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Bisphenols (BPs) are environmental pollutants with relevant DNA damage in human population; however, they are generally inactive in standard mutagenicity assays, possibly due to insufficient metabolic activation. In this study, induction of micronuclei and double-strand DNA breaks by BPA, BPF, and BPS in Chinese hamster V79-derived cell lines expressing various human CYP enzymes and a human hepatoma (C3A) (metabolism-proficient) cell line were investigated. Molecular docking of BPs to human CYPs indicated some substrate–enzyme potentials, including CYP1A1 for each compound, which did not induce micronuclei in V79-derived cell lines expressing human CYP1A2, 2E1, or 3A4 but became positive in human CYP1A1-expressing (V79-hCYP1A1) cells. In V79-hCYP1A1 and C3A cells, all compounds induced double-strand DNA breaks and micronuclei formation, which were blocked/significantly attenuated by 1-aminobenzotriazole (CYP inhibitor) or 7-hydroxyflavone (selective CYP1A1 inhibitor). Coexposure of C3A cells to pentachlorophenol (sulfotransferase 1 inhibitor) or ketoconazole (UDP-glucuronosyltransferase 1A inhibitor) potentiated micronuclei induction by each compound, with thresholds lowered from 2.5–5.0 to 0.6–1.2 μM. Immunofluorescence staining of centromere protein B with micronuclei formed in C3A cells by each compound indicated pure clastogenic effects. In conclusion, BPs are potently clastogenic in mammalian cells, which require activation primarily by human CYP1A1 and are negatively modulated by phase II metabolism.

Benzene is a human carcinogen that requires metabolic activation. We previously observed that benzene and its hydroxylated metabolites induce micronuclei in mammalian cells expressing human CYP2E1. This study was initially aimed to study another endpoint, the induction of gene mutations by those compounds in the same cell models. A V79‐derived cell line expressing human CYP2E1 and sulfotransferase (SULT) 1A1 (V79‐hCYP2E1‐hSULT1A1) pretreated with ethanol (a CYP2E1 stabilizer) was used in the hprt gene mutagenicity assay. Phenol, hydroquinone, catechol, and 1,2,4‐trihydroxybenzene all induced gene mutations, while they were inactive, or only weakly positive (hydroquinone), in parental V79‐Mz cells. Unexpectedly, benzene was non‐mutagenic in both cell lines, but it became positive in V79‐hCYP2E1‐hSULT1A1 cells using regimes of short exposure/long recovery without ethanol pretreatment, for both gene mutations and micronuclei formation. In silico molecular simulation showed binding energies and positions favorable for each compound to be oxidized by human CYP2E1, benzene demonstrating the highest affinity. By tunnel analysis, ethanol binding did not limit benzene to pass tunnel S, which was specifically active for benzene. However, its end product, acetic acid, decreased the occurrence of tunnel S from 5.4 to 2.2% and extended the length of its bottleneck from 5.5 to 9.0 Å. With residual ethanol molecules still being present in CYP2E1 for a period of time after benzene exposure, the acetic acid formed could limit the entrance of benzene, thus inhibit its metabolic activation. In summary, ethanol may interfere with the activation of benzene to mutagenic metabolites, at least in cultured cells.

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