Human CYP2E1‐dependent mutagenicity of benzene and its hydroxylated metabolites in V79‐derived cells: Suppression and enhancement by ethanol pretreatment

Abstract

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.

Potent Clastogenicity of Bisphenol Compounds in Mammalian Cells—Human CYP1A1 Being a Major Activating Enzyme

“…Understandably, although CYPs are a superfamily common for metabolic activation of environmental compounds, the formed metabolites are not necessarily conferred a toxic effect, rather they depend on several factors, including the toxicity and concentration of each metabolite. Probably both relevance and uncertainty are true in guiding the choice of candidate enzymes/chemicals in relevant metabolism-based genotoxicity assays. , Nevertheless, considering the tremendously reduced time and other costs consumed in molecular docking as compared to conventional genotoxicity assays, substrate prediction by in silico simulation may be still valuable in sharpening the size of work paid in the subsequent genotoxicity assays.…”

“…Probably both relevance and uncertainty are true in guiding the choice of candidate enzymes/chemicals in relevant metabolism-based genotoxicity assays. 35,52 Nevertheless, considering the tremendously reduced time and other costs consumed in molecular docking as compared to conventional genotoxicity assays, substrate prediction by in silico simulation may be still valuable in sharpening the size of work paid in the subsequent genotoxicity assays.…”

Influence of Bisphenol Compounds at Nanomolar Concentrations on Chromosome Damage Induced by Metabolically Activated Carcinogens in HepG2 Cells

“…In brief, HepG2 cells were seeded onto 96-well plates at a density of 2 × 10 3 cells per well, each containing 100 μL of the medium. At 24 h, cells were pretreated with each BP compound at varying concentrations for 48 h. Then, the medium was changed, and after 2 h, each carcinogen (other than benzene) was added for another 48 h. Alternatively, a regime of 6 h exposure/42 h recovery was applied to the treatment with benzene, as persistent exposure to benzene may lead to false-negative results . In some experiments, known inducers of NRs, that is, PCB 126 (40 nM) for AhR, RIF (10 μM) for PXR, and CITCO (1 μM) for CAR, were used for pretreating the cells; while in some other experiments, known inhibitors of each NR, that is, BAY-218 (700 nM) for AhR, KET (10 μM) for PXR and CYP3A enzymes, , and CINPA1 (5 μM) for CAR, were present during both the pretreatment and treatment (98 h in total); alternatively, the CYP inhibitor ABT (60 μM) was present in some cultures immediately after the removal of BPs until the end of treatment (50 h in total).…”

Phenylalanine Residues in the Active Site of CYP2E1 Participate in Determining the Binding Orientation and Metabolism-Dependent Genotoxicity of Aromatic Compounds

“…Protein and ligands were described by Charmm36 force field and CgenFF (the official CHARMM general force field server), respectively [ 25 , 32 ]. The canonical molecular dynamics with periodic boundary condition was performed by using GROMACS (Version 2018.4), with parameters being set according to previous descriptions [ 19 ]. Briefly, short-range nonbonded interactions were cut off at 1.2 nm, with long-range electrostatics calculated using the particle mesh Ewald (PME) algorithm.…”

“…Benzene and its hydroxylated metabolites (phenol and hydroquinone) are simplest aromatic substrates for human CYP2E1, thereby they are activated for mutagenic effects [ 8 ]. However, the PHEs in the active site appear to have little effect on these small ligands during the metabolism process [ 19 ]. It is likely that the PHEs in the active site are primarily important for directing the binding of larger (such as multiple phenyl groups-containing) ligands.…”