibodies to the WEEV E1 viroporin at 12 and 48 hours post-infection, whereupon they cleared WEEV and remained viable for at least two months. Levels of viral replication were monitored by in situ bioluminescence imaging for the entire eight weeks of infection. Brain tissue was fixed and cryosectioned for 3D design-based stereology. CLARITY imaging revealed wide distribution of RFP-expressing WEEV throughout the CNS. Intranasal inoculation with recombinant WEEV showed high specify for the SNpc, significant DA cell loss, glial cell activation, protein aggregation and a gene expression profile consistent with PD-like pathology. We also examined the capacity of infection with WEEV to exacerbate the neurotoxicity of the heavy metal, Mn, which causes PD-like pathology in humans and animal model. This study provides a new viral-based animal model that closely replicates the neuroanatomical features of PD that can be used to investigate interactions between infection and environmental neurotoxins that can lead to neurodegenerative disease.
Keywords: Neurotoxicology, Neuroinflammation, Virology, Manganese, Glia, Neurodegeneration, Animal Models
Weight of evidence Skin sensitization safety assessment using the Bayesian Net Integrated Testing Strategy (#113)
P. S. Kern1, C. Ryan2, E. Deconinck1, J. Jaworska3, G. Dameron2
1 Procter & Gamble Services Company NV, GPS, Strombeek-Bever, Belgium
Skin sensitization safety assessments nowadays rely heavily on using non-animal data from assays addressing steps along the Adverse Outcome Pathway (AOP). The non-animal data are generally integrated following a defined approach to predict a skin sensitization hazard or potency which can then be used as point of departure (No expected sensitization induction level NESIL) for a quantitative risk assessment. Often, those data are further combined with information on eg. (skin) metabolism, bioavailability or structural analogs in a weight of evidence safety assessment approach.
Data integration remains challenging as determination of sensitization potency and underlying uncertainty of the prediction cannot be easily determined. The Bayesian Net integrated testing strategy (BN-ITS) represents the underlying mechanistic processes of the AOP and allows the integration of data from three validated alternative methods (Direct Peptide Reactivity (DPRA) KeratinoSens®and Human Cell Line Activation Test (hClat)), along with bioavailability, physical chemical properties and in silico inputs to predict skin sensitization potencies. In the provided case studies, covering eg. fragrance materials, the BN-ITS was used as a method to derive NESILs either in form of a sensitization potency category or as a predicted pEC3 values. The predicted pEC3 values provide an advantage over the potency category predictions as they are in line with data provided by LLNA testing. The pEC3 value most appropriate to represent the predicted distribution (e.g. 50th, 70th, 90th percentile) was explored. The uncertainty of the potency prediction is assessed by conversion of the probability-based predictions to Bayes factors (BF). The case studies will illustrate how to deal with data discrepancies and how to factor in data from related structural analogs to further reduce uncertainties eg. to extrapolate from LLNA predictions to human thresholds, as well as guiding the safety assessor. The BN ITS sensitization potency prediction, combined with read across information from analogs represents a step forward in progressing the use of non-animal methods from simple hazard identification to use in safety assessment.
Keywords: Bayesian net, skin sensitization, integrated testing, safety assessment, potency
Mitigation of hypothetical on-target mechanosensory transduction risk in a TRPC6 antagonist project by evaluation of auditory function in the rat (#121)
M. Liljevald1, S. Purbrick2, R. Tapp3, M. Rolf1, W. Redfern2
1 AstraZeneca, Drug Safety and Metabolism, Mölndal, Sweden
Background: One of our small-molecule projects aims to target TRPC6 with an orally available antagonist. A key safety concern is that TRPs play a role in mechanosensory transduction. Auditory deficits occur in mice with a dual knockout of TRPC6 and TRPC3, but not with TRPC6 knockout alone (Quick et al., 2012; Sexton et al., 2016). We tested a representative AZ compound on auditory function in the rat. AZ-1 has ~20-fold selectivity for TRPC6 (IC50 = 10 nM) over TRPC3 (IC50 = 209 nM). It was tested at its MTD dose level, with the intention of achieving good coverage for both TRPC6 and TRPC3 in the cochlea. Furosemide was included as a positive control.
Protocol: Male Han Wistar rats (222 to 337 g; 9-11 weeks of age; 10/treatment group) received a single administration of either vehicle (10 mL/kg p.o.), AZ-1 (500 mg/kg p.o.) or furosemide (40, 80 or 100 mg/kg i.v.). The Auditory Brainstem Response (ABR) was recorded via scalp electrodes under ketamine-dexmedetomidine anaesthesia. The rat was placed on a heated water blanket to assist in thermoregulation, in a sound-attenuated electrically shielded enclosure. The threshold sound level in decibels (dB) for evoking the ABR response was evaluated at 3 different frequencies (4, 10 and 20 kHz), delivered as a 15 ms stimulus, in increasing 10 dB steps. ABR was tested in both ears around the Tmax (1 h post-dose for AZ-1; 10 min post-dose for furosemide). Terminal sampling of perilymph (2 μL per ear) was undertaken to confirm local exposure.
Readout: AZ-1 had no effect on ABR at perilymph exposures of 7440 nmol/L, affording good coverage of TRPC6 (744x IC50) and TRPC3 (36x IC50). Furosemide at 40 mg/kg also had no effect, whereas at 80 mg/kg and 100 mg/kg, dose-related increases in the ABR thresholds were observed, with average thresholds increases of 27.0 dB, 27.3 dB, and 46.5 dB (80 mg/kg) and 38.2 dB, 39.5 dB, and 55.2 dB (100 mg/kg) at 4, 10, and 20 kHz respectively (P < 0.01).
Outcome: Pharmacological inhibition of TRPC3 and TRPC6 did not lead to the auditory deficits reported after knockout of these proteins. This study was therefore able to mitigate a hypothetical risk identified during a literature-based target safety assessment and build confidence in the safety of TRPC6 inhibition as a therapeutic mechanism.
Quick K et al. (2012) Open Biol 2: 120068.
Keywords: TRPC6, Mechanosensory, Auditory Brainstem Response (ABR)
TOXICITY ASSAYS USING EXPANDED LIVER CELLS PROMOTE THE REDUCTION OF ANIMAL USE IN PRE-CLINICAL RESEARCH (#125)
N. Nagy1, A. Noerenberg1, T. Evenburg1, T. Johannssen1
1 upcyte technologies, Hamburg, Germany
Alternatives to animal testing in toxicity studies such as in vitro models were proposed to overcome some of the drawbacks associated with animal tests. Current in vitro models exhibit several disadvantages such artificial conditions focusing on a single cell type in 2D culture. The use of primary cells in vitro is compromised by limited cell numbers and restricted proliferation. upcyte technologies has developed a unique technique to push isolated human primary cells back into proliferation without altering the cells most relevant tissue-specific characteristics. Here we describe the most recent data of upcyte® hepatocytes and liver sinusoidal endothelial cells (LSECs) to demonstrate their suitability to replace animal models.
First, the expression of typical markers was investigated. Both upcyte® hepatocytes and LSECs expressed characteristic adult marker proteins. Secondly, tissue specific activities were identified. upcyte® hepatocytes expressed metabolizing enzymes of phase I as well as phase II. Furthermore, functional expression of drug transporters OATP2B1, NTCP and OCT1 was quantified. In upcyte® LSECs the mannose and FcɣR receptors (CD32b) were highly expressed as well as used in the functional test of receptor-mediated endocytosis.
Thirdly, toxicity studies were performed on upcyte® hepatocytes and LSECs alone as well as in co-culture. Acute and repeated-dose toxicity was investigated first in upcyte® hepatocytes with acetaminophen (APAP) for 24 h or 1 week. As a result, no effect was observed after 24 h treatment, whereas after 1 week, apoptosis and levels of intracellular Ca2+, ROS and mitochondrial superoxide were significantly increased. Then, LSECs in 2D monolayers were challenged with APAP and the toxicity was compared to expanded hepatocytes. Interestingly, LSECs showed an increased sensitivity towards APAP when compared to upcyte® hepatocytes. Furthermore, co-culture in 3D scaffolds was the most sensitive model to detect acetaminophen-induced toxicity.
Overall, upcyte® hepatocytes and LSECs combine many features of primary cells with unlimited availability to replace the use of animals in pre-clinical studies. Furthermore, co-culture of these cells in toxicity studies could boost the predictability of such assays and thereby reduce subsequent animal in vivo studies.
Keywords: hepatic toxicity, drug-induced toxicity, liver sinusoidal endothelial cells, hepatocytes, in vitro assay
Developmental Toxicity Assessment of Thalidomide and its Derivatives Lenalidomide and Pomalidomide in Zebrafish Embryos (#162)
I. Gonzalez1, M. Ipiñazar1, A. Altuna1, C. Quevedo1
1 Biobide, San Sebastian, Spain
More than half a century ago, Thalidomide was widely prescribed to pregnant women as a sedative and antiemetic. The teratogenic effects associated with its use were soon discovered along with multiple and severe birth defects. The Thalidomide disaster demonstrated for the first time that species differences exist in drug reaction/response. Mice are less sensitive to Thalidomide than other species like non-human primates and rabbits. Deleterious effects of Thalidomide have been also demonstrated in non-mammalian species as chicken, Xenopus and some fish species.
There is a worldwide trend to move away from animal testing for the human and environmental safety assessment of chemicals. Zebrafish embryo model is highly popular in toxicology, both in research, industry and potential regulatory applications. In compliance with international animal welfare regulations (e.g. Dir 2010/63/UE), the fish embryo models provide an ethically acceptable small-scale analysis system with the complexity of a complete organism.
The main objective of this study is to test the teratogenicity potential of Thalidomide in zebrafish embryos. For this purpose, embryos were exposed to this compound from 3-5 hpf (hours post- fertilization) to 4 dpf (days post-fertilization) and the evaluation of survival and morphological alterations was carried out during this period. Internal compound exposure in the larvae was determined after this analysis by liquid chromatography and mass spectrometry. Although clear morphological alterations were not observed at the concentrations analyzed, a decrease in the embryo length was consistently observed at 800 µM. Higher concentrations could not be assayed since Thalidomide precipitated from this concentration in the embryo water. Due to its hydrophilic nature, low uptake of Thalidomide (estimated internal concentration ̴ 20 µM) was confirmed after bioanalysis. Then, in spite of Thalidomide solubility limitation and its low penetrance, toxicity manifestations were already caused at 20 µM. Similar experiments were conducted for Thalidomide derivatives Lenalidomide and Pomalidomide to compare the developmental toxicity potential of these related compounds.
Keywords: Developmental toxicity, zebrafish, teratotoxicity, thalidomide, alternative models
Capturing the current use and applicability of drug transporter data for chemical safety assessment within a non-animal kinetic-based approach. (#164)
A. Paini1, S. Coecke1, A. Worth1, L. - A. Clerbaux1
1 European Commission Joint Research Centre, Directorate Health, Consumers and Reference Materials, Ispra, Italy
Membrane transporters represent the functional part of the physiological barriers of the body as they mediate the transport of compounds into and out of cells. Thereby, they play critical roles in absorption, distribution and excretion of a substance, driving kinetics. In recent decades, the drug development field has placed particular effort on studying transporters of clinical relevance. Transporter data could be a key determinant within a toxicokinetics-based risk assessment strategy. However, a global overview of the use of transporters data for chemical safety assessment is currently missing.
Keywords: Transporters, Toxicokinetics, Alternatives, ECVAM survey, chemical safety assessment
IATA used the in chemico, in vitro and in silico methods to evaluate the skin sensitization potentials of cosmetic ingredients (#187)
M. Choi1, S. - A. Cho1, S. - R. Park1, S. An1
1 AMOREPACIFIC, Safety & Microbiology Lab, Yongin, Republic of Korea
Since March 2009, using animal for testing cosmetics has banned by The Council of the European Union, and also has banned in Republic of Korea since February 2017. According to the trend of animal testing ban, many alternative animal test methods for testing toxicity of cosmetic ingredients and products have been developed. Many animal alternatives for skin sensitization is developed according to AOP (Adverse Outcome Pathway) concepts, such as protein binding (KE1), keratinocyte activation (KE2) and dendritic cell activation (KE3).
DPRA(Direct Peptide Reactivity Assay; KE1), ARE-Nrf2 luciferase test(KeratinoSens™; KE2), and h-CLAT(human Cell Line Activation Test; KE3) are the major test methods used to predict skin sensitization based on AOP concepts. Generally, these three test results are integrated to predict sensitization. Searching flexible and suitable tools for toxicological decision making called IATA (Integrated Approaches to Testing and Assessment) is important concept to predict skin sensitization to improve accuracy of chemicals. Recently, there is a trend to comprehensively predict for skin sensitization with a combination of in chemico, in vitro methods and in silico tools. And, many studies have been conducted to improve the accuracy of the prediction using in silico tools.
The purpose of this study was to compare the accuracy between in silico/vitro/chemico and in vitro/ chemico test to make our own prediction strategy of cosmetic raw materials for skin sensitization. We compared the accuracy of using only three tests with the accuracy of using the in silico tool (OECD QSAR toolbox) in addition. The results of study showed higher accuracy when using the in silico tool than if it were not used.
Keywords: in chemico, in vitro, in silico, Skin sensitization, cosmetic ingredient
Human gut-on-a-chip model as an improved intestinal barrier model to predict compound bioavailability and toxicity (#201)
H. Bouwmeester1, K. Kulthong1, 3, M. Grouls1, L. Duivenvoorde2, D. Rijkers2, G. ten Dam2, L. de Haan1, M. van der Zande2
1 Wageningen University, Toxicology, Wageningen, Netherlands
Human intestinal epithelial cells grown in a microfluidic gut-on-a-chip model have been proposed as an improved in vitro model that better recapitulates the human intestinal functions. Growing human cells under microfluidic flow conditions allow an accurate control of the extracellular chemical and physical microenvironment. Here, we developed a gut-on-a-chip model with high relevance to in vivo conditions. Intestinal epithelial cells (Caco-2) were (co-)cultured with HT29-MTX mucus secreting cells on a porous polyester membrane that was tightly clamped between two glass slides to form two separate flow chambers. The chip was mounted with a quick locking mechanism in a chip holder constructed for connecting external tubing to the chip via specific ferrules to ensure tight connections and a leaking-free system. Both above and below the membrane layer two fluid flows (25 µL/h) were applied using a pump system. Confocal microscopy analysis of the cellular monolayer, Lucifer Yellow translocation and ALP activity levels indicated good cell layer integrity and cellular differentiation. We determined intestinal barrier function by measuring the transport of Ketoprofen, Amoxicillin, Antipyrine and Digoxin resembling different groups of well-known pharmacy and a mixture of 17 dioxin congeners, food contaminants. After 24 h incubation, the amount of transported dioxins was similar in both the dynamic gut-on-a-chip and static Transwell model ranging from 0.6% to 3.3% and 0.2% to 4.4%, respectively. Dioxins detected in cellular fraction were also comparable between two models, ranging from 22 to 66%. The transport of individual congeners corresponded with their structures as revealed by molecular weight-transport relationship. In summary, the dynamic microenvironment in our gut-on-a-chip model influences growth, differentiation, and the translocation properties of intestinal Caco-2 cells which might be future developed for kinetic bioavailability studies.
Keywords: Microfluidics, Gut-on-a-chip, Organ-on-a-chip, Dioxin, Transport, Bioavailability
Non-animal vaginal irritation method admitted as nonclinical assessment model (NAM) in the Incubator Phase of the United States Food and Drug Administration (US FDA) Medical Devices Development Tool (MDDT) (#212)
E. Hill1, J. Brown2, E. - G. Costin1
1 Institute for In Vitro Sciences, Gaithersburg, Maryland, United States of America
As of December 2015, personal lubricants must receive pre-market approval from the US Food and Drug Administration (FDA) Center for Devices and Radiological Health (CDRH) in order to be sold in the US. Part of the agency-recommended biocompatibility testing battery includes the in vivo Rabbit Vaginal Irritation (RVI) test. We have created an Industry Consortium comprised of personal lubricants manufacturers and are working collaboratively with stakeholders and the US FDA to develop an in vitro testing approach that can reliably be used to replace the RVI in pre-market submissions. Our Validation Program will analyze paired in vivo-in vitro data for vaginal irritation utilizing commercially available human reconstructed vaginal tissue models. A Prediction Model will be proposed that can be used for the safety assessment of personal lubricants and vaginal moisturizers in their final, undiluted formulations with chemical and physical properties within the boundaries of products included in the qualification package (e.g., formulation, viscosity, pH, osmolality). Our Validation Program proposal has been accepted as a Nonclinical Assessment Model (NAM) in the Incubator Phase of the US FDA CDRH Medical Device Development Tool (MDDT) Program and is currently under review for consideration to advance to the Pre-Qualification stage.
Keywords: alternatives, in vitro, vaginal irritation, Medical Device, IVIVE, non-animal testing, personal lubricants
Proteomics and AllerCatPro analysis for the identification of low allergenic proteins (#224)
N. L. Krutz1, J. Winget2, C. A. Ryan2, R. Wimalasena2, S. Maurer-Stroh3, R. Dearman4, I. Kimber4, G. F. Gerberick2, 5
1 NV Procter & Gamble Services Company SA, Strombeek-Bever, Belgium
The use of botanicals and natural substances in consumer products has increased in recent years. These substances can contain protein and therefore may be a concern for the potential risk to cause immediate type (IgE-mediated) respiratory allergic reactions. Assessing the allergenic potential of proteins and protein extracts is challenging, since some proteins may cause allergic reactions, while many others are of less or no concern. Available data on well-researched proteins considered as low or non-allergenic is insufficient to be used for calibration of new and existing assessment paradigms. Here we generated a data set of proteins predicted as low allergenic based on the lack of evidence for their allergenic potential.
We chose protein extracts from sources that are considered to have less allergenic potential than the “big 8” most common food allergens, including Corn, Spinach, Potato, Rice, Tomato as well as Wheat, as one of the “big 8”. These extracts were analyzed to identify and semi-quantify all proteins by a label-free proteomics analysis conducted with QExactive HF mass spectrometer. Proteins with a relative high abundance representing <10% of the total number of all identified proteins were analyzed for their predicted allergenicity potential by AllerCatPro. This in silico model compares proteins on a sequence and 3D structure level against a reliable dataset of >3000 protein allergens and performs with 84% accuracy.
Each extract revealed 1224 (Spinach) to 1875 (Tomato) proteins with most abundant proteins per extract comprising 4.5% (Polygalacturonidase-2 in Tomato) up to 33% (small chain of RuBisCO in Spinach). As expected, the most abundant proteins identified in Wheat were predicted with strong evidence for allergenic potential such as well-known allergens belonging to the Alpha-amylase/trypsin inhibitor, Glutenin and Gliadin families. Within the relatively high abundant proteins of all six analyzed extracts, 52 proteins were predicted with no evidence for allergenic potential based on the protein structure.
This generated panel of well-researched low allergenic proteins provides a pragmatic approach to aid the development of alternative methods for more robust testing strategies to distinguish between proteins of high and low allergenic potential to assess the risk of novel proteins.
Keywords: Protein allergens, Type I Allergy, Risk Assessment, label-free proteome analysis, in silico allergen prediction model
Is there a relationship between bioaccessibility and bioavailability for metals? (#263)
Y. W. Lowney1, V. Verougstraete2, A. Oller3
1 Alloy LLC, Boulder, Colorado, United States of America
Purpose of the study:
Bioavailability refers to the portion of a chemical that is absorbed by an organism and reaches the central (blood) compartment. For most metals and complex metal-containing materials, bioavailability is a key driver of toxicity: systemic health effects will manifest only if exposures result in bioavailable metal levels above the threshold values for these effects. Bioavailability is based on results from animal (in vivo) studies. However, where it is not desirable to determine this in vivo (e.g. animal welfare considerations, more rapid throughput), studies performed in vitro (bioaccessibility or bioelution tests) can provide a measure of the solubility of metals in surrogate biological fluids. Bioavailability and bioaccessibility data can be used in many applications, ranging from grouping and read-across of metals, risk assessment from soils and hazard identification. However, before using the in vitro results as an alternative to in vivo testing, it is necessary to establish if a relationship can be defined between bioaccessibility and bioavailability. A literature review was set up to assess information on the relationship between in vivo relative bioavailability (RBA) and in vitro bioaccessibility (IVBA) for many metals.
Material and methods
A literature search of studies reporting on in vivo bioavailability (i.e. RBA) and/or in vitro extraction test results (i.e. IVBA) was conducted. The data were evaluated for evidence of a relationship between IVBA tests and RBA of metals for studies of paired samples. The study also assessed the components or conditions of IVBA methods (e.g. pH, temperature, contact time, agitation, particle size) that affect the results of extraction testing, to understand what conditions provide conservative estimates of bioavailability.
39 studies were reviewed; 22 of them included information on both relative bioavailability RBA and IVBA. The data provide strong evidence that soil- and site-specific factors exert control on the bioavailability of metals from soil relative to metal bioavailability from soluble forms. Strong predictive relationships between RBA and IVBA emerged for Pb and As, with fewer available data for Sb, Cd, Co, Ni, and Hg, and no data for Be and Se.
Keywords: metals, bioavailability, read-across, in vitro
Establishment of the human Cell Line Activation Test (h-CLAT) (#280)
H. Kim1, M. H. Hong1, I. S. Jo1, I. Ahn3, T. S. Kim1, J. H. Kim1, J. S. Yi1, K. Y. Ko1, J. Y. Kim1, J. K. Lee2, K. S. Park1
1 Ministry of Food and Drug Safety, Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Cheongju-si, Republic of Korea
A number of alternative methods targeting different key events of the adverse outcome pathway (AOP) for skin sensitization have been developed. However, it is difficult that any individual alternative methods fully replace currently accepted animal tests since biological events are very complex after exposure foreign substances. Therefore, many approaches, such like Integrated Approaches to Testing and Assessment (IATA) and Defined Approaches (DAs), to integrating information from multiple alternative methods have been developed for overcoming the limitations of the individual alternative methods.
The aim of this study is to establish the human Cell Line Activation Test (h-CLAT), which was newly approved as OECD TG 442E, and find an applicable DA to skin sensitization hazard prediction in Korea. The h-CLAT is an in vitro test method for identifying of skin sensitisers and non-sensitisers in accordance with the UN GHS. This method addresses the third key event of the skin sensitization AOP by quantifying chemical-induced changes in the expression of CD86 and CD54 protein markers on the surface of the human monocytic leukemia cell line, THP-1. As a first step, we prepared standard operating procedures (SOPs) for the h-CLAT on the basis of the procedures suggested by OECD TG 442E and EURL ECVAM DB-ALM protocol No. 158. To demonstrate technical proficiency for the method, nine proficiency chemicals including 5 sensitisers and 4 non-sensitisers listed in TG 442E were evaluated using the method. The results of h-CLAT using those test chemicals were identified to classify with the UN GHS categories and to be within the range of acceptance criteria.
Moreover, we reviewed the internationally developed DAs and analyzed each DA to compare accuracies. Therefore, we developed a new sequential testing strategy for hazard identification to predict skin sensitization potency for the murine local lymph node assay (LLNA) and human outcomes. The study results suggested that the DAs would be a promising tool for increasing the accuracy of skin sensitization assessment by addressing the limitations of each alternative method.
Funding source: This research was supported by the grants (16181MFDS521) from the Korean Ministry of Food and Drug Safety in 2017.
Keywords: Alternative methods, h-CLAT, OECD TG 442E, Defined Approach (DA)
In vitro permeability assay using an epithelial model of Caco-2/HT29-MTX/Raji-B cells: enhancer aspects of a papain-cyclodextrin complex (#306)
P. S. Lopes1, F. G. Corazza1, F. A. N. Nambu1, J. V. Ernesto1, G. H. C. Varca2, V. R. Leite-Silva1, N. Andréo-Filho1
1 UNIFESP, Departamento de Ciências Farmacêuticas, Diadema, Sao Paulo, Brazil
The oral route is one of the main routes for administration of drugs, however, the gastrointestinal tract is a hostile environment due to pH variation, presence of several digestive enzymes and the intestinal barrier that undermines the permeation of drugs. The aim of this study was to evaluate the ability of papain complexed with β-cyclodextrin to enhancer the permeation of furosemide, as a model drug, in a triple co-culture of Caco-2, HT29-MTX and Raji cells. Papain and the papain-β-cyclodextrin complex were evaluated at 0.3, 0.7 and 1.0 µM and the biophysical integrity of the cell layer was evaluated by RET (Transepitelial Electrical Resistance) at 0, 4, 24, 48 and 72 hours. The epithelium was also stained using DAPI and Alexa Fluor™ 488 Phalloidin. HPLC was employed to quantification of furosemide. The RET results at initial time for all the samples and control were in a range of 267.63 to 318.28 Ω*cm2 and after 72h this values were raised to a range of 365.14 to 492.64 Ω*cm2. There was a decrease of RET after samples’ application, nevertheless, the results showed that the epithelium presents a recovery, proportional to the time of cell replication, and that this recovery occurs in all samples tested with no significant statistical difference. The RET recovery implies that papain, complexed or not, was not able to kill the cells, corroborating the hypothesis that the action mechanism is the disruption of the tight junction. In addition, the triple co-culture presents a higher resistance to papain action, in comparison with the Caco-2 monolayer assays, emphasizing the importance of testing new drugs, potential candidates for oral formulations, in epitheliums that faithfully mimics what actually happens in in vivo systems. The fluorescent microscopy observation of the cells stained with DAPI and the junctions stained with Alexa Fluor™488 Phalloidin, showed that co-culture exhibits microvilli inherent to the intestinal tissue. The results obtained in the triple co-culture model bi-directional transport experiments confirmed the significant increase in furosemide transport indicating the importance of the paracellular route. In conclusion, the triple co-culture model was successfully standardized and papain complexed with β-cyclodextrin acts probably over the tight junctions enhancing the permeation of furosemide.
Keywords: triple co-culture; in vitro model; permeation; enhancer; intestinal absorption
Assay ready frozen THP-1 cells can be used like a reagent in a human Cell Line Activation Test (h-CLAT) to measure the skin sensitizing potential of chemicals. (#320)
V. Mazurov1, J. Walbech1, A. Loa1, O. Wehmeier1
1 acCELLerate GmbH, Hamburg, Germany
Recently a cell based in vitro model has have been approved by the OECD (Test N° 442E) to assess the skin sensitizing hazard of chemicals. One of the tests applied is the human Cell Line Activation Test (h-CLAT) which uses monocytic THP-1 cells as a surrogate for dendritic cells. These cells express CD86 and CD54 upon activation through sensitizing molecules at subtoxic concentrations.
A major obstacle of the assay is the cultivation of the THP-1 cells. The concentration rage at which skin sensitization can be measured without significantly reducing viability, is very tight. A healthy and highly viable culture of THP-1 cells is therefore of the essence. THP-1 are known to recover badly from suboptimal cultivation or cryopreservation. It usually takes at least a week of intensive care until the cells regain an acceptable viability. Because the overall fitness of the cells has a significant impact on their sensitivity to sensitizers, reproducibility of the h-CLAT very much depends on the cell culture quality and is therefore difficult to control.
We here present an optimized cryopreservation protocol for THP-1 cells. These assay ready cells recover from cryostock at a high and stable viability of greater than 90%. They can be applied in a h-CLAT skin sensitization testing immediately after resuscitation. No prior cultivation or passaging is required which eliminates the cell culture factor from the assay. By applying different reference substances like NiSO4 or DNCB we demonstrate the assay ready frozen THP-1 are equally susceptible to skin sensitizers like cells from a continuously passaged maintenance culture and provide a better reproducibility.
Keywords: skin sensitization, h-clat, THP-1, assay ready cells
Human In Vitro Models for Respiratory Toxicology: Evaluation of Goblet Cell Hyperplasia, Mucus Production, and Ciliary Beating Assays (#354)
B. Gilbert2, E. Hill2, M. Aragon2, S. Frentzel1, J. Hoeng1, S. Ito3, S. Ishikawa3, J. Budde4, A. Maione5, P. Hayden5, W. Fields6, B. Keyser6, L. Haswell7, D. Azzopardi7, H. Behrsing2
1 Philip Morris International, Neuchatel, Switzerland
Robust non-animal models and assays for pulmonary toxicology are required to make competent product development and risk assessments for new materials requiring toxicity testing. Three in vitro assays (goblet cell hyperplasia [GCH], ciliary beat frequency [CBF], and MUC5AC quantitation) were evaluated for performance and reproducibility. To assess these assays, 6 laboratories contributed data using a common protocol utilizing IL-13 as an inducer of adverse mucociliary-relevant tissue changes. MatTek EpiAirway™ and Epithelix MucilAir™ 3D tissue models were used to evaluate endpoints using histology for GCH, software-based applications, Cilia FA and SAVA, for CBF, and ELISA assay for MUC5AC. Continuous 10 ng/mL IL-13 (GCH, MUC5AC) exposures or one hour 10 µM procaterol (CBF) exposures prior to day 7 and 14 time-points were included as positive controls. Quality control endpoints (e.g. adenylate kinase tissue content and trans-epithelial electrical resistance) were also evaluated. Multi-fold increases (ranging from 2.6 to 33-fold, and 1.5 to 238-fold) in MUC5AC-stained goblet cells were measured in both tissue models after exposure with IL-13 after 7 and 14 days induction, respectively. For CBF, procaterol caused a significant increase, and IL-13 elicited a significant decrease as expected. However, the MUC5AC ELISA did not yield consistent results when frozen apical rinse samples were thawed and assayed. These results suggest these non-animal test systems may provide consistent, human-relevant data corresponding to key events involved in respiratory disease. A streamlined protocol using these controls will be applied toward additional testing. These assays, utilized in a pragmatic manner with other in vitro assays have the potential to be included in a Reduced Risk Product assessment framework.
Keywords: in vitro, airway, respiratory, non-animal, pulmonary
AllerCatPro – Prediction of protein allergenicity potential from the protein sequence (#357)
S. Maurer-Stroh1, N. L. Krutz2, P. S. Kern2, V. Gunalan1, M. N. Nguyen1, V. Limviphuvadh1, G. F. Gerberick3, 4
1 Bioinformatics Institute, Agency for Science, Technology and Research, Singapore, Singapore
Due to the risk of inducing an immediate type (IgE-mediated) respiratory allergic responses, proteins intended for use in consumer products must be investigated for their allergenic potential before introduction into the marketplace. Previous FAO/WHO guidelines for computational assessment of allergenic potential of proteins based on single hexamer peptide hits and linear sequence window identity thresholds produce a large number of proteins misclassified as allergens. We here describe the in silico model AllerCatPro, which is an extension of the existing approach with updated short peptide statistics as well as 3D surface comparison against known allergens.
We created the most complete dataset of 5334 unique allergenic protein sequences to date by determining the consensus among the major databases from FARRP, COMPARE, IUIS, UniProtKB and Allergome and manually curating unique entries for evidence of allergenic potential. We extend the hexamer hit rule by removing peptides with high probability of random occurrence measured by sequence entropy as well as requiring 3 or more hexamer hits consistent with natural linear epitope patterns in known allergens. This is complemented with a Gluten-like repeat pattern detection. We also switch from a linear sequence window similarity to a B cell epitope-like 3D surface similarity window which becomes possible through extensive 3D structure modelling covering the majority (76%) of allergens. In case no structure similarity is found, the decision workflow reverts to the old linear window rule.
Using a benchmark set of known allergens and likely non-allergens sharing the same structural fold, we show that the 3D epitope similarity method increased accuracy of classification by 2-fold compared to the classical linear window approach. The adjusted hexamer hit approach led to a 6-fold reduction of false positives. The overall accuracy of the method is 84% at highest sensitivity with other current methods ranging from 55 to 74%.
AllerCatPro is a novel computational workflow considering the sequence and 3D structure of proteins for a more comprehensive assessment of the allergenic potential of proteins and thus a more accurate prediction to improve the risk assessment process for Type I allergy.
Keywords: Protein allergens, Type I Allergy, Risk Assessment, in silico, allergenicity prediction
Using the SENS-IS assay, to assess skin sensitization hazard of commercial cosmetics finished products. (#372)
F. Cottrez1, E. Boitel1, H. Groux1
1 ImmunoSeaarch, Grasse, France
The safety of a cosmetic product can be substantiated through the toxicological analysis on individual ingredients. Although satisfactory toxicological data may exist for each ingredient of a cosmetic product, it will still be necessary to conduct some toxicological testing with the complete formulation to assure adequately the safety of the finished cosmetic. One of the preferred skin sensitization safety test today is the Human Repeated Insult Patch Test (HRIPT). However the assay is now more and more widely criticized for ethical reasons but also scientific relevance.
We therefore analyzed the possibility to use the SENS-IS assay to assess skin sensitization hazard of finished products. The SENS-IS assay is an in vitro assay that uses a 3D reconstituted human epidermis model as test system and a genomic signature of 62 biomarkers as read-out. The genomic signature allows the discrimination between irritation versus sensitization risks onto human skin. To adapt the protocol of the SENS-IS assay developed for ingredients a preliminary study was performed on 6 finished products known to have induced skin allergic reactions. Using these finished product we introduced two incubation period, one of 15 min followed by the removal of excess of product and a post incubation of 6 hrs and another without a washing step for a total incubation time of 6hrs and 15 min. Using that protocol 5 out of the 6 products provided a positive response in the SENS-IS assay confirming the human response. The 6th product was detected as widely irritant.
We used this modified protocol to test 21 cosmetics products on the shelf from 15 different brands. 3 were dedicated to babies, 3 soaps (liquid or foam), 3 make-up removers, 3 body lotion, one shaving gel and 10 body or facial skincare. Most of the products were negative and the 3 products for babies completely negatives (less than 2 sensitization biomarkers overexpressed). However, 3 finished products were slightly positive with one extremely positive (the shaving gel). Not surprisingly all the soap were irritant except the one dedicated to babies.
We are currently extended our analysis to other cosmetic finished product (blushes, hair dyes…).
This analysis shows that the SENS-IS assay could be used to test cosmetic products before or in replacement of human testing.
Keywords: Skin Sensitization, in vitro assays, Finished cosmetics products
Assessment of Pre/Prohaptens and Surfactants using the SENS-IS assay, to measure skin sensitization potency (#374)
F. Cottrez1, E. Boitel1, H. Groux1
1 ImmunoSearch, Grasse, France
The ability to identify chemicals that induce a skin sensitization reaction is of high importance for the chemical and cosmetic industry. The local lymph node assay (LLNA) was developed as a practical option for assessing skin sensitization potential of chemicals as, i.e. provide a measure of relative sensitization potency, an information of importance for the management of human health risks. To evaluate skin sensitization potency but also to be able to analyze products and mixtures of different solubility and physical states and mimic skin metabolism to identify potential prehaptens and prohaptens we developed the SENS-IS assay using 3D reconstituted human epidermis. In this study 27 pre- and pro-haptens and 10 surfactants, including SLS, were analyzed. All 27 pre/pro- haptens provided true positive results in the SENS-IS assay whereas only 13 were true positive in DPRA, and a combination of DPRA, keratinosens and hCLAT using a 2 out of 3 weight of evidence approach, gave only a sensitivity of 81% (22 out of 27). Among the chemicals positively detected by SENS-IS and not by combination of 3 assays were 3-aminophenol, resorcinol, 2-methoxy-4-methylphenol, diethylenetriamine and N,N-dibutylaniline. We also tested 12 non sensitizing surfactants, although all but one of them gave a positive LLNA response. They all gave a negative of very weak (not classified) response in the SENS-IS assay. This study shows that the SENS-IS assay is suitable to classify the sensitization potency of pre/prohaptens and surfactants.
Keywords: skin sensitization, in vitro assay, prohaptens, surfactans
Skin sensitisation assessment of 2 biocide formulations: comparison of 3 alternative methods (#427)
S. CATOIRE1, A. THELU1, B. PAGE1, T. CREUSOT1, S. SOUM1, L. BEAUDEQUIN1, B. LOPEZ2, S. MARTIN2, F. BREE2, H. FICHEUX1
1 THOR Personal Care, In Vitro Toxicology, Compiegne, France
With the increasing prevalence of Allergic Contact Dermatitis (ACD), reliable identification and evaluation of the sensitising potential of new compounds is crucial. Recognising the problem of extrapolation from animal models to human, as well as ethical and regulatory pressure to avoid the use of animal models for safety testing, there is a need for the cosmetic industry to develop alternative methods. Skin sensitisation is a complex mechanism which involves different cell types, therefore a combination of various in vitro methods is required. Integrated testing strategies (ITS) are expected to combine various information sources efficiently in a quantifiable fashion to satisfy a regulatory safety assessment requirement.
We have selected a battery of tests for skin sensitisation focused on three in vitro methods that cover the first three Key Events (KE) of the AOP with DPRA (OECD 442), h-CLAT (OECD 442E) and SENS-IS. SENS-IS uses a reconstructed human skin model (RhE) avoiding some specific limitations (solubility, metabolism…) which may be encountered in other methods.
We present a case study evaluating skin sensitisation of a specific biocide, documented as a skin sensitiser (1A) but formulated in 2 different ways: in solution (Formulation A) or with a carrier used to modify the bioavailability of the molecule and to provide a long lasting effect (Formulation B).
The first objective of the study was to compare the prediction of each single test (DPRA, h-CLAT and SENS-IS) regarding the modified formulation of the same compound. The second objective was to investigate how the combination of the 3 tests are able to pick-up the differences between the 2 formulations. In parallel, cytotoxicity was measured in both cases.
The results for Formulation A for the 3 single tests were positive, whereas Formulation B induced positive results with DPRA and h-CLAT but a negative response with SENS-IS. Moreover, the dose finding assay CV75 with h-CLAT was 5.05 and 134.45 µg/mL with A and B respectively. The decreased bioavailability of B correlates not only with a reduced cytotoxicity effect but also lower sensitisation potential with SENS-IS.
The SENS-IS method, after topical application on a RhE, seems to be the most appropriate method to forecast changes in sensitisation potential due to modified bioavailability.
Keywords: allergic contact dermatitis, sensitisation, in vitro methods, DPRA, h-CLAT, SENS-IS
The effect of diet-induced obesity on general and explorative toxicity parameters in Sprague-Dawley rats (#461)
J. M. Rojas1, F. Bolze1, I. Thorup1, J. Nowak1, C. M. Dalsgaard1, M. Skydsgaard1, L. O. Berthelsen1, K. A. Keane1, H. Søeborg1, I. Sjögren1, J. T. Jensen2, M. Dalgaard1
1 Novo Nordisk A/S, Toxicology, Safety Pharmacology and Pathology, Maaloev, Denmark
Employing disease models as an adjunct to the conventional healthy animal in the safety evaluation of novel pharmaceuticals has been recommended by regulatory agencies, when relevant. An obese rodent model that closely mimics human obesity may result in improved predictability and understanding of toxicities encountered with anti-obesity drug candidates. To establish the polygenic diet-induced obese (DIO) Sprague-Dawley (SD) rat model for safety testing, we generated historical control data encompassing standard and explorative toxicity parameters in both female and male SD rats fed either a 45% Kcal high fat diet (HFD) or a normal chow diet (NCD) for up to 33 weeks of age.
Following 29 weeks of HFD feeding, females and males achieved an 8 and 9% increase in body weight, respectively, as well as a 2-fold increase in whole-body fat mass compared to animals on NCD, indicating that these animals were obese. The obese phenotype was associated with the development of metabolic derangements i.e. hyperleptinemia, hyperinsulinemia, insulin resistance, and mild dyslipidemia. The histological examination revealed that HFD feeding induced hypertrophy in brown fat, as well as subcutaneous white fat, and adipocyte accumulation was found in pancreas, skeletal muscle and the parotid salivary glands. In the liver, a mild micro- and macrovesicular fatty change was seen. DIO rats exhibited an increased incidence of chronic progressive nephropathy, whereby females were most affected. µCT analysis of the proximal tibia metaphysis revealed that HFD-feeding induced expansion of the bone marrow area coupled with trabecular bone loss. The trabecular loss was also reflected in the histological examination. In males on HFD, circulating levels of follicle-stimulating hormone and luteinizing hormone were attenuated, and a slight reduction in testosterone was accompanied by a decreased weight of left epididymis. There was no difference in reproductive hormones or estrus cycle length when comparing females on HFD with females on NCD.
Altogether, SD rats fed a HFD developed an obese phenotype that has similarities with polygenic human obesity. Thus, our data in the DIO SD rat suggest that this disease model may be useful as an adjunct to healthy normal weight rodents in safety testing of anti-obesity drug candidates.
Keywords: Diet-induced obesity, Sprague-Dawley rat, toxicology, spontaneous background findings, non-clinical safety assessment
Creation of a stable and highly functional pluripotent stem cell derived hepatocyte model for drug metabolisation and toxicity screening (#510)
M. Kumar1, F. Chesnais1, R. Boon1, A. Krebs1, C. Curtiss1, B. Toprakhisar1, F. Jacobs3, B. Schmidt1, M. Leist2, C. Verfaillie1
1 KU Leuven, SCIL, Dept. of Development and regenration, Leuven, Belgium
As in vitro hepatic and/or in vivo models cannot fully predict liver injury caused by chemicals, high drug attrition still occurs at different stages of their development. It is believed that hepatocyte progeny from induced pluripotent stem cells (iPSCs) may enhance drug discovery and development by providing simple, reproducible, and economically effective tools for compound toxicity screening. However, most PSC derived hepatocyte models do not have a mature hepatocyte metabolism, and have poor metabolisation and toxicity of different chemicals assessment features.
Therefore, we established a robust differentiation protocol for the generation of hepatocyte-like cells from iPSCs, wherein PSC were engineered to overexpress 6 transcription factors (named HC6X) and metabolomics-based medium optimisation (named M5). The HC6X-PSC hepatic progeny is gluconeogenic, use oxidative phosphorylation for production of energy and building blocks, and have a very high ability to metabolize benzyloxy-trifluoromethylcoumarin (BCF; ± 30 percent of primary human hepatocytes, which is stable for ± 2 weeks).
We tested if the optimised HC6X-progeny in M5 would be suitable for toxicity and metabolisation studies of different chemical entities. The initial studies assessed the acute (24h) toxicity of APAP, Amiodarone and Rotenone showing IC50 similar to freshly-isolated PHH. In addition, we assessed the effects of 30 known or non-hepatotoxicants, provided by the EU-ToxRisk consortium and 11 compounds from the MIP-DILI consortium list (Sison-Young R, Arch Toxicol. 2), demonstrating that the HC6X/M5 hepatic progeny can correctly identify hepatotoxins. We also demonstrated that HC6X-progeny in M5metabolises midazolam at levels similar to PHHs, and dextromethorphan, phenacetin, and tolbutamide at levels also seen in e.g. HepaRG cells.
To conclude, we developed a robust culture system consisting of hepatic progeny that displays long term stable metabolisation characteristics and accurate toxicity detection of known hepatotoxicants, at levels similar to freshly isolated hepatocytes. Further studies are ongoing, in house, to validate the suitability of the current improved progeny for metablisation and screening of hepatotoxicants in both single and multiple exposure settings, and to have the assay validated by partners from EU-ToxRisk.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002.
Keywords: Liver Injury, Pluripotent stem cells, Hepatotoxicants, in vitro toxicity, Hepatotoxicity
Empowering scientists in the use of new approach methods: the Altertox Academy (#511)
G. A. Tenie1, I. Prachkovski1, F. Busquet1
1 Altertox Academy, Altertox Academy, Bruxelles, Belgium
There is nowadays a great number of new approach methods (NAM) developed by CROs, SMEs as well as validated Test Guidelines under OECD or ICATM collaboration. Non-animal testing methods and approaches such as in vitro, in silico, in chemico, adverse outcome pathways (AOPs) and read-across fall under the category of NAM. International and governmental bodies such as the European Commission and EU Member States are supporting the training in NAMs through research programs and/or grants. Available resources (webinars, scientific meetings, customized trainings, e-learning) also regularly provide an insight and training into these new test methods.
Despite active efforts, the dissemination of the emerging technologies can be further improved. Altertox Academy has made it its aim to bridge the gap between NAMs and the scientific community for their intensive use and implementation in EU countries and worldwide. The Academy organises tailor-made training activities linked with the 3Rs (reduction, refinement, replacement) principles, on various topics in laboratories on in vitro and/or in silico methods for researchers and toxicologists of various levels of experience. The format of a two-days training session consists of 20% lectures and 80% hands-on-training in the lab. Starting from 2016, Altertox Academy (previously operating under CAAT Academy) has held more than 20 trainings in Europe with 30 unique partners, having an approximate number of 200 lab and webinar participants of 20 different nationalities. Additionally, Altertox intends to build on this accumulated experience to act as work package leader in training under EU framework research programs and /or similar activities.
Thanks to empowering and familiarising toxicologists with the new technologies as well as critical steps which are not described in OECD Test Guidelines, Altertox Academy intends to share through this presentation feedback and lessons learned for the scientific community and therefore facilitate organising and performing successful trainings elsewhere.
Keywords: training, 3Rs, in vitro, in silico, in chemico
Addressing the exceptions of the limitation for highly volatile substances in the Short Time Exposure (STE) test method and the predictive performance for assessing eye irritation potential (#542)
T. Abo1, 2, T. Yuki1, R. Xu1, D. Araki1, Y. Takahashi1, H. Sakaguchi1, H. Itagaki2
1 Kao Corporation, Safety Science Research, Kanagawa, Japan
The Short Time Exposure (STE) test method is an in vitro alternative test method for eye irritation potential based on the cytotoxicity by 5-min treatment in Statens Seruminstitut Rabbit Cornea (SIRC) cells. The STE test method has been adopted as test guideline (TG) 491 by the Organisation for Economic Co-operation and Development (OECD) and has an applicability domain. When used to identify Globally Harmonized System (GHS) No Category, one of the chemical categories that is excluded from the applicability domain of the STE test is highly volatile substances with a vapor pressure over 6kPa (25˚C). Possible reason why these substances are not part of applicability domain is due to volatilization of substance during test solution preparation or exposure. The standard protocol employs saline as the solvent and the reduction of the highly volatile substances in solvent may lead to the false negatives. Mineral oil has the potential to lower the volatilization rate of the test substances compared to saline. We evaluated the ability of mineral oil to be a better solvent for highly volatile substances.
Highly volatile substances, which GHS classification was assigned to, were correctly evaluated without false negatives by the modification of the solvent to mineral oil. The predictive performance was improved from the accuracy 75.0% (15/20), the false positive rate 7.7% (1/13) and the false negative rate 57.1% (4/7) to the accuracy 95.0% (19/20), the false positive rate 7.7% (1/13), and the false negative rate 0% (0/7) respectively.
Furthermore, the predictive performance was verified including highly volatile substances by the modification of the protocol. The substance dataset was constructed in reference to STE Summary Review Document by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM, 2013) and the Draize eye test Reference database by Barroso et al. (2017). As a result, the accuracy 86.6% (194/224), the false positive rate 18.8% (27/144), and the false negative rate 3.8% (3/80) were obtained. In conclusion, the STE test method is suitable for testing the GHS NC substances.
Keywords: Short Time Exposure, Eye irritation, Alternative method, Highly volatile substance, Draize eye test Reference Database
A novel microtissue-based 3D human liver NASH model for drug discovery (#567)
J. Rupp1, T. Strassfeld1, S. Steiert1, P. Guye1, S. Messner1, R. Kostadinova1
1 InSphero AG, Schlieren, Switzerland
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease affecting around 30% of the population and can progress to non-alcoholic steatohepatitis (NASH), defined as hepatic steatosis with inflammation. NASH frequently further develops into fibrosis, liver cirrhosis and liver failure. At present, there is no approved, safe therapy for NASH. Thus, we aimed to develop a human-relevant in vitro 3D Liver NASH model of lipotoxic stress which incorporates key physiological aspects such as steatosis and inflammation, and to characterize this model by comparing it to established mechanisms for pathogenesis of NASH. 3D Human Liver Microtissues were engineered to incorporate all the relevant primary human liver cell types responsible for the development of the disease: hepatocytes, HSCs, Kupffer cells (KCs) and liver endothelial cells (LECs). The resulting 3D InSight™ Human Liver NASH model has been induced by treatment of the tissues with Free Fatty Acids (FFA) such as Oleic and Palmitic acids (2:1) and LPS for 7 days. We characterized the tissues under basal and induced liver disease conditions on morphological and phenotypic levels by Nile-Red staining of incorporated lipids using confocal microscopy, immunostaining techniques, gene-expression analysis, and cytokine release. Exposure of the tissues to FFA plus LPS for 7 days induced high lipid accumulation in the hepatocytes as well as increased secretion of IL-6 and TNF-a. We demonstrated that FFA plus LPS treatment induced liver inflammation and steatosis in vitro, making this model an ideal tool for anti-NASH drug discovery.
Keywords: NASH, 3D Human Liver Microtissues
Optimization of the Vitrocell 24/48 in vitro inhalation exposure system for nanoparticles (#602)
E. Frijns1, J. Van Laer1, A. Jacobs1, S. Verstraelen1
1 Flemish Institute for Technological Research, Health, Mol, Antwerpen, Belgium
Epidemiological and animal studies are both widely used approaches to investigate potential adverse effects and conduct risk assessments on inhaled chemicals and drugs. However, results are not always easy to interpret or reproduce and such experiments are extremely expensive, time consuming and use large numbers of animals. For these reasons, there is an increasing demand for the development of alternative approaches that make use of reliable in vitro inhalation testing strategies. These testing methods will require realistic lung cell models, realistic inhalation exposure systems and proper dosimetry techniques to increase the predictive ability of in vitro cell models and therefore accelerate the shift from in vivo towards in vitro testing.
The exposure of mammalian cells or tissues to substances which can be inhaled is frequently performed under submerged conditions. In doing so, the test substances are dispersed in liquid and dosed into the culture medium. A more realistic direct exposure of mammalian cells or tissue to airborne substances is exposure at the air-liquid interface (ALI) where the cell systems are not covered with culture medium.
The Vitrocell® 24/48 in vitro inhalation exposure system has been specifically designed and engineered to perform a complete dose-response profile in one run. Seven dilutions with 6 inserts each, are used for exposure to the substances and 6 inserts in the same system for clean air control.
Experiments with the 24/48 system were performed to test different humidification systems, trumpet heights, flows and cell models (A549, CALU3) to find the most optimal settings for nanoparticle exposure. The results of these experiments will be presented as well as the results of the exposure of CALU3 cells to nano-Y2O3.
The Vitrocell 24/48 In Vitro Inhalation Exposure Systems was awarded to VITO by the PETA International Science Consortium.
Keywords: Air-Liquid-Interface, in vitro, exposure, inhalation, nanoparticle
Use of tests on Hydra attenuata and Brachydanio rerio for teratogenic tests (#609)
R. E. Sornat1, D. Krakowian1, M. Napora-Rutkowska1, J. Faron1, K. Gruszka1, M. Wołany1, I. Mrzyk1, A. Kropidło1, M. Paleczny1, H. Rzodeczko1
1 Institute of Industrial Organic Chemistry, Department of Toxicological Studies, Psczyna, Poland
The study was an attempt of use of Hydra attenuata and Brachydanio rerio embryos in teratogenic studies. The aim was to determine the suitability of these species in a such type of researches.
Eleven active substances with different degree of influence on prenatal development were used in experiment: two with no adverse effect, five with toxic effect visible only at the level of maternal toxicity and four approved as strong teratogenic.
Hydra attenuata. Acute toxicity and regeneration test were performed for each substance. Tests were performed to obtain LC50 value for acute and for regeneration test. The teratogenic index was estimated. Depending on the size of the index, the appropriate point value was assigned: 0, 1 or 2 (0 – no teratogenic potential, 2 – higher teratogenic potential).
Brachydanio rerio embryos. Different concentrations of substances were used in the study to observe the concentration with toxic effects and lethal effects. The occurrence of changes and lethal effects in the embryos was marked as an appropriate point values. Lack of changes was valued as 0. The maximum point value for changes and lethal effects could be 4.
Study reveals usefulness of these animals as indicators for strong teratogenic. The final grade for each substance was the sum of the points in the study on hydra and embryos. All used in experiment strong teratogenic reached the highest value of points (5 to 6) and are clearly higher then values of substances defined as not teratogenic (0 to 4).
Keywords: Animals models, Hydra attenuate, Brachydanio rerio, teratogenic effect
A new in vitro approach to verify the moisturizing activity of topical cosmetic products (#626)
E. Regola1, R. Vicini1, A. Buzzella1, 2, F. Riva3, M. Mori1, S. Giorgetti4, C. Angelinetta1, O. Pastoris2
1 Bio Basic Europe S.r.l., Milano, italy, Italy
The moisturization of the skin is regulated by the balance between transepidermic water loss and skin ability to retain water.
This function is supported by the presence of specifical structures: Occludin which play a key role in maintaining the cutaneous barrier integrity in thight junctions, and Aquaporins (AQPs), transmembrane proteins that facilitate the transport of water and, in some cases, small solutes across cells membrane.
According to the results of in vivo moisturizing tests currently used, the aim of this study was to develop an in vitro moisturizing test based on the evaluation of expression of Occludin and AQP3.
Topical products, tested in vivo and in vitro, were divided into 3 different categories based on the active moisturizing ingredients: category A containing Glycerin and hyaluronic acid, category B containing Urea and category C containing Glycerin.
For the in vivo study the products were applied on 20 female subjects and the instrumental measurements of moisturization was performed before the beginning of treatment (T0, basal value) and after 15-30 min for short-term evaluations or after several days of continuous use for long-term evaluations.
The in vitro tests were performed on keratinocytes (HaCaT) cell cultures.
The cells were treated for 24-48-72 hours with concentrations of products choosen after a preliminary cytotoxicity test (MTT): the expression of AQP3 and of occludin was evaluated by western blot.
To further support the results the expression of AQP3 was also evaluated by immunofluorescence.
RESULTS and CONCLUSIONS
We observed an increase in skin moisturization in vivo for all tested products (particularly evident for category A and B products).
This effect is confirmed by in vitro evaluation of AQPs and occludin expression.
The expression of AQPs in the cells treated with the category A products coincide with the increase in short-term in vivo moisturization (increase of 44% after two hours from the application on skin). The slowest in vitro action of category B products reflects the long-term effects obtained following in vivo treatment for 7 days.
The moisturizing power of category C products was observed only in vivo, probably due to the humectant characteristics of glycerol present in them rather than to the increase of the expression of specific moisturizing markers.
Keywords: hydratation of skin, occludin, AQPs, in vitro moisturization test
Effects of exposure to e-cigarette aerosols compared with cigarette smoke on 3D human buccal and small airway cultures: a systems toxicology assessment (#707)
A. R. Iskandar1, S. Sandro1, M. Shoaib1, A. Kondylis1, Y. Xiang1, F. Zanetti1, S. Frentzel1, F. Martin1, N. V. Ivanov1, M. C. Peitsch1, U. Doshi2, W. McKinney2, M. Lee2, J. Hoeng1
1 Philip Morris International R&D, Neuchatel, Switzerland
Considerable attention has been given toward the potential reduced harm of e-cigarettes (e-cigs). Most in vitro studies have focused on testing e-liquid formulations directly on submerged 2D cultures. Here, we examined the effects of exposure to whole e-cig aerosols compared with mainstream cigarette smoke (CS) using human 3D organotypic cultures. Buccal and small airway cultures were exposed at the air-liquid interface over 28 minutes to 112 puffs of undiluted aerosols generated from an e-vapor product containing various e-liquids (“CARRIER” containing humectants alone, “BASE” containing humectants and 4% nicotine, and “TESTMIX” containing humectants, 4% nicotine, and flavors) or to diluted CS in Vitrocell® exposure systems. Nine independent exposures were conducted for a robust assessment. Concentrations of the deposited nicotine and carbonyls in the exposure chamber were measured each time as markers of exposure. Biological endpoints investigated include histology, cytotoxicity, inflammatory mediators, and gene microarray. Alterations in morphology and cytotoxicity were not observed in buccal cultures exposed to undiluted e-cig aerosols despite the two-fold nicotine deposition compared with the deposition following exposure to diluted CS. Similarly, morphological changes and cytotoxicity were not observed in small airway cultures following e-cig aerosol exposure despite the 10-fold nicotine deposition compared with the deposition after exposure to diluted CS. In both cultures, many more inflammatory mediators in the media were altered following exposure to CS than were altered following exposure to e-cig aerosols. In addition, compared with exposure to e-cig aerosols, CS exposure showed greater biological impact in global gene expression, including mechanisms of cell fate, proliferation, stress, and inflammatory response. In small airway cultures, these impacts reverted to the levels of the air-exposed cultures after 24 hours. In small airway and buccal cultures, e-cig aerosols caused significantly less impact than CS. Overall, we did not observe significant differences between the exposure to CARRIER, BASE, and TESTMIX aerosols.
Keywords: systems toxicology, e-cigarettes, omics, organotypic cultures
Schmidtea mediterranea as an alternative model to predict carcinogenicity via its stem cell responses (#720)
J. - P. Ploem1, A. Wouters1, M. Willems1, T. Artois1, K. Smeets1
1 Hasselt University, Zoology: Biodiversity and Toxicology, Diepenbeek, Limburg, Belgium
The need for accurate and reliable carcinogenicity assays poses an important issue within the field of toxicology. Cancer risks are directly associated with the underlying mechanism of action of the carcinogenic compound (e.g. genotoxicity). Currently, the two-year rodent carcinogenicity assay is still considered as the gold-standard regarding risk assessment of potential carcinogens while the ‘Three Rs’ principle already exists for over half a century.
The presented assay provides a simple, rapid and inexpensive alternative method to predict genotoxicity, but also has the potential to identify non-genotoxic compounds. Our methodology entails the exposure to carcinogens during the early stages of the regeneration process of a flatworm species: Schmidtea mediterranea. During this regeneration period, the tested compounds induced unique stem cell responses for the different types of carcinogens (non-genotoxic vs genotoxic).
We are currently assessing more compounds to further validate the concept, i.e. a decrease proliferating stem cells during genotoxic exposure and an increase during non-genotoxic exposure. In addition, we focused on direct and indirect DNA damage and the activated repair mechanisms of the pluripotent stem cells. Here we observed differences, between genotoxic and non-genotoxic exposure, in the type and regulation of the activated repair gene groups, e.g. non-homologous end joining (ku80), homologous recombination (rad51), post-replicative DNA mismatch repair system (msh2) and nucleotide excision repair (xpa).
Unique responses will be evaluated with more compounds as additional parameters to increase the assay’s sensitivity and specificity. The ability to discriminate between genotoxic and non-genotoxic compounds in an in vivo alternative model, makes this approach unique and with significant added value to current research and drug development.
Keywords: Carcinogenicity assay, in vivo, alternative models, Stem cells
RE-Place: centralizing information on alternative methods to animal testing to promote knowledge sharing (#69)
M. Van Mulders1, 2, B. Mertens1, V. Rogiers2
1 Sciensano, SD Chemical and Physical Health Risks, Service Health Risk and Health Impact Assessment, Elsene, Brussels, Belgium
Over the last years, many valuable in chemico, in silico and in vitro methods have been developed as alternatives to animal testing. Unfortunately, expertise on these alternative methods is scattered and communication on the topic between experts from different domains is often limited. In order to enhance the use of alternative methods to animal testing, knowledge sharing needs to be facilitated between the different parties involved (e.g. scientists, regulators, industry, ethical committees, animal welfare bodies and the general public).
In 2017, the RE-Place project was initiated by the Flemish and Brussels government with the aim to centralize the existing knowledge and increase its accessibility. The project, coordinated by Sciensano and the Vrije Universiteit Brussel (VUB), consists of 3 phases: (1) an exploratory survey to identify the experts that are currently using alternative methods to animal testing, (2) the set-up of an online tool (available via www.RE-Place.be) to collect details on alternative methods, and (3) the compilation of an easy to use database, integrating all the acquired information. The RE-Place project is performed in close collaboration with the members of the RE-Place steering committee, which consists of experts from academia, industry and government institutions, all with extensive know-how in the field of alternative methods to animal testing. Additionally, the European Union Reference Laboratory for alternatives to animal testing (EURL-ECVAM) is also involved in the RE-Place project.
Since the online tool is publicly accessible, experts from different fields will be able to connect with peers and engage in new collaborations. Centralization of the information will also allow to identify the gaps in the field of alternative methods to animal testing. Importantly, in a later stage, the RE-Place database can be further extended to a broader platform, stimulating the development of new techniques, methods and strategies and supporting education and training.
Keywords: 3R, replacement, alternative methods, database
Developmental expression of genes involved in the metabolism and transport of xenobiotics in the zebrafish from the embryonic until the juvenile stage (#522)
C. Bars1, E. Verbueken1, J. Periz-Stanaćev2, L. Vergauwen2, E. Michiels2, E. Stinckens2, I. Gabriëls2, D. Knapen2, C. Van Ginneken1, S. Van Cruchten1
1 University of Antwerp, Applied Veterinary Morphology, Wilrijk, Belgium
In recent years zebrafish, Danio rerio, embryos have gained a lot of interest as a “whole organism” for toxicity assessment of xenobiotics. Aside from diverse economic, morphological and handling advantages, it is a non-protected organism until the free-feeding stage (120 hours post fertilization) according to the EU Directive 2010/63/EU. However, the biotransformation capacity at such an early stage of development (embryonic and larval stage) remains a point of debate. This is important for a correct interpretation of toxicity data and especially when these data are used for human risk assessment. We, therefore, thoroughly characterized the transcript profiling of enzymes involved in the metabolism of xenobiotics, i.e. phase I (CYPs 1A; 1B1; 1C1; 1C2; 2K6; 3A65; 3C1) and phase II (UGT1A; SULT1) enzymes, and one drug transporter related protein (ABCB4) from 1.5 hours post fertilization (hpf) until 32 days post fertilization (dpf). RNA was extracted (NucleoSpin® RNA) from 10-30 whole organisms, depending on the age, from each of the 25-time points between 1.5 hpf to 32 dpf and stored upon use at -80°C. After synthetizing cDNA from the RNA samples (Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit), quantitative PCR was performed with Brilliant II SYBR® Green QPCR Master Mix on a Mx 3005P instrument (Agilent technologies). The mRNA relative quantity of each time point was determined after analysis of the raw data by the qBase+ software. Generally, low CYP1-3 expression was noted during zebrafish organogenesis until 3 dpf, followed by an increase of expression after this stage of development. An interesting pattern of expression of the CYP1-3 enzymes was also observed between 6 and 10 dpf. The expression of the phase II enzymes and the drug transporter seemed to be low as well until 3 dpf, highly increased from 3 dpf until 5 dpf and then stabilized. The correlation of the gene expression data with the activity of these proteins, in both the early phase of development and the adult stage of the zebrafish, will allow for a better interpretation of toxicity studies in this model.
Keywords: Ontogeny, Cytochrome P450, qPCR, In vitro model, Zebrafish
Optimization of the use of Caenorhabditis elegans (C.elegans) in reproductive toxicology testing: describing the most optimal test conditions (#768)
S. Bressers1, M. Tobor-Kaplon2, M. Beekhuijzen3
1 Radboud University, Biomedical Sciences, Nijmegen, Netherlands
Caenorhabditis elegans (C. elegans) has emerged as a promising alternative model to screen for reproductive toxicity as it is cheaper and faster when compared to tests with mammals, and complies with the ethical framework of the 3R’s (i.e. reduce, refine and replace). The methodology of these reproductive toxicity assessments used by different groups varies greatly. To set a first step in implementation of the C. elegans model for reproductive toxicity, optimization of procedures is crucial. A thorough analysis of published peer-reviewed manuscripts on the use of C. elegans in reproductive toxicity testing was performed to compare and select all test conditions that are considered to impact the outcome. Test conditions such as selection of nematode strain, culture media (liquid or agar), temperature, developmental stage at the start of exposure, use of solvent, method of exposure (agar vs liquid medium; in presence or absence of bacteria), exposure length, type of test chamber, availability of food (alive, inactivated or in absence of food) and study endpoints were reviewed. Based on the outcome of this literature review and based on in-house experimental data, the most optimal study design regarding these test conditions is proposed.
Keywords: Caenorhabditis elegans, optimization, reproduction, reproductive toxicity, 3R’s
An ontology-driven testing strategy for developmental neurotoxicity testing (#776)
E. Hessel1, Y. Staal1, A. Piersma1
1 National Institute for Public Health and Environment (RIVM), Center for Health Protection (GZB), Bilthoven, Netherlands
Developmental neurotoxicity entails one of the most complex areas in toxicology. Development of the central nervous system involves many different events within strictly controlled timeframes creating a different windows of vulnerability to chemical exposure. OECD test guidelines for DNT (TG 426 and 443) are only occasionally carried out and the predictivity of these in vivo animal tests for human health effects may be limited.
A multitude of alternative models have been developed over the years, providing insights into mechanisms of action. The development and application of mechanistically driven in vitro tests in relation to human brain development may facilitate a better prediction of DNT in the future. Given the impressive progress in mechanistic knowledge of human biology and toxicology, the time is right for a conceptual approach for designing testing strategies that cover the integral mechanistic landscape of developmental neurotoxicity. The ontology approach provides a framework for defining this landscape, upon which an integral in silico model for predicting toxicity can be built. It subsequently directs the selection of in vitro assays for rate-limiting events in the biological network, to feed parameter tuning in the model, leading to prediction of the toxicological outcome. Therefore, we generated an overview of fundamental processes of neurodevelopment based on existing knowledge of the biology of neural tube formation, brain development and neural specification. In parallel, we made an inventory of the wealth of alternative methods in this area and matched them to biological processes that need assessment for chemical perturbation. This inventory will feed into an ontology approach towards building comprehensive testing strategies for developmental neurotoxicity.
Keywords: Developmental neurotoxicity, 3r, alternatives for animal testing
Toxicogenomics of gold nanoparticles in a marine fish: linkage to established biomarkers (#138)
M. Teles1, 2, F. Reyes-López2, J. C. Balasch2, L. Guimarães1, L. Tort2, M. Oliveira3
1 CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal
In the present study, we used a species-specific enriched oligonucleotide microarray platform (SAQ), previously developed by our research group, in order to explore the underlying effects of gold nanoparticles (AuNP, spheres, citrate coated, ~40 nm) on gilthead sea bream (Sparus aurata) hepatic function after a short-term (24 h) waterborne exposure to two distinct concentrations (0.5 and 50 μg/L) of AuNP. The transcriptional profile was complemented with outcomes at higher levels of biological organization, including biotransformation, antioxidant response, hepatic health indicators, and oxidative damage indicators. The DNA damaging potential of AuNP was assessed in whole peripheral blood using the comet assay (measures DNA strand breaks) and by scoring the erythrocytic nuclear abnormalities, ENA (measures chromosome damage). Chemical quantification of AuNP was carried out in water samples and in S. aurata livers. The overall genetic response showed a differential hepatic transcriptional profile both in the number and intensity of differentially expressed genes (DEG). Concerning the functional pathways affected, main changes were found for genes encoding proteins involved in the response to xenobiotics, oxidoreductase activity, immunomodulation, DNA repair and programmed cell death types I and II. Moreover, AuNP exposure caused DNA strand breaks, however, without causing clastogenesis or aneugenesis, since no ENA were detected. In light of results obtained for all the organizational levels of response assessed, we conclude that short-term waterborne AuNP exposure has a direct effect on gene expression modulation in S. aurata liver and affects genetic function in fish.
Keywords: Nanoparticles, Fish, Transcriptional profile, Genotoxicity
Analytical and biological evaluation of cardiotoxicity safety biomarkers in the rat (#219)
T. Erkens1, J. Boonen1, N. Goeminne1, A. Kegels1, L. Van den Sande1, D. Marien1, M. van Heerden1, P. Vinken1
1 Janssen Research & Development, a division of Janssen Pharmaceutica NV, Beerse, Belgium, Preclinical Development & Safety, Beerse, Belgium
Cardiotoxicity is a major concern during drug development and is a major factor contributing to the high attrition rates. Therefore, it is important to detect cardiac toxicity early in the development process. The objective of our study was to evaluate both the analytical and biological performance of a number of safety biomarkers related to cardiotoxicity in a preclinical setting.
Serum samples were collected from male Sprague-Dawley rats prior to treatment, and 6 and 24 hours after subcutaneous single dose administration of 0.1-0.5 mg/kg isoproterenol, which is a known cardiotoxic agent. The Milliplex rat cardiac injury panel 1 (Merck) was used to measure troponin I (TnI), troponin T (TnT), creatine kinase muscle (CKM), fatty acid binding protein 3 (FABP3), follistatin-like protein 1 (FSTL1), myosin light chain 3 (MYL3) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Rat myoglobin was analyzed by Elisa (Life Diagnostics), and a panel of miRNAs was analyzed using the Abcam-FirePlex technology on a flow cytometer.
For the multiplex and myoglobin immunoassays, precision was considered acceptable for all parameters with a CV often below 20%. Parallelism was observed, although the minimum required dilution varied with parameter in the multiplex panel. For the FirePlex miRNA assay, precision results improved with signal intensity, with a CV generally ranging between 10-30%.
Histopathology confirmed the presence of heart damage in the treated rats. Except for FSTL1, increases were detected in all parameters from the cardiac multiplex panel and myoglobin, with fold changes ranging between 2- and more than 150-fold, depending on the biomarker. In addition, an increase in a number of miRNAs was observed, such as miR-133a-3p, 133b-3p, 1-3p and 208a-3p.
Although our experience with the FirePlex platform at this stage is limited and the data in this study are based on a small sample size, results indicated that the evaluated assays are able to detect biomarker changes in rats with cardiac injury. Therefore, these safety biomarker assays could be of added value in preclinical toxicity studies in the rat.
Keywords: biomarker, cardiotoxicity, preclinical, rat, safety
A multi-biomarker assessment of chronic effects of an insecticide Acer 35 EC on Nile Tilapia Oreochromis niloticus under laboratory conditions (#239)
N. L. GUEDEGBA1, 2, I. IMOROU TOKO2, P. T. AGBOHESSI2, N. OREINS1, L. FRANÇOIS1, O. PALLUEL5, S. N. M. MANDIKI1, J. - M. PORCHER5, B. SCHIFFERS3, M. - L. SCIPPO4, P. KESTEMONT1
1 University of Namur, Research Unit in Environmental and Evolutionary Biology (URBE)/ Institute of Life-Earth-Environment (ILEE), Namur, Belgium
Acer 35EC is an insecticide (20 g/L lambda-cyhalothrin + 15 g/L acetamipride) widely used in pest control in many West African countries, and particularly in cotton culture in north Benin. This study aimed to investigate under laboratory conditions the chronic effects of Acer 35EC on Nile tilapia Oreochromis niloticus using a multi-biomarker approach. For this purpose, juveniles of Nile tilapia were exposed to sublethal concentrations of Acer 35 EC (0, 1 and 10 % of LC50- 96h value) in glass aquariums during 56 days. After 0, 28 and 56 days of exposure, several biomarkers including enzymatic activities (indicators of detoxification and oxidative stress, neurotoxicity and immune responses) sex steroid hormones (Testosterone, Estradiol-17β and 11-Keto-testosterone) and histological alterations of liver, kidney and gonads were measured in males and females. A biomarker index (BI) was calculated based on the Integrated Biological Responses (IBR) developed by Beliaeff and Burgeot (2002).
The results showed that Acer 35EC reduced cholinesterase activity in muscle of treated fish of both sexes. Females displayed high levels of testosterone and 11-Keto-testosterone after 56 days of exposure. Regarding immune biomarkers, intracellular superoxide anion production decreased in both sexes after 56 days of exposure. Oxidative stress biomarkers were not influenced by Acer-35EC exposure, regardless of sex and concentration. However, liver of females exposed to 1 and 10 % of LC50- 96h value was significantly (p<0.05) affected after 28 and 56 days of exposure, while no histological differences were recorded in kidney between control and exposed fish. After 28 and 56 days of exposure, significant differences in the ovarian development were observed as the diameter of vitellogenic oocytes in exposed females differed from the one of the control females, indicating an increase in oocyte growth during the 1st month of exposure to Acer-35EC, followed by a regression during the 2nd exposure period. No differences were recorded in male gonads. Based on a large set of biomarkers, the insecticide Acer-35EC seems to impair different physiological functions in Nile tilapia juveniles on a time-dependent manner, with a stronger impact on females than in males.
Keywords: Acetamiprid, Lambda-cyhalothrin, Biomarkers, Nile Tilapia, Chronic effects
The effects of a plant-based antidiabetic supplement on the antioxidant capacity of serum and serum lipoproteins in diabetic rats (#274)
A. Ungurianu1, O. Șeremet2, D. Grădinaru1, C. Ionescu-Tîrgoviște3, R. Dănciulescu-Miulescu3, D. Margină1
1 “Carol Davila” University of Medicine and Pharmacy, Department of Biochemistry, Bucharest, Romania
The objective of our study was to compare the antioxidant capacity and redox alterations of serum samples and isolated lipoproteins from type 2 diabetes mellitus (T2DM) rats treated with either metformin, gliclazide or Retinofort – a plant-based antidiabetic dietary supplement.
Wistar male rats with alloxan-induced (130mg/kg, i.p.) T2DM were split into 4 groups which received different treatment: distilled water (p.o.) for control (n=8), metformin (n=6) 150 mg/kg, 1,6% solution, p.o, gliclazide (n=6) 10 mg/kg, 0,1% suspension, p.o and Retinofort (n=7) 100 mg/kg, 1% suspension, p.o. Rats were sacrificed and serum was separated. High and low density lipoproteins were isolated from serum with the heparin-citrate method and, respectively, the phosphotungstic acid-MgCl2 one. Antioxidant capacity was determined with a ABTS based method and lipid peoxidation was assessed with a previousely optimised Amplex Red assay, on both serum and lipoprotein preparations.
Total serum antioxidant capacity was significantly higher in the Retinofort group (p<0,001), compared to Metformin, Glicazide or control, with no significant difference between the latter three. The antioxidant capacity of HDL was similar in the four groups, however the LDL one was significantly higher compared to control for the Metformin (p=0,031) and Retinofort (p=0,039) groups.
As for the serum lipid peroxidation, compared to control, the mean values were lower for Metformin and Retinofort groups and higher for Gliclazide. Nonetheless, serum peroxidation in the Gliclazide group was significantly higher than in the Metformin one (p=0,037). HDL lipid peroxidation was significantly higher in the Retinofort and Gliclazide groups versus the Metformin group (p=0,005 and p=0,007, respectively). Regarding LDL peroxidation there was no significant differences between the four groups, however a strong positive correlation of this marker with glycemia throughout the treatment period was observed.
Retinofort treated rats exhibited superior antioxidant defence of serum and LDL compared to the other groups, accompanied by similar lipid peroxidation versus the Metformin one. Lipid peroxidation of HDL in Retinofort rats was significantly higher compared to Metformin, but the antioxidant capacity was similar.
Keywords: type 2 diabetes mellitus, antioxidant capacity, redox imbalances
Evaluation of biomarkers for haemocomopatibility of polymer biomaterials (#287)
A. Miyajima-Tabata1, K. Komoriya1, M. Tanaka2, H. Hiruma1, R. Kato1, Y. Haishima1
1 National Institute of Health Sciences, Division of Medical Devices, Kawasaki, Japan
Purpose: Haemocompatibility is a major concern associated with using medical devices or materials that contact with blood; it is greatly influenced by the physical properties of the material surface. Although some representative evaluation methods are established in ISO 10993-4, appropriate biomarkers for the evaluation of haemocompatibility have not yet been identified. 2-Hydroxyethyl methacrylate polymer (PHEMA), 2-methoxyethyl acrylate polymer (PMEA), and 2-methacryloyloxyethyl phosphorylcholine polymer (PMPC) show good haemocompatibility, and in particular, PMEA and PMPC are used as surface modification agents for blood-contacting medical devices. In this study, we evaluated the biomarkers for haemocompatibility of these biomaterials.
Methods: Both sides of a polycarbonate (PC) sheet were coated with the biomaterials. Heparinized human whole blood was incubated with polymer-coated and uncoated PC sheets for 1-4 hours in various testing tubes with gentle shaking. The levels of biomarkers, such as thrombin-antithrombin complex (TAT), platelet granule release product β-thromboglobulin (β-TG), and complement activation products (C3a, C5a and SC5b-9) were measured by ELISA. The effects of priming and air contact were also compared.
Results and discussion: No haemolysis was observed in all cases. The levels of TAT, β-TG, and SC5b-9 incubated in polypropylene (PP)-low bind tubes were lower than those in polyethylene terephthalate (PET) or uncoated PP tubes. Priming and air contact did not affect the assessment of the haemocompatibility of these biomaterials. The levels of these biomarkers increased after incubation with uncoated PC sheets. The levels of TAT and β-TG decreased in blood samples incubated with the PC sheets coated with PMEA or PMPC compared with PHEMA and uncoated PC sheets. There was no remarkable difference of the levels of C3a, C5a, and SC5b-9 in all sheets. These results indicated that TAT and β-TG could be appropriate biomarkers for evaluating the haemocompatibility of polymer biomaterials.
Keywords: haemocompatibility, polymer biomaterial
Short-term exposure to ambient motor vehicle emissions perturb the human circulating miRNA genome (#328)
J. Krauskopf1, F. Caiment1, K. van Veldhoven2, M. Chadeau-Hyam2, R. Sinharay3, K. Fan Chung3, P. Cullinan3, P. Collins3, B. Barratt4, F. J. Kelly4, R. Vermeulen5, T. M. de Kok1, J. C. S. Kleinjans1
1 Maastricht University, 1Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht, Netherlands
Motor vehicles emit large quantities of a complex mixture of harmful pollutants including several species of particulate matter (PM) as well as gaseous pollutants such as nitrogen dioxide (NO2). These traffic-related emissions have been linked to the pathogenesis of several diseases including multiple types of cancer, pulmonary and cardiovascular diseases and more recently also to neurodegenerative diseases. In search of an early diagnostic biomarker for improved air pollution related health risk assessment, recent studies have shown that certain circulating miRNAs are altered upon e.g. PM exposure in humans. In this study we assessed real-time personal exposure to vehicle-emitted air pollutants of a human population walking at a high and low traffic site, and consequently analyzed the global plasma circulating miRNA genome of all participants by next-generation sequencing. The study revealed a profile of circulating miRNAs to be dose- and pollutant species-dependently associated with multiple ambient vehicle-emitted pollutant species including PM10, PM2.5, black carbon, ultrafine particles and NO2. Our bioinformatics analysis suggests that the signature of circulating miRNAs reflect the consequences of motor vehicle emission-induced toxicity in target tissues such as the lung, heart and brain already after 2 hours. Consequently, our study suggests that signatures of circulating miRNAs present a novel class of biomarkers to be used in health risk assessment and potentially allow to interrogate organ-toxicity from miRNA based ‘liquid biopsies’.
Keywords: microRNA, biomarker, diesel, particulate matter, health-risk assessment
Apoptosis activity on the vitamin and mineral deficiency model under CCl4 intoxication (#379)
S. I. Shestakova1, N. V. Tyshk1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The model of adaptive potential reduction was tested on male Wistar rats in the 64-days experiment with carbon tetrachloride (CCl4) intoxication. Animals with an initial body weight of 85.6±1.0 g were divided into three control and three test groups, 30 males in each. Control group rats received diets (AIN-93) with 75%, 30% and 19% content of vitamins B1, B2, B3 and B6 and minerals (Fe3+ and Mg2+) (groups K-75, K-30 and K-19, respectively). Besides similar diets (O-75, O-30 and O-19), the test group rats were intraperitoneally administered CCl4 once a week throughout the experiment (8 injections). In total each animal received CCl4 in the amount of 6.5 g/kg body weight. In addition to a standard for toxicological research set of hematological, biochemical and morphological parameters, there was also used single-cell gel electrophoresis (DNA-comet) method to study the apoptosis activity in the liver, whereby the apoptotic index (AI, the number of apoptotic cells per 100 liver cells).
The analysis of the data revealed a significant increase in apoptosis activity correlated to the level of nutrient deficiency, with AI values in control groups K-75, K-30 and K-19 of 2.04, 2.26 and 5.97, respectively. The test group animals demonstrated the similar dynamics in programmed cell death intensity increasing amid the vitamins and minerals decline: the AI values in groups O-75, O-30, and O-19 were 4.68, 4.48 and 7.36, which is by 129%, 98% and 23% higher than in the respective control groups. It worth noting that the deepening nutrients shortage led to decrease difference between the test and control groups.
According to the research results, it can be concluded that the apoptosis activity indicators, which are sensitive biomarkers of toxic effects, appear to be inappropriate for application on alimentary deficiency models.
This work was supported by Russian Science Foundation grant No. 16-16-00124.
Keywords: apoptosis, biomarkers, toxicological studies, CCl4 intoxication
Discovery of urinary biomarkers for early detection of diabetic- induced chronic kidney diseases (#403)
K. S. Kim1, J. S. Kim1, H. S. Kim1
1 sungkyunkwan university, school of pharmacy, suwon, Republic of Korea
Diabetic kidney disease (DKD) is the leading of chronic and end-stage-renal disease worldwide. After development of overt diabetic nephropathy, patients will progress to end-stage renal disease (ESRD), which is associated with high morbidity and mortality, and high treatment costs. Recently, microalbumin has historically been measured as an early marker for advanced CKD. However, detection of microalbumin in the urine is not adequately predicting DKD from non-albuminuric DKD. Therefore, additional biomarkers of glomerular and/or tubular injury have been proposed to uncover early renal dysfunction and structural lesions. In this study, we identified reliable on non-invasive urinary biomarkers for early detecting DKD. We identified protein-based new biomarkers in the kidney of high fat diet-induced ZDF rats. Among the identified proteins, selenium binding protein-1 (SBP-1) and pyruvate kinase M2 (PKM2) were candidated as potential biomarker for early detecting DKD. These biomarkers were validated in urine of diabetic patients. Our results indicated that the marked increases in SBP-1 and PKM2 levels were observed in the urine of diabetic patients. Thus, these new biomarkers may have potential as alternatives to traditional biomarkers for the efficient and sensitive assessment of DKD. Further, novel biomarkers can be used as sensitive diagnostic kits in diabetic patients for early time prevention of ESRD.
Keywords: BIomarker, Diabetic nephropathy
EFFECTS OF PHENOBARBITAL (PB) ACUTE EXPOSURE ON MINIPIG LIVER GENE EXPRESSION (#493)
J. Silvano1, P. Ancian1, C. Bansard1, J. Boje Nielsen2, P. Singh1, R. Forster1
1 Citoxlab France, Evreux, France
Phenobarbital is a widely used anti-seizure medication. Treatment of laboratory animals with PB results in hepatomegaly and induction of CYP450 xenobiotic metabolizing enzymes, and long term treatment of rats and mice results in hepatic tumours. While PB has been extensively studied in rodents, little is known about the impact of PB treatment on the gene expression profile in minipig liver. For that purpose, 3 male Göttingen minipigs aged 4-5 months were treated orally with vehicle or 15 mg/kg/day PB for 6 days. On day 7, animals were euthanized and an 80 mg section of liver was snap frozen in liquid nitrogen. Total RNA was extracted from liver samples using a combined Trizol/RNeasy® method and showed adequate quality for Affymetrix target preparation using the 3’-IVT Plus kit. Labeled material was then hybridized to Affymetrix GeneChip® Porcine Arrays. The differentially expressed genes were filtered using an absolute fold-change threshold at 1.5 with a corrected p-value lower than 0.05. A total of 161 and 139 probesets were found to be significantly down and upregulated in minipig liver by PB treatment, respectively. CYP2A19, CYP2B22, CYP2C42, CYP3A39 and CYP3A46 transcripts were up-regulated with mean fold changes varying between 31.2 and 1.5. Among the top 20 most regulated genes, CYP2A19, CYP2B22, FADS2, tsukushin-like, FADS1, HMGCS2, and SULT1A1 were up-regulated and SDS, GARNL3, CLDN14, acetoacetyl-CoA synthetase-like were down regulated. Pathways and ontology classification using the DAVID software revealed that the oxidoreductase, retinol metabolism, chemical carcinogenesis and steroid hormone biosynthesis pathways were significantly enriched, with modulated genes such as CYPs, SULT1A1 (Sulfotransferase Family 1A Member 1), as well as fatty acid desaturases FADS1 and FADS2. These data will be correlated with additional analyses such as CYPs and UGT expression and ex-vivo enzyme activities in liver microsomes. In conclusion, this study aids understanding the mechanisms/modes of action of PB exposure on minipig liver and the identification of potential biomarkers of exposure and response.
Keywords: Phenobarbital, liver, minipig, Gene Expression profiling, CYP450
Calcium supplementation of pied flycatcher females in a metal-polluted environment: protective effect against oxidative stress? (#496)
S. Espín1, 2, P. Sánchez-Virosta1, 2, S. Ruiz2, T. Eeva2
1 University of Murcia, Toxicology Group / Department of Sociosanitary Sciences, Murcia, Spain
Calcium (Ca) may have a protective effect against metal toxicity by modulating metal-induced oxidative stress. The goal of this study is to assess the effects of Ca availability and metal pollution on oxidative stress biomarkers in adult female pied flycatchers (Ficedula hypoleuca). We performed a Ca-supplementation experiment in 2014 in a Ca-poor and metal-polluted area in SW Finland. We measured metal concentrations in feces, Ca levels in plasma, and antioxidant molecules (GSH, GPx, GST, CAT and SOD) and biomarkers of oxidative damage (lipid peroxidation, TBARS, and protein carbonylation, PC) in red blood cells. Pied flycatcher females showed higher metal concentrations in feces in the polluted zone. The Ca supplementation and zone had no effect on the oxidative stress biomarkers. Generalized linear models were also performed by choosing fecal metal concentrations (as a proxy of dietary levels) and Ca levels in plasma instead of pollution zone and treatment in an effort to investigate more accurately associations between metal exposure and Ca levels in the organism. Interestingly, antioxidant levels (tGSH levels, GSH:GSSG ratio, GPx and GST activities) and TBARS levels changed over the range of metal concentrations depending on the Ca levels in plasma. When plasma Ca levels were higher than 10 mg/dl, antioxidants and TBARS levels increased and GSH:GSSG ratio decreased with increasing metal concentrations. This suggests that birds are able to upregulate their antioxidant capacity to cope with higher metal exposure from a certain level of Ca concentrations in plasma. Thus, Ca concentrations in plasma may have a protective effect against metal-related oxidative stress. However, Ca could also be involved in lipid damage in a context of increased metal concentrations. Further studies are needed to better understand this mechanism. Acknowledgements: We thank J. Nurmi and M. Rainio. This study was financed by the Academy of Finland (project 265859 to T.E.), University of Turku Graduate School-UTUGS and Societas pro Fauna et Flora Fennica (P.S.-V.), and Fundación Séneca (20031/SF/16 to S.E.).
Keywords: metals, calcium, antioxidant, lipid peroxidation, insectivorous passerines
EFFECTS OF RAPAMYCIN ON IN VIVO AND EX VIVO CELL-MEDIATED IMMUNE RESPONSES IN CYNOMOLGUS MONKEYS (#517)
P. Ancian1, E. Grosdidier1, A. Decheix-Nguyen1, S. Sarlang1, J. Descotes1
1 Citoxlab France, Evreux, France
Inconsistent delayed-type hypersensitivity (DTH) responses have been reported in non-human primates, hence the need to study alternative endpoints. For this purpose, 3 male and 3 female purpose-bred Cynomolgus monkeys aged 33-36 months received a human dose of tetanus vaccine containing aluminum hydroxide IM on days 1 and 14, and were treated orally with 1 mg/kg/day rapamycin (RPM) from days 36 to 66. DTH was induced by an intradermal challenge of tetanus toxoid (TTx) and aluminum hydroxide on days 28 and 63. Injection sites were observed before challenge and after 24, 48 and 72 hours. Then, skin biopsies were taken and stained with anti-CD3, CD4, CD8, CD68, Ki67, FoxP3 antibodies. Blood was collected on days 0, 28, 31 and 35 to measure anti-TTx IgG using ELISA; on day 0 and then weekly, to study standard hematology; finally, at pre-test and on days 28, 35, 59 and 63, to quantify IFN-g secreting cells by ELISPOT and cytokine levels using a multiplex assay. No clinical signs were noted. Hematological findings were consistent with known effects of RPM. RPM blood levels measured by LC-MS/MS were within the immunosuppression range. The expected anti-TTx humoral response was seen in all animals prior to RPM treatment, and a 3-8 fold drop in anti-TTX IgG levels was measured after 4 weeks of RPM treatment. DTH reactions in animals prior to RPM treatment were slight and inconsistent and no DTH inhibition could be observed after RPM treatment. CD4+ and CD8+ T-cells, and CD68+ macrophages infiltrates at the injection site were observed after the first DTH reaction, and markedly decreased after RPM treatment. Despite inter-animal variability, the number of anti-TTX IFN-g secreting cells surged after the first DTH response, and markedly declined at the end of RPM treatment. No clear trend in any cytokine level was noted after the first DTH response, but all levels declined after RPM treatment. This study confirms that DTH in Cynomolgus monkeys requires further study and that a better understanding of DTH related events may be gained using ex vivo endpoints (cytokine analysis, immunohistochemistry etc), and in particular IFN-g ELISPOT.
Keywords: Non human primate, tetanus toxoid, delayed-type hypersensitivity, rapamycin, immunosuppression
NEOGENE Project – Task I: Characterization of in utero exposure to environmental tobacco smoke: using cotinine as a biomarker (#647)
A. I. Silva1, J. Madureira1, 2, A. T. Reis1, 2, B. Lage1, 2, F. C. Esteves1, 2, F. Barbosa Júnior3, J. P. Teixeira1, 2, C. Costa1, 2
1 Portuguese National Institute of Health, Environmental Health Department, Porto, Portugal
The NEOGENE project, Impact of transplacental exposure to tobacco smoke in the DNA of newborn. Genetic and epigenetic effects, aims to analyse both genetic damage and DNA methylation of mothers and their newborns exposed to environmental tobacco smoke (ETS) taking into consideration other possible co-exposures relevant to genetic and epigenetic alterations, namely PAHs, phthalates, brominated polyphenylene and metals.
In the last year, 500 volunteer parturients seeking prenatal care on Centro Hospitalar de São João, in Porto, Portugal have been enrolled in the study. At birth, maternal blood and urine, umbilical cord blood and placenta samples have been collected. In addition, each participant provided information on demographics, clinical history, lifestyles and exposures, including exposure to ETS before and during pregnancy, through an individual questionnaire. Within the framework of NEOGENE, characterization of ETS exposure was accomplished by measuring cotinine in maternal urine by solid phase competitive ELISA. Cotinine concentrations were determined in maternal urine as it was found to be more sensitive than the remaining samples to second hand exposure (paired samples of 15 participants were analysed to confirm matrix selection). A comparison between urinary nicotine concentrations and quantitative measurement of ETS exposure measured through questionnaire was also performed to identify possible underreport of smoking and exposure behaviors.
Smoking cessation during pregnancy has for long been an international priority to protect the health of the developing fetus. Moreover, tobacco control policies have been implemented in many countries to promote health, save healthcare costs and generate revenues for government services. Accurate assessment of fetal exposure to smoking through the objective measure of a biomarker is essential in this project, as the ultimate goal is the investigation of the effects of prenatal exposures to pollutants, including tobacco smoke, in genetic and epigenetic alterations.
This work is supported by FCT and FAPESP (FAPESP/19914/2014); Carla Costa and Joana Madureira are supported by FCT (SFRH/BPD/96196/2013 and SFRH/BPD/115112/2016 grants, respectively).
Keywords: Environmental tobacco smoke, cotinine, in utero exposure, questionnaires
Analysis of toxicologically relevant biomarkers in pesticide-treated HepaRG cells by MS-based immunoassays (#658)
F. F. Schmidt1, A. E. Steinhilber1, H. S. Hammer3, A. Mentz4, J. Kalinowski4, D. Lichtenstein2, A. Braeuning2, P. Marx-Stoelting2, A. Lampen2, T. Joos1, O. Pötz3, 1
1 Natural and Medical Sciences Institute at the University of Tübingen, Protein Analytics, Reutlingen, Germany
Consumers are exposed to multiple residues of different pesticides via their diet. The toxicological properties of these substances are generally determined individually, hence there is not much data available on potential mixture effects. Two European Union regulations (Reg No 1107/2009; Reg No 528/201) require analyzing potential cumulative or synergistic effects due to exposure to multiple pesticide residues. Due to the increasing number of available substances and thus the increase of potential multiple residues in food, analysis of mixture effects by traditional toxicological methodology would increase animal testing. Therefore, it is important to develop in vitro methods for the assessment of pesticide combinations to reduce animal experiments.
We developed mass spectrometry-(MS) based immunoassays to investigate the influence of single substances and combinations at the protein level of toxicologically relevant biomarkers. These assays include an antibody-based immunoprecipitation step to enrich the analytes of interest, followed by UHPLC-MS readout. In this approach peptides are analyzed as surrogates for the protein that can be identified and quantified via MS. This project focused on 24 toxicologically relevant proteins like cytochrome P450 enzymes (CYPs, phase I enzymes), UDP-glucuronosyltransferases (UGTs, phase II enzymes), transporters (Phase 0 and III) and others.
HepaRG are a well-established human cell model system for human hepatocytes. We used this cell system to analyze the protein profile after pesticide treatment. Cells were perturbated for 24h with single pesticides and combinations thereof. Rifampicin and CITCO served as prototypical inducers. We analyzed 24 proteins quantitatively and six of these proteins showed induction effects depending on the applied pesticide. For TNFRSF12A a 3.5-fold induction effect was observed after high dosage treatment with prochloraz and S100P protein level was increased by a factor of 7.5 after treatment with flusilazole suggesting two independent induction pathways.
In summary, we developed a very robust and accurate platform for the analysis of toxicological effects in HepaRG cells. The protein amount of CYP1A1, CYP1A2, CYP3A4, MDR1, TNFRSF12A and S100P increased depending on the chemical structure of pesticides.
Keywords: Biomarkers, Mass spectrometry, Immunoassay, Pesticides
Monitoring of triclosan and parabens in amniotic fluid. (#664)
V. Karzi1, 2, I. Katsikantami1, 2, M. N. Tzatzarakis1, E. Vakonaki1, P. Xezonaki3, A. Stratidakis1, E. Iliaki1, S. Sifakis3, A. Rizos2, A. M. Tsatsakis1
1 University Of Crete, Laboratory of Toxicology and Forensic Sciences, Heraklion, Greece
Introduction: The growing need of producing longer lasting and better quality products has led to the increasing use of antimicrobial agents like triclosan (TCS) and parabens (PBs). These compounds are mainly used in pharmaceutical products, make up and personal care products, like toothpastes, shampoos, deodorants and baby wipes. It is recorded that TCS and PBs affect the reproductive system of males and females, the thyroid gland and other tissues like the liver. In our study, we determined the concentration levels of TCS and four PBs (methyl- (MePB), ethyl- (EtPB), butyl- (BuPB), benzyl- (BePB)) in amniotic fluid collected from fifty pregnant women.
Keywords: triclosan, parabens, monitoring, amniotic fluid, pregnant women
Polymethoxyflavones prevent benzo[a]pyrene/dextran sodium sulfate-induced colorectal carcinogenesis through modulating xenobiotic metabolism in ICR mice (#63)
M. - H. Pan1, J. - C. Wu1, Y. - J. Wang2, C. - T. Ho3
1 National Taiwan University, Institute of Food Science and Technology, Taipei, Taiwan
Several studies have shown that polymethoxyflavones (PMFs) are effective in preventing carcinogen-induced colorectal cancer (CRC) or colitis. Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental carcinogenic pollutants and they have become an important issue in food contamination. Dietary intake of PAHs has been recognized as a major route of human exposure. However, the mechanisms behind dietary PAH-induced CRC remain unclear. In this study, we investigated the preventive effect of PMFs on benzo[a]pyrene/dextran sulfate sodium (BaP/DSS)-induced colorectal tumorigenesis in ICR mice. We found that PMFs significantly prevented BaP/DSS-induced colorectal tumor formation. BaP mutagenic metabolite and DNA adducts were found to be reduced in colonic tissue in the PMF-treated groups through the modulation of BaP metabolism. At the molecular level, the results of RNA-sequencing indicated that PMFs ameliorated BaP/DSS-induced abnormal molecular mechanism change including activated inflammation, downregulated anti-oxidation targets, and induced metastasis genes. Additionally, consumption of PMF extracts also altered the composition of gut microbiota and made it similar to that in the control group by increasing butyrate-producing probiotics and decreasing CRC-related bacteria. BaP in combination with DSS significantly induced colorectal tumorigenesis through induced DNA adduct formation, abnormal gene expression, and imbalanced gut microbiota composition. PMFs were a powerful preventive agent that suppressed BaP/DSS-induced CRC via modulating multiple pathways as well as ameliorating autophagic defect. These results demonstrated for the first time the chemopreventive efficacy and comprehensive mechanisms of dietary PMFs for preventing BaP/DSS-induced colorectal carcinogenesis.
Keywords: Polycyclic aromatic hydrocarbons, Chemoprevention, Polymethoxyflavones（PMFs）, Microbiota, Colorectal carcinogenesis
EXPRESSION OF LINE-1 RETROTRANSPOSON INDUCED BY ENVIRONMENTAL POLLUTANTS IN BREAST CANCER CELLS (#105)
N. Miret1, D. Zappia2, L. Zárate1, C. Pontillo1, F. Chiappini1, M. Lasagna3, C. Cocca3, F. Monczor2, A. S. Randi1
1 Universidad de Buenos Aires, Facultad de Medicina, Departamento de Bioquímica Humana, Laboratorio de Efectos Biológicos de Contaminantes Ambientales, Buenos Aires, Argentina
Breast cancer is the most common cancer worldwide in women and different studies link pesticide exposure to the risk of developing this disease. The pesticides hexachlorobenzene (HCB) and chlorpyrifos (CPF) are weak ligands of the aryl hydrocarbon receptor (AhR), a transcription factor involved in apoptosis and proliferation. Transforming growth factor-β1 (TGF-β1) is activated during cancer development. We have postulated that alterations in AhR/TGF-β1 crosstalk might contribute to breast cancer progression. Epigenetic changes include retrotransposons like the long interspersed nuclear element 1 (LINE-1), which is activated in mammary tumors. Ligands of AhR can induce activation of LINE-1 and epithelial to mesenchymal transition, through the TGF-β1/Smad pathway. Our aim was to study the effect of HCB and CPF on LINE-1 expression and to elucidate whether the mechanism of action is related to the AhR/TGF-β1 interaction in human breast cancer cells MDA-MB-231. We have reported that HCB (0.005-5 μM) activates the AhR/c-Src signaling, and induces Smad pathway, promoting MDA-MB-231 migration and invasion. Our results showed that LINE-1 mRNA levels (RT-qPCR), were enhanced at 0.005 μM HCB (p<0.01) after 24 h of exposure. However, no alterations were observed in LINE-1 protein levels (Western Blot). On the other hand, CPF increased c-Src phosphorylation after 5 and 15 min at 0.5 μM (p<0.05), and activated Smad2/3 at 15 min at 0.05, 0.5, 5 and 50 μM (p<0.01). CPF enhanced AhR protein expression (p<0.05) at 24 h and LINE-1 mRNA content after 48 h (p<0.01) at 0.05, 0.5, 5 and 50 μM. In contrast, 50 μM CPF reduced LINE-1 protein expression at 48 h (p<0.05). In conclusion, we demonstrated that the pesticides HCB and CPF modulate the AhR/TGF-β1 crosstalk and LINE-1 expression in MDA-MB-231, depending on the dose and the time of exposure. These findings could be linked to alterations previously observed in process like breast cancer cell migration, invasion or metastasis.
Keywords: LINE-1, hexachlorobenzene, chlorpyrifos, breast cancer
WATCH OUT! PESTICIDE EXPOSURE CONTRIBUTES TO TUMOR ANGIOGENESIS IN OUR BREASTS (#108)
L. Zárate1, C. Pontillo1, A. Español2, N. Miret1, F. Chiappini1, C. Cocca3, L. Alvarez1, D. Kleiman de Pisarev1, M. E. Sales2, A. Randi1
1 Universidad de Buenos Aires, Facultad de Medicina, Bioquímica Humana, Laboratorio de Efectos Biológicos de Contaminantes Ambientales, Buenos Aires, Argentina
Hexachlorobenzene (HCB) is a fungicide detected in maternal milk, and Chlorpyrifos (CPF) is a pesticide currently used in agriculture. Breast cancer is by far the most frequently diagnosed cancer in women. HCB and CPF act as endocrine disruptor in rat mammary gland. Vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) promote tumor growth and angiogenesis. Nitric oxide (NO) and abnormal expression of nitric oxide synthases (NOS1, NOS2, NOS3) are implicated in tumor progression. Our aim was to examine the action of HCB and CPF on angiogenesis in mammary carcinogenesis in vivo and in vitro. In a breast cancer xenograft model with MCF-7 cells, HCB (3 mg/kg bw) and CPF (0.1 mg/kg bw) stimulate angiogenic switch (number of vessels/mm2) and increase VEGF expression (p<0.05) by Western Blot (WB) in mice skin. In MCF-7, HCB (0.005 µM) increases COX-2 and VEGF levels at 3 h, while at 0.005-5 µM at 24 h (p<0.01). CPF (0.05 µM) enhances COX-2 and VEGF expression at 6 h, while at 50 µM at 24 h (p<0.05). In addition, HCB (0.005 µM) at 3 h increases NOS2/NOS3 and at 24 h enhances NOS1, while at 5 µM decreases NOS expression (WB, p<0.05). CPF (0.05 µM) increases NOS levels at 6 h, while at 50 µM enhances at 24 h (p<0.05). The pesticides increase the production of NO at lower doses, however reduces it at higher doses (HCB, CPF, p<0.05). Besides, the pretreatment of MCF-7 with an inhibitor of NOS prevents the increased expression of VEGF and COX-2 by HCB or CPF, suggesting that pesticides enhance angiogenic factors through NO-dependent pathway. Our results demonstrate that both pesticides increase angiogenesis and VEGF expression in vivo and in vitro. In MCF-7, at low doses, the pesticides enhance VEGF, COX-2 and NOS expression, and NO production. At high doses, increase VEGF and COX-2 levels, but differential effects on NOS expression were observed. In conclusion, our findings suggest that HCB and CPF may be a risk factor for human breast cancer progression.
Keywords: Hexachlorobenzene, Chlorpyrifos, breast cancer, angiogenesis, nitric oxide
Tobacco cigarette nicotine and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced normal breast epithelial cells transformation through activation of the a9-nicotinic acetylcholine receptor-mediated signaling pathways (#111)
Y. S. Ho1, 2, F. - S. Abdul Fattah1, 2
1 Taipei Medical University, Graduate Institute of Medical Sciences, Taipei, Taiwan
Introduction: Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nitrosamine carcinogens, including nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been detected in the second hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether nicotine and NNK act together to induce breast carcinogenesis using an in-vitro breast cell carcinogenesis model.
Methods: Immortalized non-tumorigenic breast epithelial cell line, HBL-100, was used for a time course assay exposed to either a very low level of nicotine or NNK, or both. The time course assay consisted of 23 cycles of nitrosamine carcinogen treatment. For each cycle, HBL-100 cells were exposed to 1 pmol of Nicotine or/and 100 fmol of NNK for 48 hours. Cells were passaged every 3 days and harvested after 5, 10, 15 and 23 cycles.
Results: Our results demonstrated that tumorigenecity of HBL-100, such as soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co-exposure to physiologically SHS achievable doses of Nicotine and NNK. In addition, a9-nACHs signaling activation which plays an important role in cellular proliferation and cell survival was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24- cells and mamosphere forming ability were also observed.
Conclusion: Our results indicated that chronically exposure to environmental carcinogens, such as nicotine and NNK from tobacco smoke, is able to induce breast cell carcinogenesis even at a very low dose.
Keywords: NNK, Second hand smoke, Carcinogenesis, Breast cancer
Effects of Nicotinamide Riboside Supplementation on Benzo[a]pyrene Induced BEAS-2B Cell Transformation (#221)
E. W. F. Cordeiro1, J. Oliveira1, M. R. Santos1, G. C. Silva1, T. F. Oliveira2, M. H. G. Medeiros3, P. Di Mascio3, A. P. M. Loureiro1
1 University of São Paulo, Department of Clinical and Toxicological Analyses, São Paulo, São Paulo, Brazil
Introduction: Benzo[a]pyrene (B[a]P) is a complete carcinogen belonging to the class of polycyclic aromatic hydrocarbons. In studies of our group using BEAS-2B cells exposed to B[a]P, it was possible to observe changes in the NAD+ and NADH levels, subsequent epigenetic alterations, and the induction of the tumor phenotype. Objective: To evaluate the effect of NAD+ modulation by means of nicotinamide riboside (NR) supplementation in the model of cellular transformation induced by B[a]P. Methods: NR was obtained by HPLC-PDA purification of the hydrolysis product of nicotinamide mononucleotide. NR cytotoxicity was monitored in the 250 – 1 µM range by crystal violet dye and XTT or MTT assays, every 24 h along 168 h. Subsequently, the same assays were used to verify the effect of 1 µM NR on the cytotoxicity induced by 1 µM B[a]P. Cell transformation was assessed by the soft agar assay after 168 h of exposure to 1 µM B[a]P in the presence and absence of 1 µM NR. The levels of 5-methyl-deoxycytidine (5-mC) and 5-hydroxymethyl-deoxycytidine (5-hmC) in DNA were determined by HPLC-ESI-MS/MS. Results: Concentration and time-dependent cytotoxicity were observed for NR in the 250 – 5 µM range. The cytotoxic effect was not observed for 1 µM NR along 168 h. Cells exposed to 1 µM NR + 1 µM B[a]P showed greater growth arrest (or death) in the 72 – 168 h period and higher tetrazolium salt (MTT) reducing activity at 96 h and 120 h than the cells exposed to 1 µM B[a]P alone. Cells exposed to B[a]P were the ones with the highest growth in soft agar, which returned to the control level when they were exposed to NR+B[a]P. At the same time, B[a]P induced DNA hypermethylation, which was prevented by the concomitant incubation with NR. On the other hand, the levels of 5-hmC remained higher in the B[a]P and NR+B[a]P groups than in the control and NR groups. Conclusions: NR (1 µM) was selectively cytotoxic against the cells exposed to B[a]P, without changing the viability of the normal cells. We found a protective effect of NR against cell transformation and DNA hypermethylation induced by B[a]P. The ongoing investigation of the profiles of intermediate metabolites in this model will highlight key changes that may be associated with the shift from the transformed to the non-transformed phenotype, opening new possibilities to test and establish chemopreventive approaches. Financial support: FAPESP, CNPq, CEPID Redoxoma
Keywords: Chemoprevention, benzo[a]pyrene, nicotinamide riboside, cell transformation, epigenetics
What is "Overexpression"? Example: Glycoprotein (MUC 1) in Lung Cancer Carcinoma (#295)
H. G. Bode1, M. Paulitschke2, K. Joehrens3
1 University Medical Center, Institute for Pharmacology & Toxicology, Goettingen, Lower Saxony, Germany
Material and methods
Overall results and conclusion:
It can be assumed that the enormous variety of genetic disposition of tumor cells influence the expression of such antigens, their glycosylation, their polymorphic variable number of tandem repeat domains or alternate splicing, all resulting in multiple transcript variants.
Keywords: lung cancer, immunhistochemistry, MUC 1, definition of overexpression
Chances for reducing Rodent Carcinogenicity Testing of Human Pharmaceuticals (#301)
J. W. Van der Laan1, G. Bode2, M. Pasanen3
1 Medicines Evaluation Board, Section on Pharmacology, Toxicology and Kinetics, Utrecht, Netherlands
Multi-hit and multi-step long-term reactions by pharmaceutical and chemical compounds can induce neoplasms. Drug Regulatory Authorities (DRA’s) request therefore in International Conference on Harmonization (ICH S1A) to conduct rodent carcinogenicity studies when human treatment is necessary for longer than 3- 6 months. ICH Guideline S1A defines conditions where long-term bioassays are not needed (e.g. infrequent use or short term duration, unequivocal genotoxicity, limited life-expectancy, topically applied drugs with poor systemic exposure, endogenous peptides or protein substances). The need for bioassays continues to be questioned because of too many positive outcomes. In addition, retrospective analyses of various datasets (PhRMA, FDA, JPMA, and EU) concluded that the outcome of these studies could be predicted: Negative predictions can be made when signals are absent and positive predictions are possible when signals (i.e.hyperplasia and/or pharmacology) are present. In between compounds remain for which the outcome is equivocal and where experimental studies may add value. These hypotheses stimulated ICH to test in an ongoing exercise by DRA’s and Industry if the number of rodent bioassays can be reduced. Such prospective evaluation is necessary to justify any revision of the present recommendations of the ICH S1. Sponsors are strongly encouraged to submit Carcinogenicity Assessment Documents (CADs) to DRA’s for all investigational pharmaceuticals with ongoing or planned 2-yr rat carcinogenicity studies. The CAD would address the overall carcinogenic risk of the investigational drug based on available pharmacology and toxicology data and a rationale for why the conduct of long-term studies would not add value to that assessment. DRA’s independently review the submitted documents and evaluate the degree of concordance with Sponsors. During this prospective evaluation period waiver requests will not be granted, the data are collected solely for real world experience. Submitted CADs will finely be compared to the real outcome of the 2-yr carcinogenicity studies and/or any other factors of a weight of evidence evaluation. Main objective will be the assessment of accuracy of the predictions. This paper will inform about the present details of this retrospective analyses. More than 40 cases have been collected, the aim is 20 out 50 virtual waivers. First evaluations of 22 cases are available, and results are generally as expected.
Keywords: carcinogenesis, pharmaceuticals, regulatory testing, Prediction
Apoferritine-encapsulated ellipticine – construction and properties (#313)
R. Indra1, T. Cerna1, Z. Heger2, V. Adam2, S. Dostalova2, T. Eckschlager3, M. Stiborova1
1 Charles University, Department of Biochemistry, Faculty of Science, Prague 2, Czech Republic
Adverse effects constitute an important problem in cancer treatment. One way to mitigate these effects is drug encapsulation inside a suitable nanocarrier capable of targeting. Apoferritin, iron-free form of ferritin, is naturally occurring protein that has potential to move through body without any immune response. Furthermore, it is possible to modify apoferritin with ligands to achieve tumour-specific targeting.
The simple-to-use encapsulation protocol (creating Apo-Elli) was developed. Shortly, apoferritin was decomposed by low pH in the presence of ellipticine and after several minutes re-associated by neutral pH. The nanocarrier exhibits narrow size distribution and nearly no haemolysis in comparison to free ellipticine. Ellipticine is released from Apo-Elli at acidic (6.5; more than 80 %) and neutral pH (7.4; less than 20 %) after 48 hrs incubation. The presence of membrane particles accelerates release of ellipticine from Apo-Elli and makes it possible to be transferred into microsomes even at pH 7.4. Microsomal cytochrome P450 are capable of oxidizing free ellipticine and/or its Apo-Elli form to its metabolites and generating ellipticine-derived DNA adducts, both under pH 7.4 and 6.5. The form of ellipticine plays practically no role in these processes. The Apo-Elli is also toxic to UKF-NB-4 neuroblastoma cancer cells but exhibits significantly lower toxicity for non-malignant cells (non-malignant fibroblasts, HDFn cells).
This work was supported by GACR 17-12816S and GAUK 998217.
Keywords: apoferritin, ellipticine, neuroblastoma, fibroblasts, nanomaterials
LincRNA-RoR regulates the apoptotic function of p53 via targeting miR-204/MDM2 in human esophageal squamous cell carcinoma (#399)
R. Liu1, T. Wang1, M. Shang1, X. Wang1, P. Zhang2
1 Southeast University, School of Public Health, Nanjing, China
Objective: It is well known that MDM2 has a critical role in negative regulation of p53 through the ubiquitin-proteasome pathway. However, it is unclear whether there is an additional mechanism that can keep stress-induced p53 level under control. This study was expected to reveal the regulatory effects of lincRNA-RoR(linc-RoR) on p53-mediated cell cycle arrest and apoptosis in human esophageal cancer cell(ESCC) via targeting miR-204/MDM2.
Methods: Expression levels of linc-RoR, miR-204 and MDM2 in ESCC cell lines and tissues were determined by qRT-PCR. The regulatory relationship between linc-RoR,miR-204 and MDM2 was verified in ESCC cells by qRT-PCR, dual luciferase reporter(DLR) assay, RNA immunoprecipitation(RIP) and western blot. Cell cycle and apoptosis were detected by flow cytometry.
Results: Over-expression of linc-RoR down-regulated miR-204 by 73% which was proven with RIP, DLR assay, and qRT-PCR. Furthermore, the qRT-PCR results showed that miR-204 could down-regulate MDM2. Whether miR-204 could target MDM2 3'UTR was verified by RIP and DLR. The western blot results also showed that miR-204 inhibited MDM2 expression. All above showed that MDM2 was a down-stream target of miR-204. Then, we conducted the DLR and confirmed that linc-RoR could up-regulate MDM2 by competitively binding the miR-204. At the same time, the level of p53 protein was down-regulated by over-expressing linc-RoR in ESCC cells, and p53-mediated apoptotic signaling was repressed. Consequently, we observed that S phase cell arrest was induced and apoptosis of ESCC cells was suppressed.
Conclusion: The present evidence showed that the human linc-RoR is a negative regulator of p53. Linc-RoR may regulates the apoptotic function of p53 by targeting miR-204/MDM2 in ESCC.
Keywords: Linc-RoR; p53; MiR-204; MDM2; Apoptosis
ONCOGENIC BIOMARKERS IN THE SAFETY ASSESSMENT OF NOVEL FOOD SOURCES (#497)
S. I. Shestakova1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
In the framework of safety assessment of novel food sources, such as genetically modified organisms of plant origin (GMO), a special attention is focused on specific types of toxicity, when it concerns the study of the native product. According to established practice the employed approach uses a wide complex of methods that provide the most complete and reliable information on the potential genotoxic, immunotoxic and allergenic effects. Inclusion of carcinogenicity testing in the list of obligatory studies is an important and necessary step in improving of the GMO safety assessment system. But despite the advent of new methods for carcinogenicity research, which allow with a high degree of confidence to predict the potential effect of studied substances, the enhancement of used approaches and search of modern and informative indicators remains an urgent problem of toxicology.
One of the main tasks of the study was the search of possible oncogenic markers, which already applied in clinical practice, for studying of the potential carcinogenicity in toxicological tests. It can be both tumor markers, allowing to detect tumors at early stages of development, and biomarkers, the so-called ‘tumor precursors’, allowing to reveal the carcinogenicity of the testing substance before the appearance of tumors. The most promising oncogenic markers may be indicators of DNA expression and methylation, microRNA, oncofetal proteins (AFP, oncodevelopmental antigen etc.), hormones and their receptors; as well as indicators of apoptosis activity, which indirectly characterizes the proliferation processes. Although the potential diagnostic significance of the above-mentioned biomarkers their inclusion in the list of official methods is only possible after comprehensive in vivo / in vitro studies and the reliable evidence base formation. In accordance with the established scientific practice, such studies should include a series of model experiments in laboratory animals with tumor induction by various carcinogens with different mechanisms of action; the study of the indicators changes should be performed at the different stages of oncogenesis.
The ultimate goal of the research will be the introduction of modern clinical methods and markers into carcinogenicity testing toxicological practice, and the formation of bio- and tumor markers panel with a high diagnostic significance.
This work was carried out within the state assignment of FASO Russia (theme No. 0529-2014-0047).
Keywords: oncogenic markers, GMO safety assessment, carcinogenicity testing
The cytotoxicity of doxorubicin in human mammary cells is modulated by the conditioned medium of mesenchymal stem cells (#507)
B. C. Antunes1, S. P. Camões1, M. Castro1, N. G. Oliveira1, J. P. Miranda1
1 Faculty of Pharmacy of the University of Lisbon, Research Institute for Medicines (iMed.ULisboa), Lisboa, Portugal
Mesenchymal Stem Cells (MSCs) have been triggering an increased interest in biomedicine, showing inherent immunomodulatory properties and a homing capacity to tumors that makes it an attractive target towards the development of innovative cancer therapies. However, the role of MSCs in cancer is still contradictory, with literature reporting both cancer promoting and inhibitory effects. Furthermore, the study of MSCs and their secretome in the context of chemotherapeutic drugs has not yet been fully explored. Three-dimensional (3D) cell cultures have been reported as more in vivo resembling, in aspects of morphology, viability and functionality, presenting also a paracrine activity with greater therapeutic properties, already reported for instance, in tissue regeneration. This work aimed to investigate the effect of the secretome of a specific population of human neonatal umbilical cord matrix derived MSCs (hnMSCs) in human breast cancer cells. Conditioned medium from hnMSCs (MSCs-CM) was produced and collected from both 2D monolayer cultures (CM2D) and 3D dynamic spinner flask suspension cultures (CM3D) and concentrated 20x using 3kDa protein concentrators. Cell viability/proliferation under treatment with MSCs-CM (1x and 10x concentrated) either alone or in combination with the chemotherapeutic doxorubicin (Dox, 0.1-1.0 μM) was evaluated through MTS assay (n=3) in two human breast cell lines: MDA-MB-231 (malignant; invasive) and MCF10A (non-malignant). For the MCF10A cell line, both CM2D 10x (p<0.05) and CM3D 10x (p<0.001) significantly decreased the cytotoxicity induced by Dox at 0.1 μM, while for MDA-MB-231 cells, CM2D and CM3D treatments only slightly increased cancer cell viability in the presence of Dox (n.s.). Overall, this study revealed a protective effect of the MSCs-CM against the cytotoxic effect of Dox in non-tumor breast cells, which was more marked for CM3D. Further studies are needed to better understand the underlying mechanisms of hnMSCs-CM in breast cancer.
Keywords: breast cancer, 3D cultures, conditioned medium, human neonatal mesenchymal stem cells, doxorubicin
Decreased telomeres length in patients with glioblastoma multiform. (#557)
E. K. Vakonaki1, G. A. Vaki1, K. Tsiminikaki1, A. Alegkakis1, K. Kalliantasi1, P. Fragkiadaki1, M. Flamourakis1, D. Tsoukalas1, I. Fragkiadoulaki1, M. N. Tzatzarakis1, A. M. Tsatsakis1
1 Medical School, University of Crete, Laboratory of Toxicology, Heraklion, Crete, Greece
Introduction: Glioblastoma Multiforme (GBM) is the most common and lethal of human primary central nervous system tumours, poor survival (14-16 months) despite optimal treatment options such as surgery, radiation and chemotherapy. The poor prognosis of the disease is attributed to the relative inaccuracy of current prognostic markers that are notably based on clinical and pathological variables. The aim of the study is to associate decreased telomere length with glioblastoma multiform.
Methods: Blood samples were collected from 8 patients with GBM. Metaphase spread leukocytes were isolated from peripheral blood. Telomere’s length was measured by Q-FISH with (C3TA2)3 PNA probe. Were measured 10 metaphases of each individual and analyzed by Image-J. Basic statistical tests such as independent samples t-test were used.
Results: Age of participants was 50.4 ± 18.3 ranged from 21-75 years old. Four of them were under radiotherapy and temozolomide administration. The median length and their quartiles of telomeres from the whole chromosome was 7.58 (5.62-9.76) Kb while the corresponding values of the short (<20th quartile) telomeres was 4.53 (4.12-5.02) Kb. Control median of length from the whole telomeres was significantly greater than the GBM group (p<0.01) and the same results was observed for the short telomeres (p<0.01). The proportion of extremely short telomeres (<3 Kbs) was ranged from 0.0-51.8%. No signficant differences were found when median telomere length was competed between when patients with or without therapy (p=0.715 for whole, p=0.219 for short telomeres).
Conclusion: These are preliminary results of a developing database on telomere length. Although a bigger sample of GBM patients was needed, results shows that telomere length is extremely lower than healthy participants.
Keywords: Telomeres, Glioblastoma
An autopsy case of diphenhydramine poisoning (#26)
1 Shiga University of Medical Science, The department of Legal Medicine, Otsu, Japan
Introduction: Diphenhydramine is an ethanolamine derivative actiong as a histamine H1-recepter antagonist. As diphenhydramine also has sedative and anticholinergic properties, it is used widely in numerous over-the-counter preparations.
Keywords: Diphenhydramine poisoning, Over-the-counter product, Autopsy case
The possibility use of psyllium as appropriate alternative for allopurinol in treatment of hyperuricemic patient: A case report (#215)
A. Ebadollahinatanzi1, G. Arabrahmatipour2
1 Agricultural Research, Education and Extension Organization (AREEO), Institute of Technical and Vocational Higher Education, Agriculture Jihad, AREEO, Department of Medicinal Plants, Imam Khomeini Higher Education Center, Karaj, Alborz, Iran (Islamic Republic of)
Purpose: The synthetic drugs used in hyperuricemic patients can make some adverse effects such as hepatotoxicity. Therefore, the use of natural based medicine for curing this kind of diseases has been grown worldwide. Our before study showed that psyllium and allopurinol, together, could be effective in the treatment of hyperuricemia. In this study, the possibility use of psyllium separately for treatment of a patient with increased serum uric acid levels has been investigated.
Case: The case was a 51-year-old man with pain in the joints, whose biochemical tests in several times showed an increased serum uric acid levels with average of 10.5 ± 0.3 mg/dL. He was prescribed with daily use of 5 grams of psyllium seeds in 150 ml water. This treatment continued for a 20 day period. Then, this treatment was followed up for another 20 days with the same dose which was taken by patient as alternate. Quantitative diagnostic kits and auto analyzer instrument were used to analyze serum.
Results: There were seen significant differences in uric acid levels of the patient treated with psyllium when compared with values obtained from before treatment(p <0.01). The mean serum uric acid levels of the patient were reached up to 8.1 ± 0.2 mg/dL and 6.8 ± 0.1 mg/dL 20 days and 40 days after treatment, respectively. Considering the normal range of uric acid which is 3.6-8.2 mg/dL; it can be concluded psyllium, as an alternative for allopurinol, is capable to lower serum uric acid level in hyperuricemic patient.
Keywords: Psyllium, allopurinol, hyperuricemia
Levels of heavy metals and essential minerals in hair samples of children with autism in Algeria: a case–control study (#335)
Y. Zebbiche1, 2, M. S. Bencherif2, H. Boughazi2, S. Aries1, A. Amziane1, 2, R. Djidjik2, 3, B. Alamir1, 2
1 National Center of Toxicology, Algiers, Algeria
Background & Objectives: Reported rates of autism have increased sharply all over the world, one possible factor underlying these increases is a higher exposure to environmental pollution there for a higher exposure to metals. In recent years, it has been theorized that children with autism may be poor detoxifiers compared to normally developing children. The theory is sometimes referred to as the “poor excretor theory”. The aim of this work is to compare the concentration of metals and metalloids (toxic and essentials) in the hair of autistic and non-autist children.
Methods : After the optimization and validation of a dosage method using an ICP-MS, Lead, Arsenic, Cadmium, Chromium, Selenium and Copper concentrations were measured in hair samples of 30 autistic children, aged 3 to 9 years, and compared to a control group.
Results: By comparing hair concentration of autistic vs non-autistic children, lower hair concentrations were found among the autistic children for the following toxic metals lead (p=0.010 for n=29,), cadmium (p=0.044 for n=29) and chromium (p=0.016 for n=27,) the differences were statistically significant, but the difference was not statistically significant for the arsenic (p=0.725 for n=28,), and higher hair concentrations of the essential minerals selenium (p=0.010 for n=30,) and copper (p=0.033 for n=26,) the differences were statistically significant.
Conclusion: The results of this study converge to incriminate the association between metal exposure and autism spectrum disorders.
Keywords: Autism, Environment, Metals, Essential elements, Hair, ICP-MS
Prospective follow-up study on battery ingestion in children in the Netherlands (#365)
J. J. Nugteren1, C. C. Hunault1, A. J. van Riel1, I. de Vries1
1 University Medical Center Utrecht, Dutch Poisons Information Center, Utrecht, Netherlands
In the 1990-2000s, several large North-American studies increased the awareness about the risks of button ingestion in children. Identified risk factors for severe complications were lithium-type button batteries, large diameter cells and age <4 years. We aimed to study the current frequency and circumstances of (severe) complications after battery ingestion in children in the Netherlands, in recent years.
A prospective follow-up study was conducted in 2011-2015 at the Dutch Poisons Information Center (DPIC). All consecutive cases of battery ingestion in children <6 years were included. Follow-up was performed by using a standardized questionnaire, over the telephone. The primary outcome was the occurrence of major complications (i.e. severe symptoms and/or death).
During a 4-year period, 1026 cases of human battery exposure were reported to the DPIC (annual incidence of 15.3 potential battery ingestion per million population). N = 405 exposures in which the patient was <6 years and had ingested (witnessed or suspected) at least one battery, were included in the study. In 229 children (57%), a battery was detected (spontaneous passage, positive X-ray or endoscopic removal). Button batteries with a diameter <15 mm were most often involved (54% of the batteries). Batteries with a diameter ≥15 mm were more often located in the oesophagus/throat than batteries with a smaller diameter (p < 0.001). All button batteries with a diameter ≥15 mm located in the oesophagus/throat were endoscopically removed (N=8). No severe complications or fatalities were reported; the confidence interval (CI) for the occurrence of a severe complication was [0%, 1.3%]. Local injury was noticed in 9 patients (3.9%; CI: [1.9, 7.6]).
In this study, the annual incidence of battery ingestion per million population in the Netherlands is comparable to the North-American annual incidence reported between 1985 and 2009 (between 6.3 & 15.1). Batteries with a diameter ≥15 mm are more often located in the oesophagus/throat than batteries with a smaller diameter and therefore pose a higher risk for local injuries or severe complications. The frequency of occurrence of severe complications after battery ingestion in children is low, but not negligible.
Keywords: battery ingestion, button battery ingestion, children, risk of injury
Benzylpiperazine (BZP) additively increases Methylenedioxymethamphetamine (MDMA) cardiotoxicity in H9c2 cells, an effect potentiatted by hyperthermia (#389)
F. Carvalho1, M. D. L. Bastos1, S. O. Silva1, H. Carmo1, D. D. D. Silva1
1 Faculty of Pharmacy University of Porto, Laboratory of Toxicology, Porto, Portugal
Synthetic psychotropic substances, including ecstasy (3,4-methylenedioxymethamphetamine, MDMA) and benzylpiperazine (BZP) are frequently used in combination at recreational settings that exacerbate their hyperthermic effects. Considering that each of these drugs can lead to severe cardiotoxicity, and that hyperthermia is a well-known condition that potentiates the toxicity of amphetamines, our hypothesis was that exposure of cardiac cells to this mixture would reveal responses that potentially differ from single drug toxicities, especially under increased temperature settings.
Therefore, we assessed the in vitro cardiotoxic effects of the single drugs and their combination in H9c2 cardiomyocytes. Cells were exposed to individual drugs and their mixture (MDMA:BZP 8:2 ratio based on concentrations found in blood of intoxicated patients) for 24h, at 37ºC and at 40.5ºC. After exposure, viability was recorded by the MTT assay and mixture expectations were calculated using independent action (IA) and concentration addition (CA) models. Changes in lisossomal integrity, intracellular glutathione (GSH/GSSG), ATP, reactive species (ROS/RNS), and mitochondrial membrane potential (ΔΨm) were also evaluated.
Our data showed that MDMA (EC50 1.74 mM at 37ºC and 1.17 mM at 40.5ºC) was more toxic than BZP (EC50 2.72 mM at 37 ºC and 1.93 mM at 40.5 ºC). Toxicities predicted by both IA and CA models were coincident and accurately estimated the experimentally observed additive mixture effects. Overall, hyperthermia aggravated the cardiotoxic effect of both drugs, individually and in mixture, affecting the cellular redox status, with increased generation of ROS/RNS and GSSG, depletion of GSH and ATP, decreased lisossomal uptake, and mitochondrial depolarization. Apoptosis also increased under hyperthermic conditions. From a clinical perspective, the observed additive cardiotoxicity raises concern about a potential deterioration of the health of abusers, specially under recreational conditions that favour hyperthermia.
Keywords: cardiotoxicity, H9c2, Mixture, MDMA, BZP
Metabolic dysfunction and obesogenic effects in individuals with the acute and chronic intoxication of pesticides (#662)
N. Bubalo1, G. Balan1, P. Zhminko1, V. Bubalo1, T. Usenko1, I. Rashkivska1
1 L.I.Medved's Research Center of Preventive Toxicology, Food and Chemical Safety Ministry of Health, Ukraine, "Institute of Experimental Toxicology and Biomedical Research", Kyiv, Ukraine, Ukraine
Over the last decade, there has been a significant increase in the incidence of obesity in all age groups, but the mechanisms of its development and progression have not studied in full. The studies of the contribution of some xenobiotics to the development of obesogenic effects show the role of pesticides, which called obesogens. In this study are agricultural workers with the acute poisoning of 2,4-D pesticides. In this case, we observed patients for a long period of time from 2001 until nowadays. The screening has been done 2001, 2003, 2012, 2018 years. Along with neurological disorders, the toxic liver damage was detected, followed by the formation of hepatosteatosis in 35.8% of cases in patients with acute poisoning with 2,4-D herbicides, which was associated with the formation of metabolic disorders: dyslipidemia with a predominance of low-density lipoprotein (LDL), triglycerides and total cholesterol, and hyperinsulinemia. After 3 years of dynamic observation was determined the formation of obesogenic effects was observed (with an increase in the body mass index in 48% of cases in patients with the acute poisoning with 2,4-D herbicides). At the last time, only 23 patients have been investigated. That way we will compere only this 23 patients with a control group ( 9 office workers). We focused on such indicators as and received subsequent results within such ranges: triglycerides 0,78-3,72mmol/L, cholesterol 4,04-8,61 mmol/L, high-density lipoproteins (HDL) 0,97-2,64mmol/L, low-density lipoprotein (LDL)1,2-5,04 mmol/L, very low-density lipoproteins (VLDL) 0,36-1,71, atherogenic index of plasma (AIP) 1,1-7,36 and adiponectin 2,59-22,23. Anthropometric parameters were measured in patients and the body mass index (BMI) have been calculated ( ranges 27-43). There is an imbalance of fatty tissue hormones (adiponectin) was revealed in patients who underwent acute poisoning of 2,4-D herbicides.
Keywords: obesogenic effects, pesticides, adiponectin, 2_4-D
INTEREST OF METHEMOGLOBINE DOSAGE IN PATIENTS UNDER DAPSONE (#706)
S. Fatima Zohra1
1 Faculty of medecine, Pharmacy, Oran, Algeria
Introduction :Dapsone is a sulfone antibiotic and potent anti-inflammatory which was traditionally an antileprosy drug, but the it use has expanded into the treatment of other dermatological pathology.
Dapsone metabolites N-hydroxylated are responsible of its adverse effects dose-dependent: hemolysis and methemoglobinaemia .That why the implementation of this treatment and the modification of the dose requires, by consequence an attentive clinical and biological surveillance.
This work aims to show the interest of dosage of the methemoglobin in the take charge of patients under dapsone.
Material and Methods: We have analyzed twenty samples of nine patients followed at the service of dermatology at 1st November 1954 hospital of Oran, Algeria. These patients are under dapsone or in whom the doctor intends to introduce this treatment.The measurement of methemoglobin is made by the Evelyn Malloy modified method on Varian spectrophotometer .
Discussion and Conclusion: All patients followed have a normal initial methemoglobinaemia (less than 2%) with an NSF normal witch allowed the indication of the dapsone. But almost half of these patients (44%) have a pathological methemoglobinaemia (more than 2%) after taking the dapsone at a dose of 100mg /d. While one case that presents an abnormal value after taking only a dose of 50 mg/d during eight days.According to the methemoglobinemia at day 8, the dose of dapsone in these patients was either maintained or regressed.
These results show that the dosage of the methaemoglobin has a great interest both in the decision-making by the dermatologist to prescribe the dapsone and in the monitoring of patients under this treatment.
Keywords: Dapsone, methemoglobin, monotoring, dose, dermatology
Impact of diabetes on gabapentin pharmacokinetics in patients with neuropathic pain (#717)
A. C. C. Costa1, P. A. Yamamoto2, J. R. L. Benzi1, G. R. Lauretti3, N. V. de Moraes2
1 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
Introduction: Gabapentin (GBP), an anticonvulsant used in the treatment of neuropathic pain and diabetic neuropathy, is primarily eliminated by renal excretion, partially depending on organic cation transporter 2 (OCT2) activity. Type 2 diabetes reduces the level of OCT2 transporters by 50% in rat kidneys. Purpose: the aim of this study was to determine whether the diabetes alters GBP pharmacokinetics in patients with neuropathic pain. Methods: The clinical protocol was approved by the local Ethics Committee (CAAE: 34175314.3.0000.5403). Patients with neuropathic pain score ≥ 4 on visual analogue scale due to cervical/lombar disc herniation (n=11) or due to type 2 diabetes (n=13) were included. Participants were treated with a single oral dose of 300 mg GBP. Serial blood and urine samples were collected up to 36 hours after administration. Pharmacokinetic parameters were estimated using non-compartmental analysis. Results: Type 2 diabetes maximum plasma concentration (Cmax) (Cmax = 2.45 µg/mL vs. 1.49 µg/mL, p=0.0008), without changes in the time to reach Cmax (tmax) and in the area under the curve plasma concentration versus time (AUC) extrapolated to infinity (AUC= 25.3 µg.h/mL vs. 15.8 µg.h/mL, p=0.1163;). Higher apparent total clearance (Cl/F= 18.9 L/h vs. 11.8 L/h, p=0.0159) and higher apparent volume of distribution (Vd/F= 155.3 L vs. 101 L, p=0.0061) were observed in diabetes comparing to control. No statistical differences were observed on renal clearance (ClR) between groups (ClR=5.3 L/h vs. 4.9 L/h; p=0.8516). Conclusion: Type 2 diabetes decreased Cmax by 42% and increased gabapentin apparent total clearance and apparent volume of distribution in patients with neuropathic pain. These alterations were attributed to the effects of diabetes on absorption and distribution mechanisms, since the renal clearance of GAB was similar between groups.
Keywords: gabapentin, OCT2, pharmacokinetics, neuropathic pain, type 2 diabetes
Use of in silico methods in IATA for skin corrosion and irritation (#177)
A. Abe1, T. Sezaki1, K. Kinoshita1
1 Mitsui Chemicals, Inc., Chemicals Safety Department, Chiba, Japan
For the appropriate risk assessment of chemicals, reliable information on the chemicals is indispensable. As for hazard information, from the viewpoint of animal welfare, the use of various alternative methods, such as in chemico, in vitro and in silico methods, is desirable. However, the issue here is that it is difficult to obtain reliable data using just a single alternative method.
The Integrated Approach on Testing and Assessment (IATA) is considered to be one of the promising ways to overcome this difficulty. In this approach, information from various sources such as existing information, in silico, in chemico and in vitro data is integrated and a comprehensive assessment is conducted to obtain data with a higher reliability.
The concept of IATA is made clear regarding skin corrosion and irritation assessment in the OECD Guidance Document No. 203, published in 2014. However, compared to clearly defined in vitro and in vivo tests in Part 3 of “Additional testing”, there still remains a lack of clarity in Part 1 & 2 of “Existing information and WoE”. For example, to actually conduct assessments, it is necessary to determine more specific measures such as what sources to refer to when gathering existing information, what software to use, and what kind of predictions to make with in silico methods.
In our poster, we report on a case in which we consider specific prediction procedures using the QSAR Toolbox and measures in order to obtain reliable prediction results with regards to the Read-Across from similar compounds.
Keywords: in silico, QSAR Toolbox
Large-scale in silico screening of compounds contained in printing inks for food packaging materials (#181)
M. Smieško1, C. Don1, R. Meuwly2, S. Kucsera2, B. J. Brüschweiler2
1 University of Basel, Pharmaceutical Sciences, Basel, Basel-Stadt, Switzerland
Providing sustainable and safe nutrition to the population is one of the main tasks of the modern food industry. While food packaging materials fulfill their invaluable protective role, certain substances can migrate from them into food. In order to identify potentially harmful chemical compounds contained in printing inks for food packaging materials we performed a virtual screening study along with a complex toxicokinetic characterization.
We compiled a list of 3481 chemicals unambiguously identified by the CAS number and accompanied by the the SMILES code. Next, structure conversion routines and on-line services were used to obtain a 3D computer representation of each chemical. The raw structures were further processed using software LigPrep . The resulting structures were refined using MacroModel  and forwarded as input for the evaluation of toxicokinetic parameters and descriptors using QikProp  software as well as for the virtual screening using the VirtualToxLab  technology. In total, 3075 compounds have been successfully screened by flexible docking and pose scoring for their binding towards 16 human anti-targets: 10 nuclear receptors, 4 cytochromes P450, the aryl hydrocarbon receptor and the hERG ion channel. For each structure, the overall toxic potential was calculated and evaluated along with toxicokinetic properties. Finally, compounds fulfilling the criteria for oral availability, aqueous solubility and simultaneously showing a significant binding affinity at screened off-target(s) were clustered into substance classes by a fingerprint-based similarity search.
The obtained in silico data allow identification of single potentially harmful compounds as well as decode general trends in substance classes.
 Schrodinger LLC.
 Biographics Laboratory 3R
Keywords: in silico screening, molecular docking, printing ink, food packaging material, toxicokinetic characterization
BioCelerate Toxicology Data Sharing initiative: Development of a centralized, searchable Preclinical Data Repository for the Biopharmaceutical Industry (#225)
T. G. Bjerregaard1, M. Graziano2, E. Vock3, G. Kahlbaugh4, W. Houser5, T. Page6, K. Mera7, T. Fukushima7, M. Kuzumoto7, S. Frahm8
1 Novo Nordisk, Copenhagen, Denmark
BioCelerate, a subsidiary of TransCelerate, is a preclinical industry consortium driving initiatives to increase efficiency and productivity in early stage R&D. Motivated in part by the FDA's Strategic Plan for Regulatory Science and binding guidance requiring CDISC SEND standard format for nonclinical data, the Toxicology Data Sharing Initiative was launched to enable participants to make more informed decisions on compound progression based on increased understanding of on-target and off-target toxicity.
Via access to a cloud-based data sharing platform, DataCelerate, participants have the ability to share, search for, visualize, and export de-identified unstructured (PDF format) and structured (SEND-compliant format) data sets stored in curated toxicology and background control data repositories. Long term, the platform will support and link TransCelerate and BioCelerate data sharing initiatives, facilitating valuable insights across the entire drug development continuum. Presented here are the core capabilities, use cases, and system architecture for the data sharing platform as well as the principles guiding data sharing and collaboration amongst BioCelerate participants.
Keywords: Computational Toxicology
Ligand based and structural based modeling for the understanding, classification and prediction of mitochondrial toxicity (#232)
J. C. Gomez-Tamayo1, F. Troger2, M. Pastor1, U. Norinder3, B. Zdrazil2, G. Ecker2
1 University Pompeu Fabra, DCEX, Barcelona, Spain
Mitochondrial toxicity is an extremely important endpoint, involving a large number of mechanisms. In
Keywords: computational, machine learning, structural modeling, ligand based modeling, mitochondrial toxicity
Imaging and computational modelling to unravel the dynamics of the oxidative stress pathway upon repeated perturbation (#270)
I. A. Kuijper1, L. J. M. Bischoff1, D. Noort2, J. P. Langenberg2, B. van de Water1, J. B. Beltman1
1 Leiden University, LACDR, Division of Drug Discovery and Safety,, Leiden, Netherlands
Drug induced liver injury (DILI) is a risk to human health, and makes the development of safe drugs time-consuming and costly. The oxidative stress pathway is an important pathway in protecting the liver against DILI. To mechanistically understand the protective role of this pathway, we imaged reporter cell lines for both a transcription factor (Nrf2) and a downstream target (Srxn1) with confocal microscopy. Specifically, we mapped the dynamics of these pathways upon repeated exposure to the chemicals DEM and tBHQ, which are known to induce oxidative stress. During the first exposure, the peak values of Nrf2 and Srxn1 were linearly related, albeit with a different slope for the two studied chemicals. However, this linear relationship was lost during the second exposure. Moreover, the peak value of Nrf2 was slightly higher during the first than during the second exposure at the same concentration, whereas the downstream target Srxn1 exhibited opposite behaviour, i.e., the Srxn1 peak after the second exposure was up to 4 times higher compared to the first exposure.
In order to understand the mechanisms underlying these experimental observations, we developed an ordinary differential equation (ODE) model. Our initial model could describe the dynamics of the pathway upon first exposure, yet could not fully capture the dynamics observed after the second exposure. We are currently extending the model with additional pathway elements based on gene expression profiling. Such dynamic modelling of cellular stress pathway activation and its relation to onset of cellular adversity in the presence of different chemicals during repeated exposure scenarios is expected to play an important role in the prediction of DILI in the future.
Keywords: oxidative stress, ODE model, Nrf2 pathway, repeated exposure, computational model, drug induced liver injury
Development of a Robust In Silico Profiler to Screen for Nephrotoxicity Endpoints (#300)
J. Pletz1, D. J. Ebbrell1, S. J. Enoch1, J. W. Firman1, J. C. Madden1, S. D. Webb2, M. T. Cronin1
1 Liverpool John Moores University, School of Pharmacy and Biomolecular Sciences, Liverpool, United Kingdom
Approximately 20% of cases of acute renal failure (ARF) in critically ill patients are attributed to the administration of drugs. Computational (in silico) toxicology approaches can assist in the screening, and hence identification, of compounds with the potential to induce adverse effects, including nephrotoxicity. Structure-activity relationships, captured as structural alerts, are a useful means of rationalising such organ level toxicity, but are poorly organised for effects to the kidney. The purpose of this study was to compile structural alerts for kidney toxicity and assess their robustness and sensitivity / specificity. The structural alerts obtained were rationalised to remove duplication and coded into SMARTS strings. The alerts were tested against a dataset of more than 180 known nephrotoxicants and a smaller number of non-nephrotoxicants. The alerts were evaluated for robustness and specificity by the frequency of their occurrence in nephrotoxicants. In total, over 200 structural alerts were obtained and coded. Some of these were general in nature (referring to common molecular scaffolds) or related to individual molecules, whereas others related to specific molecular fragments. The initial set of alerts identified all but 11 of the nephrotoxicants which allowed for further alerts to be created (e.g. for inorganic compounds). Due to the general nature of some alerts, the sensitivity (identification of potential nephrotoxicants) of this computational profiler is high, however the specificity is low. The sensitivity rate makes the profiler suitable as a basis for grouping and read-across and, if used as a conservative indicator, for hazard assessment. The analysis indicates the need for refinement of the general alerts and an improved data set, especially of non-nephrotoxicants.
Keywords: Structural alerts, kidney, nephrotoxicity, profiler, in silico
Predicting in silico the Direct-Peptide-Reactivity-Assay (DPRA) within the Allergic Contact Dermatitis framework: A Module Accounting for Test Variability and Abiotic Transformations (#329)
A. Detroyer1, H. Ivanova2, J. Eilstein1, C. Piroird1, S. Imbert1, A. Del Bufalo1, I. Popova2, C. Kuseva2, I. Karakolev2, S. Dimitrov2, O. Mekenyan2
1 L'Oréal, R&I, Aulnay-sous-Bois, France
Allergic Contact Dermatitis (ACD) depends, amongst other parameters, on the ability of chemicals to covalently bind with skin proteins. Thus, in chemico methods like Direct-Peptide-Reactivity-Assay (DPRA) were developed as one of the alternatives to the animal ACD tests, i.e Local-Lymph-Node-Assay (LLNA).
In this context, mechanistically based in silico DPRA models were build, to potentially be used for the screening/design of new chemicals and to help evaluate experimental DPRA results at the tests’ limits (low solubility chemicals,…).
Today, based on our DPRA and LLNA data variability study*, our models contain mechanistically justified Cysteine and Lysine peptide alerts at the 13% and 42% reactivity level with a 95% confidence. They are applied on chemicals and their oxidative derivatives generated by abiotic activation transformations, predicting the worst case scenario. These models present good predictive performance (ex. by external validation) and high transparency (justifications, applicability domain).
* Journal of Applied Toxicology: ‘Accounting for data variability, a key factor in in vivo/in vitro relationships: application to the skin sensitization potency (in vivo LLNA versus in vitro DPRA) example’; S. Dimitrov, A. Detroyer, C. Piroird, C. Gomes, J. Eilstein, T. Pauloin, C. Kuseva, H. Ivanova, I. Popova, Y. Karakolev, S. Ringeissen and O. Mekenyan; DOI:0.1002/jat.3318 http://onlinelibrary.wiley.com/doi/10.1002/jat.3318/abstract
Keywords: Computational toxicology, Direct-Peptide-Reactivity-Assay, in silico, allergic contact dermatitis, alternative methods
ToxicBlend: Virtual Screening of Toxic Compound with Ensemble Predictors (#339)
M. Zaslavskiy1, S. Jegou1, E. W. Tramel1, G. Wainrib1
1 Owkin, Paris, France
Background: Timely assessment of compound toxicity is one of the biggest challenges facing the pharmaceutical industry today. A significant proportion of compounds identified as potential leads are ultimately discarded due to the toxicity they induce. In silico models for molecular activity prediction have become critical for drug discovery and other biotechnology industries. These methods also prove very important for the prediction of intrinsic chemical properties (such as solubility or acidity constant) or target affinity prediction, including potential toxicity effects, a crucial step in the development of new drugs.
Keywords: machine learning, in-vitro toxicity, webserver, predictive toxicology, in-silico toxicology
Development of an in silico model to evaluate in vivo chromosome damaging potential of chemicals (#345)
M. Van Bossuyt1, 2, G. Raitano3, E. Van Hoeck1, V. Rogiers2, E. Benfenati3, B. Mertens1
1 Sciensano, Chemical and Physical Health Risks, Brussels, Belgium
In silico methods are vastly gaining recognition and importance in various regulatory fields. The major advantage of these computer-based techniques, in contrast to in vivo and in vitro methods, is their ability to make fast, resource-saving and reproducible predictions for virtually any (toxicological) endpoint. In chemical safety assessment, genotoxicity is a key toxicological endpoint due to its link with a range of adverse human health effects including cancer. In silico tools are increasingly used to investigate the genotoxic potential of compounds, particularly when screening large numbers of substances. Robust in silico models are currently available for (Ames) mutagenicity, however, not yet for chromosome damage. The present study involves the development of an in silico prediction model for the latter genotoxic endpoint. More specifically, public and private in vivo micronucleus test data originating from various regulatory domains (cosmetics, industrial chemicals, etc.) were collected and curated. Next, SARpy modelling was applied on a final dataset of 718 substances in order to build multiple statistical (QSAR) models by using parameter variation. Based on the validation results, the QSAR model with the best accuracy/coverage balance is associated with a prediction accuracy of 68% for a coverage of 90%. Prediction performance can be further increased through combination with expert knowledge. In a later stage, the model will be integrated in LifeSPHERA, the first in silico risk assessment tool that is being developed.
Keywords: computational toxicology, in silico, genotoxicity, screening, QSAR
DynOVis: a new tool to visualize dynamic perturbations of biological networks after toxic exposure (#347)
T. Kuijpers1, J. Kleinjans1, D. Jennen1
1 Department of Toxicogenomics, GROW-School for Oncology and Developmental Biology, Maastricht University, Maastricht, Netherlands
The development of high throughput sequencing techniques provides us with the possibilities to obtain large datasets, which capture the effect of toxic dose and time on cellular processes. However, because of the dynamic nature of these cellular processes, the analysis of the results is challenging. Therefore there is a great need for new bioinformatics tools that address this problem.
Here, we present a new visualization tool that provides dynamic network visualization
DynOVis offers a force-directed graph network style, involving multiple network analysis methods such as degree threshold, but more importantly, it allows for node expression animations as well as a frame-by-frame view of the dynamic exposure.
DynOVis offers a tool for the visualization of dynamic changes that occur in biological networks which increases the understanding of the cellular response to perturbations. The animation and frame-by-frame view enable the user to investigate the effect of changes in the network across time and to identify important nodes. This is an important step forward for unravelling the effect of dynamic changes in biological systems.
Keywords: biological networks, visualization tool, dynamic effects, ‘omics data
iSafeRabbit® QSAR To Predict Skin And Eye Irritation Potency Of Organic Chemicals (#434)
M. DELANNOY1, C. CHARMEAU-GENEVOIS1, 2, F. BAUER1, F. SAHIGARA1, P. BICHEREL1, N. MAYER1, 2, P. THOMAS1, 2
1 KREATiS, L'Isle d'Abeau, France
In agreement with the 3R’S commitment (replacement, reduction and refinement of bioassays performed on laboratory animals) iSafeRabbit®1 is a model that predicts skin and eye irritation potency of chemicals aiming to replace animal testing for the chemicals in its applicability domain.
The model follows the OECD 4042 and 4053 guidelines for in vivo studies and is based on the calculation of a value for the absorption of chemicals by the skin and the eye tissue respectively as a function of exposure time. This absorption value is calculated using the physico-chemical properties of the chemicals, and it is translated into a cell burden value causing cytotoxicity and thus irritation. These values are plotted as a function of the water solubility (or sub-cooled water solubility in the case of eyes) of the chemicals. The irritation potency results are quantified using a scoring method developed and proposed by KREATiS, i.e., Simplified Irritation Index: SIISKIN and SIIEYE, which is based on erythema scores for the skin and on corneal opacity for the eye. These SIISKIN and SIIEYE scores are related to threshold values which are transcribed to CLP/GHS classification.
iSafeRabbit® models are trained with over 20 chemical families, comprising over 220 substances using only the highest quality, validated physico-chemical and in vivo data for model development and validation. So far, the predictions have an average true positive/negative rate of 50% ranging from 13% to 100% depending on the chemical family. This poster will briefly outline the models development, validation strategy and current actions taken by KREATiS to improve the sensitivity and specificity of iSafeRabbit® models.
1 Winner of 2015 NC3Rs CRACK-IT QSARs Mix Challenge - https://www.crackit.org.uk/challenge-19-qsars-mix
2 OECD (2002). Test No. 404: Acute Dermal Irritation/Corrosion, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
3 OECD (2012). Test No. 405: Acute Eye Irritation/Corrosion, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
4 OECD principles for the validation, for regulatory purposes, of (Quantitative) Structure-Activity Relationship models - http://www.oecd.org/chemicalsafety/risk-assessment/37849783.pdf
Keywords: Skin, eye, irritation, QSAR, model
Identification of Flavouring Substances of Genotoxic Concern Present in E-Cigarette Refills (#437)
S. Barhdadi1, 2, B. Mertens1, M. Van Bossuyt1, 2, M. Canfyn1, P. Courselle1, V. Rogiers2, E. Deconinck1, T. Vanhaecke2
1 Sciensano, Scientific Direction of Chemical and Physical Health Risks, Brussels, Belgium
Over the last years, the popularity of electronic cigarettes has increased significantly. An important contributor to this trend is the availability of a wide variety of flavours used in e-liquid refills. However, the impact of the use of these flavouring components on the potential toxicity of e-cigarette vapours remains unclear. Considering the large number of e-liquid flavours available on the market, there is an urgent need to establish an efficient screening strategy to prioritize the substances of highest concern for human health. In this context, genotoxicity is a key toxicological endpoint as it is related to a broad range of adverse human health effects including cancer. Therefore, in this study, a prioritization strategy based on a combination of analytical screening, in silico prediction and literature consultation was developed for identifying potentially genotoxic substances in e-liquid flavours.
77 e-liquids, representative for the different flavour categories, were collected on the Belgian market and screened for their chemical composition using GC-MS. By using the National Institute of Standards and Technology (NIST) research library, 436 individual components could be identified. Next, the genotoxic potential of these individual components was investigated in silico with two complementary (quantitative) structure-activity relationship ((Q)SAR) models (Derek Nexus, Sarah Nexus). In total, 57 flavouring components were identified with a structural alert for genotoxicity in at least one of the two (Q)SAR models. For these substances, genotoxicity data was collected from previous European safety evaluations in different regulatory domains (e.g. by the European Chemical Agency (ECHA) and the European Food Safety Authority (EFSA)). Genotoxicity could be excluded for only 12 of the 57 components, whilst for 2 of them there is a clear concern for genotoxic potential. Data for the remaining components was missing or ambiguous and hence additional toxicological data is required in order to be able to exclude genotoxic potential.
The above findings indicate that the use of flavoring components might pose a potential health risk for e-cigarette users. Further research is needed to explore to which extent these flavouring substances are transferred from the e-liquid into the e-cigarette vapours.
Keywords: Electronic cigarette, Flavours, Genotoxicity, (Q)SAR, GC-MS
Computational approaches for predicting Molecular Initiating Events (#442)
T. E. H. Allen1, J. M. Goodman1, M. N. Grayson1, S. Gutsell2, P. J. Russell2
1 University of Cambridge, Department of Chemistry, Cambridge, Cambridgeshire, United Kingdom
The Adverse Outcome Pathway (AOP) provides a framework to encapsulate the chemical and biological processes that can lead to toxicological outcomes. The Molecular Initiating Event (MIE) is the first key event in an AOP, and an MIE can be thought of as a chemical interaction between a toxicant molecule and a biological system. If we can understand the chemistry behind these processes we can link the chemistry of molecules to their biological activity at specific targets.
We have attempted to do this in several ways. Firstly, 2D chemical structure encodes much of the information about the shape and interaction potential of molecules. We have utilized chemical informatics approaches to computationally construct structure-activity relationships for over 100 human MIEs. Open source data from ChEMBL is used and maximal common substructure searches performed to return structural features associated with biological activity at each MIE. These structural alerts are coded in SMILES to allow for the rapid in silico processing of large numbers of chemicals to identify their potential MIEs.
Secondly, we have utilised quantum mechanical density functional theory calculations to probe covalent bond forming reactions that are associated with the MIE for DNA binding. DNA is able to directly react with an electrophilic chemical, modifying its structure and causing damage that can lead to genotoxic adverse outcomes. By computationally modelling the transition state of these reactions and calculating the activation energy we can establish why some molecules are able to react with DNA and return a positive Ames test, and why some chemically similar molecules cannot.
Finally, we have begun to investigate the use of chemical and biological similarity through machine learning and artificial intelligence algorithms to predict MIEs. Neural networks and deep learning provide a strong platform for MIE prediction due to their high rates of predictivity. By incorporating large amounts of ChEMBL and ToxCast data and a variety of chemical descriptors we aim to use these powerful approaches to predict MIEs.
The in silico prediction of, and increased understanding of, these MIEs is extremely important for the future of AOP risk assessment. By incorporating more computational approaches we can improve the efficiency of safety decision making.
Keywords: Molecular Initiating Event (MIE), Adverse outcome Pathway (AOP), Computational Toxicology, Chemistry, Human Risk Assessment
Identifying Potential Analogues for Read-Across in Physiologically-Based Kinetic (PBK) Modelling (#444)
J. C. Madden1, D. Ebbrell1, M. T. D. Cronin1
1 Liverpool John Moores University, School of Pharmacy and Biomolecular Sciences, Liverpool, United Kingdom
Physiologically-Based Kinetic models are employed in many sectors (for example pharmaceuticals, food, cosmetics and environmental sciences) to provide a realistic indication of the overall internal exposure to chemicals of interest in humans and animals. In recent years, there has been a significant increase in the number of such models being published for a range of chemicals. This provides an opportunity to exploit the known data for these chemicals to assist in PBK model building for other “similar” chemicals. The read-across paradigm in toxicity prediction and safety-assessment is now well established; predictions for unknown (target) chemicals are based on information available for known (source) chemicals. In order for robust predictions to be made, a clear rationale must be provided to justify the selection of “similar” source chemicals from which data may be obtained. Lu et al (2016) have developed a particularly useful resource - a Physiologically-Based Pharmacokinetic (PBPK) knowledgebase derived from data for 307 chemicals for which PBPK models have been published in the literature. These authors presented cases studies using physico-chemical descriptors to identify analogous chemicals that could be used to provide a template for the derivation of models for similar chemicals. However, as indicated by the authors, there is no consensus as to which methods of assessing similarity is the most suitable; it is not possible to determine absolute similarity of one chemical to another – chemicals can only be considered similar with respect to a given property. In the present study the chemical space of the 307 chemicals in the knowledgebase, was compared to that of pharmaceuticals, food additives and cosmetic ingredients, to ascertain the coverage of the models and identify where there are gaps in knowledge. In addition, chemoinformatics-based measures of similarity were applied as an alternative approach to defining suitable analogues to use as source chemicals. This analysis enabled the chemical space coverage of existing models to be established and demonstrated how analogue selection was influenced by the similarity assessment metric applied.
Lu, J et al (2016) PLOS Computational Biology; DOI: 10.1371/journal.pcbi.1004495
Keywords: PBK, similarity, Read-Across
A Mechanism of Action classification method unifying toxicology and ecotoxicology (#458)
P. THOMAS1, 2, F. BAUER2
1 CEHTRA, L'Isle d'Abeau, France
To predict the toxicity of substances using Read-Across or (Q)SAR models with confidence and reliability, the Mechanism of Action (MechoA) of substances, i.e. the molecular mechanism by which they exert their toxicity, must be identified. In the field of ecotoxicology, several classification methods to predict the Mode of Action (MoA) of substances exist; however, MoAs are generally not clearly defined. For toxicology, only prediction schemes for specific endpoints exist (e.g. carcinogenicity, mutagenicity, sensitisation) (Benigni et al., 2007, 2013, Enoch et al., 2008). We have developed a universal method of classification of MechoAs for use in both fields, applicable to the vast majority of organic chemicals and to various species, mainly rats and fish (but also human).
In the Adverse Outcome Pathways (AOP) framework, MechoAs are equivalent to Molecular Initiating Events (MIE). AOPs between species may differ (e.g. a substance which is an acetylcholinesterase inhibitor in a rat may be a baseline toxic for algae due to the lack of AChE receptors in plants) but toxicity can be predicted once the key events (or final endpoint value) for each species are known. The first step in understanding and predicting toxicity therefore resides in defining completely all possible MechoAs. The next step is to use empirical data to determine the ultimate outcome for each AOP (with a QSAR per MechoA sub-class and per endpoint).
Using accumulated knowledge on MechoAs from hundreds of molecules in different species, we developed a set of structural alerts in a classification scheme based on 6 general MechoAs (currently divided into 23 sub-classes), which predicts separate MechoAs for different species whenever applicable. These MechoA classes are designed to cover all possible chemical interactions leading to toxicity, while new sub-classes can easily be added when discovered. Our decision tree obtained 92.0% correct classification for training set and 92.3% for validation set. This model is simpler and performs better than our previous model (Bauer et al., 2018) and could be used in hazard assessment to strengthen mechanistic interpretation, read-across justification and weight of evidence and QSAR approaches. This model will be continuously enhanced with the addition of new rules and minor corrections as needed.
Keywords: mechanisms of toxic action, classification, ecotoxicology, human toxicology, structural alerts, decision tree
Meta-analysis for genotoxicity prediction using data from multiple human in vitro cell models (#479)
J. R. Bayjanov1, J. C. Kleinjans1, D. G. Jennen1
1 Maastricht University, Toxicogenomics, Maastricht, Limburg, Netherlands
Whole genome transcriptional profiling allows global measurement of gene expression changes induced by particular experimental conditions. Toxic treatments of biological systems, such as cell models, may perturb interactions among genes and, in toxicogenomics, such perturbations assessed by transcriptional profiling are used to predict impact of toxic compounds.
Form early days on, this toxicogenomics-based approach for predicting apical toxicities, has been dedicated to the purpose of improving predictions of genotoxicity and carcinogenicity in vivo. Over the past decade large amounts of transcriptional profiling data have been generated for in vitro study models using various chemical compounds, across different doses and time points as well as different organisms. As part of the H2020 EU project OpenRiskNet, we propose a large-scale integrative analysis approach using these data sets for predicting genotoxicity and carcinogenicity in vivo.
From the diXa Data Warehouse, NCBI GEO, and EBI ArrayExpress we collected gene expression data for human in vitro liver cell models exposed to 125 compounds with known genotoxicity information at different time points and dosages resulting in 822 experiments. We analyzed these data sets using ten different classification algorithms, thereby using 80% of the data for training and 20% for testing. Support Vector Machines algorithm had the best accuracy for predicting genotoxicity in vivo at 92.5% with 95% specificity and 87% sensitivity. Upon identifying deregulated gene-gene interaction networks by applying ConsensusPathDB, the top 5 of affected pathways are related to p53-centered pathways. The results from our meta-analysis demonstrate both high accuracy and robustness of transcriptomic profiling of genotoxicity hazards across a large set of genotoxicants and across multiple human liver cell models. We propose that these assays can be used for regulatory purposes, certainly when applied in combination with the traditional genotoxicity in vitro test battery. Next, we want to perform similar analyses on rat and mouse in vitro data and identify core orthologous genes among the three different species that are potential predictive targets for assessing genotoxicity and carcinogenicity across different biological systems.
Keywords: genotoxicity, transcriptional profiling, in vitro, classification, human
Category assessment of repeated dose hepatotoxicity of phenolic benzotriazoles for OECD IATA case studies project in 2016 (#529)
T. Yamada1, M. Matsumoto1, S. Kitajima2, K. Aisaki2, J. Kanno2, 3, A. Hirose1
1 National Institute of Health Sciences, Division of Risk Assessment, Biological Safety Research Center, Kawasaki, Japan
Phenolic benzotriazoles (PBTAs) are UV absorbers added to various polymer products to protect against UV degradation. Several substances of this group have been described as emerging contaminants with properties of concern for environmental and human health. The PBTA category was previously assessed by U.S. EPA High Production Volume Challenge Program in 2009 and NTP Chemical Information Review in 2011. A weight-of-evidence approach was used to assess the environmental persistency of certain data-poor substances, but has not yet been attempted for assessing human health endpoints. Oral toxicity studies showed that hepatotoxicity is the primary adverse effect of these compounds but that toxicity level varies markedly depending on the type and number of substituents. In this study, transcriptomic profiles of mouse liver obtained by the Percellome Project protocols were analyzed for some category substances and then integrated into the category assessment. All the profiles showed that activation of nuclear receptor pathways and/or induction of oxidative stress are likely associated with the observed liver effects of PBTAs. Moreover, even minor structural substituent differences affected transcriptomic profiles. These results suggest that transcriptomic data could support selection of source chemical with similar mode of action. However, transcriptomic data are not available for all the category substances and AOP of the liver toxicity is not well described yet due to limited resources for testing and data analysis. Further information including gene expression and toxicokinetics for bridging target and source chemicals is required to reduce uncertainties in the read across assessment. This presentation is based on the study in the OECD IATA Case Studies Project in 2016.
Keywords: phenolic benzotriazole, toxicogenomic data, category, read-across, repeated dose toxicity
An Extended Mechanistically-Based In Silico Profiler for Liver Toxicity (#555)
M. T. Cronin1, B. Bienfait2, D. Ebbrell1, S. J. Enoch1, J. W. Firman1, J. C. Madden1, A. Mostrag-Szlichtyng3, J. Rathman3, 4, V. Vitcheva3, C. Yang2, 3
1 Liverpool John Moores University, School of Pharmacy and Biomolecular Sciences, Liverpool, United Kingdom
The ability to identify the capability of a chemical to cause harm to the liver is one of the most important considerations in the safety assessment of many substances including cosmetic ingredients, pharmaceuticals, pesticides, food additives and other industrial compounds. However, assessing repeated low dose exposure to substances in man is a complex and time-consuming process. In silico profilers are based around the ability to capture chemistry in the form of structure-activity relationships that are associated with toxicity and may assist in this task. Such profilers may be used to predict toxicity directly or to as a means to group molecules as a pre-cursor to read-across. The problems in the development of in silico models for organ level toxicity, especially to the liver, are well established. They include the lack of a complete mapping of mechanisms and a paucity of suitable data to model. Despite this, some of the limitations for the development of in silico models have recently been resolved. There is better access to high quality in vivo data through databases such as COSMOS DB; the Adverse Outcome Pathway (AOP) paradigm has provided the justification of the mechanistic basis to modelling; and new high content data resources, highly applicable to Molecular Initiating Events (MIEs), are freely available. This study focussed on the compilation of mechanistically derived structural alerts for liver toxicity utilsing these new resources. The alerts have been developed from either data from historical in vivo experimentation or in vitro assessment of the MIEs. The in silico profiler includes alerts for reactive hepatotoxicity, phosolipidosis, steatosis, mitochondrial effects and other general toxicities to the liver. Capturing alerts in this manner has created a valuable computational resource. However, evaluation of the performance of the profiler indicates areas where improvements are required and could be supported by current AOPs, notably for metabolically activated toxicants, immune and idiosyncratic effects and other specific mechanisms. The further unravelling of mechanisms of liver toxicity, supported by appropriate data and AOPs, has ensured that the profiler has evolved and been extended.
Keywords: liver, toxicity, in silico, read-across
Systems toxicology approach for the assessment of zebrafish cardiotoxicity (#561)
R. Li1, 2, C. vom Berg1, M. Talikka2, S. Madan3, J. Dörpinghaus3, J. Fluck3, J. Szostak2, F. Martin2, M. C. Peitsch2, J. Hoeng2, A. Zupanic1
1 Swiss Federal Institute of Aquatic Science and Technology, UTOX, Dübendorf, Zürich, Switzerland
Having firmly established itself as an excellent model in the developmental biology field, the zebrafish is rapidly gaining popularity in toxicological studies. This is in part due to external development, amenability to high-throughput assays, and compliance with the 3R (replacement, reduction, refinement) initiative. Crucially, the toxicological and teratological effects of chemicals are remarkably similar in zebrafish and humans. Taking advantage of these benefits, we have selected zebrafish larvae for a comprehensive systems toxicology assessment of anthropogenic chemicals found in Swiss freshwaters.
To undertake this assessment, we have developed a biological network model describing cardiotoxicity in zebrafish larvae. The model is a computable representation of data curated from scientific literature describing molecular pathways that lead to cardiotoxic outcomes in zebrafish. Key signalling nodes in the network model are linked to information about downstream gene expression. Differential expression of downstream genes obtained through transcriptomics analyses can be used to infer activity of the upstream protein. This in turn leads to mechanistic hypothesis generation and gives a quantifiable measure of network perturbation.
In parallel to the network approach, we present toxicity results for 2,3-butanedione monoxime (a known effector of cardiac function in fish) and citalopram, imidacloprid, and oxazepam (chemicals introduced into freshwaters in Switzerland by human activity) according to the Organisation for Economic Co-operation and Development fish embryo toxicity test. We then report results from chemically exposed larvae in functional cardiac assays. Finally, we describe the utility of the network model in interpreting transcriptomics data to gain mechanistic insight into the molecular events initiated by a given chemical.
In conclusion, cardiac apical endpoints and computational network scoring provide a comprehensive method for linking molecular events to organ toxicity.
Keywords: zebrafish, systems toxicology, cardiotoxicity
Enhancing Compound Safety Assessment using “Multitask” Deep Neural Nets (#566)
J. Wenzel1, L. Anger1, A. Amberg1, H. Matter2, G. Hessler2, N. Griesang3, K. Mertsch3, A. Czich1, F. Schmidt1
1 Sanofi-Aventis Germany GmbH, R&D Preclinical Safety, Frankfurt, Germany
Novel pharmaceutical candidate substances need to be stringently characterized and evaluated for potential safety hazards. In this respect, Deep Neural Nets (DNN) qualify neatly for application in drug discovery and may even have the potential to outperform classical machine learning approaches. Deep learning is a successful and highly active field of artificial intelligence research which has become prominent by Big Data analysis, such as image or voice recognition.
Quantitative computational DNN models for numerous ADMET endpoints have been derived from up to 50k (non-)/proprietary datapoints, including lipophilicity (logD), passive permeability (Caco-2/TC7), microsomal lability and clearance, but also classification models for toxicology endpoints such as clastogenicity, phototoxicity and preclinical/clinical hepatotoxicity. Throughout the study, the predictive performance outperformed established benchmark methods, while prospective performance also improved and squared correlation coefficients r2test ranged between 0.87 (logD), 0.65 / 0.69 / 0.68 (human / mouse / rat metabolic lability) and 0.71 (passive permeability).
Moreover, “Multitask”-DNNs can be tweaked to describe multiple endpoints simultaneously in a single framework, further improving the performance of individual models. By combining metabolic lability data from human, mouse, rat and various other species in a single DNN, the predictivity for new substances could be substantially improved (r2test = 0.70 / 0.78 / 0.77). The addition of passive permeability data further stabilized the model (r2test = 0.71 / 0.78 / 0.78). Likewise, the computational prediction of preclinical and clinical hepatotoxicity was reinforced by adding ADMET data (preclinical accuracy improved from 78% to 82% / clinical accuracy from 69% to 75%). The maximum achievable accuracy for joint training of preclinical and clinical hepatotoxicity data was 85% and 81%, respectively. Similarly, we also found a net improvement of compound clastogenicity prediction facilitated by joint computational modelling of micronucleus formation data with protein kinase inhibition data.
Apparently, “Multitask” DNNs allow for exploitation of hidden trends or correlation even between orthogonal ADMET assays or endpoints. This feature can be particularly rewarding when working with small toxicology datasets. Our models are managed and deployed in Googles TensorFlow environment, which also enables broad application to novel research compounds.
Keywords: Hepatotoxicity, Clastogenicity, Computational modelling, Deep learning, admet
In Silico Approaches In The Selection Of A Series Of Aroylhydrazones Bearing Melatonin Moiety For Neuropharmacological Testing (#570)
P. Gateva1, K. Ramanathan2, V. Shanthi2, J. Nivya2, K. Kamenova1, V. Angelova3, J. Tchekalarova4
1 Medical University of Sofia, Department of Pharmacology and Toxicology, Sofia, Bulgaria
Our aim was to perform in silico prediction of the chemical properties and biological activities of a series of hybrid molecules bearing aroylhydrazone and melatonin moieties as candidates for further neuro-pharmacological testing.
From the test series, we preselected 10 substances predicted to be 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) enhancers (PASS module of Way2Drug suite) and their basic chemical properties were further analyzed with QikProp module of Schrodinger suite. Acute toxicity was predicted by GUSAR module of Way2Drug suite. Repeated Doses Toxicity (RDT) was predicted by Hazard Evaluation Support System (HESS). Docking was further performed using GLIDE algorithm of Schrödinger suite, to evaluate the binding affinity of the molecules towards ionotropic glutamate receptor, GluR2. Finally, the structural similarity of the tested molecules was examined with respect to the reference ligands for GluR2 taken from Stitch database.
From the tested series, 5 substances were found to be able to cross the blood-brain barrier. For intraperitoneal, intravenous, peroral and subcutaneous route of application predicted acute rodent toxicity for all tested substances indicated class 4 or 5 by OECD. All tested substances belong to the US-EPA new chemical category of hydrazine and related compounds, and all have toxic hazard classification by Cramer high (class III), DNA binding, oncogenic, mutagenic and carcinogenic potential. Three of the tested molecules are similar to indomethacin; from which – one also similar to serotonin and one other similar to methylclofenapate; one is similar to curcumin; and two are similar to lobenzarit. According to this, the RDT is predicted as indicative for hepatotoxicity or nephrotoxicity.
Further the tested molecular structures were docked against GluR2 receptor and seven molecules were found to possess significant activity against the receptor (lesser binding energy than reference antagonist, perampanel). Of note, three out of the seven hits were also found to possess significant neuro-pharmacological effect which is a primary requisite for GluR2 antagonists.
This work is supported by the Research fund of Bulgaria Contract ДH13/16 from 21.12.2017.
Keywords: in silico, aroylhydrazones, GluR2 receptor
An automated workflow for Adverse Outcome Pathway hypothesis generation (#632)
T. Doktorova1, T. E. Exner1, B. Hardy1, O. Noffisat1
1 Douglas Connect GmbH, Basel, Basel-Stadt, Switzerland
The field of toxicology is evolving from a purely descriptive to a highly data-rich science. To structure this large amount of data and at the same way to make the usage more effective, computational approaches for data integration should be applied. In the ADVaNCE project we aim to establish an automated workflow for Adverse Outcome Pathway hypothesis generation. By combining data coming from diverse data sources, we demonstrate in practice how to use computational approaches for integration of pre-existing knowledge. As a proof-of-principle, a computationally predicted network of Adverse Outcome Pathways (AOPs) characteristic for non-mutagenic carcinogen exposure is generated to provide better understanding of the mode-of-action (MoA) of non-mutagenic carcinogen-induced hepatocellular carcinoma (HCC).
The integrated data sources so far are : (i) ToxCast database for identification of chemical-gene and chemical-biological category relationships; (ii)Comparative Toxicogenomics Database (CTD) for identification of chemical-gene, chemical-pathway and chemical-disease relationships; and (iii) Reactome for identification of disease-pathway relationships.
The final goal is to offer a hypothesis generation tool which can be freely exploited by the scientific community and will eventually guide the development of an integrated testing strategy completely based on in silico and in vitro data to be used in regulatory settings.
Keywords: computationally predicted Adverse Outcome Pathways, non-genotoxic carcinogens, hepatocellular carcinoma
An AOP-based Ontology for Spina Bifida Caused by Disturbance in Retinoic Acid Signaling (#661)
Y. C. Staal1, N. C. Baker2, L. D. Burgoon3, G. Daston4, T. B. Knudsen5, A. H. Piersma1
1 RIVM, Bilthoven, Netherlands
Retinoid signaling plays an important role in embryo-fetal development and its disruption is teratogenic. The retinoic acid (RA) pathway includes elements in retinoid metabolism and nuclear receptor (RAR, RXR) activation and thus serves as an excellent prototype for adverse outcome pathway (AOP) elucidation associated with developmental defects. The biology of the RA pathway, leading to defects in neural tube closure was the basis for the construction of an ontology for developmental toxicity. The prototype ontology describes an AOP network that incorporates feedback-loops for retinoid homeostasis and putative molecular initiating events in chemical teratogenesis. Basic elements in the ontology are subjects (enzymes, receptors, cell types) and their quantitative relationships (response-response relationships), together forming a network of biological interactions that can be mapped to a vulnerable window for teratogen-induced neural tube defects, specifically spina bifida. We have used the available data in the literature, which was searched using text-mining tools that allowed rapid identification of relevant literature related to human developmental biology, to map known molecular interactions, genetic signals and responses that: (a) play a crucial role in cellular differentiation; (b) establish anterior-posterior gradients (FGF and RA signaling) and dorsal-ventral gradients (zinc factors (Zic) and BMP signaling) for regional specification. Molecular initiating events important for RA balance (like CYP26 enzymes and RALDH2) potentially affected by xenobiotic compounds (using high-through-put screening data), will be connected with toxicological data on the development of posterior neural tube defects. Ultimately, this network can be dynamically modeled in silico, providing an integrated computational systems model with which toxicity predictions can be made at the level of adverse outcomes in the intact individual. A battery of cell-based in vitro assays can be used to monitor the critical rate-determining steps in the network, providing a tiered testing strategy to collect data feeding into the systems model. Integrating the dynamic model with information from exposure and kinetic models allows quantitative hazard and risk assessment while avoiding animal testing.
The views presented in this abstract do not necessarily reflect current or future opinion or policy of the U.S. Environmental Protection Agency and the U.S. Army Corps of Engineers.
Keywords: Ontology, computational toxicology, fetal development, retinoic acid, spina bifida
OpenRiskNet, an open e-infrastructure to support data sharing, knowledge integration and in silico analysis and modelling in risk assessment (#679)
T. E. Exner1, J. Dokler1, D. Bachler1, L. R. Farcal1, C. Evelo2, E. Willighagen2, D. Jennen3, M. Jabocs4, P. Doganis5, H. Sarimveis5, I. Lynch6, G. Gkoutos6, S. Kramer7, C. Notredame8, O. Spjuth9, P. Jennings10, T. Dudgeon11, F. Bois12, B. Hardy1
1 Douglas Connect GmbH, Basel, Switzerland
OpenRiskNet (https://openrisknet.org/) is an EU funded infrastructure project with the main objective to develop an open e-infrastructure providing resources and services to a variety of industries requiring risk assessment, including chemicals, cosmetic ingredients, drugs and nanomaterials.
The OpenRiskNet approach is to work on different case studies to test and evaluate requirements to overcome the fragmentation of data and tools and to provide solutions improving the harmonization of data, usability and interoperability of application programming interfaces (APIs) and the deployment into virtual infrastructure. The cases present real-world settings such as systems biology approaches for grouping compounds, read-across applications using chemical and biological similarity, and identifying areas of concern based only on alternative methods approaches.
We discuss our progress on the OpenRiskNet goal of harmonizing data and metadata within APIs that can be added to already existing analysis and modeling services and data warehouses. We also demonstrate how these APIs can easily be used towards the generation of full risk assessment workflows either using scripting languages or workflow managers. Finally, we show the first approaches to make these APIs semantically rich by annotating data with human- and computer-readable data schemata.
OpenRiskNet has initiated the Associated Partners Programme strengthening the working ties between the OpenRiskNet members and other organizations within the scientific community.
Keywords: computational infrastructure, data harmonization, interoperability, workflows, risk assessment, predictive toxicology
Development of a state-of-the-art multiple regression KY-method corresponding to the big data era and its application to fish toxicity (#681)
1 In Silico Data, Ltd., Chiba,, Japan
Introduction: Even when the number of samples to be analyzed is extremely large, we have developed a multiple regression method which has high analytical reliability. In the case of evaluating the toxicity of a compound using a computer, a multivariate analysis / pattern recognition method is applied. However the conventional multivariate analysis / pattern recognition method is not designed to handle a large amount of sample data such as big data. We developed the "multiple regression KY-method" as a data analysis method corresponding to the big data era and analyzed fish toxicity data. Method: In the KY-method, the sample group to be analyzed is divided into two groups. One is an in-lier group and the other is an out-lier sample group. The correlation coefficient (R) value of the in-lier sample group is greatly improved compared with the normal method.
Experiment and results: Fish toxicity data analysis was performed by two methods. One was normal linear multiple regression and the other was the regression KY-method.
1. Result by the normal linear multiple regression method using all samples.
Number of total samples: 779, Number of parameters: 28, Reliability index: 27.8,
R: 85.3, R2: 72.8, F value: 71.7, CV: 69.6
2. Results by the “multiple regression KY-method”.
a) Number of in-lier samples; 398, Number of parameters: 22, Reliability index: 18.1,
R; 98.1, R2; 96.2, F value: 428, CV: 94.4
b) Number of out-lier samples: 393, Number of parameters: 29, Reliability index: 13.6,
R: 80.4, R2: 64.7, F value: 22.9, CV: 57.5
Summary: Currently, we have entered the era of big data where the number of samples extremely large. However the data analysis method currently deployed can not correspond to the big data era. Therefore, it is necessary to develop new and state-of-the-art data analysis methods. The multiple regression KY-method discussed in this poster is developed as a data analysis method corresponding to the big data era. Details will be discussed in the poster.
Keywords: Computational Toxicology, Regression KY-method, Big data, Fish toxicity
ToxGPS, a Solution Guiding Read-Across Workflow Based On Chemoinformatics and Safety Assessment (#683)
C. Yang1, 4, B. Bienfait1, M. T. Cronin2, E. Fioravanzo3, M. Gatnik3, T. Kleinoeder1, J. Park4, J. Liu4, T. Magdziarz1, J. Marusczyk1, A. Mostrag4, M. Mulcahy4, J. F. Rathman4, 5, O. Sacher1, C. Schwab1, A. Tarkhov1
1 Molecular Networks, Nürnberg, Germany
Read-across is one of the alternative methods considered for regulatory purposes to fill data gaps encountered in product safety assessments. Although considerable experience has helped define how to properly apply these analogue and category approaches for regulatory programs, acceptance remains context-dependent and reliant on expert judgement. Recent activities from European Chemicals Agency (ECHA), Cosmetics Europe (CE), and European Food Safety Authorities (EFSA) have developed conceptual frameworks to identify and document sources of evidence, estimate uncertainty, and combine all information for the final outcome. Addressing these issues using mechanistic information within Adverse Outcome Pathways (AOPs) is under development, but still is at an early stage. Rigorous/objective metrics to evaluate the performance or quantify uncertainties of read-across results have not been the main focus of many current computational tools. To address these deficiencies, there is a clear need to develop read-across approaches that are more robust and reproducible whilst maintaining the original rationales. Recent EFSA guidelines for read-across specified the process for assembling evidence of similar types, weighing, and integration. In this study, we introduce the new solution ChemTunes•ToxGPS, which builds on these concepts and attempts to reduce uncertainty by identifying analogues that are biologically meaningful and relevant to the target toxicity endpoints. Information is combined by applying a weight of evidence method to predict the outcome and estimate uncertainties. These criteria have been captured in a chemoinformatics system as a read-across workflow. Three use scenarios are presented: 1) mechanistic knowledge or compound mode-of-action is available; 2) hypothesis generation is necessary; 3) assessing metabolites or reaction degradants. Using ToxGPS, examples are presented to address the issues related to collection of relevant evidence, consistent treatment, weighing the information, and estimating the uncertainty quantitatively, ultimately leading to reproducible and defendable final outcomes.
Keywords: Read Across, Safety Assessment, Risk Assessment, Weight of Evidence, Uncertainty
Prediction of Drug-Induced Liver Injury (#698)
T. Ghafourian1, M. Baricicova1
1 University of Sussex, Life Sciences, Falmer, United Kingdom
Drug Induced Liver Injury (DILI) is a serious drug toxicity event responsible for many failures in drug development and drug withdrawals from the market that is difficult to identify during pre-clinical studies. DILI concerns have been linked with in vitro biomarkers such as drug-induced mitochondrial damage, oxidative stress, and intracellular glutathione levels (Xu et al., 2008), and clinical factors such as drug dose and extent of metabolism (McEuen et al., 2017). This research pursued two goals: a) to find if there is a relation between DILI concern of drugs and the literature reported mitochondrial dysfunction effect of drugs, and 2) to build quantitative structure-activity relationship models for the prediction of DILI. In this investigation, a list of drugs from FDA‘s Liver Toxicity Knowledge Base (LTKB) comprising 70 drugs labelled as “most-DILI-concern” and 55 drugs labelled as ”no-DILI-concern“ were used for the modelling (Shah et al., 2015).
All of the 125 drugs were searched for literature evidence of causing mitochondrial dysfunction in any of the following ways: increasing production of reactive oxygen species (ROS), decreasing mitochondrial membrane potential, inhibiting any of the five respiratory complexes or uncoupling oxidative phosphorylation. Molecular Operating Environment (MOE) was used for the molecular modelling and the calculation of molecular descriptors. Various classification algorithms including decision trees and random forest were used in WEKA (version 3.8.0) for the classification of compounds based on DILI concern. Decision trees indicated that the most significant parameter for the prediction of DILI propensity of compounds was literature evidence of mitochondrial dysfunction as defined above. The drug dose and molecular attributes such as lipophilicity and number of aromatic rings were among the most significant parameters of the model. The best prediction was achieved using a random forest model with test set prediction sensitivity and specificity of 0.765 and 0.786, respectively.
Keywords: DILI, Drug-Induced Liver Injury, Mitochondrial dysfunction, QSAR, Liver toxicity
Pharmacological properties of a thrombin-like, inducer of platelet aggregation, purified from Cerastes cerastes venom: Elucidation of activated pathway signaling (#132)
C. FATAH1, S. SAOUD1, F. LARABA-DJEBARI1
1 USTHB, Faculty of Biological Sciences;, Department of Cellular and Molecular Biology, Algiers,, Algeria
A new thrombin-like proteinase designed Cc3-SPase, with pI 5.98, Mw of 30 083.51 Da and a sequence of 271 amino acid residues, was isolated from Cerastes cerastes venom. When submitted to multiple alignment, Cc3-SPase showed high degrees of homology and revealed highly conserved peptides with other related serine proteinases. Cc3-SPase cleaved natural and synthetic proteins including fibrinogen without affecting fibrin clots. Its catalytic activity was fully abolished when incubated with ion chelators (PMSF, EDTA and EGTA), whereas, aprotinin, antithrombin III, heparin and 1,10-phenanthroline were ineffective which may indicate the involvement of Ca2+ for both protease activity and structure stability. Conversely, Cc3-SPase was susceptible to heparin when combined to anti-thrombin III. Sensitivity of Cc3-SPase to PMSF and affinity to benzamidine indicated the presence of serine and aspartate residues in the catalytic site as confirmed by its 3D-modeled structures and molecular docking of Cc3-SPase to benzamidine. Molecular docking results yielded an interaction between benzamidine and Cc3-SPase with two hydrogen bonds. The first hydrogen bond was established between the hydrogen (H1) of the ligand and the amino acid (ASN 211) of the studied protease with a bond length equal to 1.907 Ǻ while in the second hydrogen bond was established between the hydrogen (H8) of benzamidine and the amino acid (SER 230) of Cc3-SPase with a bond length equal to 2.179 Ǻ. Molecular mechanisms revealed that Cc3-SPase acted as a pharmaco-biological agent to promote dysfunctional platelet aggregation via two signaling pathways mediated by the G-coupled protein receptors PAR1, PAR4 and αIIbβ3 integrin. Cc3-SPase used as a substitute for missing factors in deficient plasmas, it replaced FII, FVII and FVIII but not FX, since it is involved in both extrinsic and intrinsic coagulation pathways.
Keywords: Serine proteinases, Missing factors, Deficient plasma, Platelet receptors, 3D modeling, Molecular Docking
Safety evaluation of a human chimeric monoclonal antibody that recognizes the extracellular loop domain of claudin-2 (#139)
Y. Hashimoto1, T. Hata1, M. Tada2, M. Iida1, A. Watari1, Y. Okada1, T. Doi1, H. Kuniyasu3, K. Yagi1, M. Kondoh1
1 Osaka University, Graduate School of Pharmaceutical Sciences, Osaka, Japan
Claudin-2 (CLDN-2), a pore-forming tight-junction protein with a tetra-transmembrane domain, is involved in carcinogenesis and the metastasis of some cancers. Although CLDN-2 is highly expressed in the tight junctions of the liver and kidney, whether CLDN-2 is a safe target for cancer therapy remains unknown because a CLDN2-specific binder has not been developed. Here, we used DNA immunization to generate a monoclonal antibody (mAb) that recognized an extracellular loop of CLDN2, and we generated a rat monoclonal antibody (mAb, clone 1A2) that recognizes the extracellular domains of human and mouse CLDN-2. Then, we investigated the safety of CLDN-2-targeted cancer therapy by using 1A2 as a model therapeutic antibody. Because most human therapeutic mAbs are IgG1 subtype that can induce antibody-dependent cellular cytotoxicity, we generated a human–rat chimeric IgG1 form of 1A2 (xi-1A2). xi-1A2 activated Fcg receptor IIIa in the presence of CLDN-2-expressing cells, indicating that xi-1A2 likely exerts antibody-dependent cellular cytotoxicity. At 24 h after its intravenous injection, xi-1A2 was distributed into the liver, kidney, and tumor tissues of mice bearing CLDN-2-expressing fibrosarcoma cells. Treatment of the xenografted mice with xi-1A2 attenuated tumor growth without apparent adverse effects, such as changes in body weight and biochemical markers of liver and kidney injury. These results support xi-1A2 as the lead candidate mAb for safe CLDN-2-targeted cancer therapy.
Keywords: anti-cancer agent, biodistribution, claudin-2, efficacy, safety
Impact of liposomal encapsulation on the phototoxicity, photogenotoxicity and photodegradation of ciprofloxacin in the range of ocular applied concentrations (#144)
A. Zgadzaj1, J. Giebułtowicz2, M. Podbielska1, J. Gubernator3, G. Nałęcz-Jawecki1
1 Medical University of Warsaw, Department of Environmental Health Sciences, Faculty of Pharmacy, Warsaw, Poland
Liposomal drug delivery systems are well known as a way to improve the biodistribution of many medical compounds by overcoming difficulties in their cellular uptake in the target tissues or increasing the stability of pharmaceuticals. Moreover, liposomal formulations may protect the molecular integrity of therapeutic compounds from light or have an impact on the way of their photodegradation process. However, there are lack of research on the impact of liposomes on the phototoxicity or photochemical genotoxicity of photolabile drugs. In this work we evaluated the influence of liposomal encapsulation on the photodegradation, phototoxicity and photochemical genotoxicity of ciprofloxacin, a photolabile drug often used in ocular formulations. We investigated two types of liposomes: multilamellar vesicles and large unilamellar vesicles; and two different drug to phospholipids ratios. All liposomal formulations were compared with an antibiotic solution in equal concentration ranges. The differences in the photodegradation products of ciprofloxacin were evaluated by HPLC-MS technique. The phototoxicity and photogentotoxicity of irradiated samples were assessed with a set of in vitro bioassays with mammalian cells: the neutral red uptake test, MTT assay, and the micronucleus test. The irradiation of samples was performed in the sunlight simulator in different variants: short light exposition in the presence of viable cells or extended light exposition before the cell culture treatment to evaluate the differences in the cytotoxicity of short and long living photodegradation products of ciprofloxacin. The results of our project confirmed a significant influence of liposomal encapsulation on the photodegradation process of ciprofloxacin. The differences were also visible between various types of liposomes. Moreover, the liposomal encapsulation modified the drug phototoxicity in vitro.
The project was financed by the Medical University of Warsaw, Faculty of Pharmacy from the Grant for Young Scientists managed by Anna Zgadzaj in years 2017-2018 (FW14/PM1/17).
Keywords: liposomes, ciprofloxacin, phototoxicity, photodegradation
Development of a High Content Imaging Assay to Assess Drug Induced Lysosome Perturbation. (#154)
I. A. Baines1, L. J. Davis2, M. G. Lennon1, C. G. Taylor1, T. O. Dare1, N. A. Bright2, J. P. Luzio2, J. D. Parry1, S. L. Gresham1, G. J. Dear1
1 GSK Research and Development Ltd, Investigative Safety and Drug Metabolism, Ware, United Kingdom
Over 50% of commercially available drugs contain at least one basic amine group (Goldman et al. 2009, Bioanalysis, 1: 1445-1449). The acidic environment of lysosomes causes ionisation and trapping of weakly basic compounds, termed lysosomotropism. This phenomenon can lead to phospholipidosis, an over-accumulation of phospholipids, and impairment of lysosomal function (Shayman & Abe 2013, Biochim. Biophys. Acta, 1831: 602-611). Inhibition of lysosome function may also play a wider role in organ toxicity (Lu et al. 2016, Lysosomes: Biology, Diseases, and Therapeutics, ISBN: 978-1-118-64515-4, 445-485). Lysosome function is regulated by the co-ordinated lysosome expression and regulation (CLEAR) gene network, which encodes lysosome proteins and regulates processes such as lysosome biogenesis, endocytosis, mitophagy and autophagy. This gene network is regulated primarily by the master transcription factor TFEB (Settembre et al. 2012, EMBO J, 31: 1095-1108). Lysosomotropic drugs can induce TFEB activation and translocation from the cytoplasm to the nucleus, representing a potential marker for drug-induced perturbation of lysosome function (Lu et al. 2017, PloS ONE, 12: e0173771). A HEK293 cell line stably expressing TFEB coupled to GFP was generated. Ten compounds with differing physicochemical and functional properties were selected for assay validation. Data were expressed as the concentration of test compound causing 50% TFEB nuclear translocation (TFEB50 values). The overall rank order of potency was Chloroquine > Amiodarone > Fluoxetine = Gefitinib > Ketoconazole > Verapamil > Diclofenac = Propanolol >> Acetaminophen = Cetirizine. These data demonstrate that a TFEB nuclear translocation assay has been validated which represents a novel tool to investigate lysosome perturbation.
Keywords: Lysosome, TFEB, Lysosomotropism, Cell Imaging, Nuclear Translocation
Identification of novel natural inhibitors of snake venom nucleotidases using a structure-based molecular docking study (#156)
S. saoud1, F. Chérifi1, F. Laraba-Djebari1
1 University of Sciences and Technology Houari Boumediene, Faculity of biological sciences, Algiers, Algeria
In the current study we report the identification of novel natural inhibitors of snake venom nucleotidases. Therefore, components deriving from plants endowed by inhibitory effect against snake venom nucleotidases were explored. The selected compounds include the terpenoids betulin and lupeol, ascorbic, ursolic and ellagic acids, gingerol and linalool. To assess their eventual inhibitory effect against snake venom nucleotidases, these compounds were submitted to molecular docking with Cc-5’NTase a 5’-nucleotidase purified from Cerastes cerastes venom. Our results show that among the studied compounds only betulin didn’t interact with Cc-5’NTase. In fact, lupeol interacts with Cc-5’NTase by a hydrogen bond established with Tyr376 while ursolic acid interacts with the residues Arg202 and Glu207 by two hydrogen bonds. Moreover, ellagic acid and gingerol targeted residues Asp129 and His126 of Cc-5’NTase. In the other hand, the interaction with ascorbic acid takes place via two hydrogen bonds established with the residues Leu192 and Asp129, although, linalool is linked to the nucleotidase by two hydrogen bonds formed with Val265 and Phe254. Hence, ascorbic acid and linalool show a competitive inhibition of Cc-5’NTase enzymatic activity appearing in their interaction with Leu192 and Val265 involved in Cc-5’NTase interaction with its substrates ATP and ADP, respectively. Besides of this, among all compounds the most potent complexes was established with ascorbic acid and linalool as they show the most negative energies of interaction. Taken together, our results suggest an eventual inhibitory effect of compounds interacting with Cc-5’NTase and they may therefore be responsible of the inhibitory effect of their native plants against snake venom 5’-nucleotidases.
The current study opens perspectives for the design of new snake venom-5’nucleotidase inhibitors and the improvement of envenomation therapy.
Keywords: Natural inhibitors, Snake venom, Nucleotidases, Molecular docking
Thrombocytopenia after Repeated Administration of Oligonucleotides in Cynomolgus monkeys can be treated with steroids: A Case Report (#209)
B. Korbmacher1, F. Ludwig1, L. Mecklenburg1, C. Hoffmaster2, T. - W. Kim2, P. Naranayan2, S. Henry2
1 Covance Preclinical Services GmbH, Muenster, Germany
Introduction: Reduction in platelet (PLT) counts has been reported in monkeys treated with 2¢-O-methoxyethyl modified (2¢-MOE) antisense oligonucleotides (ASOs) (Henry et al., 2017). In most cases, the reduction in PLT count is gradual and PLT counts remain in the normal range with continued treatment. However, in a subset of monkeys, thrombocytopenia can be severe (PLT count <50,000 cells/μL), and is often not reproducible between studies with the same ASO (Henry et al., 2017). The incidence appears to be dose-dependent and PLT counts recover upon drug withdrawal, and in some cases drop again when re-challenged. Increases in anti-PLT IgG/IgM and/or anti-PF4 IgM secondary to innate immune cell activation leading to increased PLT clearance could explain this severe phenotype (Narayanan et al., 2018). Herein we describe two cases of severe thrombocytopenia in monkeys that had been repeatedly treated once weekly with two different ASOs, and in which treatment with methylprednisolone (MP) was successful.
Method: Both monkeys were part of subcutaneous dose toxicity studies and had shown a PLT count below 100,000 cells/µL under ASO dosing. Treatment with MP at 80 mg/dose was initiated. Animal 9481M was treated twice with MP and Animal 19896M was treated three times during the study.
Results: In one animal (19481M), given ASO1, PLT count was 8,000 cells/µL on Day 65 (48 hours after dosing) and therefore, MP treatment was initiated immediately. The PLT count increased to 145,000 cells/µL on Day 70 of the study. The animal was dosed without a dosing holiday and the second treatment of MP was given on Day 72 (48 hours after ASO dosing on Day 70). ASO administration was continued on Days 77, 84, and 91. However, following recurrence of thrombocytopenia on Day 92 (PLT count of 6,000 cells/µL), this animal was euthanized as scheduled on Day 93. In another animal (19896M), given ASO2, PLT count was 106,000 cells/µL on Day 135 (48 hours after dosing) and MP treatment was performed on Days 147 and 154. Since the PLT count decreased again to 62,000 cells/µL on Day 184, MP treatment was performed again on Day 184. The PLT count increased then to 149,000 cells/µL on Day 233 and remained stable until end of the study on Day 275 (118,000 cells/µL).
Conclusion: Treatment with MP on low PLT count animals was very effective, and it can reverse the thrombocytopenia in ASO treated monkeys.
Keywords: cynomolgus monkeys, preclinical safety, platelet count, steroid treatment, thrombocytopenia, mean platelet volume, oligonucleotide
Ursodeoxycholic acid modulates intracellular redox state and sensitizes colon cancer cells to the cytotoxicity induced by epigenetic agent vorinostat (#220)
N. Pavlovic1, K. Stankov1, B. Stanimirov1, M. Djanic1, G. Bogdanovic1, S. Golocorbin-Kon1, M. Mikov1
1 Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia
Vorinostat is the first HDAC inhibitor approved for the treatment of malignant diseases, but it is inactive against cancer cells with high intrinsic oxidative stress. Recent studies have shown that antioxidants may sensitize malignant cells to vorinostat-induced cytotoxicity. Ursodeoxycholic acid (UDCA) exerts antioxidant and cytoprotective effects and it has been extensively investigated as a chemopreventive agent. We aimed to analyze the impact of UDCA on cytotoxic activity of vorinostat towards colon cancer cells and to evaluate the underlying molecular mechanisms. Human colon adenocarcinoma HT-29 cells were used to assess the cytotoxicity of vorinostat and UDCA, alone or in combination, using the colorimetric MTT assay. Multiple drug effects were examined by calculating the combination index (CI) using CompuSyn software. Gene expression analysis was performed by SybrGreen-based real-time qPCR assay. Vorinostat exhibited a modest cytotoxic activity (IC50 = 5.1 μM), while UDCA was shown to be non-toxic towards HT-29 cells (IC50 = 352 μM). The co-incubation of cells with clinically relevant concentrations of vorinostat (1 μM and 2 μM) and non-toxic concentrations of UDCA (50 μM) over 48h resulted in significant increase of cytotoxicity. Calculated CIs of 0.049 and 0.039 for UDCA, in combination with 1 μM and 2 μM vorinostat respectively, demonstrated that UDCA exerted strong synergistic activity with vorinostat in concentrations that can be achieved in vivo. We observed the significant changes in expression of the mitochondrial pro-apoptotic Bax and anti-apoptotic Bcl-2 mRNA levels following the treatment of HT-29 cells with vorinostat, alone (1 μM) or in combination with UDCA (50 μM) over 24h. The increase in Bax/Bcl-2 ratio in vorinostat-treated cells was significantly reduced after co-treatment with UDCA. Induction of oxidative stress is the established mechanism of vorinostat-induced cytotoxicity, and we showed that vorinostat suppressed the expression of Nrf-2 gene, which regulates cellular resistance to oxidants. This effect was even more pronounced after co-treatment with UDCA. Therapeutic efficacy of vorinostat may be improved by combining it with UDCA through the mechanisms including modulation of redox status in colon cancer cells.
Acknowledgement: This work is supported by Ministry of Education, Science and Technological Development of Serbia, Grant No. 41012.
Keywords: vorinostat, epigenetics, apoptosis, oxidative stress, bile acid
Cytotoxic activity of Zn(II)/Au(I), Zn(II)/Ag(I) and Ru(III) complexes with Schiff base Salen in human cancer and non-cancer cells (#242)
D. R. Dinev1, Z. Petrova1, T. Zhivkova1, R. Spasov1, M. Georgieva3, G. Miloshev3, L. Patron2, G. Marinescu2, D. - C. Culita2, R. Alexandrova1
1 Bulgarian Academy of Sciences, IEMPAM-BAS, Sofia, Bulgaria
The aim of our study was to evaluate comparatively the cytotoxic activity of three complexes of Zn(II)/Ag(I), Zn(II)/Au(I) and Ru(III) with Schiff base obtained by the condensation reaction between salicylaldehyde and ethylenediamine (Salen).
The following permanent human cell lines were used as model systems in our study: MDA-MB-231 (triple negative breast cancer), MCF-7 (luminal A type breast cancer) HeLa (cervical carcinoma) and Lep-3 (non-tumor embryonic fibroblasts). The investigations were performed on monolayer cell cultures with short-term experiments (24 – 72 h) using 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. Long-term experiments (25-30 days) with colony-forming method were carried out to examine the influence of the compounds on 3D growth of cancer cells.
The obtained results reveal that applied at concentrations in the range 0.1 – 10 µg/ml (for Zn(II)/Au(I) and Zn(II)/Ag(I) complexes) and 5-100 µg/ml (for Ru(III) complexes) the investigated complexes decrease significantly viability and/or proliferation of the treated cells in a time- and concentration-dependent manner. ZnSalenAu is more toxic for Lep-3 non-tumor fibroblasts whereas cancer cells are relatively more sensitive to the cytotoxic activity of ZnSalenAg. Tested independently, the ligand Salen does not significantly inhibit cell viability and growth.
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 and Grant № ДКОСТ 01/16 from 17.08.2017); COST Action CA15135 (“MuTaLig”); bilateral project between Bulgarian Academy of Sciences and Romanian Academy.
Keywords: Cell lines. Cancer. Cytotoxicity.
A pharmacologic increase in activity of plasma transaminase derived from small intestine in animals receiving an acyl CoA: diacylglycerol transferase (DGAT) 1 inhibitor (#256)
H. Yokoyama1, A. Kobayashi1, K. Kondo1, S. Oshida1, T. Takahashi1, T. Masuyama1, T. Shoda1, S. Sugai1
1 JAPAN TOBACCO INC., Toxicology Research Lab., Central Pharmaceutical Research Institute, Hadano, Kanagawa, Japan
Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the relationships between the feeding status of the animals and the increase in plasma transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. As a result of measuring the transaminase levels in the liver and the small intestine mucosa after corn-oil loading in the JTT-553-treated rats, the transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was suppressed by co-administration of Actinomycin D, a nucleic acid synthesis inhibitor, and orlistat, a lipase inhibitor. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to liver injury but to be phenomena resulting from enhancement of enzyme protein synthesis and accumulated fatty acid in the small intestine due to the pharmacological action of JTT-553 in this organ.
Keywords: DGAT1 inhibitor, small intestine, transaminase, fatty acid
Validation and Utility of iPS-derived Vascular Smooth Muscle Cells as an in vitro Vascular Model for Cardiovascular Safety Assessment (#276)
C. R. Archer1, M. Budnik-Zawilska1, A. Pointon1, K. Gray1
1 AstraZeneca, Safety and ADME Translational Sciences, Drug Safety and Metabolism, IMED Biotech Unit, Cambridge, United Kingdom
Drug-induced changes in blood pressure are a common and undesirable side effect of novel compounds. There are currently limited in vitro cellular phenotypic models available to predict vasoactivity applicable to early safety screening in drug discovery and development. Cellular approaches are limited by a lack of phenotypic consistency between primary human vascular smooth muscle cells (VSMC). A human induced pluripotent (iPS)-derived VSMC model has been evaluated to provide a cellular system amenable for in vitro vascular safety assays.
Here we describe the validation and characterisation of the hiPS-VSMC model by determining the expression of specific VSMC markers and pharmacological responses to vasoactive compounds. As the mechanisms that regulate vascular tone are varied, we have developed a black box assay to detect global changes in calcium that may represent vasoactivity.
We confirm that hiPS-VSMCs express the vascular smooth muscle cell specific proteins calponin and SM22. Assessing calcium transients as a surrogate of vasoactivity, we validate the model using a panel of 26 reference compounds, including 20 vasoactive and 6 non-vasoactive compounds. 7 vasoconstrictors and 2 vasodilators showed a concentration-dependent increase in calcium transients, with negative effects observed for all of the non-vasoactive compounds. For example, the vasoconstrictor Carbachol exhibited activity with an EC50 of 5 µM (CI 1.4-17.8) with no calcium flux observed for the non-vasoactive compound Acyclovir. We hypothesise that the absence of calcium transients for some vasoactive compounds may be due to absent or low expression of the respective target receptor in this model, this was supported by gene expression data. For example, there is very low expression of the Thromboxane A2 receptor and concomitantly no pharmacological response to U46619 in the hiPS-VSMC model.
The iPS-VSMC model system demonstrates potential utility as an in vitro model for cardiovascular safety assessment. To improve this model, co-culture with endothelial cells is currently being explored to allow detection of vasoactivity via both smooth muscle and endothelial cell dysregulation.
Keywords: Drug induced vascular injury, cardiovascular safety, human induced pluripotent stem cells, smooth muscle cells, in vitro models, safety assessment
Effect of rocuronium on duration of ventilation after organophosphorus insecticide poisoning – A pilot randomised controlled trial (#284)
H. K. J. Dhanarisi1, I. Gawarammana1, 2, F. Mohamed1, 3, M. Eddleston1, 4
1 South Asian Clinical Toxicology Research Collaboration, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka
Purpose: Organophosphorus (OP) insecticide self-poisoning causes >100,000 deaths every year. 35% of patients in developing countries require intensive care and ventilation. We aimed to collect preliminary data on whether addition of the competitive nicotinic antagonist rocuronium to standard therapy for OP poisoned patients requiring intubation and ventilation might be clinically feasible and lead to reduced duration of ventilation.
Methods: A pilot three-arm dose response phase II trial was set up to compare bolus doses (0.3 mg/kg) of rocuronium bromide titrated to produce >95% inhibition or 50% inhibition of NMJ function, measured using acceleromyography, plus standard treatment, with standard treatment alone. Patients receiving bolus rocuronium then received infusions of rocuronium (1.5 mg/kg/h) for a maximum of 120 h (due to safety reasons). Eligible patients had clinical features of anticholinesterase poisoning and reduced consciousness requiring intubation and ventilation, with NMJ function conserved to >50% of normal. The primary outcome was duration of intubation; secondary outcomes included dose of atropine required and case fatality. Plasma butyrylcholinesterase (BuChE) activity was measured in patients.
Results: Forty-five patients (64%, profenofos) were recruited and randomised: rocuronium to 95% NMJ inhibition (n=15), to 50% NMJ inhibition (n=14), and control (n=16). The groups were similar at baseline. Median (IQR) duration of ventilation was longer in the rocuronium 50% (278 [121 to 378 h) and rocuronium 95% (227 [186.5 to 355.5] h) arms compared to the control arm (89 [51 to 168] h, p = 0.0956 and p=0.0465, respectively). Case fatality was 9/45 (20.0%); it was non-significantly higher in the rocuronium 50% arm (4/14, 28.6%) than rocuronium 95% (2/15, 13.3%) and control (3/16, 18.8%) arms (p=0.5840). BuChE activity remained severely inhibited for the duration of the study for most patients.
In this small feasibility study, we found no indication that rocuronium benefited ventilated OP- poisoned patients. However, as most patients were poisoned by profenofos which persisted, further research is required to assess whether this approach might work for OP insecticides that are more rapidly eliminated from the body.
Keywords: organophosphorus insecticide, rocuronium, randomised controlled trial, self-poisoning, intubation and ventilation
Fluoxetine affects midbrain neuronal specification and neurogenesis in vitro (#292)
D. Lupu1, M. Varshney2, I. Nalvarte2, J. Insunza2, F. Loghin1, J. Rüegg3, 4
1 Iuliu Hatieganu University of Medicine and Pharmacy, Faculty of Pharmacy, Dept. of Toxicology, Cluj-Napoca, Romania
Recent meta-analyses found an association between prenatal exposure to the antidepressant fluoxetine (FLX) and an increased risk of autism in children. This developmental disorder has been related to dysfunctions in the brains’ rewards circuitry, which, in turn, has been linked to dysfunctions in dopaminergic (DA) signalling. This study aimed to investigate if FLX affects processes involved in dopaminergic neuronal differentiation as a possible mechanism underlying this link, possibly via interference with the estrogen system.
Mouse neuronal precursors (wild-type (WT) and estrogen receptor β knock-out (BERKO)) were differentiated into midbrain dopaminergic precursor cells (mDPCs) and concomitantly exposed to clinically relevant doses of FLX. Subsequently, dopaminergic precursors were evaluated for expression of differentiation and stemness markers using qPCR.
In WT cells, FLX treatment led to a significant increase in early regional specification markers Orthodenticle homeobox 2 (Otx2) and Homeobox engrailed-1 and 2 (En1 and En2). On the other hand, two transcription factors essential for midbrain dopaminergic (mDA) neurogenesis, LIM Homeobox transcription factor 1 alpha (Lmx1a) and Paired-like homeodomain transcription factor 3 (Pitx3) were significantly down-regulated by FLX treatment. The stemness marker Nestin (Nes) was significantly increased, whereas the neuronal differentiation marker β3-tubulin (Tubb3) was significantly decreased. Additionally, we observed a significant down-regulation of the expression of the estrogen receptors (ERs) α and β after FLX treatment. Further investigations using BERKO neural precursor cells showed that FLX had no or even opposite effects on the genes involved in mDPC specification and on Nes expression.
These findings suggest that FLX affects the differentiation of the dopaminergic system by increasing production of dopaminergic precursors, yet decreasing their maturation, partly via interference with the estrogen system. Further studies are needed to link these molecular events to development of the dopaminergic system and address if these findings could partly underlie the association between prenatal FLX exposure and increased autism risk in children.
Keywords: fluoxetine, midbrain, dopaminergic, in vitro, developmental
In vitro Cardiotoxicity evaluation on hiPSC using phenotypic analysis (#312)
A. Perrier1, B. Postic1, M. Martins1, L. Rutault2, A. Buvat2, N. Bécourt-Lhote2, N. Claude3, H. Aerts1
1 Servier group, In vitro toxicology, Gidy, France
A large percentage of drugs fails in late development due to cardiac toxicity. Therefore, research has been focused in elucidating such toxicities at early stages of drug discovery, helping decision making process, reducing late attrition, laboratory animal use and drug development costs. To improve the early prediction of this serious adverse event, we developed in vitro tests for the detection and prediction of human cardiac toxicity using High Content Analysis (HCA) on human iPSC cardiomyocytes.
Twenty reference compounds with known adverse effects on human heart via various mechanisms (global toxicity, oxidative stress, cytoskeleton toxicity, steatosis, phospholipidosis) and ten non cardiotoxic drugs as negative controls were tested on human iPSC cardiomyocytes. Specific targets were evaluated with appropriate stainings and quantified using HCA after 24 h or 72 h of exposure to 8 concentrations of each compound. Treatments were performed between 6 and 8 days after seeding, based on ultra-structural maturity observed after immuno-histochemical stainings. All the readouts were then qualified using Receiver Operating Characteristic (ROC) curves and a ranking algorithm was developed to balance each specific toxicity in an overall multiparametric evaluation for each compound.
Based on the developed ranking algorithm, the sensitivity and specificity of HCA cardiotoxicity tests were 0.75 and 1.00 respectively, showing a good predictivity of the model toward human cardiac adverse effects. This cardiotoxicity phenotypic analysis will be used for compound screening and early safety evaluation in the drug development process. ROC AUC were 0.87, 0.61, 0.64, 0.71, 0.72 and 0.93 for global toxicity, oxidative stress, actin, tubulin, steatosis and phospholipidosis, respectively, showing that such specific tests could be also used as mechanistic tools to document a specific cardiac signal emerging at later stages of drug development.
Keywords: cardiotoxicity, hiPSC, phenotypic analysis, High Content Analysis, 3Rs
Impact and Necessity of a Kinase Safety Strategy for CNS Indications (#321)
M. Lemper1, C. Schramm1, R. Class1, K. Tilmant1, J. Neres3, D. Swinnen2, M. Geraerts3, V. De Wever3, I. Melas4, M. Camargo5, Z. Sands6, A. Valade2, G. Cebers1, J. - P. Valentin1
1 UCB, Development Science, Nonclinical Safety, Braine l'Alleud, Belgium
M. Lemper*, C. Schramm*, R. Class, K. Tilmant, J. Neres, D. Swinnen, M. Geraerts, V. De Wever, I. Melas, L. M. Camargo, Z. Sands, A. Valade, G. Cebers and J.P. Valentin
The development of kinase inhibitors for neurology indications is relatively new and demands a stringent safety profile. A cross-functional approach is needed to provide a global strategy to assess kinase selectivity and potential safety issues. Ideally the first step should be identifying target features that enable selectivity by design. This requires the availability or creation of crystal structures of ideally both target and related kinases. Once structure enabled approaches can be exploited, compounds with improved profiles are synthesized and the arising potent inhibitors should be screened in kinase selectivity panels. Unified kinase profiles and data analysis across projects enable a more holistic view of the relationship between kinase selectivity, potency and toxicity of the compounds. Tools to correlate adverse events to certain off-target kinase hits and pathways involved are extremely valuable to determine a path forward. At this point off-target profiles should be revisited to identify common kinase hits, build a deeper understanding of their safety profile and their potential impact on the program. Additional SAR cycles should be undertaken to guide chemistry to avoid unwanted kinase hits. Only once sufficient selectivity has been achieved, an early in vivo toxicology study should be performed to inform further developability of the compound. Important to note is that early in vivo toxicology studies with non-selective compounds are not useful considering the difficulties deconvoluting in vivo adverse effects to off-target kinases. This global safety strategy aims to enable the development of kinase inhibitors with an acceptable risk-benefit profile for neurology indications.
Keywords: Kinase inhibitors, safety strategy, selectivity, CNS
Evaluation of 3D-culture methods for the hepatic differentiation of human skin-derived stem cells (#363)
A. Natale1, K. Vanmol2, A. Arslan3, S. Van Vlierberghe2, 3, P. Dubruel3, J. Van Erps2, H. Thienpont2, J. De Kock1, V. Rogiers1, R. M. Rodrigues1, T. Vanhaecke1
1 Vrije Universiteit Brussel (VUB), In Vitro Toxicology and Dermato-Cosmetology (IVTD), Brussels, Belgium
Human skin precursors (hSKPs) are adult stem cells that can be easily isolated from small skin segments. These cells display a multi-lineage differentiation potential and high plasticity due to their ability to expand in vitro and to differentiate in all three germ layers. Our lab demonstrated that hSKP can differentiate towards hepatic cells in a two-dimensional (2D) set up by sequential exposure to growth factors involved in liver development. However, the obtained hepatic cells (hSKP-HPC) show a mixed phenotype of immature hepatic progenitors and mature hepatocytes. Traditional 2D in vitro systems rely on single adherence to flat substrata, contributing to the loss of cellular morphology, polarity and functionality. In this study we evaluate whether three-dimensional (3D) culture strategies can further improve the hepatic differentiation of hSKP-HPC and better replicate the properties of hepatocytes in vivo. Hence, a 3D-nanoprinted scaffold is developed using two-photon polymerization, which is an advanced optical fabrication technology that allows high-precision polymerization of photopolymers into structures with any desired geometry. Scaffolds with interconnecting baskets of cuboidal or hexagonal shape and correct hepatocellular dimensions were fabricated, mimicking the 3D-orientation of hepatocytes in vivo. Integration and distribution of hSKP in these scaffolds was confirmed by scanning electron microscopy and confocal microscopy. A microfluidic device, on which the scaffolds will be printed to ensure efficient transportation of nutrients is being tested in parallel. hSKP-HPC seeded directly on this chip were cultured under dynamic flow conditions and acquired a more polygonal morphology after few days in culture. Furthermore, preliminary data suggests that hSKP-HPC cultured under perfusion exhibit a tendency to store more glycogen than cells cultured in static conditions. By combining the 3D-scaffold into a microfluidic device, we hope to generate a liver-on-a chip device that accurately recapitulates hepatic functionality and could potentially be used for hepatotoxicity testing.
This work was supported by grants from the ‘Chair Mireille Aerens for alternative methods development’ and the Leefmilieu Brussel (BIM).
Keywords: Hepatic differentiation, microfluidics, 3D -scaffold
Evaluation of Zebrafish as a model for neurotoxicity early-screening (#610)
J. - M. Girard1, V. Gervais1, N. Rogez2, N. Ribeiro-Palha2, A. Dekeyne2, N. Bécourt-Lhote1, N. Claude1
1 Servier group, Toxicology department, Gidy, France
Challenges await in an era dedicated to the Reduce, Refine, Replace policy on animal experimentation and the need, from basic research to drug screening and lead optimization, for suitable models. As for the later, early neurotoxicity drug screening is based on cell-based in vitro assays, but results can only go so far as to yield basic information on the toxic effect, missing the big picture. Effects of a drug on the complex neuronal networks, accessing to the global consequences needs to be evaluated through an additional step, moving to expensive low throughput animal models. Consequently, neurotoxic effects are rarely used during early screens and neurotoxic effects would only be characterized at later stages of drug development, involving important losses if a drug had to be abandoned. Zebrafish larvae, an in vivo model, could fulfill that gap. At first glance, it seems far-fetched to seek a human counterpart in a fish but we are both vertebrates and 70% of protein-coding human genes are related to genes found in the zebrafish. This animal model have been used for over a decade and led to discoveries in heart diseases, cancer research, organ developments and many more. The focal point of this study is to set up a medium-throughput method to meet the need for pharmaceutical industry during early-stage drug screening and issue warnings on potential adverse neurological effects, rather than go/no go. We will show how a simple carefully set protocol and appropriately analyzed results can provide a deep insight into various drugs neurological issues.
Keywords: Zebrafish, neurotoxicity, screening
Development of high-throughput liver-on-a-chip for drug toxicity screening (#623)
K. Wilschut1, R. J. Annida1, Z. Wang1, C. Ng1, V. Shevchenko2, C. Chesne2, J. Joore1
1 Mimetas BV, Leiden, Netherlands
In vitro tissue models have been used for decades as a tool to understand the mechanism of the drug toxicity. However, the current 2D and most of the 3D models lack the physiological relevance and tissue complexity to that of the in vivo situation. Better predictive in vitro screening models early in the drug discovery pipeline are critically required to increase success of drug candidates, reduce development costs of new medicines, and reduce animal studies. Here, we developed a 3D liver-on-a-chip model comprised of HepaRG and primary human hepatocytes using the OrganoPlate® platform. This microtiter-based platform consists of 40 to 96 individual microfluidic chips supports the development of an human hepatocyte co-culture with hepatic non-parenchymal cell in a membrane-free and pump-free perfusion system. Besides having long-term high viability, this liver-on-a-chip model also shows liver functionalities such as synthesis of albumin, detoxification of ammonia, formation of bile acid and metabolism of drugs by cytochrome P450. Next to that, this model is applicable to determine concentration-dependent cytotoxicity and mitochondrial dysfunction upon the exposure to acetaminophen drug. The hepatic cultures show polarization leading to the formation of rhythmically contracting bile canaliculi that are able to disrupted upon the exposure of the cholestatic drug, a Y-27632 ROCK inhibitor analogue, fasudil. Moreover, this model also shows the accumulation of lipids after exposed to the steatosis-inducing drug cyclosporine A. These findings indicate that this model is suitable for understanding compound-specific hepatic toxicity and applicable for disease-modelling studying drug efficacy.
Keywords: organ-on-a-chip, liver toxicity
High content in vitro assessment of chemically induced liver injury: Lipid accumulation and mitochondrial dysfunction (#685)
F. A. Müller1, M. Stamou1, S. Sturla1
1 ETH Zürich, Department of Health Science and Technology, Institute of Food, Nutrition and Health, Zürich, Zürich, Switzerland
Non-alcoholic fatty liver disease (NAFLD) encompasses a complex disease spectrum ranging from benign steatosis to non-alcoholic steatohepatitis, which can progress to cirrhosis and liver failure. The prevalence of NAFLD and its link to obesity and metabolic disorders suggests that environmental exposures could contribute to NAFLD. Systematic assessments of chemicals as NAFLD risk factors have not been carried out in part due to a lack of validated in vitro testing strategies. In order to address this gap we developed a high content imaging assay that allows quantification of mechanism-based NAFLD markers in metabolically competent human liver cells. HepaRG cells were treated in vitro with increasing concentrations of compounds, selected on their mechanism of action involved in NAFLD pathophysiology including free fatty acids, T0901317 (liver-X-receptor agonist) and rotenone (complex I inhibitor in mitochondrial respiratory chain). Cells were labeled with a fluorescent lipid dye, a fluorescent mitochondrial dye, and with a marker for cell nuclei, and imaged on a high content imaging system. An algorithm was developed for automated image processing and lipid droplet number, size and intensity together with mitochondria number and intensity were quantified. The results indicated a time and concentration dependent increase in lipid accumulation. Data concerning mitochondrial dysfunction emphasized a concentration dependent disruption of mitochondrial function and integrity. Future research will aim to integrate additional endpoints into a high throughput and high content screening assay that recapitulates the cellular hallmarks of NAFLD.
Keywords: Non-alcoholic fatty liver disease, High Content Imaging, HepaRG, in vitro toxicology, hepatotoxicity
Oleanolic acid: a potential chemotherapeutic agent weakened by P-glycoprotein (#689)
M. Kayouka1, E. Saliba1, 2, D. Landy2, H. Greige1
1 Lebanese University, Chemistry and Biochemistry department, Fanar, Lebanon
Oleanolic acid (OA) is a triterpenoid, widely found in several plants and has been reported to possess numerous biological activities, including hepatoprotective, anti-inflammatory and anticancer activities. In this study, the cytotoxic effect of OA was evaluated on two cancer cell lines: human breast cancer MDA-MB-231 and human liver cancer HepG2, using the trypan blue exclusion test and the MTT assay.
Results showed that OA reduced breast cancer cell viability. After 72 h of treatment, only 28.4 % of cells remained viable at 60 μM. Whereas, no remarkable effect was observed on liver cancer cells, with 63.74 % cell survival at 60 μM. Moreover, the cytotoxicity studies have been conducted in the presence of verapamil, a P-glycoprotein (P-gp) inhibitor, to check if OA is a substrate of P-gp. OA exhibited the same effect on breast cancer cells in the presence of verapamil. However, the cytotoxicity was greatly enhanced for HepG2 cells, in the presence of verapamil (cell viability was 24.97% after 72 h at 60 μM). The difference obtained between MDA-MB-231 and HepG2 is possibly due to the fact that liver cancer cells express high levels of P-gp RNA, while low or undetectable levels were found in breast cancer cells. These results demonstrate that OA is a potential chemotherapeutic agent, but its action is weakened by the multidrug resistance protein, P-gp.
Keywords: Oleanolic acid, Cancer, P-glycoprotein, MDA-MB-231, HepG2
Drug transporter expression in non-parenchymal liver cells in comparison to hepatocytes (#697)
C. Joseph1, V. Rönnpagel1, A. Ullrich2, D. Runge2, M. Grube1
1 Universitätsmedizin Greifswald, Institut für Pharmakologie, Greifswald, Germany
Primary hepatocyte cultures have been long used as standard in-vitro drug screening models followed by in-vivo screening and clinical trials. However, only 5-10 % drug candidates finally get through the clinical trials. One reason for this poor prediction value was that the primary hepatocyte cultures alone do not mimic the native in-vivo environment in the liver and moreover, a rapid decrease of hepatic expression profile occurs in vitro over time. Hence, in order to improve drug screening in-vitro models, not only hepatocytes but also non-parenchymal liver cells (NPC) are used currently in coculture approaches. In the present study, we characterized these NPCs (mainly kupffer and endothelial cells) in comparison to hepatocytes concerning the expression of hepatic drug uptake and efflux transporters as important factors affecting drug elimination and toxicity in the liver.
We, therefore, isolated hepatocytes and NPCs from rat and mice and characterized the respective cells for cell type-specific markers on mRNA level. In addition, we further separated the murine NPC fraction into endothelial and kupffer cells by a magnetic sorting method using an antibody against Clec4f to mark kupffer cells and against Tie1 for endothelial cells. Following which, the mRNA expression of about 40 drug uptake genes (SLC- and SLCO-family) and efflux (ABC-family), as well as cell type-specific marker genes, were analyzed using a TaqMan low-density array.
As expected, hepatocyte-specific transporters like Ntcp (slc10a1), Bsep (abcb11) and Mrp2 (abcc2) were specifically expressed in hepatocytes. In contrast, transporters like Cnt3 (slc29a1) and Oatp2a1 (slco2a1) were found in each fraction in a comparable level, while others including Mrp1 (abcc1), Mrp3 (abcc3), Mrp4 (abcc4) and Octn2 (slc22a5) were detected in significantly higher amounts in NPCs, with differential expression patterns in both kupffer and endothelial cells as compared to hepatocytes. For selected transporters (e.g. Octn2) this data was confirmed at the protein and functional (carnitine uptake) levels.
In the present study, we were able to find the expression of clinically relevant drug transporters in NPCs. Interaction of drug/drug candidates with these transporters may partly explain hepatotoxic effects, which were previously not observed by focusing on only hepatocytes.
Keywords: Non-parenchymal cells, Hepatocytes, drug transporters
Toxicological evaluation of the interaction between circadian rhythm activators and general anesthetics (#702)
A. Aydın1, F. Kelleci1, M. Hamitoğlu1
1 Yeditepe University Faculty of Pharmacy, Toxicology, Istanbul, Turkey
The circadian rhythm lasts about 24 hours and is constituted by 4 main genes/proteins; Circadian Locomotor Output Cycle Clock (Clock), Brain-Muscle-Arnt-Like protein-1 (Bmal1), Cryptochrome (Cry) and Period (Per). Many pathologies, diseases, and medications including general anesthetic agents are thought to cause a change in these genes/proteins. On the other hand the activation or inhibition of the genes/proteins can contribute to the reduction of the oxidative stress potential of anesthetic agents. We investigated the effect of combination therapy of KL001 with isoflurane. Twenty-four mice were randomly divided into 4 groups of 6 animals each as a control, KL001, isoflurane and KL001 plus isoflurane group. Animals were exposed to isoflurane for 4 hours and KL001 was administered of 100 mg/kg at night hours. The Cry levels and oxidative stress parameters including malondialdehyde (MDA) level and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activities were investigated in liver, kidney, brain, and erythrocyte. Our results showed decreased MDA level in erythrocyte and liver, increased GSH-Px activity in liver and brain, increased SOD activity in erythrocyte, kidney, and brain, decreased CAT activity in liver in isoflurane group. According to our findings, isoflurane changed the oxidative stress parameters remarkably in brain. We also found a decrease of Cry level in the plasma, liver, and brain in isoflurane group. KL001 increased Cry level inhibited by isoflurane and decreased the isoflurane-induced oxidative stress. In conclusion, our data indicated that we can reduce the potential oxidative stress of isoflurane or other anesthetic agents by triggering the Cry level with a synthetic activator or by arranging drug administration time at certain period of the day. Especially in early hours of the morning, KL001 may be protective against isoflurane-induced oxidative stress. In further research, the effects of Per, Clock, and Bmal1 on anesthetic agents toxicity can also be investigated. It can be determined at which time the operations are performed to create the least toxicity.
Keywords: Biological clock, isoflurane, cryptochrome, KL001
In vitro antiproliferative effects of Fe3O4_BA loaded liposomes (#719)
C. G. Farcas1, 2, E. A. Moaca2, D. Coricovac2, C. Dehelean2, F. Loghin1
1 "Iuliu Hatieganu" University of Medicine and Pharmacy, Toxicology, Cluj-Napoca, Romania
Betulinic acid (BA) is a phytocompound which has been reported to exhibit multiple pharmacological activities, such as: antitumoral effect with selectivity on tumor cells, anti-inflammatory, antioxidant, antimalarial and antimicrobial properties. However, BA is a highly hydrophobic compound. This property limits its bioavailability and makes it imposible for in vivo administration.
The present study proposes a new formulation of BA as Fe3O4_BA loaded liposomes, which might improve its membrane permeability. Fe3O4 nanoparticles serve as carriers and as a method to augment BA efficacy by making the liposomes suitable for hyperthermia therapy.
The liposomes containing magnetic nanoparticles (Fe3O4) and BA were synthetized by thin film dispersing method and characterized by standard methods, such as: FTIR, DSC, zeta potential, TEM (transmission electron microscopy) in terms of active compound content, size and stability.
The cytotoxic effects of the novel liposomes was evaluated on human healthy and breast cancer cell lines (MCF7, MDA-MB-231) by MTT and LDH assays.
The Fe3O4_BA loaded liposomes obtained were stable and with the sizes in the range recommended for biological use. Moreover, these liposomes exhibited a higher antitumor activity when compared with BA liposomes and BA in dimethylsulfoxide, what indicates a synergistic effect of BA and Fe3O4 formulated as liposomes.
These data will be used as background for further in vivo studies to demonstrate their efficacy as antitumor agents in breast cancer animal models.
This research was supported by a grant of “Iuliu Hatieganu” University of Medicine and Pharmacy, project number 1300/23/13.01.2017.
Keywords: betulinic acid, thermosensitive magnetoliposomes, hyperthermia, breast cancer
Evaluation of the cytotoxicity of cocaine and adulterants in precursor cells of the glia (#728)
R. A. Rocha1, J. Figueroa1, R. Manzo2, D. Diaz2, A. Vasquez2, D. Soto2, B. Duffau1, I. Triviño1
1 ISP, Análisis de Ilícitos, Santiago, Chile
Cocaine hydrochloride is a highly addictive illicit drug, which generates harmful effects on the health of population, specifically in the nervous system. A high percentage of the cocaine consumed in our country is adulterated with substances such as caffeine or levamisole. In relation to these adulterants substance it is unknown if such substances are less or more harmful than cocaine itself. The objective of this study has been to evaluate the cytotoxicity of cocaine and adulterants using the line human fetal glial cells from brain SVG-P12, through the use of viability, apoptosis and cell necrosis assays.
The results show that the exposure to cocaine for 24 hours (≥ 5 mM) generates a decrease in cell viability and an increase in apoptosis and cell necrosis (≥ 0.5 mM). With respect to tested adulterants, a decrease in cell viability was observed due to the exposure of caffeine (≥ 15 mM) and levamisole (≥ 7.5 mM);observing that adulterants produce a decrease in cell viability at higher concentrations than cocaine. In the same way as for cocaine, an increase in apoptosis and cell necrosis was observed (dose-dependent) for caffeine and levamisole (≥ 2 mM).
From this study it can be concluded that the harmful effects of cocaine in precursor cells of the glia occur at lower concentrations, than levamizol and caffeine, and that it is of interest to evaluate the effects of the mixtures of these drugs.
Keywords: Cocaine, levamisole, caffeine, in vitro, cells of the glía
Maternal smoking and changes in drug metabolising enzymes in the human fetus (#741)
A. Zafeiri1, P. Filis1, U. Soffientini2, B. Lucendo-Villarin3, A. Douglas4, S. Shaw5, J. Iredale6, M. J. Swortwood7, M. A. Huestis8, M. Bellingham2, R. T. Mitchell9, D. C. Hay10, P. J. O'Shaughnessy2, P. A. Fowler1
1 University of Aberdeen, School of Medicine, Medical Sciences & Nutrition, Aberdeen, United Kingdom
The placental feto-maternal interface allows for most drugs to freely diffuse and reach the developing fetus. Major phase I and II metabolising enzymes are expressed in the human fetal liver which is the primary site for metabolism of drug compounds. Maternal smoking passes ~7,000 toxicants to the fetus, and is associated with multiple adverse consequences for peri/post-natal health and wellbeing in the offspring. Disruption of the drug metabolising machinery in the fetal liver also poses risks for drug toxicity. This study analysed drug metabolising enzymes (DME) expression levels in smoke-exposed human fetuses.
80 human fetal livers from elective terminations of normal pregnancies, at 12-19 weeks of gestation, were collected ((NHS) Grampian Research Ethics Committees (REC 04/S0802/21)). RNA was extracted, prepared for sequencing, and the Illumina NextSeq platform was used to produce 76 bp single end RNA sequencing reads. Reads were quality controlled, aligned to the human reference genome and quantified at gene regions. Statistical analyses used a generalised linear model with a three-way interaction between fetal sex, fetal age and validated maternal smoking status.
The transcript levels of human DMEs were examined via data-mining our RNAseq data. Compared with fetuses from non-smoking mothers, smoke exposure is associated with: (1) male-specific decrease in CYP3A4 at ≥16 weeks, (2) male-specific increase in UGT2B7 at 11-13 weeks, (3) female-specific increase in CYP2E1 at 14-16 weeks, followed by a decrease at 17-20 weeks. CYP3A4 is a major cytochrome p450 superfamily member metabolising drugs such as paracetamol and aspirin. CYP2E1 substrates include permutagens found in cigarette smoke and paracetamol. UGT2B7 is a UDP-glucuronosyltransferase involved in the conjugation and elimination of ibuprofen metabolites. These findings are being confirmed using 260 human fetal livers.
Disruption in the expression of these DMEs could contribute to variations in major drug metabolism and detoxification processes. Identification of the exact point/s of the effect would further our current knowledge of how toxicants can disrupt the human fetal liver and provide critical data for the development of effective drug compounds for the mother that pose minimal risks for the fetus.
Keywords: maternal smoking, drug metabolising enzymes, human fetal liver
Development of a human autologous 3-cell cytokine release assay that models the vascular wall in vitro (#53)
R. Kawai1, 2, B. Ahmetaj-Shala1, C. C. Shih1, 3, I. Marei1, K. Bhatti1, N. S. Kirkby1, J. A. Mitchell1
1 Imperial College London, National Heart and Lung Institute, London, United Kingdom
The current gold-standard method for identification and characterization of cytokine release assay for biologics use only peripheral blood mononuclear cells (PBMCs) with immobilized biologics to plastic. This platform is used as a surrogate for the commonly required binding of the Fc region of biologics, such as TGN1412, to receptors on endothelial cells in the blood vessel. Here we describe a novel in vitro cytokine release assay using autologous 3 types of important vascular cells in a co-culture system. The assay consists of (i) PBMCs, (ii) blood outgrowth endothelial cells (BOECs), and (iii) blood outgrowth smooth muscle cells (BO-SMCs). Since cytokine storm co-culture assays to date have relied on the use of human cells from different donors, the novel concept of obtaining differentiated cells from blood progenitors allows for autologous cells to be used.
We have now obtained data demonstrating that BOECs and BO-SMCs express the requisite endothelial CD31 and smooth muscle cell a-SMA cell markers, respectively, without expressing fibroblast marker, S100A4. Under control culture conditions mono-cultures of PBMCs, BOECs and BO-SMCs released relatively low levels of cytokines which were increased when each cell type was stimulated individually with LPS. However, as the 3-cell-type co-culture system was built up by combining the cells in individual wells, LPS stimulation was amplified compared to responses of cells in mono-culture.
Whilst the relevance and utility of this assay to a broad spectrum of therapeutic antibody testing is unclear we suggest that it will be useful for testing both efficacy and toxicity of cytokine-storm potential in biological therapies designed to target blood vessels and cardiovascular disease.
Keywords: TGN1412, Cytokine release assay, BOECs, BO-SMCs, LPS
Development of Highly Sensitive and Specific in vitro Renal Solute Carrier (SLC) Uptake Cell Models Using Normal Human Adult Renal Proximal Tubule Epithelial Cells for Drug Transporter Interaction Studies (#102)
R. Menth1, C. Zou1, L. Romero1, E. Turner1, K. Huang3, A. Gibson3, P. McWilliams-Koeppen1, B. Chase2
1 ATCC Cell Systems, Gaithersburg, United States of America
The disposition and clearance of drugs by the kidney relies largely on a well-characterized subset of membrane transport pumps known as solute carrier (SLC) proteins. Among the SLC family proteins, OAT1, OCT2 and OAT3 are considered the most important transporters in kidney tissue, and are recommended by the FDA, ITC and the EMA as targets for drug-drug interaction studies. Therefore, there is a need for in vitro kidney transporter models that have normal human kidney origin, functioning transporters, clinical predictability, and consistent data output for drug interaction studies. Unfortunately, primary renal epithelial cells lose OAT1, OCT2 and OAT3 transporter expression quickly in culture. Transiently expressing these transporters in primary renal epithelial cells yields large variations between experimental models making data hard to interpret. In our study, we have generated kidney transporter cell models using well-characterized hTERT-immortalized primary Renal Proximal Tubule Epithelial Cells (RPTECs) that stably overexpress either the OAT1, OCT2 or OAT3 gene. After confirming the SLC mRNA expression for each gene by RT-PCR, we performed immunostaining that showed that OAT1, OCT2 and OAT3 are correctly trafficked to the plasma membrane. Notably, those clones show typical epithelial morphology, functionality, and expression of the appropriate epithelial and kidney tissue specific markers. Most importantly, we verified that the overexpressed transporters have normal transport activities using 6-CF and EAM-1 uptake assays in a high throughput format. We also show that uptake of these compounds are blocked in a dose dependent manner by well-known SLC inhibitors. Overall, our data demonstrates that these modified RPTECs maintain kidney transporter expression over time, and provide physiologically relevant, highly sensitive and specific data regarding human kidney transporter functions, particularly for high throughput kidney drug toxicity screening
Keywords: in vitro model, Renal Proximinal Tubule Epithelial Cells, Drug Transport, kidney
Real-time cell analysis of the cytotoxicity of γ-ray radiation and the radioprotective effect of TLR5 agonist (#134)
T. Shi1, R. Zhang1, X. Chen1, C. Wang1, H. Jiang1, L. Li1
1 /, State Key Laboratory of NBC Protection for Civilian, Beijing, China
PURPOSE: The aim of this study was to establish a method to evaluate the cytotoxicity of γ-ray radiation and the radioprotective effect of TLR5 agonist in vitro. METHODS: To evaluate the cytotoxicity of γ-ray radiation, HEK293 cells were irradiated by 60Co-γ ray, and then cells were re-plated into E-plate (5,000 cells per well) depending upon radiation dosage and cell viability was measured with the real-time cell analysis (RTCA) system for 120 h. To evaluate the radioprotective effect of TLR5 agonist, HEK293-N-T cells, that over express human TLR5, were generated by lentiviral transfection of HEK293 cells. The HEK293-N-T cells were treated with different doses of CBLB502 (0, 0.8, 4, 20, and 100 ng/ml, respectively) at 6 h prior to 16 Gy radiation. After irradiation, 5,000 irradiated cells of different groups were seeded in E-plate respectively and real-time monitored for 120 h. Normal cells without any treatment were used as a control. Data was analyzed for significant differences between experimental groups by performing Analysis of Variance (ANOVA) with Tukey’s post hoc multiple comparison test. RESULTS: (1) After 12 h of irradiation, HEK-293 cells were showed reduced cell viability (compared with the control group), and the damage effect of irradiation on cell vitality enhanced with the increase of radiation dose, showed obvious dose-response relationship (p<0.05). (2) 12 h after exposure, irradiation induced a gradually decline in the cell surviving rate. The Cell Index (CI) value of irradiated cells without CBLB502 treatment reduced to zero at 68 h after IR exposure, which indicated that no cells adhered to the microelectrodes. Pre-treatment with CBLB502 of different concentrations have shown improved viability of irradiated cells in a dose-dependent manner (p<0.05). CONCLUSIONS: The present study established a RTCA-based method for assessing the cytotoxicity of γ-ray radiation and the radioprotective effect of TLR5 agonist in vitro by real-time measurements. In contrast with the evaluation method of living animals, this method has many advantages, such as low sample usage, high throughput and short time. Thus, the method developed here could be adopted for the screening and evaluation of new radioprotectants.
Keywords: γ-ray, cytotoxicity, CBLB502, radioprotective effect, RTCA
Real-time monitoring of oxygen consumption of hepatocytes in a microbioreactor (#146)
K. Uhlig1, C. P. Gehre1, S. Kammerer2, J. - H. Küpper2, D. Coleman3, G. Püschel3, C. Duschl1
1 Fraunhofer IZI-BB, Department of Molecular and Cellular Bioanalytics, Potsdam, Brandenburg, Germany
An important strategy for the improvement of toxicity testing is based on the development of approaches that provide information on molecular mechanisms at the cellular level. Such information is crucial for a fundamental understanding of the effects of toxic compounds. However, from most methods currently available, detailed insights are difficult to obtain as they rely on end-point measurements. This approach has considerable limitations as it does not provide dynamic information as obtained by real-time measurements.
Recently, together with others we developed a novel microbioreactor [1,2]. In this reactor, cells are continuously perfused with medium and it has been demonstrated that hepatocytes embedded in a collagen matrix in microwells can be cultivated for one month. As an important novelty, we inserted sensor microbeads together with cells into the microwells. These microbeads contain chromophores, whose phosphorescence life time depends on the local oxygen concentration. By carefully controlling the oxygen intake through the medium in respect to the number of cells embedded in the microwells, we are able to continuously measure the oxygen consumption of the cells through a very robust optical detection scheme. This allows the metabolic activity of cells in the reactor to be noninvasively monitored over weeks with high temporal resolution under near-physiological conditions.
We will present results that demonstrate that mitochondrial dysfunction of hepatocytes can be distinguished from other cellular response upon exposure of the cells with acetaminophen. We compare measurements obtained from primary mouse hepatocytes, hepatocytes that are expanded form primary cells using the upcyte® technology and hepatocytes from the cell line HepG2. In addition, we show how we increase throughput by parallelisation.
 S. Prill et al., Archives of Toxicology 90 (2015) 1181.
 D. Bavli et al., PNAS 113 (2016) E2231.
Keywords: toxicity testing, oxygen sensing, microbead sensors, acetaminophen, metabolic activity
Primary human hepatocyte spheroid generation and performance in different culture systems (#165)
S. Buesch1, M. Bunger2, J. Schroeder1, M. Stosik1
1 Lonza Cologne GmbH, Cologne, North Rhine-Westphalia, Germany
In the field of toxicology, there is a strong need for in vitro human liver model systems that support the long-term culture of primary human hepatocytes and closely mimic in vivo conditions of liver tissue. Compared with 2D culture, spheroids generated out of primary human hepatocytes allow for more in-vivo-like cell organization, including extensive contact of adjacent cells. Agitation on a shaker can imitate the movement of fluids within the body at least to some extent. In this study, we analyzed and optimized the formation, culture and performance of primary human hepatocyte spheroids in different cell culture systems and under various culture conditions.
We used different methods for the formation of primary human hepatocyte spheroids, including ultra-low attachment V-bottom and U-bottom plates as well as hanging drop culture. Different amounts of primary human hepatocytes were seeded with or without serum supplementation and cultured with or without shaking for up to 7 days until spheroid formation occurred. We characterized specific hepatocyte functions of the resulting spheroids immediately after spheroid formation and during following weeks of culture. The spheroids were analyzed for cell viability, bile canaliculi formation, albumin secretion and CYP3A4 activity.
Successful spheroid generation without addition of any supporting cell types was observed. This was independent of the culture vessel type used. The presence of serum was required for spheroid formation, both under gentle agitation and without shaking. Spheroids displayed typical hepatocyte activity, including sustained albumin production, basal CYP3A4 activity, CYP3A4 inducibility and bile canaliculi formation within spheroids.
We developed simple protocols for the successful in vitro generation of primary human hepatocyte spheroids in a variety of culture systems. We demonstrated that standard analysis methods like cell viability or cytochrome P450 enzyme activity assays can be easily applied to spheroid culture. The resulting in vitro spheroid cultures offer more in vivo like conditions for long term exposure and repeated dosing toxicology studies.
Keywords: hepatocytes, liver, spheroids, in vitro, microtissue, cytochrome P450
In vitro cytotoxicity assessments of persistent organic pollutants using cetacean fibroblasts (#178)
M. Ochiai1, N. Kurihara2, A. Matsuda3, S. Nakagun4, A. Shiozaki5, A. Nakata6, T. Matsuishi3, T. Kunisue1, H. Iwata1
1 Ehime University, Center for Marine Environmental Studies, Matsuyama, Ehime, Japan
Cetaceans accumulate high levels of persistent organic pollutants (POPs). Known effects of POPs include endocrine disruption, immune dysfunction and behavioural changes. In this study, we established an in vitro assay using fibroblasts derived from cetaceans, and evaluated the toxicological risks of POPs at the cellular level. Fibroblasts were cultured from six cetacean species (finless porpoise, harbour porpoise, bottlenose dolphin, Risso’s dolphin, killer whale and Ginkgo-toothed beaked whale) that were stranded or bycaught along the Japanese coasts. The blubber tissue was also collected to determine the concentrations of POPs. Seven compounds including 2,3,7,8-TCDD, CB126, CB118, CB138, CB153, CB187 and p,p'-DDE were exposed to the cells at a serially diluted concentration set of 7–8 doses, and three parameters of cellular responses (cytotoxicity, apoptosis and reduced viability) were measured. The total concentrations of POPs were found in the following descending order: killer whale > finless porpoise > Risso's dolphin > Ginkgo-toothed beaked whale ≈ bottlenose dolphin ≈ harbour porpoise. The killer whale accumulated the highest concentration of DDTs (45 μg/g lipid weight). On the other hand, higher PCB concentrations were found in the finless porpoise (21 μg/g lipid weight), which were 1–2 orders of magnitude higher than in other species. In vitro assays revealed that 2,3,7,8-TCDD and dioxin-like PCBs (CB126 and CB118) had higher cytotoxicity potentials in all the cetaceans tested. Among the six cetaceans, the finless porpoise had the highest EC50 values for all the POPs. Since the finless porpoise is a nonmigratory resident in semi-enclosed seas (e.g. Seto Inland Sea, Japan) and has chronically been exposed to higher levels of POPs, this species may have acquired resistance to the exposure to POPs. Additionally, blubber extracts including POPs were also exposed to the cells to examine the effects of POPs mixture. The percentage of cells showing cytotoxicity and apoptosis were high; 72% and 27% for the killer whale and 48% and 17% for the harbour porpoise, respectively. These results suggest that the accumulation of POPs induces cytotoxicity in cetaceans and the risk at the cellular level is of concern.
Keywords: in vitro, cytotoxicity, POPs, cetacean, marine mammal
Novel differentiation of human induced pluripotent stem cell-drived intestinal organoids (HiOs) for evaluation of intestinal fibrosis (#180)
D. Onozato1, T. Akagawa2, Y. Kida2, I. Ogawa2, T. Hashita1, 2, T. Iwao1, 2, T. Matsunaga1, 2
1 Nagoya city university, Department of clinical pharmacy, Graduate school of pharmaceutical sicences, Nagoya, Japan
Diverse functions of the intestine include absorption, metabolism, and immunity. Immune-related diseases can feature a complex pathology, such as intestinal fibrosis. Intestinal organoids can be established from human iPS cells, and recent advances in the culturing of organoids have led to the development of various 3D intestinal disease models with in vivo physiology. However, it is difficult to generate sufficient numbers of organoids for high-throughput studies using the current culture methods, and drug development applications are hindered by insufficient knowledge of the pharmacokinetic functions. In addition, the current in vitro or in vivo intestinal fibrosis models have many limitations. Here, we improved the culture of human iPS cell-derived intestinal organoids (HiOs), and explored the potential of HiOs as a model for intestinal fibrosis, and as a tool for screening anti-fibrotic drugs.
Human iPS cells were induced to differentiate into the midgut, and then were seeded on a patterning plate to form uniform spheroids. The floating spheroids were stimulated to differentiate into intestinal organoids using small molecule compounds. The intestinal organoids were treated with TGF-β1 and TNF-α to evaluate their phenotypic change.
We successfully generated organoid suspensions without scaffolds for easier harvest and use in assays. The expression of the intestinal markers and pharmacokinetics-related genes were markedly increased by the small molecule compounds. HiOs displayed polarized epithelium, and contained the various cells that constitute the small intestinal tissues. In addition, the HiOs exhibited P-gp efflux transport activity, CYP3A4 activity, and inducibility. TGF-β1 and TNF-α induced mRNA expression of mesenchymal and fibrosis markers in the HiOs. Immunofluorescence staining confirmed the epithelial-mesenchymal transition. In addition, caspase-3 positive cells were increased, the apoptotic cells were excreted in the lumen of intestinal organoids, and the epithelial cells were thickened. On the other hand, treatment with TGF-β RI Kinase Inhibitor, SB431542 suppressed these fibrosis responses in HiOs.
We were able to generate pharmacokinetically functional HiOs that are applicable for high-throughput studies. The HiOs could be useful to model intestinal fibrosis, and test the effectiveness of anti-fibrotic drugs.
Keywords: intestinal fibrosis, in vitro model, iPS cells, intestinal organoid
3D Spheroid Cultures from Human Astrocyte- and Neuronal- like Cells: New In Vitro Models to assess Magnetite Nanoparticle-Induced Adverse Effects on CNS (#194)
U. De Simone1, F. Caloni2, M. Roccio3, L. Gribaldo4, A. Spinillo3, T. Coccini1
1 ICS Maugeri SpA-BC, IRCCS, Laboratory of Clinical and Experimental Toxicology, Toxicology Unit, Pavia, Italy
Several lines of evidence demonstrate that nanoparticles (NPs) can translocate to brain and impact on the highly vulnerable central nervous system (CNS). The distribution of NPs in the bloodstream raises a particular concern of NP transfer from placenta to the fetal CNS, and since BBB develops gradually in the fetal brain, a direct exposure to NP in utero,that may have the most detrimental consequences. A recognized need is to combine nanotoxicology and neurology knowledge and calls for novel specific tools and investigation methods for this discipline including the integration of new in vitro models into safety assessment strategies.
In this study two types of CNS cell spheroids have been developed and optimised in combination with standard assays (viability readout and cell morphology) to test neurotoxic effects caused by magnetite NPs (Fe3O4NP), as NP-model, after short- (24-48h; 1-100 µg/ml) and long-term repeated exposure (up to 30 days; 0.1-25 µg/ml).
Three-dimensional (3D) spheroids were generated from human D384 astrocyte- and SH-SY5Y neuronal-like cells: they formed reproducible well-rounded spheroids, with homogeneous size distribution, viability and functionality over long period.
Short-term exposure to Fe3O4NPs induced cytotoxicity in both 3D astrocytes and neurons spheroids, starting at 10 and 25 µg/ml, respectively.
After long-term repeated dose regimen, cell spheroids showed concentration- and time-dependent accumulation of Fe3O4NPs. Cell mortality appeared at 10 and 0.5 µg/ml, for D384 and SH-SY5Y, respectively, indicating a higher susceptibility, to Fe3O4NPs, of neurons compared to astrocytes. Both spheroid types failed to maintain their canonical shape and displayed cell disaggregation occurring within the first week of treatment beginning at ≥ 0.1 µg/ml (cumulative total exposures of ≥ 1.2 µg/ml) and becoming considerably evident at higher concentrations and over the 30 day-period.
Recreating the 3D spatial environment of CNS allows cells to behave in vitro more closely to the in vivo situation, therefore providing a model that can be used as stand-alone test or as a part of integrated testing strategies. These models could drive an improvement in the species-relevant predictivity of toxicity testing.
Keywords: Fe3O4NPs, in vitro screening, D384 cells, SH-SY5Y cells, neurotoxicity, nanotoxicology
Exploration of the GARD applicability domain – Accurate sensitization assessment of UVCBs (#213)
U. I. Torstensdotter Mattson1, C. Humfrey2, O. Larne1, H. Johansson1, L. Sweet3
1 SenzaGen, Lund, Sweden
Chemical sensitization, also referred to as chemical allergy or allergic contact dermatitis (ACD), is a disease state induced by the human immune system in response to chemical sensitizers. It is estimated that ACD affects 20% of the western population, and the disease is associated with loss of life quality and is a significant economic burden for society. Therefore, regulatory authorities require safety testing for accurate assessment, classification, labelling and appropriate risk management of chemical sensitizers.
GARD – Genomic Allergen Rapid Detection – is a state of the art assay for assessment of chemical sensitizers. It is based on a dendritic cell like cell line, thus mimicking the cell type involved in the initiation of the response leading to sensitization. Recent data from an inter-laboratory ring trial demonstrates that GARD is a powerful tool for assessment of chemical sensitizers, with a predictive accuracy of 94% and excellent reproducibility between laboratories.
Almost 12000 substances were registered to ECHA by January 2017 and ~21% of those were substances of Unknown or Variable composition, Complex reaction products or of Biological materials (UVCBs). While GARD was developed for the assessment of pure, low-molecular weight chemical test substances, the interest in testing mixtures and UVCBs has prompted the exploration of such compounds’ compatibility with the current GARD SOP.
Here, we present results from the assessment of four UVCBs, additives used in gear and engine oils, using GARDskin and/or GARDpotency. In addition to vehicles defined by the SOP (water and DMSO), we explored the use of polar and non-polar vehicles (glycerol and DMF) and mixtures thereof for solubilisation of UVCBs.
It was shown that complex test substances with poor water solubility, initially considered outside of the GARD applicability domain or generating false negative results, were correctly classified based on previous in vivo data using these experimental vehicles, in three out of four samples.
The importance of incorporating flexibility in the choice of vehicles in assay development was highlighted in this study. Remarkably, poorly water-soluble UVCBs, previously considered incompatible with submerged cell cultures were correctly classified, demonstrating the broad applicability domain of the GARD assay.
Keywords: skin sensitization, GARD, UVCB
An in vitro approach to e-cigarette toxicity testing (#218)
L. Simms1, L. Czekala1, M. Stevenson1, G. Phillips1, R. Tilley2, K. Rudd1, T. Walele2
1 Imperial Tobacco LTD, Product Stewardship, Bristol, UK, United Kingdom
Following “Toxicity Testing in 21st Century: A Vision and a Strategy” a new toxicological paradigm was created, focusing on the use of human derived cells lines/tissues and the disruption of key cellular pathways. In keeping with these principles Fontem Ventures have sought current assays for the biological assessment of our products. Due to the evolving regulatory landscape and dynamic nature of innovation with e-cigarettes, new assays are required to quickly determine the biological response of these products. The published literature reveals that e-cigarette aerosols display a lack of significant cytotoxic and genotoxic responses in the CORESTA in vitro test battery.
For novel e-liquid ingredients, we screen all ingredients for Carcinogenic, Mutagenic and Reproductive (CMR) properties and respiratory sensitising potential, from the scientific literature and using in silico predictions. If no major alerts are detected, the e-liquids are assessed in a panel of biologically relevant assays. Examples of these assays include High Content Screening (HCS), in vitro primary human cell biomarkers, dermal and respiratory sensitisation and irritation assays.
The poster will present some of the data generated so far for e-liquids with or without nicotine, the impact of flavours on a range of cellular endpoints using lung cells employed in HCS (NHBE) and a 3D lung model. An in vitro human primary cell biomarker assay could detect increases in nicotine concentration in an e-liquid formulations. Characteristic fingerprint responses have been detected for certain e-liquid flavours, suggesting that flavours can play a role in the in vitro biological responses. An in vitro 3D organotypic lung model was exposed at the air-liquid interface to understand cytotoxic, inflammatory and oxidative response of the aerosol. Morphological evaluation was conducted using H&E staining and cell membrane integrity via measurement of the tight junctions using trans-epithelial electrical resistance (TEER). Results suggest a lack of cytotoxicity and inflammatory responses to e-cigarette aerosols .
These in vitro assays can greatly contribute to our current knowledge of e-liquid ingredients and aerosols and should form part of a larger weight of evidence approach including clinical studies for the assessment of this category of products.
Keywords: Respiratory toxicology, in vitro, e-cigarette, 3d lung models, Biomarkers, High Content Screening
Characterization of a lung/liver organ-on-a-chip model (#234)
D. Bovard1, A. Sandoz1, M. Morelli1, K. Trivedi1, D. Marescotti1, S. Frentzel1, K. Luettich1, J. Hoeng1
1 Philip Morris Products S.A, PMI R&D, Necuhâtel, Neuchâtel, Switzerland
Until few years ago, in vitro models could only poorly mimic the processes observed in the human body. During drug development, their inability to mimic complex physiological processes was an important reason for drug withdrawals. The combination of 3D in vitro models with an engineered microenvironment, resulting in the so-called “organ-on-a-chip” technology, allows these limitations to be overcome. This technology enables the study of complex organ interactions, better simulating processes occurring in vivo and therefore leading to better prediction of drug-associated toxicity.
With the aim of creating a model able to assess the toxicity of aerosols accurately, Philip Morris International recently developed a new lung/liver-on-a-chip device combining a bronchial tissue at the air-liquid interface with HepaRG™ liver spheroids. A specifically designed peristaltic pump allows for a continuous medium circulation and thereby enables lung-liver crosstalk. Stability of both tissues in the chip over 28 days, alone and in combination, was first evaluated. At the end of the experimental period, key liver (albumin secretion, cytochrome P450 activity) and bronchial tissue (transepithelial electrical resistance (TEER), cilia beating frequency, morphology) characteristics were comparable to control tissues that were maintained in the incubator. Using this lung/liver-on-a-chip platform, we further demonstrated the role of the liver compartment in metabolizing and inactivating a pulmonary toxicants: aflatoxin B1. When only bronchial tissues were exposed to this compound, a severe decrease of TEER values and adenosine triphosphate content was observed, along with an increased number of apoptotic cells. Conversely, in the presence of liver spheroids, bronchial tissues were unaffected by the compound. In parallel, specific inhibitors of enzymes involved in the metabolism of this compound were used to demonstrate the toxicity of the parent compound and their metabolites.
Our results demonstrate that the lung/liver-on-a-chip platform can be extremely beneficial for future toxicological analysis.
Keywords: Organ-on-a-chip, bronchial tissues, 3D organotypic, Liver spheroids, Aflatoxin B1, Microfluidic, In-vitro toxicology
Non in vivo skin sensitization strategy under REACh in practice: hurdles that need attention to minimize the need for in vivo testing. (#258)
S. Pelgrom1, H. Barentsen1, S. Jonis1, W. Westerink1, J. Rijk1, J. Paulussen1
1 CRL, 's-Hertogenbosch, Netherlands
For the endpoint skin sensitization an Adverse Outcome Pathway (AOP) has been developed, describing all crucial events involved in the cascade of skin sensitization induction. For registration of industrial chemicals under REACh, the in vivo test for the endpoint skin sensitization has been replaced by non-animal tests since October 2016. In vivo testing for skin sensitizing is permitted as last resort only. For the events involved in the AOP, the non in vivo tests include the DPRA (key event 1), the KeratinoSens (key event 2), and the the USens or hClat (key event 3). None of these individual tests alone can predict skin sensitizing properties adequately, and it is promoted by ECHA that 3 events from the AOP need to be tested, the overall conclusion being based on a 2 out of 3 approach. In a WoE statement arguments need to be given to demonstrate the reliability of the tests/strategy and overall outcome.
Since October 2016 a lot of industrial chemicals have been tested using this non in vivo approach. At CRL chemicals are tested following a step-wise approach, starting with solubility testing, and information on skin corrosion and pH, and information on substance composition (UVCB, metal). In general the preferred test strategy is DPRA together with DEREK (Q)SAR prediction (probable information on metabolism and/or potency) and KeratinoSens. Based on these results the need for a third test is considered. However, from the results obtained with non in vivo testing, several obstacles have been observed that need attention in order to reduce the need for an LLNA in addition to the non in vivo test strategy.
Due to issues including low solubility, high partition coefficient, inconclusive test result, and limitations (applicability) of some test systems for UVCBs and metals, no definite conclusion on skin sensitizing potential can be drawn based on the test results. Moreover, positive results from the non in vivo tests do not allow a conclusion on the potency (cat 1a or 1b), as required for classification and labelling.
From a dataset of 33 substances ~ 29% was conclusive with the non in vivo tests. Due to limitation of test systems no reliable conclusion for UVCB’s was possible, and 40% tests required further testing being overall inconclusive/equivocal. Reduction in in vivo testing will significantly improve with a potency test for skin sensitization that is acceptable for classification and labelling.
Keywords: skin sensitization, non in vivo, REACh, AOP
Cell-type specific effects of using two valproic acid isomers in the neural embryonic stem cell test (#261)
V. C. de Leeuw1, 2, E. V. Hessel1, A. H. Piersma1, 2
1 National Institute for Public Health and the Environment (RIVM), Center for Health Protection, Bilthoven, Netherlands
Non-animal testing methods, in vitro and in silico, are indispensable for improving human risk assessment of chemicals and reducing the number of laboratory animals used. Such an approach requires a series of complementary in vitro and in silico methods to study effects of chemicals on complex processes, such as embryonic development. The murine neural embryonic stem cell test (ESTn) may be able to mimic a number of processes in early differentiation of the embryonic neuroectoderm and brain. In previous research in our lab a biomarker gene set was derived based on testing a range of compounds in ESTn. The present study aims to further explore the response characteristics of the ESTn by exposing it to two isomers, valproic acid (VPA) and 2-ethylhexanoic acid (EHA), to investigate whether these can be distinguished based on gene expression. It is hypothesized that by using a limited number of markers for the various cell types differences in potency of the compounds can be observed. Based on morphological scoring a concentration of 0.1 mM was chosen to analyse gene expression effects, which was in between the ID50 of VPA and EHA. At this concentration, the gene expression of preneural tube marker Cdh1 was increased after 1 and 3 days of exposure. This timing corresponds to the occurrence of the gene as Cdh1 is highly expressed in early, before neural tube closure. Stem cell marker Fut4 was only downregulated after 10 days of exposure. Neuronal markers (Nes, Tubb) on the other hand were virtually unaffected, pointing to normal development of neurons once initial neural differentiation has taken place. A reduction in markers for neural crest cells (Msx2, Snai2) was observed, whereby Snai2 was affected after 4 and 10 days of exposure, while Msx2 was downregulated only after 10 days. Epithelial-to-mesenchymal differentiation (Snai2) may therefore be affected earlier than young neural crest cell differentiation (Msx2). Astrocyte expression (Gfap) was decreased after 10 days of exposure, which concurs with the late expression of Gfap. Interestingly, all gene expression responses to VPA and EHA went into the same direction, although with different effect sizes. Findings on gene expression were confirmed with immunostainings. These effects on cell type markers point to a similar mode of action with different potencies between VPA and EHA.
Keywords: Developmental neurotoxicity, reproductive toxicity, in vitro, valproic acid, embryonic stem cells
Cell-based screening of drug-induced metabolic dysfunction (#298)
M. Schwalfenberg1, R. McGarrigle1, I. Hayes1, C. Carey1, J. Hynes1
1 Luxcel Biosciences Ltd., Cork, CORK, Ireland
Recent years have seen a growing appreciation of the importance of the mitochondria and associated metabolic machinery as sites for off-target drug effects contributing to safety-related attrition and post-market drug withdrawals. High-throughput in vitro cell metabolism assays are therefore of particular importance in the identification and delineation of such metabolic perturbation. Assessing the impact of drug treatment on metabolic perturbation can however require both acute and long-term exposures. Acute measurements reveal direct effects on mitochondria and associated nutrient supply, while longer-term treatments can reveal toxicity involving transporters, MMP-mediated accumulation, CYP activity, and impaired mitogenesis. Long-term treatments also require the use of non-proliferative cell types which exhibit the relevant transporter and CYP activities; competencies which are particularly important when investigating hepatotoxicity.
The most direct measure of mitochondrial function is to assess cellular oxygen consumption, as it informs directly on the activity of the ETC. To facilitate the use of this parameter in the examination of both acute and long-term drug-induced metabolic toxicity, we here describe a 96 well plate-based oxygen consumption assay (MitoXpress®-Xtra) capable of the examination of such drug-induced metabolic perturbation. Method performance is characterised using a compound panel including Antimycin, Flutamide, Nefazodone, and Cycloheximide while HepaRG cells are used to incorporate assay sensitivity to transporter and CYP mediated toxicities. The method also incorporates measurement of both basal and maximal respiration to reveal drug-induced erosion of metabolic capacity. Importantly, we also validate that oxygen consumption measurements can be multiplexed with a cell membrane integrity read-out (Calcein-AM) to better contextualise observed metabolic alterations subsequent to long term drug treatments. Compatibility with 3D-cultures in 96 well plate format is also shown using the RAFTTM system.
Keywords: Mitochondrial Toxicity, in vitro assays, Hepatotoxicity, Cellular Metabolism, Oxygen Consumption
Neural crest related gene transcripts could serve as future targets for biomarker selection in the cardiac embryonic stem cell test (#319)
G. Mennen1, J. Pennings1, A. Piersma1, 2
1 National Institute for Public Health and the Environment (RIVM), Centre for Health Protection, Bilthoven, Netherlands
The cardiac Embryonic Stem cell Test (ESTc) was designed to detect embryotoxic compounds by their interference with the formation of beating cardiomyocytes. Molecular biomarkers could refine embryotoxicity assessment, e.g. within one compound class. The presence of neural crest (NC) cells in the ESTc was used as a targeted approach to look for gene transcripts as biomarkers. The ESTc is a heterogeneous culture, and in vivo, NC cells migrate into the heart in heart development where they contribute to heart formation. Additionally, NC cell migration is influenced by retinoic acid (RA), which is also important in heart development. Valproic acid is known to affect RA homeostasis and was tested together with its analogues 2-ethylhexanoic acid and 2-ethylhexanol. The ESTc cultures were scored for beating cardiomyocytes on differentiation day 10 and stained for NC related markers at different time points using immunocytochemistry. Expression levels of RA and NC related genes were analysed at different time-points using RT-qPCR. Whereas the three compounds did not show clear differences in effects on beating cardiomyocytes, gene expression changes indicated valproic acid as the most potent compound. Its analogues 2-ethylhexanoic acid and 2-ethylhexanol showed similar potencies. Valproic acid downregulated the expression levels of Cyp26a1 and Aldh1a2, which play a role in RA homeostasis. The immunostainings showed presence of NC cell clusters throughout the ESTc cultures. Also NC related gene transcripts (like Ap2α and Ngfr) were detected and showed mixed effects in up- or downregulation. Valproic acid stimulated Epithelial-to-Mesenchymal Transition (EMT) genes related to NC cells early in the differentiation phase. These gene transcript results suggest NC cells could play an important role in the ESTc and may be of interest for biomarker selection.
Keywords: embryonic stem cell test, neural crest, retinoic acid, valproic acid
The THP-1 Toolbox: a new method that integrates the 4 key events of skin sensitization. (#413)
E. Clouet2, C. Raffalli1, M. - H. Damiens1, M. Pallardy1, P. - J. Ferret2, S. Kerdine-Römer1
1 Université Paris-Sud, Paris Saclay, INSERM UMR-996, Châtenay-Malabry, France
Allergic contact dermatitis (ACD) is an adverse health effect that develops following repeated exposure to skin sensitizers. In the European Union, an animal testing ban has been applied under the Cosmetics Regulation, leading to development of reliably predictive non-animal methods. An adverse outcome pathways (AOP) for chemical induced skin sensitization has been already proposed in 2012 by OECD. AOPs outline causally linked key steps between a direct initiating event leading to an adverse health outcome. Four different key events (KE) have been identified and associated in the AOP for skin sensitization: (1) protein-binding reactions, reactivity, and metabolism, (2) epidermal inflammatory response, (3) DC activation and (4) T-cell proliferation. Different in vitro chemistry-based assays have been developed and allow the evaluation of sensitization hazards.
Since DC play a key role in the skin sensitization phase leading to the development of ACD, we propose to combine different tests covering all KE defined by AOP in a same cell line,the THP-1 cell, acting as a DC.
We decided to study the ROS production and GSH depletion as cellular oxidative stress for KE1, Nrf2 activation pathway and gene expressions for KE2, phenotype modifications as cell-surface markers and cytokines production for KE3, and T cell proliferation for KE4. All of those measurements were performed on the THP-1 cell-line, after exposure to a variety of chemicals, including irritants, non-sensitizers and allergens (pro/prehaptens).
Results showed early ROS production and reduction of intracellular glutathione are correlated with the potency of the chemicals such as cinnamaldehyde or methylisothiazolinone. Those chemicals as well as antioxidants specifically activate the Nrf2-Keap1 pathway, which were measured by western blot and a Nrf2 DNA-binding ELISA. They also strongly induced phenotype maturation of THP-1 cell-line with CD54 and CD86 expression at cell-surface and specific cytokine production such as IL-8, IL-18. All sensitizers were able to induce the T cell proliferation while non-sensitizers and irritants did not.
In the present study, we have demonstrated that the three main KE of skin sensitization AOP can be addressed in a same cell line as well as lymphocyte activation.
Keywords: THP1, skin sensitization, integrated AOP
Characterization of human liver organoids cultured on a newly developed microfluidic chip with improved hepatic functions (#421)
H. - J. Han1, J. H. Byeon3, J. - Y. Lee1, D. - J. Jung3, H. - B. Lee1, S. - J. Lee1, E. - H. Cho1, G. S. Jeong3, W. - C. Son1, 2
1 University of Ulsan College of Medicine, Department of Biomedical Sciences, Seoul, Republic of Korea
Hepatotoxicity is one of the major causes of drug withdrawal, thus predicting hepatotoxic potential of a drug in vitro in an early stage of drug development is highly significant. Since human hepatocytes in a traditional 2D culture show limited hepatic functions and lose their characteristics rapidly, 3D culture that shows improved hepatic functions and maintains its originalities better and longer has been widely investigated. To mimic physiological condition better, microfluidic system rather than a static condition has been applied to 3D culture. Instead of primary hepatocytes which had proved not appropriate in long-term culture, organoids using stem cells including human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), and adult stem cells (ASCs) from liver have been studied in recent years. In this study, we attempted to culture human liver organoids using adult bipotent stem cells on a simple microfluidic chip with minimal fabrication procedures and compare them to those cultured in a static condition. Bipotent stem cells were obtained from samples of liver cancer patients. Compared to the existing static culture using extracellular matrix on a 24-well plate, organoids were formed and visible in a relatively short time (2-3 days earlier) on the microfluidic device. The size and morphology of the organoids in the microfluidic culture were less variable than in the static culture. After switching to differentiation stage, the levels of albumin and urea secretion increased faster in the microfluidic condition than in the static condition. Also, the levels of hepatic marker expression including albumin, HNF4α, CYP450 enzymes and transporters (BSEP, MRP2 and NTCP) were higher in the microfluidic condition. The activity of CYP1A2 and 3A4 was also improved, which was demonstrated by phenacetin and testosterone metabolism, respectively. We concluded this newly employed, simple microfluidic device enables to culture liver organoids more efficiently with improved hepatic functions, suggesting a new tool for hepatotoxicity screening.
Keywords: 3D organoids, human bipotent liver stem cells, characterization, microfluidic culture system
Development of a cell-laden 3D hydrogel scaffold assessing neuronal function through microelectrode array recordings (#430)
I. Lauria1, F. Bendt1, J. Hartmann1, L. Nimtz1, A. Blaeser2, S. Rütten3, E. Fritsche1
1 IUF - Leibniz Research Institute for Environmental Medicine, Modern risk assessment and sphere biology, Düsseldorf, North Rhine-Westphalia, Germany
In the future, novel in vitro neurotoxicological screening methods to monitor the function of neuronal networks are crucial for safety assessment of compounds, i.e. chemicals and pharmaceuticals. These tests should be of human origin, standardized, reproducible, well characterized and allowing a medium-throughput. The three-dimensional (3D) bioprinting technology may realize these requirements by producing organ-like neural structures containing neurons and glia cells. However, 3D bioprinting of functional neuronal networks is challenging due to the complexity of brain tissues. Tailored bioinks combining 3D printability and providing the optimal neural cell environment are needed. Therefore, in this study, we aim at the development of a neural cell-compatible bioink, which allows neural progenitor cells (NPCs), neurons and glia cells to survive and form 3D electronically active neuronal networks on microelectrode arrays (MEAs). By mitochondrial activity and investigation of cytotoxicity we confirm that human NPCs are viable within laminin/alginate-based hydrogel blends. Hydrogels were analyzed by scanning electron microscopy for cell morphology and gel pore sizes and by rheology for their viscosity and elastic properties. Morphology of differentiated human NPCs was investigated by immunostaining and confocal fluorescence imaging using specific neuronal and glia markers, i.e. b(III)tubulin and GFAP. We demonstrate that human NPCs are able to differentiate into wide spread neurons and astroglia within the hydrogels. As a control, rat NPCs were embedded within laminin/alginate hydrogels. Cells are viable and can form neuronal networks. To increase the electroconductivity, silver microparticles are incorporated into the hydrogels. Silver addition reduces cell viability in such composite hydrogel materials in a concentration-dependent manner. Current and future work aims at further establishment of bio-compatible and at the same time printable material that exerts good electroconductivity for MEA recordings of standardized and reproducible 3D bioprinted neuronal networks for neurotoxicity evaluation.
Keywords: in vitro neurotoxicology, organoids, 3D bioprinting, biomaterials, neural stem cells
COMPARISON OF 2D AND 3D CULTURES OF PRIMARY HEPATOCYTES ON HEPATOCELLULAR FUNCTIONS AND HEPATOTOXICITY (#451)
H. Dinter1, 2, A. Ullrich1, D. Runge1
1 PRIMACYT GmbH, Schwerin, Germany
Primary hepatocytes of human and animal origin are the gold standard for all
Keywords: 3D culture, hepatocytes, spheroid, hydrogel, cell functionality
Streptococcus pneumoniae inhibits Pseudomonas aeruginosa growth on nasal human epithelium in vitro (#464)
S. Constant1, P. Alouani1, S. Huang1, C. Bertinetti1, O. Verbeke1, L. Wiszniewski1, M. Caul-Futy1
1 Epithelix, Plan-les-Ouates, Genève, Switzerland
Pathogens colonizing the respiratory tract compete with a range of other bacteria (1). Pseudomonas aeruginosa (PA) infection are increasingly associated with acute exacerbations in chronic obstructive pulmonary disease. Streptococcus pneumoniae (SP), meanwhile is a main cause of pneumonia, meningitis, it can leads to infections and other respiratory diseases such as bronchitis.
We report herein the use of 3D airway epithelia reconstituted in vitro to study interactions of PA and SP on nasal mucosa. MucilAir™, a fully differentiated human airway epithelium made of a mixture of primary nasal cells from 14 donors, was used to study the effects and behaviour of PA and SP (inoculated at 3E+02 and 3E+11 CFU/cm2 respectively) cultivated separately or together over 24 hours.
Apical, basolateral and intratissular PA and SP growth were quantified by Colony Forming Unit (CFU). Impairment of epithelial homeostatic barrier function was evaluated through monitoring of tissue integrity (Trans Epithelial Electrical Resistance – TEER); cytotoxicity (LDH), cilia activity, mucin and IL-8 release.
PA infection induces a loss of TEER, 20% cytotoxicity and an increase of Il-8 (+100 ng/ml). On the contrary, SP strongly increases the mucin production. While inoculated together, a lower apical PA growth is observed (- 3E+3 CFU/cm2) suggesting an inhibition due to the presence of SP.
These results suggest that in vitro human airway epithelia is a useful model to study bacterial interaction on the human nasal mucosa.
 Siegel, S.J.; Weiser, J.N. Annu Rev Microbiol. 2015 ; 69: 425–444
Keywords: in-vitro, respiratory infection
The In Vitro assessment of Respiratory Sensitisation Potential of Electronic Cigarette Liquids (#471)
M. Stevenson1, L. Czekala1, L. Simms1, N. Tschierske2, H. Johansson3, T. Walele4
1 Imperial Tobacco Ltd, Bristol, United Kingdom
There is a general consensus amongst the scientific and public health community that e-cigarettes constitute a less harmful source of nicotine than combustible cigarettes, and that flavours play a critical role in attracting and retaining smokers into the vaping category. To provide further evidence that e-cigarettes present less harm, various toxicological considerations need to be taken in to account, i.e. Respiratory Sensitisation Potential of e-liquids. Currently, no in vitro or in vivo models are validated to identify chemical respiratory sensitizers.
The objective of this study was to assess experimental and commercial e-liquids in GARDair™; an assay which claims to detect respiratory sensitisers. GARDair measures the genomic biomarker signature of a human myeloid leukemia-derived cell line exposed to test substances. Gene expression analysis is performed using Affymetrix microarray technology and a prediction model is used to classify each sample according to its respiratory sensitizing potential.
Three experimental unflavoured (Base liquid (50:50 Propylene Glycol, Vegetable Glycerin), ± 2.4%; or 4.5% Nicotine (w/w%)) and two commercial e-liquids were assessed in the GARDair assay. Several benchmark controls were assayed simultaneously with the test articles to determine the sensitivity and specificity of the assay.
Of the seven assayed benchmark respiratory sensitizers, five were accurately classified as such by GARDair. No false positives were generated. Thus, the sensitivity and specificity were estimated as 71% (5/7) and 100% (12/12), respectively. Overall predictive accuracy was 89% (17/19). All e-liquids gave negative results for respiratory sensitisation. The results highlight the potential of this assay for future ingredient assessment strategies.
Keywords: respiratory sensitization, in vitro sensitization, e-cigarette, risk assessment
OncoCilAir™: A physiological in vitro platform to assess the efficacy and the toxicity of lung cancer therapeutics. (#475)
H. Benainous1, V. Kilin3, S. Huang2, L. Wiszniewski2, J. - P. Wolf3, L. Bonacina3, S. Constant1, 2, C. Mas1
1 OncoTheis, Plan-les-Ouates, Switzerland
Every year, lung cancer is the most frequently diagnosed cancer in men and women leading to 1 million deaths worldwide. Clearly, realistic human 3D models, which recapitulate heterologous interactions between epithelial, stromal and tumor cell types are required to improve preclinical predictivity. In this prospect we have developed OncoCilAir™, a Non Small Cell Lung Cancer in vitro microtissue model which combines a functional reconstituted human airway epithelium, human primary lung fibroblasts and lung adenocarcinoma cell lines cultured at the air-liquid-interface. Remarkably, in this 3D microenvironment tumor cells expand by forming nodules, closely mimicking human lung cancer features. In addition, multiphoton imaging experiments revealed that OncoCilAir™ replicates in vitro the ability of tumors to influence surrounding healthy tissues, underscoring the physiological relevancy of the model. Accordingly, drug screenings results demonstrated that OncoCilAir™ allows accurate ranking of drug candidates through the simultaneous assessment of their efficacy and side-toxicity. Moreover, since the model remains functional for several months and reproduces in vitro critical pitfalls of the lung airway like mucus secretion and cilia beating, we showed that it can be used to assess functionalised nanoparticles, oncolytic viruses but also inhalation therapies through controlled nebulisation. Lastly, we used genome editing tools to demonstrate the feasibility of genetically modifying in vitro engineered human microtissues. In doing so, we successfully induced carcinogenesis in smoker microtissues. In conclusion, OncoCilAir™ heralds a new generation of in vitro genetically editable human models which is expected to accelerate the development of optimal lung cancer therapies while reducing animal testing.
Keywords: lung cancer, in vitro lung model, carcinogenesis
Neurospheres for Species-Specific, Medium-Throughput Analyses of Developmental Neurotoxicity (DNT) Evaluation (#480)
E. Fritsche1, S. Masjosthusmann1, N. Förster2, J. Baumann1, F. Bendt1, K. Dach1, U. Hübenthal1, M. Schmuck1, T. Temme2, A. Mosig2
1 IUF, Leibniz Research Institute for Environmental Medicine, Düsseldorf, North Rhine-Westphalia, Germany
Testing for developmental neurotoxicity (DNT) of compounds for regulatory purposes, e.g. for screening and priorisation, by using an in vitro testing battery is currently under discussion at the OECD level. Within such a battery a variety of neurodevelopmental processes at different developmental stages needs consideration. We have been establishing a DNT high content assay based on time-matched fetal human and postnatal rodent as well as human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs), which proliferate in culture and - under differentiating conditions – migrate and differentiate into neurons and glia cells, i.e. radial glia, astrocytes and oligodendrocytes. In this so-called ‘Neurosphere Assay’ we analyzed multiple pathways for their functional implications in neurodevelopmental processes, amongst them oxidative stress, histone deacetylase inhibition or thyroid hormone disruption. For high content analyses of differentiated neurospheres, we developed the software ‘Omnisphero’ allowing simultaneous analyses of multiple neurodevelopmental endpoints like NPC migration, neuron and glia differentiation, neuronal morphology and neuronal migration. This set-up allows chemical testing in a medium throughput with the ‘Neurosphere Assay’ as part of a DNT testing battery, e.g. for industrial or regulatory usage.
Keywords: DNT, neurosphere, in vitro, testing battery
Use of a 3-D human in vitro airway model (EpiAirway®-FT) to investigate clinically relevant endpoints for pulmonary disorders: Proof of concept. (#489)
D. Kidd1, A. Woodhams1, A. Daunt2, A. Postoyalko2
1 Covance Laboratories Ltd, Genetic Toxicology, Harrogate, UK, United Kingdom
Prediction of human biological responses using in vitro test systems has long been the goal of many and over the past decade or two, a vast array of models and technologies have arisen from research groups all over the world. The introduction and uptake of these new test systems have opened doors to recapitulating in vivo biological responses in vitro and many new test systems have been adopted at the international regulatory level (OECD Test Guidelines Section 4: Health Effects).
Building on the success of the 3-D skin models, commercially available airway models are also available and used regularly. EpiAirway® (MATTEK Corporation®, Ashland, USA) is a commercial in vitro lung 3D tissue model that contains the epithelial cell types found in the upper airway in vivo including functional beating cilia. These tissues respond to hyperplasia conditions as well as other important biological pathways.
In a Proof of Concept (POC) study, we questioned whether the EpiAirway® 3D tissue model could yield relevant data for important in vivo clinical endpoints. We initially evaluated the full thickness tissue model (FT) against the standard thickness model (AIR-100) concluding that the FT model gave a better representation of the in vivo responses being observed.
We then exposed the EpiAirway® FT 3D tissue model to the Th2 cytokine IL-13 as a known inducer of goblet cell hyperplasia (GCH) in vivo, using a small range of concentrations (1, 5 and 10 ng/mL) over a 6-day period. We fixed, sectioned and stained (periodic Acid-schiff (PAS)) the tissues for GCH analysis, observing a concentration related increase and up to >5 fold increase in GCH over the control tissues. Cilia beat frequency (CBF) analysis at the start and end of the exposure period yielded no significant changes in the IL-13 treated tissues compared to the control tissues however a reduction in CBF on tissues that also showed cytotoxic damage (via trans epithelial electrical resistance measurements) was observed.
These POC results show that the EpiAirway® FT 3D tissue model is a suitable model for further investigation into in vivo pathologies in an in vitro test system and we have begun to expand our work into additional endpoints to further challenge our original hypopthesis.
Keywords: EpiAirway, goblet cell hyperplasia, in vitro, cilia beat frequency, IL-13
Comparative inhalation toxicity testing using in vitro organotypic rat and human airway epithelial models (#499)
P. Hayden1, G. R. Jackson1, J. L. Vinall2, E. Storey2, H. Simpson2, M. Debatis1, A. G. Maione1, C. Roper2, M. Klausner1
1 MatTek Corporation, Ashland, Massachusetts, United States of America
Rationale: Assessment of inhalation toxicity potential is important for developing new therapeutics and establishing safe handling, labeling and emergency response procedures for chemicals. Traditionally, animal models have been used for determining inhalation toxicity. However, in vivo models may not accurately predict human outcomes and therefore, translational information or replacement is increasingly required. To address this issue, in vitro organotypic airway tissue models constructed from primary human cells have been developed. Yet, validation of these systems is problematic because available in vivo inhalation toxicity data has been primarily produced using rodents. In the current project, a scalable in vitro organotypic model of rat mucociliary airway epithelium has been produced to allow direct comparison of chemical toxicity responses with previously developed in vitro human airway models. Methods: Conducting airways epithelial cells were isolated from CD rats, expanded in culture, and cultured at the air-liquid interface for up to 27 days. Comparison of toxicity responses in rat vs. human airway models was conducted using 14 chemicals with in vivo GHS inhalation toxicity classifications ranging from categories 1-5. The airway models were exposed to 4 doses of each test chemical for 3 hrs, followed by 21 hrs recovery prior to measurement of tissue viability. IC75 concentrations for chemical toxicity were determined from the dose response data. Results: Rat and human airway models demonstrated a high level of organotypic mucociliary differentiation including pseudostratified epithelial structure with copious development of functional cilia. Robust barrier function was demonstrated by development of transepithelial electrical resistance of up to 1000 Ω x cm2. Results of chemical toxicity experiments showed that rat and human tissue responses were similar, within the same order of magnitude, for each chemical. Future work will expand the range of chemicals tested to further define the comparative responses. Conclusions: These results suggest that in vitro rat and human airway models will be useful tools to facilitate rodent to human translation of in vitro inhalation toxicology data, and ultimately a more complete transition to human based in vitro models of inhalation toxicity.
Keywords: In Vitro Airway Models, Inhalation Toxicology
Differentiation of hepatotoxic compounds by metabolomics in HepG2 cells (#554)
S. Sperber1, B. Birk1, V. Haake2, T. Walk2, B. van Ravenzwaay1, H. Kamp1
1 BASF SE, Experimental Toxicology & Ecology, Ludwigshafen, Rhineland-Palatinate, Germany
BASF and metanomics established the database MetaMap®Tox containing the plasma metabolome of more than 800 compounds derived from 28-day studies in rats. Recently, a highly stable and reproducible liver in vitro model was established, in which the intracellular metabolome of HepG2 cells can be specifically altered through treatment with different hepatotoxicants. Within the BMBF- and ZonMW-funded project SysBioToP, different liver toxicants are currently being tested in different in vitro liver cell systems using imaging technologies, transcriptomics and metabolomics. We have analysed the intracellular metabolome of HepG2 cells treated with valproic acid, paracetamol, ciprofloxazine, and diclofenac, all described to cause liver injury in a human clinical setting. The metabolome consisted of 236 unique metabolites, thereof 35 amino acids and derivatives, 11 carbohydrates and related compounds, 54 lipids, 14 energy metabolites, 6 nucleobases, 14 vitamines and cofactors as well as other miscellaneous or unknown metabolites. Our data show that valproic acid, paracetamol, and ciprofloxazine did significantly change the intracellular HepG2 metabolome, whereas diclofenac is only hardly distinguishable from the controls. A principal component analyses shows that valproic acid and paracetamol build clusters of samples separate from control but close to each other, whereas ciprofloxacine builds a cluster which clearly separates from control, but also from the other treatments. The differences in the in vitro metabolome response might reflect the in vivo effects both in terms of the underlying mode of action as well as the potency and frequency of liver injuries in the clinical application.
Keywords: hepatotoxicity, metabolome analysis in vitro, HepG2 cells
BCOP LLBO is a promising method for eye irritation classification (#565)
A. R. Van Rompay1, E. Adriaens2, S. Verstraelen1
1 VITO NV, Health, Mol, /, Belgium
Measurement of ocular irritancy is a necessary step in the safety evaluation of industrial and consumer products. Until now, no single alternative test was capable of identifying the different ocular effects observed in the in vivo Draize eye test. The CON4EI project (CONsortium for in vitro Eye Irritation testing strategy, sponsored by Cefic) assessed the reliability of 8 in vitro test methods and computational models for a set of 80 reference chemicals covering the most important in vivo drivers of classification, balanced according to the physical form (38 liquids and 42 solids) and representing different chemical classes. The different test methods were combined into a testing strategy and the performance was evaluated. BCOP LLBO is incorporated in one of the testing strategies together with SkinEthicTM HCE EIT or EpiOcularTM EIT to address the whole spectra of eye irritation potential.
Furthermore, in the ALT4EI project (ALTernatives for Eye Irritation, Sponsored by Flemish Department of Environment), remaining data gaps for the BCOP LLBO method were filled to strengthen the data set and include BCOP LLBO in OECD Test Guideline 437. In total, 145 chemicals were tested and the performance of BCOP LLBO vs BCOP OP-KIT was compared. For Category (Cat) 1 vs No Cat 1 chemicals, the BCOP LLBO has a much higher sensitivity (80 vs 68%). A higher false positive rate (21 vs 15%) was observed for BCOP LLBO, especially Cat 2 chemicals were overpredicted (38 vs 22%). Anyway, a Cat 2 chemical in BCOP is classified as ‘No Prediction can be made’, and requires a 2nd method (SkinEthicTM HCE EIT or EpiOcularTM EIT) for classification. For No Cat vs Not No Cat, a similar specifity (66 vs 64%) was obtained compared to BCOP OP-KIT.
Currently, a validation study for BCOP LLBO (sponsored by CEFIC) is finalized to demonstrate the utility and the applicability of this device to be integrated in OECD Test Guideline 437. An SPSF was accepted by the OECD Eye Expert group. The BCOP LLBO method was trained, transferred and tested for the 13 proficiency chemicals in 3 runs by VITO, CitoxLab and Charles Rivers. Data will be submitted soon and for the first time presented at this Eurotox meeting.
This research is funded by CEFIC-LRI, Flemish Department of Environment. We acknowledge Cosmetics Europe for their contribution in chemical selection and the BCOP testing.
Keywords: CON4EI, ALT4EI, eye irritation, BCOP LLBO, in vitro eye irritation testing strategy
Effect of compounds Y on the barrier function of human iPSCs derived brain microvascular endothelial cells (#568)
M. Yamashita1, H. Aoki1, T. Hashita1, T. Iwao1, T. Matsunaga1
1 Nagoya City University, Graduate School of Pharmaceutical Sciences, Clinical Pharmacy, Nagoya, Japan
The blood-brain barrier (BBB) has an essential biological barrier function to regulate molecular transport between the circulating blood and the brain parenchyma. The BBB is composed of brain microvascular endothelial cells (BMECs), which are characterized by specialized tight junctions and high expression of multidrug efflux transporters. For drug development, in vitro models are required to evaluate BBB permeability, which is important for predicting not only the efficacy but also the safety of drugs. However, there is no ideal in vitro BBB model available currently. Although BBB permeability has been evaluated using experimental animals, accurate prediction in human is difficult because of species differences. Recently, the BBB models using human induced pluripotent stem cells (iPSCs) has been developed. However, few models have robust tight junctions and can maintain the barrier function for a prolonged period. Therefore, we attempted to enhance the characterized barrier function of human iPSCs derived BMECs (iBMECs) in this study. iBMECs were differentiated by existing methods containing compounds Y. In the results, compounds Y significantly increased the trans-endothelial electrical resistance (TEER) and decreased Lucifer Yellow (LY) permeability of iBMECs. Also, compounds Y allowed iBMECs to maintain the high TEER value and low permeability of LY for a prolonged period. The results of immunofluorescence staining confirmed that the localization of Occludin, ZO-1 and Claudin5 (the tight junction markers) to membranes were stabilized by compounds Y. The efflux transporter ABCG2/BCRP was located the apical side on iBMECs. Furthermore, Hoechst33342 permeability revealed that compounds Y increased the transport activity of ABCG2/BCRP. In conclusion, we have succeeded in discovering compounds Y improving the formation of tight junctions and the activity of efflux transporter, and maintained the barrier function of the generated BBB model for a prolonged period. Thus, it was suggested that compounds Y would be useful for developing in vitro BBB models from iPSCs.
Keywords: Human induced pluripotent stem cells, in vitro BBB models, tight junctions, Pharmacokinetics, ABCG2/BCRP
Human tissue based sensitization assessment: set-up of an accurate prediction model for distinguishing skin sensitizers using a new assay adapted to hydrophobic substances and mixtures. (#571)
E. Andres1, M. Barry1, A. Hundt1, C. Dini1, P. - J. Ferret2
1 Oroxcell, Life Sciences, Romainville, France
A new assay for skin sensitization assessment, based on the specific release of IL-18 cytokine from EpiCS recontructed epidermises in response to skin sensitizer exosure, was implemented in our lab. The prediction capacities of the assay for the identification of contact sensitizers were refined in a new prediction model.
20 references substances were tested on 10 batches of reconstructed epidermises. Each batch was verified for its basal level of IL-18 release and for its reaction to solvent. Two types of prediction model were applied to evaluate the final results of the assay, in order to optimize the predictivity of the experimental data.
The EpiCS test system provided remarkably low but stable levels of basal and solvent induced IL-18 release over 10 batches of epidermises. The prediction performances were found to be comparable to previously published results. Differences in performance between the evaluated prediction models were examined in terms of the balance of correct classification of positive and negative substances.
The results indicate that the assay provides promising prediction performance, with similar levels of sensitivity, specificity and accuracy to OECD validated assays, albeit obtained with a reduced number of substances. Therefore, further testing of a wider range of substance types is recommended.
Keywords: Safety testing, Keratinocyte, Skin, Reconstructed epidermis, Sensitizer, Sensitization, IL-18
Differentiation of human pluripotent stem cells (hPSC) into neurons: a fast, reproducible and inducible system for neurotoxicity screening (#584)
J. Aleksic1, I. Geti1, G. Apic1, A. Stoop1, M. Kotter1
1 Elpis Biomed, University of Cambridge Biomedical Innovation Hub, Cambridge, United Kingdom
The lack of reproducible and scalable physiologically relevant model is one of main reasons for neurological drug failure. We present here powerful approach for the generation of human neurons by forward programming and demonstrate their potential use in neurotoxicity screening. We used optimized inducible overexpression platform (OPTi-OX) based on targeting of each of the two elements of Tet-ON system (reverse tetracycline transactivator [rtTA] responsive to doxycycline (dox), and inducible promoter regulated by rtTA (Tet-responsive element)) into different genomic safe harbour sites. To generate neurons we created pro-neuronal transcription factor NGN2 OPTi-OX hPSCs and induced them with dox in neuronal differentiation medium. Nearby pure populations of neurons are generated within 5 days and reach synchronised network activity on multi-electrode arrays within less than three weeks. The efficiency of neuron generation remained stable over extended culture periods of the inducible hPSCs (>25 passages). qPCR showed that induction of NGN2 expression resulted in downregulation of pluripotency factors OCT4 and NANOG, and after 1 week upregulation in expression of seven genes of neuronal transcriptional program. SysWiz software was used to demonstrate the potential application of these genes in detecting neurotoxicity. SysWiz contains publicly available data on chemicals, genes, proteins and toxicities/pathologies/diseases. Using SysWiz, we mapped known neurotoxins onto these seven genes, and got predictions of multiple neurotoxic effects, like Neuronal degeneration, Neuronal atrophy, necrosis, apoptosis etc. Our results demonstrate that OPTi-OX forward programming can be used for fast and reproducible generation of neurons that can be utilized for neurotoxicity testing.
Keywords: stem cells; in vitro models; neurotoxicity; mature human neurons
High content cell monitoring of cancer and cancer treatment-related cardiomyopathy (#601)
E. Dragicevic1, K. Juhasz1, 2, O. Reinhardt3, L. Doer1, M. Beckler1, S. Stölzle-Feix1, F. Alves3, 4, N. Fertig1
1 Nanion Technologies, Munich, Germany
Changes in the impedance time course of cell-covered electrodes give profound insight in changes in cell morphology, contractility, and proliferation, even over prolonged periods of time, providing a significant advantage over standard mostly endpoint cytotoxicity assays.
Here, we have applied this technology for monitoring breast cancer cell regrowth after chemotherapy treatment in vitro. One clinical regimen is a combination of cyclophosphamide, adriamycin (doxorubicin) and 5-fluorouracil (CAF) that is administered to patients for four months. Despite the initially successful multimodal therapy, tumor recurrence remains a major cause of mortality in breast cancer patients. Here, we investigated responses from murine H8N8 and H8N8 T3.2 cells via two different assay methodologies: by using an image-based live-cell analysis system and an impedance-based cell monitoring system. The murine H8N8 cells represent an immortal mammary carcinoma cell line with tumor stem cell properties, and the H8N8 T3.2 cells represent a once-treated recurrent tumor variant. For in vitro tumor regrowth investigation, the H8N8 T3.2 cells were treated with CAF for a second time. Changes in impedance and confluency of murine mammary carcinoma cells, were used as a measure of toxicity with cell viability monitored over a time period of 500 h with a 1 ms time resolution, under in vitro physiological conditions. Intrinsic dose dependent effects of CAF could be identified consistent with other methods.
Next, we tested the implication of anticancer drugs on induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). As an emerging field Cardio-Oncology aims to find a balance between oncologic efficacy and reducing adverse cardiovascular effects. By long term monitoring of iPSC-CMs a putative cardiovascular side effect can be investigated. CAF, cyclophosphamide, doxorubicin and 5-fluouracil alone, as well as paclitaxel long-and short-term implications on cell viability will be shown.
In summary, long-term high-resolution impedance proliferation curves provide amenable insights into dynamics of cell proliferation, for in vitro investigations of adjuvant chemotherapy.
Keywords: cancer, cardio-oncology, label-free, impedance, toxicity
High-throughput 3D brain model of iPSC-derived neurons and glia for prediction of neurotoxicity and safety pharmacology (#604)
C. Ramakers1, C. Chang1, K. Wilschut1, A. Nicolas1, T. Olivier1, H. L. Lanz1, S. J. Trietsch1
1 Mimetas BV, Leiden, Netherlands
Prediction of neurotoxicity remains challenging due to the lack of relevant human-based 3D in vitro models of the brain and rely heavily on ex vivo and in vivo animal testing. We show the application of a human in vitro 3D CNS models developed in the OrganoPlate® that can be used for risk assessment during drug discovery and development.
The high-throughput OrganoPlate platform consists of 96 microfluidic individual chips supporting 3D co-cultures of human iPSCs-derived neurons and astrocytes embedded in Matrigel with an adjacent easy accessible channel for medium perfusing and compound exposures.
During long-term culturing, the 3D mature CNS cultures show the presence of both GABAergic and glutamatergic neurons with astrocytes observed by plate-based immunostainings. Calcium fluctuations were detected using a calcium sensitive dye reflecting spontaneous electrophysiological activity after one week of cell culturing. Modulation of the neuronal activity was achieved by exposure to GABA, as well as neurotoxicant Methylmercury. The seizurogenic predictivity was shown after 4-AP exposure resulting in increased bursting patterns. Together with the multiplexing of assays to determine mitochondrial membrane potentials and neuronal viability, and additional neurite integrity, this CNS platform allow us to study different classes of neurotoxicants. Using automated high content microscopy this functional 3D neuronal model can be applied to high-throughput screening of specific regions of the human brain to better predict neurotoxicity and safety pharmacology, while additionally contributing to diminish the use of animal models.
Keywords: 3D in vitro models, neurotoxicity, organ-on-a-chip
Identification of oxidative stress potential in phenolic compounds and their link to liver injury (#622)
J. P. Schimming1, L. Bischoff1, B. ter Braak1, B. van de Water1
1 Universiteit Leiden, Division of Drug Discovery and Safety, Leiden, Zuid-Holland, Netherlands
Oxidative stress is a critical components of most adverse outcome pathways related to chemical-induced liver injury, which for various chemicals involves redox cycling. In order to successfully develop next generation toxicity testing concepts, a successful identification of a compound’s capacity to induce redox-cycling based liver injury is necessary. Hydroquinone like phenolic compounds are able to elicit oxidative stress via auto-oxidation and consecutive formation of reactive oxygen species, thus contributing to the development of liver damage. We have used a large panel of data-rich phenols to define whether new approach methodologies (NAM) can be used to correctly predict redox-cycling-mediated oxidative stress and cell death. Our test set did consist of 6 hydroquinone like compounds with redox-cycling potential, 12 redox-cycling negative compounds with alkyl side chain and 3 without an alkyl side chain.
To evaluate oxidative stress and cell death we applied Nrf2 pathway HepG2 BAC-GFP reporter cell lines, that allow the single cell dynamic assessment of Nrf2 activation and induction of Srxn1, an Nrf2 target gene. Utilizing high throughput confocal microscopy ensured a rapid live cell measurements of a broad concentration time response of Nrf2 pathway activation for all phenols studied in association with the onset of cell death, providing an information-rich dataset concerning oxidative stress pathway activation. In order to identify further potential mode of action, we quantified the expression of the human S1500+ gene list V2 of the NTP’s Tox21 Working Group in HepG2 and cryopreserved Primary Human Hepatocytes (cPHH).
The Nrf2 pathway reporter cell lines of the Nrf2 pathway allowed identification of redox-cycling positive hydroquinone like compounds: a concentration- and time-dependent compound specific activation of Srxn1-GFP was observed, and all compounds that induced Srxn1 also induced stabilization and nuclear translocation of Nrf2-GFP. Cell death was induced at high concentrations for all redox-cycling phenols. The redox-cycling negative phenols were inactive in both Nrf2-GFP activation as well as Srxn1-GFP induction. Nevertheless, alkylated phenols induced onset of cell death, likely independent from oxidative stress induction. In conclusion, our current reporter cell models to quantify oxidative stress induction at the cellular level support the concept to apply innovative NAM methods to support read-across-based chemical safety testing.
Keywords: Oxidative stress, hydroquinone analogues, liver injury, Nrf2 pathway, HepG2 BAC-GFP cell stress reporter
Acute lung injury on-chip: building a disease predictive model that emulates alveolar biomechanics (#625)
N. Roldan1, A. Rapet1, A. O. Stucki1, G. Raggi1, K. Fytianos2, T. Geiser2, 3, N. Hobi1, 4, O. T. Guenat1, 4
1 University of Bern, ARTORG Center, Bern, Switzerland
Acute lung injury (ALI) is a complex lung disease in which inflammation plays a central role. The disruption of the alveolar epithelial-endothelial barrier together with an exacerbated inflammatory response lead to influx of oedema fluid into alveoli impairing gas exchange and lung mechanics. Sepsis, a severe bacterial infection, is a known trigger of ALI. For this reason, a classical approach for in vitro and in vivo studies is employing lipopolysaccharide (LPS), a component from gram-negative bacteria cell wall, to stimulate the inflammatory response, immune cell infiltration and tissue damage.
In this work, our aim was to investigate the combined role of lung mechanics, often neglected in in vitro studies, and inflammatory cells on the integrity of the endothelial barrier. With that purpose, we have employed primary pulmonary endothelial cells from a human source to reconstruct the aforementioned barrier using a lung-on-chip, a microfluidic device that reproduces the breathing motion. This system accounts for mechanical cyclic strain and enables the perfusion of circulatory immune cells.
Our results show that upon challenging the endothelial cells with LPS, the presence of immune cells led to a greater inflammatory response compared to that of pulmonary endothelial cells alone, also in terms of barrier integrity. We additionally show that this effect was not only due to cell-cell crosstalk but also to the physical interaction between immune cells and the endothelial barrier.
In conclusion, our model provides proof of the determinant role of the immune system in ALI in physiologically-mimicking dynamic conditions. These findings constitute a step towards a more accurate and clinically relevant in vitro system which in the future, could represent an alternative to animal models to design novel therapeutic approaches and evaluate drug toxicity.
*N.H and O.T.G. contributed equally to this work
Keywords: lung-on-chip, acute lung injury, inflammation, biomechanics
Predicting Skin Sensitizers with Confidence – Using Conformal Prediction to Determine Applicability Domain of GARD (#628)
H. Johansson1, A. Forreryd1, U. Norinder2, 3, T. Lindberg4, M. Lindstedt4
1 SenzaGen AB, Lund, Sweden
Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical hazard testing, and to meet European legislations, considerable efforts have been made to develop animal-free approaches to replace current animal testing.
Genomic Allergen Rapid Detection (GARD) is a state-of-the-art non-animal-based testing strategy. The assay classifies chemicals as either skin sensitizers or non-sensitizers based on the readout from a genomic biomarker signature comprising 200 genes measured in an in vitro model of dendritic cells. GARD was recently subjected to a formal validation procedure (OECD TGP 4.106) involving classification of 28 tests substances in three independent laboratories and demonstrated an excellent reproducibility within (82-89%) and between laboratories (92%), as well as an outstanding predictive performance: accuracy - 94%, sensitivity – 93%, and specificity 96%.
In addition to reporting average model performance, it is also important to provide a measure on how well an assay is expected to perform for a particular test substance. Uncertainties may arise if a model is forced to classify compounds falling outside the predictive boundaries (i.e. applicability domain) of the model. However, this measure is often neglected by test developers, and assays are instead assumed to be universally applicable for all chemicals.
Based on resent research at Lund University, conformal prediction has initially proved to be a promising tool for defining applicability domain of in vitro assays. This framework provides a measure of uncertainty associated with each prediction, as well as deliver a warning if a specific test sample is outside the applicability domain. In the current study, we implemented and developed the concept of conformal prediction to suit the assay format of GARD. Historical datasets were investigated, and results demonstrated that all hitherto tested chemical reactivity domains, as well as pre-and pro-haptens are within the applicability domain of GARD and can be classified with high amount of confidence.
The presented results demonstrates the importance of establishing applicability domain of in vitro assays. For regulatory bodies, as well as for the chemical industry, the results presented for the GARD assay facilitates in the decision making process, and allows for classification of skin sensitizers with confidence, thus fulfilling characteristics for future standalone use.
Keywords: in vitro; in vitro assay; sensitization; GARD; Genomic Allergen Rapid Detection; conformal predictions
Modeling of Ligand-Induced Acute and Chronic Inflammation in the gastrointestinal tract using In vitro 3D-human small intestinal microtissues (#635)
J. Markus1, 2, T. Landry1, Z. Stevens1, M. Klausner1, P. Hayden1, S. Ayehunie1
1 MatTek Corporation, Ashland, Massachusetts, United States of America
Intestinal epithelium is known to be involved in innate immune responses by recognizing potential pathogens through cellular pattern recognition receptors (PRRs). The purpose of this study is to investigate PRR responses following exposure of an in vitro 3D human small intestinal (SMI) microtissue to various Toll-like receptor (TLRs) and Node-like receptor (NOD) ligands. The SMI microtissues are cultured using human intestinal fibroblasts and enterocytes and their 3-dimensional polarity and morphology mimics that of native in vivo tissues. Characterization of the microtissues included evaluation of structural features, barrier properties, and expression of drug transporters and drug metabolizing enzymes. Results showed that exposure of intestinal microtissues to live bacterial or ligands to TLR4 (LPS) and NOD2 (Muramyl dipeptide; MDP) were able to induce gene expression of proinflammatory cytokines such as IL-1β, IL-6, and RANTES. Prolonged exposure of intestinal microtissues to IL-1β also resulted in reduced membrane integrity and induction of pro-inflammatory cytokines (IL-6 and CCL20) known to stimulate acquired immune cell responses by inducing cytokine release such as TNF-α and IFN-γ or by initiating the migration of inflammatory cells. All these responses may be precursors for IBD-like disease. To simulate the effect of immune cell responses on the intestinal epithelium, we also exposed the microtissues to TNF-α and IFN-γ, which resulted in the reduction of membrane integrity and the release of proinflammatory cytokines. The effect of TNF-α and IFN-γ on the intestinal epithelium was further exacerbated if antigen-presenting cells such as dendritic cells were incorporated into the 3D intestinal microtissues. In summary, our results suggest that the EpiIntestinal tissue is capable of modeling innate immune responses and can be a useful tool to study the complex interactions of human intestinal epithelium with microbiome in vitro in the induction of IBD-like disease.
Keywords: intestinal toxicity, innate immunity, inflammatory bowel disease, intestinal inflammation
Rapid and Accurate High-Throughput In Vitro Prediction of Nephrotoxicity in Humans with ProxTox-HTS (#638)
R. de Wilde1, B. Kovács1, D. Gliesche1, D. Zink2
1 SOLVO Biotechnology, Budaörs, Pest, Hungary
Renal proximal tubular cells (PTCs) are essential for the renal elimination of xenobiotics due to their role in compound transport, metabolism and secretion. Therefore, drugs and their metabolites may damage PTCs and lead to acute kidney injury or chronic kidney disease. Methods for analyzing PTC toxicity are needed for predicting potential drug mediated nephrotoxic effects. Until recently, in vitro methods for predicting nephrotoxicity in humans were not available and animal models were the methods of choice. However, animal models have a low throughput, are expensive and the outcomes often poorly predict human toxicity. Human and animal PTCs display major differences with respect to transporter and drug-metabolizing enzyme expression patterns
Recently, the first in vitro methods for the accurate prediction of nephrotoxicity in humans became available. These include a high-throughput screening (HTS) method for screening of larger compound libraries. The HTS platform was developed without using pre-defined endpoints, instead, predictive cellular changes were identified by phenotypic profiling after compound treatment. Using a HTS imaging and machine learning system, 129 phenotypic features were assessed, and groups of 4-5 predictive features were selected and used as endpoints.The predictive method has been pre-validated with 44 chemically diverse compounds, with well-characterized effects on human kidneys. The method utilizes primary human renal proximal tubule or HK-2 cells in 384-well format.
Results and Conclusion
The test balanced accuracies are 82% (primary PTCs) and 89% (HK-2 cells), respectively. The results indicate DNA damage as a key determinant for PTC toxicity, which was mediated by various nephrotoxicants directly or indirectly via ROS generation. The combination of cell-based systems with computational algorithms can be used for efficient and accurate HTS in vitro kidney toxicity predictions, and is currently applied in collaboration with the US Environmental Protection Agency for the prediction of the human nephrotoxicity of ToxCast compounds.
Keywords: Nephrotoxicity, Proximal tubule cells, High-Throughput Screening, Human, Predictive
Critical Substances in Bovine Corneal Opacity and Permeability (BCOP) versus Isolated Chicken Eye (ICE) Assays (#655)
S. Ehrenberg1, S. Burkhardt1, C. Werner1, N. Bachmann1, P. Allingham1, P. Wetzel2
1 BSL BIOSERVICE Scientific Laboratories Munich GmbH, Planegg, Bavaria, Germany
Ex Vivo tests play an important role in the attempt to significantly reduce or even to avoid animal testing of chemicals. They are part of a tiered-testing strategy for regulatory classification and labelling within a specific applicability domain. One of these tests is the BCOP (Bovine Corneal Opacity and Permeability; OECD 437) method that can be used to classify chemicals, i.e. non-irritants or substances inducing serious eye damage. Another test system to classify for these categories is the ICE (Isolated Chicken Eye; OECD 438). Classification of both tests is based on the In Vitro Irritancy Score (IVIS), which is derived from combined assessments of the cornea, following exposure to a test chemical. In the BCOP test opacity and permeability are used for scoring, while for the ICE opacity, fluorescein retention, increased thickness and surface damages are evaluated.
The differences in scoring and conduct of these two tests come along with advantages but also limitations regarding critical substances. In providing examples, we point out these advantages and limitations that could help decide which test might be the more appropriate one.
For example certain coloration or consistency (e.g. dyes or sticky chemicals) can lead to elevated or false positive values in a BCOP or an abort of the running experiment because of a strong adhesion of the test substance to the cornea tissue. In this context the ICE provides better results, based on the different technique of administration and incubation of test sample. Additionally, the evaluation of the results is generated by a larger number of measurements and therefore artefacts can be excluded easier.
Depending on the physical state of the test substance, incubation time can differ between 10 minutes for fluids and surfactants and 4 hours for solids for BCOP. In contrast, incubation time for ICE lasts only 10 seconds independent of physical state.
Additionally, the scoring system differs between the tests. While in BCOP tests opacity is measured using an opacimeter, the ICE more specific criteria are analyzed using a slit lamp observation. This allows a more individual examination and less impact of interferences.
Keywords: BCOP, OECD 437, ICE, OECD 438, Isolated Chicken Eye
In vitro Evaluation of Hepatotoxicity by Amiodarone in HepatoPac (Micropatterned Co-cultured Hepatocytes) using Liver-specific Markers. (#693)
Z. Nereda1, C. Kegyes1, R. de Wilde1, Z. Gáborik1
1 SOLVO Biotechnology, Budaörs, Pest, Hungary
Drug induced liver toxicity (DILI) is a major reason for discontinuing drug development programs and identifying potential risk for DILI is therefore important. Several in vitro tests have been developed to assess hepatotoxicity risk. Models using polarized primary hepatocytes with functional drug metabolizing enzymes and transporters, such as sandwich cultured hepatocytes, spheroids, randomly cocultured hepatocytes and micropatterned cocultured hepatocytes (HepatoPac) are because of their in vivo-like properties considered to be among the more physiologically relevant models. A drawback of many in vitro tools however is lab-to-lab variability. Micropatterned co-cultures (HepatoPac) have a tightly controlled architecture and because of this we hypothesized its lab-to-lab variability might be minimal. In addition, the model has a life-span of several weeks in culture, allowing repeat dosing of test compounds. We therefore evaluated the known hepatotoxicant amiodarone in HepatoPac and compared results to published data.
Using albumin, urea, adenosine triphosphate (ATP) and glutathione (GSH), we found TC50 (the concentration that decreases a response by 50%) values close to published values. After single dosing, similar TC50 values were obtained on day 5 and 9 using intracellular ATP and GSH levels. After dosing every 2 days, we measured TC50 values of increasing potency over time, using albumin and urea levels in the medium. No toxicity was observed for negative control acetylsalicylic acid (aspirin) at any of the conditions.
We successfully used the HepatoPac toxicity assay in our lab and demonstrated low variability between our and published data, indicating the robustness of this method between labs.
Keywords: In vitro hepatotoxicity, Hepatopac, long-term incubation, repeat dosing, Human
In vitro model for the prediction of respiratory sensitization (#700)
A. Chary1, T. Serchi1, S. Cambier1, E. Moschini1, S. Contal1, J. Hennen2, J. Ezendam3, B. Blömeke2, A. C. Gutleb1
1 LIST, ERIN, Belvaux, Luxembourg
Exposure to chemicals in occupational scenarios or at consumer levels can lead to the development of respiratory allergies. At present, no in vitro method is available for the prediction of respiratory sensitization potential of chemicals.
To mimic the alveolar-capillary barrier we developed a 3D in vitro model grown at the air liquid interface including alveolar type II epithelial cells (A549), endothelial cells (EA.hy926), macrophages like cells (PMA differentiated THP-1) and dendritic like cells (DC) (non-differentiated THP-1).
Exposure to PA and TMA leads to the upregulation of the expression of CD54 on THP-1 cells up to respectively 185% and 204% relatively compared to vehicle controls while no increase was observed after exposure to irritants. The same was observed for expression of TSLPr with an upregulation up to 162% and 151% after PA and TMA exposure. After protein allergens exposures, OX40L expression was regulated up to 150% after HDM exposure and to 160% after Betv1 exposure, while OX40L expression decreases after irritants exposure, down to 85% for MeSa and to 62% for Acr. Gene expression and cytokines release allowed discriminating sensitizers from irritants.
In conclusion, the presented in vitro model represents a useful tool the prediction, in vitro, of respiratory sensitization, allowing discrimination between respiratory sensitizers, either chemicals or protein sensitizers, and respiratory irritants.
Keywords: in vitro, sensitization, 3D model, ALI, respiratory sensitization
Characterization of drug metabolizing enzymes, transporters and permeation in a human organotypic corneal tissue model (#713)
Y. Kaluzhny2, M. Kinuthia2, T. Truong2, A. Lapointe2, M. Klausner2, S. Letasiova1, P. Hayden2
1 MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
The corneal barrier is vitally important for eye protection, but also presents a significant challenge for delivery of ophthalmic drugs. Most current studies utilize excised animal corneas that are not suitable for rapid drug screening and also have poor species extrapolation and standardization. We developed a physiologically relevant, human-based in vitro 3D corneal tissue model (EpiCorneal) and evaluated its utility for ophthalmic drug development.
Tissues were characterized by histology, confocal microscopy, barrier function, and expression of genes essential for metabolism, detoxification, and drug transport. The organotypic corneal tissue model developed tissue structure, thickness and barrier formation (1000±146 Ω•cm2) comparable to native human corneal epithelium. The 3D corneal tissue expressed tight junctions, mucins, and key corneal epithelial detoxification enzymes. qPCR arrays were utilized to investigate expression of 84 key phase I/II metabolizing enzymes and 84 drug transporter genes in 3D corneal tissue and excised human corneal epithelium. Drug metabolizing enzyme and transporter gene expression were highly correlated (r2=0.87).
Permeability was evaluated using model compounds with a wide range of hydrophobicity and molecular weight, and different formulations of glaucomatous drugs Latanoprost and Bimatoprost. Coefficients of permeation for model drugs in tissue model and excised rabbit corneas showed a high correlation (r2=0.94). As expected, Latanoprost and Bimatoprost free acids had much lower permeability (Papp=1.2x10-6 and 1.9x10-6) than the corresponding prodrugs (Papp=2.5x10-5 and 5.6x10-5, respectively). The presence of 0.02% Benzalkonium chloride in ophthalmic formulations significantly affected tissue barrier and viability.
In summary, the model demonstrates in vivo like structure, barrier and drug metabolizing and transporter gene expression. Tissue permeability, as well as effects on viability and barrier of various known ophthalmic formulations is similar to excised corneas. EpiCorneal tissue model may be useful in the evaluation of corneal drug permeability and safety during the development of new ophthalmics.
Keywords: corneal epithelium, in vitro reconstructed tissue model, biocompartibility, ocular drug delivery, alternative to animal testing, drug metabolism, drug transport
Liver-Chip Identifies Mitochondrial Dysfunction, Oxidative Stress, and Innate Immune Response as Potential Pathways of Toxicity for the GPR40 agonist TAK-875 (#721)
K. - J. Jang1, M. Otieno2, J. Ronxhi1, K. Kodella1, J. Rubins1, D. Simic2, M. Singer2, H. - K. Lim2, W. Lam2, J. Chen2, D. Petropolis1, G. Kulkarni1, P. Guzzie-Peck2, K. Karalis1, G. Hamilton1
1 Emulate, Boston, Massachusetts, United States of America
Drug-induced liver injury (DILI) is a major reason for safety-related attrition in the pharmaceutical industry. Therefore, there is a need for in vitro models that can be used to consistently and reliably select compounds that have a reduced liability for liver injury. 3D liver models have the potential to revolutionize in vitro liver toxicity testing. We describe a vascularized Liver-Chip that contains primary hepatocytes, sinusoidal endothelial cells, Kupffer, and stellate cells in a spatial configuration that recreates their organization in the liver. Albumin secretion and CYP450s activities levels are maintained in culture for ≥2 weeks. The model can detect diverse liver pathophysiology, including hepatocellular injury, lipid accumulation, and stellate activation. The fluidic component provides physiological mechanical forces and allows for ease of sampling of effluent for detection of cytokines and DILI biomarkers (miR122, a-GST, CK-18, ccCK18, and total HMGB1). These endpoints were characterized with several tool compounds (e.g., APAP, fialuridine, methotrexate). The model was also used to investigate the mechanism of DILI by the GPR40 agonist TAK-875 following daily treatment at 3, 10, and 30 μM for ~ 2 weeks. There was significant turnover of TAK-875 and formation of its reactive acylgucuronide metabolite (TAK-875AG) on Day 1 (16%) and on Days 7/14 (44%) compared to 1% turnover obtained in human hepatocytes in conventional culture system. The oxidative metabolite (M-I) was formed at 4% in the Liver-Chip compared to reports of 10% found in humans. Inhibition of MRP2 expression, perturbation of mitochondrial membrane potential, and formation of reactive oxygen species and inflammatory cytokines were observed at concentrations bridging the clinical Cmax (10 μM). Interestingly, hepatic lipid accumulation and stellate cell activation were also observed at 10 μM. These data suggest that accumulation of a reactive TAK-875AG, cell stress, and an innate immune response are likely triggers for TAK-875 mediated liver toxicity. In summary, the Liver-Chip is amenable for microscopic, biochemical, and DILI biomarker evaluation and can be useful for predicting and mechanistic investigation of DILI.
Keywords: Liver-Chip, Drug discovery, Mechanism of toxicity, organ-on-a-chip
Inter- and intra-laboratory reproducibility of the in vitro photo-toxicity test using 3D reconstructed human epidermis model (#731)
H. Kandarova1, A. Liskova1, T. Milasova1, S. Letasiova1
1 MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Assessment of the phototoxicity hazard and phototoxic potency (i.e. phototoxic risk) of compounds and mixtures is a crucial step in the safety assessment of cosmetic, pesticide and pharmaceutical products absorbing UV and visible light. The validated and regulatory accepted in vitro assay, the 3T3 NRU PT (OED TG 432), provides high level of sensitivity, however, it has been reported that it also generates high rate of false positive results due to the lack of barrier properties naturally appearing in the human skin or other targeted tissues.
In vitro reconstituted human skin models are increasingly being investigated for their usability in hazard identification and safety testing, because of their organotypic structure with a functional stratum corneum that allows for assessment of bioavailability of topically applied compounds and mixtures. An in vitro phototoxicity test using the human reconstructed epidermis model EpiDermTM (EpiDermTM H3D–PT) has been developed and pre-validated almost 20 years ago and can be used either as standalone method for the phototoxicity testing of topically applied materials, or in combination with the 3T3 NRU PT, to minimise the potentially false positive results from this assay.
In the current study we internally validated the method with six reference substances, of which four were known phototoxins (chlorpromazine hydrochloride, two types of bergamot oil and anthracene) and two compounds were UV-absorbing, but without phototoxic potential (cinnamaldehyde, p-aminobenzoic acid). In the next step the method has been transferred into the second laboratory and the test was assessed with another set of 12 coded substances. The reproducibility of the predictions between the two laboratories was 100 % confirming the robustness of the protocol and the prediction model. Submission of the data for the implementation of the EpiDermTM H3D–PT into the regulatory framework is ongoing.
Keywords: phototoxicity, reconstructed human skin model, validation, EpiDerm, regulatory toxicology
Simultaneous Measurement of Contractility, Electrophysiology and Biomarker Secretion to Determine Cardiotoxicity in hiPSC Derived Cardiomyocytes (#736)
A. Fouassier1, S. Braam1, D. K. Tessa1, V. Marijn1, F. Farbod1, L. Greg1
1 Ncardia, Leiden, Netherlands
Many novel oncology therapeutics may induce cardiotoxicity by inhibiting survival pathways which are shared by both tumors and cardiac cells. Traditional methods to assess cardiotoxicity have relied upon in vitro overexpressing human cell lines or use in vivo animal models. These models often lack the complexity of human cardiomyocytes; whereas animal models may lack predictivity due to inherent species differences. Therefore, there is a need for the development of more predictive and specific assays that allow for multiparametric assessment of potential cardiotoxic side effects of new drugs in humans.
Using proprietary hiPSC-derived ventricular cardiomyocytes (Pluricyte® Cardiomyocytes) that recapitulate a human myocyte’s contractile and electrophysiological profile, as well as mature sarcomere organization, we developed a multiparametric assay to measure potential cardiotoxic effects in vitro. Here, both acute and long-term drug effects on contraction, electrophysiology and cardiac Troponin I release are determined simultaneously from each well of a 48 well plate.
To assess the effects of anticancer drugs on the physiology of Pluricyte® Cardiomyocytes, the cells were incubated with concentration ranges of Nilotinib, Lapatinib, Doxorubicin and Ponatinib for up to 64 hours, and analyzed simultaneously using microelectrode array (MEA), impedance and cardiac Troponin I (cTnI) release assays. This resulted in defined cardiotoxicity profiles for each compound. Whereas treatment with Nilotinib and Lapatinib caused a transient, short term (functional) contractile/electrophysiological deficit, Doxorubicin exhibited a continuous/long-term toxic effect in both MEA and impedance measurements. While Lapatinib and Nilotinib did not cause structural toxicity as measured by cTnI release; however, Ponatinib and Doxorubicin induced a dose-dependent increase in cTnI release. The dose-dependent increases in cTnI release correlated with reduced cell index values obtained in the impedance assay.
These data suggest that a multiplexed analysis is crucial to investigate short and long-term cardiac liabilities as it provides a more comprehensive readout that generates mechanism-specific cardiotoxicity profiles, leading to better prediction of drug-induced cardiotoxicity.
Keywords: Cardiomyocytes, cardiac safety, hiPSC, cardiotoxicity, contractility, Troponin I, MEA, in vitro
In vitro cytotoxicity and gene expression analysis of air-liquid interface model (Mucilair™) after exposure to PAHs (#342)
T. Cervena1, 2, K. Vrbova1, J. Topinka1, P. Rössner, Jr.1
1 Institute of Experimental Medicine CAS, Department of Genetic Toxicology and Nanotoxicology, Prague, Czech Republic
The morphology and culture conditions of widely used cell monocultures do not correspond to real conditions in the lungs and thus a more realistic system is needed. MucilAir™ represents 3D in vitro model consisting of human basal, goblet and ciliated cells at an air-liquid interface featuring a fully differentiated respiratory epithelium. The aim of our study was to compare the effects of short and long-term exposure of two representative polycyclic aromatic hydrocarbons [benzo[a]pyrene (B[a]P), 3-nitrobenzanthrone (3-NBA)] using MucilAir™ as a more realistic model of human lungs. Two concentrations (0.1, 1 µM) were tested at three time points (24 h, 1 week, 4 weeks). In this study, lactate dehydrogenase cytotoxicity assay (LDH), transepithelial electrical resistance (TEER) and gene expression analysis of selected genes involved in PAH metabolism and protection against oxidative stress was performed. Our results showed deregulation of antioxidant enzymes (AKR1C2, ALDH3A1, CAT, SOD1, COX2, HMOX1) after exposure. Downregulation of CAT and SOD1 following 1-week exposure was in agreement with higher cytotoxicity (LDH leakage) and TEER data. Expression of CYP1A1 gene did not show any significant changes. In summary, we observed negative effects of PAHs treatment in the 3D in vitro model that were mostly limited to long-term exposure. This work was supported by the grant of the Czech Science Foundation (16-14631S).
Keywords: 3D cell culture, PAHs, B[a]P, 3-NBA, mRNA
Predicting Hepatotoxicity: Integration of Transport, Metabolism and Bile Acid Regulation (#390)
C. Black1, J. P. Jackson1, M. K. Palmer1, C. L. Moseley1, K. R. Brouwer1
1 BioIVT, ADME-Tox, Durham, North Carolina, United States of America
Purpose: Hepatotoxicity from many drugs can involve cholestasis and accumulation of bile acids (BA). The ability to adapt to these effects can mean the difference between hepatocyte death and survival. Sandwich-cultured human hepatocytes (SCHH) can be utilized to evaluate the adaptive response of bile acid transporters stimulated by farnesoid X receptor (FXR) activation under cholestatic conditions. Integration of the adaptive response and measurement of direct hepatotoxicity was evaluated using 49 marketed compounds with a range of hepatotoxicity.
Methods: Cryopreserved, Transporter Certified™ SCHH were cultured using QualGro ™ Media for 5 days. On Day 4 of culture, the time course of the adaptive response was determined by determining the effect of cyclosporine A (CsA) on the biliary excretion, and intracellular concentration (ICC) of BA (LCMS analysis), in parallel with FXR activation (gene expression - TaqMan® primer/probe sets). The effect of FXR antagonists (troglitazone, DY268) on the FXR mediated activation of basolateral efflux transport were also evaluated. 49 compounds with varying degrees of BSEP inhibition and hepatotoxicity (NIH LiverTox database) were evaluated (24 hr exposure) for their potential to cause direct hepatotoxicity, and/or impact the adaptive response.
Results: CsA decreased the biliary excretion of endogenous bile acids in a time dependent manner, with a parallel increase in the ICC of BA, followed by activation of FXR. FXR activation resulted in a 2X increase in the biliary efflux clearance, and a 6X increase in the basolateral efflux clearance (adaptive response). Co-administration of FXR antagonists reduced the FXR mediated response by 50 and 95% for troglitazone and DY268, respectively. Integration of the effect on the adaptive response in addition to the effect on BSEP inhibition improved the accuracy for prediction of cholestatic DILI from 22% (BSEP inhibition alone) to 93%.
Conclusion: In addition to BSEP inhibition, integration of inhibition of basolateral efflux and/or interference with the adaptive response (FXR antagonism) allows for more accurate prediction of hepatotoxicity.
Keywords: hepatotoxicity, cholestasis, in vitro, human hepatocytes, BSEP Inhibition, FXR activation
A realistic mixture of Persistent Organic Pollutants (POPs) reveals possible synergism to inhibit the transactivation activities of the rat Aryl hydrocarbon Receptor (rAhR) in vitro (#62)
Q. T. Doan1, M. Muller2, H. F. Berntsen3, K. E. Zimmer4, S. Verhaegen3, E. Ropstad3, L. Connolly5, M. - L. Scippo1
1 Université de Liège (ULiege), Department of Food Science, FARAH, Liege, Belgium
While organisms are exposed to mixtures of persistent organic pollutants (POPs), scientific studies usually focus on the toxicity of a single compound at a time and few have addressed the mixture effect. This study aims to determine how a realistic mixture of POPs can affect transactivation of the rat Aryl hydrocarbon Receptor (rAhR) in vitro. Luciferase reporter Dioxin responsive rat hepatoma cell lines (DR-H4IIE) were used to screen both rAhR agonistic and antagonistic activities of 29 compounds: six perfluorinated (PFAA), seven brominated (Br), and 16 chlorinated (Cl) compounds (seven polychlorinated biphenyls (PCBs) and nine organochlorine pesticides) listed as POPs under the 2001 Stockholm Convention. Only five (two Cl and three Br) out of the 29 compounds presented rAhR agonistic activities while 16 (13 Cl and three Br) were rAhR antagonists. No effect was observed for PFAAs.
To test possible interactions between these compounds, a mixture of these 29 POPs and six sub-mixtures (PFAA, Br, Cl, Cl + Br, Cl + PFAA and Br + PFAA), prepared based on their concentrations found in Scandinavian human blood with a normal daily intake, were tested for the same activities. Not surprisingly, POP mixture also displayed a rAhR antagonistic activity (IC50 = 371 ± 52 times the blood level) with the lowest effective concentration found at 125-time blood level. This level could be plausibly reached in humans after a food contamination incident or in highly exposed sub-populations. Testing the sub-mixtures showed that the Cl mixture is responsible for the antagonism of the POP mixture, contributing to 80% of the POP response. When DR-H4IIE cells were exposed to the Cl + PFAA mixture, the antagonist level was the same as the response of the POP mixture. This indicates that PFAAs are probably non-specific rAhR antagonists as they did not induce any antagonist response when tested alone. The IC50 of the Cl mixture calculated from the measured IC50 of all 13 active chlorinated compounds, using an additive model, was about the same as the measured IC50, 1.9 µM and 2.3 µM, respectively. This suggests that these compounds act additively in the Cl mixture. In contrast, the calculated and measured IC50 for the total POP mixture were 21.8 µM and 43.2 µM, respectively, along with non-specific rAhR antagonism of PFAA mixture, indicating a possible synergistic effect.
Keywords: Persistent Organic Pollutants, DR-CALUX, Aryl hydrocarbon Receptor, Antagonistic, Synergistic mixture effect
Are epigenetic modifications linked to BPS obesogen effect after perinatal exposure? Differential DNA methylation study in mouse livers. (#116)
A. BRULPORT1, L. LE CORRE1, D. VAIMAN2, M. - C. CHAGNON1
1 University of Burgundy/AgrosupDijon, NUtox , UMR Inserm1231,, Dijon, France
In 2002, a correlation was established between the environmental pollution increase and the exponential evolution of the obesity epidemic. A few years later, the concept of obesogen was developed. Most of the known obesogens are endocrine disruptor compounds (EDCs). EDCs act at low doses and are important factors in the concept of DOHaD (Developmental Origins of Health and Disease). Epigenetic processes are important mechanisms in DOHaD concept and contribute to environmental adaptation and evolution. Epigenetic changes during the critical period of development are known to be involved in the development of obesity. This is particularly the case for bisphenol A (BPA), a monomer used mainly for the manufacture of plastics and resins. In European Union, following restrictions on the use of BPA, bisphenol S (BPS) is often used as a substitute. BPS, like BPA, is described as an EDC exhibiting estrogenic and anti-androgenic activities. We have previously demonstrated that perinatal exposure to a low dose of BPS via the drinking water (1.5 µg/kg body weight/day) had an obesogenic effect similar to BPA in male mice fed with a high fat diet. These mice show a liver triglyceride content increase without consequence at systemic level. The aim of our study was to determine if BPS can induce epigenetic modifications in liver of overweight male mice. Using an untargeted epigenetic approach by Reduced Representation Bisulphite Sequencing (RRBS), we observed a global hypomethylation of DNA liver, which is in correlation with studies performed with BPA in mouse model. By refining the analysis, we highlighted that differentially methylated CpGs relative to this obesogenic effect are notably located in promoters of 577 genes and in exons of 2.150 genes. Analysis of the targeted gene networks shows a potential impact in genes related to carbohydrate metabolism (closely linked to the liver lipid metabolism). Further analysis at mRNA and protein levels will confirm if BPS-induced liver homeostasis disruption is associated with differentially methylated CpGs in specific genes.
Keywords: BPS, endocrine disruptor, obesogen, epigenetic
Suggestions for the OECD’s Extended One-Generation Reproductive Toxicity Study to Further Understand the Temporality of Endocrine-Disrupting Effects from Chemical Exposure: Example using Bisphenol A (#253)
J. Choi1, W. Mune2, A. Joas1
1 BiPRO GmbH, part of Ramboll, Munich, Germany
The OECD’s “Conceptual Framework for Testing and Assessment of Endocrine Disrupters” has served as a guide to indicate which available test guidelines (TGs) can be considered for evaluating chemicals for endocrine disruption (ED). However, there are currently limitations with these TGs that hinder the identification of ED effects from chemicals. For example, there are concerns that exposures causing hormone insufficiencies or inappropriate hormonal action during development may cause effects that only become apparent later in life.
To gain an understanding of the extent to which such effects might not be detected by the current OECD TGs, a targeted literature search was undertaken, and a comparative analysis was conducted between studies following OECD TGs and those with similar experimental designs but a modified temporal element, e.g. altered exposure duration or study observation.
One of the more comprehensive TGs for testing ED effects in mammals is currently the Extended One-Generation Reproductive Toxicity Study (EOGRTS). The analysis revealed that in the EOGRTS exposure to bisphenol A (BPA) in mice starting from 2 weeks before mating of F0 parents up to postnatal day (PND) 90 of F1 offspring resulted in increased anogenital distance in F1 males at PND 21 and increased uterine weights in F1 females at PND 90. On the other hand, non-EOGRTS studies have shown that perinatal exposure (i.e. a shorter exposure duration than EOGRTS) to BPA in mice can trigger reproductive effects as well as affect mammary gland development and metabolism, which are two additional endpoints not directly measured in EOGRTS. Extended observation beyond EOGRTS revealed mammary tumor growth in mice perinatally exposed to BPA at 9 months of age.
In conclusion, exposure to BPA during early-life development (a critical window of chemical exposure) can induce ED effects. Suggestions for EOGRTS to further understand the temporality of ED effects from chemicals include extending the observation period of the exposed animals beyond PND 90 to reveal more severe reproductive effects such as cancer and adding assessments related to metabolism and mammary gland.
The project was funded by the European Commission, and its outcomes can be found in the EU Publication KH-01-18-009-EN-N. This work only reflects the views of the authors and not of the Commission.
Keywords: Endocrine-disrupting chemicals, OECD, Test guideline, Bisphenol A, Extended One-Generation Reproductive Study, EOGRTS
Hexachlorobenzene exposure induces cell migration and invasion through AhR, COX-2, ER and c-Src in human endometrial stromal cells (#254)
F. A. Chiappini1, L. Ceballos1, C. Pontillo1, N. Miret1, M. Núñez2, L. Álvarez1, M. Farina3, A. S. Randi1
1 Universidad de Buenos Aires, Facultad de Medicina. Departamento de Bioquímica Humana. Laboratorio de Efectos Biológicos de Contaminantes Ambientales, CABA, Buenos Aires, Argentina
Endometriosis is characterized by the implantation of ectopic endometrial tissue outside of the uterine cavity. Is a benign gynecological disease that shares some characteristics with malignancy like migration and invasion. Although the pathophysiology is unknown, endometriosis is likely multifactorial involving interplay between genetic predisposition and environmental conditions leading to the pathogenesis. Exposure to endocrine-disrupting environmental pollutants could play a role in the disease etiology. Hexachlorobenzene (HCB) is an organochlorine pesticide found in maternal milk and in lipid foods, that induces toxic reproductive effects in laboratory animals. Recently, we demonstrated that HCB enhances cyclooxygenase-2 (COX-2) expression, metalloproteinases (MMP) 2 and 9 activation and prostaglandin E2 (PGE2) secretion in human endometrial stromal cell line T-HESC. Pathological angiogenesis is the hallmark of endometriosis. Endometriotic growth is supported by the local hormonal and inflammatory microenvironment. The present study examined the effect of HCB in T-HESC on cell migration (scratch motility assay), cell invasion (transwell invasion assay), and expression of angiogenic factor Vascular Endothelial Growth Factor (VEGF) (Western Blot). Cells were exposed to HCB (0.005, 0.05, 0.5 and 5 μM) or vehicle for 24, 48 or 72 h. Results showed that the pesticide increases cell migration (47%; 81%, 77% and 96%, p<0.001) in a dose response manner and the invasiveness potential of T-HESC (200% p<0.05) through Aryl Hydrocarbon Receptor (AhR), COX-2, c-Src activation and estrogen receptor (ER). Moreover HCB (0.005-0.05 μM) induces VEGF secretion (130-150%, p<0.05), while VEGF intracellular content decreases at HCB (0.05-0.5 μM) (40-60%, p<0.05). Our results indicate that HCB exposure could contribute to maintenance and development of endometriosis promoting cell migration, invasion, and angiogenic factors secretion in human endometrial cells.
Keywords: HCB, Endometriosis, Cell migration, Cell invasion, Angiogenesis
Assessment of reproductive toxicity of male rats through maternal exposure to dienestrol (#324)
N. González1, E. Schreiber1, M. Torrente1, 2, V. Kumar1, 3, J. L. Domingo1, M. M. Gómez1, 4
1 Universitat Rovira i Virgili, Laboratory of Toxicology and Environmental Health, School of Medicine, Reus, Spain
Dienestrol is an abortive compound used to treat menopausal symptoms. It is also a diethylestilbestrol metabolite, which is a stilbene derivative with an estrogenic activity comparable to that of the natural estrogen, estradiol-17β. Dienestrol has also been classified as an endocrine disruptor altering endocrine system functions and causing possible harmful effects on the F0 and F1 reproductive system. Therefore, the aim of this study is to determine whether dienestrol affects male reproduction through maternal exposure with its ER-agonistic activity. Female Sprague-Dawley rats (90 days) were treated daily with gavage from gestational day (GD) 6, to ensure embryonic implantation, until postnatal day (PND) 21. Pregnant rats were divided into 8 groups (n=5) and the doses given were: control with vehicle (sunflower oil), 75, 50, 12.5, 6, 3, 1.5 and 0.75 µg/kg bw/day. Results reported that high concentrations of dienestrol caused abortions in all rats (75 and 50 µg/kg bw/day) and medium concentrations still caused some miscarriages (60% for 12.5 and 20% for 6 µg/kg bw/day). Lower concentrations allowed a normal pregnancy and delivery. During postnatal period (PND 1, 4, 7, 14 and 21), pup examinations were conducted to determine if the reproductive system was affected (anogenital distance (AGD) measurement and nipple retention count). At PND 21, male pups were anesthetized by intraperitoneal injection. Liver and testis were removed and weighed and cryptorchidism was assessed. Ten male rats per dose group were kept alive until PND 90 to ensure sexual maturity. Sexual organs (testis, prostate, seminal vesicle and epididymis) and liver were removed and weighed. Biochemical parameters, sperm and spermatid count, sperm motility, viability, maturity and morphology were assessed. As expected results, we expect to find differences in these physiological and spermatic parameters between the control group and the treated groups.
Keywords: dienestrol, endocrine disruptor, reproductive toxicity, male rats, maternal exposure.
Assessment of the oxidative stress effects on the reproductive system of young male rats to n-butylparaben (#356)
E. Schreiber Bru1, T. Garcia1, M. Torrente1, 2, V. Kumar1, 3, R. P. Sharma1, 3, J. L. Domingo1, M. M. Gómez1, 4
1 Universitat Rovira i Virgili, Laboratory of Toxicology and Environmental Health, School of Medicine, Reus, Spain
Parabens are a class of preservatives widely used in cosmetics, food and pharmaceutical products. There has been growing concern about its estrogenic effects on the reproductive system. The aim of this study was to determine reproductive abnormalities through subcutaneous exposure of n-butylparaben (n-ButP) are due to an oxidative stress. Young male Sprague-Dawley rats were subcutaneously exposed to n-ButP during one spermatogenic cycle (57 days). Given doses were 0, 150, 300 and 600 mg/kg bw/day of n-ButP with a vehicle (peanut oil). Moreover, a non-vehicle control group was also added to see if there were any effects from the peanut oil itself. Antioxidant enzymes activities (superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase) and levels of glutathione peroxided and reduced were measured in testis. Also, lipid peroxidation (TBARS) was assessed by a malondialdehyde assay. Gene and protein expression of catalase, glutathione reductase (GR) and glutathione peroxidase 4 (GPx-4) were also determined. Furthermore, a physiologically-based pharmacokinetic (PBPK) model was designed to assess internal dosimetry of n-ButP concentration in testis. Results show catalase, glutathione reductase and glutathione peroxidase alterations but no differences in reduced and oxidized glutathione were observed. Higher concentrations of n-ButP showed an increased level of TBARS. As expected results, we expect to find some differences in protein and gene expression of these enzymes which will allow us to relate the sperm and histological abnormalities with oxidative stress.
Keywords: n-butylparaben, oxidative stress, male rats, reproductive toxicity
Structure-activity relationships of Baikal seal estrogen receptors and environmental phenols (#487)
Y. Yoshinouchi1, M. Hirano1, H. Nakata2, K. Nomyamai1, S. Tanabe1, E. - Y. Kim3, H. Iwata1
1 Ehime University, Center for Marine Environmental Studies, Bunkyo-cho, Matsuyama, Ehime, Japan
Baikal seals (Pusa sibirica) accumulate high levels of environmental pollutants such as persistent organic pollutants (POPs). It is known that some of environmental pollutants elicit estrogen-like responses. However, no information is available for the effect of environmental pollutants on the estrogen receptor (ER) signaling pathway in aquatic mammals. There are still unanswered questions about the structural preference of environmental pollutants by ER and the molecular mechanism of ER activation. To predict the transactivation potency of environmental pollutants via Baikal seal ERs (bsERα and bsERβ), we measured the potencies of 26 bisphenol analogs (BPs), 17 hydroxylated PCB congeners (OH-PCBs), and 17β-estradiol (E2) by using the in vitro reporter gene assay system where bsERs were transiently expressed in COS-1 cells. Furthermore, the interaction energies of these compounds with bsERs were estimated by in silico ligand docking analyses. Screening results of BPs and OH-PCBs showed that most of the compounds transactivated bsERs. The in vitro and in silico analyses of bisphenol F analogs (4,4’-BPF, 2,4’-BPF, and 2,2’-BPF) and OH-CB30 congeners (4’OH-CB30, 3’OH-CB30, and 2’OH-CB30) showed that compounds with OH group at the para-position of phenyl rings were the most potent, followed by compounds with OH substitution at the meta- or ortho-position. In silico analyses demonstrated the interaction of OH group at the para position of 4,4’-BPF with M421 of bsERα, and of 4’OH-CB30 with E353. The EC50 values for tested chemicals showed a tendency to increase with an increase in the potential ligand-ER interaction energy, demonstrating a significant relationship between ligand-dependent ER transactivation and ligand-ER binding affinity. Using this regression curve, we calculated EC50values of OH-PCBs which are accumulated in wild Baikal seals. Comparison of EC50 values of E2 for bsER-mediated responses and total E2-IEQs (E2 induction equivalents) of OH-PCBs accumulated in wild Baikal seals suggested low potential of OH-PCB congeners to induce estrogen-like responses in this species. This study suggests that the in vitro ligand-dependent bsERα transactivation potency can be predicted by in silico ligand-bsERα interaction analysis.
Keywords: Baikal seal, estrogen receptor, hydroxylated PCB, bisphenol analog, SAR
Identification of endocrine active substances in medical devices within testing strategy according to ISO 10993-1 (#562)
A. Raemisch1, A. Mueller1, A. Deters1, A. Poth1
1 Dr. Knoell Consult GmbH, Health Care, Mannheim, Baden-Württemberg, Germany
The new regulation (EU) 2017/745 on medical devices was published on 5th April 2017 and will finally enter into force in 2022. The regulation sets high standards with regard to quality and safety of medical devices. Substances of high concern including endocrine disrupting substances shall not be part of the medical device (e.g. implants) at concentrations above 0.1% w/w. Otherwise a scientific justification including the potential patient exposure has to be provided. The proceeding of the identification of such substances is still under discussion for medical devices but also for other products within our daily life.
The evaluation of the biological tolerance of a medical device is a risk based approach and is evaluated according to ISO 10993 series. The biocompatibility is evaluated including material, chemical and physical characterization. In accordance with ISO 10993-1 the relevant biological endpoints are considered based on tissue contact and duration of contact. Endocrine disrupting properties are not part of this evaluation program. The question how to evaluate the potential risk for endocrine disruption is not yet considered in the biological evaluation of a medical device.
Based on the WHO classification an endocrine disrupting substance is a substance that interferes with the endocrine system causing significant adverse effects in humans and animals. There are some special properties of endocrine disrupting substances that cannot be evaluated within the scope of standard systemic toxicity tests including developmental specific effects and non-linear effect dose relationship. There are two possibilities for the identification of endocrine disrupting substances within the biological evaluation of a medical device: Chemical and toxicological characterization and biological tests
The standard biological tests usually conducted for biocompatibility evaluation are not appropriate for the identification of endocrine disrupting properties. Therefore, the chemical characterization followed by toxicological evaluation of leachable compounds is the most promising step for the evaluation of endocrine disruption.
The presentation should give a short overview on the evaluation strategy for potential endocrine disrupting compounds identified as component of a medical device especially for leachable compounds.
Keywords: endocrine disruptors, medical devices, leacheable compounds
Effect of n-butyl paraben on fertility of perinatally exposed F1 female progeny (#592)
P. V. Maske1, V. D. Dighe1, G. R. Vanage1
1 National Institute for Research in Reproductive Health (NIRRH), ICMR, National Center for Preclinical Reproductive and Genetic Toxicology, Mumbai, Maharashtra, India
Parabens are ubiquitously used class of synthetic preservatives which are esters of p-hydroxybenzoic acid (p-HBA); which are used as antimicrobial preservatives in cosmetics, pharmaceuticals, and food products. These are used singly or in combination to exert an antimicrobial effect. However, exposure to parabens has been report to affect male as well female reproductive functioning through estrogenic and/or anti-androgenic pathway, hence there is an emerging need to delineate its plausible role as an endocrine disruptor during perinatal period. The F0 dams were given subcutaneous injections of n-butyl paraben from gestation day 6 (GD 6) to postnatal day (PND) 21 with doses of 10, 100, and 1000 mg/kg Bw in corn oil. The F1 female rats were sub-fertile with increased pre- and post-implantation embryo loss and a concomitant decrease in litter size at PND 75. We observed impaired folliculogenesis with perturbed steroidogenesis at maturity might be the reason for compromised fertility of these F1 female rats. Altered expression of steroid responsive genes resulting in an inadequate number of ovarian follicles and regression of myometrium in the uterus affected the fertility and overall reproductive success. This study signifies an anti-estrogenic effect of n-butyl paraben on ovarian steroidogenesis and folliculogenesis.
Even though there is an emerging awareness of parabens with respects to its potential role as an endocrine disruptor, still there are many unanswered questions of research which demands substantial scientific evidences for foundation of ‘Paraben free’ scenario. This study is a step towards understanding whether the use of products containing parabens can be considered safe during pregnancy.
Keywords: Endocrine Disruptor, n-butylparaben, Fertility assessment, Perinatal exposure
Antiandrogenic effects of gibberellins in the recombinant yeast androgen bioassay (#615)
S. Stypuła-Trębas1, L. Radko1, P. Jedziniak1, A. Posyniak1
1 National Veterinary Research Institute, Department of Pharmacology and Toxicology, Puławy, Poland
Gibberellins (GAs) are naturally occurring tetracyclic diterpenoid phytohormones of higher plants, as well as secondary metabolites of filamentous fungi (Gibberella fujikuroi). Gibberellic acid (GA3) and gibberellin A4 (GA4) are the most active 3β-hydroxylated C19 GAs and thus are used in commercial formulations for plant growth regulation. These applications may potentially increase natural background levels of GAs in the environment as well as in food of plant origin.
The aim of our study was to examine the influence of GA3 and GA4 on the human androgen receptor (hAR) signaling with the use of the recombinant yeast bioreporter assay (Bovee et al. 2008). The compounds were dosed at 9 concentrations ranging from 1 nM to 300 µM alone or in binary mixtures with 17β-testosterone (17βT 100 nM and 5 µM). Each bioassay was performed two times, for each concentration 6 replicates were applied. Before and after the exposure, absorbance (λ=620nm) and fluorescence (λex/em=485/520nm) were measured. The cytotoxicity, androgenic and antiandrogenic activity of the compounds were assessed.
None of the chemicals at used concentrations were cytotoxic for the yeast cells. GA3 did not show any androgenic activity in the yeast androgen bioassay, whereas GA4 showed a very weak androgenic activity (EC50=152.3mM, RTA<10%). Moreover, GA4 revealed partial, dose-dependent antiandrogenic response in the presence of 50 and 100 nM of 17βT with the strongest inhibitory effect of 15.39% at the highest concentration. Although GA3 did not show antiandrogenic activity in the presence of 50 nM 17βT, it diminished the bioassay response by 22.93%.in the presence of 100 nM of 17βT.
Our study reveals that GA3 and GA4 possess the possible antiandrogenic activity. Further research is warranted to fill current gaps in knowledge, regarding hormonal activity of gibberellins.
Acknowledgments: The study was supported by the Leading National Research Centre (KNOW), Scientific Consortium “Healthy Animal- Safe Food”, Decision of Ministry of Science and Higher Education No 05-1/KNOW/2015.
Keywords: gibberellins, antiandrogens, yeast bioreporter
Investigation of Possible Effect of Carnosic Acid (CA) on Combined Exposure to Bisphenol A (BPA) And Diethyl Hexyl Phthalate (DEHP) (#641)
S. Özdatlı1, R. Kalkan1, M. Hatimoğlu2, U. Kilic3, A. Aydın2
1 Istanbul Medipol University, REMER (Regenerative and Restorative Medicine Research), Istanbul, Turkey
DEHP and BPA, the main components of plastic products, are the important endocrine-disrupting chemicals which can mimic or disrupt the actions of estrogen by inhibiting normal functions of the body. Because of this widespread usage of DEHP and BPA, not only alone exposure but also combined exposure is considered as an important issue. Nowadays, researches are increased related to the effects on neurological functions instead of endocrine disrupting effects. This study aims to investigate combined exposure of DEHP and BPA and the possible effect of carnosic acid as an antioxidant.
In this study, C-57BL/6 mice aged 16 weeks (25-35 g body weight) were randomly divided into 15 groups (6 mice/group) as only BPA and DEHP (16, 5, 1.6 mg/kg), 20 mg/kg CA, BPA+DEHP and BPA+DEHP+20 mg/kg CA (16, 5, 1.6 mg/kg). Chemical exposure was applied for 28 days by oral gavage. Level of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities in cortex were analyzed.
The SOD activity of all dosage of BPA+ DEHP and BPA+DEHP+20CA significantly decreased as compared to control, vehicle, 16 DEHP and 5 DEHP (except 5 BPA+DEHP+20CA). Not only the activity significantly decreased in 20 CA, 1.6 BPA+DEHP and all of BPA+DEHP+20CA dosages as compare to 16 BPA but also seen in 20 CA, 16 and 5 BPA+DEHP according to 5BPA. While significantly decreased CAT activity was seen in the combined usage of 16 and 1.6 BPA+DEHP+20CA versus 16 DEHP and 20CA; combined usage of 16 and 5 BPA+DEHP caused significant increment in the activity. Moreover, insignificant decrease was observed in all dosage of BPA, 5 and 1.6 DEHP. In MDA levels, combined of BPA+DEHP caused significant decrease according to vehicle. And also, no significant changes were observed in GSH-Px activity levels of BPA+DEHP+20CA and BPA+DEHP groups.
BPA and DEHP did not have an adverse effect on SOD and GSH activity when used alone; their combined use led to prominently decrease in SOD activity. This decrease in SOD enzyme may be due to increased oxidative stress or radical scavenging activity of CA. Although a significant decrease in CAT activity was observed when BPA and DEHP were used alone, insignificant decreased CAT activity seen with combined usage of BPA+DEHP+20 CA. That is, formed hydrogen peroxide was removed from the medium by CAT instead of GSH-Px. In our study, we found that BPA+DEHP or BPA+DEHP+20 CA may show its toxicity by increasing hydrogen peroxide.
Keywords: BPA, DEHP, neurotoxicity, carnosic acid
Prediction and evaluation of the internal dosimetry of two wide spread endocrine disruptors in fetus using a physiologically based pharmacokinetic model. (#644)
M. A. Martinez1, J. Rovira1, 2, R. Prasad Sharma2, M. Nadal2, M. Schuhmacher1, 2, V. Kumar1, 2
1 Universitat Rovira i Virgili, Environmental Engineering Laboratory, Departament d'Enginyeria Quimica, Tarragona, Spain
Bisphenol A (BPA) and Di-(2-ethylhexyl) phthalate (DEHP) are two wide spread chemicals classified as endocrine disruptors (ED) and categorized as chemicals of concern by the World Health Organization. Several studies have shown that the major human exposure to BPA and DEHP is the dietary source. However, some recent studies affirm that non-dietary sources (dermal, non-dietary ingestion and inhalation) may have major contribution in aggregate exposure and internal dosimetry. The aim of this study was to estimate the prenatal exposure to BPA and DEHP through dietary and non-dietary pregnant women’s exposure. To estimate prenatal exposure, a physiologically based pharmacokinetic (PBPK) model was adapted for pregnancy to assess the internal dosimetry levels of these EDs in the fetus. Estimates of exposure from all pathways along with their relative importance were provided in order to establish which proportion of the total exposure came from diet and which from non-diet. Total non-dietary mean values were 0.002 µg/kgbw/day (0.000; 0.004 µg/kgbw/day for 5th and 95th percentile, respectively) for BPA and 0.597 µg/kgbw/day (0.116 µg/kgbw/day and 1.506 µg/kgbw/day for 5th and 95th percentile, respectively) for DEHP. Indoor environments and especially dust ingestion were the main non-dietary contributors to the total exposure of BPA and DEHP with 60% and 81%. However, as expected, diet showed the higher contribution to total exposure with >99.9% for BPA and 63% for DEHP. Although diet was considered the primary source of exposure to BPA and phthalates, non-dietary sources need to be more thoroughly evaluated because with these sources the first-pass metabolism is lacking, so these may be of equal or even higher toxicological relevance than dietary sources.
Keywords: Bisphenol-A, Di-(2-ethylhexyl) phthalate (DEHP), PBPK modeling, exposure assessment.
Multi-omics Analysis Reveals that Co-exposure to Phthalates and Metals Disturbs Urea Cycle and Choline Metabolism (#665)
D. Sarigiannis1, 2, N. Papaioannou1, N. Kapretsos1, C. Gabriel1, E. Distel3, E. de Oliveira4, S. Karakitsios1, M. Aggerbeck3, R. Barouki3
1 Aristotle University of Thessaloniki, Chemical Engineering, Thessaloniki, Greece
The aim of this study was to obtain mechanistic insight into how co-exposure to phthalates and metals causes neurodevelopmental perturbations based on in vitro assays. The HepaRG cell line, a validated model for xenobiotic metabolism, was used as in vitro model. HepaRG cells were exposed to mixtures of DEHP, DiNP, and BBzP phthalates, methylmercury and total mercury. These were the most abundant pollutants in the REPRO PL and PHIME cohorts that studied the environmental causation of neurodevelopmental disorders in neonates and children across Europe. The effective concentrations of the chemicals in vitro were estimated through extrapolation from human biomonitoring data through internal dosimetry modeling using the INTEGRA computational platform. Multi-omics analyses were performed on the treated cell models including transcriptomics, proteomics, and metabolomics. The multi-omics data were analysed using bioinformatics algorithms involving normalization, quality control, advanced statistics, and multi-omics pathway analysis with GeneSpring GX V.14.9.
Integrated pathway-level analysis of transcriptomics and proteomics data revealed that co-exposure to phthalates and heavy metals leads to the perturbation of the urea cycle due to alterations in the expression levels of arginase-1 and -2, argininosuccinate synthase, carbamoyl-phosphate synthase, ornithine carbamoyltransferase, and argininosuccinate lyase. Co-mapping of proteomics and metabolomics data revealed that their common drivers are responsible for the homeostasis of metabolic pathways related to choline, phosphatidylcholine, phospholipases and triacylglycerol metabolism. The identification of the urea, phosphatidylcholine biosynthesis I and phospholipases metabolic pathways is of particular interest since these pathways have been also identified in human samples from the REPRO PL and PHIME cohorts using untargeted metabolomics analysis and have been associated with impaired psychomotor development in children at the age of three to six. Our work reveals that co-exposure to plasticizers and metals disturb biochemical processes related to mitochondrial respiration during critical developmental stages that are clinically linked to neurodevelopmental perturbations.
Keywords: neurodevelopment, phthalates, metals, multi-omics
In Vitro Kinetics and Quantitative In Vitro-In Vivo Extrapolation of Nephrotoxicants (#730)
N. Kramer1, F. Taverne1, A. Mally2, S. Jarzina2, B. Birk3, S. DiFiore4, B. Elliger5, R. Gehring1
1 Utrecht University, Institute for Risk Assessment Sciences, Utrecht, Netherlands
The predictivity of human nephrotoxicity by animal tests is low owing to the interspecies differences in active transporters and metabolizing enzymes. Thus the need for alternative, human-relevant in vitro nephrotoxicity assays is pressing. Within the Innosytox RISK-IT project, we have developed adverse outcome pathways (AOPs) for nephrotoxicity via lysosomal overload and mitochondrial DNA-interaction by exposing human RPTEC/TERT1 and rat NRK-5E cells to, amongst others, polymyxin B and tenofovir. A selection of in vitro biomarkers has been measured over time using high-content imaging, qPCR and metabolomics. For chemical potency, cell type and assay sensitivity ranking, as well as the quantification of AOPs, changes in in vitro biomarkers over time were compared to nominal and analytically measured medium as well as cell-associated concentrations of test chemical over time. Our results indicated that expressing effect concentrations as time-weighted, cell-associated concentrations reduced the variation in effect concentrations between cell types, assays and chemicals. We subsequently constructed a physiologically based kinetic (PBK) model explicitly modelling proximal tubule concentrations of the nephrotoxicants in rats and humans. We tested the different no-observed effect concentrations (NOEC) from the suite of in vitro bioassays to estimate rat and human equivalent doses in a quantitative in vitro in vivo extrapolation (QIVIVE) exercise.
Keywords: AOP, nephrotoxicity, PBPK, QIVIVE
Research on Environmental Exposure and Health Effects in Residents around Mining Areas in Mongolia (#84)
Y. S. Hong1, J. W. Seo1, S. H. Nam2, E. Altangerel3, D. S. Kim2
1 Dong-A University, Heavy Metal Exposure Environmental Health Center, Busan, Republic of Korea
Objective: The mining activity has accounted for more than 50% of the Mongolia industry. Mining related disease are increasing annually. Mining sector health problems pose important issues for Mongolia. We conduct health and exposure assessment of residents living near Umnugobi mining areas in Mongolia to WHO’s recommendation.
Methods: Environmental samples were collected in all 3 soum. In total: 27 water and soil samples from different sides were collected in accordance with standard method from 3 soums of Umnugobi aimag. A total of 118 children at 4th&5thgrade and 124 adults in 3 soums of Umnu gobi aimag were participated. We performed a questionaire survey and measured the level of lead, cadmium, copper, mercury, arsenic in blood and urinary arsenic, mercury in hair from all subjects.
Results: The geometric mean concentrations of blood lead in 3 soums adults(Bayandalai, Tsogttsetsii, Khanbogd) were 5.05ug/dl, 4.78 ug/dl and 4.56 ug/dl, respectively. The geometric mean concentrations of blood lead in 3soums children(Bayandalai, Tsogttsetsii, Khanbogd) were 7.42ug/dl, 5.13 ug/dl and 4.78 ug/dl, respectively. But, the level of As, Cu, Cd, and Hg in 3soums adults and children showed relatively low level compared to the international criteria.
Conclusion: Biomonitoring result shows that blood lead level significantly in all soums. It is considered that there is a need for such survey in other Mongolia mining areas.
Keywords: Mongolia, blood lead concentration, mining area, biomonitoring
Prenatal phthalate exposure levels in the Flemish 3xG cohort (#594)
M. De Soomer1, 2, N. Lambrechts1, S. Remy1, 3, E. Govarts1, B. Morrens4, E. Den Hond5, V. Nelen5, A. De Decker5, I. Loots4, G. Schoeters1, 2
1 VITO, Unit Environmental Risk and Health, Mol, Antwerpen, Belgium
Phthalates are known endocrine disruptors to which we are exposed daily since they are present in many consumer products. While experimental studies have demonstrated the harmful effects of phthalates, real life exposure during pregnancy and its implications for a developing child are less well studied. We assessed the prenatal exposure to 11 phthalate metabolites in a Belgian birth cohort (3xG, N = 300, sample collection 2011-2015). Second trimester pregnancy spot-urine levels were measured using Ultra Performance Liquid Chromatography (UPLC) coupled to Water Xevo TQ-S tandem mass spectrometer. We measured 4 metabolites of di-2-ethylhexyl phthalate (DEHP): mono-ethylhexylphthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP); two metabolites of Diisononyl cyclohexane-1,2-dicarboxylate (DINCH): Cyclohexane-1,2-dicarboxylate-mono-(7-hydroxy-4-methyl)octyl ester (OH-MINCH), Cyclohexane-1,2-dicarboxylate-mono(oxo-isononyl) ester (oxo-MINCH); and mono-n-butyl phthalate (MnBP), mono-benzyl phthalate (MBzP), mono-isobutyl phthalate (MiBP), mono-methyl phthalate (MMP). Especially OH-MINCH, oxo-MINCH and MMP have not been frequently studied before. The median (25th, 75th percentile) concentrations for the urinary metabolites corrected for urine dilution with creatinine (µg/g crt) were: MEHP 2.54 (1.5, 4.43), 5OH-MEHP 8.18 (5.45, 12.41), 5oxo-MEHP 6.03 (4, 9.05), MnBP 28.57 (20.22, 42.05), MBzP 6 (3.26, 10.95), MiBP 44.12 (28.46, 71.31), MMP 3.54 (1.96, 6.05), 5cx-MEPP 4.27 (3.01, 5.74), OH-MINCH 0.35 (0.22, 0.73) and oxo-MINCH 0.32 (0.19, 0.65). The proportion of samples above the LOQ was at least 88%. In comparison to the concentrations observed in the Polish cohort REPRO_PL which started in 2007 (urine spot samples in week 30 – 34 of pregnancy), the values observed in 3xG are higher for all phthalate metabolites. Compared to the INMA birth cohort set up in Sabadell (Catalonia, Spain, 2004-2008, mean concentration spot urine samples of 1st and 3d trimester) the observed concentrations are lower for all metabolites except for MiBP. In both cohorts DINCH and DMP were not measured. We are now further exploring the immune effects and the association with allergies of these compounds as well as the variables that might be related to phthalate exposure in pregnant women.
3xG is funded by NIRAS and the local partnerships MONA and STORA.
Keywords: phthalates, allergies, immune system
Biomonitoring of phthalate metabolites and bisphenols in amniotic fluid from pregnant women in Greece (#684)
I. Katsikantami1, 2, V. Karzi1, 2, M. N. Tzatzarakis1, S. Sifakis3, 4, P. Xezonaki3, E. K. Vakonaki1, A. Rizos2, B. Izotov5, T. Burykina6, A. M. Tsatsakis1
1 University of Crete, Medical School, Laboratory of Toxicology and Forensic Sciences, Heraklion, Greece
Introduction: The possible associations between prenatal exposure to endocrine disrupting chemicals and developmental effects in fetus triggered the investigation for the presence of seven phthalate metabolites (PMs) (MEP, MEHP, MEHHP, MEOHP, MiBP, MnBP, MBzP) and three bisphenols (BPA, BPF and BPS) in amniotic fluid.
Methods: A total of 100 amniotic fluids were collected from pregnant women during 2016-2017. Enzymatic hydrolysis was carried out (10 μL E. coli, phosphate buffer 250 μL, pH 6.8) in 1 ml of amniotic fluid and incubated at 37οC for 90 min. Samples were acidified and extracted three times with 2 ml ethyl acetate for 20 min. Organic extracts were combined, evaporated to dryness and reconstituted in 100 μL methanol prior to instrumental analysis with LC-MS.
Results & Discussion: Recoveries for PMs ranged from 79.5% (MnBP) to 116.1% (MiBP) and for bisphenols from 92.0% (BPA) to 93.4% (BPF). LODs for PMs ranged from 0.016 (MEHHP) to 0.853 (MEP) ng/ml and for bisphenols from 0.013 (BPS) to 0.075 (BPA) ng/ml. The 4% of the samples were positive for BPF (median: 1.0 ng/ml, range: 0.4-3.7 ng/ml), 17% for BPA (1.7 ng/ml, 1.2-5.8 ng/ml), 22% for MEHP (2.3 ng/ml, 1.4-7.0 ng/ml) and 5% for MnBP (10.7 ng/ml, 2.4-24.5 ng/ml). Positive samples for MBzP, MiBP and MEOHP were below 3% and MEP, MEHHP and BPS were not detected in any sample. Despite their low detection frequencies, high levels were detected for MiBP, MBzP and MnBP (102.9, 20.2, 24.5 ng/ml) The 25% of the samples were detected with one PM and 4% with two. Only 3 samples were burdened with both bisphenols and PMs indicating their different sources of exposure.
Conclusion: BPA was the mostly detected bisphenol as it is most commonly used in consumer products. DEHP is a ubiquitous phthalate and its primary metabolite (MEHP) was the most abundant, although its oxidative metabolites (MEHHP, MEOHP) were not detectable. Placenta tissue is a barrier for endocrine disruptors, although not totally effective because some compounds are transferred from mother and reach the fetus.
Keywords: phthalate metabolites, bisphenols, amniotic fluid, pregnant women, biomonitoring
A refuted association between soil contamination from past antimony mining and exposure of children to inorganic arsenic (#703)
L. Perharic1, M. Martinčič1, V. Zadnik2, S. Uršič1, D. Mazej3, Z. Šlejkovec3, M. Šter4, A. France-Štiglic4, A. Kukec1
1 National Institute of Public Health, Centre for Envronmental Health, Ljubljana, Slovenia
In 2016, we determined urinary arsenic (As) in children aged 3-5 years from two areas of the Zagorje upon Sava municipality (central Slovenia) in order to assess an association between soil contamination related to previous antimony (Sb) mining and exposure to inorganic As (IAs). The national Medical Ethics Committee approved the study. We recruited the participants by on site presentations and personal letters, adverts in the media and social websites. Following the parental informed consent, we obtained detailed health, environmental and dietary history. The concentrations of total As, its species (arsenite-AsIII, arsenate-AsV) and metabolites (monomethylarsonic acid-MMA and dimethylarsinic acid-DMA) were determined using inductively coupled plasma mass spectrometry, and coupled high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry, respectively. Levels of quantification for total As were 0.3 µg/L; for AsIII and MMA 0.9 µg/L; for AsV and DMA 1.5 µg/L. Levels of detection for total As were 0.1 µg/L; for AsIII and MMA 0.3 µg/L; for AsV and DMA 0.5 µg/L. Urinary creatinine was determined by the Jaffe's reaction; the acceptable concentrations were between 0.3 and 3.0 g/L. The results were judged as statistically significant at p ≤ 0.05. In total 72 children of both genders were enrolled. In spring, 45 first morning urine spot samples were obtained, in autumn 69. Due to over-dilution, insufficient volume or seafood consumption within 72 h prior to sampling, 20 urine samples were excluded from statistical analyses. In spring, total urinary As concentrations (TUAsC) were 4.1 (50th percentile-P50) and 8.4 µg/L (75th percentile–P75) in the eastern and northern (EN) area with history of Sb mining, and 6.5 (P50) and 7.9 (P75) µg/L in the western and southern (WS) area with no history of Sb mining. In autumn, TUAsC were 3.7 (P50), 4.9 (P75) and 19.6 (95th percentile-P95) µg/L in the EN, and 4.1 (P50), 5.3 (P75) and 11.3 (P95) µg/L in the WS. In spring, inorganic urinary As concentrations (IUAsC=∑ AsIII+AsV+MMA+DMA) were 3.6 (P50) and 4.4 (P75) µg/L in the EN; 3.1 (P50) and 3.9 µg/L (P75) in the WS. In autumn, IUAsC were 3.4 (P50), 4.7 (P75) and 8.9 (P95) µg/L in EN, and 3.7 (P50), 4.5 (P75) and 7.7 (P95) µg/L in WS. The exposure to IAs did not differ significantly geographically or seasonally. We concluded that soil contamination with As due to past Sb mining was not associated with increased IAs exposure in children.
Keywords: urinary arsenic, environmental exposure, cross-sectional study, human biomonitoring
Chronic exposure to low concentration of di-(2-ethylhexyl) phthalate adversely affects aging indicators in Caenorhabditis elegans (#55)
V. H. - C. Liao1, C. - M. How1, P. - L. Yen1
1 National Taiwan University, Bioenvironmental Systems Engineering, Taipei, Taiwan
Purpose: There is an increasing concern regarding the contamination of phthalates in food. Among phthalates, di-(2-ethylhexyl) phthalate (DEHP) is one of the most commonly used plasticizers. There are growing evidences indicating that DEHP might be released from food packaging and other plastic products, thereby posing a potential health risk and food safety. Herein, this study investigated whether chronic exposure to DEHP at low concentration affects aging in the model organism Caenorhabditis elegans.
Methods: Wild-type N2 C. elegans at L1 larvae were exposed to a low concentration of DEHP (1.5 mg/L) to adult at 20oC, followed by examining aging indicators including lifespan, locomotion (head threshes), pharyngeal pumping rate, and defecation cycle.
Results: The results showed that chronic exposure to DEHP significantly decreased the lifespan of C.elegans (DEHP-treated vs. control, p < 0.001). In addition, DEHP exposure caused a more significant decline in head threshes with aging than the untreated control worms at day-0, -4, and -8 adulthood. Moreover, chronic exposure to DEHP accelerated the decrease of pharyngeal pumping rate by 13% and 10% comparing with the control ones at day-4 and day-8 adulthood, respectively. Finally, chronic exposure to DEHP significantly extended the defecation cycles of C. elegans (65.4 and 83.0 sec) comparing with the untreated control ones (59.2 and 76.1 sec) at day-4 and day-8 adulthood, respectively.
Conclusions: Results from this study indicated that chronic exposure to low concentration of DEHP (1.5 mg/L) adversely affects aging indicators which might potentially accelerate aging process. This study implies that chronic exposure to a low concentration of DEHP cause a higher risk of premature aging.
Keywords: chronic exposure, aging indicator, C. elegans, DEHP
Refined assessment and perspectives on the cumulative risk resulting from the dietary exposure to pesticide residues in the Danish population (#126)
M. O. Larsson1, F. Laporte1, N. Bjerre1, V. S. Nielsen1, N. Cedergreen2
1 Bayer Crop Science, Crop Science, Copenhagen, Denmark
Relatively few studies are available on realistic cumulative risk assessments for dietary pesticide exposure.
Despite available studies showing low risk, public concern remains. A method to estimate realistic residue levels
based on information from spraying journals and supervised residue trials was described in a previous publication.
The present article proposes a new method to estimate average residue levels in imported foods based
on residue monitoring data and knowledge about agronomic practices. The two methods were used in combination
to estimate average pesticide residue levels in 47 commodities on the Danish market. The chronic consumer
exposure was estimated in six Danish diets. The Hazard Index (HI) method was used to assess consumer
risk. Despite the conservative (cautious) risk assessment approach, low HI values where obtained. The HI was
16% for adults and 44% for children, combining the risk of all pesticides in the diet. Conclusion: the present
study adds support to the evidence showing that adverse health effects of chronic pesticide residue exposure in
the Danish population are very unlikely. The hazard index for pesticides was lower than that of commonly occuring mycotoxins, and substantially lower than hazard quotients for caffeine and alcohol. Hazard Quotients for caffeine and alcohol were calculated based on average Danish consumption data, and with endpoints and safety factors similar to what is normally used for pesticides and mycotoxins.
Keywords: pesticide residue, cumulative risk assessment, food safety, dietary pesticide exposure
Investigation of pyrrolizidine alkaloids – cytotoxicity tests using primary rat hepatocytes and HepG2 cells: Role of cytochrome P450 3A (#142)
L. Gao1, L. Rutz1, K. - H. Merz1, D. Schrenk1
1 University of Kaiserslautern, Food Chemistry and Toxicology, Kaiserslautern, Rhineland-Palatinate, Germany
Background: Pyrrolizidine alkaloids (PAs) are secondary metabolites occurring in a wide range of plant species. Some 1,2-unsaturated PAs exert toxic effects through metabolic activation which form the corresponding dehydropyrrolizidine derivatives, primarily in the liver, catalyzed by cytochrome P450 monooxygenases. Due to their hepatotoxicity, genotoxicity and carcinogenicity, the presence of PAs as contaminants in food and feed, depending on the dose, has become a relevant safety issue.
Objectives: In order to assess potential risks and confirm the connection between structure and in vitro toxicity, we generate data firstly concerning cytotoxicity of some food-relevant PAs. In addition, we also wanted to test in vitro the role CYP3A plays in metabolic activation of PA-induced cytotoxicity.
Methods: After 24 h and 48 h exposure, cytotoxicity of the selected PAs was determined at concentrations ranging from 1 to 300 µM by the Alamar blue assay in primary rat hepatocytes and in the human HepG2 cell line. A kinetic assay analyzing 7-benzyloxyresorufin-O-dealkylation (BROD) was used for measuring the activity of CYP3A enzymes.
Results: A structure dependent cytotoxicity was demonstrated with rat hepatocytes in primary culture. Lasiocarpine, an open-chained di-ester with 7S-structure, proved to be the most cytotoxic followed by the other di-esters echimidine, retrorsine, seneciphylline and senecionine. The mono-esters heliotrine, indicine, europine and lycopsamine were much less cytotoxic. On the contrary, failure to detect cytotoxicity in HepG2 cells is possibly due to the lack of CYP3As. In primary rat hepatocytes, the CYP3A activity decreased rapidly during the culture, therefore, the time to incubation significantly affects the cytotoxicity. These data confirm that CYP3A plays a critical role in PA-induced hepatotoxicity.
Keywords: pyrrolizidine alkaloids, food contaminants, metabolic activation, structure-dependent cytotoxicity, Alamar blue assay
Estimation of theoretical oral exposure of insecticides residues in grain stocks (#148)
S. Sergeiev1, M. Prodanchuk1, O. Kravchuk1, A. Grynko1, N. Kolontaeva1, V. Lyshavskyi1
1 L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Kyiv, Ukraine
Insecticides belonging to pyrethroid (deltamethrin) and organophosphorus compounds (pyrimiphos-methyl and chlorpyrifos-methyl) are used in Ukraine for pest control of grain stocks (GS). Treated GS can be a significant source of insecticides residues that can affect humans at oral intake. Oral exposure can be considered as a function of hazard, residual quantities (RQ) and exposure duration. In turn, the hazard is determined by the parameters of toxicity that form the acceptable daily intake (ADI), which is a criterion for assessing the hazard. RQ in products forms the theoretical daily intake (TDI) into body or magnitude of exposure. The possible duration of exposure is characterized by the period of retention of insecticide in foodstuff.
The purpose of the study. Integral estimation of theoretical oral exposure of insecticides RQ in GS.
Materials and methods. Analyzed the following indicators: ADI of active ingredient (AI), residues of AI in GS (gas-liquid chromatography), TDI of AI with grain in human body, dissipation periods of AI in grain (DT50) obtained with study of AI decline in grain stocks. The axonometric method (geometric addition of vectors) is applied: each indicator (ADI, TDI, DT50) or vector evaluated in marks, then calculate the geometric sum of marks – integral exposure vector (R). R was evaluated according to the proposed scale.
Results. It was found that deltamethrin is characterized by low, pyrimifos-methyl – high, and chlorpyrifos-methyl – very high R. In last two cases, pyrimiphos-methyl has the highest mark of TDI due to high RQ, and chlorpyrifos-methyl has the highest marks for all three vectors – ADI, TDI and DT50. Proposed method for establishing an integral exposure vector allows predicting the oral exposure of insecticides RQ after pest control of grain stocks. Exposure is proportional to the insecticides toxicity, amount of residues and duration of their dissipation period in food. Integral exposure vector has an indicator value and can serve as basis for next evaluation of insecticides residues in grain processing products and exposure when consuming these products.
Keywords: grain stocks, insecticides residues, oral exposure
Hierarchical Bayesian exposure assessment of dietary polycyclic aromatic hydrocarbons in 3-6 year old Finns (#179)
T. M. Hirvonen1, M. Hokkanen1, P. Pasonen1, L. Uusitalo2, M. Erkkola2, L. Korkalo2, P. Tuominen1, A. Mikkelä1
1 Finnish Food Safety Authority Evira, Risk Assessment Unit, Helsinki, Finland
Polycyclic aromatic hydrocarbons (PAHs) are genotoxic carcinogens. Concentrations of PAH compounds (Benzo[a]pyrene (B[a]P), chrysene, benz[a]anthracene, benzo[b]fluoranthene), and their sum (PAH4) were determined in bread, ready-to-eat breakfast cereals, fats and oils, and traditionally smoked meat and fish using a gas chromatography-tandem mass spectrometry (GC-MS/MS) method. PAH concentrations from literature were also used in food concentration estimation. Hierarchical Bayesian approach was used to estimate PAH concentrations in foods. Consumption of different food products was modeled using a 1-5 days dietary recall of 815 children. Daily consumption was modeled using log-normal distribution, and the consumption frequency using a binomial model to take into account the variation between different individuals in consumption. Finally, the chronic intake (B[a]P and PAH4) resulting from consumption of each food product was obtained as a combination of the mean concentration and long-term consumption. The resulting posterior distribution of Bayesian model describes the variation and uncertainty in the chronic intake and other quantities of interest. BMDL10 of 0.07 mg/kg bw./day for B[a]P and 0.34 mg/kg bw./day for the PAH4 were used for risk characterization1. Margin of exposure (MOE) was calculated by dividing BMDL10 by exposure. Total mean exposure of B[a]P was 0.83 ng/kg bw./day and for PAH4 5.4 ng/kg bw./day. Major proportions of exposures for B[a]P were bread (25%), sausage (21%), and fats and oils (20%) and for PAH4 bread (27%), sausage (25%), and fats and oils (17%). 97.5th percentile of total exposure for B[a]P was 1.5 ng/kg bw./day and for PAH4 9.7 ng/kg bw./day, respective MOEs being 47 000 and 35 000. In conclusion, dietary PAH exposure in 3-6 year old Finns was at acceptable level.
Keywords: polycyclic aromatic hydrocarbons, exposure, food, Bayesian modelling
Safety evaluation of plants collected from the wild served as food in Danish restaurants. (#237)
M. M. Egebjerg1, P. T. Olesen1, F. D. Eriksen1, G. Ravn-Haren1, L. Bredsdorff1, K. Pilegaard1
1 Technical University of Denmark, National Food Institute, Kgs Lyngby, Denmark
Within the last decade the New Nordic Cuisine has received much media coverage. The restaurants have focused on increased use of locally grown plant food, including wild plants collected from the countryside. In addition, many cookbooks and guided nature walks have assisted interested consumers in the search for wild plants for culinary purposes. As part of a control campaign running from May-October 2016, the Danish food authorities investigated the use of plants picked from the wild, cultivated in private gardens or market gardens in restaurants and local food producers. Here we present examples of safety evaluations of some of the 50 plant species identified from this campaign based on published phytochemical investigations and toxicological data in humans. In the period from February to October, 2017, searches were performed in databases on bibliographic information using the preferred scientific name, and if relevant also synonyms. The full scientific papers were obtained if abstracts described ethnobotanical studies on food use in European countries prior to 15 May 1997 (the date the first novel food regulation came into force), constituents (especially if toxicological relevant), experimental laboratory animal studies on the toxicological effects of the plants, or cases of intoxications in humans or animals exposed to the individual plants. For the majority of the plants no or very limited phytochemical and safety information were available. Additionally, we found that of the 50 plants reviewed almost half contained compounds with toxic or potentially toxic effects if eaten. For many of the remaining plants, the data was insufficient to establish a safe edible amount. Many of the species may be considered novel food according to the EU regulation, since a food use to a significant degree in EU member states prior to 15 May 1997 could not be established. This review has demonstrated a strong need for better information on novel food status and safety of plants picked from the wild or plants previously mainly cultivated e.g. for ornamental use but now introduced as food, so that food producers, chefs and writers of cookbooks also in future have a stronger attention on whether the plants are safe to eat.
Keywords: Novel food, wild edible plants, review, food safety, toxicology
The safety assessment of proteins in food: where do we stand? (#268)
A. Fernandez Dumont1, C. Paoletti1, J. Gomez Ruiz1, M. Ardizzone1, A. Lanzoni1
1 European Food Safety Authority, Parma, Italy
Purpose: the risk assessment of proteins is critical for food safety authorities worldwide. The adverse effects from exposure to proteins can be life-threatening as in case of anaphylactic reactions. While various guidelines detailing how to deal with the assessment of protein safety are available, no global consensus exists. Scientific advances and the evolution of risk assessment tools should be used to re-think the protein safety assessment strategy, upon which a global consensus could be achieved. Here we provide state-of-the art considerations paving the road to do so.
Methods: The safety assessment of proteins relies on information of different nature providing the necessary weight-of-evidence to estimate risks. However, the current paradigm borrows the classical principles and methodologies developed for assessing “chemicals”, i.e. small molecules. But proteins are large and complex biological molecules, needing dedicated considerations for hazard and exposure assessments.
Results: the safety assessment of proteins could be streamlined. First, more informative tools for protein analysis and a clearer framing of exposure-scenarios are necessary to better inform the risk assessment strategy to follow. For example, improved in silico analysis approaches, coupled with thorough criteria to interpret outcomes should be pursued. Targeted in vitro testing tools should be further developed and validated in robustness and reliability to integrate and possibly replace animal models. A promising way forward is the development of more informative in vitro digestion tests. Second, other tools and in particular animal models should be used to obtain final decisive evidence on protein safety only when the initial protein and exposure assessment is not sufficient to conclude on the safety. Currently, well-established acute, 28-day, 90-day toxicity studies designed for “chemical” assessment are used, but their relevance for protein safety assessment is questioned. A deep revision of the use of animal models for protein safety is needed to establish when they are informative and what parameters should be considered. Finally, innovative ways to link the outcome of the protein assessments with post market realities are necessary to better inform and continuously improve the strategy used.
Keywords: food safety, protein assessment, allergenicity, toxicity
In vitro study of the cytotoxic effects of microcystin-LR, cylindrospermopsin and their mixture in the neuronal SH-SY5Y cell line (#272)
M. G. Hinojosa1, D. Gutierrez-Praena1, A. I. Prieto1, L. Espinar-López1, A. M. Cameán1, A. Jos1
1 University of Sevilla, Area of Toxicology. Faculty of Pharmacy, Sevilla, Spain
Anthropogenic activities and climate changes are contributing to the increase of the appearance of cyanobacterial blooms, capable of producing harmful secondary metabolites known as cyanotoxins, such as microcystin-LR (MC-LR) and cylindrospermopsin (CYN). Despite being mainly considered as a hepatotoxin and a cytotoxin, respectively, both toxins seem to be able of causing neurotoxicity in different experimental models, although studies concerning this aspect are still scarce, especially with CYN. In the present work, the human neuroblastoma SH-SY5Y cell line was used to assess the cytotoxicity induced by MC-LR and CYN pure standards and their mixture, since their simultaneous occurrence is frequent in nature. For this purpose, cells were exposed to 0-100 µg/mL MC-LR and 0-5 µg/mL CYN for 24 and 48 hours. Cytotoxicity was evaluated using the MTS tetrazolium salt reduction (MTS), the neutral red uptake (NR) and the total protein content (PC) assays. The MTS assay was performed to evaluate the mixture at EC50, EC50/2 and EC50/4 concentrations for both periods of time. The results of this combination were also evaluated using the CalcuSyn software, in order to establish the type of interaction of the mixture (additive, synergistic or antagonistic effect). In the MTS assay, an EC50 value of 32.21±1.89 µg/mL after 24 hours of exposure and 20.8±2.08 µg/mL after 48 hours was obtained for MC-LR. In the case of CYN, the EC50 values were 0.87±0.13 µg/mL and 0.32±0.08 µg/mL after 24 and 48 hours, respectively. Regarding to the mixture, an antagonistic effect was observed at low concentrations at both exposure periods, although a synergistic trend was observed at the highest concentrations assayed after 24 and 48 hours. Therefore, our results show the neurotoxic potential of both cyanotoxins, showing for the first time in vitro, the high sensitivity of neuronal cells to CYN and its combination with MC-LR.
Acknowledgments: Spanish Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE).
Keywords: microcystin, cylindrospermopsin, neurotoxicity, SH-SY5Y cell line
Health effects of red meat products containing phytochemicals and reduced levels of nitrite: the PHYTOME project (#290)
S. G. van Breda1, T. M. de Kok on behalf of the PHYTOME consortium1
1 Maastricht University Medical Center, GROW School for Oncology and Developmental Biology, Department of Toxicogenomics, Maastricht, Netherlands
Purpose: It has been proposed that endogenously formed N-nitroso compounds (NOCs) are partly responsible for the link between red meat consumption and colorectal cancer (CRC) risk. As nitrite has been indicated as one of the critical factors in the formation of endogenous NOCs, it is of high importance to reduce the nitrite levels in meat. Therefore, the PHYTOME project was initiated (Phytochemicals to reduce nitrite in meat products; www.phytome.eu), a major EU funded research project aiming to develop innovative meat products in which the food additive nitrite has been replaced by natural compounds originating from fruits and vegetables.
Methods: A human dietary intervention study was conducted in which healthy subjects consumed 300 grams of meat for two weeks, in subsequent order: normal processed red meat, white meat, and red processed meat with normal or reduced levels of nitrite and added phytochemicals (normal-nitrite or low-nitrite PHYTOME meat, respectively). Before and after each intervention period, colorectal biopsies, urine and faeces were collected for analyses of different markers of effect.
Results: Apparent total N-nitroso compounds (ATNC) in faecal water, as measure of total NOCs, were significantly higher following processed red meat intake when compared to intake of white meat, normal-nitrite - and low-nitrite PHYTOME meat. In addition, DNA strand breaks induced in ex-vivo faecal water exposed CACO-2 cells and O6-methyl-guanine adducts levels in colonic DNA were significantly higher after consumption of normal processed red meat as compared to white meat intake. Furthermore, normal-nitrite and reduced-nitrite PHYTOME meat intake resulted in reduced levels of these genotoxic markers; however, these effects were not statistically significant. Whole genome gene expression analyses identified differentially expressed genes and co-expressed genes which are related to molecular pathways which explain cancer risk initiation after intake of processed red meat and cancer risk prevention after intake of the PHYTOME meat. Together these results indicate that addition of natural extracts to conventional processed red meat products results in reduced formation of NOCs, and may therefore contribute to a reduced risk of CRC, which is mechanistically supported by gene expression analyses.
Keywords: colorectal cancer, phytochemicals, N-nitroso compounds, prevention, red meat and nitrite
A proof-of-concept study: Evaluating the Applicability of High-Throughput Screening Data and Read-Across Tools for Food Relevant Chemicals (#355)
J. L. Nguyen5, 5, A. Maier2, J. Ovesen2, N. Kleinstreuer3, R. Judson4, M. Krishan5
2 University of Cincinnati, Department of Environmental Health, Cincinnati, Ohio, United States of America
Alternative toxicity methods to characterize the hazards of chemical substances have been proposed to reduce animal testing and efficiently screen thousands of chemicals. Relevant resources include large in vitro datasets from efforts such as the high-throughput screening (HTS) Tox21/ToxCast programs and read-across tools such as the Organization for Economic and Cooperation Development (OECD) QSAR toolbox. The goal of this work is to compare the results from traditional toxicity studies with predictions from these alternative testing methods for food relevant chemicals in ToxCast. Computational models developed using Tox21/ToxCast HTS data were used to predict the activity of food relevant chemicals against the estrogen receptor (ER) and androgen receptor (AR) pathways. We identified 94 putatively estrogen- or androgen-active food relevant chemicals in ToxCast. To reduce possible confounding from cytotoxicity and cell stress, the ER and AR model results were filtered based on observed in vitro cytotoxicity, which resulted in 89 putatively active, non-cytotoxic food chemicals. As a proof of concept, we further shortlisted these 89 chemicals based on the availability of in vivo data related to developmental and reproductive toxicity (DART). This resulted in 10 putatively active, non-cytotoxic, endocrine disrupting chemicals. Structural similarity and similar mode of action were used to identify potential analogues for the 10 chemicals. We used read-across approaches to compare the pattern and potency of DART for the analogues to that of the target chemical. These methods identified 3 target chemicals for which the analogue approach and computational models successfully predicted the DART potential of the food relevant chemicals. This study demonstrates that Tox21/ToxCast HTS assay data can be used for prioritization along with weight of evidence from read-across tools to evaluate food relevant chemicals, although the limitations in the approaches are evident. This abstract does not necessarily reflect EPA policy.
Keywords: Read-across, OCED Toolbox, Food and Chemical Safety, DART, ToxCast
Hepatotoxicity of Pyrrolizidine Alkaloids in Human Hepatocytes and Endothelial Cells (#371)
J. Glück1, J. Ebmeyer1, J. Waizenegger1, C. Luckert1, A. Braeuning1, A. Lampen1, S. Hessel-Pras1
1 Federal Institute of Risk Assessment, Food Safety, Berlin, Berlin, Germany
Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites produced in a wide variety of different plant species as a defense mechanism against herbivores. Humans are exposed to these substances via the consumption of contaminated food. PA intoxication in humans causes severe hepatotoxicity: while acute intoxication leads to veno-occlusive disease, hepatomegaly, ascites and liver hardening, chronic PA intoxication is characterized by hepatic necrosis, fibrosis and cirrhosis. It is considered that PA toxicity is based on PA metabolites formed during metabolic activation in the liver. The molecular mechanisms of PA toxicity are not well understood.
In this project we aimed to elucidate the mode of action of several structural different PA. To identify signaling pathways affected by PA a whole transcriptomics µ-array was performed with four PA in human primary hepatocytes, comprising all structural characteristics of hepatotoxic PA. Though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure.
Since there were hints for induction of cholestasis and apoptosis, these pathways were investigated in detail. PA exposure showed structure- and concentration-dependent cytotoxicity in HepaRG cells. An apoptotic potential of PA was revealed for two retronecine-type PA. PA structure- and time-dependently induced depolarization of the mitochondrial membrane potential, nuclear as well as DNA fragmentation and also an increase in pro-caspase cleavage and activity. Effects on the cholesterol and bile acid metabolism were shown in down-regulation of various transporters and metabolizing enzymes.
However, effects on the endothelial cells were not included in these studies. Therefore, we used HUVEC cells to simulate sinusoids of the liver. PA were bio-activated by using S9 fraction resulting in a decrease in HUVEC viability, an increased expression of the FAS receptor and of different interleukins, along with opposite-directed regulation of the two genes PTGIS and PTGS2 that encode enzymes involved in prostanoid synthesis. Non-metabolized PA produced no relevant effects in gene expression analysis.
Keywords: pyrrolizidine alkaloids, hepatotoxicity, cholestasis
Transcriptome responses in Caco-2 cells to micro- and nanoparticles in food-grade titanium dioxide (E171) and potential impact on colon cancer development (#382)
J. Briede1, 2, H. Proquin1, 2, T. M. de Kok1, 2, H. van Loveren1, 2
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
Titanium dioxide (TiO2) is used as a food additive, known as E171 in Europe, and provides a white colour. Recent studies showed that E171 ingestion causes a significantly increased number of colorectal tumours in colorectal cancer mouse model while in rats it also induces inflammatory responses in the intestines. E171 consists of particles of different size fractions that can be distinguished in nanoparticles (NPs) and microparticles (MPs). In order to obtain more insight into the cellular responses related to colon cancer development evoked by E171 at the transcriptome level, including the relative contribution of the NPs and MPs, was investigated by whole genome microarray analyses. Furthermore, the capacity of E171, TiO2 NPs and MPs to induce oxidative stress, DNA damage was assessed by using electron spin spectroscopy, the comet assay and the micronucleus test. The capacity for ROS generation in a cell-free environment was the highest for E171 followed by NPs and MPs. Only MPs were capable to induce ROS formation in exposed Caco-2 cells. E171, MPs and NPs all induced single-strand DNA breaks. Chromosome damage was shown to be induced by E171, as tested with the micronucleus assay in HCT116 cells. Transcriptome analysis showed that E171, NPs, and MPs induce gene expression changes related to pathways involved in signalling, inflammation, immune system, transport, and cancer devleopment. Exposure to NPs showed similar effects as E171 resulted in gene expression changes in TLR cascade, MHC class I and II presentation, late cornified envelope, potassium channels, and cell cycle. Exposure to MPs induced similar changes as E171 in gene expression related to Hedgehog family, α-defensins, cadherin and cholinergic receptors. Overall gene expression changes were associated with the immune system and inflammation induced by E171, but also by the MPs and NPs, suggesting the creation of a favourable environment for cancer development, without showing a clear distinction between the two different size fractions.
Keywords: Titanium dioxide, E171, Transcriptomics, colon cancer, nanoparticles
3-MCPD-induced organ toxicity is based on oxidative stress (#388)
K. Schultrich1, T. Buhrke1, C. Henderson2, R. Wolf2, A. Braeuning1, A. Lampen1
1 German Federal Institute for Risk Assessment, Department of Food Safety, Berlin, Berlin, Germany
3-Chloro-1,2-propanediol (3-MCPD) fatty acid esters are food contaminants which are formed during the refinement of vegetable oils. After ingestion, the esters are hydrolyzed in the gastrointestinal tract to release free 3-MCPD. 3-MCPD has been classified to be possibly carcinogenic to humans (category 2B) and is therefore in the focus of food safety authorities.
Previous studies suggested that oxidative stress plays a role in 3-MCPD toxicity. In the present study, so-called HOTT reporter mice were employed to elucidate the impact of 3-MCPD on cellular oxidative stress in more detail. These transgenic reporter mice contain a gene cassette in their genome in which the lacZ reporter is under the control of the heme oxygenase 1 (HO-1) promoter which is responsive to oxidative stress induced by reactive oxygen species (ROS). ROS-mediated lacZ gene expression can be visualized by LacZ staining of tissue slices by using X-Gal. HOTT reporter mice were daily treated with 0, 1, 10, 100 mg 3-MCPD per kg of body weight over a period of 28 days. Subsequently, the mice were sacrificed, and tissue slices were prepared from kidneys, testes and brain and subjected to LacZ staining.
In the mouse kidney cortex a dose-dependent increase of blue stain was observed that was exclusively visible in the renal cortex and not in the renal medulla. In mouse testes LacZ positive cells were detected on the basal membrane in the seminiferous tubules. The LacZ staining in the tubules was intensified with increasing 3-MCPD doses. In mouse brain a dose-dependent increase in LacZ positive cells was detected in specific areas within the cerebellum, midbrain and pons. In all three organs, the amount of irreversibly oxidized DJ-1 protein which is a biomarker for high ROS burden was dose-dependently increased by 3-MCPD. 3-MCPD-mediated ROS induction in mouse kidney, testes and brain was furthermore confirmed by a dose-dependent induction of gene expression of ROS-responsive genes such as heme oxygenase 1 or catalase and by the observation of an increased amount of 3-hydroxynonenal-induced protein adducts indicating increased lipid peroxidation.
Taken together, these data demonstrate that 3-MCPD induces ROS formation in mouse kidney, testes, and brain. Further studies will focus on the molecular mechanisms of 3-MCPD-mediated ROS induction.
Keywords: 3-MCPD, food contaminant, oxidative stress
Effects of some controversial food additives on zebrafish embryonic development (#420)
D. Duy Thanh1, F. Van den Bossche1, 2, N. Lai Thanh3, M. Muller1
1 University of Liege, Laboratory for Organogenesis and Regeneration, GIGA Institute, Liege, Belgium
Background information: The rising concerns about potential hazardous properties of food additives requires extensive, yet animal-minimized, testing strategies. For that purpose, the zebrafish embryo is the ideal model system which is amenable to high-throughput in vivo assays.
Method: In this study, we exposed zebrafish wildtype and transgenic fluorescent embryos to different concentrations of ten controversial food additives: the preservative Sodium benzoate (SB); the flavour enhancer Monosodium glutamate (MSG), the sweetener Aspartame (Asp), and colouring agents Tartrazine (TTZ), Quinoline yellow (QY), Sunset yellow (SY), Azorubine (Az), Ponceau 4R (P4R), Allura red AC (AR), and Brilliant blue (BB). Data was recorded and analysed for morphological and lethal effects, remarkable biological/functional outcomes are further investigated with emphasis on neurodevelopment and neurobehaviour.
Results: Effects of each substance on zebrafish embryonic morphology and lethality were determined as well as the corresponding concentration-response relationship. Calculated toxicological indexes (LC50, EC50, and teratogenic index TI) revealed that SB belongs to Cat.3 aquatic toxicity class, while QY and BB are highly teratogenic. Remarkably, adverse effects of BB and P4R present a multiphasic dependence on concentration, with BB appearing to weaken the embryonic yolk sac. SB exposure decreased the zebrafish motoneuron differentiation rate, while Az exposure stimulated the hatching rate. Behavioural studies revealed a synergistic effect on locomotive activity due to a mixture of Asp/MSG.
Conclusion: Our results demonstrate that the zebrafish embryo is a cost-effective model for high throughput screening of food additives’ safety and toxicity. This in vivo model allows systematic detection of biological effects, especially those unexpected by targeted in vitro and in silico models. Also, our data suggest the need to reconsider the safety of food additives SB, BB, and QY as well as other controversial food additives in further studies.
Keywords: food additives, zebrafish embryo, developmental toxicology, in vivo
A tool to integrate scientific risk assessment principles into the management and prioritization of chemical contaminants in raw materials. (#460)
T. STROHEKER1, G. SCHOLZ1, P. MAZZATORTA1
1 Nestle Research Center, Lausanne, Vaud, Switzerland
We have developed a new globally applicable scientific tool for the assessment and prioritization of chemical hazards in food raw materials. The tool is based on the four principles of risk assessment, considering the overall exposure to chemical contaminants. From these calculations, and the application of a decision tree, the tool provides the user with a level of risk (or “likelihood to cause harm”). For each individual contaminant, a severity grade was set according to the use of an additional decision tree based on toxicological characteristics of the chemical (e.g. carcinogenicity, reprotoxicity or developmental toxicity). Both decision trees implies scientific, objective and transparent selection criteria. Taken together, severity and risk are positioned in a matrix informing on the prioritization level of each combination of chemical hazard and raw material. The proposed model is intended to be adequately protective for consumer’s health, as it considers a conservative food intake scenario, as well as various sources of contaminant exposure. The model’s design is flexible and can easily be adapted to the needs of different food product categories and scenarios. The model was tested using several examples, the results of which are consistent with existing data in the literature.
Keywords: Chemical contaminants, risk assessment, Food raw material, HACCP, Food safety
Animal-free strategies to prioritize substances of concern: case studies with food contact materials (#512)
B. Mertens1, M. Van Bossuyt1, 2, T. Vanhaecke2, V. Rogiers2, E. Van Hoeck1
1 Sciensano, Chemical and Physical Health Risks, Brussels, Belgium
Humans are exposed, intentionally or not, to a large variety of substances most of which have not (recently) been evaluated for their safety. Environmental pollution and contaminated food are important sources of non-intentional exposure to non-evaluated substances. A detailed characterization of the complete toxicological profile of all these substances is not feasible from an economic and ethical (animal welfare) point of view. In silico models, however, are useful tools to assign priority to those substances for which a comprehensive safety evaluation is most urgently needed. In several recent studies, we have illustrated the potential of in silico tools for the priority setting of substances that can be used in different types of food contact materials (FCM). Despite their multiple benefits in food handling and processing, the safety of these FCM should be assessed as migration of compounds from FCM into the food has been reported. However, the safety assessment of FCM substances is currently hampered by the lack of a clear and harmonized legislative framework. For non-plastic FCM types for example, no specific regulation on the starting products exists. Furthermore, guidance on the assessment of non-intentionally added substances (NIAS) (e.g. impurities, oligomers, degradation products and neoformed compounds), which can migrate both from plastic and non-plastic FCM, is currently missing.
An overview of different strategies combining in silico tools with literature data and results from in vitro experiments to prioritize FCM substances will be presented using case studies. For the prioritization of starting products in non-plastic FCM, the advantages and limitations of the approaches developed for coatings and printed paper and board FCM will be summarized and discussed. In addition, a strategy that was developed to assign priority to substances migrating from plastic baby bottles will be used to demonstrate the potential of in silico based strategies for the assessment of NIAS. The different case studies clearly show the potential of animal-free prioritization strategies, not only for FCM but also in numerous other domains where there is a need to identify substances of concern to human health.
Keywords: in silico, food contact materials, safety assessment
Pathogenic gene profiles of Listeria monocytogenes isolated from food and environment in Korea (#530)
Y. Y. Bae1, Y. S. Lee1, B. J. Kim1, M. R. Choi1, J. H. Hwang1, S. H. Kim1, H. S. Kwak1
1 Ministry of Food and Drug Safety, Food Microbiology Division, Cheongju, Republic of Korea
In order to identify causative agents of foodborne outbreak, genetic characteristics of isolated strains could be critical information as well as rapid isolation and identification of pathogen from suspected foods. Listeriosis is serious infection usually caused by eating food (soft cheeses, sprouts and cantaloupe etc.) contaminated with the Listeria monocytogenes.
Keywords: Salmonella, Foodborne Pathogen, Pathogenic gene profiles, Genetic characteristics
Acute and sub-acute oral toxicity study of fermented platycodon extract in rats (#536)
J. - D. Heo1, J. Choi1, J. Kim1, S. - J. Lee1, Y. - G. Moon1, W. - S. Kim1, T. - K. Tak1, J. - H. Lee1, K. - H. Hwang1
1 KOREA INSTITUTE OF TOXICOLOGY, Gyeongnam Department of Environmental Toxicology & Chemistry, Jinju-si, Republic of Korea
The platycodon is referred as the root in Korea. The roots are commonly used for treating bronchitis, asthma, tuberculosis, diabetes, and other inflammatory diseases.The present study aims to evaluate the safety of ferment platycodon extract (fP) for the development of health food or natural therapeutics. We performed acute and sub-acute oral administration (2 weeks repeated) in SD rats. In sub-acute study, the fP was administered to male and female SD rats at oral doses of 2000 mg/kg (acute study) and 0, 250, 500 and 1000 mg/kg (sub-acute study). Experimental animals were monitored for the mortality, body weight changes, food intake, clinical signs and gross findings during observation or administration period. Sub-acute toxicity study was also accompanied by hematological and biochemical analyses. In the above studies, we concluded that fP has weak toxicity in SD rats.
Keywords: Platycodon, General toxicity
Zearalenone (mycotoxin) metabolism in urine of farmed pigs and cattle (#541)
P. T. Koivisto1, M. Kolmonen1, P. Peltola1
1 Finnish Food Safety Authority Evira, Chemistry, Helsinki, Finland
Materials and methods
Zearalenone is hormonally active mycotoxin that may occur in feed (and food). Zearalenine is metabolized to analogous resorcylic acid lactones zeranol and taleranol among others. Most sensitive animal is pig, but also human exposure is possible through grain. Human exposure has been estimated small. Metabolism of zearalenone is well understood. Regulatory limits for zearalenone in grain is 100 ug/kg in pig feed and 500 ug/kg for cattle.
Urine samples were from official monitoring of banned hormones zeranol and taleranol on year 2017. Analysis of pig and cow urine sample was performed with UHPLC-MS/MS technique from purified urine samples after deglucuronidation.
On average, most abundant metabolite was β isomer of zearalenol (10 µg/L) in cows urine, while in pig urine was α isomer (2.3 µg/L) respectively. Zeranol concentration was 0.57 and 0.07 (cow/pig) and taleranol 1.8 and 0.14 ug/L respectively. Zearalenone mycotoxin was also detected (5.2 pig and 4 µg/L in cow) in most of the samples which indicated that sources of metabolites was most probably through mycotoxin contamination on feed. Zearalenone level in feed samples has been below regulatory limits. One sample was analyzed without enzymatic hydrolysis, in which there was not metabolites detected.
Metabolites exists mainly as conjugated forms (glucuronides), which indicates less harm of mycotoxin contamination to farm animals.
Decreasing of harmful metabolites can be made by decreasing levels of zearalenone in feed.
Keywords: mycotoxin, metabolism
Concern for adverse effects of huperzine A when sold as an ingredient in food supplements (#550)
L. Bredsdorff1, K. Pilegaard1
1 Technical University of Denmark, National Food Institute, Kgs. Lyngby, Denmark
Huperzine A is marketed as a cognitive enhancer for healthy individuals when sold as an ingredient in food supplements. Huperzine A is, however, an acetylcholinesterase (AChE) inhibitor with potential for serious adverse health effects if administered in the doses recommended for food supplement use (up to 900 µg/d). Huperzine A acts by inhibiting AChE, thus preventing the degradation of endogenous acetylcholine, which leads to disrupted neurotransmission that may result in a variety of symptoms such as salivation, defecation, muscle fasciculation, convulsions and death.
Purpose: The aim of this study was to make a risk assessment of huperzine A from food supplements based on the available literature. Method: A systematic review of the literature covering the bibliographic databases SciFinder®, PubMed and Web of ScienceTM was performed to search for toxicological data on huperzine A. Results: Very few toxicological studies were identified in the literature search. However, several articles cite a series of unpublished studies submitted to the FDA as part of an investigational new drug submission. The study information is sparse and only provides the species, dose range, duration and a short summary of end-points. None of the studies are longer than 180-days. The observed adverse effects can all be related to AChE inhibition but due to the limited information none of the studies are suitable for establishing a health-based guidance value such as an Acute Reference Dose (ARfD) or Acceptable Daily Intake (ADI). One study in humans showed that administration of 200 µg huperzine A resulted in an erythrocyte AChE-inhibiting activity >20% for almost 5 hours post-dose. A specific cut off value of 20% for brain or erythrocyte AChE inhibition is considered to differentiate between adverse and non-adverse effects. Huperzine A is under investigation for use as a drug for Alzheimer’s disease. Adverse effects like diarrhoea, nausea and vomiting have been reported in these studies, where the investigated doses generally are half the highest dose recommended by vendors of food supplements. Whereas, a certain degree of side effects is accepted for drugs, the same effects should be unacceptable from food supplements intended for healthy individuals.
Keywords: Huperzine A, acetylcholinesterase, food supplement, toxicity
Food contaminant mycotoxin altertoxin II: cell membrane as target (#572)
G. Del Favero1, D. Marko1
1 University of Vienna, Faculty of Chemistry, Department of Food Chemistry and Toxicology, Vienna, Wien, Austria
Cell membrane can be seen as the first interface of the cells with the extracellular environment. As such, it plays a prominent role in mediating the first contact with toxicants. Food contaminant mycotoxins are no exception and, regardless the exposure route, cell membrane can be considered a very relevant toxicological target at single cell level. In spite of that, for some mycotoxins little is known about the effects on cellular membrane. Among the emerging mycotoxins there are the secondary metabolites of Alternaria alternata, which can be found as contaminants in several food commodities, especially after storage. Alternaria alternata molds produce hundreds of different metabolites, but the comprehension of the mechanism of action of altertoxin II (ATXII) is particularly relevant due to its mutagenic and genotoxic potential [1, 2]. In this study, the effect of ATXII was investigated on two different intestinal cell models HCEC-1CT human colonic epithelial cells and HT-29 intestinal adenocarcinoma cells. The effect on the membrane was measured with confocal microscopy and live cell imaging, as well as functionally, with protocols of biomechanical stimulation. HCEC-1CT cells appeared to be more sensitive than HT-29 cells to the effect of the toxin, thus undergoing severe and concentration dependent morphological alterations after incubation with ATXII. Moreover, alteration of the membrane morphology was accompanied by a higher sensitivity to biomechanical stimulation (i.e. shear stress) that down-streamed in the intracellular compartment, ultimately affecting actin cytoskeleton. In conclusion, cell membrane proved to be an extremely sensitive target for ATXII and its study opens new perspectives in the comprehension of the toxic potential of food borne mycotoxins.
Keywords: Altertoxin II (ATXII), mycotoxins, cell membrane
Fusarium mycotoxins exposure assessment through diet and urine analytical study. (#590)
Y. Rodriguez-Carrasco1, D. Carballo1, E. Ferrer1, G. Font1, H. Berrada1
1 Valencia University, Food Science Toxicology, Burjassot, Valencia, Spain
Fusarium is regarded as one of the most important mycotoxigenic fungi genera. Fusarium spp. can produce several mycotoxins of which the trichothecenes, fumonisin and zearalenone are the most important ones based on occurrence and toxicity. The research toward the analytical determination and occurrence of Fusarium toxins in several food commodities as well as in human urine from different population groups are very useful tools to carry out exposure population to mycotoxins. In the developed work, a multi-mycotoxin method based on gas chromatography-tandem mass spectrometry was developed and optimized with high resolution and sensitivity for determining fifteen mycotoxins and metabolites in selected food and human urine. Moreover, a QuEChERS-based extraction taking profit of high recovery and sample throughput, high speed and simplicity, the use of low-solvent as well as the low glassware usage and bench space.
A considerable number of matrices (n= 500) including wheat-based products, different types of bread beer and wine were analyzed. Additionally, mycotoxins urinary levels from 54 individuals were also investigated. The methodologies employed in each investigated matrix were properly validated according to current legislations.
Results showed that up to 80% samples were contaminated at least with one mycotoxin being deoxynivalenol (DON) the most frequently detected toxin. Moreover, HT-2 co-occurred in less than 10% of total samples. The overall average of all positive samples was 22.5 µg/L for DON and 2.5 µg/L for HT-2.
Despite the important incidence, average values obtained were much lower than maximum limits. The exposure assessment carried out in several food commodities showed that probable daily intake of the studied mycotoxins was below the tolerable daily intakes (TDI). Nonetheless, when mycotoxin urinary levels were employed to assess the exposure to mycotoxins higher exposure data were obtained , and even when consuming a contaminated food with DON below legal limit, the TDI could be exceeded being children the most susceptible group.
Ministry of Economy and Competitiveness (AGL2016-77610-R) and Government Scholarship program “Carlos Antonio López – Paraguay”
Keywords: mycotoxins, wheat based food, urine, exposure assessment
CO-PRESENCE OF MYCOTOXINS IN TUNISIAN FEED SAMPLES (#595)
C. Juan1, S. Oueslati2, 3, A. Juan-García1, J. Mañes1, H. Berrada1
1 University of Valencia, Laboratory of Food Chemistry and Toxicology, Burjassot, Valencia, Spain
A performed QuEChERS method was applied to analyze the co-presence of twenty-one mycotoxins (ENs, BEA, AFBs, OTA, AME, AOH, TENT, DON, 3ADON, 15ADON, NIV, NEO, DAS, T-2 and HT-2 toxin) in 122 Tunisian market feed samples, including for poultry (n=43), cattle (n=35), rabbit (n=12), sheep (n=16) and horse (n=16). It was previously validated according to the European Commission Decisions (2002/657/EC) and applied. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) were used for the detection. For the first time, mycotoxins presence was evaluated in animal feed from Tunisia intended for different animal species.
The validated method presented good linearity (R2>0.985) and sensitivity, the limits of quantification ranged from 0.1 ng g-1 (ENA1) to 225 ng g-1 (3ADON). Average recoveries (n=3) for all studied feed samples were ranged between (82±28) % for OTA to (104±27) % for BEA. Intra-day (n = 6) and inter-day precision (n = 9), expressed as relative standard deviation, were lower than 16% and 20%, respectively.
The 85% of analyzed feed samples were positive for at least one mycotoxin. Poultry (n=43) and rabbit (n=12) feed samples were the most contaminated. ENB was the most prevalent mycotoxin with values ranged between 0.5 ng g-1 for horse feed and 40 ng g-1 at poultry feed, followed by DON detected from 16 ng g-1 in cattle feed to 250ng g-1 in chicken feed. None positive sample exceeded the maximal amounts set by EU legislations for animal feed (Directive 2003/100/EC; Recommendation 2006/576/EC; Recommendation 2013/165/EU). Mycotoxins co-occurrence was observed in all positive samples at least by three different mycotoxins (21%). A relatively high rate of feed contamination especially by-fusarotoxins was registered. Even if no toxicological concern was revealed, the contamination is a fact and the co-contaminated samples might exert adverse effects due to the mycotoxins potential interactions.
Keywords: Mycotoxins, QuEChERs, feed, co-occurrence, mass spectrometry
Toxicity and metabolism of deoxynivalenol in human hepatoma (HepG2) cell line (#613)
L. Radko1, A. Tkaczyk1, P. Jedziniak1, S. Stypuła-Trębas1, A. Posyniak1
1 National Veterinary Research Institute, Department Pharmacology and Toxicology, Puławy, Poland
Introduction and aim. Contamination of deoxynivalenol (DON), a trichothecene mycotoxin, in cereal grains is becoming a major problem in agricultural and food industry. The potential risks of DON on human and animal health may occur after ingestion of contaminated foods or feeds. The better understanding of molecular responses of human cells to DON and metabolic responses of in vitro system to the exposure to the mycotoxin was the main goal this study.
Methods. The cytotoxic potential of the mycotoxin was investigated using human hepatoma (HepG2) cell line. The four biochemical endpoints were assessed by means of mitochondrial (MTT) and lysosomal (NRU) activity, total cell protein content (TPC), and membrane integrity (LDH) after 72 incubation with DON at concentrations ranging from 0.1 to 100 µg/ml. Additionally, concentration of DON in the medium from HepG2 cultures were determined using liquid chromatography coupled with mass spectrometry (LC-MS/MS).
Results. The cytotoxicity of DON was concentration- and assay- dependent. The mycotoxic in the lowest used concentration (0.1 µg/ml) significantly decreased viability of the cells. The degree of DON toxicity strongly varied, depending on the cytotoxicity parameter evaluated. DON compromised viability according to the parameters of lysosomal activity, mitochondrial activity, membrane integrity and total protein content. The EC50 values for DON in HepG2 cultures were <10 µg/ml. DON was poorly metabolized by HepG2 cells.
Conclusion. The toxicity of DON was higher in comparing in other studies on HepG2. The increase of risk for human health related to DON due to its presence in food commodities all over the world, highlights the importance of these studies in toxicology and in food science. Among that, studies on interaction DON with other mycotoxins and xenobiotics are needed to better clarify the metabolism and toxicity of study mycotoxin.
Funding source: The study was supported by the Leading National Research Centre (KNOW), Scientific Consortium “Healthy Animal- Safe Food”, Decision of Ministry of Science and Higher Education No 05-1/KNOW/2015.
Keywords: deoxynivalenol, cytotoxicty, metabolism, HepG2 cells
90-day toxicity and genotoxicity studies with high-purity phenylcapsaicin (#618)
S. Stiller1, T. R. Paulsen2, K. Weber3, C. Donath4, G. Schreib4, K. H. Jensen2
1 BSL BIOSERVICE Scientific Laboratories Munich GmbH, Planegg, Bavaria, Germany
The use of capsaicin in agriculture, food industries and pharmacology is limited by cost and availability but synthetic capsaicin analogues, such as phenylcapsaicin, can solve problems related to both quality requirements and volumes needed by the different industries.
To evaluate the safety of the synthetic capsaicin analogue phenylcapsaicin (7-phenylhept-6-yne-acid-hydroxy-3-mathoxylbenzylamide, CAS No. 848127-67-3), an initial 28-day dose range finder experiment revealed mortality and severe clinical findings on animals orally dosed with 500 and 1000 mg/kg bw/day. Animals administered with doses up to 250 mg/kg bw/day developed only mild or local clinical findings.
Based on that data a 90-day repeated dose oral gavage 0, 30, 100 or 250 mg/kg bw/day toxicity study with a 28-day recovery period was conducted in Wistar rats. Examinations of clinical signs, body and organ weight, haematology, urinalysis, clinical chemistry, food consumption and macroscopic, as well as histopathological tissue examinations were carried out for signs of toxicity. Moreover, a Reverse Mutation and a Micronucleus assays were also performed in order to evaluate genotoxic capabilities of phenylcapsaicin.
Degenerative, but reversible changes in the liver at 250 mg/kg bw/day, and local irritating effects in the stomach at 100 and 250 mg/kg bw/day were found. These findings were associated with phenylcapsaicin-related clinical symptoms, i.e., diarrhoea, salivation and moving of bedding material. Similar to our observations, capsaicin has also been suggested to increase salivary secretion and it is known to be a strong irritant to gastric mucosa resulting in severe gastritis and diarrhoea. Interestingly, capsacin and its analogues have been reported to have a gastro-protective effect against ulcerogenic injuries at low doses, but could increase rat stomach mucosal damage at high dosages which is in line with our observation at higher dose. No persistent effects of phenylcapsaicin were observed in food consumption or body weight gain. Phenylcapsaicin was considered not genotoxic.
The NOAEL of phenylcapsaicin for systemic toxicity is considered to be at 100 mg/kg bw/day which is based on degenerative changes in the liver. Due to irritating effects in the stomach, the NOAEL for local effects was established at 30 mg/kg bw/day.
Keywords: Phenylcapsaicin, oral dose toxicity, food, repeated dose, Ames, micronucleus, rats, in vivo
Prevalence of Food Allergy among United Arab Emirates (UAE) Population, a Meta- analysis Study (#687)
N. E. M. Mustafa1
1 University of Sharjah, Department of Environmental Health Sciences/ College of Health Sciences, Sharjah, United Arab Emirates
Purpose: Food allergy (FA) is a common health problem that affects substantial number of people around the world and has significant psychological effects that may negatively affect the quality of life for children, adolescents and adults. Severe FA cases could lead to life-threatening immunoreactions that can initiate respiratory and/or cardiovascular problems. These reactions could be immune system mediated (food allergy) or non-immune mediated reactions (food intolerances). UAE community is dynamic and diverse; its population has representatives from all over the world. Hence, its dietary systems is highly internationalized. This meta-analysis study is intended to highlight the prevalence of FA in UAE and describes the types of the food reported as source of allergens.
Methods: Data were retrieved for the period from 2006 to 2016 and refined according to PRISMA 2009 Flow Diagram (Preferred Reporting Items for Systematic Reviews and Meta-Analyses). The words: food, allergy and UAE were used interchangeably to search Google Scholar and PubMed databases, from each, 1230 and 17 records were retrieved respectively. Non-applicability of data to food consumption represents the main exclusion criteria. Eight records were assessed for eligibility, only five were qualified for meta-analysis and the rest were excluded.
Results: FA cases that belong to four years were analyzed: 397 in 2006, 103 in 2013, 255 in 2014 and 14 in 2016. Most of them are school children (n=411), university students (n=255) and asthmatic children (n=103).They are from cities and administrative units scattered all over UAE and belongs to different ethnic and cultural groups. Types of food encountered in FA cases are tree nuts, peanuts, sesame, eggs, milk, fish, fruits, tomatoes and wheat.
Among children and infants, FA prevalence is gender-equal. However, among university students, female reported cases (12%) doubled those reported by their male colleagues. Parent-reported FA cases represent 8% of investigated populations while SPT checks applied by health practitioners among asthmatic children show nearly the same percentage for FA cases, whereas self-reporting was the main source of FA data among university students. In general, no significant differences in FA prevalence observed among included studies. Population-wide and more community-representing studies are recommended.
Keywords: Food, Allergy, UAE, Prevalence, Meta- analysis
HPLC-FLD and LC-MS/MS determination of Aflatoxin M1 from milk after biofixators usage (#696)
M. Ivešić1, Z. Kuharić1, Z. Pavlek1, Z. Jakopović2, I. Čanak2, J. Frece2, K. Markov2, J. Bošnir1
1 Andrija Stampar Teaching Institute of Public Health, ZAGREB, Croatia
The toxicity of aflatoxin M1 (AFM1) has a high importance for the food quality and possible entrance in human body. Mycotoxin-binding agents (MBA) could be used for fixation of AFM1 to minimise exposure through dairy products. The purpose of this study was to determine unbounded amounts of AFM1 after adding of lactic acid bacteria (LAB) and β-glucan (cellular yeast components) added firstly for binding then removing aflatoxin AFM1 from milk. LAB were isolated from fresh cow's milk and traditional dairy products such as: fresh cow cheese and cream. All milk samples used in the experiment were first analysed and verified for AFM1 contamination free, than spiked with known amount of AFM1. Membrane filtration method and Centricon Plus-70, a MWCO 100 kDa were selected for removal of resulting biofixator-AFM1 complex from milk.The concentration of AFM1 residues in the samples were determined by high performance liquid chromatography using a fluorescent detector (HPLC-FLD). Previously obtained values were confirmed by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Before HPLC or LC-MS/MS analysis the samples were purified on immunoaffinity columns. Both methods were fully validated by determination of following validation parameters: selectivity, linearity, precision, accuracy, recovery, limits of detection and quantification. Validation data proved that both methods are suitable for determination of AFM1 residues in milk. Obtained results of AFM1 after removed biofixator-AFM1 complex from milk shown very good correlation between both methods.
Keywords: Aflatoxin M1, Food safety, Mycotoxin-binding agents, HPLC-FLD, LC-MS/MS
Prospective hazards of fenvalerate in extensive pollution influence the apoptosis sensitivity (#733)
Z. - G. Cui1, Y. - J. Jin1, L. Sun1, S. A. Zakki1, Q. Feng1, H. Inadera1
1 University of Toyama, Department of Public Health, Faculty of Medicine, Toyama, Japan
Aim: Fenvalerate (Fen) is a widely used synthetic pyrethroid insecticide in agricultural, domestic, and veterinary applications. Fen induces abnormal cell proliferation and apoptosis which correspond its hazardous effects. Cadmium (Cd) is a potential environmental hazard that induces disorders in various organs, it has been classified as a human carcinogen by the International Agency for Research on Cancer. In the present study, to explore health hazards for combined pollution of Fen and Cd, the effects of Fen on Cd-induced apoptosis were investigated in human myeloid leukemia U937 cells.
Keywords: Insecticide, Apoptosis, Cancer
Safety assessment of biotechnologically produced 2'-Fucosyllactose, a novel food additive (#781)
J. Salverda1, D. van Berlo1, A. Wallinga1, F. van Acker1, D. Delsing2
1 Triskelion, Zeist, Netherlands
Breastfeeding is best, but not always possible and in certain suboptimal circumstances (e.g. maternal malnutrition) breastmilk is not sufficiently nutritious. In such cases, infant formula is the best alternative. The third most prevalent component of human breast milk is a fraction known as human milk oligosaccharides (HMO). The most abundant HMO is 2’-Fucosyllactose (2’-FL).
HMO are considered very important for the development of intestinal microbiota of infants and have been found to protect against infant diarrhea. Ingestion of HMO has shown to prevent pathogen adhesion to the intestinal epithelium, hence reducing the chance of infection. The beneficiary effect of 2’-FL on the health of infants has been supported by epidemiological studies.
The aim of this study was to assess the safety of biotechnologically produced 2’-FL using a genetically engineered E. coli K12 strain as a processing aid.
2’-FL was tested in an Ames test (OECD TG 471), an in vitro micronucleus test (OECD TG 487) and in a sub-chronic (13-week) oral toxicity study in rats (OECD TG 408).
In the bacterial reverse mutation test and the in vitro micronucleus test, 2’-FL did not induce genotoxicity.
In the sub-chronic oral toxicity study, juvenile Wistar rats (25-days old) were exposed shortly after weaning to dietary levels of 3, 6 and 10% (w/w) 2’-FL. Control animals received regular cereal feed. A 6.4% decrease in food consumption was observed in female rats of the high-dose group, but this did not result in lower body weight. In high-dose males, an 8.3% increase in liver weight was found in the absence of liver pathology. Both findings were considered as non-adverse. Changes in absolute and relative weight of the filled and empty cecum were observed in animals of the low-, mid- and high-dose groups and were ascribed to the high content of non-digestible carbohydrates in the experimental diets containing 2’-FL.
In conclusion, the No Observed Adverse Effect Level (NOAEL) for 2’-FL in rats was ≥ 10% in the diet, corresponding to greater than 7.2 and greater than 7.8 g/kg body weight/day in males and females, respectively.
Keywords: 2’-Fucosyllactose; human milk oligosaccharide; food safety; infant formula; 90-day toxicity
Genotoxicity of molybdenum(III) and nickel(II) and interactions between these microelements. (#46)
S. Terpilowska1, A. K. Siwicki2
1 The John Paul II Catholic University of Lublin, Institute of Environmental Engineering, Laboratory of Environmental Biology, Lublin, Poland
The transition elements: molybdenum and nickel are essential micronutrients for human, animals and plants. Micronutrients in human and animal organisms play a crucial role in prevention and treatment of various diseases. They also play an important role in the optimization of physical and mental functions. Moreover, the elements mentioned above are components of biomaterials.
The aim of presented work was to investigate the genotoxicity of nickel(II) and molybdenum(III) and interactions between these elements on BALB/3T3 and HepG2 cell lines.
The BALB/3T3 and HepG2 cells were cultured at 2 x 105 cells/mL as adherent monolayers in plastic tissue-culture dishes. After 24 hours of incubation the medium was exchanged for the fresh supplemented with NiCl2 x 6H20 or MoO3 at final concentrations as follows: 100, 200, 400, 600, 800, 1000, 1200, 1400 μM. The cells exposed to iron(III) and nickel(II) simultaneously were similarly plated at a density of 2 x 105 cells/mL and incubated for 24 hours. Next the medium was exchanged for fresh supplemented with NiCl2 x 6H20 at concentration of 200 μM and MoO3 at concentration of 1000μM or NiCl2 x 6H20 at concentration of 1000 μM and MoO3 at concentration of 200 μM. After 24 hours of incubation comet and micronucleus assays were performed.
The incubation of BALB/3T3 and HepG2 cells with nickel(II) and molybdenum(III) induce the increase of number of comets and micronuclei. Microscopic analysis were showed increase the number of polynuclear and giant cells after treatment with molybdenum(III) and nickel(II). Moreover, the treated cells displayed characteristic apoptosis in comparison to control cells. Nuclear convulsion and fragmentation, cytoplasmic blebbing and cytoplasmic vacuolation were observed. The results obtained from both cell lines show that BALB/3T3 cells are more sensitive when compared to the HepG2 cells after incubation with nickel chloride. However, HepG2 cells are more sensitive when compared to the BALB/3T3 cells after incubation with molybdenum trioxide.
Additions of Ni(II) at 200 μM plus Mo(III) at 1000 μM showed synergistic effect in increase of number of comets and micronuclei. The same effects was observed in pair Ni(II) at 1000 μM plus Mo(III) at 200 μM.
Keywords: genotoxicity, molybdenum(III), nickel(II)
Estimation of the non-genotoxic effect level in vivo in bone marrow micronucleus tests for a rare disease drug candidate (#60)
L. Mueller1, A. Zeller1, M. Guerard1, A. Poirier1
1 F. Hoffmann-La Roche, Pharmaceutical Sciences, Basel, Switzerland
A rare disease drug candidate induced elevated micronucleus frequencies in the bone marrow of rats in juvenile and adult rats. Thorough in vitro and in vivo investigation on the mode of action supports a mechanism secondary to cell cycle interaction and excludes direct DNA-reactivity. In order to support clinical evaluation of this candidate for single doses in healthy volunteers and repeat doses in patients with a rare disease (including pediatric patients), a reliable point of departure with respective confidence limits needed to be established across studies. We applied the benchmark dose (BMD) concept using a critical effect size (CES) based on statistical analysis of historical negative control distributions to support an exposure-based dosing in the clinic. The calculated maximum dose level avoids exposures against a critical genotoxic effect level that interferes with chromosome integrity in the bone marrow (or that of other tissues) but would still be predicted as sufficiently efficacious. The BMD concept requires the toxicologist to a priori define a small increase over controls that is “acceptable” to be induced by a non-DNA-reactive genotoxic test substance and thereby define limits for safe (or very low risk) exposures for certain conditions such as clinical trials. To render the metrics calculated from the data of animals treated with the genotoxicants applicable for risk assessment, the chosen CES should represent a low but measurable response level, reflecting an effect that is negligible or non-adverse. On the basis of a large historical database we have determined a suitable CES, which allowed to define safe/low-risk exposure limits under which this compound could be tested in the clinic. These conditions were laid out in the clinical trial protocols and were positively reviewed by the competent Health Authorities.
Keywords: Micronucleus in vivo, cell cycle, rare disease, clinical trial
Chromosomal aberrations and micronuclei structures after dermal exposure to polycyclic aromatic hydrocarbon and ultraviolet radiation (#86)
A. Malkova1, J. Smejkalova1, K. Hamakova2, J. Kremlacek3, T. Svadlakova4, L. Borska3, Z. Fiala1
1 Charles University, Faculty of Medicine, Institute of Hygiene and Preventive Medicine, Hradec Kralove, Czech Republic
Goeckerman therapy of psoriasis (GT) includes dermal exposure to crude coal tar ointment (CCT) and ultraviolet-radiation (UVR). The CCT contains polycyclic aromatic hydrocarbons (PAHs), some of them are immunosuppressive, genotoxic and carcinogenic. According to IARC both factors (CCT and UVR) are classified as human carcinogens. Combined exposure to PAHs and UVR can induce increase of mutations at chromosomal level (chromosome aberration) and creation of micro-nuclear structures and could increase the risk of malignant transformation of cells. This study describes the presence of chromosomal aberrations and various micronuclei structures in lymphocytes of psoriatic patients treated by dermal exposure to CCT and UVR.
The group of patients with psoriasis (n=32, male n=15, smokers n=13) was treated by GT (4% CCT applied topically and UVB whole body irradiation). The blood samples were collected before the first and immediately after the last procedure. We investigated (1) the number and distribution of micronuclei (MN), nuclear buds (gen amplification) and nucleoplasmic bridges (dicentric chromosome) in 1 000 binucleated lymphocytes (BNL) and (2) the structural aberration (SAL) and numerical aberration (NAL) in 100 metaphasical lymphocytes.
Total number of BNL with MN (p<0.001), BNL with 1 MN (p<0.001), BNL with 2 MN (p<0.05) and BNL with ≥3 MN (p<0.01), total number of aberrated cells (SAL+NAL; p<0.001) and SAL (p<0.001) were significantly increased after GT. The smokers had significantly higher total number of aberrated cells (p<0.05) and number of smoked cigarettes correlated with total number of aberrated cells (rho=0.38, p<0.05). We also found correlation between age and BNL with MN (rho=0.55, p<0.001) and correlation between range of psoriatic lesions and total number of aberrated cells (rho=0.37, p<0.05).
In conclusion, combined therapeutic exposure to CCT and UVR (Goeckerman therapy) increases the levels of the micronuclei and aberrations in lymphocytes of psoriatic patients and therefore increases the probability of malignant transformation of cells.
Supported by projects PROGRES Q40/09 and SVV-2018.
Keywords: Polycyclic aromatic hydrocarbons, Ultraviolet-radiation, Chromosomal aberrations, Micronuclei structures, Genotoxic effect
Occupational exposure to bitumen fumes (#90)
L. Borska1, C. Andrys2, J. Kremlacek1, A. Malkova3, T. Svadlakova2, P. Borsky3, Z. Fiala3
1 Charles University, Faculty of Medicine, Institute of Pathological Physiology, Hradec Kralove, Czech Republic
The road workers are exposed to bitumen fumes including benzo[a]pyrene (BaP). After absorption, the BaP is metabolized into reactive metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) which attacks structure of DNA and creates DNA-adducts. DNA adducts become antigenic and induce expression of antibodies against BPDE-DNA adducts (anti-BPDE-DNA) which reflect the intensity of exposure. Exposure to mutagenic/carcinogenic BaP induces mutations at the chromosomal level (chromosome aberration) and elevation of oxidative stress, associated with creation of various oxidized forms of nucleobases and nucleosides (consequent repair processes release multiple oxidized guanine species into the serum).
The present study focused on occupational exposure to bitumen fumes/ BaP with the aim of contribution to exposure assessment (anti-BPDE-DNA) and to evaluation of associated genotoxic effects (chromosomal aberration and oxidative stress).
The exposed cohort consisted of 42 road workers exposed to bitumen fumes. Their blood samples were taken during the first and the sixth months of the summer road-working season. Serum level of anti-BPDE-DNA (IgG/IgM) and serum level of 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, and 8-hydroxyguanine were analyzed by ELISA methods. The standard microscopic analysis (total number of aberrated cells) was used for determination of chromosomal aberration in peripheral lymphocytes.
In the blood samples taken during the sixth month of the exposure we found (compared with the first month) non-significantly elevated level of anti-BPDE-DNA, significantly elevated level of oxidative stress (p<0.001) and significantly elevated level of chromosomal aberration in peripheral lymphocytes (p<0.05).
In conclusion, the seasonal exposure of the observed group of road workers (bitumen fumes/ BaP) significantly increased the level of oxidative stress and the likelihood of genotoxic effects.
Supported by projects PROGRES Q40-09.
Keywords: Road workers, Bitumen fumes, Polycyclic aromatic hydrocarbons, anti-BPDE-DNA, Genotoxic effects
EVALUATION OF DNA DAMAGE AND MODULATION OF GENE EXPRESSION IN THE HEART OF RATS THAT RECEIVED VITAMIN D3 SUPPLEMENTED OR DEFICIENT DIETS (#155)
L. G. Antunes1, C. S. Machado2, A. F. Aissa1, D. L. Ribeiro2
1 University of Sao Paulo, School of Pharmaceutical Sciences of Ribeirao Preto, Ribeirao Preto, Sao Paulo, Brazil
Vitamin D3 deficiency has been associated with the altered expression of genes associated with increased blood pressure; however, the role of vitamin D3 supplementation in the genetic mechanisms of hypertension remains unclear. Thus, the aim of this study was investigate how vitamin D3 supplemented or deficient diets regulate the expression of genes related to hypertension pathways in heart cells of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) controls. An additional aim was to assess the impact of vitamin D3 on DNA damage and oxidative stress markers. The animals were fed a vitamin D3 control diet (1,000 IU/kg) supplemented diet (10,000 IU/kg) or deficient diet (0 IU / kg) during 12 weeks. The gene expression profiles were evaluated by PCR array, the DNA damage was assessed by comet assay, and the oxidative stress markers thiobarbituric acid reactive substances and glutathione were assessed via biochemical analysis. Vitamin D3 supplementation or deficiency did not induce DNA damage or oxidative stress in heart cells. Our results showed that the vitamin D3 supplemented diet downregulated the expression of genes involved with increased blood pressure, such as Ace and Ren in SHR rats and Kcnma1, Scnn1a and Scnn1g in normotensive WKY controls. In turn, vitamin D3 deficient diets upregulated the expression of genes involved with increased blood pressure, including the Ace and Cacna1c genes in the SHR and WKY rats, and this diet induced lipid peroxidation in SHR rats. Vitamin D3 supplemented diets altered the expression of genes associated with maintaining blood pressure, while vitamin D3 deficient diets induced oxidative stress and increased the expression of genes directly associated with hypertension. The results of this study contribute to a better understanding of the role of vitamin D3 in the genetic mechanisms of hypertension.
Financial Support: CNPq (302290/2015-0) and FAPESP (2012/04325-9)
Keywords: Vitamin D, comet assay, genotoxicity, SHR, supplementation
Investigation of the Potential Non-Genotoxic Mode of Action for 1,2-Dichloroethane (DCE)-Induced Mammary Tumors in Female Rats (#167)
D. R. Boverhof1, J. A. Hotchkiss1, M. J. LeBaron1, L. M. Segal3, C. Howick2
1 The Dow Chemical Company, Toxicology & Environmental Research & Consulting, Midland, Michigan, United States of America
1,2-Dichloroethane (DCE) is a chlorinated hydrocarbon used as a chemical intermediate including in the synthesis of vinyl chloride which is used to make poly vinyl chloride. While DCE has induced tumors in both rats and mice, the overall weight of evidence for genotoxicity suggests that DCE is a non-genotoxic carcinogen. The present study was conducted to further assess the in vivo genotoxicity of DCE and explore a potential mode of action for tumor formation in rat mammary tissue. Fischer 344 rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (six hours/day, seven days/week) for 28 days. This concentration of DCE is approximately 20% higher than that reported to induce mammary tumors in rats. Endpoints examined included DNA damage (via Comet assay), GHS/GSSG levels, tissue adduct levels, cell proliferation and serum prolactin levels. One mechanism of Action (MoA) hypothesis was that alterations in serum prolactin and cell proliferation were potential non-genotoxic key events leading to tumor formation. Exposure to DCE did not have an effect on serum prolactin levels or cell proliferation in mammary epithelial cells. DNA adducts were identified including the N7-guanylethyl glutathione (GEG) cross-link adduct which was considered as a biomarker of exposure, with higher adduct levels measured in liver (non-target tissue) compared to mammary tissue isolated from the same rats. The results of the comet assay in mammary epithelial cells indicated that DCE did not result in increased DNA damage while the positive control N-Nitroso-N-methylurea resulted in a statistically significant increase relative to controls. While the results of this study do not identify a specific MoA for DCE-induced mammary tumors in rats, the lack of any exposure-related genotoxic effects in the Comet assay or relevant target-tissue specific DNA adducts further contributes to the weight of evidence for DCE as a non-genotoxic carcinogen.
Keywords: Dichloroethane (DCE), in vivo genotoxicity, Fischer 344 rat, Comet assay, mammary epithelial cells
Impact of the S9% in the Ames test revertant colonies on the strains of Salmonella typhimurium TA100 and TA98 (#200)
A. T. C. Paulino1, S. Mateus1, I. Sardo1, C. Pires1
1 Ascenza Agro SA, Microbiology and Celular Biology Laboratory, Setubal, Portugal
The Ames test, also known as Reverse Mutation Assay, is a biological assay used to detect compounds that can lead to genetic mutations. This test uses Salmonella typhimurium strains that carry mutations involved in histidine synthesis. Thereby, if the compound is mutagenic, the number of revertant colonies per plate increases when compared with number of revertant colonies per plate induced spontaneously in the absence of a mutagen.
The bacteria used for an ames test are incapable to enzymatically metabolize chemicals, so it is necessary to include an exogenous system of mammalian metabolic activation, a post-mitochondrial fraction of a rat liver (S9), prepared in a mixture, the S9-mix.
According with the OECD guideline for testing of chemicals nº 471 Bacterial Reverse Mutation Test, adopted on 21st July 1997: “The post-mitochondrial fraction is usually used at concentrations in the range from 5 to 30% v/v in the S9-mix.” Considering that, it was performed an ames test, using the methods stated in the guideline nº 471, with several percentages of S9 (5%, 15%, 25% and 30%), in the S9-mix, with the strains TA100 and TA98 to test dimethyl sulfoxide (solvent control), 7,12-dimethylbenzanthracene (positive control) and three substances used as active ingredients in plant protection products: deltamethrin, clethodim and imidacloprid.
The results revealed that in the case of the strain TA100, with the increase in the S9 in the S9-mix, the number of spontaneously induced revertant colonies decreases with all the tested compounds, including the controls.
For the strain TA 98 the results revealed that there are no significate changes in the number of revertant colonies with the increase of the S9 in the S9-mix. There’s however to remark a minor decrease in the number of revertant colonies of the positive control and a minor increase in the number of revertant colonies for clethodim, comparing results between the S9 at 5% in the S9-mix and the other percentages of S9. These observations have no impact in the evaluation of each substance as mutagenic or non-mutagenic under the conditions applied in the trials.
Further investigation is necessary in order to assess if the range mentioned in the OECD guideline nº 471 should be narrowed.
Keywords: Ames, metabolic activation
Pouteria ramiflora (Sapotaceae) leaves extract increases the antiproliferative and pro-apoptotic effects of cisplatin in HepG2 cells (#250)
I. M. S. Cólus1, K. Tuttis1, J. M. Serpeloni1, E. A. Varanda2, L. C. Santos3, W. Vilegas4, W. Martínez-Lopez5
1 Londrina State University, Department of General Biology, Londrina, Paraná, Brazil
Pouteria ramiflora (Mart.) Radlk., popularly known as curriola, is commonly used as medicinal plant for treatment of worm infection, dysentery, pain, and inflammation. Extracts of medicinal plants of this genus are being studied in order to ensure the safe use and effectiveness of these plants and to investigate possible new biological activities. Thus, this study evaluated the Pouteria ramiflora extract for antiproliferative activity and induction of cell death in human hepatocellular carcinoma cell line (HepG2) in order to verify the safety of its use and assess if its association with cisplatin interferes in the cellular proliferation and cell death processes. To assess the antiproliferative effect we used flow cytometry and immunocytochemistry, while cell death induction was investigated by apoptosis/necrosis triple staining assay. In addition, the modulation of genes related to cell cycle control and mechanism of cell death was investigated. The results demonstrated that P. ramiflora extract at different concentrations (0.5, 1.0 and 2.0 µg/mL) presented antiproliferative effect with cycle arrest in S and G2/M phase and induced death by apoptosis at concentrations of 1.0 and 2.0 µg/mL. The association of the extract at the concentration of 1.0 µg/mL to cisplatin potentiated its antiproliferative effect with arrest in S phase via CDK2 gene modulation and decreasing the number of cells stained with anti-cyclin D1 antibody (G1 phase). Also was observed potentiation of cytotoxic effect from cisplatin with increase of apoptotic cell death and BAX gene expression. These results demonstrate that P. ramiflora extract should be used with caution for therapeutic purpose and encourage further studies that can prove its use as an adjuvant in liver cancer treatment in the future.
Financial support: FAPESP, CAPES/DS, CNPq
Keywords: medicinal plant, apoptotic, cyclin D1, PCNA
Assessment of the sensitivity of the Ames Assay to pick up true mutagens at concentrations lower than the maximum OECD TG471 guideline dose (#266)
D. Holland1, S. Pfuhler2
1 Procter & Gamble Services NV/SA, Global Product Stewardship, Strombeek-Bever, Belgium
When assessing chemicals for genotoxicity, the Ames test data generated often stems from testing mixtures. Normally such data cannot be accepted for individual components in the mixture since the OECD TG471 required top dose of 5000 µg/plate is not achieved. Previous studies looking at the potential of the Ames test to detect mutagenic impurities in active pharmaceutical ingredients suggest that the assay is sufficiently sensitive; it was estimated that 85% of the total mutagens detected with the standard protocol are also detected at 5% of the regular top dose (≤ 250 µg/plate). To investigate whether a similar level of sensitivity would hold true for a different chemical database we gathered Ames data for 351 chemicals from the EURL ECVAM Genotoxicity & Carcinogenicity Consolidated Database of Ames Positive Chemicals. Where available, three positive studies per chemical were compiled and summarized. After initial cleaning of the database eliminating chemicals with insufficient or inconclusive data, 252 chemicals and 592 studies were left for further review.
The lowest positive dose exceeded 500 µg/plate (or 10% of the OECD TG471 required top dose) for only 22/252 chemicals. This means that 91 % of Ames positive compounds would be picked up also if the top dose in the Ames assay would have been lowered to 500 µg/plate. It further indicates that mixture data might be acceptable for the assessment of individual chemicals within the chemical space covered by the ECVAM database. To further analyze the impact of functional groups, a chemical class analysis has also been conducted.
Reference: Kenyon, M.O., Cheung, J.R., Dobo, K.L., Ku, W.W. (2007) An evaluation of the sensitivity of the Ames assay to discern low-level mutagenic impurities, Regulatory Toxicology and Pharmacology 48 (2007) 75–86.
Keywords: Ames assay, Genotoxicity
IN VITRO GENOTOXICITY ASSESSMENT OF CYLINDROSPERMOPSIN-MICROCYSTIN-LR MIXTURES BY THE MICRONUCLEUS AND COMET ASSAYS. (#273)
L. Diez-Quijada Jiménez1, M. Puerto1, A. I. Prieto1, A. Jos1, A. M. Cameán1
1 University of Sevilla, Area of Toxicology/Faculty of Pharmacy, Sevilla, Spain
At present, the study of the potential toxic effects of mixtures of cyanotoxins such as Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are of interest due to their growing co-occurrence. It is also a reason of concern for the European Food Safety Authority due to the human exposure through contaminated water and food. Among the toxicological consequences of their exposure, the potential genotoxicity is of main importance and it has been not yet investigated for CYN+MC-LR mixtures. For this reason, in this work, the in vitro genotoxicity of CYN and MC-LR mixtures has been studied by the micronucleus test (MN) with presence/absence of the microsomal fraction S9 (OECD 487) in the L5178Y Tk +/- cell line, and the standard and modified comet assay to evaluate oxidative DNA damage in the Caco-2 cell line. The exposure concentrations were selected based on previous studies in which the EC50 of these toxins were determined. The ratio CYN:10 MC-LR was selected, taking into account the higher abundance of MC-LR in the environment. In the MN test the concentrations evaluated were 0.125-2 μg/ml CYN and 1.25-20 μg/ml MC-LR, whereas in the comet assay the concentrations were 0.625 -2.5 μg/ml CYN and 6.25-25 μg/ml MC-LR. The results obtained in the MN test do not show a genotoxic effect in absence of S9 mix. However, genotoxic effects were shown in the presence of S9 at high concentrations. On the other hand, both, the standard and the modified version of the comet assay, after 24 or 48h of exposure to the mixtures did not show genotoxicity. In short, the results obtained showed that the genotoxicity observed can be due to CYN and MC-LR metabolites although further studies are needed.
Acknowledgments: Spanish Ministry of Economy and Competitiveness for financing the project (AGL2015-64558-R, MINECO / FEDER, UE). Leticia Diez-Quijada Jiménez also thanks for the grant BES-2016-078773 associated with this project (MINECO / FEDER).
Keywords: Genotoxicity, Cylindrospermopsin, Microcystin-LR, Micronucleus Test, Comet assay
UV-A and UV-B damage: the protective effect of Vitis vinifera L. extract on HaCaT cell line (#455)
1 University of Milan, Department of pharmacological and Biomolecular Sciences, Milan, italy, Italy
Exposure to the ultraviolet components of sunlight UV-B (280-315 nm) and UV-A (315-480 nm) is the major cause of skin damage. UV-B components have a low wavelength being absorbed almost completely by the epidermis; however, they interact directly with DNA causing molecular rearrangements. Longer wavelength UV-A penetrates deeply into the dermis generating reactive oxygen species (ROS) that can indirectly damage DNA. Recently, different polyphenol-enriched extracts have been proposed for the prevention of UV-mediated skin damage. Here it has been evaluated the capability of aqueous extracts of Vitis Vinifera L., validated for the contents of anthocyanins, flavonoids and caffeic acid, to interfere with UV damage protecting human keratinocyte (HaCaT) cell line. The treatment with the extract of Vitis Vinifera L. (100µg/ml, 1h in serum-free media) was followed by the exposure to UV-A (2.5-5-10-20 J/cm2) or UV-B (20-30-40-80-160-320-640 mJ/cm2) radiation in PBS. The extract inhibits DNA damage as indicated by the alkaline comet test, ɣH2AX and micronucleus assays, evaluated at T0 after the exposure. Only for the UV-A damage a correlation with the reduction of ROS synthesis is seen, and this suggests that the extract could have the ability to neutralize ROS, acting as a scavenger and/or also by activating antioxidant systems. Moreover, it seems that the extract could protect from the direct damage induced by UV-B, consisting in the formation of cyclobutane pyrimidine dimers (CPD); oxidative damage by UV-B is found only at dosages 10x higher than those for direct damage. Finally, it has been evaluated if these protective effects are related to a mitigation of the inflammatory response, by measuring IL-8 release and activation of the nuclear transcription factor NF-kB.
Keywords: genotoxicity, UV radiation
Genotoxic damage among Bolivian farmers exposed to a complex mixture of pesticides (#466)
J. X. Barron Cuenca1, 2, N. Tirado2, J. Barral2, P. Almaraz2, U. Stenius1, M. Berglund1, K. Dreij1
1 Karolinska Institutet, Institute of Environmental Medicine, Stockholm, Sweden
Pesticides are well known as chemicals that can increase the risk to develop cancer by induction of DNA damage and oxidative stress, especially in populations exposed to them for long periods of time. Bolivian farmers have been increasing the use of mixtures of pesticides during the last decades to increase their production and their economy. Chronic exposure to these chemicals, their combined effects and gene polymorphism can increase the risk of genotoxic damage, mutagenicity and development of chronic disease.
A cross-sectional study in 297 volunteers from three different Bolivian agricultural communities was conducted. A survey was used to assess pesticide exposure. Blood samples were collected and micronucleus and Comet assays were performed to evaluate the genotoxic damage. Frequency of (glutathione transferase) GST null genotypes (GSTM1 and GSTT1) were measured to evaluate the impact on DNA damage levels.
We found that 75% of the farmers combined several pesticides. Organophosphates were the most used pesticides (78%), followed by bipyridyls and pyrethroids (48% and 23%, respectively). Higher frequency of micronuclei was found in women compared to men (4.52 vs 3.57, p<0.05). Farmers who had worked in the field >8 years had higher frequency of micronuclei compared to farmers who had worked <8 years (4.23 vs 2.94, p<0.05). Significant differences in levels of DNA damage between the three communities were also found. Notably, for all communities, farmers that obtained information about how to handle pesticides from the seller had higher levels of DNA damage compared to those that got information from other sources (13.1% tail DNA, p<0.05). There was no correlation between lack of personal protection equipment and levels of DNA damage. We could not find any correlation between DNA damage levels and GST genotype (GSTM1 and GSTT1: 21% and 96% null, respectively).
In conclusion, organophosphates are the most commonly used pesticides. Gender, years as farmer and information source are important determinants for risk of genotoxic damage among Bolivian farmers.
Keywords: Pesticides, Micronucleus assay, Comet assay, Genotoxic damage, GST genotypes
Integrated genotoxicity testing (Pig-a and MNT) within 28-day mouse study – A case study with Meptyldinocap metabolite, X12335709 (#485)
M. Aggarwal1, L. Murphy2, C. Terry2, E. M. Donner3
1 Corteva Agriscience™, Agriculture Division of DowDuPont™, Toxicology, R&D, Abingdon, United Kingdom
Meptyldinocap, a fungicide, is predicted to produce very low levels of a ground water metabolite, X12335709. Meptyldinocap was found to be non-genotoxic when tested in both in vitro (bacterial reverse mutation test (Ames test), mammalian cell gene mutation test (HPRT) and mammalian chromosomal aberration test (HLCAT)) and in vivo (mouse micronucleus test, (MNT)). On the other hand, X12335709 was negative in the HPRT and HLCAT but positive in the Ames test. Therefore, an in vivo mutagenicity study was needed to confirm the in vitro mutagenicity seen in bacteria with X12335709, and a repeated dose rodent study was required in order to compare the systemic toxicity of X12335709 with that of meptyldinocap. These studies were also needed for the identification of an appropriate reference dose (RfD) for human health risk assessment (HHRA). To address all these issues, an integrated 28-day repeat dose dietary mouse study was performed at two dose levels which equate to LOAEL and NOAEL for meptyldinocap. Expression levels of Pig-a protein (as CD59 marker; Gollapudi et al., 2015) and induction of micronucleus in peripheral blood were measured in treated and untreated males. Appropriate positive controls for the Pig-a assay and MNT were included in the study. The colour of the urine was similar to the colour of X12335709, indicating that animals had systemic exposure to X12335709. Adverse effects were not observed at any dose level, and both the Pig-a assay and MNT was negative. Taken all data together, X12335709 was considered to be non-genotoxic and meptyldinocap RfD (e.g., ADI) can be used for HHRA to X12335709. Although a valid OECD test guideline for Pig-a assay is currently only pending, the assay is described in the literature sufficiently to be conducted in a standardised and validated manner. Integrating the Pig-a assay (and MNT) within a repeat dose rodent study is perhaps better option for understanding in vivo mutagenicity than using the currently available comet (OECD 489) and transgenic rodent (OECD 488) assays, both from scientific and 3Rs (Replacement, Reduction, and Refinement) perspectives.
Keywords: In vivo mutagenicity, Genotoxicity, Pesticide, Agrochemcial, Integrated testing
Combined application of comprehensive DNA analysis for DNA modification and reporter gene mutation assay to investigation of the mechanisms underlying hepatocarcinogenicity of elemicin (#545)
Y. Ishii1, L. Shi1, S. Takasu1, A. Kijima1, K. Ogawa1, T. Umemura1
1 National Institute of Health Sciences, Division of Pathology, Kanagawa, Japan
[Introduction] DNA adductome analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS) enables comprehensive monitoring of chemical-specific DNA adducts. Therefore, combined application of DNA adductome analysis and reporter gene mutation assay using transgenic rodents is a powerful tool for investigating the underlying mechanisms of chemical carcinogenesis. Elemicin is one of natural alkoxybenzene compounds, and is a flavor component of several plant species such as nutmeg and mace. Our previous data demonstrated that significant increases in the number and areas of GST-P-positive foci were apparent in 400 mg/kg elemicin group, indicating its hepatocarcinogenicity. However, the underlying mechanisms remain uncertain. In the present study, to clarify the hepatocarcinogenesis of elemicin, we carried out DNA adductome analysis and gpt assay in the livers of gpt delta rats treated with elemicin by gavage at doses of 0, 25, 100 or 400 mg/kg/day for 13 weeks.
[Results] Comprehensive DNA adduct analysis demonstrated the existence of several spots as putative DNA adducts at m/z434, 458, 474 and 496. In addition, MS spectrum analyses showed that all spots are including the fragment ion peaks showing loss of deoxyribose ([M+H-116]+) and cleavage of elemicin (m/z207), indicating elemicin-specific DNA adducts. Peaks of all adducts were detected from low-dose group by multiple reaction monitoring (MRM) analysis, and those areas were increased in a dose-dependent manner. Significant increases in gpt mutant frequencies in the livers were detected in rats given 100 mg/kg bw and above.
[Discussion] Characteristic fragment ions observed in MS spectrum analyses strongly suggested that all spots originated from elemicin-specific dG, dA and dC adducts. It is highly probable that these DNA adducts formation is responsible for increases in gpt mutant frequencies. The present data imply the involvement of genotoxic mechanisms in hepatocarcinogenesis of elemicin.
Keywords: gene mutation, elemicin, DNA adduct
The ToxTracker reporter assay detects indirect genotoxicity caused by high levels of oxidative stress. (#551)
P. van Rossum1, R. Derr1, P. Racz1, N. Moelijker1, I. Brandsma1, G. Hendriks1
1 Toxys BV., Leiden, Netherlands
ToxTracker is a mammalian stem cell-based reporter assay that detects activation of specific cellular signaling pathways upon exposure to compounds (Hendriks et Real, Tox Sci 2016). ToxTracker contains six different GFP-tagged reporters that allow the discrimination between the induction of DNA damage, oxidative stress and protein damage in a single test.
We first performed an extensive validation using 62 compounds from the recently updated ECVAM compound library. ToxTracker classified the genotoxic carcinogens as genotoxic with a sensitivity of 94%. The non-genotoxic carcinogens and non-carcinogens were classified as non-genotoxic by ToxTracker with a specificity of 95%. Interestingly, various compounds that give misleading positive results in the conventional in vitro genotoxicity assays did not activate the DNA damage reporters but did induce high levels of oxidative stress or protein damage in ToxTracker.
To investigate potential indirect genotoxic effects of compounds caused by high levels of oxidative stress, a selection of different genotoxic and oxidative compounds was tested in ToxTracker in the presence of ROS scavengers. For each compound, three concentrations that induce 10/25/50% cytotoxicity in the reporter cell lines were tested in combination with increasing concentrations of the ROS scavengers. In the absence of the ROS scavengers, all tested compounds activated the Srxn1-GFP and Blvrb-GFP reporters for oxidative stress. In the presence of ROS scavengers, the activation of these reporters was reduced. The complete absence of induction of the Bscl2-GFP DNA damage marker, that is activated by DNA replication stress and induction of bulky, promutagenic DNA lesions, upon exposure to oxidative agents, indicates that the tested compounds did not react directly with the DNA. Also the cytotoxicity of the oxidative compounds was reduced by the addition of a ROS scavenger, suggesting that induction of oxidative stress contributes to cytotoxicity.
In conclusion, the ToxTracker assay in combination with a ROS scavenger can discriminate between direct DNA reactivity and indirect genotoxicity caused by oxidative stress.
Keywords: ToxTracker, Genotoxicity, Oxidative stress, Antioxidant, Mechanistic Toxicology
Polycyclic aromatic hydrocarbons in herbal medicinal products (#556)
T. Vavvas1, 3, G. Bode2, 3
1 Bionorica SE, Division Drug Regulatory Affairs, Neumarkt, Bavaria, Germany
Polycyclic Aromatic Hydrocarbons (PAH) are ubiquitous contaminant-mixtures that are formed in natural or anthropogenic combustion processes. By dry or wet atmospheric deposition or by the formation in thermal processing methods, PAHs can contaminate herbal substances that are used in herbal medicinal products (HMP).
Many PAH substances have genotoxic and carcinogenic effects; others may act as synergists. Different scientific organisations analysed the risk for human health of PAHs in food and the environment. This risk is reflected in the legislation concerning food, environment and other products, but not for HMPs. Committee on Herbal Medicinal Products (HMPC) called involved parties in a reflection paper to join the scientific discussion and collect data for a further evaluation and the development of an appropriate guidance.
A literature research was performed discussing different risk assessment approaches, as the risk evaluation for toxic mixtures and contaminants in plants is particularly challenging. In addition PAH contamination data from different studies in medicinal plants, spices, tea and herbs was evaluated and compared with each other and with data from the food sector. PAH was detected in most of the samples in a broad range of values, showing a need to discuss appropriate protection measures.
Overall results and conclusion
A future risk management needs a structured data collection under comparable conditions generating a broad database and the definition of appropriate threshold values, which consider contamination levels of herbal substances, transfer rates for herbal preparations and the overall consumption rate. PAH contamination can be reduced if harvesting in industrial areas and bad drying practices are avoided. Washing was described to reduce PAH content on the surface of plants. For producers and suppliers of herbal substances and herbal preparations, the Guideline on Good Agricultural and Collection Practice (GACP) provides general recommendations that minimise the risk of contaminated plant material.
If contamination sources can´t be identified and avoided, only regular testing embedded in reasonable risk management will reliably detect non-acceptable exposures.
Keywords: Genotoxicity, Carcinogenesis, Safety Assessment of Mixtures
Genetic toxicological comparison of Extract of Acer tegmentosum (#606)
E. Cho1, Y. Kim1, J. Yoon1, S. Kim3, D. Kim3, B. Kang1, 2
1 Seoul National University Hospital, Department of Experimental Animal Research, Biomedical Research Institute, Seoul, Republic of Korea
Acer tegmentosum is commonly used as a traditional medicine for treatment of liver diseases in the east Asia region. An active flavonoid compound of the Acer tegmentosum extract has been known to have protective effect to liver damage caused by alcohol consumption, but, little is known about its toxicity. In this study, the genetic toxicity of extract of Acer tegmentosum was confirmed by Ames test, chromosome aberration test and in vivo micronucleus test. In the Ames test, Acer tegmentosum extract did not induce mutagenicity in either Salmonella typhimurium or Escherichia coli with or without metabolic activation. In the chromosome aberration test, there was no significant increase in chromatid breaks and exchanges. In the mouse micronucleus test, there was no significant increase in the occurrence of micronucleus polychromatic erythrocyte. These results suggest that the extract of Acer tegmentosum induce no genotoxicity under the conditions of this study.
Keywords: Acer tegmentosum extract, genetic toxicity, Ames test, chromosome aberration test, micronucleus test
GENOTOXICITY TESTING OF LAMBDA CYHALOTHRIN IN FLUCTUATION AMES ASSAY AND MICE BONE MARROW MICRONUCLEUS TEST (#612)
O. Kravchuk1, M. Prodanchuk1, N. Nedopytanska1, T. Tkachuk1, V. Bubalo1, O. Tkachuk1, O. Zubko1, O. Kostik1
1 L. I. Medved's Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine, Kyiv, Ukraine
The synthetic pyrethroids comprise one of the major group of insecticides that are routinely used against indoor and agricultural pests for more than 30 years. Lambda cyhalothrin is highly toxic to insects and many fish and aquatic invertebrate species. Toxicological properties of lambda cyhalothrin are well studied, but the published mutagenicity data are highly contradictory.
Study of mutagenicity is a mandatory for the toxicological assessment to justifying pestices usage in Ukraine. For this reason we conduct full test battery, which includes: in vitro: Ames assay (OECD 471) and MN assay (OECD 487); in vivo: CA assay (OECD 475), MN assay (OECD 474) and comet assay (OECD 489). Following OECD guidelines we modified our SOPs for rapid mutagenicity screening of well known generic pesticides comply with GLP.
The aim of the study was to research the potential mutagenic effects of lambda cyhalothrin technical (purity 97,1%) in screening mice bone marrow MN assay and fluctuation Ames assay using S.typhimurium TA98, TA100 with/without metabolic activation, preincubation was 90 min. Selection of concentrations in Ames test were based on preliminary experiment which was performed before the main test. In the absence of cytotoxicity and precipitation in preliminary experiment the following concentrations (2.5; 0.5; 0.1; 0.02; 0.004; 0.0008 mg/ml ) were defined.
MN test was conducted on certificated CD1 healthy young mice, male obtained from the SPF animal breeding center of our center. Acclimatization took 5 days before dosing. One treatment by gavage was in the doses: 5.0; 1.0; 0.2 mg/kg/bw. Exposure time - 24 hours.
Obtained experimental data of positive and negative controls were ranged with own historical control. The lambda cyhalothrin in the MN assay in high concentrations was shown significant (p≤0.05) increase in the frequency of micronucleated polychromatophilic erythrocytes in compation to the negative control and historical control data. Ames assay results showed statistically significant absence of the mutagenic effect.
Keywords: fluctuation Ames assay, micronucleus test, lambda cyhalotrin, pesticides, genotoxicity
Cytogenetic activity of 2,6-dimethylpyridine-N-oxide in a test for induction of chromosome aberrations in mouse bone marrow cells in vivo (#629)
O. Vasetska1, O. Zubko1, M. Prodanchuk1, O. Kravchuk1, P. Zhminko1
1 L.I.Medved`s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Ukraine (State Enterprise), Department, Kyiv, Ukraine
The cytogenetic activity of 2,6-dimethylpyridine-N-oxide was investigated in accordance with OECD 475 on young adult mice CD-1 (males, n=5 for each group). In the first experiment, CD-1 mice were orally administered 2,6-dimethylpyridine-N-oxide at doses: 710.0, 71.0, 7.1, 0.71, 0.071 mg/kg and in the second - the classical inducer of chromosome aberrations Cyclophosphamide in dose 40 mg/kg and followed by a single oral administration of 2,6-dimethylpyridine-N-oxide in the same doses. Negative control group received distilled water. Positive control group received Cyclophosphamide (40 mg/kg orally).
It`s established, the average frequency of metaphases with chromosome aberrations in mouse bone marrow cells of the negative control was 0.20 ± 0.19%, positive control - 14.00 ± 1.55% (P ≤ 0.001). Under the influence of 2,6-dimethylpyridine-N-oxide in 2 lower doses, it was 0.20 ± 0.19%, in doses of 7.1; 71.0 and 710.0 mg/kg - 0.40 ± 0.28%, 0.60 ± 0.35% and 0.80 ± 0.40%, respectively (P> 0.05). Under the action of Cyclophosphamide with 2,6-dimethylpyridine-N-oxide at a dose of 710.0 mg/kg, it did not differ from positive control. With doses decrease of 2,6-dimethylpyridine-N-oxide, a significant reduction (P ≤ 0.001) of the average frequency of metaphases with chromosome aberrations was observed (at 71 mg/kg to 7.80 ± 1.20%; at 7.1 mg/kg - up to 6.20 ± 1.08%; 0.71 mg/kg - up to 5.00 ± 0.97%; 0.07 mg / kg - up to 3.80 ± 0.86%).
Thus, 2,6-dimethylpyridine-N-oxide in the studied doses doesn`t induce chromosome aberrations in the mouse bone marrow cells. On the background of Cyclophosphamide, 2,6-dimethylpyridine-N-oxide reduced the number of aberrant cells in doses from 71.0 mg/kg to 0.071 mg/kg with the greatest effect at 0.071 mg/kg (75,86%). This indicates that the 2,6-dimethylpyridine-N-oxide reduces the mutagenic effect of cyclophosphamide.
Keywords: 2.6-dimethylpyridine-N-oxide, Cytogenetic activity, chromosome aberrations, cyclophosphamide
Evaluation of Inhaled Low Dose Formaldehyde Induced DNA Damage by Liquid Chromatography-Tandem Mass Spectrometry (#633)
J. Leng2, 3, C. - W. Liu2, H. J. Hartwell2, R. Yu2, Y. Lai4, K. Lu2, E. Leibold1, J. A. Swenberg2
1 BASF SE, Product Safety, Ludwigshafen, Germany
As a high volume chemical, formaldehyde toxicity and carcinogenesis have been controversial. Previously, we have demonstrated that exogenous formaldehyde induced DNA monoadducts accumulate in a highly nonlinear manner. However, the effect of low dose exogenous formaldehyde exposure in the range of regulatory limit values is still unclear. In this study, both exogenous and endogenous DNA monoadduct (N2-HOMe-dG) and DNA-protein crosslink (DPC, dG-Me-Cys) were measured to assess the accumulation of DNA adducts arising from the inhalation to 0, 1, 30, 300 ppb [13C2H2]-formaldehyde for 28 days (6 hours/day). Ultrasensitive nano-liquid chromatography mass spectrometry, including triple quadruple and high resolution Orbitrap mass spectrometer, was used to improve the sensitivity and detection of DNA monoadducts and DPC. Our data clearly show that low exogenous formaldehyde exposure did not cause detectable amounts of exogenous DNA monoadducts or DPC in any tissue of exposed rats. In contrast, endogenous formaldehyde adducts were detectable in all tissues analyzed, with mean levels ranging from 2.35 to 5.06 adducts per 107 dG and 1.52 to 8.03 adducts per 108 dG for DNA monadducts and DPC in different tissues, respectively. These novel findings substantiate the threshold mode of action of carcinogenesis and will further improve risk assessment of low formaldehyde exposures in the range of regulatory limit values.
Keywords: Formaldehyde, carcinogenesis, DNA-adducts, risk assessment
Investigation of the effects of infrared rays on DNA strand in hot environment workers by comet assay (#656)
S. Kanbur1, A. Beceren2, S. Aydemir3, M. F. Uzun4
1 Gedik University, Department of Occupational Health and Safety, Health Sciences Faculty, Istanbul, Turkey
IR rays extend from the nominal red edge of the visible spectrum at 700 nm to 1 mm. Occupational exposure of infrared (IR) radiation can emerge in many industrial processes such as bakery, food processing, glass and porcelain productions, iron foundry and mining. IR radiation has beneficial uses for industrial, scientific, and medical applications; however, its possible unhealthy outcomes are unclear. When the thermal comfort conditions are not provided for the bakery employees, especially in the working environments with high temperature values, effects such as fatigue, cramps and heat stroke are observed in the early period. These health problems arise, in particular, from shorter IR wavelengths. In addition, there are not enough studies on the genotoxic risks of bakery workers. In the present study, we aimed to investigate the DNA damage in bakery employees occupationally exposed to the IR rays in hot environment.
This study was performed with the employees of a bakery company in Istanbul. A total of 80 male individuals (n=30, control; n=50, subject) were included and all of them were volunteers. Control group consisted of healthy individuals not exposed to IR rays. Fresh blood samples were collected from participants and conduct with the comet assay within the same day to evaluate the DNA damage. The standard protocol for alkaline comet assay was operated for the lymphocytes of the participants with minor modifications of Singh et al, 1988. Randomly selected 100 cells per sample (two duplicate sample slides, 50 cells for each) were scored to count the percentage DNA in tail (%DNAT) using BAB Bs200Pro image analysis software (BAB LTD., Ankara, Turkey).
DNA damage assessed in 80 subjects showed that the mean %DNAT was significantly high (p<0.001) in occupationally exposed subjects as 31,61 ± 4,43 compared with unexposed controls (15,62 ± 3,18). Nonsmoker subjects also showed higher level of the mean %DNAT (21,79 ± 1,98) respect to controls. In conclusion, we suggested that, exposure to IR rays may induce genotoxic effect in the peripheral lymphocytes and smoking has synergistic effect on DNA damage.
Keywords: Genotoxicity, Comet assay, IR rays, Occupational exposure
Sericin nanocarriers loaded with doxorubicin induce DNA damage in breast cancer cells (#669)
A. Hudita1, V. Lavric1, I. C. Radu2, B. Galateanu1, C. Zaharia2, C. Negrei3, O. Ginghina4, 5, M. Costache1
1 University of Bucharest, Department of Biochemistry and Molecular Biology, Bucharest, Romania
Doxorubicin is a well-known effective chemotherapeutic agent administrated especially in breast cancer treatments. Despite its efficacy, doxorubicin causes severe toxicity to most major organs, especially irreversible cardiotoxicity. In order to manage this dangerous toxicity, a smart therapeutically approach would be the use of drug – delivery systems for doxorubicin targeted delivery. More, using a drug – delivery system for doxorubicin could protect the drug from degradation, improve its therapeutic concentration and significantly increase its bioavailability. In this view, the aim of our study was to develop and in vitro validate a nanosized drug delivery system for doxorubicin based on the natural polymer silk sericin.
After nanoparticle synthesis, the sericin nanocarriers obtained were characterized in terms of size and morphology. The doxorubicin uptake was determined by UV – VIS spectrophotometry and the drug release potential was investigated using UV – VIS spectroscopy. Using the MCF – 7 breast cancer cell line, the lethal dose 50 (DL50) of the sericin nanoparticles was determined. Moreover, the toxicity of the unloaded and doxorubicin loaded nanoparticles was evaluated in MCF – 7 cells after 12h and 24h of treatment in terms of cell viability and proliferation potential, as well as by highlighting the morphological changes that the treatment regimen triggers in the breast cancer cells. Additionally, the genotoxic potential of the sericin nanoparticles was also investigated by measuring the DNA damage induced by treatment through comet assay.
Our results show that we obtained small nanoparticles that presented the typically round shape morphology. The treatment with the unloaded sericin nanoparticles did not exert any cytotoxic effects on MCF – 7 cells as it did not affect MCF – 7 cell viability, proliferation potential and morphology and no comets that indicate DNA damage were observed after comet assay. However, the treatment of the same cells with doxorubicin loaded sericin nanoparticles dramatically decreased cell viability and proliferation potential, altered the extracellular matrix shape and induced serious DNA damage as numerous comets could be observed after the treatment.
Doxorubicin loaded sericin nanoparticles exert cytotoxic effects on breast cancer cells and could be further used for in vivo studies on animal models in order to better describe their mechanism of action and to determine their distribution pattern and cleareance.
Keywords: nanocarrier, nanotoxicity, breast cancer, doxorubicin, personalized medicine
Safety profile characterization of a cosmetic silanol compound in vivo (#709)
L. MARTINO-ROARO1, A. VERGARA-CASTAÑEDA1, R. SANCHEZ-HUESCA1, E. VERGARA2, 1, J. C. SANCHEZ-CARRILLO3, U. LOPEZ-GARDUÑO1
1 UNIVERSIDAD LA SALLE, GRUPO DE INVESTIGACIÓN EN CIENCIAS BÁSICAS Y CLÍNICAS DE LA SALUD. FACULTAD DE CIENCIAS QUÍMICAS, MEXICO, Mexico
The Preclinical Phase needs to be completed to initiate Clinical Phases, which are specially regulated in the research and development of new drugs. For substances commercialized differently to a medicine as a cosmetic, cosmeceutical, supplement or food there is a void of regulation. Common sense indicates that the safety assessment is necessary for all types of substances that interact with humans. Our interest is to characterize the safety of a common active compound used in cosmetics, the caffeine-alginate methylsilanetriol (CAMeSi) whose mechanism of action is inhibiting the lipoprotein-lipase and direct stimulation to G-coupled receptor to increase the adenylate cyclase enzyme. In two previous studies, we determined the DL50 in mice that classified CAMeSi as a non-toxic compound according to the OCDE guidelines, and a non-genotoxic agent with the micronucleus test in mouse blood cells. The objective was to determine if CAMeSi has effects over the cell proliferation through the ratio of polychromatic erythrocytes (PE)/normochromic erythrocytes, this ratio reflects the cytotoxicity. 5 groups of male mice (n=5) received a single dose i.p. of: 1) 0.3mL of butylene glycol-distilled water (negative control), 2) 0.02 mL/Kg CAMeSi, 3) 0.28 mL/Kg CAMeSi, 4) 2.86 mL/Kg CAMeSi and 5) 60 mg/Kg of ifosfamide, a cytotoxic alkylating agent. Kruskal-Wallis nonparametric test and Dunn's multiple comparison test were applied to data.
Results indicated that the butylene glycol-water mixture was not a good negative control as it decreased 30% the number of PE, ifosfamide on the contrary, showed the greatest cytotoxic effect at 72 h (80.5%). On the effect of CAMeSi administered in the 3 chosen doses, the compound modified PE production after 24 h. The maximum cytotoxic effect was obtained at 72 h with the higher dose (70.5%), the low and medium doses had a slight recovery, but the damage on cell proliferation persists (35.4% and 51.2%). Thus, the CAMeSi showed an evident cytotoxic effect at a biological level. It is important to carry out other cytotoxicity tests exposing more animals, since these results suggest the compound presented variability intersubjects. More studies on CAMeSi are necessary to clarify the risk of exposing population to this substance.
Keywords: citotoxicity, caffeine-alginate methylsilanetriol
Genotoxicity assessment of caffeine-alginate methylsilanetriol with the mammalian erythrocyte micronucleus test (#711)
A. Vergara-Castañeda1, L. Martino-Roaro1, R. Sanchez-Huesca1, E. Vergara1, 2, J. C. Sánchez-Carrillo3, M. Martínez-Briseño1
1 Universidad La Salle, Grupo de Investigación en Ciencias Básicas y Clínicas de la Salud. Facultad de Ciencias Químicas, Ciudad de México, Mexico
Actually, the toxicological evaluation of new compounds is mandatory by all regulatory agencies related to health. Due to the impact of the results of tests that evaluate DNA damage, it is necessary not only to investigate new molecules but also the existing ones, especially those that are active ingredients of medicines, cosmetics and other consumer products. Caffeine-alginate methylsilanetriol (CAMeSi) is a complex of organic silanol and caffeine, whose synergic activity enhances lipolysis in the adipose tissue. This compound is part of innumerable formulations of multiple commercial brands in cosmetic, cosmeceutical and health-care products, then, contact with general population is already a fact and there’s no evidence of its possible genotoxic effects, therefore, it is very important to perform research in this topic.
The aim of this study was to assess the genotoxicity of CAMeSi in vivo. The experiment was done according to the OECD 474 guideline. 25 male mice were organized in 5 groups with 5 individuals each and were administered intraperitoneally with: 0.3mL of butylene glycol-distilled water (negative control), three other groups with CAMeSi (0.02 mL/Kg, 0.28 mL/Kg y 2.86 mL/Kg) and the last group was treated with 60 mg/Kg of ifosfamide, an antineoplastic alkylating agent known as a MN inductor. A blood sample from the tail of each mouse was obtained prior administration, and at 24, 48 and 72 h post-administration; then, were smeared onto slides, fixed with methanol, stained with Giemsa solution and observed microscopically to score the rate of micronucleated polychromatic erythrocytes (MNPE) in 1000 polychromatic erythrocytes per mouse. The obtained data was statistically analyzed with Kruskal-Wallis (p<0.05) and Dunn’s multiple comparison post hoc tests.
Ifosfamide increased the rate of micronuclei (MN) from 24 h (24.7 ± 1.45 MN) and showed the highest effect at 48 h (26.7 ± 1.93 MN). The CAMeSi doses evaluated did not modify the frequency of MN compared with the negative control group throughout the experiment (3.85 vs 2.8 MN respectively), therefore it was not a genotoxic compound under the conditions of the present study. Based on the results obtained, we suggest to continue with the characterization of the CAMeSi safety profile.
Keywords: Genotoxicity, Caffeine-alginate methylsilanetriol, micronucleus, in vivo
Compilation of historical negative and positive control group data of CHO-HPRT assay as per OECD guidelines. (#735)
K. K. MISHRA1, K. D. VASHI1, I. M. BARAD1, R. M. NAGANE1
1 Jai Research Foundation, Toxicology/Mutagenicity, Vapi, Gujarat, India
The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines (OECD 476) emphasize the importance of historical negative controls both for data quality and interpretation of results. The objective of the present paper is to discuss various methods applied to present the historical control data. In this paper data obtained from more than 20 individual experiments conducted in CHO-K1 cell line, as per the recent revision of OECD 476 were used for historical control data compilation. Briefly the cultures were exposed for 4-6 hour exposure period in the absence and presence of metabolic activation in duplicates. Following treatment the cultures were subcultured at regular interval and subjected to selective medium for selection of mutant colonies. At the end of incubation period of 16 days spontaneous mutation frequency were enumerated. In the present abstract we have discussed the method to calculate 95% CI and quality control (QC) methods (i.e. C-Chart).
The laboratory maximum-minimum limits obtained through mean control limits or range without transformation, affects interpretation of results (or biological relevance) when the study results are higher than the concurrent control but within the maximum and minimum mean control limits. Therefore historical control data transformation using 95% CI was performed.
Results shows that historical control data presentation using 95% CI is more precise and meaningful for acceptance of study and interpretation of the results.
Keywords: CHO, HPRT Assay, Historical negative control data
Translational regulation of Ercc5 after benzo[a]pyrene exposure in mouse hepatocytes (#323)
E. Smit1, R. Haerkens1, J. Kleinjans1, T. van den Beucken1
1 Maastricht University, Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht, Netherlands
Translation of mRNAs is an important part of gene expression. By controlling mRNA translation, rapid cellular alterations in protein expression can be achieved under stressful conditions. Here we aimed to identify translationally regulated mRNAs after benzo[a]pyrene (BaP) exposure and validate the relevance of a translationally altered candidate gene. We have exposed primary mouse hepatocytes (PMHs) to 10µM BaP for 24h. Differentially translated mRNAs were captured and RNAseq was used to assess changes in mRNA abundance and mRNA translation. From this analysis, Errc5 was identified as a translationally upregulated gene after BaP exposure. Errc5 is part of the nucleotide excision repair (NER) mechanism that removes DNA adducts which for example are formed after BaP exposure. Ercc5 cuts on the 3’ end of the lesion and contributes to the removal of the adduct-containing DNA. Next, we confirmed translational regulation of Ercc5 in immortalized mouse hepatocytes (AML-12). Exposure to 10 or 20µM BaP for 24h increased mRNA translation of Errc5 without affecting total Ercc5 mRNA levels, confirming the findings in the PMHs. To study the biological relevance of Errc5 for the cellular response to BaP we have established AML-12 cell lines with stable Ercc5 knock-down using two different lentiviral small hairpin RNAs (shRNAs). Our preliminary results suggest that shErrc5 cells are more sensitive to BaP exposure compared to cells expressing a control shRNA when using cell survival and viability as an endpoint. We are currently conducting studies with shErrc5 cells to determine the impact on DNA adduct levels after exposure to BaP. Furthermore, we will determine which upstream signaling events trigger increased mRNA translation of Errc5 in response to BaP.
Keywords: mRNA translation, Ercc5, Benzo[a]pyrene, mouse hepatocytes