Expression Of Glutathione S-transferase Isozymes Research Articles

Putative Role of Glutathione S‐transferases as Mediators of Cell Growth, Development, and Differentiation in the Eukaryotic Model Organism, Dictyostelium discoideum

In addition to their role in detoxification, Glutathione S‐transferase (GST) enzymes influence signal transduction, development, and differentiation in many eukaryotic cells. Transcriptome analysis of the cellular slime mold, Dictyostelium discoideum (strain AX4) expresses five GST (α1‐α5 class) transcripts whose function(s) remain unknown. This study characterized the activity and expression of GST isozymes in D. discoideum at various stages of growth and development. A GSH‐agarose pull down assay of the lysates from vegetative amoebae identified two protein species of ~24kDa; mass spectrophotometric analysis identified GSTα3> GSTα1>GSTα2 as predominant species. Measurements of GST activity in amoeba using the substrate 1‐chloro‐2‐4‐dinitrobenzene (CDNB) revealed functional enzyme activity, which was significantly reduced by as much as 50% in the presence of 500 nM ethacrynic acid (EA, a GSTs inhibitor). Ethacrynic acid also delayed the formation of aggregates and multicellular development. Levels of GSTs activity differed significantly between vegetative amoebae and multicellular stages of development (aggregate, slug, culmination, fruiting body), indicating the importance of GSTs to influence cell signaling and gene expression during morphogenesis and differentiation. Current studies aim to determine: a) the levels of each isozyme and their substrate specificities; b) the impact of GST null mutants on growth and development of the organism. These studies will contribute to elucidating the expanding the role and function of GST enzymes in eukaryotic signal transduction that regulates cell growth, chemotaxis, and differentiation.

Selective induction of glutathione S-transferases in round spermatids from the Brown-Norway rat by the chemotherapeutic regimen for testicular cancer

Chemotherapeutic drugs can affect DNA in male germ cells, thereby impacting on the integrity of the genome transmitted to offspring. Drug metabolizing enzymes can protect cells from xenobiotic insult. We analyzed the expression pattern of such enzymes in isolated round spermatids from rats exposed to drugs used to treat testicular cancer: bleomycin, etoposide, and cisplatin (BEP). The number of isozymes expressed and the overall relative expression values were highest for the glutathione S-transferases (GSTs). Moreover, BEP treatment significantly increased the expression of 8 GSTs and 3 aldehyde dehydrogenases. Increased expression of GST isozymes was confirmed by qRT-PCR and Western blot analysis. Although Gst genes can be targets for epigenetic modifications, promoter DNA methylation was not affected by BEP treatment. As GSTs are involved in drug resistance mechanisms, we hypothesize that BEP induction of GST expression may lead to the survival of damaged germ cells and the production of abnormal sperm.

Glutathione S-transferases and S-glutathionylation in cancer

CN04-02 The glutathione-S-transferase (GST) super family comprises multiple isozymes (Alpha, Mu, Pi, Omega, Theta, and Zeta) with evidence of functional polymorphic variations. Correlative studies have shown that genetic differences of GST isozyme expression may contribute to cancer susceptibility and treatment. Over the last two decades, a body of data has accumulated linking aberrant expression of GST isozymes with the development and expression of resistance to cancer drugs. In particular, GST Pi (GSTP) is over-expressed in a number of different tumors compared to normal tissues (including ovarian, non-small cell lung, breast, colon, liver, pancreas and lymphoma). Moreover, a significant range of anticancer drugs can cause an increased expression of GSTP in drug resistant selected cell lines (and drug treated patients). From a functional viewpoint, not all drugs used to select for resistance are substrates for catalysis by GSTP. In fact, catalytic constants for GSTP conjugation reactions with virtually all standard cancer drugs are poor. So why is GSTP so frequently over-expressed in these situations? There is recent evidence that GST isozymes, and GSTP in particular, have multiple functions in cells, many unrelated to thioether bond catalysis with chemical moieties. These include: (1) ligand biding and transport of heme, bilirubin, nitric oxide; (2) protein: protein interactions with possible chaperone like functions; (3) regulation of mitogen activated protein kinases, particularly c-jun NH2 terminal kinase (JNK); (4) mediation of the forward reaction of the post-translational process of S-glutathionylation. Of these, aberrant kinase signaling pathways and altered protein S-glutathionylation patterns are both characteristic of the malignant phenotype. What happens in the absence of GSTP? GSTP null animals have essentially normal development and life spans. Mouse embryo fibroblast (MEF) cells isolated from wild type or GSTP null animals differ in a number of characteristics related to signaling and growth. For example, the doubling time for wild type cells is 33.6 h compared to 26.2 h for GSTP null. Both early passage and immortalized MEF cells from GSTP null animals express significantly elevated activities of extracellular regulated kinases (ERK1/ERK2). Null animals had constitutively elevated c-jun NH2-terminal kinase (JNK) activity compared to wild type and this is correlated with altered regulation of genes downstream of JNK. As a whole, the genetic absence of GSTP influences the capacity of stress kinases to regulate gene expression and this can have an impact on cell proliferation pathways. The non-lethality of the deletion points to possible functional redundancy and implies that other GST (or other redox proteins) may substitute for the absence of GSTP. This conclusion would seem to be supported by the data suggesting general redundancy of function amongst, and within, this protein cluster. Cysteine residues in proteins provide a nucleophilic site for a number of disparate post-translational modifications. For example, disulfide bonds between vicinal thiols have major implications for three dimensional protein structures. Contingent upon the steric properties and local environment of the cysteine, lipidation of these residues may occur through S-isoprenylation, S-farnesylation, S-geranylgeranylation or S-palmitoylation. The direct addition of GSH to cysteines with low pKa’s creates an S-glutathionylated residue, resulting in an increase in both molecular weight (of 605) and negative charge (from the glutamic acid residue). The scheme in the Figure summarizes some components of this reaction. GSTP can facilitate the forward reaction and glutaredoxin, sulfiredoxin or thioredoxin can contribute to the reverse reaction. The reversible nature of this pathway is important in facilitating a sulfur based regulatory pathway that can expedite response to stress conditions. Importantly, a number of phosphatases can be regulated by S-glutathionylation and this provides a link and conduit with phosphate based signaling pathways. Because the structure, function and cell distribution of proteins can be affected by S-glutathionylation, the importance of GSTP in mediating this reaction could have significant consequences and may be a contributory factor in the high expression levels of GSTP in many tumors. These multiple functionalities contribute to the recent rational efforts to target GSTs with novel small molecule therapeutics. While the ultimate success of these attempts remains to be shown in the clinic, at least three drugs are in late-stage clinical testing. Two of these (NOV-002 and Telintra) are being tested therapeutically as small molecule myeloproliferative agents. As the field progresses, the concept of designing new drugs that might interfere with protein:protein interactions between GSTs and regulatory kinases provides a novel approach to identify new targets in the search for cancer therapeutics. Relevant

Effect of naturally occurring phenolic acids on the expression of glutathione S-transferase isozymes in the rat

Naturally occurring plant phenols, protocatechuic and tannic acids, have been reported to be inhibitors of chemical mutagenesis and carcinogenesis in experimental models. Our previous studies, have shown that these compounds modulate the activity of phases 1 and 2 enzymes in rodents. The aim of the present study was to investigate whether these compounds affect protein levels of rat hepatic and renal glutathione S-transferase (GST) isozymes. Male Wistar rats were treated intraperitoneally with protocatechuic or tannic acid at 50 mg/kg body weight five times during 14 days. 3-Methylcholanthrene (MC) was administered at 20 mg/kg body weight on day 13 (the last treatment with phenolic compounds) and on day 14. Tissues were obtained from rats terminated 24 h after the last treatment. Western blot analysis with specific antibodies showed significant differences in the effect of the phenolic compounds in the liver and kidney. In the liver, protocatechuic acid significantly increased the constitutive GSTμ, while tannic acid reduced the GSTα protein level by 60%. Both plant phenols decreased all classes of constitutive GST isozymes in the kidney including GSTπ, and also the MC-induced GSTα and/or π protein levels. These results, as well as our previous reports, suggest that protocatechuic and tannic acids interfere with the pathways related to xenobiotic toxicities and carcinogenesis. This effect may be important for chemoprotective activity of these plant phenols.

Hepatic and extrahepatic expression of glutathione S-transferase isozymes in mice and its modulation by naturally occurring phenolic acids

A simple plant phenolic acid, protocatechuic acid and a polyphenol, tannic acid are potential chemopreventive agents which inhibited the chemically induced carcinogenesis in many experimental models. We previously demonstrated that those compounds modulate the activity of xenobiotic detoxifying enzymes, including GST in mouse liver, kidney and epidermis. Intraperitoneal (i.p.) treatment with protocatechuic acid in the dose of 80 mg/kg for three consecutive days increased the GST activity in liver and kidney. In case of tannic acid the same effect was observed in kidney after i.p. administration of the single dose of 80 mg/kg. Topical application of phenolic acids resulted in enhancement of epidermal GST activity. The focus of this study was to further investigate the effects of these phenolic acids on the protein levels of GST isozymes in the same tissues using the treatment protocols used in our previous studies. The results confirmed the expression of GST alfa, mu, pi and theta in mouse liver, kidney and epidermis. Treatment with protocatechuic acid resulted in an increase of the expression of GST class mu in liver, but did not affect this isoform in skin and kidney. This compound inhibited the level of kidney GST pi by 35%. Tannic acid decreased the expression of GST mu, alpha and theta in liver. Application of the equimolar doses of both phenolic acids on mouse skin resulted in reduced level of the GST alpha protein. The results of our study indicate that, although moderate, the effect of protocatechuic acid and tannic acid on GST subunits in mice may play certain role in biological activity of these compounds. Of special importance could be the increased expression of GST mu in liver which is involved in detoxification of many carcinogens including polycyclic aromatic hydrocarbons.

Glutathione S-transferase polymorphisms: cancer incidence and therapy.

The super family of glutathione S-transferases (GSTs) is composed of multiple isozymes with significant evidence of functional polymorphic variation. Over the last three decades, data from cancer studies have linked aberrant expression of GST isozymes with the development and expression of resistance to a variety of chemicals, including cancer drugs. This review addresses how differences in the human GST isozyme expression patterns influence cancer susceptibility, prognosis and treatment. In addition to the well-characterized catalytic activity, recent evidence has shown that certain GST isozymes can regulate mitogen-activated protein kinases or can facilitate the addition of glutathione to cysteine residues in target proteins (S-glutathionylation). These multiple functionalities have contributed to the recent efforts to target GSTs with novel small molecule therapeutics. Presently, at least two drugs are in late-stage clinical testing. The evolving functions of GST and their divergent expression patterns in individuals make them an attractive target for drug discovery.

Changes in expression and activity of glutathione S-transferase in different organs of schistosoma haematobium-infected hamster.

Schistosomiasis is a major health problem in many subtropical developing countries, causing a number of serious pathologies, including bladder cancer. Most of the toxic compounds formed as a result of these infestations are derived either exogenously or formed endogenously and can be conjugated with glutathione (GSH) via glutathione S-transferase (GST). The present study investigates the effect of Schistosma haematobium infection on the activity of GST and glutathione reductase (GR) and levels of glutathione and free radicals (measured as thiobarbituric acid reactive substances) in different organs of the male hamster. The total activity of GST was increased in several organs; in kidney by 50 and 46% at 6 and 10 weeks postinfection, respectively, and in bladder tissues by 169, 23, and 130% at 2, 4, and 6 weeks postinfection, respectively. In support of this, the expression of GST isozymes was also induced in kidney and bladder tissues at early stages (2, 4, and 6 weeks) and reduced at the later stages of infection (8 and 10 weeks). In contrast, the expression of these isozymes was decreased in the spleen and liver at 2, 4, 6, 8, and 10 weeks postinfection. Also, such activity was decreased in lungs by 74 and 78% and in bladders by 65 and 72% at 8 and 10 weeks postinfection, respectively. GSH levels increased in lungs by 95, 40, and 56% at 2, 4, and 6 weeks and in spleen by 26 and 74% at 4 and 6 weeks, respectively, but decreased at later stages of S. haematobium infection in these organs. The depletion of GSH levels also occurred in bladders by 72 and 54% at 8 and 10 weeks postinfection, respectively. The activity of GR was increased in the livers, lungs, and kidneys of the S. haematobium-infected hamster. TBARS also increased in the lung by 14, 65, 53, 828, and 624% and in the kidney by 64, 29, 87, 190, and 111%, and in the bladder by 216, 23, 1468, 528, and 1025% at 2, 4, 6, 8, and 10 weeks postinfection, respectively. This study indicates that low GST expression and high levels of free radicals could provide new evidence for damage to the bladder and other organs as a result of S. haematobium infection.

Cancer drugs, genetic variation and the glutathione-S-transferase gene family.

The glutathione-S-transferase (GST) super family comprises multiple isozymes (Alpha, Mu, Pi, Omega, Theta, and Zeta) with compelling evidence of functional polymorphic variation. Over the last two decades, a significant body of data has accumulated linking aberrant expression of GST isozymes with the development and expression of resistance to cancer drugs. Clinical correlation studies show that genetic differences within the human GST isozymes may play a role in cancer susceptibility and treatment. The initial confusion was presented by the fact that not all drugs used to select for resistance were substrates for thioether bond catalysis by GSTs. However, recent evidence that certain GST isozymes possess the capacity to regulate mitogen activated protein kinases presents an alternative explanation. This dual functionality has contributed to the recent efforts to target GSTs with novel small molecule therapeutics. While the ultimate success of these attempts remains to be shown, at least one drug is in late-stage clinical testing. In addition, the concept of designing new drugs that might interfere with protein:protein interactions between GSTs and regulatory kinases provides a novel approach to identify new targets in the search for cancer therapeutics.

Depletion of colonic detoxication enzyme activity in mice with dextran sulphate sodium-induced colitis.

The increased risk of colonic malignancies in individuals with ulcerative colitis has prompted a search for early biomarkers of disease progression. To characterize Phase II detoxication enzyme expression during acute and chronic colitis. The mouse model of dextran sulphate sodium (DSS)-induced colitis represents a relevant system with which to sequentially evaluate the spectrum of biochemical changes associated with colorectal cancer risk. Acute and chronic colitis were induced in Swiss Webster mice by administering DSS in the drinking water (5%) for 1-4 cycles. Each cycle consisted of 7 days DSS and 14 days of water. The glutathione S-transferase (GST) activity, gamma-glutamylcysteine synthetase (gamma-GCS) activity and glutathione content of the colonic tissues were determined at various time points throughout the experiment. Alterations in GST isozyme expression were confirmed by Western and Northern blot. GST activity was reduced significantly in the colon by the end of Cycle 1 (84% of control values). Specific activities continued to decrease with subsequent cycles of DSS exposure. By the end of Cycle 4, glutathione levels and gamma-GCS activity had reached 29% and 56% of control, respectively. These data suggest that detoxication enzyme depletion is associated with both acute and chronic colitis and may be an important event in the progression of ulcerative colitis to colon cancer.

Identification of glutathione S-transferase isozymes and gamma-glutamylcysteine synthetase as negative acute-phase proteins in rat liver.

Because acute infection and inflammation affect drug metabolism and drug-metabolizing enzymes, the effect of the acute-phase response on the expression of glutathione S-transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute-phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene-specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute-phase response as judged by the increased expression of positive and decreased expression of negative acute-phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1-chloro-2,4-dinitrobenzene was also significantly decreased. mRNA expression of gamma-glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn-Superoxide dismutase expression was increased 13-fold, and thioredoxin level was elevated 3-fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute-phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug-metabolizing enzymes known to be decreased during the acute-phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.

Over-expression of glutathione S-transferase Yp isozyme and concomitant down-regulation of Ya isozyme in renal cell carcinoma of rats induced by ferric nitrilotriacetate.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage, a consequence of iron-catalysed Fenton-like reactions, that finally leads to a high incidence of renal cell carcinoma (RCC) in rodents. Glutathione S-transferase (GST) is a family of enzymes that play an important role in detoxification of hydrophobic and electrophilic molecules, and has been associated with putative preneoplastic foci of rat hepatocarcinogenesis and chemotherapy-resistance of human cancers. Our previous study revealed an induction of pi-class glutathione S-transferase (Yp) mRNA in the kidney 3 h after administration of Fe-NTA. In the present study, expression of GST isozymes were further investigated in the Fe-NTA-induced RCCs of rats which are characterized by (1) high incidence of metastasis and invasion, (2) high incidence of tumour-associated mortality, and (3) possible involvement of reactive oxygen species in carcinogenesis. In the Fe-NTA-induced RCCs, the levels of alpha-class GST (Ya) mRNA and proteins were markedly decreased with no apparent change in the copy number of the gene. In contrast, GST-Yp mRNA and proteins were significantly increased in the RCCs while the total GST enzymatic activity was decreased. Immunohistochemical analysis revealed intense staining of GST-Yp not only in the primary RCCs and its metastatic sites, but also in their non-tumorous part of proximal tubules. The contrastive expression of GST isozymes in this renal carcinogenesis model suggests an alteration of its transcription mechanisms and warrants further investigation of this particular detoxifying enzyme from the viewpoint of reactive oxygen species-induced carcinogenesis.

Methionine and Cysteine Affect Glutathione Level, Glutathione-Related Enzyme Activities and the Expression of Glutathione S-Transferase Isozymes in Rat Hepatocytes , ,

Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and γ-glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P< 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.

DIFFERENTIAL EXPRESSION OF GLUTATHIONE-S-TRANSFERASE ISOZYMES IN MURINE AND HUMAN SKIN TUMORS

Glutathione S-transferases (GSTs) are a family of widely distributed multifunctional enzymes present in a number of organs and species. GSTs participate in the detoxification of a wide variety of xenobiotics and their metabolites. Mammalian GSTs have been grouped into three major classes alpha, mu and pi. The differential expression of specific GST isozymes has been reported as a phenotypic marker for the preneoplastic lesions and neoplastic tissues in liver and other extracutaneous organs of rat and other species including humans. However, the expression of GST isozymes in skin cancer is not known. In the present study, therefore, employing Western blot analysis, we assessed the GST isozyme expression in experimentally-induced murine skin tumors and clinically obtained human skin basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). As compared to normal murine skin showing high levels of GST-pi, significantly lower expression of GST-pi was observed in murine skin papillomas and carcinomas induced chemically in SENCAR mice or by chronic ultraviolet B radiation in SKH-1 hairless mice. Similar results were also observed for GST-mu isozyme. On the other hand, compared to normal murine skin where the expression of GST-alpha isozyme was not detectable, significant levels of this GST isozyme were observed in all the murine skin papillomas and carcinomas. In case of human skin BCCs and SCCs, variable GST isozyme patterns were evident, however in each case, GST-pi was found to be significantly underexpressed as compared to that in skin from healthy subjects. Our results suggest that altered phenotypic expression of GST isozymes may be a useful marker for skin cancer.

Protection by transfected glutathione S-transferase isozymes against carcinogen-induced alkylation of cellular macromolecules in human MCF-7 cells.

Increased expression of glutathione S-transferase (GST) isozymes has been correlated with development of resistance both to cytotoxic anticancer agents and to genotoxic carcinogens. While most anticancer agents are poor GST substrates, the model alkylating carcinogen 4-nitroquinoline-1-oxide (NQO) is a good substrate for human pi class GST (hGSTP1-1) and murine GST mu-1 (mGSTM1-1), but not human GST alpha-2 (hGSTA2-2). We investigated whether expression of these GST isozymes in stably transfected clonal cell lines could protect against the genotoxic and cytotoxic effects of NQO. Compared to parental MCF-7 or pSV2neotransfected control cell lines, covalent labeling of total cellular macromolecules by [3H]NQO (0.1-1.0 mM) was reduced by 70% and 92% in hGSTP1-1- and mGSTM1-1-transfected cell lines, respectively, but was not affected in the hGSTA2-2 expressing line. The observed protection was closely correlated with the relative specific activity of each cell line for conjugation of NQO by the transfected GST isozymes and this protection was reversible by pretreatment of cells with the GST inhibitor ethacrynic acid. Similar results were obtained when covalent labeling of total cellular nucleic acid or DNA was measured. However, clonogenic survival assays indicated that the sensitivity of these cell lines to the cytotoxic effects of NQO was similar for the control and GST-transfected MCF-7 cell lines. Thus, while expression of hGSTP1-1 and mGSTM1-1 (but not hGSTA2-2) was highly protective against alkylation of cellular macromolecules by NQO, this protection was not effective against cytotoxicity induced by NQO as measured by clonogenic assay. These results indicate that expression of GST isozymes can protect differentially against the acute genotoxic and potentially mutagenic effects, as compared to the cytotoxic effects, of electrophiles that are detoxified by glutathione conjugation.

Difference in Glutathione S-Transferase Response to Oxidative Stress Between Porcine and Bovine Lens

Porcine and bovine lens GSTs were compared in the stability against various oxidative stress which is a major factor of cataract formation in order to clarify the role of lens glutathione S-transferase (GST) and its relation to cataractogenesis. Class π porcine lens GST was inactivated reversibly by biological disulfides, cystine and cystamine, and also inactivated by active oxygen species such as O 2 - generated through xanthine-xanthine oxidase system and H 2O 2. On the other hand, class μ bovine lens GST was insensitive to such applied oxidative stress. Furthermore, 1,2-naphthoquinone, which is a metabolite of naphthalene and an actual inducer of naphthalene cataract, strongly inactivated porcine lens GST though it did not affect bovine enzyme. Thus, porcine and bovine lens GSTs had different sensitivity to various oxidative stress which could induce cataract formation. The results suggest that the differential expression of GST isozymes among animals may explain the variation in the cataract formation caused by oxidative stress.

Effects of altered calcium homeostasis on the expression of glutathione S- transferase isozymes in primary cultured rat hepatocytes

The effects of altered Ca 2+ homeostasis on glutathione S-transferase (GST) isozyme expression in cultured primary rat hepatocytes were examined. Isolated hepatocytes were cultured on Vitrogen substratum in serum-free modified Chee's essential medium and treated with Ca 2+ ionophore A23187 at 120 hr post-plating. GST activity increased slightly, albeit significantly, in a concentration-dependent manner in A23187-treated hepatocytes relative to untreated controls. Western blot analysis using GST class α and μ specific antibodies showed an ∼1.6-and 1.5-fold increase in the class α, Ya and Yc subunits, respectively, whereas no significant increase (∼1.2-fold) in class μ GST expression was observed following A23187 treatment. Northern blot analysis revealed an ∼5-fold increase in GST class α and an ∼7-fold increase in class μ GST mRNA levels in ionophore-treated hepatocytes compared to untreated cells. Results of the Western and Northern blot analyses of the ionophore-treated hepatocytes were compared with those obtained for tert-butyl hydroperoxide-treated cells. Inununoblot analysis showed a significant increase in the expression of GST class α, Ya and Yc subunits, ∼1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class μ GSTs. Northern blot analysis showed ∼3- and 2-fold increases, respectively, in class α and μ GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca 2+ homeostasis produced by either Ca 2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.

Glutathione S-Transferases in Human and Rodent Skin: Multiple Forms and Species-Specific Expression

The glutathione S-transferases (GST) are a family of widely distributed multifunctional detoxification enzymes that catalyze the reaction between reduced glutathione and a variety of electrophiles. Of interest is the fact that several extracutaneous tissues exhibit a distinct spectrum of isozymes that are expressed in a highly controlled fashion. Despite the fact that the skin is continuously exposed to numerous injurious agents, little is known about the expression of GST isozymes and their role in metabolism of physiologic and xenobiotic substrates in cutaneous tissue. Using specific polyclonal antibodies to the Alpha, Mu, and Pi classes of GST, we identified their expression in rat, mouse, and human skin cytosol. In each species, GST isozymes expressed activities towards 1-chloro-2,4-dinitrobenzene, benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, leukotriene A4, and ethacrynic acid, but not towards bromosulfophthalein and cumene hydroperoxide. Western blot analysis indicated the predominant expression of Pi isozyme in all three species. Alpha class of isozyme(s) was present only in human skin, whereas Mu class of isozyme(s) was detected only in rat and mouse skin. Similarly, in normal and transformed cultured human keratinocytes Pi was the predominant isozyme. In situ localization studies using immunohistochemical techniques confirmed the observations of Western blotting. In mouse skin, Pi and Mu isozyme(s) were found to be predominantly localized in sebaceous glands, whereas no reactivity was observed with the Alpha class of isozymes. Our data show that multiple forms of GST exist in rodent and human skin and that GST Pi is the predominant isozyme in each species. Furthermore, cutaneous GST can metabolize both endogenous substrates and foreign compounds.

Glutathione S-transferase isozymes in Aedes aegypti: Purification, characterization, and isozyme-specific regulation

Glutathione S-transferase (GST) isozymes were purified from the GG strain of Aedes aegypti, a strain having ≥4-fold higher total GST activity compared to the wild-type lab strain. Purification involved S-hexyl-glutathione affinity chromatography in high salt buffer, and GST specific elution with S-( p-bromobenzyl)-glutathione. Final purification was accomplished on DEAE-Sepharose. Two isozymes, GST-1b and GST-2 were purified using this procedure, and an additional isozyme, GST-1a, was partially purified. The GST-2 isozyme has one of the highest specific activities reported for a GST, with a specific activity of 739 μmol/min/mg using 1-chloro-2,4-dinitrobenzene (CDNB), and 16.4 μmol/min/mg using 3,4-dichloronitrobenzene (DCNB) as substrates. GST-2, GST-1a, and GST-1b were analyzed for amino acid composition and subjected to N-terminal sequencing. All three GSTs showed amino acid differences, especially among the nonpolar and polar amino acids. The amino acid composition of GST-1b was found to be more similar to GST 1-1 from Drosophila melanogaster than to GST-2 or GST-1a from Aedes aegypti. Only GST-2 gave N-terminal sequence data, raising the possibility that GST-1a and 1b are N-terminally blocked. The A. aegypti GST-2 showed amino acid sequence identity or similarity in all but one residue between residue numbers 31 through 41 compared to the D. melanogaster and Musca domestica GST 1-1 isozymes. The pattern of GST isozyme expression was analyzed in various tissues and stages of development of the GG and wild type strains using isozyme-specific antisera and substrates. GST-1a was constitutively overexpressed in all tissues examined in the GG strain compared to the wild type strain. The expression of GST-1b was similar in both strains for all tissues and developmental stages examined. GST-2 was constitutively overexpressed in head, thorax and abdomen, but was not detected in ovaries of the GG strain. These results suggest that elevated GST activity in the GG strain is due to constitutive overexpression of GST-2 and GST-1a. GST-1a, GST-1b and GST-2 apparently are the products of 3 independently regulated genes and appear to be expressed in a tissue-specific manner.

Purification and characterization of unique glutathione S-transferases from human muscle

Results of studies designed to investigate the origin of the diversity of glutathione S-transferase (GST) isozymes in human tissues indicated that human muscle has at least three forms of GST with p I values of 5.0, 5.1, and 5.2 that are distinct from GST isozymes characterized so far. The major muscle isozyme which was expressed in all the six samples analyzed in this study was a unique GST of p I 5.2 that was designated as GST ζ. It had a blocked N-terminal and did not correspond to any of the known three classes (α, μ, or π) of human GST as evidenced by its immunological properties and substrate specificities. The N-terminal regions of human muscle GST 5.1 and 5.0 had identical amino acid sequences except at residue 5, but demonstrated significant differences in amino acid composition and substrate specificities. These two isozymes showed homology with the μ class of human GST in their N-terminal region and were also immunologically related to the μ class of human GST although their subunit Molecular weight values ( M r 23,000) were lower than that reported for GST ψ. The substrate specificities of these isozymes were also significantly different from those of other human GST isozymes characterized so far. Significantly, muscle tissue did not express the a class of GST isozymes; however, two other isozymes were identified, GST 4.8 and GST 4.5, which had identical N-terminal amino acid sequences that were similar to that reported for the π class of human GST. GST 4.8 was present in all six samples analyzed in this study whereas GST 4.5 was present in only two of these samples, indicating a possibility of polymorphism at the GST3 locus. This study indicated the occurrence of at least three distinct isozymes in muscle tissue, providing further evidence for tissue specific expression of GST isozymes in humans.