P21-09 Cocktail Effects of Endocrine Disruptors and Carcinogenic Chemicals on DNA Alterations in a Human Liver Organoid Model

Chronic liver diseases, such as metabolism-associated steatotic liver disease (MASLD) and hepatocellular carcinoma, are increasing, potentially linked to daily exposure to complex mixtures of environmental contaminants within the chemical exposome. Our research focuses on two contaminant families of concern for human health: endocrine disrupting chemicals (EDC), specifically per- and polyfluoroalkyl substances (PFAS), and heterocyclic aromatic amines (HAAs), which are pro-carcinogens found in food and air. EDCs may disrupt lipid and glucose metabolism, aggravating MASLD, while HAAs are suspected to induce mutations in the liver. Our goal is to characterize the combined metabolic and genotoxic effects of PFAS and HAA mixtures in human liver to elucidate the associated toxicological mechanisms. This includes determining if these contaminants have additive, synergistic, or antagonistic interactions through shared molecular targets, particularly nuclear receptors. To ensure accurate toxicity prediction in human, and to replace, refine or reduce animal testing in line with the 3Rs principle, experiments were conducted using the Hepoid® model, an innovative 3D human liver cell model where hepatocytes cultivated in a defined matrix are concomittantly differenciated and proliferative for many days (Rose et al. 2021) , (Rose et al. 2022) . After 14 days of treatment, neither PFAS nor HAAs, used at human-relevant doses, exhibited cytotoxicity, as determined by ATP measurement. Moreover, PFAS (PFOA, PFOS, PFHxS) induced significant lipid droplet accumulation in hepatocytes regardless of dose as well as HAAs (AaC, IQ). Based on these findings, conditions mimicking steatosis were established to perform genotoxicity and mutagenicity assays. Primary DNA lesions were measured by gH2AX immunolabelling and comet assay, while chromosomal aberrations were estimated using the micronucleus test. No synergistic or additive genotoxic effects were observed, suggesting efficient DNA repair mechanisms. To investigate molecular mechanisms underlying PFAS and HAA interactions, RT-qPCR was used to measure the expression of genes involved in lipid, glucose, xenobiotic metabolism as well as DNA damage response. Moreover, siRNA transfections have partially eliminated several nuclear receptors and reduced the expression of target genes, enabling us to study molecular mechanisms in greater depth.Our innovative approach elucidates the toxic effects of contaminant mixtures on human health, in particular chronic liver diseases by mimicking MASLD, and provides new insights for risk assessment and prevention.