Purification and characterization of hepatic glutathione transferases from an insectivorous marsupial,the brown antechinus (Antechinus stuartii)

1. Five unique glutathione transferase isoenzymes were purified from the hepatic cytosol of an insectivorous marsupial, the brown antechinus. The purified GSTs were characterized by structural and catalytic properties including apparent molecular weight andisoelectricpoint,specificity towards modelsubstrates,kineticparameters,sensitivityto inhibitors and cross-reactivity with antisera raised against human GSTs. 2. An alpha class GST, Antechinus GST 1-1, predominated in the hepatic cytosol, representing 71% of the total GST purified. The substrate specificity of Antechinus GST 1-1 was similar to that of other alpha class GSTs, particularly with respect to its high activity with cumene hydroperoxide. The mu class was represented by three GST isoenzymes, Antechinus GST 3-3, GST 3-4 and GST 4-4. These isoenzymes represented 8, 2 and 10% of the total GST purified respectively. A single GST, Antechinus GST 22, belonged to the pi class of GSTs and represented 12% of the total GST purified. The hepatic GST isoenzyme ratio (by class) observed in the brown antechinus was more similar to that observed in the human than in rat. 3. A previous study investigating a herbivorous marsupial, the brushtail possum (Trichosurus vulpecula) also identified a predominant hepatic GST belonging to the alpha class and displaying peroxidase activity. The evolutionary conservation of a similar predominant GST isoenzyme in these marsupials suggests that they play an important role in the detoxication metabolism of these unique mammals.     

Selective expression of the three classes of glutathione S-transferase isoenzymes in mouse tissues

The suitability of mouse as an animal model for studying the glutathione S-transferase (GST)-mediated detoxification mechanisms has been studied by analyzing the expression of the alpha, mu, and pi classes of glutathione S-transferase isoenzymes in mouse brain, heart, kidney, spleen, liver, and muscle. Individual isoenzymes from each of these tissues have been purified, characterized, and classified into the three known classes of GST. These studies demonstrate that GST isoenzymes are variably expressed in different mouse tissues, suggesting that their expression is tissue specific. A major isoenzyme, belonging to the pi class, with a pI value in the range of 8.6-9.1 and an approximate subunit Mr value of 22,500 was detected in each tissue investigated in this study. A variable number of mu class isoenzymes with subunit Mr values of 26,500 were expressed in all mouse tissues studied, except spleen and muscle. Only liver and kidney showed the expression of an alpha class isoenzyme, each having a basic pI value and subunit Mr of approximately 25,000. Another minor acidic alpha class isoenzyme, also with a subunit Mr value of 25,000, was detected in liver, kidney, and brain. While multiple GST isoenzymes were detected in all other tissues studied, only spleen showed the presence of a single isoenzyme, which belonged to the pi class. These results reveal considerable differences in the GST isoenzyme composition of mouse tissues as compared to rat and human tissues. However, several apparent similarities in mouse and human tissues exist, suggesting that the mouse model can be used to analyze the GST-mediated detoxification mechanisms in humans.     

Differences between rats and rabbits in hepatic cytosolic glutathione S-transferase expression in response to nitrogen heterocycle and other inducers.

Glutathione S-transferase (GST) expression was examined in hepatic cytosol from rats and rabbits treated with 4-picoline, pyrrole, pyridine, pyrazine, imidazole, or piperidine using enzymatic activity, SDS-PAGE, and immunoblot analyses and the results were compared to those obtained with phenobarbital and 3-methylcholanthrene. SDS-PAGE and immunoblot analyses of hepatic cytosol prepared from rats treated with pyrazine revealed the induction of class alpha (Ya and Yc) and mu (Yb) bands with a corresponding 2.4-fold increase in metabolic activity using 1-chloro-2,4-dinitrobenzene as substrate. A new class alpha band migrating in the region of the Yc band was observed in the SDS-PAGE and detected in the immunoblot of cytosol from pyrrole-treated rats, whereas treatment with 4-picoline, imidazole, or piperidine failed to alter the expression of the major classes of GST isozymes in this species. SDS-PAGE and immunoblot analyses of rabbit hepatic cytosol revealed a unique species-dependent difference in the expression of GSTs. While phenobarbital and 3-methylcholanthrene induce class alpha and mu GST expression in rat hepatic cytosol, one of the most interesting observations was that neither of these agents stimulated GST expression in the rabbit. Immunoblot analysis of cytosol isolated from 4-picoline-treated rabbits using GST class alpha-specific IgG showed the appearance of a novel class alpha 28-kDa GST band and the concomitant disappearance of a class alpha 29-kDa GST band. In addition, SDS-PAGE and immunoblot analyses showed that treatment of rabbits with pyrrole, pyrazine, imidazole, or piperidine resulted in the disappearance of this class alpha 29-kDa GST band with no detectable expression of the class alpha 28-kDa GST band; the level of the class alpha 29-kDa band was unaffected by pyridine treatment. In contrast, immunoblot analyses of hepatic cytosol revealed that a 25.5-kDa class mu GST band disappeared following treatment with pyridine, but was unaffected by treatment with other nitrogen heterocycles. The Vmax of glutathione conjugation to the substrate 1-chloro-2,4-dinitrobenzene decreased by 52, 36, 59, 41, 37, and 32% in hepatic cytosol isolated from 4-picoline-, pyrrole-, pyridine-, pyrazine-, imidazole-, and piperidine-treated rabbits, respectively. The results suggest that nitrogen heterocycles differ in their ability to modulate glutathione S-transferase isozyme expression in rat and rabbit hepatic tissue and that rabbit hepatic GSTs are refractory to induction by agents such as pyrazine, phenobarbital, or 3-methylcholanthrene and hence these xenobiotics do not appear to be bifunctional inducers in this species.     

Inhibition of human glutathione S-transferases by bile acids

Glutathione S-transferase (GST) isoenzymes isolated from various human tissues are differentially inhibited by bile acids. Trihydroxy bile acid (lithocholate) was found to be more inhibitory to all the human GST isoenzymes tested in this study, as compared to the monohydroxy (cholate) and dihydroxy (chenodeoxycholate) bile acids. Among the three major classes of GST, mu class isoenzymes are generally inhibited to a greater extent than the alpha and pi class isoenzymes. The results of this study also indicate that differential inhibition of GST by various bile acids may be used to distinguish closely related GST isoenzymes within the mu class of GST isoenzyme. Likewise, the pi class or the anionic isoenzymes of human kidney, placenta, and erythrocytes can be distinguished using bile acid inhibition studies. These studies also provide further support for tissue-specific expression of GST isoenzymes in humans.     

Mechanisms of inactivation of Schistosoma mansoni and mammalian glutathione S-transferase activity by the antischistosomal drug oltipraz.

Glutathione S-transferase (GST) purified from Schistosoma mansoni or human placenta was inhibited by the antischistosomal drug oltipraz (OPZ) in a time- and concentration-dependent manner. Inhibition of placenta GST was complete at a low concentration of drug, whereas that of parasite GST was incomplete and relatively high amounts of OPZ were needed to reach 50% inhibition. Complete reactivation of GST from placenta was achieved with dithiothreitol (DTT) and other sulfhydryl-containing compounds, while the inactivation of parasite GST was irreversible. The oxy-derivative of OPZ (RP 36,642), in which the thione sulfur is replaced with oxygen, did not inhibit GST activity. There were no differences between OPZ and RP 36,642 in their patterns of binding to the hydrophobic non-substrate site of GST. GST from the placenta incorporated much higher levels of [14C]N-ethylmaleimide compared to schistosome GST. The incorporation of [14C]N-ethylmaleimide by GST was inhibited by OPZ but not by RP 36,642. Yeast and S. mansoni hexokinases were similarly inhibited by OPZ but not by RP 36,642. Both hexokinase preparations recovered their activity following incubation with DTT. These data suggest that the inactivation of these enzymes by OPZ is a result of its interaction with their SH groups. Thus, the antischistosomal activity of OPZ may be accounted for by its interaction with the SH groups of macromolecules in general.     

Differential effects of oltipraz and its oxy-analogue on the viability of Schistosoma mansoni and the activity of glutathione S-transferase

Adult worms of Schistosoma mansoni recovered from mice treated with oltipraz (OPZ) showed a significant diminution in their ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan, a measure of parasite viability. Incubation of glutathione S-transferase (GST) from S. mansoni with OPZ resulted in a time- and concentration-dependent inhibition of enzyme activity. RP 36,642 (an inactive oxy-derivative of OPZ) had a minimal effect on the viability of the worms and no effect on GST activity. The structural integrity of OPZ, particularly the thione sulphur, appears to be necessary for expression of the antischistosomal effects of the drug. OPZ-induced inhibition of GST was non-competitive with either reduced glutathione (GSH) or 1-chloro-2,4-dinitrobenzene (CDNB), indicating that the drug is not a substrate for GST-catalysed conjugation reactions. In addition, the inhibition of GST could not be reversed by dialysis or repurification of the enzyme via a GSH-agarose affinity column. The effects of OPZ on GST activity could render the parasite vulnerable to damage by host-derived reactive oxygen species and aldehydic products of lipid peroxidation. The effects of OPZ on GST activity may play a role in the antischistosomal action of OPZ.     

Differential induction of rat hepatic glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate. Comparison with induction by phenobarbital and 3-methylcholanthrene

Male Wistar rats were treated with hexachlorobenzene, benzyl isothiocyanate, phenobarbital or 3-methylcholanthrene. Hepatic cytosolic glutathione S-transferase (GST) activity was determined with the substrates 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one. Cytosolic glutathione peroxidase activity was measured with cumene hydroperoxide. GST activity toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and ethacrynic acid was enhanced by all compounds, hexachlorobenzene and 3-methylcholanthrene causing the largest and the smallest increase respectively. Trans-4-phenyl-3-buten-2-one-conjugating activity exhibited only small changes, while peroxidase activity with cumeme hydroperoxide was not changed by any of the inducing agents. GST isoenzymes were purified on S-hexylglutathione Sepharose 6B and separated by means of FPLC-chromatofocusing, to evaluate effects on the GST isoenzyme pattern. Hexachlorobenzene and phenobarbital both caused an increase in the relative amounts of subunits 1 and 3 when compared with subunits 2 and 4 respectively. For 3-methylcholanthrene only induction of subunit 1 was observed, possibly due to the relatively low induction levels of total GST activity. In benzyl isothiocyanate-treated animals, an induction of subunit 3 was found as well as an increase in the relative amount of subunit 2. Thus, benzyl isothiocyanate behaves differently from hexachlorobenzene, phenobarbital and 3-methylcholanthrene as an inducing agent of rat hepatic glutathione S-transferases.     

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.     

Glutathione transferases in human nasal mucosa

Glutathione transferase (GST) was investigated with 1-chloro-2,4-dinitrobenzene as substrate in tissues speciments of human nasal mucosa. The average ±(SD) of GST activity in the cytosol was 76.8 ±21 nmol/min/mg with a range of 47–113. Using affinity chromatography and isoelectric focusing, the isozymes of GST from human nasal mucosa have been purified and characterized. On the criteria of isoelectric point, substrate specificities, apparent subunit molecular weight, sensitivity to characteristic inibitors and immunological properties the major GST purified (about 85% of total activity) can be identified as class pi GST. Although a limited amount of class alpha GST was expressed by human nasal mucosa, no class mu isoenzymes was noted. In addition, we have also identified a GST subunit that cannot be related to any of three major classes of human GST.     

Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide

As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.     

Glutathione conjugation of styrene 7,8-oxide enantiomers by major glutathione transferase isoenzymes isolated from rat livers

Male Sprague-Dawley rat liver cytosol mediated regioselective conjugation of styrene 7,8-oxide (STO) enantiomers with glutathione in completely trans-ring-opening manner to afford (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2R)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 22:1 for (R)-STO and also to afford (1R)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 12:1 for (S)-STO. In the above cytosolic reactions, (R)-STO was conjugated 1.8 times faster than (S)-STO, while the (R)- to (S)-ratio in rate of the conjugation was 2.7 when racemic STO was used as a substrate. A kinetic study, carried out by using six major glutathione transferase (GST) isoenzymes isolated from the cytosol, indicated that GSTs 3-3, 3-4 and 4-4 (class mu enzymes) had much higher Kcat/Km values towards both STO enantiomers than the other three major isoenzymes, GSTs 1-1, 1-2 and 2-2 (class alpha enzymes). All the class mu enzymes mediated preferential glutathione conjugation of (R)-STO to (S)-STO. On the contrary, the class alpha enzymes catalysed the conjugation of (S)-STO preferentially to (R)-STO. The kinetic study strongly suggested that GSTs determining the higher enantioselectivity towards (R)-STO in the rat liver cytosol were the class mu enzymes, especially GST 3-3, which had the highest Kcat/Km value towards (R)-STO as well as the highest (R) to (S) ratio in the enantioselectivity among the six isoenzymes examined. GST 7-7, isolated as a major enzyme from the liver cytosol of the animals bearing hepatic hyperplastic nodules which were induced by chemical carcinogens, catalysed preferential GSH conjugation of (S)-STO to (R)-STO.     

Glutathione transferase theta 1-1-dependent metabolism of the water disinfection byproduct bromodichloromethane

Bromodichloromethane (CHBrCl(2)), a prevalent drinking water disinfection byproduct, was previously shown to be mutagenic in Salmonella that express rat GSH transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the kinetics of CHBrCl(2) reactions mediated by GST in different species as well as the isoform specificity and reaction products of the GST pathway. Conjugation activity of CHBrCl(2) with GSH in mouse liver cytosol was time- and protein-dependent, was not inhibited by the GST alpha, mu and pi inhibitor S-hexyl-GSH, and correlated with GST T1-1 activity toward the substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. Conjugation activities in hepatic cytosols of different species toward CHBrCl(2) followed the order mouse > rat > human. As compared with CH(2)Cl(2), the catalytic efficiency (k(cat)/K(m)) of conjugation of CHBrCl(2) with GSH by pure recombinant rat GST T1-1 was approximately 3-6-fold less. Taken together, this suggests that GST T1-1 is the primary catalyst for conjugation of CHBrCl(2) with GSH and that flux through this pathway is less than for CH(2)Cl(2). The initial GSCHCl(2) conjugate formed was unstable and degraded to several metabolites, including GSCH(2)OH, S-formyl-GSH, and HCOOH. Addition of NAD(+) to cytosol did not alter the rate of conjugation of CHBrCl(2) with GSH; however, it did increase the amount of [(14)C]HCOOH produced ( approximately 10-fold). A similar result was seen in a reaction containing pure rat GST T1-1 and GSH-dependent formaldehyde dehydrogenase, indicating that GSCH(2)OH was formed as a precursor to S-formyl-GSH. The half-life of synthetic S-formyl-GSH in pH 7.4 buffer was approximately 1 h at ambient temperature and decreased to approximately 7 min in pH 9.0 buffer, and it does not react with deoxyguanosine. In conclusion, GST T1-1 conjugation of CHBrCl(2) has been definitively demonstrated and the kinetics of conjugation of CHBrCl(2) with GSH characterized in mouse, rat, and human hepatic cytosols. The significance of this GST pathway is that reactive GSH conjugates are produced resulting in possible formation of DNA adducts. Comparisons with CH(2)Cl(2) suggest that the reactive intermediates specific to GSH conjugation of CHBrCl(2) are more mutagenic/genotoxic than those derived from CH(2)Cl(2).     

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 80mg/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 80mg/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.     

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

The effects of altered Ca2+ 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 Ca2+ 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 alpha and mu specific antibodies showed an approximately 1.6- and 1.5-fold increase in the class alpha, Ya and Yc subunits, respectively, whereas no significant increase (approximately 1.2-fold) in class mu GST expression was observed following A23187 treatment. Northern blot analysis revealed an approximately 5-fold increase in GST class alpha and an approximately 7-fold increase in class mu 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. Immunoblot analysis showed a significant increase in the expression of GST class alpha, Ya and Yc subunits, approximately 1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class mu GSTs. Northern blot analysis showed approximately 3- and 2-fold increases, respectively, in class alpha and mu GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca2+ homeostasis produced by either Ca2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.     

Direct comparison of the nature of mouse and human GST T1-1 and the implications on dichloromethane carcinogenicity

Dichloromethane (DCM) is a hepatic and pulmonary carcinogen in mice exposed to high doses by inhalation. It has been shown previously that the incidence of liver and lung tumors does not increase in rats or hamsters exposed to the dihaloalkane under conditions similar to those that produced tumors in mice. The biological consequences of DCM exposure to humans is therefore uncertain. The carcinogenic effects of DCM in the mouse are caused by the interaction with DNA of a glutathione (GSH) conjugate that is produced by the class theta glutathione S-transferase T1-1 (GST T1-1). The species specificity is thought to be due to the greater amount of transferase activity in mouse target organs and specific nuclear localization of GST T1-1 in target cells. This paper directly compares the relative capacity and locality of DCM activation in mouse and human tissues. The results show that mouse GST T1-1 is more efficient in catalyzing the conjugation of DCM with GSH than the orthologous human enzyme. In addition, the mouse expresses higher levels of the transferase than humans in hepatic tissue. Histochemical analysis confirmed the presence of GST T1-1 in the nucleus of mouse liver cells. However, in human liver GST T1-1 was detected in bile duct epithelial cells and hepatocyte nuclei but was also present in the cytoplasm. Taking this information into account, it is unlikely that humans have a sufficiently high capacity to activate DCM for this compound to be considered to represent a carcinogenic risk.     

Expression of human mu or alpha class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents.

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.     

Effects of ethanol on glutathione conjugation in rat liver and lung

The ability of ethanol to alter glutathione (GSH) conjugation and its dependence upon duration of administration were investigated in rats in correlation with lipid peroxidation and the induction of microsomal enzymes. Significant decreases in hepatic GSH and glutathione-S-transferase (GST) activity in both liver and lung were found in rats treated acutely with ethanol (4 g/kg body weight 6 hr prior to killing). These decreases were accompanied by an increased loss of both GSH and GST into the plasma and increased hepatic lipid peroxidation. On the other hand, there was a dose-dependent increase in hepatic GSH after chronic administration of ethanol in drinking water (5 and 10%) for 3 weeks. This increase in hepatic GSH may be due to increased synthesis of GSH in the liver. No significant induction of GST by chronic ethanol treatment was observed in either organ. Ethanol was compared with the well-known inducers phenobarbital and beta-naphthoflavone. Although there was some evidence of increases in lipid peroxidation and/or microsomal enzyme activity with the inducers, no simple link between these increases and the induction of GST activity was identified.