Inhalation exposure of transgenic Big Blue® mice: Qualification of nasal and lung tissues

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Abstracts / Toxicology Letters 258S (2016) S62–S324

P16-028 Low level arsenic exposure during pregnancy in the 3xG cohort in Flanders: Less efficient detoxification is associated with higher oxidative stress

P16-029 Alteration on DNA methylation of rassf1, cyclin D2 and P16 genes and global DNA methylation and histone modifications in human hepatocarcinoma cells in response to BPA

N. Lambrechts 1,∗ , E. Govarts 1 , A. Colles 1 , S. Remy 2 , B. Morrens 3 , M. Leermaekers 5 , E. Van De Mieroop 4 , E. Den Hond 4 , V. Nelen 4 , I. Loots 3 , G. Schoeters 1

E.F. Karaman 1,∗ , M. S¸enyıldız 1 , S. Sancar Bas¸ 2 , P. Arda Pirinc¸c¸i 2 , S. Özden 1 1

1

Environmental Risk and Health, Flemish Institute for Technological Research (VITO), Mol, Belgium 2 University of Antwerp, Department of Biomedical Sciences, Antwerp, Belgium 3 University of Antwerp, Department of Social Sciences, Antwerp, Belgium 4 Department of Health, Provincial Institute for Hygiene (PIH), Antwerp, Belgium 5 Department of Analytical, Environmental and Geochemistry (AMGC), Vrije Universiteit Brussel (VUB), Brussels, Belgium Increasing epidemiologic evidence indicates that even low level arsenic (As) exposure affects adverse pregnancy outcomes. It is well known that As metabolism changes during pregnancy by means of detoxification. This study investigates whether urinary As species and As methylation efficiency are associated with oxidative stress in 151 pregnant women of the 3xG cohort in Flanders, Belgium (2012–2015). Urine samples were collected between 25 and 35 weeks of gestation and different As species (inorganic As (iAs; As(III), As(V)), monomethylAs acid (MMA) and dimethylAs acid (DMA)) were measured using HPLC-IC-MS. Multiple linear regression analyses were performed to evaluate the associations between As exposure and oxidative stress, measured by the amount of 8-hydroxy2 -deoxyguanosine (8-OHdG, ln-transformed) in the same urine samples (ELISA). The associations were adjusted for age and smoking before sample collection. Increased 8-OHdG levels values were associated with higher MMA levels: for each ␮g/l increase in MMA, 8-OHdG increased with 24% (␮g/l) (95% CI = 0.5–51.7%). Further, a decreasing trend between the secondary methylation index (=DMA/MMA) and 8OHdG was found (ˇ = 0.998 (95% CI = 0.996–1.000)). No significant associations were observed for iAs, DMA and the primary methylation index (=MMA/iAS) with 8-OHdG. The information in this study indicates that in a region with a relatively low environmental As burden, the toxic metabolite MMA is associated with increased oxidative stress in pregnant women. Furthermore, mothers with less efficient detoxification of As had higher levels of 8-OHdG. The 3xG study is part of the cAT project, funded by NIRAS and the local partnerships MONA and STORA. http://dx.doi.org/10.1016/j.toxlet.2016.06.1867

Department of Pharmaceutical Toxicology, Istanbul University, Faculty of Pharmacy, Istanbul, Turkey 2 Department of Molecular Biology, Istanbul University, Faculty of Science, Istanbul, Turkey Bisphenol A (BPA), as synthetic monomer used in industry in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Continuous release of free BPA into food, beverages, and the environment has resulted in a widespread human exposure to this chemical. Defining the hazard identification under the risk assessment of chemicals is an important step in the elucidation of the mechanism of toxicity. Recently, showing the role of endocrine effects of environmental chemicals on the changes in gene expression may be associated with epigenetic mechanisms such as DNA methylation and histone modifications are emerging as an alternative way. The aim of the study was to investigate dose-related effects of BPA (0, 100 nM, 1 and 10 ␮M for 48 and 96 h) on DNA methylation in human hepatocarcinoma (HepG2) cells. We also investigated expression profiles of p16, cyclineD1 and Rassf1 genes and global histone modifications (H3K9me3, H3K9ac, H3K27me3). IC50 value of BPA was determined as 134 and 180 ␮M in HepG2 cells for 24 h by MTT and neutral red uptake tests. We observed decrease on the global levels of 5methylcytosine and 5hydroxymethylcytosine at 1 and 10 ␮M after 96 h BPA exposure. BPA caused alterations on the expression of p16, cyclineD1 and Rassf1 genes. Further investigations on the effects of BPA on histone modifications are underway in order to give more insight to the role of epigenetic mechanisms of BPA in HepG2 cells. In this study we suggest that BPA may disrupt epigenetic events by altering global and gene-specific DNA. http://dx.doi.org/10.1016/j.toxlet.2016.06.1868 P16-030 Inhalation exposure of transgenic Big Blue® mice: Qualification of nasal and lung tissues R. Young ∗ , H. Dinesdurage Department of Toxicology, BioReliance, Rockville, MD, USA Inhalation is a significant route of human exposure to chemicals. As a result, in vivo inhalation genotoxicity and carcinogenicity studies are often part of chemical safety/risk assessments of gasses and volatile chemicals. OECD Test Guideline 488, Transgenic Rodent (TGR) Mutation Assay, permits assessment of upper respiratory tract mutagenicity following inhalation exposures. Qualification of the Big Blue® (BB) mouse assay required modification and optimization of nasal cavity and lung tissue collection and DNA isolation, as well as generating historical control data from untreated and positive control exposed animals. Nasal tissue was collected from left and right nasal cavity by removing respiratory and olfactory epithelium with associated turbinates and septum. Modified DNA isolation methods were developed for nasal epithelium that included liquid nitrogen-cooled pulverization, homogenization, digestion and phenol chloroform extraction. Lung was extracted

Abstracts / Toxicology Letters 258S (2016) S62–S324

using standard homogenization, digestion and dialysis methods using RecoverEase kits from Agilent. Background mutant frequencies (MF) from five studies were consistent with other somatic tissues. Statistically significant increases in cII MF were observed in lung (57.4 ± 15.0 × 10−2 in controls vs 154 ± 24.0 × 10−2 in ENU treatment groups) and nasal samples (54.4 ± 10.8 × 10−2 vs 416 ± 54.0 × 10−2 ). In comparison, similar background MF and increases were seen with liver (4.2-fold), glandular stomach (18.6fold) and bone marrow (14.5-fold). These results extend the range of the BB TGR mouse mutation assay to upper respiratory tract tissues thereby demonstrating robustness of the assay and the assay’s suitable for use with inhalation exposure. http://dx.doi.org/10.1016/j.toxlet.2016.06.1869 P16-031 Alkylating agents activate chemosensing Transient Receptor Potential A1 cation channels B. Stenger 1,∗ , D. Steinritz 2 , A. Tsoutsoulopoulos 2 , A. Breit 1 , H. Thiermann 2 , A. Schmidt 2 , H. John 2 , T. Popp 2 , T. Gudermann 1 1

Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilian-Universität München, Germany 2 Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Alkylating agents have been used for chemical warfare since World War I. Recent reports from Syria and Iraq on the use of sulfur mustard (SM) by militant groups underline the acute threat by these compounds. Exposure to SM results in painful injuries affecting the respiratory system, eyes and skin. Despite decades of medical research, there is neither a specific therapy nor an antidote currently available. Toxicity provoked by highly reactive compounds has been considered as an unspecific process resulting in uncontrollable cell damage. However, recent research identified Transient Receptor Potential A1 channels (TRPA1) as cellular chemosensors for a plethora of noxious substances implicating a substantial role in molecular toxicology. TRPA1 is expressed in different human tissues including skin, lung and neuronal tissue. Here we demonstrate that SM and the monofunctional alkylating agent CEES (model substance for SMpromoted effects) are both able to activate TRPA1 channels, that results in the elevation of intracellular calcium concentrations. The TRP-channel blocker AP18 was able to prevent the CEES/SMinduced calcium response. In HEK293 cells overexpressing TRPA1, CEES cytotoxity was significantly enhanced compared to wildtype cells. TRPA1 blockers attenuated cytotoxicity, especially in the low-dose range of CEES. Similar results were found in A549 lung epithelial cells, endogenously expressing TRPA1. These cells also responded after CEES/SM challenge with a distinct calcium influx that could be diminished by AP18. In summary, TRPA1 channel are involved in the pathophysiology of alkylating compounds and drugs addressing these channels may represent a promising approach to mitigate alkylating agent induced cell damage. http://dx.doi.org/10.1016/j.toxlet.2016.06.1870

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P16-032 CeO2 nanoparticle attenuates paraquat-induced liver injury by decreasing oxidative stress and DNA damage A. Ranjbar ∗ , H. Heidary Dartoti, S. Soleimani Asl, F. Firozian, M. Taheri Azandariani Cerium oxide nanoparticle (CeNPs) is one of the most widely used and most important nanoparticles in addition to having strong antioxidative properties and inhibiting free radicals. Paraquat (PQ), an effective and widely used herbicide, has been proven to be safe when appropriately applied to eliminate weeds. The purpose of this study was to investigate the effect of CeNPs against PQ-induced liver injury in association with its antioxidant activity. The male rats were treated intraperitoneally daily with PQ (50 mg/kg/day) and CeNPs (15, 30 and 60 mg/kg/day) were administered alone or in combination for 14 days. After treatments, serum and liver tissue samples were collected from all rats. Oxidative stress biomarkers include total antioxidant capacity (TAC), lipid peroxidation (LPO), total thiol groups (TTG), DNA damage and NO levels were carried out. The results have shown that PQ induces oxidation stress and hepatic tissue damage in comparison to control group, CeNPs especially in 15 mg/kg dose has antioxidative effect and taking this combination with PQ compromises the damage. Oxidative stress and consequently hepatic damage induced by PQ can be reduced by CeNPs in lower doses. http://dx.doi.org/10.1016/j.toxlet.2016.06.1871 P16-033 Molecular mechanisms of alkylphenol-mediated endocrine disruption in Leydig cells P. Labohá 1,∗ , T. Jambor 2 , A. Yawer 1 , N. Lukáˇc 2 , I. Sovadinová 1 1

Research Centre for Toxic Compounds in the Environment (Recetox), Faculty of Science, Masaryk University, Brno, Czech Republic 2 Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic Recently, a dramatic decline of male reproduction function has been reported, associated with impairment of testicular functions and fertility. Nonylphenol (NP) and octylphenol (OP) are widespread man-made alkylphenolic environmental contaminants with endocrine disruptive properties listed among the EU priority hazardous substances. NP and OP have been shown to decrease sperm fertilizing ability and functions of male reproductive system. Both these alkylphenols interfere with estrogen and androgen receptors, and thus with proper functioning of natural hormones that are essential for normal development of male reproductive system. However, there is still a limited knowledge on different molecular mechanisms underlying their adverse effects on male reproduction system. Our study focused on epigenetic mechanisms that could be involved in NP and OP toxicity, namely on gap junctional intercellular communication (GJIC) through connexin (Cx) channels. Cx43-based GJIC between Leydig cells plays crucial roles in proper testicular steroidogenesis and hormonal control of spermatogenesis. We determined the effects of NP and OP on viability and proliferation of mouse Leydig TM3 cells (ATCC) and assessed their ability to disrupt GJIC in TM3 cells and alter Cx43 expression and phosphorylation. Since TM3 cells express estrogen receptor (ER), we used specific inhibitors of different signalling pathways to determine if the effects on GJIC are mediated via ER or via different