Suppression of glutathione S-transferases in rat seminal vesicles or pituitary glands

We have studied the tissue-specific expression of GSH S-transferases in rat seminal vesicles and pituitary glands by in vitro translation and immunoprecipitation. The major GSH S-transferase subunit expressed in rat seminal vesicles belongs to the Yb mobility class whose expression diminishes when the rats are treated with pentobarbital. The pattern of GSH S-transferase expression in the pituitary gland is very similar to that of the rat brain with Yb size subunit(s) predominant. The Y beta size subunit is also expressed together with the Yc and Y delta subunits. The expression of GSH S-transferases was drastically reduced in pituitary gland poly(A) RNAs from diethylstilbestrol-treated, ovariectomized female rats. Xenobiotics such as phenobarbital, 3-methylcholanthrene, and trans-stilbene oxide induce rat liver GSH S-transferase activities, especially the Ya- and Yb-subunit containing isozymes. Induction of GSH S-transferases by a combination of the three xenobiotics is neither additive nor synergistic, however. Our results clearly demonstrate that GSH S-transferase expression in seminal vesicles and pituitary glands can be suppressed by phenobarbital and diethylstilbestrol, respectively. Our findings suggest that different GSH S-transferase isozymes respond differently to various xenobiotics. Both induction and suppression occur in rats treated with xenobiotics. This notion helps to explain the lack of additive or synergistic induction in rats treated with more than one xenobiotic.     

Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver.

Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity.     

Induction by butylated hydroxytoluene of rat liver gamma-glutamyl transpeptidase activity in comparison to expression in carcinogen-induced altered lesions

Butylated hydroxytoluene (BHT) at concentrations of 300-6000 ppm in the diet caused a dose-dependent increase in gamma-glutamyl transpeptidase (GGT) activity in normal F344 male rat liver at 18 weeks. However, the activities of glutathione S-transferases (GSTs) of rat liver cytosol were enhanced only at concentrations of 3000 or 6000 ppm BHT. Histochemically, the enhanced GGT activity was localized to hepatocytes surrounding the portal areas. Autoradiographic measurements of DNA synthesis showed that dietary BHT did not increase the level of cell proliferation and the GGT-positive hepatocytes did not exhibit different rates of DNA synthesis from those of GGT-negative cells. Feeding of the liver carcinogen N-2-fluorenylacetamide (FAA) induced foci and nodules of GGT-positive altered cells which exhibited higher rates of DNA synthesis than those of surrounding GGT-negative hepatocytes. Following iron loading, the periportal GGT-positive hepatocytes produced by BHT accumulated cellular iron, whereas the cells in FAA-induced lesions excluded iron. These results suggest that dietary BHT induces GGT activity in periportal hepatocytes without proliferation of the cells and that induction does not represent fetal expression or a preneoplastic alteration.     

Rat lung glutathione S-transferases: subunit structure and the interrelationship with the liver enzymes

Six forms of glutathione S-transferases designated as GSH S-transferase I (pI 8.8), II (pI 7.2), III (pI 6.8), IV (pI 6.0), V (pI 5.3) and VI (pI 4.8) have been purified from rat lung. GSH S-transferase I (pI 8.8) is a homodimer of Mr 25,000 subunits; GSH S-transferases II (pI 7.2) and VI (pI 4.8) are homodimers of Mr 22,000 subunits; and GSH S-transferases III (pI 6.8), IV (pI 6.0) and V (pI 5.3) are dimers composed of Mr 23,500 and 22,000 subunits. Immunological properties, peptide fragmentation analysis, and substrate specificity data indicate that Mr 22,000, 23,500 and 25,000, are distinct from each other and correspond to Ya, Yb, and Yc subunits, respectively, of rat liver.     

Subunit structure of human and rat glutathione S-transferases

In rat tissues different forms of glutathione (GSH) S-transferases represent various dimeric combinations of at least four different classes of subunits categorized on the basis of their Mr values as seen on polyacrylamide gels. These subunit types represent heterogeneous populations and the actual number of subunits in rat GSH S-transferases may be far more than is known at present. Human GSH S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits which can be classified into three types, A (Mr 26,500), B (Mr 24,500) and C (Mr 22,500). There is evidence for considerable charge heterogeneity in each of these subunit types.     

Constitutive and Aroclor 1254-induced hepatic glutathione S-transferase, peroxidase and reductase activities in genetically inbred mice

1. Constitutive and Aroclor 1254-induced hepatic glutathione (GSH) S-transferases, GSH peroxidase and GSH reductase activities were determined in 12 strains of 8-10 week-old inbred male mice. 2. The constitutive GSH S-transferase activity varied from 2.5 (SJL/JCR) to 8.9 (C57BL/6N) mumol/min/mg protein and the corresponding values for the Aroclor 1254-treated mice were in the range of 7.1-23.0 mumol/min/mg protein. Aroclor 1254 significantly induced GSH S-transferase activity in all mice, however, significant interstrain differences were found in inducibility. 3. Aroclor 1254-treatment caused a 4.2-fold induction of GSH S-transferase in NFS/NCR but only a 1.4-fold increase in AKR/NCR mice. Aroclor 1254 significantly induced GSH reductase in all strains studied while GSH peroxidase activity decreased in these mice. 4. The range of hepatic GSH levels in control and Aroclor 1254-treated mice was relatively narrow for both groups (6.59-11.25 microM/g wet tissue).     

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.     

Rat glutathione S-transferases supergene family. Characterization of an anionic Yb subunit cDNA clone

High multiplicity of GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Subunit composition analysis of rat liver GSH S-transferases indicated that heterodimer associations were not random, limiting the generation of GST isozyme multiplicity. We have analyzed a Yb subunit cDNA clone, pGTR187, that may correspond to an anionic Yb subunit sequence. Comparison with other GSH S-transferase cDNA sequences and blot hybridization results indicates that the multiple Yb subunits are encoded by a multigene family. This Yb subunit sequence has very limited homology to Ya and Yc subunit cDNAs, but slightly more sequence homology to the Yp subunit cDNA. More consistent sequence homology is found at the amino acid level with 28% conservation throughout the coding sequences. These results and results published from other laboratories clearly indicate that rat GSH S-transferases are products of at least four different gene families that constitute a supergene family. Conceptually, the supergene family may encode GSH S-transferases of very different structures that are essential to metabolize a multitude of xenobiotics in addition to serving other physiologically important functions.     

The enzymatic defluorination of fluoroacetate in mouse liver cytosol: the separation of defluorination activity from several glutathione S-transferases of mouse liver

The liberation of free fluoride ion from fluoroacetate (FAc) proceeds as an enzyme-catalyzed dehalogenation reaction in the soluble fractions of several organs of the CFW Swiss mouse. Liver contained the highest FAc defluorinating activity. The enzyme activity in other organs decreased in the order kidney greater than lung greater than heart greater than testes. No activity was detected in the brain. Experiments were designed to characterize and identify the enzyme species responsible for FAc metabolism in liver. Enzyme activity was dependent on the concentration of glutathione (GSH) in the assay mixture, with maximal activity occurring above 5 mM. The dehalogenation of FAc had an apparent Km of 7.0 mM when measured in the presence of a saturating concentration of GSH. An increase in the pH of the assay mixture enhanced fluoride release in both phosphate and borate buffer. The defluorination activity was reduced to negligible levels when stored for 24 h at 4 degrees C. The addition of either GSH, dithiothreitol, or 2-mercaptoethanol increased stability, with the latter providing protection for greater than 150 h at a concentration of 15 mM. DEAE anion-exchange chromatography separated the defluorinating activity from 90% of the soluble GSH S-transferase activity measured with 1-chloro-2,4-dinitrobenzene. FAc defluorination activity did not bind to a GSH affinity column which selectively separates it from a group of anionic GSH S-transferases. The GSH-dependent enzyme which dehalogenates FAc has unique properties and can be separated from the liver GSH S-transferases previously described in the literature.     

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.     

A subclass of glutathione S-transferases as intracellular high-capacity and high-affinity steroid-binding proteins.

The distribution of glucocorticoids incubated with rat liver cytosol preparations or administered in vivo to adrenalectomized rats was analysed by chromatographic procedures. Corticosterone or dexamethasone was co-eluted with Yb-type GSH S-transferases in anion-exchange and gel-permeation chromatography systems, and these glucocorticoids also were bound to Yb forms in analyses by immunoadsorbent and lysyl-GSH affinity matrices. Pretreatment of cytosol with lysyl-GSH to extract GSH S-transferases or incubation with excess bilirubin, which is expected to compete with steroids for binding to the protein, yielded preparations that were devoid of this major steroid-binding component. In mixtures of the multiple rat GSH S-transferases, corticosterone preferentially interacted with Yb forms rather than Ya and Yc subgroups. All of the multiple Yb forms resolved by chromatofocusing procedures retained the steroid-binding capacity. It is suggested that these abundant proteins can account for a considerable share of intracellular glucocorticoid binding and represent a high-affinity non-saturable binding component with potential to function in steroid-hormone metabolism and action.     

Immunocytochemical localization of the major glutathione S-transferases in adult Schistosoma mansoni

Indirect immunofluorescence was used to investigate the tissue distribution of the major isoenzymes of Schistosoma mansoni glutathione S-transferase (GSH S-transferase). When polyclonal rabbit antisera against GSH S-transferase isoenzymes SmGST-1, -02, and -3 were applied to cryostat or plastic-embedded sections of fixed adult worms, a punctate pattern of enzyme distribution was observed that was restricted to the parenchyma. Labeling was much more pronounced in males than females, consistent with the biochemically determined distribution of these enzymes between the sexes. Intense immunolabeling was noted within the subectocytoplasmic core tissue of the tubercles of the male that appeared to be connected to deep parenchymal cells by immunoreactive cell processes. Immunofluorescence could be blocked completely by prior incubation of antisera with affinity-purified enzyme. Although schistosome GSH S-transferases have been reported to be protective antigens, no immunoreactivity was detected within or on the tegument, including the dorsal spines of the male. The lack of tegumental immunoreactivity was confirmed by immunoblotting of tegumental membrane preparations following SDS-PAGE. Muscle fibers, vitelline cells, and cecal epithelium also failed to react. The fact that the GSH S-transferases were not uniformly distributed among all parenchymal cells suggests the existence of subpopulations of parenchymal cells that are preferentially involved in the conjugation of electrophiles with glutathione.     

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.     

Lipid Peroxidation Scavengers Prevent the Carbonylation of Cytoskeletal Brain Proteins Induced by Glutathione Depletion

In this study, we investigated the possible link between lipid peroxidation (LPO) and the formation of protein carbonyls (PCOs) during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH depletor diethyl maleate (DEM) in the absence or presence of classical LPO scavengers: trolox, caffeic acid phenethyl ester (CAPE), and butylated hydroxytoluene (BHT). All three scavengers reduced DEM-induced lipid oxidation and protein carbonylation, suggesting that intermediates/products of the LPO pathway such as lipid hydroperoxides, 4-hydroxynonenal and/or malondialdehyde are involved in the process. Additional in vitro experiments revealed that, among these products, lipid hydroperoxides are most likely responsible for protein oxidation. Interestingly, BHT prevented the carbonylation of cytoskeletal proteins but not that of soluble proteins, suggesting the existence of different mechanisms of PCO formation during GSH depletion. In pull-down experiments, beta-actin and alpha/beta-tubulin were identified as major carbonylation targets during GSH depletion, although other cytoskeletal proteins such as neurofilament proteins and glial fibrillary acidic protein were also carbonylated. These findings may be important in the context of neurological disorders that exhibit decreased GSH levels and increased protein carbonylation such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.     

Amino acid substitutions in the human glutathione S-transferases confer different specificities in the prostaglandin endoperoxide conversion pathway

The human glutathione S-transferases 1-1 and 2-2, which differ from each other by 11 amino acids, have different catalytic activities against cumene hydroperoxide and t-butyl hydroperoxide. Using prostaglandin H2 as the peroxide substrate, we found that GSH S-transferase 1-1 catalyzed the transformation of prostaglandin H2 to prostaglandin F2 alpha and E2 at a 4:1 ratio whereas GSH S-transferase 2-2 produced primarily prostaglandin D2 and F2 alpha at a 4:1 ratio. Our results indicate that GSH S-transferases catalyze the reduction and isomerization of prostaglandin H2 endoperoxide in vitro. We suggest that the amino acid substitutions between these two isozymes may be responsible for the difference in catalytic specificities. We propose that these isozymes are important reagents for the biosynthesis of various prostaglandins.     

Human glutathione S-transferases. The Ha multigene family encodes products of different but overlapping substrate specificities

The human glutathione S-transferase cDNAs encoding subunits 1 and 2 contain intrinsic ribosome-binding sites in their 5'-untranslated regions for direct expression in Escherichia coli. We show that functional human GSH S-transferases 1-1 and 2-2 are synthesized from lambda gt11 cDNA clones lambda GTH1 and lambda GTH2 in phage lysates of E. coli Y1090, in lysogens of E. coli Y1089, and from the plasmid expression constructs in pKK223-3. The E. coli-expressed human GHS S-transferases 1-1 and 2-2 do not have blocked N termini in contrast to those directly purified from human livers. These two isozymes, with 11 amino acid substitutions between them, are similar in their Km values for GSH and 1-chloro-2,4-dinitrobenzene and Kcat values for this conjugation reaction. The human GSH S-transferase 2-2, however, is a more active GSH peroxidase than transferase 1-1 toward cumene hydroperoxide and t-butyl hydroperoxide. Our results indicate that different members of a GSH S-transferase gene family with limited amino acid substitutions have different with limited amino acid substitutions have different but overlapping substrate specificities. We propose that accumulation of single amino acid replacements may be an important mechanism for generating diversity in GSH S-transferases with various xenobiotic substrates. In situ chromosomal hybridization results show that the GSH transferase Ha genes are located in the region of 6p12.     

Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label

The glutathione S-transferases are a family of dimeric enzymes that catalyze the reaction between GSH and a variety of electrophiles. Two closely related isozymes, referred to as YaYa and YcYc, were purified from rat liver. A radiolabeled azido derivative of glutathione (S-(p-azidophenacyl)[3H]glutathione) was prepared and used to label covalently the active site of the above two glutathione S-transferases. The noncovalently bound affinity label was a competitive inhibitor of glutathione S-transferase YaYa toward both 1-chloro-2,4-dinitrobenzene and GSH. The covalently labeled enzymes no longer bound to a GSH-affinity column, and covalent labeling was reduced in the presence of GSH and S-(dinitrophenyl)glutathione. These results suggest that the affinity label was binding at the active site. The covalently labeled enzymes were digested with trypsin, and the labeled peptides were purified by HPLC and then sequenced. A single-labeled peptide was identified in the tryptic digest of the YaYa isozyme, whereas two labeled peptides were present in the tryptic digest of YcYc. The Ya peptide sequence was identical with the published deduced sequence of amino acids between residues 212 and 218 and the sequences of the two peptides purified from Yc were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Ya peptide and the smaller peptide purified from Yc came from the same region of the Ya and Yc subunits. This common region and a second region of the Yc subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.     

Modulation of the cytotoxicity and mutagenicity of benzo[a]pyrene and benzo[a]pyrene 7,8-diol by glutathione and glutathione S-transferases in mammalian cells (CHO/HGPRT assay)

Biologically reactive metabolites of benzo[a]pyrene (BP) and benzo[a]-pyrene 7,8-diol (BP-diol), formed by the mixed-function oxidase (MFO) system, are substrates for conjugation and detoxication by glutathione (GSH) when catalyzed by glutathione S-transferases (GSHT). We have investigated the detoxication of BP- and BP-diol-induced cytotoxicity and mutagenicity with GSH by supplementing the S9 mix used in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) assay with GSH (6.5 mM) or GSH plus GSHT. The addition of GSH to the S9 mix resulted in a reduction of BP- and BP-diol induced cytotoxicity. GSH plus GSHT eliminated BP-induced cytotoxicity and reduced the mutagenicity of BP. GSH inhibited the mutagenicity at low (essentially non-lethal) concentrations of BP-diol, but did not do so at toxic concentrations. GSH plus GSHT inhibited the cytotoxicity and mutagenicity of BP-diol at concentrations not affected by GSH alone. These studies indicate that biochemical mechanisms of detoxication can affect the biological activity of a carcinogen, such as BP or BP-diol as profoundly as bioactivation by the MFO system.     

A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses

Glutathione (L-γ-Glutamyl-L-Cysteinylglycine) appears as the major nonprotein thiol compound in yeasts. Recent advances have shown that glutathione (GSH) seems to be involved in the response of yeasts to different nutritional and oxidative stresses. When the yeast Saccharomyces cerevisiae is starved for sulfur or nitrogen nutrients, GSH may be mobilized to ensure cellular maintenance. Glutathione S-transferases may be involved in the detoxification of electrophilic xenobiotics. Vacuolar transport of metal derivatives of GSH ensure resistance to metal stress. Growth of methylotrophic yeasts on methanol results in the formation of an excess formaldehyde that is detoxified by a GSH-dependent formaldehyde dehydrogenase. Growth of yeasts on glycerol results in the accumulation of methylglyoxal detoxified by the glyoxalase pathway. Glutathione per se can react with oxidative agents or is involved in the oxidative stress response through glutathione peroxidase.     

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.