Genetic Analysis of Glutathione S-transferase A1 Polymorphism in the C
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《毒物学科学杂志》
Department of Pharmacology, Basic Medical College of Wuhan University, Wuhan 430071, China
Department of General Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China
2 To whom all correspondence should be addressed at Department of Pharmacology, Basic Medical College of Wuhan University, 185, DongHu Road, Wuhan 430071, China. Fax: 0086-27-87331670. E-mail: clbwhcbd@yahoo.com.
ABSTRACT
Genetic polymorphisms of human glutathione S-transferases (hGSTs) have important implications for drug efficacy and cancer susceptibility. hGSTA1 is the most abundant subfamily of hGSTs. The aim of this study was to investigate the distribution of hGSTA1 genetic polymorphism in the Chinese population and whether there exists the potential activity alterations caused by this polymorphism. Therefore, genomic DNA was extracted from peripheral blood of 140 healthy Chinese people and 11 normal liver tissues obtained from patients who had undergone liver surgery. Two variants in the promoter region of the hGSTA1 gene were identified by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP). Activities toward selected substrates of the wild type (hGSTA1A) and variant (hGSTA1B) were measured spectrometrically using S-9 fractions from liver samples. 5-androstene-3,17-dione (AD), cumene hydroperoxide (CuOOH), and 1-chloro-2,4-dinitrobenzene (CDNB) were used as marker substrates toward hGSTA1, hGSTA, and hGST, respectively. The kinetic parameters (Km, Vmax, and Vmax/Km) of hGSTA1 were determined with different concentrations of AD. The results showed that, in the study population, the proportions of hGSTA1A/A, hGSTA1A/B, and hGSTA1B/B genotypes were 75.0, 24.3, and 0.7%, respectively, and the allele frequencies of hGSTA1A and hGSTA1B were detected to be 87.1 and 12.9%, respectively. The variant hGSTA1 showed a significantly decreased activity for AD isomerization as compared to the wild type. Kinetic analyses revealed that the Vmax value of the variant hGSTA1 was 48% of that of the wild type despite a Km value of 62% (p < 0.01). This means that the Vmax/Km in the variant hGSTA1 was 76%. These data indicate that the distribution of hGSTA1 gene is polymorphic in Chinese and is different from those in other racial populations. The promoter sequence polymorphism of the hGSTA1 gene is associated with decreased Km and Vmax values of the enzyme in individuals with the variant allele. This variant is also associated with a decrease in hGSTA1 activity toward preferred substrates. This altered activity, however, is accompanied by significant individual variation in the variant population.
Key Words: glutathione S-transferases; polymorphism; promoter; catalytic activity; enzyme kinetics.
INTRODUCTION
Human glutathione S-transferases (hGSTs) are a large family of phase II drug-metabolizing enzymes and at least six distinct hGST isoenzymes have been identified in humans, which are referred to as class Alpha (A), Mu (M), Pi (P), Theta (T), Kappa (K), and Zeta (Z) based on immunological, functional, and physical properties (Hays and Pulford, 1995; Hayes and Strange, 2000). hGSTs are involved in the metabolism of a large range of endogenous and exogenous compounds, and the variation in hGSTs activity is known to have a major impact on the sensitivity of cells to these substances (Guy et al., 2004; Hayes and Strange 2000; Strange et al., 2001). This is assumed to result in differences in individual susceptibility to illnesses related to such substances, including various carcinogens. In addition, the efficacy of therapeutic drugs is affected by individual variation in the ability of these enzymes to metabolize them (Hayes and Strange, 2000).
Interindividual variation in the activities of hGSTs enzymes may be due to environmental influences, including diet, lifestyle (e.g., drug use), and exposure to toxins in the environment, but it is likely that genetic variation also plays a role. An increasing body of evidence suggests the importance of several hGSTs polymorphisms. In particular, mutational genotypes of hGSTs have been associated with an increased susceptibility or worse outcome in diseases. There are many known genetic sequence variants with potential phenotypic effects (Hayes and Strange, 2000). For example, homozygous null hGSTM1 individuals can not express the respective protein and appear to be at increased risk of several cancers (Strange et al., 2000).
Of all the hGSTs, the Alpha class isoenzymes are the most abundant hGSTs found in the human liver, and are notably responsible for metabolizing the nitrogen mustard group of some anticancer drugs, and for binding endogenous compounds such as bilirubin in the liver. Members of the Alpha class possess high glutathione peroxidase activity and play an important role in protecting cells against reactive oxygen species and the products of peroxidation (Whalen and Boyer, 1998). The alpha class genes are localized on chromosome 6p12 containing five functional genes (hGSTA1 hGSTA5) and seven pseudogenes (Morel et al., 2002). hGSTA1 constitutes approximately one-half of the total amount of alpha class hGSTs in human liver (Mulder et al., 1999). The polymorphism in hGSTA1 consists of five single nucleotide polymorphisms (SNPs), one of which is a silent base substitution in the exon 5 (Tetlow et al., 2001). The other four mutational sites are apparently linked on the proximal promoter. The variants are, therefore, named as such: hGSTA1A (–631G/T, –567T, –69C, –52G) and hGSTA1B (–631G, –567G, –69T, –52A) (Coles et al., 2001a). This polymorphism is of particular interest because the hGSTA1B allele, which is associated with lower hepatic expression of hGSTA1, appears to confer susceptibility to colorectal cancer, possibly as a result of inefficient hepatic detoxification of N-acetoxy-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, a selective hGSTA1 substrate (Coles et al., 2001b; Sweeney et al., 2002). On the other hand, Bredschneider et al. (2002) have studied the effects of several hGSTA1 promoter polymorphisms and found no correlation between them and either hGST protein abundance or enzymatic activity. Since the effect of the hGSTA1 genetic polymorphism is still obscure and the frequencies of the allelic variants of hGSTA1 gene in the Chinese population have not been reported, we determined the frequencies of the hGSTA1 genotypes in 140 healthy Chinese subjects in the present study. Then the enzymatic assays were performed spectrometrically on liver S-9 fractions from 11 normal livers. 5-androstene-3,17-dione (AD), cumene hydroperoxide (CuOOH), and 1-chloro-2,4-dinitrobenzene (CDNB) were used as marker substrates toward hGSTA1, hGSTA, and total hGST, respectively, to analyze the potential activity alterations caused by the genetic polymorphism in the Chinese.
MATERIALS AND METHODS
Chemicals.
Genomic DNA preparation kit was purchased from Vitagene Co. (Hangzhou, China). Oligonucleotide primers were customly synthesized by Sangon Biological Engineering Technology Co. (Shanghai, China). Deoxyribonucleoside triphosphate (dNTP) and Taq DNA polymerase were purchased from Tianyuan Co. (Wuhan, China). Restriction enzyme Ear I was obtained from New England Biolabs (MA). AD was obtained from Steraloids Co. (Newport, RI). CuOOH, CDNB, reduced glutathione (GSH) and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma Co. (St. Louis, MO). All other chemicals and reagents were of analytical grade.
Subjects and liver tissues.
A population of 140 unrelated healthy students (51 females and 89 males) in the medical college of Wuhan University came from 25 provinces of China and all are ethic Han origin (Chinese majority, 95% of the population). Their age ranges from 17 to 23. All subjects were asked to fill out a detailed questionnaire and were defined as healthy, not having a history of cancer, and not exposed to any known mutagen. Venous blood samples were collected from each of the 140 volunteers using 1.5 mg/ml edetate sodium as the anticoagulant. Samples were kept at 4°C until further analysis.
Eleven normal human liver tissues were obtained from patients of Chinese origin who had undergone liver surgery, as nontumorous tissue surrounding surgically removed liver tumors or metastases or material surgically resected for other reasons. Tissues were immediately frozen at –80°C until use. All patients' records were screened to include only patients who were not taking any drugs that might affect hepatic expression of hGSTs. The study was approved by the Ethical and Research Committee of Wuhan University, China, and written informed consent was obtained from each patient.
Preparation of subcellular fractions.
Genomic DNA was isolated from blood or liver samples according to the manufacturer's instructions. The concentration and purity of DNA were determined by UV-1601 ultraviolet spectrophotometer (Shimadzu, Japan). DNA samples were stored at –20°C until used as template DNA for polymerase chain reaction (PCR).
S-9 fractions were prepared as below. In brief, the piece of liver was homogenized (1:4, w/v) in ice cold Tris-HCl buffer (50 mM, pH 7.4) containing 0.1 mM EDTA. The homogenate was centrifuged at 9000 x g for 20 min at 4°C. The supernatant was collected and stored at –80°C for the further assays. S-9 protein concentrations were determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin (BSA) as standard.
Genetic analysis of hGSTA1.
For hGSTA1 genotyping, the analysis of the C-69T SNP was carried out using PCR-restriction fragment length polymorphism (RFLP). Genomic sequences of the hGSTA1 gene were retrieved from public databases (Genbank Accession No. L13269 and X67663). Specific oligonucleotide primers for PCR were designed by Primer Primer 5.0 (U.S.). Then we inputted the primer sequences to the BLAST database to compare the homology with other hGSTs and ensured that the primers were unique to hGSTA1. A 400 bp fragment was amplified with a forward primer (F: 5'-GCA TCA GCT TGC CCT TCA-3') and reverse primer (R: 5'-AAA CGC TGT CAC CGT CCTG-3'). PCR was performed in a total volume of 50 μl with 0.1 μg genomic DNA, 0.1 μM each primer, 0.2 mM each dNTP and 1.6 units Taq DNA polymerase in 1x buffer as supplied with the polymerase. Thermocycler parameters included an initial 5 min denaturation step at 95°C, followed by 30 cycles of 1 min for each step: denaturation at 95°C, anneal at 64°C and extension at 72°C. A final 6 min extension was performed before cooling reaction to 4°C. All amplification steps were completed using the Thermolyne Amplitron II Thermal Cycler (U.S.). PCR products were electrophoretically separated on a 1.5% agarose gel and visualized with ethidium bromide to determine successful amplification and adequate signal. Automated sequencing of the PCR fragment using "BigDye" terminators (U.S.) confirmed that the expected sequence of hGSTA1 proximal promoter was amplified from genomic DNA with the primers.
For RFLP analysis, digestions (20 μl) were performed for 4 h at 37°C using 5 units restriction enzyme Ear I and 4 μl unpurified PCR product in 1x buffer as supplied with the restriction enzyme. Aseptic distilled water substitutes the restriction enzyme acted as the negative control. The digested products were separated on 3% agarose gel stained with ethidium bromide for 1 h. The wild type allele (C) had no Ear I site and was still 400 bp. Ninety-two bp nucleotide was removed from the variant allele (T), which yielded a 308 bp fragment.
Enzyme activity assays.
Catalytic activities were determined for 11 preparations of S-9 fraction isolated from samples derived from wile type and variant individuals. Each preparation was derived from tissue from a different individual. The potential functional alterations of hGSTA1 caused by the SNPs were characterized by enzyme activity assays, AD, CuOOH, and CDNB were used as selective substrates toward hGSTA1, hGSTA, and hGST, respectively.
hGSTA1 activity.
Specific hGSTA1 activity measurement with AD was performed spectrophotometrically by some modifications of the assay procedure of Pettersson and Mannervik (2001). The reaction mixture consisted of 0.1 M Tris-HCl (pH 8.0), 1% (v/v) methanol as solvent for the substrate, 1 mg S-9 protein, 0.1 mM AD, and 1 mM GSH. The specific activity of hGSTA1 was determined by the increased absorption at 248 nm, which was produced by the product of the AD isomerization reaction, 4-androstene-3,17-dione (248 = 16.3 mM/cm). A correction for the spontaneous reaction between GSH and AD in the absence of enzyme was made.
hGSTA activity.
hGSTA has high substrate specificity toward CuOOH. Catalytic activity towards CuOOH was measured in S-9 fractions using modifications of a method described previously (Romero et al., 2002). Reaction was performed in 0.1 M potassium phosphate (pH 7.0) in a total volume of 1 ml. S-9 protein (1 mg) was incubated with CuOOH (1.5 mM), GSH (1 mM), NADPH (0.2 mM) and glutathione reductase (0.5 unit), and the time dependent disappearance of NADPH was monitored at 340 nm (340 = 6.2 mM/cm). Blank reaction with enzyme source replaced by distilled water was subtracted from each assay.
hGST activity.
hGST activity was measured with CDNB as described by Habig et al. (1974) with slight modifications. In short, the reaction of 1 mM CDNB with 1 mM GSH in the diluted human liver S-9 protein (1 mg) was monitored spectrophotometrically by recording the increase in absorbance at 340 nm (340 = 9.6 mM/cm). The final concentration of ethanol in the incubation mixture was 1% (v/v). A correction for the spontaneous reaction between GSH and CDNB in the absence of enzyme was made.
Kinetic studies.
On the assumption that the variant might show altered enzyme kinetics, the kinetic parameters of hGSTA1 were determined with different concentrations of AD. S-9 proteins of wild type and variant hGSTA1 were incubated at a 1 mg/ml protein concentration with five different concentrations of AD (5, 10, 20, 50, 100 μM). The kinetic parameters (Km, Vmax, and Vmax/Km) were obtained by fitting of the Michaelis-Menten equation to the data by non-linear regression analysis. The analysis was carried out in 6 different S-9 fractions prepared from six liver samples with known genotypes.
Statistical analysis.
All data were analyzed using the statistics package GraphPad Prism version 4.0. Allelic and genotype frequencies in Chinese and other populations were compared statistically by Pearson 2 test. Statistical significance of observed compared to expected Hardy-Weinberg genotype frequencies was assessed. Comparison of means between the wild type and the variant hGSTA1 was made by Student's t-test. p value of 0.05 was considered statistically significant.
RESULTS
hGSTA1 Genotype Analysis
Genotype and allele frequencies of 140 healthy Chinese were analyzed for the hGSTA1 polymorphism. Three different genotypes (CC, CT, and TT) reported previously (Coles et al., 2001a) were observed after digesting the amplified DNA fragment of 400 bp with the restriction enzyme Ear I. The enzymatic restriction products of the hGSTA1 gene were of the expected size (Fig. 1). Three different banding patterns were obtained: the wild type (CC) produced only a band at 400 bp, homozygous mutant genotype (TT) is characterized by 308 and 92 bp products, while heterozygous genotype (CT) yielded three bands (400, 308, and 92 bp).
Out of the 140 healthy individuals in Chinese having been genotyped for hGSTA1, 75.0% (105/140) were homozygous hGSTA1A, 24.3% (34/140) were heterozygous hGSTA1 (hGSTA1A/B), whereas 0.7% (1/140) were homozygous hGSTA1B. The frequency distributions of hGSTA1A and hGSTA1B alleles were 87.1% and 12.9%, respectively. There was no significant difference between male and female genotype frequencies (Table 1). Comparisons of the observed distributions of hGSTA1 genotype and those predicted by allele frequencies by 2 analyses showed that the population, in this study, was consistent with the Hardy-Weinberg equilibrium, indicating that the subjects were sufficiently random and representative.
Of the selected 11 liver samples, 7 individuals were wild homozygous hGSTA1A, the others were heterozygous hGSTA1. No subject with homozygous hGSTA1B genotype was found.
Enzymatic Activity Analysis
Three typical alpha class substrates, including AD, CuOOH, and CDNB, were used to determine and compare the catalytic activities of the two enzymes, hGSTA1A/A and hGSTA1A/B, encoded by the wild type hGSTA1 and the variant, respectively. AD isomerization activity of the wild type hGSTA1 was 46.1 ± 12.5 μmol/min/mg. The variant catalyzed AD at a rate of 24.1 ± 13.1 μmol/min/mg, which was about 52.3% of that of the wild type activity (p < 0.05). For CDNB and CuOOH, no statistically significant changes in activity were obtained from the variant hGSTA1. The percentages of the wild type enzymatic activities were 180 and 120% of the variant hGSTA1 with CDNB and CuOOH, respectively (Fig. 2).
Enzyme Kinetics
The kinetic properties of the proteins encoded by the wild type hGSTA1 and the variant were studied further in which AD concentrations were varied at 5 different concentrations. As shown in Figure 3 and Table 2, the Vmax value of the variant hGSTA1 was 48% of that of the wild type despite a Km value of the variant of 62% (p < 0.01). This means that the Vmax/Km in the variant hGSTA1 was 76%. These results indicated a slight reduction in the enzymatic activity of the variant for the isomerization of AD.
DISCUSSION
hGSTs are detoxifying enzymes that catalyze the conjugation reactions between GSH and a variety of electrophilic compounds, including carcinogens and anticancer drugs. It has been documented that genetic differences play an important role in the individual variation in constitutive and/or inducible hGSTs levels, which influence the drug efficacy, and susceptibility to environmental mutagens and carcinogens. The alpha class hGSTs is the predominant class in the human liver, contributing about 65 to 80% of the total liver hGST concentration. hGSTA1 expression in human liver was reported to be about 5-fold higher compared with hGSTA2, whereas other isoforms of the alpha class appear to be expressed at even lower levels (Bredschneider et al., 2002). The core control element for the hGSTA1 gene, including the DNA polymerase II binding site, are located within the proximal promoter gene that is situated upstream of the start of transcription. This region, therefore, is an important region to study in the search for human variation affecting gene expression (Coleman et al., 2002). Coles et al. (2001a) screened the whole proximal promoter sequence of hGSTA1 and found two alleles, hGSTA1A and hGSTA1B, containing four linked base substitutions at position –631, –567, –69, and –52. The polymorphism of hGSTA1 has been found to be widespread in Caucasians, African-Americans, Hispanics, and Japanese. In our study, we sequenced the PCR products and confirmed that the linkage disequilibrium between the –69 base pair and the other three variant sites noted in Coles's study populations also exists in our population. The PCR-RFLP assay for the base change at –69 provides a method that can be used to explore the effects of hGSTA1 polymorphism.
Genetic polymorphisms of hGSTs class differ significantly among racial groups and residential populations in different parts of the world (Nazar-Stewart et al., 2003). The results of our study, when compared to those of other studies, illustrate the marked variation in genotype and allele frequencies of the hGSTA1 gene among various world populations. A striking difference in the distribution of the variant genotype and allele has been observed among the Chinese population and Caucasian, African-American, and Hispanic populations, these latter groups sharing allele frequencies as high as 39%, as determined by Coles et al. (2001a). Chinese population demonstrated a similar allele frequency to the description in a Japanese population (Matsuno et al., 2004). These data implicated that Asian populations (Chinese and Japanese) have lower mutant allele frequency (13 and 16%) of hGSTA1 gene as compared to the Caucasian (26%), African-American (38%), and Spanish (39%).
To examine functional alterations due to the SNPs in hGSTA1 gene, the activities of wild type (hGSTA1A/A) and variant (hGSTA1A/B) were examined with three typical alpha class substrates: AD, CuOOH, and CDNB. The variant hGSTA1 showed a significant decrease in catalytic ability toward AD as compared to the wild type, however, the activities toward CDNB and CuOOH were found to be similar. It should be mentioned that large individual difference existed in the hGST and hGSTA enzyme activities of the 11 liver samples studied, which are 12- and 1.5-fold variability, respectively.
Variation in hGSTs activity between individuals has been proposed to underlie susceptibility to a number of illnesses and contribute to population variance in the responses to therapeutic drugs (Strange et al., 2001). The source of this variation in enzyme activity may be due to a number of environmental factors, but may also be influenced by genetic variation. Given this, several studies have previously sought to identify functional polymorphism that could be responsible for variation in enzyme activity. For example, polymorphisms in the hGSTM1, hGSTM3, hGSTT1, hGSTP1 genes are related to differing susceptibilities in the incidence of lung and breast cancers (Strange et al., 2001). However, information about enzymatic function alterations of the variant hGSTA1 is poorly documented. Coles et al. (2001a) found two haplotypes of the hGSTA1 promoter with four polymorphic variants, the haplotype that they designed hGSTA1A had approximately 4-fold higher activity in their assay. Consistently, we have confirmed that polymorphism of the hGSTA1 gene significantly attenuates the specific hGSTA1 activity accompanied by significant interindividual difference. Furthermore, on the assumption that the variant might show altered enzyme kinetics, the kinetic parameters of hGSTA1 were determined with different concentrations of AD and a significantly lower Km value of variant hGSTA1 was observed. Although the Vmax value between the wild type and the variant was not statistically significant possibly due to the small number of liver samples, the extent of decreased Vmax value was larger than those of the wild type. This resulted in a reduction in the catalytic activity of the variant hGSTA1.
Genetic polymorphisms are known to occur in the population and some of these can be directly related to changes in the protein abundance and other phenotypes. For example, a null mutation of the hGSTM1 gene has been associated with a variety of carcinomas (Strange et al., 2000; Wu et al., 2002). However, the phenotypic relevance of changes in enzyme activity is a controversial subject and in vitro effects such as those we reported may have no in vivo counterpart due to a variety of other factors that influence enzyme activity. Bredschneider et al. (2002) have studied the effects of several hGSTA1 promoter polymorphisms and found no correlation between them and either hGST protein abundance or enzymatic activity. However, that study also highlighted the large variations, up to 8-fold, in enzyme activity and abundance between individuals, which would make it very difficult to detect small variation in a small group of samples. These large variations in hGST activities may be the result of environmental effects as well as both cis and trans acting genetic polymorphism that have not yet been determined. However, the presence of additional phenotypic influences does not negate the potential clinical significance of the functional polymorphisms that we and others have described.
In conclusion, our study clearly indicated that the distributions of the hGSTA1 gene are polymorphic in the Chinese population and different from those in other racial populations. This is the first report on hGSTA1 allelic variants and genotypes in the Chinese population. We also demonstrated that the variant hGSTA1 is associated with attenuated specific hGSTA1 activity accompanied by large individual variation. It is reasonable to speculate that the variant of hGSTA1 might be associated with a deleterious biological consequence in human susceptibility to some diseases and in the clinical use of anticancer drugs via its effect on hGSTA1 activity.
NOTES
1 The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
This study is supported by a grant from the Pre-research Foundation of Wuhan University, China (No. 301270050) and Scientific Research Initiation Foundation for Abroad Returning by the Ministry of Education, China (No. 2004).
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Department of General Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China
2 To whom all correspondence should be addressed at Department of Pharmacology, Basic Medical College of Wuhan University, 185, DongHu Road, Wuhan 430071, China. Fax: 0086-27-87331670. E-mail: clbwhcbd@yahoo.com.
ABSTRACT
Genetic polymorphisms of human glutathione S-transferases (hGSTs) have important implications for drug efficacy and cancer susceptibility. hGSTA1 is the most abundant subfamily of hGSTs. The aim of this study was to investigate the distribution of hGSTA1 genetic polymorphism in the Chinese population and whether there exists the potential activity alterations caused by this polymorphism. Therefore, genomic DNA was extracted from peripheral blood of 140 healthy Chinese people and 11 normal liver tissues obtained from patients who had undergone liver surgery. Two variants in the promoter region of the hGSTA1 gene were identified by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP). Activities toward selected substrates of the wild type (hGSTA1A) and variant (hGSTA1B) were measured spectrometrically using S-9 fractions from liver samples. 5-androstene-3,17-dione (AD), cumene hydroperoxide (CuOOH), and 1-chloro-2,4-dinitrobenzene (CDNB) were used as marker substrates toward hGSTA1, hGSTA, and hGST, respectively. The kinetic parameters (Km, Vmax, and Vmax/Km) of hGSTA1 were determined with different concentrations of AD. The results showed that, in the study population, the proportions of hGSTA1A/A, hGSTA1A/B, and hGSTA1B/B genotypes were 75.0, 24.3, and 0.7%, respectively, and the allele frequencies of hGSTA1A and hGSTA1B were detected to be 87.1 and 12.9%, respectively. The variant hGSTA1 showed a significantly decreased activity for AD isomerization as compared to the wild type. Kinetic analyses revealed that the Vmax value of the variant hGSTA1 was 48% of that of the wild type despite a Km value of 62% (p < 0.01). This means that the Vmax/Km in the variant hGSTA1 was 76%. These data indicate that the distribution of hGSTA1 gene is polymorphic in Chinese and is different from those in other racial populations. The promoter sequence polymorphism of the hGSTA1 gene is associated with decreased Km and Vmax values of the enzyme in individuals with the variant allele. This variant is also associated with a decrease in hGSTA1 activity toward preferred substrates. This altered activity, however, is accompanied by significant individual variation in the variant population.
Key Words: glutathione S-transferases; polymorphism; promoter; catalytic activity; enzyme kinetics.
INTRODUCTION
Human glutathione S-transferases (hGSTs) are a large family of phase II drug-metabolizing enzymes and at least six distinct hGST isoenzymes have been identified in humans, which are referred to as class Alpha (A), Mu (M), Pi (P), Theta (T), Kappa (K), and Zeta (Z) based on immunological, functional, and physical properties (Hays and Pulford, 1995; Hayes and Strange, 2000). hGSTs are involved in the metabolism of a large range of endogenous and exogenous compounds, and the variation in hGSTs activity is known to have a major impact on the sensitivity of cells to these substances (Guy et al., 2004; Hayes and Strange 2000; Strange et al., 2001). This is assumed to result in differences in individual susceptibility to illnesses related to such substances, including various carcinogens. In addition, the efficacy of therapeutic drugs is affected by individual variation in the ability of these enzymes to metabolize them (Hayes and Strange, 2000).
Interindividual variation in the activities of hGSTs enzymes may be due to environmental influences, including diet, lifestyle (e.g., drug use), and exposure to toxins in the environment, but it is likely that genetic variation also plays a role. An increasing body of evidence suggests the importance of several hGSTs polymorphisms. In particular, mutational genotypes of hGSTs have been associated with an increased susceptibility or worse outcome in diseases. There are many known genetic sequence variants with potential phenotypic effects (Hayes and Strange, 2000). For example, homozygous null hGSTM1 individuals can not express the respective protein and appear to be at increased risk of several cancers (Strange et al., 2000).
Of all the hGSTs, the Alpha class isoenzymes are the most abundant hGSTs found in the human liver, and are notably responsible for metabolizing the nitrogen mustard group of some anticancer drugs, and for binding endogenous compounds such as bilirubin in the liver. Members of the Alpha class possess high glutathione peroxidase activity and play an important role in protecting cells against reactive oxygen species and the products of peroxidation (Whalen and Boyer, 1998). The alpha class genes are localized on chromosome 6p12 containing five functional genes (hGSTA1 hGSTA5) and seven pseudogenes (Morel et al., 2002). hGSTA1 constitutes approximately one-half of the total amount of alpha class hGSTs in human liver (Mulder et al., 1999). The polymorphism in hGSTA1 consists of five single nucleotide polymorphisms (SNPs), one of which is a silent base substitution in the exon 5 (Tetlow et al., 2001). The other four mutational sites are apparently linked on the proximal promoter. The variants are, therefore, named as such: hGSTA1A (–631G/T, –567T, –69C, –52G) and hGSTA1B (–631G, –567G, –69T, –52A) (Coles et al., 2001a). This polymorphism is of particular interest because the hGSTA1B allele, which is associated with lower hepatic expression of hGSTA1, appears to confer susceptibility to colorectal cancer, possibly as a result of inefficient hepatic detoxification of N-acetoxy-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, a selective hGSTA1 substrate (Coles et al., 2001b; Sweeney et al., 2002). On the other hand, Bredschneider et al. (2002) have studied the effects of several hGSTA1 promoter polymorphisms and found no correlation between them and either hGST protein abundance or enzymatic activity. Since the effect of the hGSTA1 genetic polymorphism is still obscure and the frequencies of the allelic variants of hGSTA1 gene in the Chinese population have not been reported, we determined the frequencies of the hGSTA1 genotypes in 140 healthy Chinese subjects in the present study. Then the enzymatic assays were performed spectrometrically on liver S-9 fractions from 11 normal livers. 5-androstene-3,17-dione (AD), cumene hydroperoxide (CuOOH), and 1-chloro-2,4-dinitrobenzene (CDNB) were used as marker substrates toward hGSTA1, hGSTA, and total hGST, respectively, to analyze the potential activity alterations caused by the genetic polymorphism in the Chinese.
MATERIALS AND METHODS
Chemicals.
Genomic DNA preparation kit was purchased from Vitagene Co. (Hangzhou, China). Oligonucleotide primers were customly synthesized by Sangon Biological Engineering Technology Co. (Shanghai, China). Deoxyribonucleoside triphosphate (dNTP) and Taq DNA polymerase were purchased from Tianyuan Co. (Wuhan, China). Restriction enzyme Ear I was obtained from New England Biolabs (MA). AD was obtained from Steraloids Co. (Newport, RI). CuOOH, CDNB, reduced glutathione (GSH) and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma Co. (St. Louis, MO). All other chemicals and reagents were of analytical grade.
Subjects and liver tissues.
A population of 140 unrelated healthy students (51 females and 89 males) in the medical college of Wuhan University came from 25 provinces of China and all are ethic Han origin (Chinese majority, 95% of the population). Their age ranges from 17 to 23. All subjects were asked to fill out a detailed questionnaire and were defined as healthy, not having a history of cancer, and not exposed to any known mutagen. Venous blood samples were collected from each of the 140 volunteers using 1.5 mg/ml edetate sodium as the anticoagulant. Samples were kept at 4°C until further analysis.
Eleven normal human liver tissues were obtained from patients of Chinese origin who had undergone liver surgery, as nontumorous tissue surrounding surgically removed liver tumors or metastases or material surgically resected for other reasons. Tissues were immediately frozen at –80°C until use. All patients' records were screened to include only patients who were not taking any drugs that might affect hepatic expression of hGSTs. The study was approved by the Ethical and Research Committee of Wuhan University, China, and written informed consent was obtained from each patient.
Preparation of subcellular fractions.
Genomic DNA was isolated from blood or liver samples according to the manufacturer's instructions. The concentration and purity of DNA were determined by UV-1601 ultraviolet spectrophotometer (Shimadzu, Japan). DNA samples were stored at –20°C until used as template DNA for polymerase chain reaction (PCR).
S-9 fractions were prepared as below. In brief, the piece of liver was homogenized (1:4, w/v) in ice cold Tris-HCl buffer (50 mM, pH 7.4) containing 0.1 mM EDTA. The homogenate was centrifuged at 9000 x g for 20 min at 4°C. The supernatant was collected and stored at –80°C for the further assays. S-9 protein concentrations were determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin (BSA) as standard.
Genetic analysis of hGSTA1.
For hGSTA1 genotyping, the analysis of the C-69T SNP was carried out using PCR-restriction fragment length polymorphism (RFLP). Genomic sequences of the hGSTA1 gene were retrieved from public databases (Genbank Accession No. L13269 and X67663). Specific oligonucleotide primers for PCR were designed by Primer Primer 5.0 (U.S.). Then we inputted the primer sequences to the BLAST database to compare the homology with other hGSTs and ensured that the primers were unique to hGSTA1. A 400 bp fragment was amplified with a forward primer (F: 5'-GCA TCA GCT TGC CCT TCA-3') and reverse primer (R: 5'-AAA CGC TGT CAC CGT CCTG-3'). PCR was performed in a total volume of 50 μl with 0.1 μg genomic DNA, 0.1 μM each primer, 0.2 mM each dNTP and 1.6 units Taq DNA polymerase in 1x buffer as supplied with the polymerase. Thermocycler parameters included an initial 5 min denaturation step at 95°C, followed by 30 cycles of 1 min for each step: denaturation at 95°C, anneal at 64°C and extension at 72°C. A final 6 min extension was performed before cooling reaction to 4°C. All amplification steps were completed using the Thermolyne Amplitron II Thermal Cycler (U.S.). PCR products were electrophoretically separated on a 1.5% agarose gel and visualized with ethidium bromide to determine successful amplification and adequate signal. Automated sequencing of the PCR fragment using "BigDye" terminators (U.S.) confirmed that the expected sequence of hGSTA1 proximal promoter was amplified from genomic DNA with the primers.
For RFLP analysis, digestions (20 μl) were performed for 4 h at 37°C using 5 units restriction enzyme Ear I and 4 μl unpurified PCR product in 1x buffer as supplied with the restriction enzyme. Aseptic distilled water substitutes the restriction enzyme acted as the negative control. The digested products were separated on 3% agarose gel stained with ethidium bromide for 1 h. The wild type allele (C) had no Ear I site and was still 400 bp. Ninety-two bp nucleotide was removed from the variant allele (T), which yielded a 308 bp fragment.
Enzyme activity assays.
Catalytic activities were determined for 11 preparations of S-9 fraction isolated from samples derived from wile type and variant individuals. Each preparation was derived from tissue from a different individual. The potential functional alterations of hGSTA1 caused by the SNPs were characterized by enzyme activity assays, AD, CuOOH, and CDNB were used as selective substrates toward hGSTA1, hGSTA, and hGST, respectively.
hGSTA1 activity.
Specific hGSTA1 activity measurement with AD was performed spectrophotometrically by some modifications of the assay procedure of Pettersson and Mannervik (2001). The reaction mixture consisted of 0.1 M Tris-HCl (pH 8.0), 1% (v/v) methanol as solvent for the substrate, 1 mg S-9 protein, 0.1 mM AD, and 1 mM GSH. The specific activity of hGSTA1 was determined by the increased absorption at 248 nm, which was produced by the product of the AD isomerization reaction, 4-androstene-3,17-dione (248 = 16.3 mM/cm). A correction for the spontaneous reaction between GSH and AD in the absence of enzyme was made.
hGSTA activity.
hGSTA has high substrate specificity toward CuOOH. Catalytic activity towards CuOOH was measured in S-9 fractions using modifications of a method described previously (Romero et al., 2002). Reaction was performed in 0.1 M potassium phosphate (pH 7.0) in a total volume of 1 ml. S-9 protein (1 mg) was incubated with CuOOH (1.5 mM), GSH (1 mM), NADPH (0.2 mM) and glutathione reductase (0.5 unit), and the time dependent disappearance of NADPH was monitored at 340 nm (340 = 6.2 mM/cm). Blank reaction with enzyme source replaced by distilled water was subtracted from each assay.
hGST activity.
hGST activity was measured with CDNB as described by Habig et al. (1974) with slight modifications. In short, the reaction of 1 mM CDNB with 1 mM GSH in the diluted human liver S-9 protein (1 mg) was monitored spectrophotometrically by recording the increase in absorbance at 340 nm (340 = 9.6 mM/cm). The final concentration of ethanol in the incubation mixture was 1% (v/v). A correction for the spontaneous reaction between GSH and CDNB in the absence of enzyme was made.
Kinetic studies.
On the assumption that the variant might show altered enzyme kinetics, the kinetic parameters of hGSTA1 were determined with different concentrations of AD. S-9 proteins of wild type and variant hGSTA1 were incubated at a 1 mg/ml protein concentration with five different concentrations of AD (5, 10, 20, 50, 100 μM). The kinetic parameters (Km, Vmax, and Vmax/Km) were obtained by fitting of the Michaelis-Menten equation to the data by non-linear regression analysis. The analysis was carried out in 6 different S-9 fractions prepared from six liver samples with known genotypes.
Statistical analysis.
All data were analyzed using the statistics package GraphPad Prism version 4.0. Allelic and genotype frequencies in Chinese and other populations were compared statistically by Pearson 2 test. Statistical significance of observed compared to expected Hardy-Weinberg genotype frequencies was assessed. Comparison of means between the wild type and the variant hGSTA1 was made by Student's t-test. p value of 0.05 was considered statistically significant.
RESULTS
hGSTA1 Genotype Analysis
Genotype and allele frequencies of 140 healthy Chinese were analyzed for the hGSTA1 polymorphism. Three different genotypes (CC, CT, and TT) reported previously (Coles et al., 2001a) were observed after digesting the amplified DNA fragment of 400 bp with the restriction enzyme Ear I. The enzymatic restriction products of the hGSTA1 gene were of the expected size (Fig. 1). Three different banding patterns were obtained: the wild type (CC) produced only a band at 400 bp, homozygous mutant genotype (TT) is characterized by 308 and 92 bp products, while heterozygous genotype (CT) yielded three bands (400, 308, and 92 bp).
Out of the 140 healthy individuals in Chinese having been genotyped for hGSTA1, 75.0% (105/140) were homozygous hGSTA1A, 24.3% (34/140) were heterozygous hGSTA1 (hGSTA1A/B), whereas 0.7% (1/140) were homozygous hGSTA1B. The frequency distributions of hGSTA1A and hGSTA1B alleles were 87.1% and 12.9%, respectively. There was no significant difference between male and female genotype frequencies (Table 1). Comparisons of the observed distributions of hGSTA1 genotype and those predicted by allele frequencies by 2 analyses showed that the population, in this study, was consistent with the Hardy-Weinberg equilibrium, indicating that the subjects were sufficiently random and representative.
Of the selected 11 liver samples, 7 individuals were wild homozygous hGSTA1A, the others were heterozygous hGSTA1. No subject with homozygous hGSTA1B genotype was found.
Enzymatic Activity Analysis
Three typical alpha class substrates, including AD, CuOOH, and CDNB, were used to determine and compare the catalytic activities of the two enzymes, hGSTA1A/A and hGSTA1A/B, encoded by the wild type hGSTA1 and the variant, respectively. AD isomerization activity of the wild type hGSTA1 was 46.1 ± 12.5 μmol/min/mg. The variant catalyzed AD at a rate of 24.1 ± 13.1 μmol/min/mg, which was about 52.3% of that of the wild type activity (p < 0.05). For CDNB and CuOOH, no statistically significant changes in activity were obtained from the variant hGSTA1. The percentages of the wild type enzymatic activities were 180 and 120% of the variant hGSTA1 with CDNB and CuOOH, respectively (Fig. 2).
Enzyme Kinetics
The kinetic properties of the proteins encoded by the wild type hGSTA1 and the variant were studied further in which AD concentrations were varied at 5 different concentrations. As shown in Figure 3 and Table 2, the Vmax value of the variant hGSTA1 was 48% of that of the wild type despite a Km value of the variant of 62% (p < 0.01). This means that the Vmax/Km in the variant hGSTA1 was 76%. These results indicated a slight reduction in the enzymatic activity of the variant for the isomerization of AD.
DISCUSSION
hGSTs are detoxifying enzymes that catalyze the conjugation reactions between GSH and a variety of electrophilic compounds, including carcinogens and anticancer drugs. It has been documented that genetic differences play an important role in the individual variation in constitutive and/or inducible hGSTs levels, which influence the drug efficacy, and susceptibility to environmental mutagens and carcinogens. The alpha class hGSTs is the predominant class in the human liver, contributing about 65 to 80% of the total liver hGST concentration. hGSTA1 expression in human liver was reported to be about 5-fold higher compared with hGSTA2, whereas other isoforms of the alpha class appear to be expressed at even lower levels (Bredschneider et al., 2002). The core control element for the hGSTA1 gene, including the DNA polymerase II binding site, are located within the proximal promoter gene that is situated upstream of the start of transcription. This region, therefore, is an important region to study in the search for human variation affecting gene expression (Coleman et al., 2002). Coles et al. (2001a) screened the whole proximal promoter sequence of hGSTA1 and found two alleles, hGSTA1A and hGSTA1B, containing four linked base substitutions at position –631, –567, –69, and –52. The polymorphism of hGSTA1 has been found to be widespread in Caucasians, African-Americans, Hispanics, and Japanese. In our study, we sequenced the PCR products and confirmed that the linkage disequilibrium between the –69 base pair and the other three variant sites noted in Coles's study populations also exists in our population. The PCR-RFLP assay for the base change at –69 provides a method that can be used to explore the effects of hGSTA1 polymorphism.
Genetic polymorphisms of hGSTs class differ significantly among racial groups and residential populations in different parts of the world (Nazar-Stewart et al., 2003). The results of our study, when compared to those of other studies, illustrate the marked variation in genotype and allele frequencies of the hGSTA1 gene among various world populations. A striking difference in the distribution of the variant genotype and allele has been observed among the Chinese population and Caucasian, African-American, and Hispanic populations, these latter groups sharing allele frequencies as high as 39%, as determined by Coles et al. (2001a). Chinese population demonstrated a similar allele frequency to the description in a Japanese population (Matsuno et al., 2004). These data implicated that Asian populations (Chinese and Japanese) have lower mutant allele frequency (13 and 16%) of hGSTA1 gene as compared to the Caucasian (26%), African-American (38%), and Spanish (39%).
To examine functional alterations due to the SNPs in hGSTA1 gene, the activities of wild type (hGSTA1A/A) and variant (hGSTA1A/B) were examined with three typical alpha class substrates: AD, CuOOH, and CDNB. The variant hGSTA1 showed a significant decrease in catalytic ability toward AD as compared to the wild type, however, the activities toward CDNB and CuOOH were found to be similar. It should be mentioned that large individual difference existed in the hGST and hGSTA enzyme activities of the 11 liver samples studied, which are 12- and 1.5-fold variability, respectively.
Variation in hGSTs activity between individuals has been proposed to underlie susceptibility to a number of illnesses and contribute to population variance in the responses to therapeutic drugs (Strange et al., 2001). The source of this variation in enzyme activity may be due to a number of environmental factors, but may also be influenced by genetic variation. Given this, several studies have previously sought to identify functional polymorphism that could be responsible for variation in enzyme activity. For example, polymorphisms in the hGSTM1, hGSTM3, hGSTT1, hGSTP1 genes are related to differing susceptibilities in the incidence of lung and breast cancers (Strange et al., 2001). However, information about enzymatic function alterations of the variant hGSTA1 is poorly documented. Coles et al. (2001a) found two haplotypes of the hGSTA1 promoter with four polymorphic variants, the haplotype that they designed hGSTA1A had approximately 4-fold higher activity in their assay. Consistently, we have confirmed that polymorphism of the hGSTA1 gene significantly attenuates the specific hGSTA1 activity accompanied by significant interindividual difference. Furthermore, on the assumption that the variant might show altered enzyme kinetics, the kinetic parameters of hGSTA1 were determined with different concentrations of AD and a significantly lower Km value of variant hGSTA1 was observed. Although the Vmax value between the wild type and the variant was not statistically significant possibly due to the small number of liver samples, the extent of decreased Vmax value was larger than those of the wild type. This resulted in a reduction in the catalytic activity of the variant hGSTA1.
Genetic polymorphisms are known to occur in the population and some of these can be directly related to changes in the protein abundance and other phenotypes. For example, a null mutation of the hGSTM1 gene has been associated with a variety of carcinomas (Strange et al., 2000; Wu et al., 2002). However, the phenotypic relevance of changes in enzyme activity is a controversial subject and in vitro effects such as those we reported may have no in vivo counterpart due to a variety of other factors that influence enzyme activity. Bredschneider et al. (2002) have studied the effects of several hGSTA1 promoter polymorphisms and found no correlation between them and either hGST protein abundance or enzymatic activity. However, that study also highlighted the large variations, up to 8-fold, in enzyme activity and abundance between individuals, which would make it very difficult to detect small variation in a small group of samples. These large variations in hGST activities may be the result of environmental effects as well as both cis and trans acting genetic polymorphism that have not yet been determined. However, the presence of additional phenotypic influences does not negate the potential clinical significance of the functional polymorphisms that we and others have described.
In conclusion, our study clearly indicated that the distributions of the hGSTA1 gene are polymorphic in the Chinese population and different from those in other racial populations. This is the first report on hGSTA1 allelic variants and genotypes in the Chinese population. We also demonstrated that the variant hGSTA1 is associated with attenuated specific hGSTA1 activity accompanied by large individual variation. It is reasonable to speculate that the variant of hGSTA1 might be associated with a deleterious biological consequence in human susceptibility to some diseases and in the clinical use of anticancer drugs via its effect on hGSTA1 activity.
NOTES
1 The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
This study is supported by a grant from the Pre-research Foundation of Wuhan University, China (No. 301270050) and Scientific Research Initiation Foundation for Abroad Returning by the Ministry of Education, China (No. 2004).
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