Polymorphisms within the phase II metabolizer enzymes GST T1, GST M1 and GST P1 affect the body's ability to detoxify a range of potential leukaemogens encountered in the environment. Using PCR, GST T1, GST M1 and GST P1 genotypes were determined in 557 adults with acute leukaemia and 952 age, sex and geographically matched controls. The strongest association with acute leukaemia was observed for the GST T1 null genotype, which occurred among 19% of cases and 14% of controls [odds ratio (OR) 1.45, 95% confidence interval (CI) 1.09-1.93]. A slightly higher proportion of cases (53%) than controls (49%) displayed the GST M1 null genotype, although the difference was not statistically significant (OR 1.22, 95% CI 0.98-1.52). No effect was observed for the GST P1 genotype and no interaction between the GST T1 and GST M1 genotypes was evident. Acute myeloid leukaemia (AML) was weakly associated with both GST T1 null (OR 1.32, 95% CI 0.97-1.79) and GST M1 null (OR 1. 24, 95% CI 0.98-1.56), whereas acute lymphoblastic leukaemia (ALL) was associated with GST T1 null (OR 3.28, 95% CI 1.31-8.26). No associations between smoking and disease risk in relation to GST T1 and GST M1 polymorphic status were found.

OBJECTIVE

To test whether genetic deletions of glutathione-S-transferase (GST) are associated with squamous cell carcinoma (SCC) of the larynx. GST are a group of detoxifying enzymes that may help reduce the risk of developing cancer in response to environmental carcinogens. Polycyclic aromatic hydrocarbons, found in high concentration in cigarette smoke, are known carcinogens especially for SCC of the larynx. Individuals with absolute or relative deficiency of the GST enzyme system may therefore be at a higher risk of developing laryngeal carcinoma.

METHODS

Genotyping for GST-M1 and GST-T1 was performed using polymerase chain reaction (PCR) assay on fresh frozen tissue specimens of 20 patients with SCC of the larynx and on 20 control subjects with a similar smoking history. Because this assay results in the absence of a PCR product in individuals expressing the GST-M1/GST-T1 null genotype, oligonucleotide primers that amplify a portion of the albumin gene were included in a multiplex PCR as a positive control for DNA quality and PCR conditions. The chi-square test was used for statistical analysis.

RESULTS

GST-M1 gene was deleted in 80% of patients with laryngeal SCC and in 50% of control subjects (P <.05). No statistically significant difference was observed in the incidence of GST-T1 gene deletion in patients with SCC of the larynx and control subjects.

CONCLUSIONS

GST-M1 gene deletion was significantly associated with SCC of the larynx and may produce a risk for this particular disease.

We investigated glutathione S-transferase (GST) P1 Ile (105) Val, T1, and M1 polymorphisms in 45 patients with documented cryptogenic cirrhosis and 56 healthy control subjects. Polymerase chain reaction-based procedures were performed in the studied populations to confirm the genotypes of GSTT1, M1, and P1. Ile/Val and Val/Val GSTP1 genotypes were more frequent in the patients with cirrhosis (n=39, 87%) than in the control subjects (n=10; 18%) (odds ratio [OR] 34.04; 95% confidence interval [CI] 10.70 to 108.31, P<0.001). Among these patients with cirrhosis, 16 were heterozygous and 23 were homozygous, whereas only one person in the control group was homozygous. The GSTM1 null genotype was also more prevalent in cirrhotic patients than in healthy control subjects (OR 6.83, 95% CI 2.53 to 18.42, P<0.001). The rate of GSTT1 deletion did not show a significant difference between the two groups (OR 2.35, 95% CI 0.76 to 7.28, P=0.111). To our knowledge, this is the first evidence that GSTP1 and GSTM1 polymorphisms may be related to the development of cirrhosis by unknown mechanisms. The significant association of cryptogenic cirrhosis with Val/Val GSTP1 genotype encoding a low detoxification activity protein implicates this polymorphism as a risk factor for the occurrence of the disease.

Campylobacter is the leading cause of foodborne diarrhoeal illness in the developed world and consumption or handling of contaminated poultry meat is the principal source of infection. Strategies to control Campylobacter in broilers prior to slaughter are urgently required and are predicted to limit the incidence of human campylobacteriosis. Towards this aim, a purified recombinant subunit vaccine based on the superoxide dismutase (SodB) protein of C. jejuni M1 was developed and tested in White Leghorn birds. Birds were vaccinated on the day of hatch and 14 days later with SodB fused to glutathione S-transferase (GST) or purified GST alone. Birds were challenged with C. jejuni M1 at 28 days of age and caecal Campylobacter counts determined at weekly intervals. Across three independent trials, the vaccine induced a statistically significant 1 log10 reduction in caecal Campylobacter numbers in vaccinated birds compared to age-matched GST-vaccinated controls. Significant induction of antigen-specific serum IgY was detected in all vaccinated birds, however the magnitude and timing of SodB-specific IgY did not correlate with lower numbers of C. jejuni. Antibodies from SodB-vaccinated chickens detected the protein in the periplasm and not membrane fractions or on the bacterial surface, suggesting that the protection observed may not be strictly antibody-mediated. SodB may be useful as a constituent of vaccines for control of C. jejuni infection in broiler birds, however modest protection was observed late relative to the life of broiler birds and further studies are required to potentiate the magnitude and timing of protection.

Oxidative stress contributes to hepatitis C virus (HCV)-induced liver damage. The activity of antioxidant glutathione S-transferases (GSTs) T1 and M1 is polymorphic. The GSTT1 and GSTM1 genotypes were identified in 139 HCV-infected patients and in 329 healthy individuals. Among patients, there was an excess of GSTT1 (odds ratio [OR], 2.76 [95% confidence interval [CI], 1.77-4.30]; P<.001) and GSTM1 (OR, 1.54 [95% CI, 1.02-2.35]; P=.032) null genotypes and of double-null haplotypes (OR, 3.65 [95% CI, 1.98-6.75]; P<.001). The GSTT1 null genotype, particularly if associated with the GSTM1 null genotype, may facilitate HCV infection becoming chronic.

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase. We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.

OBJECTIVE

Data from several studies suggest that oxidative stress may play a role in the pathophysiology of tardive dyskinesia (TD). Glutathione S-transferase (GST) enzymes play important roles in protecting cells against oxidative stress. In the present study, we investigated the hypothesis that polymorphisms in genes for these detoxifying enzymes can influence susceptibility to TD in patients with schizophrenia.

METHODS

The GST-M1, GST-T1, and GST-P1 loci were analyzed by polymerase chain reaction (PCR)-based methods in 83 schizophrenic patients with TD and 126 schizophrenic without TD who were matched for antipsychotic drug exposure and other relevant variables. The multifactor dimensionality reduction (MDR) approach was used to analyze gene-gene interactions.

RESULTS

There were no significant differences in the distributions of the GST-M1, GST-T1, and GST-P1 genotypes between the TD and non-TD groups (p>> 0.05). However, in comparison of the severity of TD among genotypes using Poisson regression showed that Ile/Ile genotype of GST-P1 had higher AIMS score compared to Ile/Val + Val/Val genotypes (X(2) = 7.13, p = 0.008). MDR analysis did not show a significant interaction between the three GST gene variants and susceptibility to TD (p>> 0.05).

CONCLUSIONS

These results suggest that GST gene polymorphisms do not confer increased susceptibility to TD in patients with schizophrenia but TD severity might be related with GST-P1 variants.

Prostate cancer is the most common urologic malignancy, involving multiple factors. There is evidence that suggests that detoxification enzymes and growth factors may play a role in its development . The glutathione S-transferase (GST) enzymes detoxify several carcinogens and genetic polymorphisms in GSTM1, T1, and P1 (Ile105Val) have been reported to be associated with prostate cancer, mainly from blood samples. As expression studies suggest differential expression of different genes in tissues, we hypothesize that polymorphic status may be differently expressed for GSTM1, GSTT1 and GSTP1 gene in blood and tissues of prostate cancer patients and BPH controls, impacting on the development of prostate cancer. To study this, we extracted DNA from blood and tissue samples of patients undergoing biopsy procedures or transurethral resection of prostate tissue. Genotyping for GSTM1 and T1 was conducted by multiplex PCR and for GSTP1 by the PCR-RFLP method. Our results suggested no significant differences in frequency distribution of M1, T1 and P1 between blood and tissue samples of patients and controls, but in a few patients differences in polymorphic status were observed. However, they were not significant. Furthermore, we observed a significant risk of prostate cancer with null allele of GSTT1 and GSTM1 and Val allele of GSTP1, supporting our previous findings. A study with large sample size using radical prostectomy tissue now needs to be performed to attain a specific conclusion.

A case-control study was carried out to examine the relation between genetic polymorphisms of five genes, cigarette smoking and colorectal cancer risk. We collected blood samples from 106 colorectal cancer patients and 100 healthy persons, then analyzed them to identify genotypes for glutathione S-transferase (GST) M1, T1, P1, N-acetyltransferase (NAT) 1 and 2 by the PCR method. We also collected smoking history data from all participants by questionnaire. From statistical evaluation on various combinations of genotypes, we observed that the cancer risk of those who have both GSTM1 present genotype and GSTP1 Adenine/Adenine homozygous genotype was significantly less than those who have other combinations of genotypes for two genes. For other combinations of genes, there was no significant association between genotype and cancer risk. There was also no significant association between amount of cigarette smoking and the cancer risk. These findings suggest that it is valuable to study cancer risk when examining genotypes of more than two genes at the same time. For further study, we need to collect more samples to increase statistical reliability, and besides cigarette smoking, include the nutrition data as an environmental factor.

OBJECTIVE

Glutathione S-transferase (GST) plays a key role in the detoxification of xenobiotic atherogens generated by smoking. We investigated whether functional GST gene polymorphism might be associated with acute myocardial infarction (AMI) and smoking. No such investigation has previously been conducted among North Indians.

METHODS

230 patients with AMI and 300 healthy controls of North Indian ethnicity were enrolled in the study. GSTM1/T1/P1 gene polymorphisms were examined using restriction fragment length polymorphism.

RESULTS

When GST polymorphism was analyzed in patients with AMI, GSTM1 null genotype frequencies were 0.24 and 0.21 among cases with coronary artery disease and controls, respectively. The respective GSTT1 null genotype frequencies were 0.10 and 0.20 (p = 0.001). GSTP1 variant Val/Val allele frequencies were 0.02 and 0.07 with p = 0.03. After risk factor adjustment, only GSTP1Val/Val was found to be protective against disease. Considering the effect of GST (T1, M1, and P1) gene polymorphism on smoking, subjects were further divided into smokers and nonsmokers. However, GSTT1 null as well as GSTP Val/Val genotypes were protective only in nonsmokers (p = 0.01 and p = 0.04).

CONCLUSIONS

A significant protective effect of GSTT1 null and GSTP1 Val genotype against disease was observed in patients with AMI although protection was limited to nonsmokers.

Many cyanobacterial species are able to produce cyanotoxins as secondary metabolites. Among them, microcystins (MC) are a group of around 80 congeners of toxic cyclic heptapeptides. MC-LR is the most studied MC congener, in view of its high acute hepatotoxicity and tumor promoting activity. Humans may be exposed to cyanotoxins through several routes, the oral one being the most important. The accepted pathway for MC-LR detoxication and excretion in the urine is GSH conjugation. The GSH adduct (GS-MCLR) formation has been shown to occur spontaneously and enzymatically, catalyzed by glutathione transferases (GSTs). The enzymatic reaction has been reported but not characterized both in vitro and in vivo in animal and plant species. No data are available on humans. In the present work, the MC-LR conjugation with GSH catalyzed by five recombinant human GSTs (A1-1, A3-3, M1-1, P1-1, and T1-1) has been characterized for the first time. All GSTs are able to catalyze the reaction; kinetic parameters K(m), k(cat), and their relative specific activities to form GS-MCLR were derived (T1-1>> A1-1>> M1-1>> A3-3 ≫ P1-1). In the range of MC tested concentrations used (0.25-50 μM) GSTT1-1 and A1-1 showed a typical saturation curve with similar affinity for MC-LR (≈80 μM; k(cat) values 0.18 and 0.10 min(-1), respectively), A3-3 and M1-1 were linear, whereas GSTP1-1 showed a temperature-dependent sigmoidal allosteric curve with a k(cat) = 0.11 min(-1). The enzymes mainly expressed in the liver and gastrointestinal tract, GSTA1-1, T1-1, and M1-1, seemed to be mainly involved in the MC-LR detoxification after oral exposure, whereas P1-1 kinetics and location in the skin suggest a role related to dermal exposure. Considering the high frequency of some GST polymorphism, especially M1 and T1 gene deletion, with complete loss in activity, this information could be the first step to identify groups of individual at higher risk associated with MC exposure.

Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide substrates. In the present study, combined quantum mechanical/molecular mechanical (QM/MM) simulations of the conjugation of glutathione to phenanthrene 9,10-oxide, catalyzed by the M1-1 isoenzyme from rat, have been carried out to obtain insight into details of the reaction mechanism and the role of solvent present in the highly solvent accessible active site. Reaction-specific AM1 parameters for sulfur have been developed to obtain an accurate modeling of the reaction, and QM/MM solvent interactions in the model have been calibrated. Free energy profiles for the formation of two diastereomeric products were obtained from molecular dynamics simulations of the enzyme, using umbrella sampling and weighted histogram analysis techniques. The barriers (20 kcal/mol) are in good agreement with the overall experimental rate constant and with the formation of equal amounts of the two diastereomeric products, as experimentally observed. Along the reaction pathway, desolvation of the thiolate sulfur of glutathione is observed, in agreement with solvent isotope experiments, as well as increased solvation of the epoxide oxygen of phenanthrene 9,10-oxide, illustrating an important stabilizing role for active site solvent molecules. Important active site interactions have been identified and analyzed. The catalytic effect of Tyr115 through a direct hydrogen bond with the epoxide oxygen of the substrate, which was proposed on the basis of the crystal structure of the (9S,10S) product complex, is supported by the simulations. The indirect interaction through a mediating water molecule, observed in the crystal structure of the (9R,10R) product complex, cannot be confirmed to play a role in the conjugation step. A selection of mutations is modeled. The Asn8Asp mutation, representing one of the differences between the M1-1 and M2-2 isoenzymes, is identified as a possible factor contributing to the difference in the ratio of product formation by these two isoenzymes. The QM/MM reaction pathway simulations provide new and detailed insight into the reaction mechanism of this important class of detoxifying enzymes and illustrate the potential of QM/MM modeling to complement experimental data on enzyme reaction mechanisms.

This study was designed to investigate the association between null polymorphisms of glutathione S-transferase (GST) M1 and T1 genes and idiopathic male infertility in a Russian population including 203 infertile and 227 fertile men. The nondeletion genotype of the GSTT1 gene was found to be strongly associated with the increased risk of idiopathic male infertility and asthenozoospermia.

OBJECTIVE

Most drugs are developed based on data from European-derived 'reference' populations; however, clinically relevant DNA polymorphisms often demonstrate population-specific patterns of allele frequencies. Given that the knowledge of the frequency distribution of functional polymorphisms in a population may guide national planning for selection of therapeutic options, in the present study we examined the allele frequencies of enzymes responsible for drug disposition in Portugal.

METHODS

Using PCR- and Pyrosequencing-based methods, the current study assessed the frequencies of 15 key polymorphisms from genes encoding enzymes involved in Phases I, II and III of drug metabolism, DNA repair and intracellular metabolism in 135 healthy individuals from Portugal.

RESULTS

Allele frequencies were derived for cytochrome P450 (CYP)2C9*2 (13.2%), CYP2C9*3 (8%), CYP2C19*2 (14%), CYP3A4*1B (7%), CYP3A5*3C (87.5%), glutathione S-transferase (GST)M1*0 (77.9%), GSTP1 313A>G (33%), inosine triphosphatase 94C>A (7%), UDP-glucuronosyltransferase (UGT)1A1*28 (28%), UGT1A1 -3156G>A (23%), ATP-binding cassette (ABC)B1 1236C>T (46%), ABCB1 2677G>A/T (2 and 42%), ABCG2 421C>A (8%), excision repair cross-complementing rodent repair deficiency 2 2251A>C (3%) and thymidylate synthetase 1494del (31%).

CONCLUSIONS

Although, on the whole, the frequency distributions among the Portuguese fitted the patterns commonly found in other Europeans well, evidence for some degree of African influence was observed. This is the most comprehensive study on pharmacogenetically relevant variations in Portugal to date, and the baseline of pharmacogenetic data might be important for determining policy guidelines for cancer prevention and drug treatments in the Portuguese population.

The development of gastric cancer is a multistep process that is multi-factorial. An association with the Helicobacter pylori infections, gastric atrophy and gastric cancer has received recent attention. The objective of this study was to elucidate the risk factors for gastric cancer by using molecular epidemiological techniques for genetic susceptibility, gastric atrophy and serum markers including H pylori infection. We used an age- and gender-matched case-control study, where patients with benign gastric lesions were the controls. Low serum pepsinogen I levels (cut-off < 50 ng/mL) and low pepsinogen I/pepsinogen II ratios (cut-off < 3.0) were significantly associated with the risk of gastric cancer (odds ratio [OR] = 3.53: 95% confidence interval [CI] = 2.46-5.09 and OR = 4.73: 3.26-6.88, respectively). However, seropositivity of serum immunoglobulin G (IgG) antibody against H pylori (OR = 1.09: 0.74-1.61) was not associated with gastric cancer, even when analyzed by age greater than or less then 50 years. Specific genotypes of the cytochrome p450 2E1 (CYP2E1) RsaI polymorphism and glutathione-S-transferase (GST) M1 gene deletion were determined but were not associated with gastric cancer; however, a Lmyc genetic polymorphism was associated with gastric cancer (OR = 1.55: 1.03-2.34). Therefore, in this Japanese study, atrophic mucosal change, indicated by serum pepsinogen levels, is a possible risk factor for gastric cancer.

In preclinical pharmacokinetic studies and in in vitro rat, dog, and human primary hepatocyte incubations, the sulfonamide (-NH-SO(2)-) bond of a potent inhibitor of the HIV-1 protease containing the p-cyanopyridinyl moiety (PNU-109112), undergoes metabolic cleavage to form the corresponding amine metabolite (PNU-143070). Strikingly, a compound, PNU-140690, obtained by substituting the cyanopyridinyl group of PNU-109112 with a trifluoropyridinyl moiety, was stable under the same in vivo and in vitro conditions used for PNU-109112. The apparent "sulfonamidase activity" present in liver was localized to the cytosolic fraction and shown to be an enzyme-mediated reaction requiring reduced glutathione (GSH). The enzyme responsible was purified in a single step on a GSH immobilized gel and was identified as glutathione-S-transferase (GST) by sequence analysis of peptides obtained by tryptic digestion of the purified protein. Moreover, a mixture of GST isoenzymes purified from rat liver, and three recombinant human GST isoforms, A1-1, M1-1, and P1-1, were active toward PNU-109112 sulfonamide cleavage; the three isoforms exhibited differential rates of PNU-109112 cleavage, demonstrating isoenzyme selectivity.

BACKGROUND

This study quantified the impact of drug pathway-associated genetic variants on the pharmacokinetics (PK) of gemcitabine and cisplatin in patients with advanced non-small-cell lung cancer (NSCLC).

METHODS

Thirty-seven patients with advanced NSCLC were sampled for plasma concentrations of gemcitabine, difluoro-deoxy uridine (dFdU), intracellular gemcitabine triphosphates (dFdCTP), and unbound platinum concentrations after gemcitabine 1,250 mg/m(2) i.v. followed by cisplatin 75 mg/m(2). We analyzed 13 germline single nucleotide polymorphisms and one deletion-glutathione S-transferase (GST) M1-within six drug pathway-associated genes (GSTM1, GSTP1, cytidine deaminase (CDA), solute carrier (SLC) 28A1, SLC28A2, and deoxycytidine kinase). PK models were fitted to the data using nonlinear mixed-effects modeling, and genetic data were tested on drug PK and hematological toxicity.

RESULTS

Patients carrying the nonsynonymous CDA SNP 79A>>C (CDA*2) had a 21% lower gemcitabine clearance as compared to wild-type patients (outcomes and complications.0.0009), but the risk for chemotherapy-associated neutropenia (61% vs. 32%, P = 0.07) and severe neutropenia (17% vs. 5%, P = 0.26) was not significantly higher. Other gene polymorphisms were not associated with drug PK parameters or hematological toxicity. The known functional mutant variant CDA*3 was not found in any of the patients.

CONCLUSIONS

Although the mutant CDA*2 allele results in an increased exposure to gemcitabine in Caucasian patients, this study gives no definite conclusion on the clinical relevance of this finding. Further studies should look into the relationship between CDA genotypes, plasmatic CDA activity, and clinical outcome in patients receiving gemcitabine-based chemotherapy.

OBJECTIVE

The interplay between genetic susceptibility and exposure to carcinogens has been shown to be involved in the etiology of many solid tumors. We studied the frequency and clinical correlates of polymorphisms resulting in deletions of two genes involved in the detoxification of potentially carcinogenic agents, glutathione S-transferase (GST)-M1 and GSTT1 in patients with Hodgkin's lymphoma (HL).

METHODS

The prevalence of gene deletions in 90 patients with HL was compared with a case-matched cohort of 176 normal blood donors. GST gene polymorphisms were studied using a multiplex PCR method, including the BCL2 gene as an internal control.

RESULTS

Deletions of the GSTT1 gene were more frequent in cases compared with controls (28.9 versus 17.6%, P = 0.04), resulting in an increased risk for HL in individuals with the GSTT1-null genotype (odds risk, 1.9; 95% confidence interval 1.04-3.46). The GSTT1-null genotype particularly increased the HL risk in females aged <45 years (odds risk 6.1, 95% confidence interval 1.6-23, P = 0.008). Correlating patient characteristics to genotype, we found an association between the GSTT1-null genotype and a limited stage of disease (I/IIA versus IIB-IV, 40.6 versus 19.6%, P = 0.047) and an erythrocyte sedimentation rate of <50 mm/h (P = 0.02). Patients with at least one GST deletion (GSTM1- or GSTT1-) had a significant better disease-free survival when compared with those with undeleted GST genes (GSTM1+/GSTT1+; P = 0.012).

CONCLUSIONS

The GSTT1-null genotype may increase the risk for HL and is associated with favorable prognostic factors, and the presence of at least one GST deletion indicates an improved disease-free survival.

A human small cell lung cancer cell line, U-1906, developed altered functional properties upon continuous in vitro cultivation. Cells obtained at late (U-1906 L) and early (U-1906 E) passages of cultivation differ in drug resistance to the cytostatic therapeutic agents cisplatin and doxorubicin. The U-1906 L cells are 1.6-fold and 1.3-fold more resistant to cisplatin and doxorubicin respectively, than are the U-1906 E cells. In the more resistant U-1906 L cells, the total glutathione (GSH plus GSSG) level is 40% lower, whereas the activities of GSH-linked enzymes such as GSH peroxidase and GSH transferases are significantly higher. Quantitative analysis with isoenzyme-specific ELISAs demonstrated increased concentrations of all three of the measurable GSTs, M1-1, M3-3 and P1-1, in the more resistant cells. The intracellular protein expression patterns of the U-1906 E and the U-1906 L cells are very similar as revealed by two-dimensional denaturing electrophoresis, but show significant alterations in the concentrations of some components. Two 35 kDa proteins of different pI values, the concentrations of which are increased in the U-1906 L cells, were both identified as glyceraldehyde-3-phosphate dehydrogenase, either by N-terminal or by internal amino acid sequence analysis. The present study demonstrates that the increased resistance of the U-1906 L cells may involve multiple detoxification mechanisms and that the contribution of the GSH-linked detoxification can be ascribed to the elevation of cytosolic GST isoenzymes, GSH peroxidase and glutathione reductase, rather than to the intracellular GSH concentrations.

The human glutathione S-transferase (GST) P1 alleles coding for Val(105) (hGSTP1*B and/or P1*C) are over- represented in lung cancer patients. However, the corresponding recombinant Val(105) protein variants tend to show higher catalytic activity than the Ile(105) variants towards bay-region diol epoxides that are thought to be etiological agents in lung cancer. We have examined 29 normal human lung samples with respect to several factors that could confound relationships between hGSTP1 allele type and cancer susceptibility, namely, inter-individual and allele-specific variation of hGSTP1 expression, and differences between the catalytic properties of the native and recombinant hGSTP1-1 variant protein products. hGSTP1 expression varied 7-fold among individuals but was independent of hGSTP1*A, P1*B or P1*C allele type. hGST subunits A1, A2, M1 and M3 were minor components, similarly variable in expression. Despite this variability of expression, the levels of hGSTP1 expression linearly correlated with those of the next most highly expressed GST, hGSTM3, even though the genes for these GSTs are on different chromosomes. Differences between the native protein variants, using 1-chloro-2,4-dinitrobenzene and (+)-anti-benzo[a]pyrene diolepoxide as substrates, were more marked than those between the recombinant variants. However, the order of differential catalytic specificity was the same for native and recombinant variants. Neither the expression of the hGSTP1 alleles nor the catalytic properties of the protein variants appears to provide a simple mechanistic rationale for the observed over-representation of the hGSTP1*B and/or 1*C alleles in lung cancer.

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are bioactivated by the cytochrome P450 (P450) 1A family of enzymes to metabolites that are capable of covalently binding to DNA, a critical step in the initiation of carcinogenesis. We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P4501A expression for up to 45 days. Here, we tested the hypothesis that MC elicits persistent induction of other genes that are regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 micromol/kg) ip once daily for 4 days, and gene expression patterns were investigated using total liver RNA isolated from animals at 1, 15, and 28 days after MC withdrawal. Gene expression was studied by cDNA microarray analyses using 4608 unique clones from liver-derived expressed sequence tag (EST) libraries fortified with clones of known liver genes representing approximately 4000 genes. Several phase I (P4501A1, -1A2) and phase II [e.g., glutathione-S-transferase (GST)-M1, UDP-glucuronosyl transferases (UGT)] genes were persistently induced (3-10-fold) by MC for 15-28 days. The persistent induction of P4501A1 gene expression was confirmed by real time reverse transcriptase polymerase chain reaction (RT-PCR) experiments. MC also elicited a 5-fold persistent augmentation of acute phase genes such as orosomucoid 1 and alpha-1-acid glycoprotein (AGP), and this was accompanied by sustained liver damage and inflammation in the MC-exposed rats. In conclusion, our results strongly suggest that sustained induction of P4501A1 by MC is accompanied by persistent expression of other genes belonging to the Ah gene battery, as well as certain other genes involved in toxic responses. Elucidating the mechanisms of persistent induction of P4501A1 and other genes by MC might lead to a better understanding of the mechanisms of toxicity mediated by PAHs.

The major human Mu-class glutathione S-transferases (GST) have been purified to allow comparisons of their catalytic, physicochemical and immunochemical properties. GST isoenzymes, purified from hepatic, testicular and skeletal muscle tissue were found to comprise three distinct subunits (M1, M2 and M3) which may combine to form both homodimeric and heterodimeric proteins. Two distinct subunits, M1a and M1b, which represent allelic charge variants have been isolated but no polymorphic forms encoded at the GST M2 and M3 loci have been observed. Three GST isoenzymes (M1a-1a, M1a-1b and M1b-1b) have been purified from a single liver specimen. In addition, GST M1a-2, M1b-2, M2-2 and M2-3 have been isolated from muscle, whilst the M3-3 homodimer has been purified from human testis. The homodimeric enzymes GST M1a-1a, M1b-1b, M2-2 and M3-3 have pI values of 6.1, 5.5, 5.3 and 5.0, whilst SDS-PAGE indicated that M1a, M1b, M2 and M3 have molecular masses of 26.7, 26.6, 26.0 and 26.3 kDa, respectively. The M1, M2 and M3 subunits isolated from either liver, skeletal muscle or testis, are catalytically distinct. Both M1-type subunits (M1a and M1b) possess a high activity for trans-4-phenyl-3-buten-2-one, whereas, the skeletal muscle subunit M2 has a high activity towards 1,2-dichloro-4-nitrobenzene. By contrast, the testicular GST subunit M3 has no detectable activity towards either of these substrates. However, all three Mu-class subunits are active towards the compounds 4-hydroxynonenal and 4-hydroxydecinal, possible endogenous substrates which are produced by lipid peroxidation. The human Mu-class subunits can be distinguished immunochemically; antisera raised against the testicular GST M3-3 showed no reactivity towards either the M1 or M2 subunits. The M3 subunit has a blocked N-terminus but automated amino-acid sequencing of a CNBr-derived peptide allowed 14 residues of the M3 subunit to be identified. These data indicated that testicular GST M3-3 is likely to correspond to the brain/testis Mu-class GST cDNA described by Campbell et al. (Campbell E., Takahashi Y., Abramovitz M., Peretz M., & Listowsky I. (1990) J. Biol. Chem. 265, 9188-9193).

Direct quantitation of glutathione S-transferase (GST) isoforms [alpha (GST-A) and micro (GST-M)] in human liver cytosol was achieved by liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) analysis of signature peptides of GST-A and GST-M and their corresponding stable isotopic peptide internal standards via multiple reaction monitoring (MRM). The selection of signature peptides was performed via trypsin digestion of commercially available cDNA-expressed GST-A1 and GST-M1, followed by LC/ESI-MS/MS with an ion trap mass spectrometer and sequencing with the TurboSEQUEST application. Quantitative analysis of the selected signature peptides in the multi-reaction monitoring (MRM) mode was performed using a triple-quadruple mass spectrometer. A series of human cytosol samples was quantitatively analyzed for levels of GST-A and GST-M. The total level of GST-A and GST-M obtained from this LC/ESI-MS/MS method was well correlated with the total level of GST determined by the 1-chloro-2,4-dinitrobenzene (CDNB) method.

We investigated the polymorphic enzymes cytochrome P450 1A2 (CYP1A2), N-acetyltransferase (NAT2), glutathione S-transferase (GST) M1 (GSTM1), and T1 (GSTT1) in relation to cigarette smoking-associated urinary mutagenicity detected on YG1024 Salmonella typhimurium strain with S9 mix in 97 smokers. In each subject, cigarette smoke intake was checked by analysis of urinary nicotine plus its metabolites. NAT2 and CYP1A2 phenotypes were determined by the molar ratio of urinary caffeine metabolites detected by high-performance liquid chromatography, and GSTT1 and GSTM1 genotypes were determined by PCR. An increase in urinary mutagenicity was significantly related to levels of exposure to cigarette smoke and CYP1A2 N-hydroxylation activity (linear multiple regression analysis t = 4.51 and P < 0.001 and t = 3.09 and P = 0.003; F = 6.31, P < 0.001). Urinary mutagenicity was significantly higher in CYP1A2 extensive metabolizer smokers (n = 49) than in CYP1A2 poor metabolizer ones (n = 48; 2176 +/- 1525 versus 1384 +/- 1206 revertants/mmol creatinine, Mann-Whitney U-test, z = 2.65, P < 0.001). The highest mutagenic activity was seen in subjects CYP1A2 extensive metabolizer/NAT2 slow acetylators (n = 29) with respect to the other phenotype combinations (n = 68; 2392 +/- 1660 versus 1525 +/- 1238 revertants/mmol creatinine, Mann-Whitney U-test, z = 2.37, P = 0.017). NAT2 acetylation activity was slightly but inversely related to urinary mutagenicity, and the association was not significant. No effect of GSTM1 and GSTT1 genotypes in lowering (detoxifying) urinary mutagens was found. The significant enhancement of urinary mutagenicity associated with increased CYP1A2 activity, as already seen for diet-caused urinary mutagenicity, allows for many analogies between the process of mutagen formation derived from cooked meat and that from cigarette smoke condensate. In conclusion, the intensity of tobacco smoke exposure, modulated by CYP1A2 activity, is the major determinant of mutagenic urine among smokers, whereas GSTM1 and GSTT1 genotypes have no influence on this biomarker. This study suggests that CYP1A2 should definitely be determined in future studies involving urinary mutagenicity in cases in which smoking is a factor.