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Use of the γH2AX/pH3 in vitro genotoxicity method for genotoxicity mode of action determination in the context of chemical risk assessment.
October 17, 2024, 10 a.m. ET
Abstract: The development of in vitro genotoxicity assays as an alternative to animal carcinogenicity testing is of growing interest. The improvement of fast and specific high-throughput in vitro genotoxic assays for genotoxicity and cancer hazard assessment is a major priority. The quantification of histone H2AX and H3 phosphorylation markers (γH2AX and pH3) was proposed to have an insight into the mode of action (MoA) of genotoxicity. While an increase in γH2AX is observed after cell treatment with clastogens, pH3 induction is observed after exposure to aneugens. The in vitro γH2AX/pH3 method is the first one to permit to easily and reliably discriminate the genotoxic mode of action (clastogens, aneugens and misleading cytotoxic chemicals). This method, coupled with the used of human cell lines with different bioactivation capacities, permit to differentiated direct genotoxins from bioactivated ones.
In 2023, the Organisation for Economic Co-operation and Development (OECD) endorsed the Development of a Detailed Review Paper (DRP) and a Retrospective Performance Analysis (RPA) for the in vitro γH2AX/pH3 method. For this purpose, an expert working group review 298 studies performed in the last 15 years with this test method. In total, 817 chemicals were included in the dataset comprising aneugens (4,6%), clastogen (53,1%), aneugen/clastogen (2,1%) and non-genotoxic chemicals (40,1%). Different mechanisms of action for aneugenic (kinases inhibitors, tubulin binder) and clastogenic compounds (oxidative stress, bulky DNA adducts, topoisomerase inhibitor, inter-crosslink, dNTPs pool imbalance, nucleoside analogues, alkylation, intercalating agent, DNA repair inhibitor) were covered in the dataset as well as “false” positive genotoxins (apoptosis or p53 inducers). Regular in vitro and in vivo genotoxic test results for each chemical have been also collected. In this lecture, a chemical analysis of the used dataset using multi-descriptors will be presented. Then, the performance of the in vitro γH2AX/pH3 method (sensitivity, specificity and accuracy) to detected genotoxicity will be reported and compared to the bacterial Ames (TG471), the mammalian cell in vitro mutation tests (TG476 and TG490) and the in vitro micronucleus (TG487) and chromosome aberration assays (TG473) tests. First analysis of the dataset suggested a good accuracy of the in vitro γH2AX/pH3 method for in vitro and in vivo genotoxicity detection.
Author Bio: Dr. Marc Audebert (M), PhD in Life Sciences and Toxicology, is currently Research Director at INRAE Toxalim. He is part of the “Metabolism and Xenobiotics” (MeX) team, which gained international recognition in the field of metabolic and genotoxicity studies. He is involved in OECD test guideline development for genotoxicity testing for France. MA skills in different scientific domains : genotoxicity, cell and molecular biology, DNA repair, carcinogenesis, cell signalling. His current research focuses on a novel genotoxicity assay based on the quantification of histone H2AX/H3 phosphorylation, with simultaneous examination of the cytotoxicity and genotoxicity of xenobiotics on human cells. Using this assay, he successfully evaluated the genotoxic potential and metabolism of several groups of food contaminants, using single compounds as well as mixture. He has coordinated or participated to several projects contributing to establish the scientific basis for predicting and assessing the genotoxic effects of chemicals withe NAMs. MA is board member of the French-Society of genetic toxicology and European Environmental Mutagen and Genomics Society since 2010.
DyNAMic Discussions Series
Learn more about the DyNAMic Discussions series and webinars.
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- Dr. Matthias Herzler, Towards a unified NGRA framework.
Using ‘Omics for in vitro toxicology and drug discovery
Date: November 21, 2024
Time: 10:00 – 11:30 a.m. ET
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- Dr. David Rouquie, Bayer. OASIS: a multi-stakeholder collaboration leveraging robust omics data to evaluate liver toxicity.
- Dr. Jessica Ewald, Broad Institute. Cell Painting as an emerging omics’ tool for toxicology and drug discovery.
