Methyl linoleate ozonide as a substrate for rat glutathione S-transferases: reaction pathway and isoenzyme selectivity

The 9,10-mono-ozonide of methyl linoleate was shown to be a substrate for rat hepatic cytosolic, rat lung cytosolic and rat hepatic microsomal glutathione S-transferases (GST). The activities of lung cytosol and liver microsomes with methyl linoleate ozonide (MLO) were found to be high relative to the activity demonstrated by liver cytosol, as compared with their respective activities towards 1-chloro-2,4-dinitrobenzene (CDNB). Only a slight catalytic activity towards the ozonide was noticed for rat lung microsomes. Isoenzyme 2-2 exhibited the highest specific activity (208 nmol/min/mg) when isoenzymes 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 7-7 were compared. This isoenzyme accounts for approx. 25% of cytosolic GST protein in rat lung, while in rat liver it represents approx. 9%. This may partly explain the high activity towards the ozonide noticed for rat lung cytosol. No stable conjugates were formed as products of the reaction of MLO with glutathione; although two glutathione-conjugates were noticed on TLC, they were only formed as intermediate compounds. Coupling of an aldehyde dehydrogenase assay or a glutathione reductase assay to the GST-catalyzed conjugation, demonstrated that oxidized glutathione and aldehydes are formed as the major products in the reaction. To further confirm the formation of aldehydes, the products of the GST-catalyzed reaction were incubated with 2,4-dinitrophenylhydrazine, which resulted in hydrazone formation. In conclusion, the activity of the GST towards the ozonide of methyl linoleate is similar to their peroxidase activity with lipid hydroperoxides as substrates.     

Role of cardiac glutathione transferase and of the glutathione S-conjugate export system in biotransformation of 4-hydroxynonenal in the heart

There is a remarkable difference in the isozyme pattern between cardiac and hepatic glutathione S-transferases in rat (Ishikawa, T., and Sies, H. (1984) FEBS Lett. 169, 156-160), and one near-neutral isozyme (pI = 6.9) of the cardiac glutathione S-transferases was found to have a significantly high activity toward 4-hydroxynonenal. The isozyme was inhibited by the resulting glutathione S-conjugate of 4-hydroxynonenal competitively with GSH and noncompetitively with 4-hydroxynonenal. The kinetic parameters estimated for the isozyme were: kcat = 460 mol X min-1 X mol enzyme-1, Km = 50 microM for 4-hydroxynonenal, Ki = 85 microM. When the heart was perfused with 4-hydroxynonenal, a marked decrease was observed in the intracellular GSH level, accompanied by an increase of glutathione S-conjugate of 4-hydroxynonenal in the heart. The rate of the conjugation reaction was more than 30 times the rate of the spontaneous reaction, the half-life of 4-hydroxynonenal in the heart being less than 4 s. The glutathione S-conjugate of 4-hydroxynonenal was released from the heart into the perfusion medium. Saturation kinetics were observed for the release with respect to the intracellular level of the S-conjugate (Vmax = 12 nmol X min-1 X g heart-1), and there was a competition by the S-conjugate for GSSG release. The release of the glutathione S-conjugate is considered as a carrier-mediated process and to be important not only in interorgan glutathione metabolism but also in diminishing the inhibitory effect of the S-conjugate on glutathione S-transferases and glutathione reductase.     

Effects of sulfite on glutathione S-sulfonate and the glutathione status of lung cells

A mechanistic study was performed to elucidate the biochemical events connected with the cocarcinogenic effect of sulfur dioxide (SO2). Glutathione S-sulfonate (GSSO3H), a competitive inhibitor of the glutathione S-transferases, forms in lung cells exposed in culture to sulfite, the hydrated form of SO2. Changes in glutathione status (total GSH) were also observed during a 1-h exposure. Some cells were pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. In human lung cells GSSO3H formed in a concentration-dependent manner, while glutathione (GSH) increased and glutathione disulfide (GSSG) decreased as the extracellular sulfite concentration was increased from 0 to 20 mM. The ratio of GSH/GSSG increased greater than 5-fold and the GSH/GSSO3H ratio decreased to 10 with increasing sulfite concentration. GSSO3H formed in rat lung cells exposed to sulfite, with no detectable effect on GSH and GSSG. GSSO3H also formed from cellular GSH mixed disulfides. GSSO3H formed rapidly, reaching its maximum value in 15 min. The viability of both cell types was unaffected except at 20 mM sulfite. GSSO3H incubated with human lung cells did not affect cellular viability. BCNU inhibited cellular GSSO3H reductase to the same extent as GSSG reductase. These results indicate that GSSO3H is formed in cells exposed to sulfite, and could be the active metabolite of sulfite responsible for the cocarcinogenic effect of SO2 by inhibiting conjugation of electrophiles by GSH.     

Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

Rat lung glutathione release: response to oxidative stress and selenium deficiency

We performed experiments to characterize the glutathione-dependent metabolism occurring during tert-butyl hydroperoxide infusion in isolated perfused rat lungs and to examine the effect of selenium deficiency on this metabolism. Selenium deficiency resulted in decreased lung glutathione peroxidase activity but normal glutathione reductase activity and glutathione content. Infusion of the hydroperoxide into control lungs caused a proportional increase in tissue glutathione disulfide (GSSG) concentration and release of GSSG into the perfusate up to an infusion rate of 250 nmol of tert-butyl hydroperoxide X min-1 X 100 g body wt-1. Infusion rates greater than this resulted in continued rise of tissue GSSG concentrations but GSSG release into the perfusate plateaued. Infusion of tert-butyl hydroperoxide into selenium-deficient rat lungs resulted in much lower concentrations of tissue GSSG and GSSG release into the perfusate; however, release in the selenium-deficient rat lung was also found to be saturable at infusion rates of 450 nmol of tert-butyl hydroperoxide X min-1 X 100 g of body wt-1. Selenium deficiency in the rat decreases the rate of reduction of infused tert-butyl hydroperoxide by glutathione and may predispose the lung to free radical damage.     

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.     

Indomethacin inhibition of glutathione S-transferases

Indomethacin inhibited rat liver glutathione S-transferases (EC 2.5.1.18). Its inhibition was non-competitive with respect to 3,4-dichloronitrobenzene with an apparent Ki of 5.3 X 10(-5) M and uncompetitive with respect to glutathione with an apparent Ki of 4.0 X 10(-5) M. 4-Chlorobenzoic acid and 5-methoxy-2-methylindole-3-acetic acid, two metabolites of indomethacin, were weak inhibitors of the enzymes. On the other hand, meclofenamic acid was a competitive inhibitor of the enzymes with an apparent Ki of 3.0 X 10(-4) M. Possible significance of these findings in arachidonic acid metabolism is discussed.     

Multiple glutathione S-transferase isoforms are present on male germ cell plasma membrane.

Phase II detoxification enzymes, the glutathione S-transferases (GSTs) of 24 kDa are known to be cytosolic enzymes. This study shows that multiple GST isoforms that are 24 kDa in size are present on the extracellular side of the plasma membrane of rat male germ cells. The GST activity of male germ cell plasma membranes is several folds higher than somatic cell plasma membrane GST activity. Isoform composition of the germ cell plasma membrane and the cytosolic pool differ, GSTM5 and GSTPi being absent on the plasma membranes. The molecular masses of the common isoforms are comparable between the two pools and both pools show GST and glutathione peroxidase activity.     

Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens.

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.     

Glutathione S-transferase-catalyzed conjugation of 9,10-epoxystearic acid with glutathione

The possible role of glutathione S-transferases (GST) in detoxification of fatty acid epoxides generated during lipid peroxidation has been evaluated. Present studies showed that cytosolic human glutathione S-transferases belonging to alpha, mu, and pi classes isolated from human liver and lung catalyzed the conjugation of glutathione and 9,10-epoxystearic acid. The product of enzymatic reaction, i.e., conjugate of GSH and epoxystearic acid, was isolated and characterized. The Michaelis constant (Km) values of the alpha, mu, and pi classes of GSTs for 9,10-epoxystearic acid were found to be 0.47, 0.32 and 0.80 mM, respectively, whereas the maximal velocity (V max) values for the alpha, mu, and pi classes of GSTs were found to be 142, 256, and 52 mol/min/mol, respectively. These results indicate that even though 9,10-epoxystearic acid is a substrate for all the three classes of GSTs, the mu class isozymes have maximum activity toward this substrate and may preferentially metabolize fatty acid epoxides more effectively as compared to the other classes of GSTs.     

4-Hydroxynonenal inhibits glutathione peroxidase: protection by glutathione.

4-Hydroxy-2,3-trans-nonenal, a lipid peroxidation product, inhibits glutathione peroxidase in a concentration-dependent manner. The concentration providing 50% inhibition is 0.12 mM. This inhibition can be almost completely (89%) prevented by 1 mM glutathione added to the incubation mixture 30 min before 4-hydroxy-2,3-trans-nonenal or 2,3-trans-nonenal, but not by other thiol-containing antioxidants such as 0.5 mM dithiothreitol or beta-mercaptoethanol. Again the addition of 1 mM glutathione, and not of 0.5 mM dithiothreitol or beta-mercaptoethanol, to the enzyme 30 min after incubation with 4-hydroxy-2,3-trans-nonenal restores activity to the same extent as does the preincubation with GSH. In view of the known reactivity of 4-hydroxy-2,3-trans-nonenal with lysine residues and the reversibility of the inhibition, the involvement of a lysine residue in GSH binding to glutathione peroxidase is proposed. The potential relevance of the inhibition of glutathione peroxidase by 4-hydroxy-nonenal to oxidative tissue damage is discussed with particular emphasis on neurological disorders.     

Plasma glutathione turnover in the rat: effect of fasting and buthionine sulfoximine

Hepatic glutathione (GSH) plays an important role in the detoxification of reactive molecular intermediates. Because of evidence that the intrahepatic turnover of glutathione in the rat may be largely accounted for by efflux from hepatocytes into the general circulation, the quantitation of plasma GSH turnover in vivo could provide a noninvasive index of hepatic glutathione metabolism. We developed a method to estimate plasma glutathione turnover and clearance in the intact, anesthetized rat using a 30-min unprimed, continuous infusion of 35S-labelled GSH. A steady state of free plasma glutathione specific radioactivity was achieved within 10 min, as determined by high-pressure liquid chromatography with fluorometric detection after precolumn derivatization of the plasma samples with monobromobimane. The method was tested after two treatments known to alter hepatic GSH metabolism: 90 min after intraperitoneal injection of 4 mmol/kg buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and after a 48-h fast. Liver glutathione concentration (mean +/- SEM) was 5.00 +/- 0.53 mumol/g wet weight in control rats. It decreased to 3.10 +/- 0.35 mumol/g wet weight after BSO injection and to 3.36 +/- 0.14 mumol/g wet weight after fasting (both p less than 0.05). Plasma glutathione turnover was 63.0 +/- 7.46 nmol.min-1.100 g-1 body weight in control rats, 35.0 +/- 2.92 nmol.min-1.g-1 body weight in BSO-treated rats, and 41.7 +/- 2.28 nmol.min-1.g-1 body weight after fasting (both p less than 0.05), thus reflecting the hepatic alterations. This approach might prove useful in the noninvasive assessment of liver glutathione status.     

Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively.     

Rat hepatic glutathione S-transferase-mediated embryotoxic bioactivation of ethylene dibromide.

The embryotoxic effects of ethylene dibromide (EDB) bioactivation, mediated by purified rat liver glutathione S-transferases (GST), were investigated using rat embryos in culture. Significant EDB metabolism was observed with rat liver GST purified by affinity chromatography (specific activity of 188 +/- 11.3 nmol/min/mg protein). The reaction was enzymatic in nature and the conjugation rate was proportional to the concentration of EDB (up to 0.75 mM) and the enzyme present in the reaction medium. EDB activation by 100 units (1 unit = 1 nmol of glutathione consumed per min) of purified rat liver GST caused a significant reduction in general development as measured by crown-rump length, yolk sac diameter, somite number, and the composite score for different morphological parameters (Brown and Fabro methodology). Structures most significantly affected were the central nervous and olfactory systems as well as the yolk sac circulation and allantois. The results of this study clearly indicate that under in vitro conditions, bioactivation of EDB by GST can lead to embryotoxicity.     

Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxication

Corn ( Zea mays L.) glutathione S-transferases (EC 2.5.1.18) have attracted interest, in part, due to their involvement in the metabolism of several herbicides, including atrazine and alachlor. Three corn, glutathione S-transferases have been purified, and cDNA clones have been isolated and sequenced for two of these, GST I and GST III. In addition to showing some amino acid sequence similarity to each other, the two sequenced corn glutathione S-transferases also show some similarity to rat and human enzymes. The corn glutathione S-transferases responsible for atrazine tolerance have not yet been purified or cloned, but purification attempts indicate that corn has two glutathione S-transferases with activity towards atrazine. While many glutathione S-transferases from various organisms have been detected by using 1-chloro-2,4-dinitrobenzene as a substrate, the atrazine-specific glutathione S-transferases have very little or no activity with 1-chloro-2,4-dinitrobenzene. This shows the importance of assaying with a variety of substrates when characterizing glutathione S-transferases.     

Subunit composition of rat liver glutathione S-transferases

The plasmid pGTR112 contains partial coding sequences for one of the rat liver glutathione S-transferase subunits. We have used immobilized pGTR112 DNA to select for complementary and homologous liver poly(A)-RNAs under conditions of increasing stringency for hybridization. Each fraction of selected poly(A)-RNAs was assayed by in vitro translation followed by immunoprecipitation. A total of four distinct polypeptides precipitated by antiserum against rat liver glutathione S-transferases were resolved by NaDodSO₄ polyacrylamide gel electrophoresis. They are separated into two pairs according to the sequence homology of their poly(A)-RNAs with the pGTR112 DNA. Purified rat liver glutathione S-transferases can be resolved on gradient NaDodSO₄ polyacrylamide gels into four polypeptides. There should be ten isozymes of different binary combinations from four distinct subunits for the rat liver glutathione S-transferases.     

Irreversible inhibition of hepatic glutathione S-transferase by ciprofibrate, a peroxisome proliferator

Ciprofibrate (2-[4-(2,2-dichlorocyclopropyl) phenoxy]2-methyl propionic acid) which is a hypolipidemic agent and has been shown to cause peroxisome proliferation, non-competitively inhibits glutathione S-transferase activity of rat liver, both in vivo and in vitro. Among all the glutathione S-transferases of rat liver, ligandin is maximally inhibited by ciprofibrate. Studies with the purified glutathione S-transferases of rat liver indicate that the affinities of different subunits of liver enzymes for ciprofibrate are in the order Ya greater than Yb, Yb' greater than Yc.     

Activation of rat glutathione transferases in class mu by active oxygen species

The activities of rat glutathione transferases (GSTs) 3-3, 3-4, 4-4 in Class mu towards 1-chloro-2,4-dinitrobenzene (CDNB) but not 1,2-dichloro-4-nitrobenzene were increased up to 5-fold during preincubation with 0.4 mM xanthine and xanthine oxidase in 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA. The activated GST 3-4, purified by S-hexylglutathione affinity chromatography after the treatment, had a higher specific activity (130 units/mg) than that of the nontreated (35 units/mg), the Km and Vmax values for glutathione or CDNB also were increased. Other rat GSTs in Class alpha and pi were inactivated by the same treatment. In the presence of superoxide dismutase, the activation of GST 3-4 did not occur.     

Trialkyl phosphorothioates and glutathione S-transferases

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSS-trimethyl phosphorodithioate (OSS-Me(O] and OOS-trimethyl phosphorothioate (OOS-Me(O] the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.