Cytotoxicity and mutagenicity of 5-methylchrysene and its 1,2-dihydrodiol in V79MZ cells modified to express human CYP1A1 or CYP1B1, in the presence or absence of human GSTP1 coexpression (original) (raw)
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Mutation Research-fundamental and Molecular Mechanisms of Mutagenesis, 2007
We have used V79MZ hamster lung fibroblasts stably transfected with human cytochrome P450-1A1 (hCYP1A1; cell line designated V79MZh1A1) or P450-1B1 (hCYP1B1; cell line designated V79MZh1B1) alone, or in combination with human glutathione-S-transferase (GST) alpha-1 (hGSTA1), in order to examine GST protection against cytotoxicity and mutagenicity of dibenzo[a,l]pyrene (DBP) and the intermediate dihydrodiol metabolite (±)-DBP-11,12-dihydrodiol (DBPD). At comparable expression levels of hCYP1A1 and hCYP1B1, both DBP and DBPD were more cytotoxic in V79MZ1A1 (IC50 = 2.7 and 0.7 nM, respectively) than in V79MZh1B1 (IC50 = 6.0 and 4.8 nM, respectively). In contrast, both DBP and DBPD were two- to four-fold more mutagenic in V79MZh1B1 than in V79MZ1A1. Co-expression of hGSTA1 with hCYP1A1 decreased DBP cytotoxicity two-fold compared to V79MZh1A1 with hCYP1A1 alone, and provided a small, yet still statistically significant, 1.3-fold protection against DBPD. Protection against mutagenicity of these compounds was comparable to that for cytotoxicity in cells expressing hCYP1A1. In V79MZh1B1 cells, co-expression of hGSTA1 conferred up to five-fold protection against DBP cytotoxicity, and up to nine-fold protection against the (±)-DBP-dihydrodiol cytotoxicity relative to the cells expressing hCYP1B1 alone. Co-expression of hGSTA1 also reduced mutagenicity of DBP or its dihydrodiol to a lesser extent (1.3–1.8-fold) than the protection against cytotoxicity in cells expressing hCYP1B1. These findings demonstrate that the protective efficacy of hGSTA1 against DBP and DBPD toxicity is variable, depending on the compound or metabolite present, the specific cytochrome P450 isozyme expressed, and the specific cellular damage endpoint examined.
Toxicological Sciences, 2007
Transgenic cell lines were constructed to study the dynamics of competition between activation versus detoxification of benzo[a]pyrene (B[a]P) or B[a]P-7,8-dihydrodiol metabolites. Stably transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone or in combination with human glutathione-Stransferase M1 (hGSTM1) were used to determine how effectively this GST isozyme protects against cytotoxic, genotoxic, and mutagenic effects of B[a]P or the enantiomeric dihydrodiol metabolites -7,8-diol). Expression of hGSTM1 in the presence of hCYP1A1 conferred significant 8.5fold protection against B[a]P-induced cytotoxicity, but protection against cytotoxicity of either B[a]P-7,8-diol enantiomer was not significant. Mutagenicity of B[a]P at the hprt locus was dose and time dependent in cells that expressed hCYP1A1. Mutagenicity of B[a]P was reduced by 21-32% and mutagenicity induced by the B[a]P-7,8-diols was reduced 20-58% in cells further modified to coexpress hGSTM1-1 compared to cells expressing hCYP1A1 alone. Expression of hGSTM1-1 reduced adducts in total cellular macromolecules by twofold, in good correlation with the reduction in B[a]P mutagenicity. These results indicate that while hGSTM1-1 effectively protects against hCYP1A1-mediated cytotoxicity of B[a]P, a significant fraction of the mutagenicity that results from activation of B[a]P and its 7,8-dihydrodiol metabolites by hCYP1A1 is derived from B[a]P metabolites that are not detoxified by hGSTM1.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2016
Benzo[a]pyrene (B[a]P) is a known genotoxicant and carcinogen, yet its genotoxic response at low level exposure has not been determined. This study was conducted to examine the interplay of dose and metabolic capacity on the genotoxicity of B[a]P. An improved understanding the biological significance of low level chemical exposures will help to improve human health risk assessments. The genotoxic and mutagenic effects of B[a]P were investigated using human cell lines AHH-1, MCL-5, TK6 and HepG2, with differential expression of the CYP450 enzymes (CYP1A1, 1B1 and1A2) involved in B[a]P metabolism. MCL-5 and HepG2 cells showed detectable basal expression and activity of CYP1A1, 1B1 and 1A2 as compared to AHH-1, which only show CYP1A1 basal expression and activity. TK6 cells showed negligible expression levels of all three CYP450 enzymes. In vitro micronucleus and the hypoxanthine phosphoribosyltransferase assays were conducted to determine the effect of B[a]P on chromosome damage and point mutation induction. After 24h exposure, concentration-dependent increases in micronucleus (MN) frequency were observed in all cell lines except TK6. After 4h exposure, only the metabolically competent cell lines MCL-5 and HepG2 showed MN induction (with a threshold concentration at 25.5µM from MCL-5 cells) indicating the importance of exposure time for genotoxicity. The HPRT assay also displayed concentration-dependent increases in mutant frequency in MCL-5 cells, after 4h and 24h treatments. Mutation spectra analysis of MCL-5 and AHH-1 HPRT mutants revealed frequent B[a]P induced G to T transversion mutations (72% and 44% of induced mutations in MCL-5 and AHH-1, respectively). This study therefore demonstrates a key link between metabolic capability, B[a]P exposure time and genotoxicity.
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2002
Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32 P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides Abbreviations: ara-C, cytosine arabinoside; B[a]P, benzo[a]pyrene; BNF, -naphthoflavone; C3H10T1/2, C3H10T1/2CL8; trans-B[a]P-4,5-diol, (±) trans-4,5-dihydro-4,5-dihydroxyB[a]P; BPDE, anti-trans-B[a]P-7,8-diol-9,10-epoxide; MMS, methyl methanesulfonate; PAH, polycyclic aromatic hydrocarbon; ROS, reactive oxygen species ଝ The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. (S. Nesnow). 1383-5718/02/$ -see front matter. Published by Elsevier Science B.V. PII: S 1 3 8 3 -5 7 1 8 ( 0 2 ) 0 0 2 1 8 -8
European Journal of Pharmacology: Environmental Toxicology and Pharmacology, 1993
A V79 Chinese hamster cell line stably expressing human cytochrome P450 1A1 (CYP1A1) was obtained by chromosomal integration of the human CYP1A1 cDNA under the control of the SV40 early promoter. Chromosomal integration was verified by Southern analysis, and effective transcription of the human CYP1A1 cDNA was demonstrated by Northern analysis. The CYP1A1 cDNA-encoded protein was characterized by Western analysis using anti-rat CYP1A1. Intracellular association of CYP1A1 with the endoplasmic reticulum could be visualized by in situ immunofluorescence. Crude cell lysates of the V79 derived cell line was able to catalyze 7-ethoxyresorufin-O-deethylation (EROD) with an activity of about 50 pmol min-1 mg-1 total protein, and an aryl hydrocarbon hydroxylase activity (AHH) of 25 pmol min-1 mg-1. CYP1A1 dependent cytotoxicity, measured by neutral red uptake, and genotoxicity, determined by the frequency of micronucleus formation, of benzo[a]pyrene (B[a]P) and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) could be demonstrated at substrate concentrations as low as 10 nM. Thus, this cell line presents a sensitive tool for studying CYPIA1 mediated metabolism of polycyclic aromatic hydrocarbons (PAl-I). B[a]P and the purified (+)-and (-)-enantiomers of B[a]P-7,8-diol were compared for their mutagenicity. The (-)-enantiomer was found to be 3-5-fold more mutagenic than the (+)-enantiomer. Cytochrome P450 1A1 (human); Cytochrome P450 1A1 recombinant expression vector; V79 Chinese hamster cells; Polycyclic aromatic hydrocarbons; Benzo[a]pyrene
Cancer research, 1996
The metabolism of environmentally occurring methylated polynuclear aromatic hydrocarbons by human cytochrome P450 (P450) enzymes has not been examined previously. We compared the metabolism of the tobacco smoke constituents 5-methylchrysene (5-MeC), a strong carcinogen, and 6-MeC, a weak carcinogen, in 18 hepatic and 11 pulmonary human microsomes. Major metabolites of 5-MeC were its proximate carcinogen trans-1,2-dihydroxy-1,2-dihydro-5-methylchrysene (5-MeC-1,2-diol), as well as 5-MeC-7,8-diol, bay region dihydrodiols, and phenols. 5-MeC-1,2-diol and 5-MeC-7,8-diol were formed stereoselectively, with the R,R enantiomers predominating. Major metabolites of 6-MeC were 6-MeC-1,2-diol, bay region dihydrodiols, phenols, and 6-(hydroxymethyl)chrysene. 6-MeC-1,2-diol was also formed stereoselectively in the 1R,2R configuration. All human liver samples formed the proximate carcinogenic 1,2-diols (0.2-2.3 pmol/mg protein/min for 5-MeC and 0.3-3.1 pmol/mg protein/min for 6-MeC). Comparable r...
Chemico-Biological Interactions, 2007
1,2:3,4-Diepoxybutane is hypothesized to be the main intermediate involved in mutagenicity following exposure to low levels of 1,3-butadiene (BD) in mice, while metabolites of 3-butene-1,2-diol (BD-diol) are thought to become involved in both rats and mice at higher exposures. BD-diol is biotransformed to hydroxymethylvinyl ketone (HMVK), a potentially mutagenic metabolite, and 3,4-epoxy-1,2-butanediol (EB-diol), a known mutagen. To determine the relative importance of HMVK and EB-diol in BD-diol associated mutagenesis, we have examined the dosimetry of a HMVK derived DNA adduct, as well as EB-diol derived DNA and hemoglobin adducts, in rodents exposed to BD-diol. We previously demonstrated similarities in the shapes of the dose-response curves for EB-diol derived DNA adducts, hemoglobin adducts, and Hprt mutant frequencies in BD-diol exposed rodents, indicating that EB-diol was involved in the mutagenic response associated with BD-diol exposure. To examine the role of HMVK in BD-diol mutagenicity, a method to quantify the ␣-regioisomer of HMVK derived 1,N 2-propanodeoxyguanosine (␣-HMVK-dGuo) was developed. The method involved enzymatic hydrolysis of DNA, HPLC purification, and adduct measurement by liquid chromatography-tandem mass spectrometry. Intra-and inter-experimental variabilities were determined to be 2.3-18.2 and 4.1%, respectively. The limit of detection was ∼5 fmol of analyte standard injected onto the column or 5 fmol/200 g DNA. The method was used to analyze liver DNA from control female F344 rats and female F344 rats exposed to 36 ppm BD-diol. In addition, liver samples from female Sprague-Dawley rats exposed to 1000 ppm BD were analyzed. ␣-HMVK-dGuo was not detected in any of the samples analyzed. Several possible explanations exist for the negative results including the possibility that ␣-HMVK-dGuo may be a minor adduct or may be efficiently repaired. Alternatively, HMVK itself may be readily detoxified by glutathione (GSH) conjugation. While experiments must be conducted to understand the exact mechanism(s), these results, in addition to published EB-diol derived adduct dosimetry and existing HMVK derived mercapturic acid data, suggest that EB-diol is primarily responsible for BD-diol induced mutagenicity in rodents.
Carcinogenesis, 1998
Previous studies have identified allelic variants of the human glutathione transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (k cat /K m ) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti-and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest k cat /K m value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of k cat /K m . With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased k cat /K m values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionyl-*Abbreviations: PAH, polycyclic aromatic hydrocarbons; CDNB, 1-chloro 2,4-dinitrobenzene; syn-and anti- CDE,4epoxide;h]anthracene-1,2-diol 3,4-epoxide.