Glutathione S-transferase Subunits Research Articles

Effects of microsomal enzyme inducers on glutathione S-transferase isoenzymes in livers of rats and hamsters

The effects of microsomal enzyme inducers on glutathione S-transferase (GST) isoenzymes were studied in livers of rats and hamsters using three hypolipidemic drugs of the peroxisome proliferator type and the two model substances phenobarbital (PB) and 3-methylcholanthrene (MC). The effects were investigated by immunoblot analysis of the various GST subunits using polyclonal antibodies directed to rat subunits 1–4. In untreated animals the subunit composition was different, with hamsters having a much higher content of class mu isoenzymes. Administration of all three hypolipidemic drugs reduced the protein concentration of both alpha and mu class GSTs in rats but reduced only class mu subunits in hamsters. This reduction was in good agreement with the decreased activity observed with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB) in both species. As expected, PB and MC increased GST activity together with the concentration of subunits 1 and 3 in rats. In hamsters, PB significantly increased subunit 1 and slightly reduced subunits 3 and 4, although this decrease was not significant. Total GST, measured with CDNB, was reduced by 17%. In contrast, MC slightly decreased subunit 1 and markedly raised subunits 3 and 4, resulting in a net increase in total GST activity. All drugs increased relative liver weight, microsomal protein concentration and total P450 in both species; in contrast, total cytosolic proteins were raised by all drugs in rats but not in hamsters, except for MC. The results obtained in these two species show that GST activity is not always increased by microsomal enzyme inducers. The response may depend in part on isoenzyme profile, and varies with the subunit considered.

Regulation of Glutathione S-Transferase Gene Expression and Activity by Dietary Selenium

To determine selenium′s effects on glutathione S-transferase gene expression and enzyme activity, weanling rats were fed a selenium-deficient diet, or the same diet supplemented with 0.1 (control) or 2.0 mg selenium/kg diet as sodium selenite, for 91 days. Consumption of either the selenium-deficient or high selenium diet increased activity of glutathione S-transferase, measured with 1-chloro-2,4-dinitrobenzene as substrate, compared to the control diet. Transcription of genes for glutathione S-transferase subunits was unaffected by selenium intake. Steady state levels of mRNA for glutathione S-transferase subunits were affected variably by changes in selenium intake, depending upon the tissue and subunit examined. These results suggest that the biological effects of selenium may be due in part to its regulation of gene expression for glutathione S-transferase family enzymes.

Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors.

Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic content of AFB1-AR. Both BHA and BHT were also able to induce this enzyme but, by contrast, PB was found to be a poor inducer of AFB1-AR. AFB1, 3-MC and clofibrate were unable to serve as inducers of this reductase. The presence of Alpha-class GST, including the Yc2 subunit, was examined in various rat tissues. Constitutive expression of Yc2 was found in the epididymis at levels comparable with that observed in the liver from EQ-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunological comparison of cytosolic glutathione S-transferases between rat and two strains of houseflies

Five different antisera, which include three antisera raised against rat liver glutathione S-transferases (GST), one antiserum raised against human pi GST, and one antiserum raised against housefly GST1, were used to examine their cross-reactivity with different classes of GST subunits isolated from rat liver and the housefly. Two classes of rat liver GSTs, alpha and mu, were isolated from rat liver and two classes of housefly GSTs, GST1 and GST2, were isolated from both CSMA and Cornell-R strains. Antiserum against GST 3-3 was the most reactive antiserum and reacted not only with the mu class of GSTs but also with the GST1 class from both CSMA and Cornell-R strains. Antiserum against human pi GST and antiserum against housefly GST1 had weak immunological reactivity toward the GST1 class from both strains of housefly. Antiserum against GST 4-4 and antiserum against GST 1-1 had immunological reactivity toward any class of GSTs from housefly. None of the five antisera had any immunological cross-reactivity toward subunit 2 of the alpha class of rat GST and the GST2 class of housefly GSTs from both strains.

Human glutathione S-transferases

1. 1. Multiple forms of glutathione S-transferase (GST) isoenzymes present in human tissues are dimers of subunits belonging to three distinct gene families namely α, μ and π. Only the subunits within each class hybridize to give active dimers. 2. 2. These subunits are differentially expressed in a tissue-specific manner and the composition of glutathione S-transferases in various tissues differs significantly. 3. 3. Minor GST subunits not belonging to these three classes are also present in some tissues. 4. 4. An ortholog of rat GST 8-8 and mouse mGSTA4-4 is selectively expressed in some human tissues including bladder, brain, heart, liver, and pancreas. This isoenzyme designated as GST 5.8 expresses several fold higher activity towards 4-hydroxy-2,3-trans-nonenal as compared to the routinely used substrate 1-chloro-2,4-dinitrobenzene.

Ammonium 4-Chloro-7-sulfobenzofurazan: A Fluorescent Substrate Highly Specific for Rat Glutathione S-Transferase Subunit 3

Ammonium 4-chloro-7-sulfobenzofurazan (Sbf-Cl) is a water-soluble fluorescent reagent which is highly specific for thiol groups. We describe here a simple and sensitive fluorescence assay which is highly specific for subunit 3 of the rat glutathione S-transferases. Specific activities of isoenzymes 3-3 and 3-4 were an order of magnitude greater than those of isoenzymes 1-1, 1-2, 2-2, and 4-4, with glutathione and Sbf-Cl concentrations of 1 and 2 mM, respectively. The catalytic specificity constant, kcat/Km was in the range of 104-106 M−1 s−1, indicating that Sbf-Cl is a very good substrate for rat hepatic glutathione S-transferases. The specificity constants measured for the heterodimers 1-2 and 3-4 were greater than the values predicted when dimeric independence is assumed. This suggests that binding of Sbf-Cl to the glutathione S-transferases may result in steric alterations and subsequent elevation in enzymatic activity. Sbf-Cl was shown to be at least 10-fold more sensitive for the detection of rat GST isoenzyme 3-3 than DCNB. Consequently, Sbf-Cl will have an important future role in the investigation of the active site topology of glutathione S-transferases, in addition to its obvious potential as a substrate for the detection and quantitation of rat glutathione S-transferase subunit 3.

Piperine effects on the expression of P4502E1, P4502B and P4501A in rat.

1. Treatment of rat with piperine (PIP) (1.4 mmol/kg, 3 days ip injections) resulted in an approximate two-fold increase in total liver microsomal P450 content relative to that in uninduced animals. 2. 4-Nitrophenol and aniline hyroxylase activities in the hepatic microsomes prepared from rat treated with PIP decreased by 30 and 28% respectively as compared with control. Immunoblot analyses also revealed decreased P4502E1 levels in hepatic microsomes from PIP-treated animals. 3. In contrast with P4502E1 suppression, hepatic 2B1 and 2B2 levels were significantly increased in PIP-induced animals, as evidence by both metabolic activity and immunoblot analysis of the liver microsomal fractions. The rate of hexobarbital hydroxylase activity in microsomes from PIP-treated animals was markedly elevated and was inhibited by approximately 62% in the presence of monoclonal anti-P4502B IgG. Immunoblot analyses demonstrated that P4502B1 and 2B2 levels in hepatic microsomes from PIP-treated animals were comparable with those from phenobarbital-treated animals. 4. 7-Ethoxycoumarin deethylase activity was elevated approximately two-fold in PIP-induced animals and was 17% of that derived from 3-methylcholanthrene-induced animals. 7-ethoxycoumarin deethylase activity in PIP-induced hepatic microsomes was inhibited 63% in the presence of monoclonal anti-P4501A antibody. Immunoblot analysis confirmed the increase in P4501A levels by PIP, which was 15% of that in hepatic microsomes from 3-methylcholanthrene-induced animals. 5. PIP treatment failed to affect microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GST) expression, as indicated by immunoblot analyses using polyclonal antibodies toward mEH and GST subunits Ya, Yb1, Yb2 and Yc. 6. These results demonstrate that PIP treatment suppressed P4502E1 expression and enhanced 2B and 1A expression, whereas this agent failed to affect hepatic mEH and GST expression.

Human Mu-class glutathione S-transferases present in liver, skeletal muscle and testicular tissue

The major human Mu-class glutathione S-transferases (GST) have been purified to allow comparisons of their catalytic, physicochemical and immunochemical properties. GST isoenzymes, purified from hepatic, testicular and skeletal muscle tissue were found to comprise three distinct subunits (M1, M2 and M3) which may combine to form both homodimeric and heterodimeric proteins. Two distinct subunits, M1a and M1b, which represent allelic charge variants have been isolated but no polymorphic forms encoded at the GST M2 and M3 loci have been observed. Three GST isoenzymes (M1a-1a, M1a-1b and M1b-1b) have been purified from a single liver specimen. In addition, GST M1a-2, M1b-2, M2-2 and M2-3 have been isolated from muscle, whilst the M3-3 homodimer has been purified from human testis. The homodimeric enzymes GST M1a-1a, M1b-1b, M2-2 and M3-3 have p I values of 6.1, 5.5, 5.3 and 5.0, whilst SDS-PAGE indicated that M1a, M1, M2 and M3 have molecular masses of 26.7, 26.6, 26.0 and 26.3 kDa, respectively. The M1, M2 and M3 subunits isolated from either liver, skeletal muscle or testis, are catalytically distinct. Both M1-type subunits (M1a and M1b) possess a high activity for trans-4-phenyl-3-buten-2-one, whereas, the skeletal muscle subunit M2 has a high activity towards 1,2-dichloro-4-nitrobenzene. By contrast, the testicular GST subunit M3 has no detectable activity towards either of these substrates. However, all three Mu-class subunits are active towards the compounds 4-hydroxynonenal and 4-hydroxydecinal, possible endogenous substrates which are produced by lipid peroxidation. The human Mu-class subunits can be distinguished immunochemically; antisera raised against the testicular GST M3-3 showed no reactivity towards either the M1 or M2 subunits. The M3 subunit has a blocked N-terminus but automated amino-acid sequencing of a CNBr-derived peptide allowed 14 residues of the M3 subunit to be identified. These data indicated that testicular GST M3-3 is likely to correspond to the brain/testis Mu-class GST cDNA described by Campbell et al. (Campbell E., Takahashi Y., Abramovitz M., Peretz M., & Listowsky I. (1990) J. Biol. Chem. 265, 9188–9193).

Differential induction of glutathione S-transferase subunits by phenobarbital, 3-methylcholanthrene and ethoxyquin in rat liver and kidney

The inducibility of Glutathione S-transferase (GST) in male Sprague-Dawley rats, treated with phenobarbital (PB), 3-methylcholanthrene (MC) and ethoxyquin (ETQ), was examined in detail. The subunit compositions of hepatic and renal GST were determined by using a reverse-phase HPLC technique. In liver, PB was found to induce the Yb 1, Yb 2, Ya 2, Ya 2 and Yk subunits by about 2.1-, 1.8-, 1.8-, 4.4- and 4-fold, respectively, while MC induced the Yb 2, Yc, Ya 2 and Yk subunits by about 1.5-, 1.5-, 6- and 1.7-fold, respectively, and ETQ increased the levels of Yb 1, Yb 2, Yc, Ya 2 and Yk subunits by about 2.1-, 1.7, 1.9-, 14.9-, and 1.8-fold, respectively. In contrast, kidney cytosolic GSTs were induced only by treatment with ETQ and PB and MC had little or no effect. The Pi class subunit Yp in the rat kidney was increased about 4-fold and Mu class Yb 2 was induced by about 2-fold, by the ETQ treatment.

Effects of the naturally occurring alkenylbenzenes eugenol and trans-anethole on drug-metabolizing enzymes in the rat liver

In order to study the effects of trans-anethole and eugenol on drug-metabolizing enzyme activities in vivo, male Wistar rats were treated by gavage with trans-anethole (125 or 250 mg/kg body weight) or eugenol (250, 500 or 1000 mg/kg body weight) daily for 10 days. In liver microsomes and cytosol various phase-I and phase-II biotransformation enzyme activities were determined. No effect on total cytochrome P-450 content in liver microsomes from rats treated with eugenol or trans-anethole was observed. Administration of 1000 mg eugenol/kg body weight, but not the lower doses, significantly increased cytochrome P-450-dependent 7-ethoxy-resorufin O-deethylation (EROD) and 7-pentoxy-resorufin O-depentylation (PROD); administration of trans-anethole (125 or 250 mg/kg body weight) did not alter EROD and PROD activities. In rat liver cytosol, UDP-glucuronyl transferase (GT) activity towards the substrate 4-chlorophenol was significantly increased in all treated rats, and activity towards 4-hydroxybiphenyl as substrate was significantly increased in rats treated with 250 mg trans-anethole/kg or with 500 or 1000 mg eugenol/kg. DT-diaphorase (DTD) activity was only significantly enhanced in the liver cytosol of rats treated with trans-anethole at 250 mg/kg body weight. Enhancement of cytosolic glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene was found for all eugenol- and trans-anethole-treated rats. In addition, significantly increased levels of GST subunit 2 were measured by HPLC in the liver cytosol of rats treated with eugenol (500 or 1000 mg/kg body weight) or trans-anethole (250 mg/kg body weight). It is concluded that both eugenol and trans-anethole preferentially induce phase II biotransformation enzymes in rat liver in vivo.

In vitro and in vivo reversible and irreversible inhibition of rat glutathione S-transferase isoenzymes by caffeic acid and its 2- S-glutathionyl conjugate

The reversible and irreversible inhibition of glutathione S-transferases (GST) by caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid] was studied in vitro using purified rat isoenzymes, and in vivo in male Wistar (WU) rats. The concentrations of caffeic acid that inhibited reversibly 50% of the activity of different GST isoenzymes towards 1-chloro-2,4-dinitrobenzene (CDNB) (I 50 values) were 58 (GST 4-4), 360 (GST 3-3) and 470 μ m (GST 7-7), and higher than 640 μ m for GST isoenzymes of the α class (GST 1-1 and 2-2). The major glutathione conjugate of caffeic acid, 2- S-glutathionylcaffeic acid (2-GSCA), was a much more potent reversible inhibitor of GST, with I 50 values of 7.1 (GST 3-3), 13 (GST 1-1), 26 (GST 4-4), 36 (GST 7-7) and more than 125 μ m (GST 2-2). On the other hand, caffeic acid was a much more efficient irreversible inhibitor of GST than 2-GSCA. In this respect, GST 7-7 was by far the most sensitive enzyme. The remaining activity towards CDNB (expressed as percentage of control) after incubating 1.25 μ m-GST with 100 μ m-caffeic acid for 6 hr at 37°C was 34 (GST 2-2), 24 (GST 1-1), 23 (GST 4-4), 10 (GST 3-3) and 5% (GST 7-7). Almost no irreversible inhibition of GST 1-1 and 3-3 occurred during incubation with 2-GSCA. Incubation of caffeic acid with liver microsomes from dexamethasone-induced rats catalysed the oxidation of caffeic acid about 18 times more effectively as compared with the spontaneous oxidation, as determined by the formation of GSH conjugates from caffeic acid. In vivo, the effect of single oral doses of caffeic acid (50–500 mg/kg body weight) on the cytosolic GST activity towards CDNB was studied 18 hr after dosing in the liver, kidney and intestinal mucosa. A marginal but significant linear relationship was found between the amount of caffeic acid dosed and the irreversible inhibition of GST activity in the liver, with a maximum of about 14% inhibition in the highest dose group. This inhibition coincided with a small decrease in the μ-class GST subunits, which was only significant for GST subunit 4.

The effect of hepatic regeneration on the expression of the glutathione S-transferases.

The effect of hepatic regeneration on expression of four glutathione S-transferase (GST) subunits (Ya, Yc, Yb1, Yb2) was examined in rats following partial hepatectomy (PH). mRNA levels of the Ya and Yc subunits (Alpha class) decreased and were 13% and 42% of levels in sham-operated animals respectively 12 h after surgery. mRNA levels for the Yb1 subunit (Mu class) also decreased but were not maximally reduced until 24 h after PH (22% of sham-treated level). mRNA levels of the Yb2 subunit were affected little by PH. Changes in levels of mRNA appeared to reflect a decrease in both transcriptional activity and mRNA stability. The decrease in mRNA levels was associated with a fall in enzymic activity and in protein levels of Alpha-class GSTs. Within 48 h of surgery, levels of mRNA, protein enzymic activity and transcriptional activity had all fully recovered. GSH levels also decreased in the first 6 h after PH. However, 24 h after surgery GSH levels in animals having undergone PH exceeded those in sham-treated animals by 2-fold and this difference persisted for 72 h. These findings suggest that during the early phases of hepatic regeneration, because of decreased GST and GSH levels, the liver may be unusually susceptible to injury by toxic compounds. However, by the first round of cell division (36-48 h post-surgery) the liver has fully recovered its ability to metabolize toxic electrophiles.

Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize.

The effects of the dichloroacetamide safener benoxacor on maize (Zea mays L. var Pioneer 3906) growth and glutathione S-transferase (GST) activity were evaluated, and GST isozymes induced by benoxacor were partially separated, characterized, and identified. Protection from metolachlor injury was closely correlated with GST activity, which was assayed with metolachlor as a substrate, as benoxacor concentration increased from 0.01 to 1 [mu]M. GST activity continued to increase at higher benoxacor concentrations (10 and 100 [mu]M), but no further protection was observed. Total GST activity with metolachlor as a substrate increased 2.6- to 3.8-fold in response to 1 [mu]M benoxacor treatment. Total GST activity from maize treated with or without 1 [mu]M benoxacor was resolved by fast protein liquid chromatography anion-exchange chromatography into four major activities, designated activity peaks A, B, C, and D in their order of elution. These GST activity peaks were enhanced to varying degrees by benoxacor. Activity peak B showed the least induction, whereas activity peak A was absent constitutively and thus highly induced by benoxacor. In contrast to earlier reports, there appear to be not one, but at least two, major constitutive isozymes (activity peaks A and D) having activity with metolachlor as substrate; there were at least three such isozymes in benoxacor-treated maize (activity peaks A, C, and D). The elution volumes of activity peaks A, B, C, and D were compared with those of partially purified maize GST I and GST II; also, the reactivity of polypeptides in these activity peaks with antisera to GST I or GST I/III (mixture) was evaluated. Evidence from these experiments indicated that activity peak B contained GST I, and activity peak C contained GST II and GST III. Activity peaks A and D contained unique GSTs that may play a major role in metolachlor metabolism and in the safening activity of benoxacor in maize. Isozymes present in activity peaks A and D were not detected in earlier reports because of the very low activity with the artificial substrate 1-chloro-2,4-dinitrobenzene. Immunoblotting experiments also indicated the presence of numerous unidentified GST subunits, including multiple subunits in chromatography fractions containing single peaks of GST activity; this is indicative of the likely complexity and diversity of the maize GST enzyme family.

Glutathione S-transferase isoenzymes in rat brain neurons and glia

The glutathione S-transferases (GSTs) constitute a family of cytosolic isoenzymes and a structurally unrelated microsomal enzyme that is involved in the detoxication of electrophilic xenobiotics. These enzymes also participate in the intracellular binding and transport of a broad range of lipophilic compounds including bilirubin, and hormones such as the glucocorticoids and thyroid hormones. The present investigation demonstrates that GSTs are present in neurons of the brainstem, forebrain, and cerebellum. An isoenzyme-specific distribution of GSTs was found in cytoplasm, nuclei, and nucleoli. The regional and cellular distribution of cytosolic GSTs in the brain was studied by immunohistochemistry, spectrophotometric enzyme assay, and reverse-phase HPLC. Polyclonal antibody against microsomal GST was strongly reactive with Purkinje cells throughout the cerebellar cortex, and with neurons in the brainstem and hippocampus. Nuclei of Purkinje cells and of neurons in the brainstem, hippocampus, and cerebral cortex were immunopositive for alpha-class GST 1-1 (YaYa), whereas alpha-class GST 2-2 (YcYc) antibody was consistently immunoreactive with the nucleolus, but not with the nucleus or soma. All alpha-class GST antibodies studied were reactive, to various degrees, with astrocytes and choroid plexus; however, ependymal cells of the subventricular zones were immunonegative. alpha-class GST 8-8 (YkYk) immunoreactivity was specifically localized to endothelial cells and/or astrocytic end feet associated with blood vessels. Reverse-phase HPLC indicated that there were also substantial regional differences in the pattern of alpha-, mu-, and pi-class GST subunit expression. For example, the thalamus/hypothalamus had the highest GST activity and greatest concentration of total GST protein and mu-class GST subunit 6 (Yb3), whereas the brainstem had the greatest concentration of pi-class GST subunit (Yp). This regional variation in GST expression may be reflective of regional differences in cell populations. In cerebellar cortex, the concentration of mu-class GST subunit 4 (Yb2) was greatest in the flocculus and lowest in the vermis. This is of clinical interest because the pattern of expression of mu-class GST subunit 4 (Yb2) in the cerebellum coincides with the known regional susceptibility of this structure to degeneration after exposure to toxic or metabolic insults. The vermis is most susceptible to these insults, whereas the lateral lobes and flocculus are most resistant.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental aspects of Bufo bufo embryo glutathione transferases

The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.

Effects of hyperbilirubinaemia on glutathione S-transferase isoenzymes in cerebellar cortex of the Gunn rat.

The glutathione S-transferases (GSTs) are a family of isoenzymes involved in the detoxication of a variety of electrophilic xenobiotics. The present investigation demonstrates that GST activity and the concentration of cytosolic GSTs in cerebellar cortex of Gunn rats were increased in hyperbilirubinaemic animals compared with non-jaundiced controls. Age-dependent and region-specific increases in GST isoenzymes were seen in three regions of the cerebellar cortex of jaundiced Gunn rats, whereas GST concentrations were not altered in the brainstem, thalamus/hypothalamus, cortex or liver. Cytosolic GST activity was increased 1.3-fold in the flocculus and lateral hemispheres of 20-day-old and 1.7-fold in the flocculus, lateral hemispheres and vermis of 60-day-old jaundiced (jj; homozygous) Gunn rats compared with non-jaundiced (Jj; heterozygous) Gunn rats. H.p.l.c. was used to determine the GST subunit protein concentrations in cytosolic fractions isolated from liver and brain regions of jaundiced and non-jaundiced animals. In all regions of the cerebellum from 20-day-old animals, the levels of Alpha-class GST subunits 2 (Yc1; 3.0-fold) and 8 (Yk; 2.0-fold) were increased in jaundiced rats. In 60-day-old animals, the concentrations of Alpha-class GST subunits 2 (Yc1; 5.0-fold) and 8 (Yk; 3.0-fold), Mu-class subunit 11 (Yo; 2.5-fold) and Pi-class subunit 7 (Yp; 2.0-fold) were increased in all regions of cerebellar cortex of jaundiced animals. In cerebellum of 10-, 20- and 60-day-old non-jaundiced and jaundiced Gunn rats, the flocculus had the highest concentration of Mu-class GST subunit 4 (Yb2) and vermis the lowest; hyperbilirubinaemia increased the concentration of subunit 4 (Yb2; 3- to 5-fold) in the flocculus and lateral hemispheres, but not the vermis, of 20- and 60-day-old rats. Intraperitoneal injection of sulphadimethoxine, a long-acting sulphonamide which displaces bilirubin from its albumin-binding sites and increases the bilirubin levels in tissues, further increased the already elevated concentrations of GST subunits in the lateral regions of cerebellar cortex of hyperbilirubinaemic rats. For example, the concentration of subunit 4 (Yb2) was increased 2.2-fold (compared with non-jaundiced controls) in Gunn rats injected with saline and 7.4-fold in rats injected with 100 mg of sulphadimethoxine/kg body weight. In contrast, GSTs in the vermis of jaundiced animals were not affected by sulphadimethoxine injection. Sulphadimethoxine had no effect on GST concentrations in lateral regions and vermis of heterozygous (Jj) Gunn rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Constitutive and inducible profile of glutathione S-transferase subunits in biliary epithelial cells and hepatocytes isolated from rat liver.

The constitutive and inducible cytosolic glutathione S-transferase (EC 2.5.1.18) subunit compositions of parenchymal cells (hepatocytes) and biliary epithelial cells (BEC) from rat liver have been quantitatively analysed using reverse-phase h.p.l.c. Hepatocytes, analysed in the absence of non-parenchymal cells, expressed constitutively the following subunits, in order of their concentration: 3, 4, 2, 1a, 1b, 8, 6 and 10. BEC express constitutively only four of the GST subunits expressed by hepatocytes and these are, in order of their concentration: subunits 2, 7, 4 and 3. Notable differences from hepatocytes are that BEC completely lack the Alpha-class subunits 1a and 1b that are major subunits in hepatocytes, Mu-class subunits make up a very low proportion of the total, and the Pi-class subunit 7 is a major subunit in BEC, whereas it is essentially absent from hepatocytes. For the first time, the effects of the inducing agents phenobarbitone (PB), beta-naphthoflavone (beta-NF) and ethoxyquin (EQ) have been characterized in a comprehensive and quantitative manner in both cell types. PB, beta-NF and EQ increased total GST protein in hepatocytes by approx. 2-fold, 3-fold and 4-fold respectively. Subunits significantly induced in hepatocytes were (in order of fold-induction): by PB, 1b > 8 > 3 > 2 > 4; by beta-NF, 1b > 8 > 2 > 3 > 4; and by EQ, 7 > 1b > 10 > 8 > 3 > 2 > 1a > 4. In BEC, neither PB nor beta-NF had significant effects on the total amount of GST protein, although PB did significantly induce subunit 3 at the expense of other subunits. EQ increased total GST protein nearly 5-fold in BEC, subunits 7 and 3 being induced dramatically above constitutive levels.

Interindividual differences in the in vitro conjugation of methylene chloride with glutathione by cytosolic glutathione S-transferase in 22 human liver samples

The interindividual variation in the in vitro conjugation of methylene chloride with glutathione by cytosolic glutathione S-transferase (GST) was investigated with 22 human liver samples. In three of the samples no activity towards methylene chloride was observed. Eleven samples showed an activity ranging from 0.20 to 0.41 (0.31 ± 0.08) nmol/min/mg protein, and eight samples an activity of 0.82-1.23 (1.03 ± 0.14) nmol/min/mg protein. The activities towards 1-chloro-2,4-dinitrobenzene (CDNB) of these three groups were 1.17 ± 0.25, 1.12 ± 0.35 and 1.20 ± 0.53 μmol/min/mg protein, respectively. In nine of the liver samples, the α-, μ- and π-clas GST subunits were quantified. In two of these samples, no activity was observed towards methylene chloride, while α-,μ- and ;π-class subunits were expressed in these human liver cytosolic samples. As the highest activity towards methylene chloride was still 1.4 times lower than the activity in rat cytosol, the existence of the three populations seems to be of little importance for human risk assessment.

Glutathione S-transferases in primary cultured rat hepatocytes.

The present study is aimed to elucidate the changes in glutathione S-transferase (GST) activity and GST subunit components in primary cultured rat hepatocytes. Enzyme activity was measured with 1-chloro-2,4-dinitrobenzene as cosubstrate. The activity decreased at 48 hr, and subsequently increased and returned to levels initially observed at 12 hr by 120 hr. Phenobarbital caused an induction of GST activity in culture at 72 and 168 hr. Immunocytochemical studies were performed using a peroxidase-anti-peroxidase technique with three polyclonal antibodies: anti-Ya, Yb1 and Yp. With anti-Ya, hepatocytes were persistently positive up to 144 hr in cell culture. With anti-Yb1, hepatocytes were positive at 24 hr, though positivity then gradually decreased. On the other hand, with anti-Yp, cells were almost negative at 48 hr and became obviously positive at 96 hr. Immunoelectron microscopy with anti-Yb1 using the avidin-biotin ferritin method revealed ferritin particles in the ribosomes on endoplasmic reticulum as well as in the free cytoplasmic space. In conclusion, the GST subunit components are in a state of dynamic change in cultured rat hepatocytes, and overall time-dependent increase in the total activity of the enzyme can be accounted for by increased expression of the Yp subunit. Finally, the intracellular localization of Yb1 subunit was clarified in the present report.

Glutathione S-transferase isoenzyme patterns in different subtypes of enzyme-altered rat liver foci treated with the peroxisome proliferator nafenopin or with phenobarbital.

It is known that phenobarbital (PB) and the peroxisome proliferator (PP) nafenopin (NAF) promote tumor formation by stimulating selective growth of different subtypes of liver foci. While PB enhanced the gamma-glutamyltranspeptidase (GGT)-positive eosinophilic-clear cell foci (ECF), NAF amplified the GGT-negative weakly basophilic foci (WBF). These findings provide the possibility of using the occurrence of these foci subtypes as early indicators for the carcinogenic potential of PB- and PP-type promoters. In order to improve the methods for the discrimination between ECF and WBF we studied further differences in their phenotype, as determined by the expression pattern of glutathione S-transferase (GST) subunits. GST subunits of the alpha (Ya, Yc), mu (Yb1, Yb2) and pi family (Yp), which compose different GST isoenzymes, were demonstrated by immunohistochemical methods. ECF were the only foci subpopulation that expressed GST subunit Yp, while this subunit was always absent in WBF and in another focus subtype, the tigroid foci (TF). Neither PB nor NAF changed this pattern. Thus Yp expression was rather a function of the focus type than of the promoter used. Upon PB treatment expression of the GST subunits Yb1 and Yb2 was frequently elevated in ECF, while Ya and Yc remained more or less unchanged. In NAF-treated livers large WBF, however, showed diminished expression of all investigated GST subunits of the alpha and mu family. In conclusion, PB seems to promote mostly ECF with elevated levels of mu and pi class GSTs, while low levels or absence of all GSTs tested may be associated with growth selection of WBF through the PP NAF.