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[Cancer Research 59, 2358-2362, May 1, 1999]
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[Cancer Research 59, 2358-2362, May 15, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Differential Protection against Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced DNA Damage in HepG2 Cells Stably Transfected with Allelic Variants of {pi} Class Human Glutathione S-Transferase1

Xun Hu, Christian Herzog, Piotr Zimniak and Shivendra V. Singh2

Cancer Research Laboratory, Mercy Cancer Institute, Mercy Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15219 [X. H., S. V. S.]; and Departments of Medicine [C. H., P. Z.] and Biochemistry and Molecular Biology [P. Z.], University of Arkansas for Medical Sciences and McClellan Veterans Affairs Hospital Medical Research [P. Z.], Little Rock, Arkansas 72205


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results and Discussion
 REFERENCES
 
The {pi} class glutathione S-transferase (GSTP1-1), which is polymorphic in human populations, is believed to play an important role in detoxification of the ultimate carcinogen of widespread environmental pollutant benzo[a]pyrene {(+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [(+)-anti-BPDE]}. The allelic variants of human GSTP1-1 (hGSTP1-1) differ in their structures by the amino acids in positions 104 (isoleucine or valine) and/or 113 (valine or alanine). Here, we have determined the protective effect of overexpression of allelic variants of hGSTP1-1, through stable transfection in HepG2 cells, against (+)-anti-BPDE-induced DNA modification. Clonal transfectants of HepG2 cells corresponding to the three allelic variants of hGSTP1-1 [(I104,A113), (V104,A113), and (V104,V113), denoted hGSTP1(IA), hGSTP1(VA), and hGSTP1(VV), respectively] with similar levels of hGSTP1 protein were identified and characterized for their GST activity and (+)-anti-BPDE-induced DNA modification. The glutathione S-transferase activity toward (+)-anti-BPDE was significantly higher (~3.0–3.6-fold) in cells transfected with hGSTP1(VA) [HepG2(VA)] and hGSTP1(VV) [HepG2(VV)] compared with hGSTP1(IA) transfectant [HepG2(IA)]. The formation of (+)-anti-BPDE-DNA adducts was significantly reduced in HepG2(VA) and HepG2(VV) cells compared with cells transfected with insert-free vector (HepG2-vect). Maximum protection against (+)-anti-BPDE-induced DNA damage was afforded by the hGSTP1(VV) isoform. The results of this study indicate that the allelic variants of hGSTP1-1 significantly differ in their ability to provide protection against (+)-anti-BPDE-induced DNA damage. Thus, hGSTP1-1 polymorphism may be an important factor in differential susceptibility of individuals to tumorigenesis induced by benzo[a]pyrene.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results and Discussion
 REFERENCES
 
BP3 is the prototypic member of the PAH family of environmental pollutants, which are present in cigarette smoke, automobile exhaust, and so on and are suspected human carcinogens (1) . The tumorigenic activity of BP has been linked to its metabolite (+)-anti-BPDE, which is generated during cytochrome P450- and epoxide hydrolase-mediated bioactivation of the parent compound (2 , 3) . Covalent interaction of (+)-anti-BPDE with nucleophilic centers in DNA is a critical event in the initiation of BP-induced tumorigenesis. On the other hand, GST-catalyzed conjugation of (+)-anti-BPDE with GSH is believed to be important in the cellular detoxification of this environmental carcinogen (4) . Previous studies have shown that the {pi} class isoenzyme (hGSTP1-1) is highly efficient in the GSH conjugation of (+)-anti-BPDE (4) . Although two functional GSTP genes are expressed in the mouse (5) , only one functional GSTP gene has been identified in humans (6) . On the other hand, GSTP gene has been shown to be polymorphic in human populations. We were the first to report two variants of hGSTP1-1, differing in their primary structures by a single amino acid at position 104 (isoleucine or valine), through amino acid sequence analysis of the protein (7) . Our results were independently confirmed through partial cDNA sequence analysis of hGSTP (8) . Subsequently, we have shown that these two variants of hGSTP1-1 differ in their activity toward the model substrate 1-chloro-2,4-dinitrobenzene (9) . More recently, several independent groups of investigators have not only confirmed our results but also identified another divergent site at position 113 (alanine or valine; Refs. 10, 11, 12, 13 ). Full-length cDNAs corresponding to three allelic variants of hGSTP1-1 [(I104,A113), (V104,A113), and (V104,V113)] have been isolated (10 , 13) . The hGSTP1(IA) allele is most frequent in human populations (10, 11, 12, 13) .

We have shown recently that the hGSTP1-1 variants with valine at position 104, hGSTP1(VA) and hGSTP1(VV), are relatively more active than hGSTP1(IA) in the GSH conjugation of (+)-anti-BPDE (14) . Our studies also show that the transition alanine-113->valine in the presence of valine 104 causes a significant increase in the catalytic efficiency of the enzyme toward (+)-anti-BPDE (14) . These observations led us to postulate that hGSTP1(VV) variant may be relatively more effective than either hGSTP1(IA) or hGSTP1(VA) in providing protection against (+)-anti-BPDE-induced DNA modification.

Here, we have tested the above hypothesis by determining the protective effect of the overexpression of allelic variants of hGSTP1-1, through stable transfection in HepG2 cells, against (+)-anti-BPDE-induced DNA damage. The results of this study indicate that the hGSTP1(VV) variant is significantly more effective than hGSTP1(IA) in providing protection against (+)-anti-BPDE-induced DNA damage.


   Materials and Methods
Top
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results and Discussion
 REFERENCES
 
Materials.
Radioactive and nonradioactive (+)-anti-BPDE were purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository (Chemsyn Science Laboratories, Lenexa, KS). Mammalian expression vector [pcDNA3.1(-)] and Escherichia coli competent cells (ONE Shot TOP10F') were procured from Invitrogen (Carlsbad, CA). Lipofectamine Reagent, Opti-MEM Reduced Serum Medium, cell culture media, and FCS were obtained from Life Technologies, Inc. (Gaithersburg, MD). GSH, CDNB, and epoxy-activated Sepharose 6B were purchased from Sigma Chemical Co. (St. Louis, MO). Restriction endonucleases (XbaI and BamHI) were obtained from Promega (Madison, WI).

Construction of Expression Vector.
cDNA encoding the three allelic forms of hGSTP1-1 in pET-9a vector was obtained during the course of our previous work (14) . The plasmids were digested with XbaI and BamHI, and the inserts were purified by 1% agarose gel electrophoresis and ligated into the mammalian expression vector pcDNA3.1(-), previously cut with XbaI and BamHI.

Transfection of HepG2 Cells.
HepG2 cells were transfected with allelic variants of hGSTP1-1 using Lipofectamine Reagent according to the manufacturer’s instructions (Life Technologies, Inc.). Briefly, HepG2 cells (at ~50% confluence) were rinsed with serum-free MEM and exposed to transfection mix containing 3 µg of plasmid DNA and 12.5 µl of Lipofectamine Reagent in 2 ml of Opti-MEM Reduced Serum Medium. The cells were incubated for 6 h at 37°C in a humidified CO2 incubator. The transfection mix was replaced with MEM containing 10% FCS. After 18 h of incubation, stable transfectants were isolated by selection on 800 µg/ml G418 for ~2 weeks. Alternatively, transfectants were obtained by the calcium phosphate coprecipitation technique (15) . Several G418-resistant stable clones corresponding to each allelic variant of hGSTP1-1 were selected for further characterization. Transfectants were maintained in MEM containing 10% FCS and 200 µg/ml G418. HepG2 cells were also transfected with insert-free pcDNA3.1(-) vector, and this transfectant was used as the control.

GST Activity Determination.
Cells in the logarithmic growth phase were trypsinized, washed twice with PBS, and lysed by sonication. The cell lysate was centrifuged at 14,000 x g for 40 min, and the supernatant was used for the determination of protein content (16) , GST activities toward CDNB (17) and (+)-anti-BPDE (18) , and GSH levels (19) .

Determination of hGSTP1 Level in Transfected HepG2 Cells.
Cell lysates, obtained as described above, were subjected to GSH affinity chromatography according to the method of Simons and Vander Jagt (20) for the purification of total GSTs. The recovery of the GST activity was always >90%. The GSH affinity-purified GST preparation was concentrated and subjected to reverse-phase HPLC for the quantitation of hGSTP1. Reverse-phase HPLC was performed as described by us previously (18) , with slight modifications. Briefly, a Waters Delta-Pak column (3.9 mm x 150 mm) was preequilibrated with 42.3% acetonitrile in 0.1% trifluoroacetic acid. After sample loading, the column was eluted with a 10-min linear gradient of 42.3–51.3% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Under these conditions, hGSTP1 eluted at a retention time of ~7 min.

Quantitation of GSH Conjugate of (+)-anti-BPDE (BPD-SG).
Approximately 2 million cells were plated in T25 flasks and allowed to attach overnight. The medium was changed to prewarmed serum-free MEM, and the cells were exposed to 1.0 µM (+)-anti-BPDE for 10 min at 37°C. The cells were washed twice with ice-cold PBS. The medium and the PBS washes were pooled and concentrated by solid-phase extraction using Sep-Pak Vac cartridges (Waters/Millipore, Milford, MA) according to the manufacturer’s instructions. This fraction was used to determine extracellular levels of BPD-SG. The cells were lysed with 3 ml of lysis solution containing 50% ethanol and 1% acetic acid. The cell lysate was centrifuged at 14,000 x g for 40 min. The supernatant fraction was concentrated and used to determine intracellular accumulation of BPD-SG. The BPD-SG was quantified by reverse-phase HPLC, as described by us previously (18) .

Quantitation of BPD-DNA Adducts.
Approximately 2 million cells were plated in T25 flasks and incubated overnight at 37°C in a humidified CO2 incubator. The medium was replaced with prewarmed serum-free MEM before addition of the [3H](+)-anti-BPDE. The cells were exposed to 0.1, 1.0, or 5.0 µM [3H](+)-anti-BPDE for 10 min at 37°C. Subsequently, the cells were rinsed twice with PBS, trypsinized, and collected for the isolation of DNA. The DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. The radioactivity associated with the DNA was determined by liquid scintillation counting.

Quantitation of BPD-dG.
The exposure of cells to radioactive (+)-anti-BPDE and purification of DNA were the same as described above for the quantitation of BPD-DNA adducts. Approximately 30 µg of DNA were subjected to enzymatic digestion using 30 µg each of micrococcal nuclease and bovine spleen phosphodiesterase II (Sigma) in 50 mM Tris-HCl (pH 7.2) containing 10 mM CaCl2 at 37°C for 6 h. Approximately 10 µg of digested DNA were analyzed for BPD-dG using a Waters Nova Pak C18 (3.9 mm x 150 mm) HPLC column. The BPD-dG was eluted with a 10-min linear gradient of 18–22.5% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min and monitored at 247 nm. Under these conditions, BPD-dG eluted at a retention time of about 5.2 min. Fractions of 0.5 ml each were collected and monitored for radioactivity by liquid scintillation counting. The retention time of BPD-dG was established by reverse-phase HPLC analysis of the commercially available standard (Chemsyn Science Laboratories).


   Results and Discussion
Top
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
REFERENCES
 
Several independent stable transfectants of HepG2 cells corresponding to each of the three allelic variants of hGSTP1 [hGSTP1(IA), hGSTP1(VA), and hGSTP1(VV)] were screened for hGSTP1 protein level by reverse-phase HPLC analysis of the GSH-affinity purified GST preparations (Fig. 1)Citation , and clones with comparable levels of hGSTP1 protein were selected for further studies. The hGSTP1 expression was negligible in control HepG2-vect cells. Among the transfectants, the clonal cell lines HepG2/hGSTP1(IA)-20, HepG2/hGSTP1(VA)-4, and HepG2/hGSTP1(VV)-21 expressed 1275 ± 100, 1259 ± 144, and 1232 ± 148 ng of the corresponding hGSTP1 allelic protein per mg of total cellular protein, respectively. Because these expression levels were not significantly different from each other, the above set of transfectants with matching hGSTP1 levels was used for all further comparisons. These cells are denoted HepG2(IA), HepG2(VA), and HepG2(VV) cells, respectively.



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  		Fig. 1. Reverse-phase HPLC analysis of hGSTP1 protein in control HepG2-vect cells and hGSTP1-transfected HepG2 cells.

 
The above clones were further characterized for their GST activity toward CDNB and (+)-anti-BPDE (Table 1)Citation to confirm that hGSTP1 protein was expressed in these cells in active form. The GST activity toward CDNB in the cytosol of the hGSTP1-transfected HepG2 cells was significantly higher (8–16-fold; P < 0.05) than that of control HepG2-vect cells. In addition, the GSH-CDNB conjugating activity in HepG2(IA) cell cytosol, 351 ± 26 nmol·min-1·mg-1 protein, was ~1.9-fold higher than in HepG2(VA) and HepG2(VV) cells (P < 0.05). These results are in agreement with previous kinetic studies (9 , 10) , which have shown that the hGSTP1(IA) isoform is more active in catalyzing GSH conjugation of CDNB than the hGSTP1(VA). On the other hand, GSH-(+)-anti-BPDE-conjugating activities in the cytosol of HepG2(VA) and HepG2(VV) cells were ~3.0- and 3.6-fold higher (P < 0.05), respectively, than that of HepG2(IA) cells (Table 1)Citation . No GST activity toward (+)-anti-BPDE could be detected in HepG2-vect cells. Since cellular GSH concentration can affect GST-catalyzed conjugation reactions (21) , the levels of this nonprotein thiol were also measured in the above cells. As shown in Table 1Citation , the GSH levels were found to be comparable in control- and hGSTP1-transfected HepG2 cells.


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  		Table 1 Characterization of HepG2 cell transfectants

 
To determine whether differences in GST activities toward (+)-anti-BPDE in hGSTP1-transfected HepG2 cells resulted in differential intracellular accumulation of BPD-SG, intracellular levels of this conjugate were compared following a 10-min exposure of cells to 1 µM (+)-anti-BPDE. As shown in Fig. 2ACitation , the intracellular accumulation of BPD-SG in hGSTP1-transfected HepG2 cells was significantly higher (3.7–12-fold) compared with control HepG2-vect cells. Moreover, the intracellular levels of BPD-SG in HepG2(VV) cells, 36 ± 6 pmol/106 cells, was ~3.3-fold higher (P < 0.05) than that in HepG2(IA) and HepG2(VA) cells. Because the GSH conjugate of (+)-anti-BPDE can be transported out of cells (22) , the differences in intracellular accumulation of BPD-SG among hGSTP1-transfected HepG2 cells may result from differential efflux of the conjugate. To test this possibility, we measured the extracellular levels of BPD-SG (medium plus PBS washes) (Fig. 2B)Citation . No BPD-SG conjugate could be detected in the medium of control HepG2-vect cells. For the three hGSTP1 transfectants, the total amounts of extracellular BPD-SG per 106 cells were similar and were considerably lower than the amounts of conjugate found within cells. For example, in the case of HepG2(VV) cells, only 8% of BPD-SG that formed in the course of the 10-min incubation were found in the extracellular compartment.



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  		Fig. 2. Analysis of GSH conjugate of (+)-anti-BPDE in HepG2 transfectants. A, intracellular accumulation of BPD-SG. B, extracellular levels of BPD-SG. Cells were exposed to 1.0 µM (+)-anti-BPDE for 10 min. The medium and PBS washes were pooled and used for the quantitation of extracellular BPD-SG. Columns, means of three determinations; bars, SD. a, significantly different from HepG2-vect cells (P < 0.05); b, significantly different from HepG2(IA) cells (P < 0.05).

 
Exposure of control- and hGSTP1-transfected HepG2 cells to [3H](+)-anti-BPDE for 10 min resulted in the formation of covalent BPD-DNA adducts (Table 2)Citation . The amounts of BPD-DNA adducts increased with increasing concentrations of [3H](+)-anti-BPDE in the media for both control- and hGSTP1-transfected HepG2 cells. The levels of BPD-DNA adducts were significantly lower in HepG2(VA) and HepG2(VV) cells compared with vector-transfected control cells at every concentration of [3H](+)-anti-BPDE tested in this study. While hGSTP1(IA) was least effective in preventing (+)-anti-BPDE-induced DNA damage, maximum protection was afforded by the hGSTP1(VV) isoform. Moreover, the protective potential of hGSTP1(VV) against [3H](+)-anti-BPDE-induced DNA modification was greater at low concentrations of (+)-anti-BPDE than at higher concentrations. Thus, at 0.1 µM [3H](+)-anti-BPDE, the levels of BPD-DNA adducts in HepG2(VV) cells was only 31% of that found in control cells, whereas at 5.0 µM [3H](+)-anti-BPDE, it rose to almost 60% of control (Table 2)Citation .


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  		Table 2 Formation of DNA adducts of (+)-anti-BPDE in HepG2 cell transfectants

 
Previous studies have shown that BPD-dG is the predominant DNA adduct of (+)-anti-BPDE (23) . Therefore, formation of the BPD-dG was also compared in HepG2 cells transfected with the three allelic forms of hGSTP1, and the results are shown in Fig. 3Citation . At 1.0 µM [3H](+)-anti-BPDE, less BPD-dG adduct was formed in HepG2(VA) and HepG2(VV) cells but not in HepG2(IA), compared with control HepG2-vect cells; these differences were statistically significant. Moreover, at this concentration, HepG2(VA) and HepG2(VV) cells were significantly more effective in reducing the amount BPD-dG than HepG2(IA) cells. However, at a higher [3H](+)-anti-BPDE concentration (5.0 µM), a statistically significant reduction in the formation of BPD-dG relative to HepG2-vect cells was observed only in HepG2(VV).



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  		Fig. 3. BPD-dG adduct formation in HepG2 cells transfected with allelic variants of hGSTP1. Cells were exposed to 1.0 or 5.0 µM [3H](+)-anti-BPDE for 10 min and washed with PBS. DNA was isolated and digested by micrococcal nuclease and bovine spleen phosphodiesterase II. The BPD-dG was quantified by reverse-phase HPLC followed by liquid scintillation counting. Columns, means of three determinations; bars, SD. a, significantly different from HepG2-vect cells (P < 0.05); b, significantly different from HepG2(IA) cells (P < 0.05).

 
Previous studies have suggested that the hGSTP, which is polymorphic in human populations (10, 11, 12, 13) , may play a major role in the detoxification of (+)-anti-BPDE because this isoenzyme is relatively more efficient than other classes of GSTs toward (+)-anti-BPDE (4) . Recently, Fields et al. (24) have shown that overexpression of hGSTP1-1(IA), through stable transfection in human breast cancer cells, significantly reduces anti-BPDE-induced DNA damage, although the role of other hGSTP1-1 variants in cellular protection against anti-BPDE-induced DNA modification was not examined. We reported previously that the allelic variants of hGSTP1, when purified and assayed in vitro, differ in their activity toward (+)-anti-BPDE and that the hGSTP1(VV) isoform is significantly more efficient than either hGSTP1(IA) or hGSTP1(VA) in the GSH conjugation of this dihydrodiol epoxide (14) . The results of this study indicate that hGSTP1(VV) is also more effective than other two variants in GSH conjugation of (+)-anti-BPDE in cells, i.e., in an experimental system that is closer to a physiological situation than in vitro studies but is still well defined and controlled. Despite a similar level of hGSTP1 protein, considerably more BPD-SG conjugate is formed in HepG2 cells transfected with hGSTP1(VV) than in transfectants expressing the other two variants of hGSTP1-1. At the same time, in the presence of hGSTP1(VV), the least amount of BPD-DNA adducts is formed. The intracellular accumulation of BPD-SG in HepG2 transfectants inversely correlates with the formation of BPD-DNA adducts (Fig. 4)Citation . In HepG2-vect cells, for every 15 BPDE molecules that undergo GSH conjugation, 1 molecule escapes detoxification and modifies DNA (Fig. 4)Citation . In contrast, in cells expressing hGSTP(VV), the ratio is 1 BPDE molecule reacting with DNA of 400 molecules that form the GSH conjugate. hGSTP1(IA) and hGSTP1(VA) have an intermediate effectiveness (Fig. 4)Citation . These results demonstrate that the hGSTP1 variant with highest catalytic efficiency for the GSH conjugation of (+)-anti-BPDE is also most effective in preventing (+)-anti-BPDE-induced DNA modification.



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  		Fig. 4. Correlation between BPD-DNA adduct and BPD-SG conjugate formation in HepG2 cells transfected with hGSTP1 variants. Cells were treated with 1.0 µM (+)-anti-BPDE for 10 min, and adduct and conjugate levels were determined as described in "Materials and Methods" and in Table 2Citation and Figure 2Citation . To express the level of BPD-DNA adducts per 106 cells; we assumed that the total DNA content of HepG2 cells was 5 µg/106 cells.

 
The results of this study, taken together with our earlier studies, suggest that cancer incidence may be lower in individuals which are homozygous for hGSTP1(VV) allele than in heterozygotes or hGSTP1(IA) homozygotes. Epidemiological studies, examining the distribution of hGSTP1 alleles in normal healthy subjects versus cancer cases, however, have presented contradictory conclusions (10, 11, 12 , 25 , 26) . On the one hand, some studies have shown a strong association for hGSTP1(VA) and/or hGSTP1(VV) alleles with different types of cancers (10, 11, 12) . Certain other studies, on the other hand, have reported increased frequency of hGSTP1(IA) in cancer cases as compared with control subjects (25) . Although the reasons for this discrepancy are not fully known, a possible explanation can be offered. PAHs normally occur in the environment as complex mixtures of many different compounds with varying mutagenic and carcinogenic potency (1 , 3) . Depending upon the position of the epoxide ring, the PAH diol epoxides can be classified either as bay region or fjord region (3) . In general, the PAH diol epoxides of the fjord-region class, which are nonplanar molecules, are more mutagenic and/or carcinogenic than the corresponding planar, bay-region PAH diol epoxides (27 , 28) . Recent studies from our laboratory indicate that the planarity of the PAH-diol epoxide molecule may be an important determinant of the activity of hGSTP1 variants toward this class of chemical carcinogens (14 , 30) . We have shown that the hGSTP1(VV) variant, which is significantly more efficient than hGSTP1(IA) in the GSH conjugation of planar PAH-diol epoxides like (+)-anti-BPDE, is considerably less active than hGSTP1(IA) in catalyzing the GSH conjugation of nonplanar PAH-diol epoxides such as racemic anti-diol epoxides of benzo[g]chrysene (14 , 29 , 30) . Because the PAH mixtures in various sources of environmental exposure are likely to be different, individuals with a given hGSTP1 genotype may be at a different risk for carcinogenesis, depending on the chemical composition of the PAH mixture to which they are exposed. BP and (+)-anti-BPDE have been historically used as models of a PAH and its derived ultimate carcinogen. However, in evaluating protective effects of hGSTP1, it seems essential to include other compounds, in particular the fjord-region PAH diol epoxides.


   ACKNOWLEDGMENTS
 
We thank Colleen Murphy for technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This investigation was supported in part by USPHS grants ES 09140 (to S.V.S.), awarded by the National Institute of Environmental Health Sciences, and CA 76348 (to S.V.S.), awarded by the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, Fax: (412) 232-5753; E-mail: ssingh{at}mercy.pmhs.org Back

3 The abbreviations used are: BP, benzo[a]pyrene; PAH, polycyclic aromatic hydrocarbon; (+)-anti-BPDE, (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; GST, glutathione S-transferase; GSH, glutathione; GSTP, {pi} class GST; hGSTP, human GSTP; CDNB, 1-chloro-2,4-dinitrobenzene; HPLC, high-performance liquid chromatography; BPD-DNA, DNA adduct of (+)-anti-BPDE; BPD-SG, GSH conjugate of (+)-anti-BPDE; BPD-dG, trans-7,8,9-trihydroxy-10-(N2-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene. Back

Received 1/28/99. Accepted 3/18/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 REFERENCES
 

  1. IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Polynuclear Aromatic Compounds. Part 1. Chemical, Environmental, and Experimental Data. IARC Scientific Publ. No. 32. Lyon, France: IARC, 1983.
  2. Sims P., Grover P. L. Epoxides in polycyclic aromatic hydrocarbon: metabolism and carcinogenesis. Adv. Cancer Res., 20: 165-274, 1974.[Medline]
  3. Thakker D. R., Yagi H., Levin W., Wood A. W., Conney A. H., Jerina D. M. Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens Anders M. W. eds. . Bioactivation of Foreign Compounds, : 177-242, Academic Press New York 1985.
  4. Robertson I. G. C., Guthenberg C., Mannervik B., Jernström B. Differences in stereoselectivity and catalytic efficiency of three human glutathione transferases in the conjugation of glutathione with 7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Cancer Res., 46: 2220-2224, 1986.[Abstract/Free Full Text]
  5. Xu X., Stambrook P. J. Two murine GST-{pi} genes are arranged in tandem and are differentially expressed. J. Biol. Chem., 269: 30268-30273, 1994.[Abstract/Free Full Text]
  6. Kano T., Sakai M., Muramatsu M. Structure and expression of a human class {pi} glutathione S-transferase messenger RNA. Cancer Res., 47: 5626-5630, 1987.[Abstract/Free Full Text]
  7. Ahmad H., Wilson D. E., Fritz R. R., Singh S. V., Medh R. D., Nagle G. T., Awasthi Y. C., Kurosky A. Primary and secondary structural analyses of glutathione S-transferase {pi} from human placenta. Arch. Biochem. Biophys., 278: 398-408, 1990.[Medline]
  8. Board P. G., Webb G. C., Coggan M. Isolation of a cDNA clone and localization of the human glutathione S-transferase 3 genes to chromosome bands 11q13 and 12q13–14. Ann. Hum. Genet., 53: 205-213, 1989.[Medline]
  9. Zimniak P., Nanduri B., Pikula S., Bandorowicz-Pikula J., Singhal S. S., Srivastava S. K., Awasthi S., Awasthi Y. C. Naturally occurring human glutathione S-transferase GSTP1–1 isoforms with isoleucine and valine in position 104 differ in enzymic properties. Eur. J. Biochem., 224: 893-899, 1994.[Medline]
  10. Ali-Osman F., Akande O., Antoun G., Mao J-X., Buolamwini J. Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase {pi} gene variants: evidence for differential catalytic activity of the encoded proteins. J. Biol. Chem., 272: 10004-10012, 1997.[Abstract/Free Full Text]
  11. Harries L. W., Stubbins M. J., Forman D., Howard G. C. W., Wolf C. R. Identification of genetic polymorphisms at the glutathione S-transferase {pi} locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis (Lond.), 18: 641-644, 1997.[Abstract/Free Full Text]
  12. Watson M. A., Stewart R. K., Smith G. B., Massey T. E., Bell D. A. Human glutathione S-transferase P1 polymorphism: relationship to lung tissue enzyme activity and population frequency distribution. Carcinogenesis (Lond.), 19: 275-280, 1998.[Abstract/Free Full Text]
  13. Lo H-W., Ali-Osman F. Genomic cloning of hGSTP1*C, an allelic human {pi} class glutathione S-transferase gene variant and functional characterization of its retinoic acid response elements. J. Biol. Chem., 272: 32743-32749, 1997.[Abstract/Free Full Text]
  14. Hu X., Xia H., Srivastava S. K., Herzog C., Awasthi Y. C., Ji X., Zimniak P., Singh S. V. Activity of four allelic forms of glutathione S-transferase hGSTP1–1 for diol epoxides of polycyclic aromatic hydrocarbons. Biochem. Biophys. Res. Commun., 238: 397-402, 1997.[Medline]
  15. Maruyama K., Wang H-M., Miyajima A., Takebe Y., Arai N. Karam J. D. Chao L. Warr G. W. eds. . Methods in nucleic acids research, : 283-305, CRC Press Boca Raton, FL 1990.
  16. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1976.[Medline]
  17. Habig W. H., Pabst M. J., Jakoby W. B. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249: 7130-7139, 1974.[Abstract/Free Full Text]
  18. Hu X., Benson P. J., Srivastava S. K., Mack L. M., Xia H., Gupta V., Zaren H. A., Singh S. V. Glutathione S-transferases of female A/J mouse liver and forestomach and their differential induction by anti-carcinogenic organosulfides from garlic. Arch. Biochem. Biophys., 336: 199-214, 1996.[Medline]
  19. Beutler E. Reduced glutathione (GSH) Beutler E. eds. . Red Cell Metabolism: A Manual of Biochemical Methods, : 131-132, Grune & Stratton New York 1984.
  20. Simons P. C., Vander Jagt D. L. Purification of glutathione S-transferases from human liver by glutathione-affinity chromatography. Anal. Biochem., 82: 334-341, 1977.[Medline]
  21. Hayes J. D., Pulford D. J. The glutathione S-transferase supergene family: regulation of GST* and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol., 30: 445-600, 1995.[Medline]
  22. Srivastava S. K., Hu X., Xia H., Bleicher R. J., Zaren H. A., Orchard J. L., Awasthi S., Singh S. V. ATP-dependent transport of glutathione conjugate of 7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles. Biochem. J., 332: 799-805, 1998.
  23. Cheng S. C., Hilton B. D., Roman J. M., Dipple A. DNA adducts from carcinogenic and non-carcinogenic enantiomers of benzo[a]pyrene dihydrodiol-epoxide. Chem. Res. Toxicol., 2: 334-340, 1989.[Medline]
  24. Fields W. R., Morrow C. S., Doss A. J., Sundberg K., Jernström B., Townsend A. J. Overexpression of stably transfected human glutathione S-transferase P1–1 protects against DNA damage by benzo[a]pyrene diol-epoxide in human T47D cells. Mol. Pharmacol., 54: 298-304, 1998.[Abstract/Free Full Text]
  25. Morita A., Yano M., Tsujinaka T., Ogawa A., Taniguchi M., Kaneko K., Shiozaki H., Doki Y., Inoue M., Monden M. Association between genetic polymorphisms of glutathione S-transferase P1 and N-acetyltransferase 2 and susceptibility to squamous-cell carcinoma of the esophagus. Int. J. Cancer, 79: 517-520, 1998.[Medline]
  26. Harris M. J., Coggan M., Langton L., Wilson S. R., Board P. G. Polymorphism of the {pi} class glutathione S-transferase in normal populations and cancer patients. Pharmacogenetics, 8: 27-31, 1998.[Medline]
  27. Buening M. K., Wislocki P. G., Levin W., Yagi H., Thakker D. R., Akagi H., Koreeda M., Jerina D. M., Conney A. H. Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene-7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Proc. Natl. Acad. Sci. USA, 75: 5358-5361, 1978.[Abstract/Free Full Text]
  28. Levin W., Chang R. L., Wood A. W., Thakker D. R., Yagi H., Jerina D. M., Conney A. H. Tumorigenicity of optical isomers of the diastereomeric 3,4-diol-1,2-epoxides of benzo(c)phenanthrene in murine tumor models. Cancer Res., 46: 2257-2261, 1986.[Abstract/Free Full Text]
  29. Hu X., Ji X., Srivastava S. K., Xia H., Awasthi S., Nanduri B., Awasthi Y. C., Zimniak P., Singh S. V. Mechanism of differential catalytic efficiency of two polymorphic forms of human glutathione S-transferase P1-1 in the glutathione conjugation of carcinogenic diol epoxide of chrysene. Arch. Biochem. Biophys., 345: 32-38, 1997.[Medline]
  30. Hu X., Xia H., Srivastava S. K., Pal A., Zimniak P., Singh S. V. Catalytic efficiencies of allelic variants of human glutathione S-transferase P1-1 toward carcinogenic anti-diol epoxides of benzo[c]phenanthrene and benzo[g]chrysene. Cancer Res., 58: 5340-5343, 1998.[Abstract/Free Full Text]



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