AhR activation triggers the synergistic genotoxicity between benzo[a]pyrene and aflatoxin B1 in metabolic-competent HepaRP cells.

Human dietary exposure involves multiple contaminants, raising concerns about "cocktail" effects ignored by single-compound risk assessment. Benzo[a]pyrene (BaP) and aflatoxin B1 (AFB1) are co-occurring food contaminants requiring cytochrome P450-mediated bioactivation. They both form DNA-reactive metabolites and are classified as genotoxic carcinogens. Here, BaP and AFB1 genotoxicity was evaluated individually and in binary combination in metabolically competent HepaRP cells using γH2AX as DNA damage biomarker. Concentrations were selected to reflect physiologically relevant levels (BaP: 0.125-10 µM; AFB1: 0.01-2 µM). Benchmark concentration (BMC) analysis with the PROAST covariate approach allowed deriving potency estimates, predicting mixture effects under dose addition hypothesis. Cells co-exposed to BaP-AFB1 presented a synergistic increase in γH2AX foci number compared to additive model predictions. Mechanistic investigations identified aryl hydrocarbon receptor (AhR) signaling as a key driver of this interaction. First, AhR pharmacological activation with β-naphthoflavone (BNF) recapitulated the effects of BaP, enhancing the genotoxicity induced by AFB1 exposure. In contrast AhR inhibition with CH223191 reduced DNA damage in cells treated with BaP, BaP-AFB1 or BNF-AFB1. Consistent with the metabolic interaction hypothesis, BaP or BNF alone or in mixture with AFB1 increased the expression and activity of CYP1 family enzymes, while CH223191 prevented these responses. Finally, non-genotoxic polycyclic aromatic hydrocarbons (PAHs) as fluoranthene and phenanthrene also enhanced AFB1 genotoxicity. Altogether, these results underline the importance of AhR-dependent metabolic activation in synergistic interaction within mixtures, supporting the necessity of mechanistic knowledge to improve risk assessment, notably for co-exposure to food contaminants.