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The glutathione transferases (GSTs) mediate the detoxification of a broad spectrum of electrophilic chemicals. We report here the identification and characterisation of a novel naturally occurring transition that changes codon 169 from GGC (Gly) to GAC (Asp) in the human Pi class GST, GSTP1. Expression of the variant in human HepG2 cells led to a small increase in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation compared to the wild-type protein. Asp(169) GSTP1-1 expressed at high levels in Escherichia coli displayed a small but significant increase in specific activity towards CDNB compared to Gly(169) GSTP1-1. The catalytic efficiency with CDNB was higher for Asp(169) GSTP1-1 compared to the wild-type enzyme, although the kinetic constants of the mutant and the wild-type enzyme towards glutathione were not different. Modelling indicated that the mutation does not appear to change protein conformation. The distribution of the genotypes in a normal healthy population (217 individuals) was 94.3% for the Gly/Gly genotype and 5.7% for the Gly/Asp genotype; no Asp/Asp genotypes were detected in this population. The frequency of the Asp(169) allele in the only oxidative stress-linked pathology that we have studied to date, i.e. alcoholic liver disease, was not significantly different from healthy controls. In conclusion, we have detected and characterised a novel SNP in GSTP1 that may play a role in modulating the activity of GSTP1-1.

GSTs are a family of inducible phase II enzymes that may play a neuroprotective role in Parkinson's disease (PD). GSTs may also modify PD risk by metabolizing compounds in cigarettes, as cigarette smoking is generally found to be associated with a decrease in PD risk. Using a population-based case-control study design, we examined polymorphisms of the mu, omega, pi, and theta classes of GST to elucidate the main effects and smoking-GST interactions on PD risk. From three rural California counties, we recruited 289 incident idiopathic PD cases, clinically confirmed by our study neurologist, and 270 population controls, marginally matched by age, gender, and race. We assessed main gene polymorphism associations and evaluated interactions between smoking and GST polymorphisms as departures from a multiplicative scale adjusting for age, gender, and race. We also restricted analyses to Caucasian subjects to address the potential for population stratification (n=235 cases, 220 controls). Among Caucasians, we observed a risk reduction in subjects carrying at least one variant allele for GSTO1 (OR=0.68, 95% CI: 0.47-0.98) and also GSTO2 (OR=0.64, 95% CI: 0.44-0.93); both genes were in strong linkage disequilibrium. No main gene effects were observed for the remaining polymorphisms. We noted a multiplicative interaction between ever having smoked regularly and GSTO1 (OR(interaction)=0.55, 95% CI: 0.33-0.92) and GSTO2 (OR(interaction)=0.54, 95% CI: 0.32-0.90). Results were similar when combining all races. These findings and the paucity of similar studies suggest a need for further inquiry into the association between GSTs, smoking, and PD risk.

Objectives: The zeta-class glutathione transferase GSTZ1-1 catalyses the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine catabolic pathway and the biotransformation of alpha-halo acids, including dichloroacetic acid (DCA). Genetic polymorphisms in the coding sequence of GSTZ1 result in significant changes in enzyme function, and deficiency of GSTZ1-1 in mice causes induction of a range of Phase-II enzymes. In this study, the potential for polymorphisms in regulatory sequences to alter gene transcription was investigated., Methods: A total of 10 single-nucleotide polymorphisms (SNP) were identified in African and Australian European subjects in a region extending 1.5-kb upstream of the GSTZ1 start of transcription. These SNPs formed at least 10 haplotypes and only two were shared between the two population samples. The effect of these SNPs on gene expression was evaluated by the transient expression of specific alleles fused to a luciferase reporter gene., Results: Of the 10 SNPs identified, only -1002 G>A and -289 C>T caused significant changes in promoter activity. The -1002 G>A SNP converts a v-Myb site to a S8 homeodomain (Prx2) site, and the -289 C>T SNP abolishes an Egr1 binding site., Conclusion: These SNPs may alter GSTZ1 expression, which may alter the pharmacokinetics of DCA, which is used therapeutically for the treatment of lactic acidosis.

Objectives and Methods: A combined database mining approach was used to detect polymorphisms in the mu class glutathione-S-transferase (GST) genes. Although a large number of potential polymorphisms were detected in the five genes that comprise the Mu class GSTs using sequence alignment programs and by searching single nucleotide polymorphism databases, the majority were not validated or detected in three major ethnic populations (African, Southern Chinese and Australian European)., Results: Two new polymorphisms were detected and characterized in the GSTM3 gene. A rare pG147W substitution was detected only in the Southern Chinese subjects. A more common pV224I substitution was found in each of the ethnic groups studied, and significant differences in allele frequencies were observed between each group. These two polymorphisms can combine to form four distinct haplotypes (GSTM3A [p.G147;V224], GSTM3C [p.G147;I224], GSTM3D [p.W147;V224], GSTM3E [p.W147;I224]). The four isoforms were expressed in Escherichia coli and characterized enzymatically with several substrates including 1-chloro-2,4-dinitrobenzene (CDNB), cumene hydroperoxide and t-nonenal. GSTM3-3 containing the variant p.W147 residue tended to show diminished specific activity and catalytic efficiency with CDNB. In contrast, GSTM3-3 containing the variant p.I224 residue tended to show increased specific activity and catalytic efficiency with CDNB. Interactions between the different p.147 and p.224 residues were also observed, with the GSTM3C isoform exhibiting the greatest activity with each substrate, and GSTM3E the lowest., Conclusion: These functional polymorphisms may play a significant role in modulating the ability of GSTM3-3 to metabolize substrates such as the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea.

Deficiencies of the glutathione transferase isoenzymes GSTM1-1 and GSTT1-1 have been shown to be risk modifiers in a number of different cancers but there have been no similar studies with GSTP1-1, the only member of the Pi class of glutathione S-transferases expressed in humans. Over-expression of GSTP1-1 in tumours suggests that it may be a significant factor in acquired resistance to certain anticancer drugs. We previously identified a cDNA clone with two amino acid substitutions (I105V, A114V). This clone suggests that the GSTP1 gene is polymorphic and it is possible that the different genotypes may be associated with altered cancer risk or drug resistance. In the present study, we report methods for genotyping individuals at codons 105 and 114 of GSTP1 and demonstrate that these two loci are polymorphic in several different racial groups. We also detected significant linkage disequilibrium between these two loci. To determine if either of the alleles at these two loci were associated with altered cancer susceptibility, we genotyped individuals with colorectal cancer or lung cancer. A total of 131 colorectal and 184 lung cancer patients were compared with 199 control individuals. Overall, there were no significant associations between the GSTP1 polymorphisms and either form of cancer.

A general algorithm is presented for identifying sets of positions in multiple sequence alignments that best characterize an a priori partitioning such as those determined by inhibition studies or other experimental techniques. The algorithm explores combinations of polymorphic columns in the alignment and evaluates how well these sites reflect the original input partition. Partitions across the polymorphic columns are derived using a tree building procedure with conventional amino acid substitution matrices. Elucidation of those amino acids which govern the biochemical behaviour of a protein with a given substrate or inhibitor can provide insights towards an understanding of the tertiary conformation of the protein. Since it is likely that such positions will be spatially clustered in the protein fold, these positions may give rise to useful distance constraints for substantiating model protein structures. The method is exemplified using data for a set of human mu class glutathione S-transferases. A novel aspect for predicting the behaviour of new polymorphic sequences is also discussed.

The products of three human glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) (GST) loci (GST 1, GST 2 and GST 3) were purified and their immunohistochemical localization in liver was studied with special attention to the polymorphism of GST1 (neutral isozyme). The GST1 was homogeneously stained in cytoplasm of hepatocytes throughout the lobule of liver showing GST1 1, GST1 2 and GST1 2-1 phenotypes. However, none of the hepatic tissue showing GST1 0 phenotype was stained. Immunohistochemical staining of GST2 (basic isozyme) was distributed in the cytoplasm of hepatocytes homogeneously throughout the hepatic lobule in all cases and the strong staining intensity was also demonstrated in nucleus. GST3 (acidic isozyme) was strongly stained in biliary epithelium, while staining of hepatocytes was not apparent. These results indicate that the human liver GST isozymes exhibit significant difference in their inter-individual, specific cellular and organellar distribution.