Differential induction of rat hepatic glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate (original) (raw)
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Hepatology, 1996
uid chromatography (HPLC) analysis of GST subunits. Recently, we used human hepatocytes in primary cul-HPLC also showed an induction of subunit 10 at the ture to study the effects of inducers of glutathione-Sprotein level of which the mRNA was not analyzed. Our transferases (GSTs) in the expectation that information results show that rat hepatocytes in primary culture obtained can be used to predict the value of particular prove to be a good model for the effect of inducers on inducers for use in the chemoprevention of cancer and both the expression of GST mRNA and protein levels other toxicities. However, in vitro human studies cannot in the rat liver in vivo. The demonstration of this good readily be confirmed by studies in vivo. This problem correlation in the rat with respect to increases gives supdoes not arise in experimental animals. In the current port for the use of human hepatocytes for predictive studies, the response of male rat hepatocytes in primary studies of chemoprotection in human pharmacology. culture to the following inducers of GST isoenzymes has (HEPATOLOGY 1996;23:881-887.) been determined: 3-methylcholanthrene (MC); phenobarbital (PB); 1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz (OPZ), and the results have been compared with induction obtained in livers The glutathione-S-transferases (GSTs) represented of MC-and OPZ-treated rats. Each type of inducer was by four multigene families (a, m, p, and u) provide profound to elicit a different response. In vitro, phenobarbitection against the genotoxicity and cytotoxicity of a tal increased messenger RNA (mRNA) levels of subunits number of electrophiles. 1,2 In the rat, GST class a con-1b and 3 after 12 and 72 hours, respectively; MC had tains subunits 1a, 1b, 2, 8 and 10; class m, subunits 3, a rapid effect on GST a class mRNAs (bringing about 4, 6, 9, and 11; class p, subunit 7 1 ; and class u, subunits increase after only 2 hours of treatment), increased sub-5, 12, and 13. 2,3 unit 7 mRNA slightly, and had no effect on mu class Like other enzymes of xenobiotic metabolism they mRNAs; dithiolethiones induced both subunit 1b and 7 are most abundant in the liver and are inducible by mRNAs after 4 hours and, to a much lower extent, submany xenobiotics. 4 By increasing hepatic detoxication, unit 3 mRNA after 72 hours. In vivo, MC induced significantly both subunit 1b and 7 mRNAs whereas OPZ in-inducers have the potential to prevent what might othcreased significantly subunits 1b, 3 and 7 mRNA levels, erwise be serious toxic damage. For example, rats are and to a lower extent those of subunit 2, after 3 days and usually susceptible to aflatoxin B 1 (AFB 1)-induced beyond to at least 5 days of treatment. Results obtained hepatocarcinogenesis but may become resistant after in mRNA studies were confirmed by high-pressure liqthey have been treated with chemicals that increase levels of GST. 5 This phenomenon, referred to as chemoprevention, is also believed to occur in humans. There-Abbreviations: GST, glutathione-S-transferase; AFB1, aflatoxin B1; OPZ, fore, it is possible that inducers might be of value for oltipraz; MC, 3-methylcholanthrene; PB, phenobarbital; mRNA, messenger RNA; cDNA, complementary DNA; HPLC, high-pressure liquid chromatogra-the prevention of AFB 1-associated liver cancer in huphy. mans, provided they are nontoxic and increase levels From 1 INSERM U49, Unité de Recherches Hépatologiques, Hôpital Pontof those GSTs that are effective enzymes for the carcichaillou, Rennes, France; and 2 Cancer Research Campaign, Molecular Toxicolnogenic electrophile AFB 1-exo-oxide.
Molecular and Cellular Biochemistry - MOL CELL BIOCHEM, 2000
The effect of thioacetamide (TA), an hepatotoxic and hepatocarcinogenic compound, on the expression and activity of the cytosolic enzyme glutathione-S-transferase (GST) was studied in rat liver. Four h following the administration of 14C-labeled thioacetamide (10 mg/Kg), several subunits of GST were found to be radioactively labeled. A single sublethal dose of TA (250 mg/Kg) decreased by three-fold the expression of classα GST at 24-48 h of treatment, but did not significantly affect the transcription of class μ GST. The activity of the enzyme toward 1-chloro-2,4-dinitrobenzene was mildly inhibited (66% of the control) by a 24 h TA treatment and gradually increased thereafter. It is proposed that the covalent binding of TA or its derivative to the GST subunits does not affect the activity of the enzyme. Nevertheless, GST activity inhibition is due to the deleterious effect of TA on GST transcription.
Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes
FEBS Letters, 1989
mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.
Glutathione-S-transferase (GST) is a family of enzymes involved in the detoxification of toxic and carcinogenic compounds. In the present study, the effect of thioacetamide (TA), a hepatotoxic and hepatocarcinogenic compound, on the activity and expression of GST of Wistar female rats was tested. Animals were treated with a single dose of TA (250 mg/kg) for 12, 24, 48 and 72 hours. GST activity toward the broad substrate 1-chloro-2,4-dinitrobenzene was enhanced by TA. The protein level of the GST classes alpha and mu as well as the mRNA level of several GST subunits were also positively affected by the TA treatment. Female Wistar rats of the same age but from two other different colonies had their GST activity either inhibited or not affected by TA. The basal mRNA level of class alpha and class mu GST was also tested in female Wistar rats obtained from five different sources. Differences in the basal level of class alpha mRNA were observed in rats from at least three different sources, while class mu mRNA level was distinct in two groups of animals.
Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents
Biochemical and Biophysical …, 1979
The activation of microsomal glutathione S-transferase in oxidative stress was investigated by perfusing isolated rat liver with 1 mM tert-butyl hydroperoxide (t-BuOOH). When the isolated liver was per fused with t-BuOOH for 7 min and 10 min, microsomal, but not cytosolic, glutathione S-transferase activ ity was increased 1.3-fold and 1.7-fold, respectively, with a concomitant decrease in glutathione content. A dimer protein of microsomal glutathione S-transferase was also detected in the t-BuOOH-perfused liver. The increased microsomal glutathione S-transferase activity after perfusion with t-BuOOH was reversed by dithiothreitol, and the dimer protein of the transferase was also abolished. When the rats were pretreated with the antioxidant a-tocopherol or the iron chelator deferoxamine, the increases in microsomal glutathione S-transferase activity and lipid peroxidation caused by t-BuOOH perfusion of the isolated liver was prevented. Furthermore, the activation of microsomal GSH S-transferase by t-BuOOH in vitro was also inhibited by incubation of microsomes with a-tocopherol or deferoxamine. Thus it was confirmed that liver microsomal glutathione S-transferase is activated in the oxidative stress caused by t-BuOOH via thiol oxidation of the enzyme.
Biochemical Pharmacology, 1990
Glutathione S-transferase (GST) isoenzymes of conventionally and co-cultured adult rat hepatocytes were purified and the GST subunits were separated by reversed phase HPLC in order to study the development of the GST subunit composition as a function of culture time and culture conditions. Several media conditions were tested, namely medium with and without fetal calf serum and with nicotinamide or dimethyl sulphoxide. Compared to the GST subunit composition of freshly isolated hepatocytes, changes in culture and media conditions result in a modification of the subunit profile. General observations are a decrease of subunits 1 and 2, an increase of subunit 3, a stabilization of subunit 4 and "de nouo" expression of subunit 7. When [%]methionine was added to the various culture media. and the thus labelled subunits were purified and separated. it was shown that cultured adult rat hepatocytes are able to synthesize the different GST proteins. Furthermore. the GST subunit composition, measured during various culture conditions. is probably a reflection of the "de nouo" synthesis in uitro.
Selective expression of the three classes of glutathione S-transferase isoenzymes in mouse tissues
Toxicology and Applied Pharmacology, 1990
The suitability of mouse as an animal model for studying the glutathione Stransferase (GST)-mediated detoxification mechanisms has been studied by analyzing the expression of the OL, /J, and T 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 r class, with a pZ value in the range of 8.6-9.1 and an approximate subunit M, value of 22,500 was detected in each tissue investigated in this study. A variable number of p class isoenzymes with subunit M, values of 26,500 were expressed in all mouse tissues studied, except spleen and muscle. Only liver and kidney showed the expression of an 01 class isoenzyme, each having a basic pZ value and subunit I%& of approximately 25,000. Another minor acidic 01 class isoenzyme, also with a subunit M, 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 rr 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. o 1990 Academic Press, Inc.
FEBS Letters, 1991
Previous studies, by using Northern blotting analyses, showed that phenobarbital (PB) affects the steady-state mRNA levels of glutathione S-transferase (GST) subunits 1/2, 3/4 and 7 in both conventional cultures of adult rat hepatocytes and co-cultures, with rat liver epithelial cells [Vandenberghe et al., 1989, FEBS Lett. 251, 59-64; Morel et al., 1989, FEBS Lett. 258, 99-102]. To determine whether PB acts at the transcriptional level, nuclear 'run on' experiments using cDNA probes hybridizing to GST subunits 1/2, 3/4 and 7 mRNA were performed on purified nuclei isolated from control and PB treated hepatocytes seeded under conventional and co-culture conditions. Data from this study demonstrate that the increase in steady-state mRNA levels observed in both conventional culture and co-culture after 4 days PB exposure results from an increased transcriptional activity of the GST genes. However, a substantial increase in steady-state mRNA levels in the absence of a commensurate increase in transcriptional activity at 12 days of co-culture, indicates that the barbiturate has also a stabilizing effect in vitro on the GST mRNAs.
Characterization of Rat-Liver Microsomal Glutathione S-Transferase Activity
European Journal of Biochemistry, 1980
Rat liver microsomes were shown to catalyze the conjugation of 1-chloro-2,4-dinitrobenzene with glutathione and this activity has been characterized. It cannot be removed from the microsomes by washing or other procedures which release loosely bound material from membranes. The microsomal glutathione S-transferase can be activated up to eight fold by treatment with N-ethylmaleimide. This activation also affects the apparent Km of the enzyme(s) for both glutathione and 1-chloro-2,4-dinitrobenzene. Upon subcellular fractionation of the liver the N-ethylmaleimide-activateable glutathione S-transferase distributes in the same manner as a marker for the endoplasmic reticulum and unlike markers for the other organelles and for the cytoplasm. Treatment of microsomes with proteases revealed that the enzyme is at least partially exposed on the cytoplasmic surface of the endoplasmic reticulum. Finally, three inducers of drug-metabolizing systems-i.e. phenobarbital, methylcholanthrene, and trans-stilbene oxide-all increase the activity of the cytoplasmic glutathione S-transferases, but they do not affect the microsomal activity. These and other considerations indicate that the microsomal glutathione S-transferase(s) is distinct from the cytoplasmic enzymes catalyzing similar reactions. The microsomal enzyme is likely to be involved in drug metabolism and the possibility of activating it through attack on a sulfhydryl group may represent an important physiological response to certain xenobiotics.