Effects of aging on hepatic and pulmonary glutathione S-transferase activities in male and female Fischer 344 rats

The effects of aging on cytosolic glutathione S-transferase activities were evaluated with liver and lung cytosol from male and female Fischer 344 rats 3, 12, and 24 months of age. Age-related changes were tissue-, sex-, and substrate-specific. With liver and lung cytosol from both males and females, rates of metabolism of 1,2-epoxy-3-(p-nitrophenoxy)propane and p-nitrobenzyl chloride were lower in the old group than in the young group; however, patterns of decrease differed with tissue and sex. With 1,2-dichloro-4-nitrobenzene, metabolism was affected by aging only in liver and lung cytosol from males. Finally, with 1-chloro-2,4-dinitrobenzene, metabolic rates were altered during aging only with liver cytosol from females. However, the apparent Km was higher with liver cytosol from old males; those values from lung cytosol of males and liver or lung cytosol from females were unchanged. These data indicate that changes in the cytosolic glutathione S-transferase isozymes occurred during aging.     

Effect of garlic on the hepatic glutathione S-transferase and glutathione peroxidase activity in rat

It was attempted to observe the effect of garlic on the hepatic glutathione s-transferase and glutathione peroxidase activity in this study. Glutathione s-transferase (EC 2.5.1.18) are thought to play a physiological role in initiating the detoxication of potential alkylating agents, inclnding pharmacologically active compounds. Glutathione peroxidase (EC 1.11.1.9) might play an important role in the protection of cellular structures against oxidative challenge. The activities of glutathione s-transferase and glutathione peroxidase in rat liver were increased by the treatment of garlic juice. Allicin fraction, heat-treated allicin fraction and garlic butanol fraction markedly inhibited glutathione s-transferase activityin vitro, whereas glutathione peroxidase activity was significantly increased in heat-treated allicin fraction and garlic butanol fraction.     

Modification by phenobarbital of decreased glutathione content and glutathione S-transferase activity in livers of lead-treated mice.

Lead acetate (100 mg/kg) administered i.p. to male mice decreased hepatic glutathione (GSH) content and also glutathione S-transferase (GST) activity. However, the liver GSH content of mice treated with both lead and phenobarbital (80 mg/kg, i.p.) remained unchanged, whereas their GST activities were higher than the controls. Phenobarbital antagonized the Pb-induced decrease in liver adenosine triphosphate content. Additionally, phenobarbital shortened the half-life of hepatic GSH determined using buthionine sulfoximine, an inhibitor of GSH synthesis. Acceleration of hepatic GSH turnover by phenobarbital possibly diminishes the Pb-induced impairment of GSH-conjugation of xenobiotics.     

Effect of monensin on liver glutathione, glutathione S-transferase and monooxygenases in rats.

Monensin administered ip to male rats at a dosage of 2.5 mg/kg/d for 3 consecutive days did not change the liver levels of glutathione, but depressed significantly the amount of cytochrome P-450 and the activities of aniline hydroxylase and a cytosolic CDNB-specific glutathione S-transferase. There was a marked decrease in the aminopyrine N-demethylase activity and a significant increase in the pentobarbital sleeping time in rats treated with monensin. In contrast, no change in these parameters was found 2 h after a single ip dose (7.5 mg/kg) of monensin. The results suggest that monensin-induced inhibition of the liver cytosolic glutathione S-transferase and microsomal monooxygenases is non-specific.     

Impairment of hepatic glutathione S-transferase activity as a cause of reduced biliary sulfobromophthalein excretion in clofibrate-treated rats

Administration of clofibrate reduced the maximal excretion rate of bile sulfobromophthalein (BSP) in rats but left that of phenol-3,6-dibromophthalein (DBSP) unchanged. This decrease in liver transport of BSP was due to reduced bile excretion of conjugated BSP. Hepatic uptake and storage of this dye were not impaired. Liver glutathione S-transferase activity in vitro, measured with BSP, 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2, 4-dinitrobenzene (CDNB) was significantly reduced. This alteration in liver conjugating activity was probably not related to a modification of the hepatic GSH pool, since the GSH level was unchanged or only increased slightly after clofibrate treatment. Detection of this inhibition required at least two daily doses of clofibrate. Inhibition was dose-related and lasted for several days after cessation of the drug. In clofibrate-treated rats, Lineweaver-Burk plots showed a reduced Vmax for both the BSP and GSH substrates. These results suggest that clofibrate decreases hepatobiliary transport of BSP by lowering glutathione S-transferase activity in the liver.     

Quantification of individual glutathione S-transferase isozymes in hepatic and pulmonary tissues of naphthalene-tolerant mice

Acute exposure to naphthalene produces severe bronchiolar epithelial cell necrosis in mice, whereas subchronic exposure to naphthalene (200 mg/kg/7 days) fails to produce epithelial necrosis and renders the animals tolerant to subsequent challenge doses of naphthalene. Mechanisms responsible for the development of tolerance have not been delineated. The few studies exploring naphthalene tolerance focus on expression of microsomal enzymes and have yet to delve into expression of the hepatic detoxification enzymes such as glutathione S-transferases (GSTs; EC 2.5.1.18). Glutathione conjugation catalyzed by GSTs accounts for one of the two primary routes of naphthalene detoxification. In this study, we rigorously quantify levels of individual GST isozymes expressed within the livers and lungs of mice with acquired tolerance to naphthalene. Subchronic exposure to naphthalene increases the abundance of some hepatic GSTs to levels as much as 68% greater than controls. Naphthalene-tolerant mice displayed increases in mGSTM1 (51%), mGSTM2 (58%), and mGSTP1 (66%), whereas no significant difference in mGSTA3 was observed between exposed and control mice. Extracts of pulmonary tissues from naphthalene-tolerant mice showed minor increases in levels of mGSTP1 (7%) and Peak 8 isozyme (27%) and decreases in levels of mGSTM1 (31%), mGSTM2 (17%), and mGSTA3 (8%). The total enzymatic activity for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) was 22% lower in lung extracts from naphthalene-tolerant animals than in controls. These results indicate that induction of hepatic GSTs is substantial and may be an important factor in the development of tolerance to naphthalene.     

Nitroglycerin relaxes coronary artery of the pig with no change in glutathione content or glutathione S-transferase activity.

1. The role of glutathione content and glutathione S-transferase activity in vascular relaxant responses to nitroglycerin was evaluated in potassium (30 mM)-contracted coronary artery strips of the pig by measuring changes in tension, glutathione content and glutathione S-transferase activity. 2. Prior exposure of coronary artery strips to nitroglycerin (10(-5)M or 10(-4)M for 20 min) resulted in tachyphylaxis to subsequent relaxation to nitroglycerin (10(-8)-10(-5)M). 3. The glutathione content and glutathione S-transferase activity of the arterial strips rendered tachyphylactic by prior exposure to nitroglycerin (10(-5)M for 20 min or 10(-3)M for 120 min) were not significantly different from those of control strips. 4. Treatment with diethyl maleate (10(-4)M or 10(-3)M for 60 min) markedly depleted arterial glutathione content in a concentration-dependent manner with no change in glutathione S-transferase activity. 5. The relaxant response of coronary artery strips to nitroglycerin (10(-8)-10(-5)M) was completely unaffected following treatment with diethyl maleate (10(-4)M or 10(-3)M for 60 min). 6. The results suggest that vascular glutathione content does not play an important role in vascular relaxation or tolerance development to nitroglycerin, at least in pig isolated coronary artery.     

Decreased glutathione S-transferase activity in mice livers by acute treatment with lead, independent of alteration in glutathione content

Glutathione S-transferase (GST) activity and glutathione content in livers of male mice were assayed after acute administration of lead acetate (100 mg/kg i.p.). Fall in GST activity of lead-treated mice followed the decrease in glutathione content with a delay of more than 1 day. In addition, L-methionine (250 mg/kg i.p.) pretreatment did not antagonize the fall in GST activity induced by lead. In contrast, diethyl maleate, a potent glutathione depletor, increased GST activity. Thus, lead administration reduced the ability of the phase II reaction of drug metabolism, although glutathione depletion was not necessarily a critical factor for impairmen of GST per se.     

Plasma glutathione S-transferase in carbon tetrachloride treated rats and its association to hepatic cytosolic isozymes

The effect of carbon tetrachloride (CCl4) treatment on plasma and liver cytosolic glutathione S-transferase (GST) activities was investigated in rats. CCl4 was intraperitoneally administered at a dose of 0.5 ml/kg. The elevation of plasma GST activity paralleled the increase of plasma glutamate pyruvate transaminase activity after the administration of CCl4. Liver cytosolic GST activities were significantly decreased by CCl4 treatment. To establish the relationship of plasma GST with liver cytosolic isozymes, Western blot analysis using antibodies against cytosolic GST 1-2 and 3-4 was performed. The Western blots showed the existence of GST 1-2 and 3-4 in plasma at 24 hr after CCl4 treatment. The data thus strongly suggest that cytosolic GSTs are lost from the liver to plasma as a consequence of liver damage. The Western blot analysis of plasma GST may be useful for monitoring liver damage.     

Anatoxin-a elicits an increase in peroxidase and glutathione S-transferase activity in aquatic plants

Although the toxic effects of cyanotoxins on animals have been examined extensively, little research has focused on their effects on macrophytes and macroalgae. To date only microcystins have been found to be detrimental to aquatic plants. Peroxidase activity of the free floating aquatic plant Lemna minor and the filamentous macroalga Chladophora fracta was measured after exposure to several concentrations of the cyanotoxin, anatoxin-a. Peroxidase activity (POD) was significantly (P < 0.05) increased after 4 days of exposure to an anatoxin-a concentration of 25 microg mL(-1) for both L. minor and C. fracta. Peroxidase activity was not significantly increased at test concentrations of 15 microg mL(-1) or lower. In another experiment, the effects of various concentrations of anatoxin-a on the detoxication enzyme, glutathione S-transferase (GST) in L. minor were investigated. GST activity was significantly elevated at anatoxin-a concentrations of 5 and 20 microg mL(-1). Photosynthetic oxygen production by L. minor was also found to be reduced at these concentrations. This is the first report to our knowledge of the cyanotoxin anatoxin-a being harmful to aquatic plants.     

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.     

Circadian variation of hepatic glutathione S-transferase activities in the mouse

1. The circadian variation in glutathione S-transferase (GST) activity was studied in the hepatic cytosolic fraction of the male and female mouse. A circadian variation in GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the male, the activity being higher in the light phase (07:00-19:00 h) than in the dark phase (19:00-07:00 h) during a day under normal lighting conditions. 2. The circadian variation was only existed from June to October. The difference between the lowest activity (at 01:00 h) and the highest activity (at 13:00 h) was maximum in August. 3. In both the normal and reversed light/dark cycle (lights on 07:00 and 19:00 h, respectively), reduced glutathione (GSH) content was lowest in the middle of the light period and highest in the middle of the dark period and GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) exhibited opposite peaks and troughs. GST activities toward CDNB and 1,2-dichloro-4-nitrobenzene (DCNB) during the normal lighting schedule was higher at 13:00 h than at 01:00 h, but no differences were observed under reversed lighting conditions. 4. A circadian variation in GST activity for CDNB and DCNB was also observed in the female in a similar manner to the male, but the variation in the activity for EPNP was not observed in the female. 5. Thus, the circadian variation of hepatic GST activities in mouse were dependent on the enzyme substrates used, and seemed to be reflected by the difference in each isozyme levels. The daily change in the hepatic GSH levels is also thought involved, at least in part, in the regulation of GST activity.     

Antagonism of glycerol trinitrate activity by an inhibitor of glutathione S-transferase

The in vitro spasmolytic activity of glycerol trinitrate was measured on the KCl-contraction of aorta strips from the rabbit. In the presence of sulphobromophthalein, a known inhibitor of glutathione S-transferase, the dose-activity curve for the nitrate was displaced to the right. Much smaller displacements were obtained with the control spasmolytic substances--papaverine and S-nitroso-N-acetylpenicillamine. It was confirmed that sulphobromophthalein inhibits glutathione S-transferase activity in aorta homogenates. Aorta extracts did not detectably catalyze the reaction between glutathione and sulphobromophthalein and the glutathione level was not decreased by treating the intact aorta with sulphobromophthalein. It is concluded that sulphobromophthalein acts as a specific antagonist of the spasmolytic activity of glycerol trinitrate, probably as a result of its inhibition of glutathione S-transferase. It thus seems probable that glutathione and glutathione S-transferase are involved in the pharmacological activation of the organic nitrates.     

Up-regulation of glutathione S-transferase activity in enterocytes of young children.

The relationship between age and busulfan apparent oral clearance (Cl/F) expressed relative to adjusted ideal body weight and body surface area (bsa) was evaluated in 135 children aged 0 to 16 years undergoing hematopoietic stem cell transplantation for various disorders. Busulfan plasma levels were measured by gas chromatography-mass spectrometry after the first daily dose of the 4-day dosing regimen. Cl/F expressed relative to adjusted ideal body weight (ml/min/kg) and bsa (ml/min/m(2)) was lower in 9- to 16-year-old (y.o.) compared with 0- to 4-y.o. children (49 and 30%; p<.001). We hypothesized that the greater busulfan Cl/F observed in young children was in part due to enhanced (first-pass intestinal) metabolism. Busulfan conjugation rate was compared in incubations with human small intestinal biopsy specimens from healthy young (1- to 3-y.o.) and older (9- to 17-y.o.) children. Villin content in biopsy specimens was determined by Western blot and busulfan conjugation rate was expressed relative to villin content to control for differences in epithelial cell content in pinch biopsies. Intestinal biopsy specimens from young children had a 77% higher busulfan conjugation rate (p =.037) compared with older children. We have previously shown that glutathione-S-transferase (GST) A1-1 is the major isoform involved in busulfan conjugation, and that this enzyme is expressed uniformly along the length of adult small intestine. Thus, the greater busulfan conjugation activity in intestinal biopsies of the young children was most likely due to enhanced GSTA1-1 expression. We conclude that age dependence in busulfan Cl/F appears to result at least in part from enhanced intestinal GSTA1-1 expression in young children.     

Changes in hepatic cytosolic glutathione S-transferase activity and expression of its class-P during prenatal and postnatal period in rats treated with aflatoxin B1

The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GST-P activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50–60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (~40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to transplancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation.     

Role of glutathione and glutathione S-transferase in chlorambucil resistance.

A chlorambucil (CLB)-resistant cell line, N50-4, was developed from the established mouse fibroblast cell line NIH 3T3, by multistep drug selection. The mutant cells exhibited greater than 10-fold resistance to CLB. Alterations in GSH and glutathione S-transferase (GST) were found in CLB-resistant variants. A 7-10-fold increase in cellular GSH content and a 3-fold increase in GST activity were detected in N50-4 cells, compared with parental cells, as determined by enzymatic assays. An increase in steady state levels of the GST-alpha isozyme mRNA was found in the CLB-resistant cells, as analyzed by Northern blotting. No GST gene amplification or rearrangement was shown by Southern blot analysis. To test the relative roles of GSH and GST in CLB resistance, a number of GSH- and GST-blocking agents were used. The CLB toxicity was significantly enhanced in N50-4 cells by administration of either the GSH-depleting agent buthionine sulfoximine or the GST inhibitors ethacrynic acid or indomethacin. The resistance to CLB cytotoxicity in N50-4 cells, however, was still significantly higher than that of parental cells. The resistance of N50-4 cells to CLB was almost completely abolished by combination pretreatment yielding both GSH depletion and GST inhibition. The results indicate that both increased cellular GSH content and increased GST activity play major roles in CLB resistance in N50-4 mutant cells.     

Effect of diallyl disulfide on the hepatic glutathione s-transferase activity in rat: Diallyl disulfide effect on the glutathione S-transferase

Glutathione s-transferase is thought to play a key role in initiating the detoxication of potential alkylating agents, including pharmacologically active compounds. It is widely accepted that garlic contains allin which is converted to allicin by alliinase. Allicin is easily degraded to diallyl disulfide and other components. This report attempted to observe the effect of diallyl disulfide on some biological activities. It was observed that the activity of serum transaminase was not changed by the treatment of diallyl disulfide. The liver cytosolic glutathione s-transferase was significantly increased. whereas the microsomal glutathione s-transferase was not increased.