Disadvantaged induction of gene mutations by lower chlorinated biphenyls in V79-derived cells co-expressing mouse relative to human CYP2E1, molecular docking/dynamics studies suggesting unfavored metabolic activation by the mouse enzyme.

Polychlorinated biphenyls (PCBs) have been classified as human carcinogens. Mutagenicity of lower chlorinated biphenyls as well as activation of transcription factors by some other congeners may contribute to the carcinogenicity of PCBs. Recently, we reported that human CYP2E1 activates mono- and dichlorobiphenyls to mutagens. However, mutagenicity of other PCBs and the involvement of other CYPs remained unknown. In this study, Chinese hamster V79-derived cell lines genetically engineered for expression of individual human CYP enzymes and a human hepatocyte (L-02) line endogenously expressing various CYPs were used to determine the activities of several tri- and tetrachlorobiphenyls to induce micronuclei and gene mutations. 2,3,4'-Trichlorobiphenyl, 2,3,3'-trichlorobiphenyl, 2,4,4',5-tetrachlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl efficiently induced micronuclei and/or gene mutations in V79-derived cells at low micromolar concentrations, depending on human CYP2E1, while they were inactive in parental V79-Mz cells and weakly positive or inactive in V79-derived cells expressing human CYP1A1, 1A2, 1B1 or 3A4. The induction of gene mutations in human CYP2E1-expressing V79 cells by 2,3,4'-trichlorobiphenyl and 2,4,4',5-tetrachlorobiphenyl was more potent than that of N-nitrosodimethylamine, a strong carcinogen activated by CYP2E1. As representative PCB compounds, 2,3,3'-trichlorobiphenyl and 2,3,4'-trichlorobiphenyl induced micronuclei in L-02 cells, and this effect was blocked by specific CYP2E1 inhibition, wherein the effects of benzo[a]pyrene and aflatoxin B1 (activated by some CYPs other than CYP2E1) were unaffected. This study demonstrates that some non-planar tri- and tetrachlorobiphenyls are potent mutagens in mammalian cells-more potent than previously tested mono- and dichlorobiphenyls-and that among several human CYP enzymes, CYP2E1 is most efficient in activating these environmental contaminants.

Human CYP2E1-activated mutagenicity of dioxin-like PCBs 105 and 118—Experimental data consistent with molecular docking results

Featured structure-activity relationships for some tri- and tetrachlorobiphenyls in human CYP2E1-activated mutagenicity — Impact of the extent of ortho-chlorination

The cDNA encoding the mouse Cyp2e1 protein has been isolated and sequenced, and shown to share 92%, 79%, 80% and 79% sequence similarity over the coding region with rat, human, rabbit 1 and rabbit 2 CYP2E1 cDNA sequences respectively. The predicted Cyp2e1 protein contains 493 amino acids, with a molecular mass of 56781 Da. The protein contains many features common to other cytochrome P450s, including a potentially phosphorylatable serine residue at position 129 within a canonical cyclic AMP-dependent protein kinase site. Southern blot analysis of genomic DNA prepared from C57BL/6 and DBA/2N mice suggests the presence of only a single Cyp2e1 gene. The Cyp2e1 gene was isolated and its organization was established by PCR using oligonucleotides to its predicted intron/exon boundaries. These results showed that the mouse Cyp2e1 gene is approx. 11,000 bp in length and has a similar structure to the human and rat CYP2E1 genes. Cyp2e1 protein expression was studied in a variety of tissues and a sexual dimorphism in its levels in some tissues was noted. Acetone treatment induced the Cyp2e1 protein in all of the tissues studied in both sexes, but this Cyp2e1 protein induction was not accompanied by an increase in Cyp2e1 mRNA levels. Indeed, mRNA levels were seen to be decreased on treatment, suggesting that acetone administration affects either mRNA translation efficiency or protein stability. Of a wide range of drugs known to modify other cytochrome P450 levels only diethylnitrosamine had a significant effect on Cyp2e1, causing a decrease in protein levels.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with continued public health concerns. The lower chlorinated biphenyls are supposed to be mutagenic following metabolic activation. However, in a preliminary study, we recently observed induction of micronuclei by several PCBs in a subclone of Chinese hamster V79 cell line, V79-Mz, which is deficient in xenobiotic-metabolizing enzyme activities. In this study, metabolism-free genotoxicity of PCBs was investigated, using 10 tri- and tetrachlorobiphenyls, in V79, V79-Mz, and human hepatoma (HepG2) cell lines. Among the four tetrachlorobiphenyls, 2,4,4',5- and 2,3'4,4'-tetrachlorobiphenyl-both having a noncoplanar configuration-induced micronuclei in V79-Mz cells, while their coplanar analogs 3,4,4',5- and 3,3',4,4'-tetrachlorobiphenyl were inactive. Furthermore, 2,3,3'- (PCB 20) and 2,3,4'-trichlorobiphenyl (PCB 22) started to induce micronuclei in V79-Mz cells at 10 μM and higher concentrations, demonstrating more potent effects than observed with 2,2',3-, 2,2',4-, 2,2',5, and 2,4,4'-trichlorobiphenyl. As representative compounds, PCB 20 and 22 induced micronuclei in relatively high concentrations in HepG2 cells (p53-proficient), though they did not induce Hprt gene mutations in V79-Mz cells. PCB 20 and 22 increased mitotic index and induced cell cycle arrest at the G2/M phase, with effects more potent in V79-Mz than in V79 cells. This study suggests that 2,3,4'- and 2,3,3'-substituted PCBs are micronuclei inducers and G2/M arresters among a number of trichlorobiphenyls in mammalian cell lines, though with potency lower than that observed recently in V79-derived cells expressing human CYP2E1. Similarly, some noncoplanar tetrachlorobiphenyls possess metabolism-independent chromosome-damaging potentials. Environ. Mol. Mutagen. 58:199-208, 2017. © 2017 Wiley Periodicals, Inc.

Since 1980, 2145 samples of raw milk from producers in the district of Freiburg have been analysed for polychlorinated biphenyls (PCB) during a monitoring programme. In 1983, we started to search for the source of PCB, whenever milk was contaminated above the average value (above 0.2 mg/kg fat, calculated as Clophen A60). In 7 cases, previous coats of paint in the silos proved to be the cause of the PCB contamination. Five samples of milk and 4 corresponding silages as well as 1 sample of wood (from a cowshed) were investigated for all of the individual PCB components included in the technical mixtures of Clophen A30 and A60. Two samples of silages contained both low and high chlorinated biphenyls, whereas low chlorinated biphenyls (di-, tri-, and tetrachlorinated biphenyls) were not detected in the corresponding milk samples. Pentachlorinated biphenyls were only detected in the range of 1%-2%. In the samples of wood and corresponding milk, a different PCB pattern appeared. There were remarkably high percentages of hepta- and octachlorinated biphenyls as well as a lower percentage of hexachlorinated biphenyls. The exact PCB content of the milk, determined by the addition of all single components, proved to be approximately half the value obtained by the usual calculation based on the evaluation of the three main peaks of the technical PCB mixture (Clophen A60). During 1984-1987, 607 samples of raw milk were analysed for six single PCB components, for which legal tolerance levels became valid in 1988.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Human CYP2E1-dependent mutagenicity of mono- and dichlorobiphenyls in Chinese hamster (V79)-derived cells.

Mutagenicity of N-nitrosodiethanolamine in a V79-derived cell line expressing two human biotransformation enzymes

Biotransformation enzyme-dependent formation of micronucleus and multinuclei in cell line V79-hCYP2E1-hSULT1A1 by 2-nitropropane and N-nitrosodimethylamine

Human CYP2E1-dependent and human sulfotransferase 1A1-modulated induction of micronuclei by benzene and its hydroxylated metabolites in Chinese hamster V79-derived cells

Polychlorinated biphenyls (PCBs) were monitored in surface water collected in the Selangor River basin, Malaysia, to identify the occurrence, distribution, and dechlorination process as well as to assess the potential adverse effects to the Malaysian population. Ten PCB homologs (i.e., mono-CBs to deca-CBs) were quantitated by using gas chromatography-mass spectrometry (GC/MS). The total concentration of PCBs in the 10 sampling sites ranged from limit of detection to 7.67ngL-1. The higher chlorinated biphenyls (tetra-CBs to deca-CBs) were almost not detected in most of the sampling sites, whereas lower chlorinated biphenyls (mono-CBs, di-CBs, and tri-CBs) dominated more than 90% of the 10 homologs in all the sampling sites. Therefore, the PCB load was estimated to be negligible during the sampling period because PCBs have an extremely long half-life. The PCBs, particularly higher chlorinated biphenyls, could be thoroughly dechlorinated to mono-CBs to tri-CBs by microbial decomposition in sediment or could still be accumulated in the sediment. The lower chlorinated biphenyls, however, could be resuspended or desorbed from the sediment because they have faster desorption rates and higher solubility, compared to the higher chlorinated biphenyls. The health risk for the Malaysia population by PCB intake that was estimated from the local fish consumption (7.2ngkg-1 bwday-1) and tap water consumption (1.5×10-3-3.1×10-3ngkg-1 bwday-1) based on the detected PCB levels in the surface water was considered to be minimal. The hazard quotient based on the tolerable daily intake (20ngkg-1 bwday-1) was estimated at 0.36.

1-Methylpyrene (1-MP) is a widespread pollutant that is carcinogenic in animals following metabolic activation. Previous studies have shown that benzylic hydroxylation of 1-MP, catalyzed by multiple CYP isoforms, gives rise to 1-hydroxymethylpyrene (1-HMP), which becomes bioreactive following further metabolism by various sulfotransferase (SULT) isoforms. However, the mutagenic and chromosome damaging effects of 1-MP and 1-HMP in mammalian cells have not been investigated. In this study a Chinese hamster V79-derived cell line expressing both human CYP2E1 and human SULT1A1 was used to investigate the ability of 1-MP and 1-HMP to induce cytotoxicity (using the CCK-8 assay), micronuclei and Hprt gene mutations. The role of each enzyme was investigated through co-exposure in the presence of an enzyme inhibitor. We found that at concentrations of 0.5-4 μM and 5-20 μM, under conditions where no reduction in cell viability/growth occurred, 1-HMP and 1-MP induced micronuclei in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner; however, both compounds were inactive in V79 cells. Similarly, they both caused an increase in Hprt mutant frequency in V79-hCYP2E1-hSULT1A1 cells in these concentration ranges, with 1-MP impairing cell viability/growth at 10 μM and above in the mutagenicity assay. The compounds were again both inactive in V79 cells. The effects of 1-HMP in V79-hCYP2E1-hSULT1A1 cells were blocked or reduced by addition of pentachlorophenol (PCP), a SULT1 inhibitor; the genotoxicity of 1-MP was significantly reduced by either 1-aminobenotrazole, a CYP2E1 inhibitor, or PCP. The results suggest that human CYP2E1 and SULT1A1 cooperate to activate 1-MP and cause genotoxicity in mammalian cells.

Human CYP2E1 metabolizes many xenobiotics of low-molecular weight, thereby activating various promutagens/procarcinogens. In toxicological studies in vitro, dimethylsulfoxide (DMSO) is a common vehicle for organic compounds. However, it was observed to potently inhibit CYP2E1 activity. We were interested in whether it affects CYP2E1-dependent mutagenic responses. In this study, N-nitrosodiethylamine (NDEA), which is soluble in both water and DMSO, was used as a model promutagen. It induced Hprt gene mutations and micronuclei in a Chinese hamster V79-derived cell line expressing both human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) even at low-micromolar concentrations, but was inactive in parental V79 cells. Mutagenicity of NDEA was also observed in a recombinant V79-hCYP2E1 cell line that expresses human CYP2E1 at a lower level. NDEA induced micronuclei in human L-02 hepatocytes which expressed CYP2E1 even more weakly. DMSO did not modify NDEA-induced gene mutations or micronuclei, up to 0.2% (v:v, the highest noncytotoxic concentration) in V79-hCYP2E1-hSULT1A1 cells. In parental V79-Mz cells, NDEA induced micronuclei with Aroclor 1254-induced rat liver S9 mix, and this effect was unaffected by DMSO up to 0.2%. However, it inhibited the effect of NDEA in L-02 (by 44%) and V79-hCYP2E1 cells (by 70%) at 0.2%, with the effects of NDEA remaining statistically significant. No effect of DMSO was observed on CYP2E1 protein expression in V79-hCYP2E1-hSULT1A1 or its mRNA transcripts in each cell line. We conclude that DMSO may not significantly affect CYP2E1-dependent mutagenic effects, at concentrations up to 0.2% in cells with relatively high CYP2E1 expression. Environ. Mol. Mutagen. 60:214-226, 2019. © 2018 Wiley Periodicals, Inc.

Persistent organic pollutants : aberrant DNA methylation underlying potential health effects