Distribution of Glutathione S-Transferase Isoforms in Rat Liver after Induction by β-Naphthoflavone or 3-Methylcholanthrene

Abstract: Regional differences in vulnerability to xenobiotic liver damage may relate to the distribution of the detoxication capacity of the glutathione S-transferases (GST). HPLC analysis of cell lysates obtained by digitonin infusion from either the periportal or the perivenous region revealed that the content of all the GST subunits investigated (1, 2, 3, 4 and 8) was higher in the perivenous region. The strongest perivenous dominance was observed for subunit 1 (Ya) and the α class appeared to be more zonated that the μ class. A similar perivenous dominance was observed by analysis of GST activity with either 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloronitrobenzene (DCNB) or trans-4-phenyl-3-buten-2-one (PBO) as substrate. In contrast, with cumene hydroperoxide (CuOOH) or tert-butyl hydroperoxide (tBOOH) as substarate a reciprocal twofold periportal dominance was observed. Induction by pretreatment with β-naphthoflavone reduced or abolished the perivenous dominance of the α subunits 1, 2 and 8. In contrast, after pretreatment with 3-methylcholan-threne, only the acinar gradient of subunits 2 (Yc) was abolished, while the strong perivenous gradient subunit 1 (Ya) was maintained and that of subunit 8 (Yk) increased. CDNB based assays demonstrated that β-naphtoflavone treatment reduced (from 2.1 to 1.4) while 3-methyl cholanthrene enhanced (to 2.6) the perivenous/periportal GST activity ratio. Assays based on CuOOH or tBOOH indicated that neither the Se-dependent nor the Se-independent glutathione peroxidase activity nor its acinar distribution was affected by the inducers. These results demonstrated that although the expression of all investigated members of the alpha and mu classes is higher in the perivenous region, there are marked isozyme differences, the acinar gradient being particularly prominent for subunit 1 (Ya). The distinct difference in the acinar induction pattern of GST Ya between β-naphthoflavone and 3-methylcholanthrene resembles that reported for cytochrome P450 (CYP1A1 and CYP1A2), also members of the aryl hydrocarbon (Ah) receptor genes, suggesting common regionally acting regulatory elements in the expression of these genes in the liver.     

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.     

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.     

Treatment of intact hepatocytes with the calcium ionophore A23187 perturbs both the synthesis and the degradation of the second messenger cyclic AMP. Actions on adenylate cyclase and cyclic AMP phosphodiesterase activities

The presence of the calcium ionophore A23187 augmented glucagon's ability to elevate intracellular cyclic AMP concentrations in intact hepatocytes. However, when the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine (IBMX) was added to prevent the degradation of cyclic AMP then the presence of A23187 attenuated the ability of glucagon to increase intracellular cyclic AMP concentrations. Treatment of intact hepatocytes with A23187 led to a dose-dependent persistent inhibition of the glucagon-stimulated adenylate cyclase activity expressed by a membrane fraction isolated from such ionophore-treated hepatocytes. In hepatocytes where glucagon-stimulated adenylate cyclase activity was desensitized then A23187-treatment of hepatocytes failed to exert any inhibitory action on adenylate cyclase. Treatment of isolated membranes directly with A23187 did not elicit any changes in glucagon-stimulated adenylate cyclase activity. Such actions of A23187 were blunted when Ca2+ (2.5 mM) was not added to the extracellular medium. It is suggested that treatment of hepatocytes with A23187 leads to the functional uncoupling of glucagon-stimulated adenylate cyclase activity in a manner which appears to mimic the desensitization process. A23187-treatment also exerted an overall inhibitory effect on the cyclic AMP phosphodiesterase activity displayed by intact hepatocytes. Thus treatment of hepatocytes with A23187 exerted a profound effect on cyclic AMP metabolism in these cells.     

Induction of glutathione S-transferase isoenzymes in mouse liver by 5-(2-pyrazynl)-4-methyl-1,2-dithiole-3-thione (oltipraz).

Treatment of mice with a single dose of oltipraz (OPZ) at 200 mg/kg led to a significant (P less than 0.05) increase in hepatic cytosolic glutathione S-transferase (GST) activity and content. GST activity monitored with 1,2-dichloro-4-nitrobenzene was increased 3.8-fold 3 days after treatment, suggesting the induction of mu class isoenzymes. Ethacrynic acid, a marker for pi class isoforms, showed only a slight increase in GST activity while no induction was observed with cumene hydroperoxide, an indicator for the alpha class. The increase in mu class isoenzymes was further confirmed by separation of the mouse liver affinity purified GST by chromatofocusing and also by resolving the GST subunits by reverse-phase high performance liquid chromatographic procedures. Therefore, OPZ induces mainly the mu class isoenzymes in mouse hepatic tissues.     

Mechanism of chemical-induced toxicity. II. Role of extracellular calcium

Previous studies disagree as to if chemical-induced cell death is caused by the influx and accumulation of extracellular Ca2+. To determine the role of extracellular Ca2+ in toxic cell death, the viability (leakage of intracellular K+ and lactate dehydrogenase) and total Ca2+ content of isolated hepatocytes incubated in the presence or absence of extracellular Ca2+ were determined during a toxic insult with bromobenzene, ethyl methanesulfonate (EMS), Ca2+ ionophore A23187, and adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The present study utilized the dibutyl phthalate separation technique which enabled the analysis of only viable hepatocytes for changes in intracellular Ca2+ and K+ content during toxic cell injury. The three chemical treatments, bromobenzene, EMS, and ADR-BCNU, each caused an accelerated loss of viability in hepatocytes incubated without extracellular Ca2+ as compared to cells incubated with Ca2+. Furthermore, the total Ca2+ content of viable hepatocytes incubated in the presence of extracellular Ca2+ did not increase during chemically induced cell injury as compared to control cells. In fact, a significant decline in total cellular Ca2+ was observed in viable hepatocytes incubated in Ca2+-free medium during toxic cell injury. Treatment with Ca2+ ionophore A23187 was also toxic to hepatocytes incubated in the presence or absence of extracellular Ca2+. At high concentrations of ionophore (20 microM or 4 micrograms/10(6) cells), cell death was accelerated in hepatocytes incubated with Ca2+ as compared to cells incubated in Ca2+-free medium. In contrast, after treatment with lower concentrations of ionophore (10 microM or 2 micrograms/10(6) cells), the rate of cell death was reversed with hepatocytes incubated without extracellular Ca2+ dying first. Thus, depending on the concentration of A23187 and the time of exposure, the presence of extracellular Ca2+ can be shown either to accelerate or protect against cell death. Surprisingly, reversible and irreversible cell injury were not observed in hepatocytes incubated with extracellular Ca2+ and 2 microM A23187 though this treatment resulted in an 800% increase in total intracellular Ca2+ content. We conclude that chemical-induced hepatic cell death is not caused by an increase in total cellular Ca2+ resulting from the influx of extracellular Ca2+.     

Induction of class alpha glutathione S-transferases by 4-methylthiazole in the rat liver: role of oxidative stress

The expression of glutathione S-transferase (GST) is a crucial factor in determining the sensitivity of cells and organs in response to a variety of toxicants. Expression of class alpha GST genes by methyl-substituted thiazoles was assessed in the rat liver. Northern blot analysis revealed that 4-methylthiazole (4-MT) elevated rGSTA2, A3, A5 and M1 mRNAs in the liver by 19-, 4-, 6- and 9-fold at 24 h after treatment, respectively, as compared to control. Consecutive 3-day treatment with 4-MT resulted in 4- to 7-fold increases in rGSTA and M1 mRNAs. Multiple treatments with 5-methylthiazole (5-MT) caused marginal increases in GST mRNAs in spite of the large increases in certain GST mRNAs at 24 h. Either 4, 5-dimethylthiazole (DT) or 2,4,5-trimethylthiazole (TT) minimally affected the rGSTA and rGSTM mRNA expression at 1-3 day(s). Western blot analysis showed that 4-MT induced rGSTA1/2, rGSTA3/5 and rGSTM1 proteins by 2.6-, 2.1- and 2.1-fold at 3 days, respectively, while other methylthiazoles failed to induce the GST subunits. Starving rats were treated with a lower dose of methylthiazoles to study the role of oxidative stress in the mRNA expression. The levels in rGSTA2/3/5 mRNAs were significantly enhanced by 4-MT in starving rats, whereas rGSTM1/2 mRNAs were not further increased. Other methylthiazoles were inactive in enhancing the mRNAs in starving animals. Pretreatment of starving rats with either cysteine or methionine completely prevented the increases in class alpha GST mRNAs by 4-MT. Data showed that 4-MT induces class alpha GSTs with the increases in the mRNAs, whereas 5-methyl-, dimethyl- and trimethyl-substituted thiazoles were minimally active. Increases in the class alpha GST mRNAs by 4-MT may be associated with the oxidative stress in hepatocytes, as supported by starvation and sulfur amino acid experiments.     

Regulation of glutathione S-transferase enzymes in primary cultures of rat hepatocytes maintained under various matrix configurations.

Primary rat hepatocytes were cultured under various matrix and media conditions and examined after 1 week for the expression and regulation of cytosolic glutathione S-transferase (GST) enzymes. Striking effects on cell morphology were observed in relation to the different matrix conditions, whereas media effects were less prominent. Hepatocytes cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) or modified Chee's medium (MCM) maintained similar levels of total GST protein regardless of the matrix configuration or corresponding cell integrity. However, HPLC analysis showed a differential expression pattern of individual GST subunits in both a time- and medium-dependent fashion. A variable, but pronounced, matrix and medium effect was observed on the induction of total GST expression by various prototypical inducers. Dexamethasone (10 microM) induced subunits A2, M1 and M2 in a medium- and matrix-dependent fashion, whereas phenobarbital (100 microM) induced significantly only subunit A2. beta-Naphthoflavone (50 microM) suppressed all GST subunit expression except subunit P1, which was induced in a matrix- and medium-dependent fashion. These studies show that total basal level expression of GSTs in vitro is reflective of a concomitant increase in mu and pi class subunits and a decrease in alpha class subunits. Moreover, the matrix and medium conditions influence both the basal and inducible expression of GST subunits in cultured rat hepatocytes.     

Cyclosporin A protects hepatocytes against prooxidant-induced cell killing. A study on the role of mitochondrial Ca2+ cycling in cytotoxicity.

Cyclosporin A (CsA) is a potent inhibitor of the prooxidant-induced release of Ca2+ from isolated mitochondria. In this investigation, pretreatment of hepatocytes with CsA before exposure to the prooxidants tert-butyl hydroperoxide (tBH), cumene hydroperoxide or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) prevented the loss of cell viability. HPLC analysis of adenine and pyridine nucleotide concentrations in hepatocytes treated with 3,5-Me2-NAPQI showed a rapid depletion of ATP prior to the loss of cell viability versus the maintenance of near control levels of ATP in hepatocytes treated with CsA before 3,5-Me2-NAPQI. In 3,5-Me2-NAPQI-exposed hepatocytes there was also a rapid loss of cellular NAD+ which could be accounted for initially by a transient increase in NADP+. Measurement of the intracellular Ca2+ pools showed an early depletion of the mitochondrial Ca2+ pool in hepatocytes exposed to 3,5-Me2-NAPQI, tBH or cumene hydroperoxide; this loss was prevented by CsA. In conclusion, these results show that CsA protected hepatocytes from prooxidant injury by preventing mitochondrial Ca2+ cycling and subsequent mitochondrial dysfunction. This suggests that in prooxidant injury, excessive Ca2+ cycling is an early and important event leading to mitochondrial damage and subsequently to cell death.     

Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress

To investigate the relationship between alterations of cytosolic Ca2+ concentration and development of cytotoxicity, isolated rat hepatocytes were loaded with the fluorescent indicator Quin-2 AM and then incubated with non-toxic or toxic levels of menadione (2-methyl-1,4-naphthoquinone) or tert-butyl hydroperoxide (t-BH). The resulting changes in cytosolic Ca2+ concentration were compared to those seen upon exposure of the hepatocytes to an alpha 1-adrenergic agonist, phenylephrine, as well as to those induced by menadione and t-BH in hepatocytes pretreated with agents that modify their toxicity. Exposure of hepatocytes to phenylephrine or non-toxic levels of menadione caused a moderate and transient increase in cytosolic Ca2+ (less than or equal to 0.7 microM), whereas a toxic concentration of menadione produced a marked, sustained increase in Ca2+ which fully saturated the binding capacity of Quin-2 (greater than 1.5 microM). Treatment of the hepatocytes with the protective agent, dithiothreitol, prevented both the increase in cytosolic Ca2+ and the cytotoxicity induced by menadione. On the other hand, pretreatment of cells with diethylmaleate to deplete intracellular glutathione made otherwise non-toxic concentrations of menadione cause both a sustained increase in cytosolic Ca2+ and cytotoxicity. Similarly, toxic concentrations of t-BH also caused a sustained increase in cytosolic Ca2+. The iron chelator, desferrioxamine, and dithiothreitol (DTT), which protected the cells from t-BH toxicity, also prevented the sustained elevation of cytosolic Ca2+. Our findings provide further support for the hypothesis that a perturbation of intracellular Ca2+ homeostasis is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress.     

Class I and IV antiarrhythmic drugs and cytosolic calcium regulate mRNA encoding the sodium channel alpha subunit in rat cardiac muscle.

Previous studies have shown that chronic in vivo treatment with the antiarrhythmic drug mexiletine produces an increase in sodium channel number. We examined whether chronic mexiletine treatment would similarly regulate the level of mRNA encoding the cardiac sodium channel. RNA isolated from cardiac tissue was probed with a 2.5-kilobase cRNA transcribed with T7 RNA polymerase from the clone Na 8.4, which encodes nucleotides 3361-5868 of the alpha subunit of the RIIA sodium channel subtype. Chronic mexiletine treatment produced a 3-fold increase in the level of mRNA encoding sodium channel alpha subunits. Previous studies of cultured skeletal muscle cells had suggested that chronic sodium channel blockade may mediate an increase in sodium channel mRNA by changes in cytosolic Ca2+ concentration. To address this issue, we assessed whether verapamil would also produce up-regulation of the level of mRNA encoding the sodium channel and whether the calcium ionophore A23187 would produce the opposite effect on mRNA level. Verapamil treatment increased sodium channel mRNA level up to 3-fold, whereas in vitro A23187 treatment decreased the mRNA level 5-fold. The combination of verapamil and mexiletine produced no further increase in the mRNA level, compared with that seen with the single agents, suggesting a convergent second messenger pathway for the actions of these two drugs. These data show that the level of mRNA encoding sodium channels is substantially increased during antiarrhythmic drug treatment and suggest that change in cytosolic Ca2+ concentration is the second messenger involved in the regulation of levels of mRNA encoding the alpha subunit of the cardiac sodium channel.     

Increased formation of phosphatidic acid induced with vasopressin or Ca2+ ionophore A23187 in rat hepatocytes

The effects of vasopressin and Ca2+ ionophore A23187 on phospholipid metabolism were investigated in rat hepatocytes. Vasopressin stimulated the incorporation of [32P]Pi into phosphatidic acid within 2 min but then it returned to control level after 10 min. On the other hand, the stimulation of the incorporation of [32P]Pi into phosphatidylinositol continued with incubation times up to 20 min. The Ca2+ ionophore A23187 also increased the 32P-labeling in phosphatidic acid, although it had no effect on [32P]Pi incorporation into phosphatidylinositol. Concerning the incorporation of [3H]glycerol, vasopressin did not enhance its incorporation into phosphatidic acid and phosphatidylinositol. The Ca2+ ionophore A23187 increased the incorporation into phosphatidic acid without significant effects on that into phosphatidylinositol. In the hepatocytes prelabeled with [3H]arachidonic acid, stimulated degradation of phosphatidylinositol with the addition of vasopressin and resultant formation of phosphatidic acid were observed within 5 min. The transient accumulation of diacylglycerol, the product of phosphatidylinositol hydrolysis, also occurred within 5 min with vasopressin. On the other hand, with the Ca2+ inophore A23187, stimulated degradation of triacylglycerol to diacylglycerol and the consequent formation of phosphatidic acid were observed. The Ca2+ ionophore A23187 caused a significant release of free [3H]arachidonic acid, although vasopressin had no effect.     

Induction by the calcium ionophore A23187 of a protective effect against cell injury in cultured gastric mucosal cells

An intrinsic protective mechanism against cell injury seems to exist in cultured gastric mucosal cells. Cells, isolated from the stomachs of 10- to 12-day-old rats and subcultured, were examined for damage by the erythrosine B dye exclusion test. Pretreatment with 5 microM A23187 (a calcium ionophore) diminished the cell damage induced by acidified medium (pH 3.5) or 8 mM aspirin (pH 5.0). The effect of A23187 appeared 4 hr after its addition and was reversible. Protection by A23187 against cell injury diminished in the absence of extracellular Ca2+ and was dependent on Ca2+ concentration. An increase in intracellular Ca2+ may induce cell resistance against injury in cultured gastric mucosal cells.     

Protection of rat hepatocytes from tert-butyl hydroperoxide-induced injury by catechol

Metabolism of tert-butyl hydroperoxide (TBHP, 2.0 mM) by glutathione peroxidase within isolated rat hepatocytes caused a rapid oxidation of intracellular reduced glutathione and ultimately NADPH through glutathione reductase. TBHP also caused the formation of surface blebs in the hepatocyte plasma membrane followed by the leakage of cytosolic enzymes, such as lactate dehydrogenase, into the incubation medium. Catechol (0.1 mM) protected hepatocytes from the cytotoxic effects of TBHP but did not prevent the rapid oxidation of glutathione indicating normal metabolism of TBHP through glutathione reductase. In contrast, addition of catechol to the hepatocyte incubations prevented TBHP-induced depletion of intracellular NADPH and increased the total NADP+ + NADPH concentration without altering significantly the intracellular NADP+ content or the NADPH/NADP + NADPH ratio. Catechol did not alter TBHP stimulation of the pentose phosphate pathway. Hepatocytes incubated with sublethal concentrations of TBHP (1.0 mM) did not leak lactate dehydrogenase into the medium but did lose intracellular potassium. In these experiments, TBHP caused a sustained increase in phosphorylase alpha activity suggesting that TBHP metabolism may be associated with a sustained increase in cytosolic free Ca2+. In the presence of catechol, phosphorylase alpha activity was increased by 5 min but returned toward control by 20 min. These data suggest that catechol may be protecting hepatocytes from TBHP-induced injury by preventing a sustained rise in cytosolic free Ca2+ concentration.     

Glucocorticoid regulation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferases, and NAD(P)H:quinone oxidoreductase in cultured fetal rat hepatocytes

The regulation of polycyclic aromatic hydrocarbon-inducible enzymes, cytochrome P450IA1, NAD(P)H:quinone oxidoreductase, and glutathione S-transferases, by glucocorticoids was investigated using primary fetal rat hepatocyte culture. Treatment of cells in culture with 1,2-benzanthracene (100 microM, 72 hr) resulted in 60-, 2-, and 6-fold increases in cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone reductase activities, respectively. The inductive effect of 1,2-benzanthracene on cytochrome P450IA1 and glutathione S-transferase (1-chloro-2,4-dinitrobenzene conjugation) activities was potentiated approximately 3- and 2- to 3-fold, respectively, when dexamethasone (0.01-1 microM) was included in the culture medium. In contrast, 1 microM dexamethasone was found not to potentiate the induction of NAD(P)H:quinone oxidoreductase activity by 1,2-benzanthracene. Treatment of cultured hepatocytes with dexamethasone alone, at concentrations of up to 100 microM, resulted in a 2- to 4-fold increase in glutathione S-transferase and NAD(P)H:quinone oxidoreductase activity. Both the induction of glutathione S-transferase activity by high concentrations of dexamethasone alone and the potentiation of 1,2-benzanthracene induction by lower concentrations of dexamethasone were observed for other steroids of the glucocorticoid class in conjunction with a variety of polycyclic aromatic hydrocarbons. Western immunoblot analyses indicated that low concentrations of dexamethasone (0.1-1 microM) potentiated 1,2-benzanthracene-dependent induction of cytochrome P450IA1, glutathione S-transferase Ya/Yc subunit and NAD(P)H:quinone oxidoreductase content. Additionally, increased glutathione S-transferase activity in response to concentrations of dexamethasone exceeding 1 microM was associated with concomitant increases in Ya/Yc and Yb subunit content. Potentiation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone oxidoreductase protein content by low concentrations of glucocorticoids and induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase by high concentrations of glucocorticoids alone indicates the importance of these endogenous compounds in the regulation of some hepatic enzymes involved in xenobiotic metabolism.     

Hepatocytes isolated from preneoplastic rat livers are resistant to ethacrynic acid cytotoxicity

Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics, such as several cytostatic drugs, through conjugation with glutathione (GSH). Pi class GST (GST P) liver expression is associated with preneoplastic and neoplastic development and contributes with the drug-resistance phenotype. Ethacrynic acid (EA) is an inhibitor of rat and human GSTs. In addition, causes lipid peroxidation in isolated rat hepatocytes. Therefore, we decided to evaluate the role of the GST/GSH system in isolated hepatocytes from preneoplastic rat livers (IP) in the presence of EA and determine the cytotoxicity of the drug. Our results showed a resistance to the toxic effects of EA since viability and cellular integrity values were significantly higher than control. Initial levels of thiobarbituric acid reactive substances (TBARS) in IP hepatocytes were significantly higher than control and the presence of EA did not change TBARS levels. A diminution in intracellular total GSH was observed by treating with EA isolated hepatocytes from both groups. However, the initial total GSH levels were higher in IP hepatocytes than in control. Immunoblotting analysis showed the presence of GST P in IP animals only. Although alpha and mu class isoenzymes levels were decreased in IP hepatocytes, total GST activity was 1.5-fold higher than in control. In addition, multidrug-resistance protein 2 (Mrp2) showed fivefold decreased levels in IP hepatocytes. In conclusion, increased total GSH, decreased Mrp2 levels and the presence of GST P could be critical factors involved in the resistance of IP hepatocytes to the toxicity of EA.     

Elevation of pi class glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins

Increased expression of the glutathione S-transferase (GST; E.C.2.5.1.18) pi class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST pi and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalovirus immediate-early promoters. These GST pi expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST pi. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST pi. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumene hydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST pi. Southern blot analysis demonstrated the incorporation of the GST pi expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST pi RNA that was slightly larger than the endogenous GST pi RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5' leader sequence of the expression vector. The effect of increased GST pi activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-epoxide, as well as to ethacrynic acid (3.1-to 4.4-fold). Although increased GST pi expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells.

The modulation of garlic oil (GO) and three allyl compounds, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), on the antioxidation system in rat livers and red blood cells was examined. Rats were orally administered GO (200 mg/kg body weight), DAS (20, 80 mg/kg body weight), DADS (80 mg/kg body weight) or DATS (70 mg/kg body weight) three times a week for 6 weeks. Control rats received corn oil (2 ml/kg body weight) alone. GO, DADS and DATS treatment significantly increased the glutathione (GSH) content (48-84%) in red blood cells (P < 0.05). DATS displayed a greater enhancement than GO and DADS (P < 0.05). Hemolysis induced by tert-butyl hydroperoxide was not suppressed by GO or allyl compound treatment although higher GSH content was evident. Hepatic GSH was not influenced by garlic components. In rat livers, DADS and DATS significantly increased the activity of GSH reductase (46 and 54%, respectively) and of GSH S-transferase (GST) (63 and 103%, respectively), but decreased the GSH peroxidase activity (27 and 28%, respectively). In contrast, GSH reductase and GST activities in the DAS group, either 20 or 80 mg/kg body weight, were similar to the control group. A decrease of GSH peroxidase activity was observed in rats dosed with 80 mg/kg body weight (P < 0.05). An increase in GST activity and a decrease in GSH peroxidase activities were also noted in GO-treated rats (P < 0.05). In red blood cells, three GSH-related antioxidant enzyme activities were not affected by garlic oil and its organosulfur components. Immunoblot assay showed that, accompanying the increase in hepatic GST activity, GO, DADS, DAS (80 mg/kg body weight) and DATS increased the expression of GST Ya, Yb1 and Yc proteins. Results indicate that GO and three allyl compounds play a differential role in modulation of the GSH-related antioxidant system in rat livers and red blood cells.