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Exposure to environmental levels of polycyclic aromatic hydrocarbons leads to epigenetic modulation in a rat model (#45)
R. C. Duca1, N. Grova2, M. Gosh1, P. H. Hoet1, J. Vanoirbeek1, B. M. Appenzeller2, L. Godderis1, 3
1 KU Leuven, Environment and Health, Leuven, Belgium
Besides genetic mechanisms, rapidly growing evidences have linked environmental pollutants (e.g. polycyclic aromatic hydrocarbons, PAH), with epigenetic variations, including changes in DNA methylation among others. To date, studies concerning the effect of PAH exposure on the global DNA methylation are limited and contradictory results have been observed.
In this context, the present study aimed to evaluate the epigenetic alterations induced by multi-residue exposure to PAH. Female Long Evans rats were exposed to a mixture of 16 US-EPA priority PAH, 3 times per week over a 90-day period. The livers were used to assess the (hydroxy)methylation status of genomic DNA / RNA, together with reduced and oxidized forms of glutathione.
Our results showed an increase of GSH after chronical exposure to relatively low amounts of 16 US-EPA PAHs due to a stimulation of phase II detoxification mechanism through GSH conjugation in response to unbalance redox environment. This increased need of GSH leads to global DNA and RNA hypomethylation. In contrast, our results indicate an increased level of DNA methylation for the highest exposure levels. This can be explained by the positive association between global DNA methylation levels and the formed PAH-DNA adducts.
Thus, we further hypothesize that depending on the levels of exposure, PAH toxic mechanism is changing from depletion of global DNA/RNA methylation at lower levels to the formation of DNA adducts at higher levels. This hormetic response may give critical insights into the PAH epigenetic carcinogenicity.
Keywords: polycyclic aromatic hydrocarbons, epigenetic changes, DNA and RNA (hydroxy)methylation, glutathione, rat liver
Mesotheliomagenic macrophages proliferate and are immunosuppressive in CNT-induced peritoneal mesothelioma in rat (#109)
M. Orsi1, H. Kiyambu1, C. Al Hatem1, D. Brusa1, M. Palmai-Pallag1, D. Lison1, F. Huaux1
1 Université Catholique de Louvain, Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Experimentale et Clinique (IREC), Bruxelles, Brussels, Belgium
Malignant mesothelioma (MM) is a cancer affecting serous membrane of pleural and peritoneal cavities caused by carcinogenic fibers such as asbestos or carbon nanotubes (CNT). MM is still highly refractory to existing therapies. We have recently shown that, beside neutrophilic inflammation, immunosuppressive monocytes preventing efficient anti-tumor immune responses are rapidly recruited and involved in the development of MM.
Our current experimental study aims to characterize macrophages present in the peritoneal cavity after mesotheliomagenic (CNT-7 and asbestos) and not mesotheliomagenic (CNT-T) –fiber injection. In particular, our goals are to investigate the origins of peritoneal macrophages and their possible role in the establishment of an early immunosuppressive microenvironment, which determines tumor escape.
We used a Wistar rat peritoneum model which allows directly exposing mesothelial cells to asbestos or CNT, and easily sampling the mesothelial cavity for monitoring macrophage responses during the carcinogenic process.
Our data indicate that macrophages (CD11b/chi and His48int) from CNT-7 (not CNT-T) -treated rats inhibit T lymphocyte proliferation in vitro and contribute to immunosuppressive environment established before tumor establishment. Furthermore, we demonstrate that mesotheliomagenic peritoneal macrophages possess the ability of self-renewal and proliferation compared to macrophages collected from saline- or CNT-T-treated animals.
Altogether, our data demonstrate that carcinogenic fibers possess the intrinsic capacity to induce a preferential, rapid and sustained accumulation of immunosuppressive and proliferating macrophages before mesothelioma is established.
Keywords: Immunosuppression, Mesothelioma, Macrophages, carbon nanotubes
Glial-neuronal signaling mechanisms underlying the neurotoxic effects of manganese (#174)
K. A. Popichak1, M. F. Afzali1, K. S. Kirkley1, R. B. Tjalkens1
1 Colorado State University, Fort Collins, Colorado, United States of America
Overexposure to manganese (Mn) causes inflammation and neuronal injury in the cortex and basal ganglia, resulting in neurotoxicity. The mechanisms underlying neuronal death from exposure to Mn are not well understood but involve inflammatory activation of microglia and astrocytes. Expression of neurotoxic inflammatory genes in glia is highly regulated through the NF-kB signaling pathway but the key factors modulating toxic glial-glial and glial-neuronal signaling by Mn are not known. We examined the role of NF-kB in Mn-induced neurotoxicity by exposing purified cultures of microglia, astrocytes or mixed glia to varying concentrations of Mn and then incubated neurons with the glial conditioned media (GCM) of each cell type. We hypothesized that mixed glial cultures exposed to Mn (0-100mM) would enhance glial activation and neuronal death compared to microglia, wild-type astrocytes or IKK-knockout astrocytes alone or in mixed cultures. Treatment of primary cultures of pure astrocytes, microglia or mixed glia with 0-100 mM Mn for up to 24 hrs resulted in enhanced mRNA expression for inducible nitric oxide synthase (NOS2) and multiple inflammatory cytokines and chemokines, including TNF, CCL2 and IL-6, with the highest expression levels detected in mixed glial cultures. Gene deletion of the NF-κB activator, IKK2, in knockout astrocytes dramatically reduced cytokine release in Mn-treated mixed glial cultures. Neuronal viability and apoptosis was measured by flow cytometry and live cell imaging following GCM exposure from purified cultures of glia and from mixed glia containing either wildtype or IKK2 knockout astrocytes. GCM from Mn-treated mixed glial cultures induced greater neuronal death than either cell type alone. Mn-treated GCM from IKK2 knockout mixed glia cultures significantly decreased expression of inflammatory cytokines and neuronal death compared to wildtype mixed glia cultures or GCM from purified astrocytes or microglia. This suggests that NF-kB activation in astrocytes is an important regulator of cell-cell communication between glia that mediates the injurious effects of Mn to neurons.
This work was supported by grants from the National Institutes of Health (ES021656, ES021656-S1, ES026860 and ES024183).
Keywords: Glia, Inflammation, Manganese, Neurotoxicity, Nuclear Factor-kappaB
Investigation of drug-induced cholestasis in HepaRG cells using untargeted LC-MS metabolomics (#196)
M. Cuykx1, 4, C. Beirnaert2, 3, R. M. Rodrigues4, K. Laukens2, 3, T. Vanhaecke4, A. Covaci1
1 University of Antwerp, Toxicological Centre, Wilrijk, Belgium
Despite pre-clinical toxicity screening, hepatotoxicity remains a major challenge in drug development. Mechanistic data, including mode of actions (MOAs), offer important insights to improve toxicity testing. In this context, metabolomics, the study of small endogenous molecules, is a powerful tool as it gives a holistic overview of the events in cellular metabolism. Here, metabolomics is applied to identify in vitro markers of toxicity for cholestasis, which is a severe type of drug-induced liver injury (DILI).
HepaRG cell cultures were exposed to the cholestatic reference toxicant bosentan at a sub-cytotoxic (IC10) and a 1/10th lower dose for an acute (24 h) and sub-chronic (72 h) period. Untargeted metabolomics was performed on the polar and non-polar metabolites using liquid chromatography and accurate mass spectrometry. The data were analysed using multivariate analyses to select markers of toxicity.
The intracellular metabolome of cells exposed to IC10 concentrations of bosentan was different from the negative control group. Exposure to the cholestatic agent bosentan induced alterations in the carnitine levels and phospholipid composition. Markers of toxicity in the polar fraction included disruption of the basic (amine) metabolic pathways and upregulation of phosphorylated products. Other markers included pantothenic acid, creatine and nucleotide alterations. Exposure to the lower dose could not be discriminated from the negative control group. Comparison of the identified in vitro markers of cholestatic toxicity identified to those found in a previous study using the steatotic agent valproic acid revealed clear distinct pattern differences in many metabolite classes.
Cholestasis caused by drug toxicity can be investigated in vitro through the combination of HepaRG cell cultures and LC-MS metabolomics. Exposure to IC10 concentrations of bosentan invoked clear responses and several potential markers of toxicity for cholestasis could be identified, that were different from previously established markers for steatosis. These different fingerprints suggest that metabolomics could be used to discriminate between different types and MOAs of DILI.
Keywords: HepaRG, Drug-Induced Liver Injury (DILI), Liquid Chromatography-Mass Spectrometry, Metabolomics, Cholestasis
Steatotic effects of propiconazole and tebuconazole in human liver cells are PXR-mediated (#267)
C. Knebel1, D. H. D. Nguyen2, A. Lampen2, A. Braeuning2, P. Marx-Stoelting1
1 German Federal Institute for Risk Assessment (BfR), Experimental Toxicology, Berlin, Berlin, Germany
Triazoles are widely used fungicides which show liver toxicity in vivo. While enzyme induction and associated hepatocellular hypertrophy are the most prominent findings, some of the triazoles have also been reported to cause hepatocellular vacuolization indicative of steatosis.
The aim of our study was to represent the steatosis adverse outcome pathway (AOP) by in vitro assays and to elucidate the molecular mechanism of triazole-mediated steatosis. Therefore, we used the two triazoles propiconazole (Pi) and tebuconazole (Te) as test substances. We analyzed the ability of Pi and Te to activate nuclear receptors described as molecular initiators in the AOP for steatosis in HepG2 cells. To investigate the expression of marker genes associated with the steatosis AOP, we performed RT-PCR in HepaRG cells. The triglyceride accumulation which is described as key event in the steatosis AOP was measured by AdipoRed staining. The JC-1-Assay was performed to analyze the mitochondrial disruption, another key event.
Our results indicate that Pi and Te interact with human PXR, AHR and CAR. Both substances induce the expression of steatosis-related genes. Furthermore, treatment with Pi and Te leads to triglyceride accumulation and mitochondrial disruption. This triglyceride accumulation was not visible in PXR knockout HepaRG cells.
All in all, our findings demonstrate that the steatosis AOP can be covered by a battery of in vitro assays, measured in cell lines of human origin, in order to facilitate analyses of the molecular mode(s) of action of test compounds. PXR activation seems to play an important role for triazole-mediated steatotic effects.
Keywords: Liver, AOP, steatosis, in vitro methods
microRNAs as signatures of structural cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (#285)
V. Gryshkova1, J. Palmer3, A. Smith3, R. Burrier3, M. Colwell3, A. Delaunois1, A. Nogueira da Costa2, J. - P. Valentin1
1 UCB Biopharma sprl, Development Science, Braine l'Alleud, Belgium
Cardiovascular toxicity accounts for approximately a third of safety related drug attritions over the past 10 years. By combining hERG inhibition data in vitro and QTc data in vivo, certain aspects of functional cardiotoxicity such as QT prolongation and arrhythmia can be accurately predicted. However, the mechanisms that underlie structural cardiotoxicity are poorly understood and novel mechanistic biomarkers are required to establish in vitro systems for detection of potential liabilities. MicroRNAs (miRNAs) are involved in a range of diseases, including cardiovascular pathologies, and have shown their potential to be reliable biomarkers. The aim of this study was to identify miRNAs dysregulated in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) treated with a panel of 39 drugs (9 – non-cardiotoxic; 9 - functional, 9 – structural, 12 – general, i.e. both functional and structural cardiotoxic compounds). Cells were exposed to each drug for 72 hours and cell pellets were used to perform Next Generation Sequencing (NGS) of miRNAs and small RNAs (Illumina platform). The differential expression analysis was done for all treatments compared to reference-treated controls and a two-fold change cut-off was applied to identify dysregulated miRNAs. Non-cardiotoxic and functional cardiotoxic compound treatments were associated with changes only in a limited number of miRNAs, whereas treatment with general or structural cardiotoxicants resulted in strong perturbations in miRNA expression. Among general and structural cardiotoxic compounds, anthracyclines and tyrosine kinase inhibitors were shown to have the highest impact on miRNA expression. The analysis of toxicological pathways (Ingenuity Pathway Analysis, IPA) of the dysregulated miRNAs showed complex molecular changes related to cardiac fibrosis, hypertrophy, inflammation, dilation, and infarction. This study is the first to identify miRNAs as toxicity signature of hIPSC-CM-treated with a large set of drugs. miRNA panels and their gene targets could be a valuable tool for early detection, prediction, and investigation of cardiotoxicity in vitro.
Keywords: biomarkers, omics, microRNAs, cardiotoxicity
The malformation of Purkinje cells becomes the sensing tool for developmental neurotoxicity - its potential and limitation (#358)
S. Yoshida1, S. Iwamoto1, Y. Fueta2, S. Ueno2, Y. Sekino3, Y. Nomura4, Y. Kanda5
1 Toyohashi University of Technology, Environmental and Life Sciences, Toyohashi, Japan
Autism spectrum disorder (ASD), which is a severe neurodevelopmental disorder, is reported to show cerebral and cerebellar abnormalities. Multiple chemicals, for example, sodium valproate (VPA) or chlorpyrifos (CPF), have been associated with an increased risk of ASD. In early human ASD, the cerebellar abnormality is one of the pathological focus points.
We have established the ASD-model rat with the administration to embryonic day 16 p.o. (VPA; 600mg/kg or CPF; 10 mg/kg of mother weight, respectively), and observed their cerebellar development. VPA- or CPF-treated ASD animal models showed the excess development of Purkinje cells and excess folds in the V to VI lobules of the cerebellar vermis within two weeks after birth. This alteration is similar to human early ASD cerebellum. Conversely, we observed little developmental abnormality of Purkinje cells in a 2.0 mg/kg tributyltin (TBT)-administrated rat, whereas TBT is the candidate of the neurodevelopmental toxins.
Because the VPA-administrated rat shows cerebellar alteration even in mature, we observed the behavior of VPA-administrated or vehicle male rats from six to eight weeks. An animal placed on the open-field circle (80 cm in diameter) with an overhead camera, was tested with regard to its behavior, social interaction to a same- or bigger-size strange rat and target interaction to well-known or new objects for 10 minutes. All tracking data was analyzed with Tracker Video Analysis and Modeling Tool produced by D. Brown.
In the individual behavior, VPA animals moved faster and longer than the vehicle. In the social interaction with a bigger-size stranger, VPA animals showed more fear than vehicle and sometimes froze. In target behavior to a new object, VPA animals kept away. The grooming time of VPA animals was shorter than the vehicle. Consequently, VPA-administrated ASD model animals with Purkinje cell alteration showed the trends of hyperactive behavior in a reassured condition but displayed fear to a strange situation, which behavior is similar to ADHD, not ASD. We suggest the malformation of Purkinje cells would help us clarify neurodevelopmental toxicity of chemicals and the chemical-induced ASD-animal models could mimic human ASD behavior, however, the model animal of mental disorder might have some limitation of human modification.
Keywords: Autism, model animal, developmental neurotoxicity, cerebellum
A possible risk of genome editing for human gene therapy (#436)
R. Ono1, K. Tano2, S. Yasuda2, Y. Yasuhiko1, K. Aisaki1, S. Kitajima1, J. Kanno3, Y. Sato2, Y. Hirabayashi1, 4
1 National Institute of Health Sciences (NIHS), Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, Kawasaki, Japan
The CRISPR/Cas system allows the introduction of double strand breaks (DSBs) at particular loci in the genome. DSBs are subsequently repaired through non-homologous end joining (NHEJ), or homologous recombination (HR).
To evaluate the frequency of the capture of unintentional sequences associated with genome editing in human iPS cells, we introduced mutations with CRISPR-Cas9 in 10 locus over 8 genes. 2000 single clones were isolated and the capture of unintentional sequences were analyzed. 3.5 % of clones were identified with the capture of unintentional sequences, including human short interspersed nuclear elements (SINE) and CRISPR-Cas9 vector sequences. The findings of this study highlight a new possible risk of genome editing for human gene therapy.
Reference: Ono R, Ishii M, Fujihara Y, Kitazawa M, Usami T, Kaneko-Ishino T, Kanno J, Ikawa M, Ishino F. 2015. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes. Scientific reports 5: 12281.
Keywords: capture of unintentional sequences, genome editing, human iPS cells
Molecular and chemical response signatures illustrate species differences of developing neural progenitor cells from human, mouse and rat (#616)
S. Masjosthusmann1, M. Barenys1, J. Baumann1, F. Bendt1, D. Becker1, R. Deenen2, N. Förster1, A. Mosig3, U. Hübenthal1, J. Klose1, K. Köhrer2, B. Petzuch1, M. Schmuck1, T. Temme1, 3, E. Fritsche1, 2
1 Leibniz Research Institute for Environmental Medicine, Düsseldorf, North Rhine-Westphalia, Germany
Given the high vulnerability of the developing brain there is concern that many compounds that have not been tested for their developmental neurotoxic potential place our children at risk. Therefore testing for Developmental Neurotoxicity (DNT) represents an emerging issue in future risk assessment. One approach to handle this issue is the use of alternative methods that if combined in a testing battery allow reliable, fast and cheap compound screening and prioritization for regulatory purposes. An in vitro methods that could be part of a testing battery are aggregated primary neural progenitor cells (NPC) growing as neurospheres. This alternative method functionally mimic progenitor cell proliferation, migration, neuronal-, and glial differentiation and therefore represents major neurodevelopmental key events in vitro. We performed a molecular and functional characterization, compared chemical response signatures generated from a test set of 17 DNT positive and 10 DNT negative compounds and conducted a mechanistic investigation of arsenite induced DNT in developing NPC of human and rodent. Transcriptome analyses revealed that Gene ontology (GO) biological processes involved in organ and tissue development and regulation of major neurodevelopmental processes are overrepresented in developing NPC. We found that despite conservation of GO categories across species, transcriptome changes in the mRNA profile are highly species-specific with only ~10% overlap of regulated genes. These differences on the molecular level are supported by species- specific chemical response signatures over an array of 8 endpoints that are highly relevant for brain development as well as by differences in the molecular mechanism by which arsenite induces adverse effects in developing NPC. Overall, this work demonstrates, (i) that a human based alternative system should be favored to predict human toxicity, and (ii) the importance of translational approaches that, based on the knowledge of in vitro species comparisons, predict human hazard more accurately than the conventional in vivo approaches in rodents.
Keywords: DNT, in vitro toxicity testing
Nrf2 regulates the skin innate immune response to chemical sensitizers (#673)
D. G. HELOU1, B. NOEL1, F. GAUDIN2, H. GROUX3, M. PALLARDY1, S. CHOLLET-MARTIN1, 4, S. KERDINE-ROMER1
1 Université Paris-Sud, Université Paris-Saclay, INSERM UMR 996, Châtenay-Malabry, France
The skin is the largest organ containing a variety of immune cells that could be directly exposed to many aggressions, such as ultraviolet radiation and chemical compounds. Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative stress and play an anti-inflammatory role. We previously demonstrated that Nrf2 controls skin inflammation in contact hypersensitivity. In this study, we highlighted Nrf2 role in the regulation of the innate immune response to 1-chloro-2,4-dinitrobenzene (DNCB), a well-known chemical sensitizer.
Wild-type (WT) and Nrf2 knock-out (KO) mice were treated with 1% of DNCB or with acetone (vehicle). Using H/E histological stain, we compared ear thickness and revealed that DNCB induced an exacerbated skin inflammation in KO mice in comparison to WT mice. Then, a PCR-array showed a high transcription of Nrf2 target genes in WT mice skin only, suggesting an activation of Nrf2 in skin cells. Moreover, the skin of KO mice evidenced a chemokine-rich microenvironment with higher levels of CCL2, CCL4 and CCL11 as compared to WT mice. Neutrophil and inflammatory monocyte recruitment to the skin was two- to three-fold higher in KO mice, 24 hours after DNCB treatment. Interestingly, skin macrophages of KO mice displayed a downregulation of F4/80 and an important upregulation of CD86. Furthermore, DNCB application failed to upregulate CD36 scavenger in KO mice skin macrophages in comparison with WT mice. Using an in vitro macrophage model, we showed that the absence of Nrf2 lead to a reduced phagocytosis capacity (in particular of damaged neutrophils), associated with a low induction of CD36. We suggest that the exacerbated skin inflammation measured in KO mice could be related, to a decreased phagocytic function of macrophages.
This study highlights the early implication of Nrf2 in the control of the skin innate immunity and points out its role in the resolution of skin inflammation, by improving CD36-dependent macrophage clearance capacity. Thus, we are suggesting for the first time that Nrf2 is a critical target in the chemically-induced skin inflammation.
Keywords: Nrf2, DNCB, innate immunity, skin, phagocytosis