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PubMed2000

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage.

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.

Amino Acid Sequence↗
PubMed1996

Rabbit aorta glutathione S-transferases and their role in bioactivation of trinitroglycerin.

The pharmacological action of glyceryl trinitrate (GTN), a widely used drug for the treatment of angina pectoris, is thought to be mediated through release of nitric oxide (NO) during its biotransformation. Since glutathione S-transferases (GST) can utilize GTN as substrate and GST inhibitors can attenuate GTN-induced relaxation of rabbit aorta in vitro it has been suggested that these enzymes are involved in the bioactivation of GTN in rabbit aorta. Because GSTs are multifunctional enzymes and a multitude of GST isozymes with varying substrate preferences are present in mammalian tissues, the role of specific GST isozymes in bioactivation of GTN in rabbit aorta needs to be established. Therefore, during the present studies we have purified and characterized GST isozymes from rabbit aorta and evaluated their possible roles in the biotransformation of GTN. The results of these studies showed that rabbit aorta contained three GST isozymes having pI values of 9.4, 7.7, and 5.4. Structural, immunological, and kinetic studies showed that GST 9.4, GST 7.7, and GST 5.4 belonged to the alpha-, pi-, and mu-classes, respectively. The relative abundance of these enzymes in rabbit aorta was alpha > pi > mu. The alpha- and mu-class GST isozymes had similar activities toward GTN (0.71 U/mg and 0.86 U/mg, respectively) while the pi-class GST showed much lower activity toward GTN. The catalytic efficiency k(cat)/Km of the mu- and alpha-class GSTs toward GTN were similar but these activities were differentially inhibited by ethacrynic acid, its GSH conjugate, bromosulfophthalein (BSP), and hematin. These results suggest that in rabbit aorta GSTs may be involved in bioactivation of GTN, and because of their higher abundance the alpha-class GSTs may be more important for the pharmacological effects of GTN than the mu-class GSTs. The results on kinetics of inhibition by various inhibitors suggest that hematin may be an effective inhibitor to delineate the role of specific GST isozymes in the bioactivation of GTN.

Amino Acid Sequence↗
PubMed1998

The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall.

The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic alpha,beta-unsaturated aldehydes (Misra et al., Toxicol. Appl. Pharmacol. 133, 27-33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the alpha,beta-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 microM allylamine (a cardiovascular toxin that is metabolized in vivo and in vitro by VSMCs to the reactive aldehyde, acrolein) or to acrolein (2-10 microM) for 48 h; dose-cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50 by two- to threefold at 50 to 100 microM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1 mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4-4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8-8, which is known to utilize alpha,beta-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against alpha,beta-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.

Acrolein↗
PubMed2001

Surgery and prognostic factors for gastric stromal tumor.

Morphologic studies of gastric stromal tumors (GSTs) indicate that mitotic counts (MCs) and tumor size are major discriminants predictive of biologic behavior. The purpose of this study is to improve the understanding of GST behavior, including the prognostic factors and surgical treatment of GSTs. A retrospective analysis (1990--1997) of the clinical course for 116 patients with GSTs was completed, with a median follow-up of 43 months. Tumors were categorized as malignant GSTs (n = 17) when the MC was > 5/50 high-power fields (HPF) and the size > 5 cm or as benign GSTs (n = 99) when the MC was < or = 5/50 HPF and the size < or = 5 cm, MC < or = 5/50 HPF and size > 5 cm, or MC > 5/50 HPF and size < or = 5 cm. None of 99 benign tumors recurred or metastasized, whereas 7 of 17 malignant tumors recurred. MCs had a close correlation with tumor size. Immunohistochemical studies using CD34, smooth muscle actin, S-100 protein, and synaptophysin have shown positive rates of 61%, 33%, 14%, and 3%, respectively. Smooth muscle actin reactivity was more common in the benign tumors (p = 0.046) and synaptophysin reactivity in the malignant tumors (p = 0.010). Univariate analysis showed that the following clinicopathologic factors were potentially related to poor survival of patients: (1) MC > 5/50 HPF (p = 0.0001); (2) severe pleomorphism (p = 0.0062); (3) necrosis (p = 0.0173); (4) marked cellularity (p = 0.0112); (5) presence of ulceration of overlying gastric mucosa (p = 0.0091); (6) tumor size > 5 cm (p = 0.0195); and (7) exogastric growth pattern (p = 0.0344). Tumors with MC > 5/50 HPF and size > 5 cm were found to be strong indicators of an unfavorable prognosis. The type of surgery and tumor site did not affect the prognosis of the patients.

Adult↗
PubMed1999

The carbohydrate crystalean and colonic microflora modulate expression of glutathione S-transferase subunits in colon of rats.

BACKGROUND: Glutathione S-transferases (GSTs)* are an important class of phase II, predominantly detoxifying, enzymes. The supergene family is composed of several isoenzymes, hetero- and homodimers, with tissue specific distribution and levels of expression. The hypothesis is that a higher expression of individual proteins within a specific tissue may be associated with a decreased burden of exposure to reactive carcinogens and ultimately with a decreased cancer risk in this tissue. AIMS OF THE STUDY: Since nutrition is expected to contribute to the gene expression, it was the aim of this study to investigate the impact of dietary factors, especially resistant starch, and of the gut microflora, which may be influenced by diet, on the GSTs in colon cells of rats. METHODS: For this, a technique using high pressure liquid chromatography was established with which for the first time GST isoenzymes were analysed in colon cells and compared to the levels of the corresponding proteins in the liver of the same rat. RESULTS: It was found that colon cells contain mainly GST pi and low amounts of mu but not GST alpha. In contrast, the predominant form of GSTs in the liver was alpha, then mu and hardly and pi Altogether, liver cells had approximately tenfold more total GSTs than colon cells. The feeding of "Crystalean", a retrograded, high amylose starch which alters the fermentation profile and the composition of the microflora, led to higher levels of GST pi in the colon. Furthermore, the comparison of GSTs in colon cells of germ-free rats revealed they were much lower than those observed in rats with conventional microflora. CONCLUSIONS: These findings clearly demonstrate that the gut bacteria, or their metabolic products, enhance GST expression. The studies support the hypothesis that nutrition--by affecting the gut flora--may induce this potentially protective and important class of phase II enzymes in important tumor target cells.

Amylose↗
PubMed2004

Selective expression of detoxifying glutathione transferases in mouse colon: effect of experimental colitis and the presence of bacteria.

Glutathione transferases (GSTs) play a central role in the cellular defense against harmful endogenous compounds and xenobiotics in mouse and man. The gastrointestinal channel is constantly exposed to bacteria, bacterial products, and xenobiotics. In the present study the distribution of alpha, mu, and pi class GSTs was examined immunohistologically in the colon of conventional and germ-free (GF) mice subjected to experimental colitis. The tissues samples were from conventional mice with and without colitis induced by dextran sulfate sodium (DSS); GF mice treated with DSS or carrageenan; and GF mice inoculated with normal mouse bacterial flora as well as with Lactobacillus GG. In conventional as well as in GF mice the mu and pi class GSTs showed reduced intestinal expression when colitis was induced. In con-rast, the level of GSTs reacting with antibodies directed against the alpha class, in particular mGST A4-4, was elevated after induction of inflammation. Of special interest is mGST A4-4 because of its high catalytic activity with toxic products of lipid peroxidation. In the colon of conventionalized GF mice that were given mouse intestinal flora, the mGST A4-4 expression was increased with time for several weeks, but then showed a decrease to a normal level. Additionally, the inoculation of GF mice with Lactobacillus GG induced all the intestinal GSTs studied.

Animals↗
PubMed2002

Characterization of a recombinant mu-class glutathione S-transferase from Taenia solium.

A Taenia solium larval glutathione S-transferase fraction (SGSTF), composed of two proteins with Mr 25,500 (SGSTM1) and 26,500 (SGSTM2), was purified by GSH-sepharose. Its N-terminal sequence analysis revealed that both proteins are related to mammalian mu-class GST enzymes. A cDNA clone coding for SGSTM1 was isolated and the amino acid sequence analysis showed close identity with two Echinococcus GSTs and also high identity with several mu-class GSTs that have been reported. In addition, SGSTM1 presents a similar structure to mu-class GSTs, including the mu loop. The recombinant SGSTM1 is a dimeric protein with enzymatic properties clearly related to mammalian mu-class GSTs. Western blot studies indicated that SGSTM1 is not antigenically related to SGSTM2 or mammalian GSTs from rabbit, pig and rat livers. Immunization with SGSTF and SGSTM2 was highly effective in reducing cysticerci load in murine cysticercosis. In contrast, no protection was obtained using native SGSTM1 and recombinant SGSTM1 as immunogens.

Amino Acid Sequence↗
PubMed1987

Enzymatic transformation of PGH2 to PGF2 alpha catalyzed by glutathione S-transferases.

Glutathione S-transferases (GSTs) purified from both rat liver cytosol and microsomes catalyzed the direct reduction of PGH2 to PGF2 alpha. As much as 40% of the substrate was transformed into a prostanoid whose Rf value corresponded to that of PGF2 alpha. The identification of the reaction product as PGF2 alpha was confirmed by TLC and reverse-phase HPLC as well as by mass spectral analysis. In the absence of GSTs, PGH2 was found to be primarily converted to PGE2 and PGD2. Also, PGF2 alpha formation was completely abolished by decylglutathione, a potent inhibitor of both peroxidase and transferase activity associated with GSTs. These results indicate that the direct reduction of endoperoxide moiety of PGH2 to form PGF2 alpha is an enzymatic process. Interestingly, selenium-dependent glutathione peroxidase (Se-GSH-Px) showed very little PGF2 alpha formation from PGH2. However, this enzyme was very active in the reduction of PGG2 to PGH2. In contrast, GSTs were very poor in the conversion of PGG2 to PGH2. Therefore, it is possible that the relative tissue distribution of Se-GSH-Px and GSTs might play an important role in the tissue specific synthesis of PGF2 alpha.

Animals↗
PubMed1989

Glutathione conjugation of the fluorophotometric epoxide substrate, 7-glycidoxycoumarin (GOC), by rat liver glutathione transferase isoenzymes.

The fluorophotometric substrate, 7-glycidoxycoumarin (GOC), was examined for the assay of epoxide-glutathione (GSH)-conjugating activities of seven major GSH transferases (GSTs) isolated from rat liver cytosols. GST 7-7 (GST-P), isolated from the liver cytosol of rats bearing hepatic hyperplastic nodules, catalysed the GSH conjugation of GOC at a higher rate than any other examined GST isolated from the normal rat liver cytosol. GSTs 3-3, 3-4 and 4-4 (group 3-4 enzymes) had specific activities towards GOC by one fifth to one third of that of GST 7-7. GSTs 1-1, 1-2 and 2-2 (group 1-2 enzymes) had very low activities towards this epoxide. A kinetic study indicated that GST 7-7 showed the largest kappa cat/Km value for the catalytic reaction of GOC-GSH conjugation among the GSTs. In spite of their much smaller kappa cat values, group 3-4 enzymes showed much larger kappa cat/Km values for GOC than the group 1-2 enzymes, because GOC had a much higher affinity for group 3-4 enzymes than for group 1-2 enzymes. A comparative study was also done with GSH conjugations of styrene 7,8-oxide (STO) and 1-chloro-2,4-dinitrobenzene by the GSTs. Unlike GOC, the conjugation of STO was mediated at rates about twice as high by group 3-4 enzymes than by GST 7-7. STO was also a very poor substrate for group 1-2 enzymes.

Animals↗
PubMed1990

Protective activity of different hepatic cytosolic glutathione S-transferases against DNA-binding metabolites of aflatoxin B1.

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.

Aflatoxin B1↗
PubMed1993

Interactions of glutathione S-transferase-pi with ethacrynic acid and its glutathione conjugate.

Ethacrynic acid, a diuretic drug known to be an inhibitor of glutathione S-transferases (GSTs), has been shown to enhance the cytotoxicity of the alkylating agent class of chemotherapeutic drugs in cultured cancer cells resistant to alkylating agents. This action of ethacrynic acid is presumably mediated by inhibition of GSTs which are implicated in detoxification of alkylating agents. In addition to being an inhibitor of GSTs, ethacrynic acid also interacts with GSTs as a substrate for conjugation with GSH to yield an ethacrynic acid-GSH conjugate. This conjugate is formed both enzymatically and non-enzymatically and itself is a GST inhibitor. Since ethacrynic acid-GSH conjugate is itself likely to be able to mediate reversal of alkylating agents through GST inhibition, we have synthesized and purified the ethacrynic acid-GSH conjugate, studied the kinetics of inhibition of human lung pi-class GST by ethacrynic acid and the conjugate, and compared the kinetics of the enzymatic and non-enzymatic formation of the conjugate using an HPLC method. Results of our studies showed that the ethacrynic acid-GSH conjugate was a more potent inhibitor of human lung GST-pi than ethacrynic acid (Ki = 1.5 vs. 11.5 microM, respectively) and that their mechanisms for GST inhibition were distinct (competitive and non-competitive, respectively). Comparison of enzymatic and non-enzymatic rates of conjugate formation in vitro indicated that GST-pi catalyzed a rapid conjugation of ethacrynic acid with GSH at a concentration of ethacrynic acid an order of magnitude above that required to nearly completely inhibit GST catalyzed conjugation of 1-chloro-2,4-dinitrobenzene. However, because of the rapid non-enzymatic reaction, and the inhibition of GST-pi with the accumulation of the conjugate in the reaction mixture, the overall quantity of the conjugate formed after 150 min was nearly identical in the presence or absence of GST-pi. Results of these studies suggest that inhibition of GSTs by ethacrynic acid-GSH conjugate may be the main mechanism through which ethacrynic acid reverses alkylating agent resistance.

Alkylating Agents↗
PubMed1994

Inter-individual variability of human hepatic glutathione S-transferase isozymes assessed by inhibitory capacity.

Glutathione S-transferases (GSTs) are important as drug metabolizing enzymes mainly in the detoxication process. To assess individual differences in susceptibility to different chemicals, it would be useful to know the activity of specific GST isozymes in individuals. In order to study individual variability, typical substrates specific for the three major classes of GSTs were used not only as substrates but also as inhibitors using 17 organ-donor livers. Out of 17, 4 livers were low in the mu-form isozyme, judging from the depressed GST activity for trans-4-phenyl-3-buten-2-one (tPBO), a mu-class substrate. The range of tPBO activity exhibited more than 20-fold variation, while less than 2-fold variation was observed in the activities for 1-chloro-2,4-dinitrobenzene (CDNB), a non-specific substrate for alpha-, mu-, and pi-class GSTs. In addition to the conventional approach to assessing individual variability, well known substrates for GSTs, cumene hydroperoxide (CHP, alpha-class), tPBO (mu) and ethacrynic acid (EA, pi) were used as inhibitors against CDNB-GSH conjugation. CHP inhibited the CDNB-GSH conjugation in mu-depressed livers more potently than in the non-depressed livers, probably because CHP is a specific inhibitor for alpha-class GST. Inhibition by CHP may therefore be used to detect livers which are low in mu-form. The inhibition by tPBO was found to be weak and the variability was small (CV of 9% in 17 livers). EA potently inhibited GST activity with its inhibition constant (Ki) 2,000-fold lower than that of CHP. The small individual variation in inhibition by EA (CV of 6%) supported that EA is a universal but potent inhibitor against major GSTs contained in human liver.

Benzene Derivatives↗
PubMed1994

Conjugation of microsome generated and synthetic aflatoxin B1-8,9 epoxide and styrene oxide to glutathione by purified glutathione S-transferases from hamster and mouse livers.

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B1 (AFB1)-epoxide and styrene oxide has been studied with purified glutathione transferases (GSTs) from mouse and hamster liver cytosols. In hamster, with microsomally activated epoxide, the alpha group of GSTs show about 10-fold more activity than the mu group. With the synthetic AFB1 epoxide, the mu enzymes designated H3B and C show considerable activity although less than alpha, whereas H3A and D demonstrate similar ranges of activity as the alpha group. The pi class of GST could not be assayed due to its absence in the hamster liver. The mouse liver cytosols show 3.6-fold greater activity than hamster cytosol in microsome mediated assay system. The mouse alpha and mu enzymes have similar levels of activity in the microsome mediated system; this activity could not be determined with the pi GST due to shortage of this enzyme. The alpha group has 2- and 5-fold higher activity than mu and pi group of GSTs, respectively, with the synthetic epoxide of AFB1. With styrene oxide, the purified GSTs from hamster liver show total loss of activity whereas in the mouse alpha, mu and pi classes of GSTs have similar range of activity as the cytosol. The role of alpha and mu isozymes of GST in rendering these animals resistant to hepatocarcinogenecity is suggested.

Aflatoxin B1↗
PubMed1996

Expression and induction of an immunochemically related class of glutathione S-transferases in Daphnia magna.

The cytosolic glutathione S-transferases (GSTs) are dimeric enzymes that are responsible for the conjugation of glutathione to an electrophilic center of a variety of lipophilic compounds. The purpose of the present study was to characterize the GSTs of Daphnia magna with respect to enzyme multiplicity, molecular weight, isoelectric points, and immunochemical distinction and to determine the inducibility of these enzymes by the prototypical mammalian GST inducer, phenobarbital. GSTs were purified from crude cystosols prepared from daphnids by glutathione-sepharose affinity chromatography. SDS-polyacrylamide gel electrophoresis of the affinity purified GSTs revealed the presence of multiple subunits with molecular weights ranging from 26.9 to 30.2 kDa. Preparative electrofocusing separated GST activity into three major fractions having approximate isoelectric points of 4.5, 4.8 and 5.6. All of the catalytically active fractions contained a single protein band of the same molecular weight (30.2 kDa) during SDS-PAGE. A monoclonal antibody, prepared against the affinity-purified GST proteins, recognized three distinct proteins separated during analytical-scale isoelectric focusing (pI 4.6, 4.7 and 4.8). These proteins may represent a class of GSTs distinct from the GST having a pI of 5.6. Treatment of daphnids with phenobarbital elevated both GST catalytic activity and immunodetectable protein. These results demonstrate that multiple immunochemically related proteins of the same molecular weight but varying isoelectric points are responsible for most of the GST catalytic activity with the substrate 1-chloro-2,4-dinitrobenzene.

Animals↗
PubMed1994

Immunological comparison of cytosolic glutathione S-transferases between rat and two strains of houseflies.

Five different antisera, which include three antisera raised against rat liver glutathione S-transferases (GST), one antiserum raised against human pi GST, and one antiserum raised against housefly GST1, were used to examine their cross-reactivity with different classes of GST subunits isolated from rat liver and the housefly. Two classes of rat liver GSTs, alpha and mu, were isolated from rat liver and two classes of housefly GSTs, GST1 and GST2, were isolated from both CSMA and Cornell-R strains. Antiserum against GST 3-3 was the most reactive antiserum and reacted not only with the mu class of GSTs but also with the GST1 class from both CSMA and Cornell-R strains. Antiserum against human pi GST and antiserum against housefly GST1 had weak immunological reactivity toward the GST1 class from both strains of housefly. Antiserum against GST 4-4 and antiserum against GST 1-1 had no immunological reactivity toward any class of GSTs from housefly. None of the five antisera had any immunological cross-reactivity toward subunit 2 of the alpha class of rat GST and the GST2 class of housefly GSTs from both strains.

Animals↗
PubMed2004

Glutathione-S-transferase A4-4 modulates oxidative stress in endothelium: possible role in human atherosclerosis.

The role of alpha-class mammalian glutathione S-transferases (GSTs) in the protection of many cell types, including vascular smooth muscle cells, against oxidant damage has been demonstrated, but the role of GSTs in the endothelial cell is not well studied. In order to examine the role of GSTs in the endothelial cell, a stable transfection of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, mouse isozyme of GSTs with activity in vascular wall, was established. Transfected cells demonstrated significantly higher GSTs enzyme activity and expressed significantly increased resistance to the cytotoxicity of allylamine, acrolein, 4-hydroxy-2-nonenal (4-HNE), and H(2)O(2) (P < 0.05). A significantly higher rate of proliferation and lower baseline level of intracellular malondialdehyde (MDA) and 4-HNE were present when compared to wild-type or vector-transfected MS1 endothelial cells (P < 0.05). Transfection protected MS1 endothelial cells from 4-HNE and H(2)O(2) induced apoptosis by inhibiting phosphorylation of c-Jun N-terminal kinases (p-JNK) and consequent activation of p53 and Bax. In early human fibrous atherosclerotic plaques, immunohistochemical studies demonstrated marked induction of hGSTA4-4 in endothelial cells overlying plaque, and in proliferating plaque vascular smooth muscle cells. Our results indicate that endothelial cell mGSTA4-4 can play a key role in protecting blood vessels against oxidative stress and, thus, is likely to be a critical defense mechanism against oxidants that act as atherogens.

Animals↗
PubMed2006

Relation of glutathione S-transferase genotypes (GSTM1 and GSTT1) to laryngeal squamous cell carcinoma risk.

The purpose of the present study was to investigate GSTM1 and GSTT1 genotypes by using multiplex polymerase chain reaction (PCR) in patients with laryngeal squamous cell carcinoma (LSCC). The genotypes of 110 patients with LSCC and of 197 healthy subjects as the control group were determined by PCR analysis for GSTM1 and GSTT1 genes. Results showed that frequencies of GSTM1-null, GSTT1-null, and both GSTs-null genotypes were 51.8, 30, and 16.4%, respectively, in the patients with LSCC and 37.6, 15.7, and 5.6% in the control group. There was a significant difference between the genotype distributions of all GSTs in patients and in control groups (P < 0.05). The results support the hypothesis that null genotypes of GSTM1 and GSTT1 can reduce detoxification capacity of GSTs as members of the xenobiotic enzyme system. GSTM1-null, GSTT1-null, and both GSTs-null genotypes were more common in the patients with LSCC than in the control group. Patients with both GSTs-null genotypes had the highest risk for supraglottic LSCC in the early period, even if they were light-to-medium smokers. Investigation and determination of the genetic basis of LSCC may contribute to detection of risk groups and to prevent LSCC in the population.

Adult↗
PubMed2001

Identification and cloning of a key insecticide-metabolizing glutathione S-transferase (MdGST-6A) from a hyper insecticide-resistant strain of the housefly Musca domestica.

Strains of the housefly, Musca domestica, highly resistant to organophosphate (OP) and other insecticides are known because they overproduce glutathione S-transferases (GSTs). Previous work has shown that overproduction in these strains involved numerous isozymes with glutathione conjugating activities (Pesticide Biochem. Physiol., 25 (1986) 169; Mol. General Genetics, 227 (1991) 355; J. Biol. Chem., 267 (1992) 1840; Mol. General Genetics, 245 (1994) 236; J. Mol. Evol., 43 (1996) 236). The current work describes the purification and identification of a M. domestica GST isozyme (pI 7.1) broadly specific for substrates from a housefly strain, Cornell-HR, that is highly resistant against OP-insecticides, and the isolation of two new MdGST genes using the antibody made against it. This isozyme, which was identified from amongst more than 20 isoelectric forms of GSTs of the same subunit size, was highly active for conjugating GSH to the model substrate 3,4-dichloronitrobenzne (DCNB). When expressed in Escherichia coli, one of the cloned GSTs, MdGST-6A, produces an enzyme that conjugates glutathione to the insecticides methyl parathion and lindane. On indication that it was the most active isozyme toward several xenobiotics among several MdGSTs tested, we advance the notion that MdGST-6A probably plays an important role in M. domestica Cornell-HR's resistance towards OP-insecticides. MdGST-6A and a second closely related one found in this work, MdGST-6B, are members of the traditional insect class I family (theta-class) and share the greatest homologies with a cluster of Drosophila GSTs on locus 55. In addition to having the unusually broad substrate specificity, the sequence of the new group of enzymes reveals that it has a highly diverged hydrophobic motif in its active site as compared to other class I GSTs from insects.

Amino Acid Sequence↗