Induction of translationally active rat liver glutathione S-transferase B messenger RNA by phenobarbital

Liver poly(A⁺)-RNA was isolated from untreated and phenobarbital-treated rats and translated in cell-free systems derived from wheat germ and rabbit reticulocyte lysates. The primary translation product of glutathione S-transferase B was comprised of two nonidentically sized subunits which comigrated on SDS-polyacrylamide gels with the purified glutathione S-transferase B subunits. The level of translatable glutathione S-transferase B mRNA in rat liver was elevated approximately 3 to 4-fold by phenobarbital administration. Our data suggest that chronic phenobarbital administration to rats increases the amount of cytosolic glutathione S-transferase B via an increase in the functional mRNA level encoding for the enzyme.     

Selective induction of glutathione S-transferase subunits in wheat plants exposed to the herbicide acifluorfen

Exposure to the herbicide acifluorfen resulted in marked increase of glutathione S-transferase (GST) enzyme activity in wheat seedlings, primarily in shoot tissues. From the six major, constitutively expressed GST subunits found in untreated wheat shoots subunits 2 and 3 were selectively induced by acifluorfen. No new subunit could be detected. The induced subunits belong to those GST isoenzymes, which metabolize diphenyl ether herbicides.     

Pivotal role of electrophilicity in glutathione S-transferase induction by tert-butylhydroquinone

Although the induction of glutathione S-transferase (GST) activity by tert-butylhydroquinone (tBHQ) has been well-documented in several cell culture systems and rodent experiments, the exact mechanism responsible for its inducibility is still not thoroughly understood. To more precisely define the molecular mechanism of GST induction by tBHQ, we examined the one-electron oxidation and glutathione (GSH) reaction potentials of tBHQ as compared to its analogue, 2,5-di-tert-butylhydroquinone (DtBHQ). tBHQ and DtBHQ showed similar one-electron oxidation potentials, including free radical quenching (antioxidant), oxidative conversion of both compounds to a benzoquinone form, and Cu(2+)-dependent superoxide generation. On the other hand, the reduced GSH level was observed by the addition of tBHQ, but not DtBHQ, suggesting that tBHQ acts as an electrophile while DtBHQ does not. The data were consistent with the observation that tBHQ more potently induced the GSTP1 gene expression in RL34 cells than DtBHQ did. Moreover, we indeed detected the GSH-tBHQ conjugates in the cells exposed to tBHQ using an electrochemical detector-high-performance liquid chromatography technique. Thus, we conclude that an electrophilic quinone oxidation product that reacts with intracellular nucleophiles including protein thiol or GSH plays a major role in the GSTP1 gene expression.     

Selective induction of heme oxygenase-1 isozyme in rat testis by human chorionic gonadotropin

A radioimmunoassay was developed to assess the response of testicular HO-1 to agents known to increase the microsomal heme oxygenase activity. Treatment of rats with human chorionic gonadotropin (hCG) increased the microsomal heme oxygenase activity in rat testis. The following data suggest that the increase was specific to the HO-1 isozyme: (a) The elution profile of heme oxygenase activity from a DEAE-Sephacel column showed an increase in the HO-1 peak, but not in the HO-2 peak, (b) the Western immunoblot of the testis microsomes showed an increase in HO-1 protein, and (c) the amount of HO-1 protein that was present in the microsomes, when measured by radioimmunoassay, was doubled. Using radioimmunoassay, it was shown that other agents known to increase the testicular heme oxygenase, sodium arsenate and sodium arsenite, also increased the microsomal content of HO-1. An inhibitor of the testicular microsomal heme oxygenase activity, cadmium, also increased the microsomal HO-1 protein. The findings suggest that inducibility of HO-1 extends to tissues other than the liver, in this instance, the testis, and further support the possibility that HO-1 is the only inducible form of heme oxygenase.     

Metabolism of melphalan by rat liver microsomal glutathione S-transferase

One of the major problems in the treatment of human cancer is the phenomenon of drug resistance. Increased glutathione (gamma-glutamylcysteinylglycine, GSH) conjugation (inactivation) due to elevated level of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) remains uncertain. In our experiments, a combination of liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between GSH and melphalan, one of the alkylating agents. The spontaneous reaction of 1mM melphalan with 5mM GSH at 37 degrees C in aqueous phosphate buffer for 1h gave primarily the monoglutathionyl and diglutathionyl melphalan derivatives, with small amounts of mono- and dihydroxy melphalan derivatives. We demonstrated that rat liver microsomal GST presented a strong catalytic effect on the reaction as determined by the increase of monoglutathionyl and diglutathionyl melphalan derivatives and the decrease of melphalan. We showed that microsomal GST was activated by melphalan in a concentration- and time-dependent manner. Microsomal GST which was stimulated approximately 1.5-fold with melphalan had a stronger catalytic effect. Thus microsomal GST may play a potential role in the metabolism of melphalan in biological membranes, and in the development of ADR.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.     

Inhibition of rat liver glutathione S-transferase isoenzymes by acrolein

The in vitro effect of the toxin and teratogen, acrolein, on the fetal rat liver glutathione S-transferase isoenzyme, YcYfetus, was investigated and compared with acrolein's effect on some of the adult rat liver glutathione S-transferase isoenzymes. Acrolein was found to inhibit all the isoenzymes investigated and double-reciprocal plots suggest that inhibition is either noncompetitive or mixed-type noncompetitive. It is therefore attractive to suggest that should a similar situation arise in vivo, it may provide one mechanism for the teratogenicity of acrolein.     

Differential induction of rat hepatic cytochrome P-448 and glutathione S-transferase B messenger RNAS by 3-methylcholanthrene

Liver poly(A⁺)-RNA isolated from untreated and 3-methylcholanthrene treated rats has been translated in the rabbit reticulocyte cell-free system in order to determine the level of translationally active cytochrome P-448, glutathione S-transferase B and serum albumin mRNAs. Translatable cytochrome P-448 mRNA was not detected in untreated rats; however in animals treated with 3-methylcholanthrene cytochrome P-448 mRNA was elevated markedly. Functional rat liver glutathione S-transferase B mRNA was elevated 2-fold by 3-methylcholanthrene administration, whereas the serum albumin mRNA level was decreased by 50%. Our results indicate that 3-methylcholanthrene is not just a specific inducer of drug metabolizing enzymes but can alter the mRNA level encoding other polypeptides and thus affect cellular homeostasis.     

Activation of rat liver microsomal glutathione S-transferase by gallic acid

The effect of phenolic antioxidants on the rat liver microsomal glutathione S-transferase (MGST1) was investigated in vitro. When microsomes were incubated with various polyphenolic antioxidants, gallic acid (3,4,5-trihydroxybenzoic acid) markedly increased MGST1 activity and the increase was prevented in the presence of superoxide dismutase (SOD) or catalase. The MGST1 activity increased by gallic acid was decreased by further incubation with sodium arsenite, a sulfenic acid reducing agent, but was not with dithiothreitol, a disulfide bond reducing agent. The incubation of microsomes with gallic acid in the presence of the NADPH generating system which generates reactive oxygen species (ROS) through cytochrome P-450 system increased the MGST1activity in spite of scavenging the ROS and the increase was also depressed by SOD/catalase. The increase of MGST1 activity by gallic acid was prevented by co-incubation with a stable radical, 1,1-diphenyl-2-picrylhydrazyl or ferric chloride. These results suggest that the gallic acid acts as a pro-oxidant and activates MGST1 through oxidative modification of the enzyme.     

Preferential binding of steroids by anionic forms of rat glutathione S-transferase

Rat liver glutathione S-transferases with isoelectric points near 6.7 were resolved from more basic forms of the protein. This anionic fraction represented about 30% of the total activity in liver with 1-chloro-2,4-dinitrobenzene and was the preponderant form utilizing trans-4-phenyl-3-butene-2-one as a substrate. The anionic transferases are dimeric proteins composed of two subunits designated as Yb and were distinguished from the cationic transferases on the basis of structural, immunological, and binding properties. Amino acid compositions and immunological properties of the anionic protein were similar to those of glutathione S-transferases A and C. The anionic forms had substantially less ordered secondary structure than cationic forms composed of subunits Ya and Yc. Stoichiometric ratios of two high affinity binding sites per dimer, also differentiated between the anionic and all of the cationic transferases which bind only a single mole of ligand. Affinity matrices composed of corticosterone or cholate, and circular dichroism methods, were used to demonstrate selective binding of steroids and bile acids to the anionic glutathione S-transferases. Glucocorticoids and progestins were shown to bind with high affinity whereas estrogens were bound at distinct lower affinity sites. In contrast to the cationic transferases, glutathione had no effect on binding of the steroids to the anionic forms, which suggested that these proteins have the capacity to bind these substances even in a milieu with high concentrations of glutathione.     

Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper

H(2)O(2) inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu(2+)/ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu(2+) binding to protein thiol groups. In the present study, we show that nanomolar Cu(2+) in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu(2+) in the presence of ascorbate, however, inhibited this activity. Micromolar Cu(2+) in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu(2+) either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu(2+)-binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu(2+)/ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.     

Acidic glutathione S-transferases of rat testis.

In most organs of the rat the predominant forms of glutathione S-transferase have alkaline (greater than 7.0) pI values. In contrast, in the cytosol from rat testes almost 50% of the transferase activity is due to isoenzymes with acidic (less than 7.0) pI values. We have purified three acidic forms of glutathione S-transferase from rat testis cytosol. One form accounted for more than 90% of the enzymic activity in the acidic fraction. This major form was a homodimer of a new subunit, termed Yt. This subunit had an electrophoretic mobility that was different from the subunits that form the alkaline transferases. In addition, functional and immunological studies were consistent with the unique nature of the Yt subunit. The two minor acidic enzymes of rat testis appeared to be heterodimers of the Yt subunit and a subunit with an electrophoretic mobility identical with that of the Yb subunit present in some alkaline enzymes.