ToxSci Advance Access originally published online on May 24, 2007
Toxicological Sciences 2007 99(1):51-57; doi:10.1093/toxsci/kfm133
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Protective Efficacy of hGSTM1-1 against B[a]P and (+)- or (–)-B[a]P-7,8-Dihydrodiol Cytotoxicity, Mutagenicity, and Macromolecular Adducts in V79 Cells Coexpressing hCYP1A1
,1
* Department of Cancer Biology
Department of Biochemistry and Comprehensive Cancer Center, Wake Forest University, Winston-Salem, North Carolina 27157
Gen Pharm Tox, Munich, Germany
1 To whom correspondence should be addressed at Biochemistry Department, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Fax: (336)-716-7671. E-mail: atown{at}wfubmc.edu.
Received April 9, 2007; accepted May 18, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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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-S-transferase 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 (+)-benzo[a]pyrene-7,8-dihydrodiol ((+)-B[a]P-7,8-diol) and (–)-benzo[a]pyrene-7,8-dihydrodiol ((–)-B[a]P-7,8-diol). Expression of hGSTM1 in the presence of hCYP1A1 conferred significant 8.5-fold 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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The glutathione-S-transferases (GSTs) are a superfamily of detoxification enzymes that catalyze the conjugation of reduced glutathione (GSH) to a variety of electrophiles of both endogenous and exogenous origin (Coles and Ketterer, 1990
; Hayes and Pulford, 1995; Mannervik and Danielson, 1988). On the basis of primary structure, the mammalian soluble GST superfamily is currently subdivided into seven classes, designated alpha, mu, pi, kappa, omega, sigma, and theta. Among these, the alpha, mu, and pi classes are the most abundantly expressed in mammalian tissues. A putative role for GSTs in chemoprevention of cancer has been implicated based on the ability of certain GST isozymes to conjugate and thus inactivate electrophilic carcinogens and reduce the formation of mutagenic DNA adducts (Coles and Ketterer, 1990). These include the reactive diol-epoxide (DE), formed via metabolic activation by cytochrome P450 (CYP) isozymes of environmental polycyclic aromatic hydrocarbons (PAHs) that are present in smoke from cigarettes and other combustion products (Hecht, 1999). The competing dynamics of activation of PAHs by CYP versus the opposing detoxification of the reactive PAH-DE by GSTs is an issue that is presently not well understood on a quantitative basis. The studies described herein were designed to model this competition at a functional level via expression of hCYP1A1 alone or together with hGSTM1.
The human GSTP1 and GSTM1 isozymes exhibit the highest reported catalytic activities for the conjugation of the ultimate DE metabolites of the ubiquitous environmental PAH benzo[a]pyrene (B[a]P) (Robertson et al., 1986; Sundberg et al., 1997). However, while kinetic data obtained with purified enzymes are useful for comparisons among different GST isozymes, it does not necessarily predict whether expression of a specific GST will actually provide protection against specific chemical toxins in the context of viable cells or tissues. There are many potential factors that may govern GST protective efficacy, including intracellular compartmentation of the GST relative to the activation pathway, availability of a conjugate efflux pathway, and/or accessibility of the relevant target(s) associated with specific endpoints such as cytotoxicity or mutagenicity.
The role of GST in vivo is further complicated by genetic polymorphisms or deletions in some human GST genes. Many attempts have been made to address this issue by population-based molecular epidemiology studies to define the association between disease states and the presence or absence of particular GST alleles. In particular, epidemiological studies have suggested an association between the hGSTM1 null phenotype (GSTM1*0) and susceptibility to various types of cancer. Much attention has focused upon lung cancer risk in smokers harboring the GSTM1*0 genotype, as the GSTM1-1 isozyme exhibits activity for conjugation of the epoxide metabolites of the PAH present in cigarette smoke. However, although early results with limited sample sizes suggested a moderately increased risk of lung cancer in smokers with the GSTM1*0 genotype (Nazar et al., 1993; Seidegård et al., 1990), meta-analysis of the data compiled from a number of studies indicated little if any change in the overall risk of lung or breast cancer in smokers with the GSTM1*0 genotype (Houlston, 1999; Vogl et al., 2004).
In view of the difficulty of in vivo assessment of the function of detoxification enzymes in humans, we have constructed transgenic cell lines for use in examination of the role of specific CYP and GST enzymes in metabolism of PAHs and their role in determining cellular response to PAH exposure as reflected in various toxic endpoints (Townsend et al., 1998a,b). The present study is focused on the opposing roles of activation by the hCYP1A1 pathway versus detoxification by hGSTM1-mediated conjugation. Activation by hCYP1A1 was examined first in this model system due to the known inducibility of this isozyme in lung, and the correlation between the inducibility and B[a]P carcinogenesis (Shimizu et al., 2000). The V79 Chinese hamster lung fibroblast cell line is well-characterized for use in the hprt mutagenicity assay, and has no endogenous CYP expression that would otherwise complicate interpretation of results (Glatt et al., 1987).
The V79 cell line was modified to express hCYP1A1, either alone (Schmalix et al., 1993) or in combination with hGSTM1 (Townsend et al., 1998b), by stable transfection. A low level of hamster pi class GST is expressed in V79 cells, but this GST has been shown to be inactive as a catalyst for BPDE conjugation in intact cells (Sundberg et al., 2000), and hence does not contribute to the background activity for conjugation of this substrate. These cell lines represent a reconstruction of activation and detoxification pathways relevant to PAH cytotoxicity and mutagenicity, in order to examine competition between activation by CYP and protection by GST within a controlled in vitro system. The carcinogen chosen for initial study was B[a]P, a major component of the PAH present in smoke, since it is a well-known mutagen and carcinogen with a well-characterized pathway of activation via hCYP1A1. The various cell lines were exposed to B[a]P or the intermediate metabolites (+)- or (–)-B[a]P-7,8-diol ((+)- or (–)-benzo[a]pyrene-7,8-dihydrodiol) to examine protection afforded by hGSTM1 against cytotoxicity, mutagenicity, and whole-cell macromolecular alkylation. The results of these studies have important implications for the roles of CYP1A1 activation versus GSTM1 detoxification in the induction and control of PAH-mediated toxicity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Materials and chemicals.
Caution: The PAH and PAH metabolites described herein are potential chemical carcinogens and must be handled with care as outlined in the National Cancer Institute Guidelines.
DMEM (Dulbecco's Modified Eagle's Medium) and fetal bovine serum (FBS) were purchased from Gibco/BRL (Grand Island, NY). B[a]P, and the pure enantiomers (+) and (–)-B[a]P-trans-7,8-diol were purchased from Midwest Research Institute (Lexena, KS). 3H-B[a]P was purchased from American Radiolabeled Chemicals (St Louis, MO). Puregene Cell Lysis Solution and Puregene Protein Precipitation Solution were purchased from Gentra Systems (Minneapolis, MN). All other reagents were analytical grade and purchased from Fisher Scientific (Raleigh, NC) or Sigma (St Louis, MO).
Cell culture and cell lines.
The parental V79MZ (University of Mainz strain) Chinese Hamster lung fibroblast cell line and the stable transfection of the V79MZ cell line to express human CYP1A1 have been described (Glatt et al., 1987; Schmalix et al., 1993). Generation of the V79MZ cell lines expressing the human CYP1A1 and stably transfected hGSTM1 by the method of calcium phosphate-mediated transfection has also been described (Townsend et al., 1998b). All cell lines were grown and maintained in DMEM medium supplemented with 5% FBS at 37°C in 5% CO2 atmosphere and maintained under selection with 0.250 mg/ml G418. Cells were subcultured at 1:20 dilution every 2–3 days.
GST assay.
The assay used is a modification of the method described by Habig and Jakoby (1981). Cells were grown on 100-mm culture dishes, harvested by scraping into cold PBS + 5mM ethylenediaminetetraacetic acid (EDTA), pelleted at 500 x g, sonicated in 50mM Tris, pH 7.4 + 5mM EDTA, and centrifuged at 14,000 x g to remove particulates. Cytosolic supernatant (10–20 µg total protein) was assayed at room temperature in a solution of 0.1M K2PO4, pH 6.5, and 1mM GSH. The reaction was initiated by addition of 1mM (final concentration) 1-chloro-2,4-dinitrobenzene (CDNB). Change in absorbance was monitored at 340 nm for 90 s. Activity was calculated using the 
A/min and extinction coefficient, and was reported as nmol/min/mg protein. Protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) using bovine serum albumin to generate a standard curve.
CYP assay.
hCYP1A1 activity was measured as ethoxyresorufin-O-deethylase (EROD) by the method of Burke et al. (1994). Cell postmitochondrial supernatants of 150–200 µl (obtained as previously described for the GST assay) were assayed in a volume of 1 ml containing 50mM Tris/HCl (pH 7.4), 25nM MgCl2, 5mM glucose-6-phosphate, 50 U/ml glucose-6-phosphate dehydrogenase, 10µM dicoumerol, and 100µM ß-nicotinamide adenine dinucleotide phosphate, reduced, and were initiated by the addition of 5µM ethoxyresorufin. Samples were incubated at 37°C for 1 h with gentle shaking, and the reaction was stopped by the addition of 1 ml of MeOH. Fluorescence was measured on a Perkin–Elmer LS-3B spectrofluorimeter (Perkin–Elmer, Shelton, CT) with excitation at 522 nm and emission at 586nM, and compared to a standard curve constructed with known concentrations of resorufin.
Cytotoxicity assay.
Cells were plated at a density of 250 cells per well in 96-well plates, and allowed to attach and grow without G418 for 16–24 h. Cells were exposed to B[a]P or B[a]P-7,8-diol enantiomers continuously for 72 h and fixed in 5% tricarboxylic acid when control wells reached approximately 90% confluence. Plates were stained with sulforhodamine B (0.4% in 1% acetic acid), rinsed, and dried, and the dye solubilized by addition of 100 µl of 10mM Tris base (no pH adjustment), and then absorbance at 560 nm read with a microplate reader (Molecular Devices, Sunnyvale, CA) as an indirect measure of cellular density and protein. The IC50 is the dose level that reduced the cell density by 50% as compared to the untreated control.
Mutagenicity assay.
Cells were plated at a density of 5 x 105 cells per 100-mm plate in the absence of G418 selection for 48-h dose–response studies, or at 2.5 x 105 cells per 100-mm plate for time course studies. After allowing cells to adhere to the dishes overnight, cells were treated with 100, 300, or 600nM B[a]P for the 48-h dose–response study, or with 300nM B[a]P for the 72-h time course study. In the 48-h dose–response studies for mutagenicity of the dihydrodiol enantiomers, cells were exposed to 3, 10, 30, or 100nM (+)- or (–)-B[a]P-7,8-diols. The B[a]P or B[a]P-7,8-diols (or vehicle only) were added to the cell culture medium in dimethyl sulfoxide (0.1% final concentration). At the end of the exposure period, medium containing PAH was removed, and 10 ml of fresh medium was added and left on the cultures overnight. Cells were subcultured the following day for phenotypic development at their original densities, and allowed to grow for 6–7 days with one interim subculture. After this period, cells were subcultured at a density of 5 x 105 cells per 100-mm plate for incubation with 6-thioguanine (10 µg 6-TG/ml media) in order to select for hprt mutants. Selection with 6-TG was carried out for 10 days, and mutant colonies were stained with methylene blue (0.16% in methanol), rinsed in tap water, dried, and counted manually. Mutants are expressed as colonies per million cells.
Total cellular macromolecular adducts.
Cells were plated and exposed to 100nM 3H-B[a]P for the indicated times and processed as previously described for analysis of total cellular adducts (Kushman et al., 2007). Cell pellets were sonicated in 200 µl of ddH2O, and 20 µl of taken for protein analysis. To the remaining volume, 500 µl of cold methanol + 1% perchloric acid was added. The suspension was incubated on ice for 10 min, then 700 µl of hexane was added, and the hexane/MeOH suspension mixed by inversion for 15 min. Cells were pelleted at 14,000 x g and the upper hexane and lower 70% MeOH layers aspirated. Pellets were resuspended in 600 µl 70% MeOH/1% perchloric acid, followed by a 20-s sonication. Hexane (600 µl) was then added and the suspension once again mixed by inversion for 15 min. The suspension was again pelleted at 14,000 x g, the hexane and 70% MeOH layers aspirated, and the pellet was resuspended in 500 µl of 70% MeOH by sonication for 20 s. Suspensions were analyzed by scintillation counting and final DPM normalized to mg protein as determined by BCA assay. Control experiments indicated that the first extraction removes greater than 97% of the unbound 3H-B[a]P, and the second extraction removes > 90% of the remaining 3H-B[a]P. The residual 3H-B[a]P concentration in the second MeOH layer indicated a negligible contribution of trapped solvent to the nonextractable label bound to the pellet (< 100 CPM, assuming 
10 µl residual solvent).
Statistical analysis.
All data were statistically analyzed by InStat 3.0, using Student's t-test for significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Transgenic Cell Lines
The average GST specific activity in V79MZh1A1 cells expressing transfected hGSTM1-1 (clone 22, designated V79MZh1A1-Mu22) was 1430 ± 195 nmol/min/mg (Table 1). Background GST activity was 386 + 112 nmol/min/mg in V79MZ and 359 + 133 nmol/min/mg in V79MZh1A1 cells, due to expression of a hamster GSTP1 that shows negligible activity for conjugation of BPDE in V79MZ cells (Sundberg et al., 2000
). There is no endogenous mu-class GST expression in either V79MZ or V79MZh1A1 cells (not shown). The hCYP1A1 activities measured in the EROD assay were comparable for both the V79MZh1A1 (15.8 + 1.9 pmol/min/mg) and GST-transfected V79MZh1A1-Mu22 cells (14.4 + 3.3 pmol/min/mg). Parental V79MZ cells had no detectable CYP1A1 activity, and CYP1A1 is not inducible in this cell line (Swedmark et al., 1992).
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Cytotoxicity of B[a]P and Pure Enantiomeric B[a]P-7,8-diols
Cells expressing hCYP1A1 effectively metabolized B[a]P to cytotoxic species, as indicated by the 27-fold greater toxicity than with control V79MZ cells (IC50 = 0.27 vs. 7.5µM, respectively) (Table 2). Cells expressing hGSTM1-1 in addition to hCYP1A1 were 8.5-fold less sensitive to B[a]P cytotoxicity than cells expressing only hCYP1A1 (IC50 = 2.3 vs. 0.27µM, respectively; p < 0.05). Thus, the expression of hGSTM1 reversed a significant fraction of the toxicity of the reactive B[a]P metabolites conferred by hCYP1A1 expression; the V79MZh1A1-Mu22 cells were only threefold, instead of 27-fold more sensitive to B[a]P than the V79MZh1A1 cells. Cells were also exposed to either pure (+)- or (–)-B[a]P-7,8-diol enantiomers. Expression of hCYP1A1 conferred more than 100-fold enhancement of cytotoxicity relative to the V79MZ control cells (Table 3). In V79MZh1A1-Mu22 cells, protection by hGSTM1-1 against either of the B[a]P-7,8-diol enantiomers was only 1.3-fold or less (not significant, p > 0.3).
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Mutagenicity of B[a]P and B[a]P-7,8-diol Enantiomers
Mutagenicity was measured by frequency of mutation at the hprt locus following exposure of the cells to B[a]P. Exposure to 100, 300, or 600nM B[a]P for 48 h indicated that mutagenicity was dose dependent in the V79MZh1A1 cells. Mutagenicity continued to increase at higher doses, but was limited by increased cell killing above 600nM (not shown). The results demonstrated a significant 21–32% decrease in mutagenicity (or 1.3- to 1.5-fold protection) at the hprt locus in the cells coexpressing hGSTM1-1 (Fig. 1; p
0.02). The time course study demonstrated that mutagenicity at 300nM B[a]P was time dependent (Fig. 2), but that a similar degree of protection was provided by hGSTM1-1 expression at all time points (p < 0.03). The degree of protection afforded by the presence of hGSTM1-1 and the absolute mutant colony numbers at 48 h were consistent with those observed in the dose–response experiment at 300nM B[a]P. The leveling of mutant formation over the time course was reflective of consumption of the parent B[a]P from the cell culture medium. Analysis of the rate of disappearance of B[a]P from cell culture medium over a 48-h time period demonstrated metabolism of approximately half of the parent compound over the first 24 h, with a near-complete disappearance of the compound at 48 h (Kushman et al., 2007).
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Cells were also exposed to the pure enantiomers of the 7,8-dihydrodiol metabolites of B[a]P, (+)-B[a]P-7,8-diol, and (–)-B[a]P-7,8-diol in order to determine protective efficacy of GSTs against mutagenicity of these compounds. Mutagenicity of either (+)-B[a]P-7,8-diol or (–)-B[a]P-7,8-diol was dose-dependent (Fig. 3), and the (–)-enantiomer yielded a greater absolute number of mutants at each concentration tested than the (+) enantiomer. Expression of hGSTM1 provided 1.3- to 2.4-fold protection against (+)- or (–)-B[a]P-7,8-diol mutagenicity (p
0.02).
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Total Cellular Macromolecular Adducts of [3H]-B[a]P
The overall accumulation of covalent adducts formed from metabolism of [3H]-B[a]P in total cellular macromolecules (i.e., protein, RNA and DNA) was examined in cells exposed to 100nM [3H]-B[a]P for 48 h. Expression of hCYP1A1 resulted in 1.51 pmol adducts bound/mg protein, a 73-fold increase compared to 0.02 pmol/mg in the control V79MZ cells lacking CYP activity. Expression of hGSTM1-1 resulted in a 47% (1.9-fold) reduction in covalent macromolecular adducts (Table 4, p
0.001), similar to the degree of reduction in B[a]P mutagenicity by hGSTM1-1. Most of the adducts are protein linked, since DNA adducts contribute less than 5% of the total bound [3H]-B[a]P, and RNA binding is insignificant (unpublished results).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Expression of GST isozymes is generally believed to play an important role in cellular defenses via catalysis of conjugation with GSH of highly reactive metabolites formed via oxidation of certain environmental carcinogens such as B[a]P. However, although there is substantial evidence from in vitro studies with purified GST isozymes and the ultimate PAH-DE substrates to support this association, there is not a similarly strong body of direct evidence that demonstrates the functional protective significance of the presence or absence of specific GST isozymes as an isolated variable. Meta-analysis of population-based risk assessment studies of the role of homozygous deletions of the mu-class hGSTM1 and the theta-class hGSTT1 have not yielded convincing support for the notion that these genes play an important part in modulation of the relative risk of cancer in smokers, despite the high prevalence of the null alleles (Houlston, 1999
; Vogl et al., 2004). We have investigated the efficacy of expression of hGSTM1-1 for protection against cytotoxicity and mutagenicity induced by exposure to the PAH B[a]P and its 7,8-dihydrodiol metabolites when these PAH are activated by human CYP1A1. We also investigated protection conferred by GST expression against total cellular macromolecule alkylation by 3H-B[a]P. The results obtained in this study demonstrate that the protective efficacy of GSTM1-1 against the toxic effects of PAHs can vary significantly depending on the specific biological endpoint examined and whether B[a]P or the intermediate metabolites were tested. The strong protection against B[a]P and the contrasting lack of protection against the B[a]P-7,8-diol enantiomers in the cytotoxicity assay may be due to detoxification by hGSTM1-1 of metabolites formed via pathways of B[a]P activation other than through formation of B[a]P-7,8-diol. Another possibility is that, regardless of the key cytotoxic metabolite detoxified, the limitation in the rate of initial oxidation of B[a]P by hCYP1A1 to various dihydrodiol intermediates may allow the hGSTM1-1 to better accommodate the flux of the critical DE metabolites that are the most potent cytotoxic as well as mutagenic metabolites. This would seem likely based on the much greater potency and higher fold-activation ratio (i.e. ± hCYP1A1 expression) of the dihydrodiols than with B[a]P itself.
While the human GSTM1-1 was capable of providing substantial protection against cytotoxicity of B[a]P, the protection against mutagenicity as indicated by the hprt mutagenicity assay was consistently 1.5-fold or less over a range of doses and exposure times. Mutant colony formation began to plateau after 48 h in both the CYP1A1 and GSTM1-1 expressing cells, which is consistent with the greater than 90% depletion of B[a]P from the cell culture medium after 48 h, as shown in a previous study (Kushman et al., 2007). The protection against mutagenicity of the B[a]P-7,8-diols was higher than with B[a]P, up to twofold for either enantiomer studied, and is in distinct contrast to the lack of protection against B[a]P-7,8-diol cytotoxicity. The absolute mutagenicity of the (–)-B[a]P-7,8-diol was higher than that of (+)-B[a]P-7,8-diol, consistent with the fact that it gives rise to the (+)-anti-BPDE enantiomer, the most mutagenic metabolite (Wood et al., 1977). Although the efficiency for conjugation by hGSTM1 of the (+)-syn-BPDE and (–)-anti-BPDE enantiomers formed from the (+)-B[a]P-diol is reportedly higher overall than the conjugation efficiency with the (+)-anti-BPDE and (–)-syn-BPDE enantiomers formed from the (+)-B[a]P-diol (Sundberg et al., 1997), our results indicated a similar functional protection by hGSTM1-1 against the mutagenicity of the two B[a]P-7,8-diol enantiomers. This could be the result of differences in the sensitivity of hGSTM1-1 to stereoselective inhibition by the GSH conjugates of the two pairs of BPDE enantiomers, a process that could rapidly and differentially attenuate the rates of detoxification as the conjugates accumulate within the confines of the cell (Driscoll et al., 2003; Morrow et al., 1998). Supporting this possibility is our observation that GSH conjugates of 4-nitroquinoline-1-oxide accumulate rapidly and decline slowly in V79MZ cells, indicating that they most likely do not express GSH conjugate efflux transporters at functionally significant levels (data not shown).
Interestingly, protection conferred by hGSTM1-1 against total macromolecular (i.e., protein, DNA and RNA) adducts of [3H]-B[a]P was similar to that for mutagenicity, with a 50% reduction by hGSTM1-1 relative to cells expressing hCYP1A1 only. The close correlation of mutagenicity with cellular adducts also raises the possibility that damage to cellular protein may contribute to mutagenicity, for example, by reducing the fidelity of replication through inhibition or damage to key enzymes involved in DNA replication and/or repair (Driscoll et al., 2003). It will be of interest in future studies to examine levels of specific DNA adducts in this system by 32P-postlabeling or other sensitive analytical procedures for the detection of carcinogen–DNA adducts to determine if a reduction of a specific adduct, such as N2-guanine-BPDE, correlates with any of the toxic endpoints examined in this study.
Comparison of the protection by hGSTM1 with that provided by hGSTP1, reported in an earlier study in this lab (Kushman et al., 2007), indicates that hGSTP1 expression appears to confer greater resistance to mutagenesis of B[a]P and the (+)- and (–)-B[a]P-7,8-diol enantiomers than hGSTM1, even taking into account the moderately higher protein expression level of the hCYP1A1 + hGSTP1 dual-transfected cell line. Protection against B[a]P cytotoxicity by hGSTP1 was also greater than by hGSTM1, but this difference was approximately in proportion to the higher expression level of hGSTP1 (S. Kabler and A. Townsend, in preparation). The earlier study examined only the hGSTP1 variant that is most common in the human population; there are two polymorphisms in the hGSTP1 coding region that give rise to three additional variant alleles, each of which has a distinct catalytic profile for conjugation of BPDE and other PAH-DE (Hu et al., 1997). The significance of these variants warrants further study.
The results in this report support the hypothesis that hGSTM1-1 detoxifies reactive metabolites of B[a]P as a common mechanism of protection against cytotoxicity and mutagenicity, but the degree of protection varies between these endpoints, for reasons that remain unknown. Hence, while protection against parent B[a]P cytotoxicity is strong, protection against B[a]P macromolecular adducts and mutagenicity is only moderate. The finding that hGSTM1 expression conferred much greater protection against B[a]P-induced cytotoxicity than genotoxicity in intact cells suggests that this could be due in part to the preservation by hGSTM1 expression of a greater number of mutated cells than the fraction that survive of the cells that express only hCYP1A1. This would tend to offset the beneficial effects of hGSTM1 protection against mutagenicity. The major body of evidence seems to suggest that the absence of a functional GSTM1-1 gene in humans is likely a weak to moderate risk factor for cancer. Thus, the modest protection against genotoxicity observed in these cellular studies suggests that hGSTM1-1 expression alone may account for a relatively minor component of cellular detoxification of B[a]P metabolites produced via the CYP1A1 pathway. This, in turn, could explain the modest relative risk factors often reported for cancer incidence in individuals lacking the hGSTM1 gene.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institute for Environmental Health Sciences, National Institutes of Health grant (# RO1-ES-10175); and training grant (# T32-ES-007331) to M.E.K. and S.A.
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