Brian Coles

Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2 expression

The patterns of expression of glutathione S-transferases A1 and A2 in human liver (hGSTA1 and hGSTA2, respectively) are highly variable, notably in the ratio of hGSTA1/hGSTA2. We investigated if this variation had a genetic basis by sequencing the proximal promoters (-721 to -1 nucleotides) of hGSTA1 and hGSTA2, using 55 samples of human liver that exemplified the variability of hGSTA1 and hGSTA2 expression. Variants were found in the hGSTA1 gene: -631T or G, -567T, -69C, -52G, designated as hGSTA1*A; and -631G, -567G, -69T, -52A, designated as hGSTA1*B. Genotyping for the substitution -69C > T by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), showed that the polymorphism was widespread in Caucasians, African-Americans and Hispanics, and that it appeared to conform to allelic variation. Constructs consisting of the proximal promoters of hGSTA1*A, hGSTA1*B or hGSTA2, with luciferase as a reporter gene, showed differential expression when transfected into HepG2 cells: hGSTA1*A approximately hGSTA2 > hGSTA1*B. Similarly, mean levels of hGSTA1 protein expression in liver cytosols decreased significantly according to genotype: hGSTA1*A > hGSTA1-heterozygous > hGSTA1*B. Conversely, mean hGSTA2 expression increased according to the same order of hGSTA1 genotype. Consequently, the ratio of GSTA1/GSTA2 was highly hGSTA1 allele-specific. Because the polymorphism in hGSTA1 correlates with hGSTA1 and hGSTA2 expression in liver, and hGSTA1-1 and hGSTA2-2 exhibit differential catalysis of the detoxification of carcinogen metabolites and chemotherapeutics, the polymorphism is expected to be of significance for individual risk of cancer or individual response to chemotherapeutic agents.

Kinetics and equilibria of S-nitrosothiol-thiol exchange between glutathione, cysteine, penicillamines and serum albumin.

The kinetics and equilibria of S‐nitrosothiol‐thiol (SNO—SH) exchange reactions were determined using differential optical absorption. At pH 7.4 and 37°C, k 2 values ranged from 0.9 M−1 · s−1 for the reaction between S‐nitroso‐glutathione (GSNO) and N‐acetyl‐penicillamine, and up to 279 M−1 · s−1 for the exchange between S‐nitroso‐penicillamine (penSNO) and GSH. SNO—SH exchange involving GSH/GSNO and cysteine/cySNO was relatively rapid, k 2 approx. 80 M−1 · s−1 with an equilibrium constant slightly in favour of GSNO. GSNO was strongly favoured in equilibrium with penSNO, k eq 0.0039. In the case of SNO—SH exchange between S‐nitroso human serum albumin (albSNO) and GSH or cysteine k 2 values were 3.2 and 9.1 M−1 · s−1, respectively. The results show that the initial rate of SNO—SH exchange between physiological albSNO (7 μM) and venous plasma levels of GSH and cysteine is very slow, < 1%/min. On the other hand, if a nitrosothiol such as cySNO were to enter a cell, it would be rapidly converted to GSNO (43%/s).

Human Alpha Class Glutathione S‐Transferases: Genetic Polymorphism, Expression, and Susceptibility to Disease

The human alpha class glutathione S-transferases (GSTs) consist of 5 genes, hGSTA1-hGSTA5, and 7 pseudogenes on chromosome 6p12.1-6p12.2. hGSTA1-hGSTA4 have been well characterized as proteins, but hGSTA5 has not been detected as a gene product. hGSTA1-1 (and to a lesser extent hGSTA2-2) catalyzes the GSH-dependent detoxification of carcinogenic metabolites of environmental pollutants and tobacco smoke (e.g., polycyclic aromatic hydrocarbon diolepoxides) and several alkylating chemotherapeutic agents and has peroxidase activity toward fatty acid hydroperoxides (FA-OOH) and phosphatidyl FA-OOH. hGSTA3-3 has high activity for the GSH-dependent Delta(5)-Delta(4) isomerization of steroids, and hGSTA4-4 has high activity for the GSH conjugation of 4-hydroxynonenal. hGSTA4 is expressed in many tissues; hGSTA1-1 and hGSTA2-2 are expressed at high levels in liver, intestine, kidney, adrenal gland, and testis; and hGSTA3 is expressed in steroidogenic tissues. Functional, allelic, single nucleotide polymorphisms occur in an SP1-binding element of hGSTA1 and in the coding regions of hGSTA2 and hGSTA3. The main effects of these polymorphisms are the low hepatic expression of hGSTA1 in individuals homozygous for hGSTA1*B and the low specific activity of the hGSTA2E-2E variant toward FA-OOH. These properties suggest that alpha class GSTs will be involved in susceptibility to diseases with an environmental component (such as cancer, asthma, and cardiovascular disease) and in response to chemotherapy. Although hGSTM1, hGSTT1, and hGSTP1 have been associated with such diseases (on the basis of genetic polymorphisms as indicators of expression), alpha class GSTs have been little studied in this respect. Nevertheless, hGSTA1*B has been associated with increased susceptibility to colorectal cancer and with increased efficacy of chemotherapy for breast cancer. Methods for identification and quantitation of human alpha class GST protein, mRNA, and genotype are reviewed, and the potential for GST-alpha in plasma to be used as a marker for hepatic expression and induction is discussed.

Glutathione transferases in primary rat hepatomas: the isolation of a form with GSH peroxidase activity

A previously uncharacterized glutathione (GSH) transferase which is not apparent in normal liver, accounts for at least 25% of the soluble GSH transferase content of primary hepatomas induced by feeding N,N‐dimethyl‐4‐aminoazobenzene. This enzyme is readily isolated, has an isoelectric point of 6.8, is composed of two identical subunits of apparent M r 26 000 and has GSH transferase activity towards a number of substrates including benzo(a)pyrene‐7,8‐diol‐9,10‐oxide. It is unusual in that it has GSH peroxidase activity towards fatty acid hydroperoxides but not towards the model substrates, cumene hydroperoxide and t‐butyl hydroperoxide. It has been shown by tryptic peptide analysis to be distinct from GSH transferases composed of subunits 1, 2, 3,4 or 6 and has been designated GSH transferase 7‐7.

Human glutathione S-transferase A2 polymorphisms: variant expression, distribution in prostate cancer cases/controls and a novel form

Variability of expression of the major glutathione S-transferases (GSTs) of liver, GSTA1 and GSTA2, is thought to affect the efficiency of detoxification of xenobiotics, including chemical carcinogens. Polymorphism of the GSTA1 regulatory sequence determines some of the variation of hepatic GSTA1 expression, but the polymorphisms in GSTA2 (exons 5 and 7) were not thought to affect GSTA2 activity. By examining GST protein expression for a set of human liver and pancreas samples (coupled with a cloning/polymerase chain reaction-restriction fragment length polymorphism strategy), we identified a novel substitution Pro110Ser (328C>T) and the corresponding novel variant GSTA2*E (Ser110Ser112Lys196Glu210), and confirmed the presence of variants GSTA2*A (Pro110Ser112Lys196Glu210), GSTA2*B (Pro110Ser112Lys196Ala210) and GSTA2*C (Pro110Thr112Lys196Glu210). GSTA2*C occurred at 30-60% (i.e. approximately 100-fold more frequent than previously reported) and GSTA2*E occurred (heterozygous) at approximately 11%. Hepatic expression of the Ser112 variants (GSTA2*A, GSTA2*B or GSTA2*E) was approximately four-fold higher than that of the Thr112 variant (GSTA2*C). Compared to any other variant, GSTA2E had lower rates of catalysis towards 1-chloro-2,4-dinitrobenzene (CDNB), 4-vinylpyridine, and cumene-, t-butyl- and arachidonic acid hydroperoxides, although kcat/Km for CDNB were similar for all four variants. Using a prostate cancer case-control population, it was found that GSTA1*A/GSTA2 C335 and GSTA1*B/GSTA2 G335 were in linkage disequilibrium in Caucasians but not in African-Americans. However, there were no significant differences in the distribution of these polymorphisms or resultant haplotypes by case status. Nevertheless, the rare genotypes, GSTA2*E/*E and GSTA1*B/*B + GSTA2*C/*C (potential low GSTA2 activity and low hepatic GSTA1 and GSTA2 expression, respectively) could increase the risk of adverse effects of xenobiotics via compromised efficiency of detoxification.

Thymine hydroperoxide, a substrate for rat Se-dependent glutathione peroxidase and glutathione transferase isoenzymes

The thymine hydroperoxide, 5‐hydroperoxymethyluracil, is a substrate for Se‐dependent glutathione (GSH) peroxidase and the Se‐independent GSH peroxidase activity associated with the GSH transferase fraction. These enzymes may contribute to repair mechanisms for damage caused by oxygen radicals. GSH transferases 1‐1, 2‐2, 3‐3, 4‐4, 6‐6 and 7‐7 [(1984) Biochem. Pharmacol. 33, 2539–2540] are shown to differ considerably in their ability to utilize this substrate. For example, high activity is found in GSH transferase 6‐6 which is the major isoenzyme in spermatogenic tubules where DNA synthesis is so active and faithful DNA replication so important. The activity of the purified GSH transferase isoenzymes towards 5‐hydroperoxymethyluracil is comparable with their activity towards other endogenous substrates related to cellular peroxidation such as linoleate hydroperoxide and 4‐hydroxynon‐2‐enal or biologically important xenobiotic metabolites such as benzo(a)pyrene‐7,8‐diol‐9,10‐oxide.

Glutathione Transferases and Carcinogenesis

Carcinogenesis has been regarded as being divisible into at least 3 major phases: (a) initiation, a largely irreversible process which has been compared to mutation; (b) promotion, a process during which cells undergo phenotypic changes, some of which are referred to as preneoplastic, and a proportion of which attain autonomy when they can be regarded as neoplastic; and (c) progression, a period of continuing phenotypic diversification during which a selection is made of those cells that possess autonomy and advantages over the host tissue. The result is increasing malignancy and, without intervention, the death of the host (22). The selective forces involved in carcinogenesis are reminiscent of those proposed for evolution, but in the case of cancer it is evolution within a lifetime and the result is biological chaos.

The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris

1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.

Identification of 4′-sulphonyloxy-N-(glutathion-S-methylene)-4-aminoazobenzene, a compound conjugated with both sulphate and glutathione, which is a major biliary metabolite of N,N-dimethyl-4-aminoazobenzene

A major biliary metabolite in the rat of the hepatocarcinogen N,N-dimethyl-4-aminoazobenzene (DAB) has been identified as 4′-sulphonyloxy-N-(glutathion-S-methylene)-4-aminoazobenzene. This conjugate can be synthesized by the condensation of 4′-sulphonyloxy-4-aminoazobenzene with formaldehyde and glutathione (GSH).

Activation of Toxic Chemicals by Cytochrome P450 Enzymes

Cytochrome P450 (P450) enzymes are involved in the oxidations of numerous steroids, eicosanoids, alkaloids, and other endogenous substrates. These enzymes are also the major ones involved in the oxidation of potential toxicants and carcinogens such as those encountered among pollutants, solvents, and pesticides, as well as many natural products. A proper understanding of the basic mechanisms by which the P450 enzymes oxidize such compounds is important in developing rational strategies for the evaluation of the risks of these compounds.