Human glutathione S-transferases

1. Multiple forms of glutathione S-transferase (GST) isoenzymes present in human tissues are dimers of subunits belonging to three distinct gene families namely alpha, mu and pi. Only the subunits within each class hybridize to give active dimers. 2. These subunits are differentially expressed in a tissue-specific manner and the composition of glutathione S-transferases in various tissues differs significantly. 3. Minor GST subunits not belonging to these three classes are also present in some tissues. 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.     

Human glutathione S-transferases

Human glutathione S-transferases (GSTs) are a functionally diverse family of soluble enzymes of detoxification that use reduced glutathione (GSH) in conjugation and reduction reactions. Toxic electrophiles, including a variety of carcinogens, are substrates for the GSTs and after conjugation or reduction they are more easily excreted into bile or urine. Many of the GSTs have been cloned, and the three-dimensional structures of GSTs from several species, including humans, have been determined. These data have provided significant insight into how the GSTs function as enzymes. Many GST substrates are inducers of GST gene expression; nonsubstrate inducers include H2O2 and other reactive oxygen species. The regulatory elements of several human GST genes have been partially characterized, and the regulation of the GSTs in humans appears to be very different from that in rodents. Several polymorphisms of GST expression occur commonly in humans and have been associated with an increased susceptibility to certain cancers, particularly when combined with other genetic and environmental factors such as smoking. The role of GSTs in protecting cells from injury by toxic electrophiles continues to be developed.     

Binding of paracetamol metabolites to mouse liver glutathione S-transferases

In mouse liver homogenate with an intact microsomal metabolism covalent binding of [14C]-paracetamol amounted to 1 nmol/mg protein. 65% of the total radioactivity were bound to soluble protein and 35% to microsomes. In the soluble fraction the major radioactivity peak co-chromatographed with glutathione S-transferase activity on Sephacryl S-300. Two different minor labelled fractions with apparent molecular weights of 130 000 and 25 000 daltons were also found. In a second experiment in a reconstituted system of microsomes and supernatant, 86% of the radio-activity was bound to supernatant and 14% by of microsomes. Following ion exchange chromatography of the supernatant on DEAE-Sepharose, the two major radioactivity-containing fractions coincided with GSH-S-transferase activities, but not with selenium-dependent or non-selenium-dependent glutathione peroxidase. The data show that irreversible binding of paracetamol metabolites in mouse liver occurs preferentially to GSH-S-transferases.     

Age and gender dependent levels of glutathione and glutathione S-transferases in human lymphocytes

Glutathione S-transferases (GSTs) are a family of enzymes involved in the detoxification of a wide range of chemicals including chemical carcinogens. Human cytosolic GSTs are divided into four major classes; alpha, mu, pi and theta. This study was performed to evaluate the influence of age and gender on the GST isoenzyme expression and glutathione (GSH) content in lymphocytes. Blood was collected from 124 healthy controls, which were divided into age groups of 20-40 years (21 females, 20 males), 40-60 years (20 females, 21 males) and 60-80 years (20 females, 22 males). Lymphocytes were isolated by density centrifugation on Histopaque-1077. After homogenization, cytosolic fractions were isolated. Herein, GST isoenzyme levels were determined by densitometrical analysis of western blots after immunodetection with monoclonal antibodies. Total GSH content was determined by high performance liquid chromatography after conjugation with monobromobimane. Spearman rank correlation and Wilcoxon rank sum tests were used for statistical evaluation. Lymphocytic GSTmu and pi levels were not correlated with age or gender. GSTalpha was not detectable in lymphocytes. GSH contents were not different in males and females, but decreased with age in both males and females. In age group 60-80, GSH content was significantly lower as compared with age groups 20-40 and 40-60 in both sexes. Since high GSH is an essential factor in the detoxification of many compounds, these data indicate that the detoxification potential of the GSH/GST system in lymphocytes may decrease with age in man.     

Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.     

Over-expression of glutathione S-transferases, DT-diaphorase and an apparently tumour-specific cytosolic class-3 aldehyde dehydrogenase by Warthin tumours and mucoepidermoid carcinomas of the human parotid gland

A comprehensive biochemical, immunological and histological study was undertaken during different stages of experimental allergic encephalomyelitis (EAE). Wistar rats with EAE induced by sensitization with bovine myelin showed a maximum decrease of body weight 14-16 days post-inoculation (dpi), coincident with the appearance of the paralysis symptom (acute period). Quantitation of some brain components indicated a temporal dissociation among the alterations observed. The higher diminution of myelin basic protein (MBP) occurred at 6 dpi and then increased to reach 21 dpi, a normal value. Also, the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase was reduced by 40% with respect to control animals only at 6 dpi. The total lipid content was normal; however, among the individual lipids, sulfatides were principally degraded during the acute stage but the amount of cerebrosides was decreased during the recovery period (29-40 dpi). Free cholesterol was similar in both groups of animals, whereas cholesterol esters were detected in EAE animals from 14 to 40 dpi. Central nervous system meningeal and parenchymal infiltration with mononuclear cells was recognized principally at 14 dpi, but some of cells were still present at 40 dpi. Deposits of immunoglobulins in the infiltrated regions as well as in spinal cord motor neurons were observed among 14-29 dpi. Total circulating antibodies to MBP began to increase at 14 dpi, reaching a plateau at 21 dpi and then maintaining this value until 40 dpi. However, the population of anti-MBP antibodies that also recognizes the neuronal protein synapsin was only present at 14 dpi. The present results suggest that the neurological symptoms can be related to some early changes in the myelin membrane followed by alterations involving neuronal structures. The existence of immunological factors against some epitopes in MBP that also recognize a synaptosomal protein might account, at least in part, for the axonal damage and disruption of the normal interneuronal activity in EAE and lead together with the alterations in some specific myelin constituents and the concomitant CNS inflammatory process to the observed hindlimb paralysis.     

Isoform-specific biotransformation of glyceryl trinitrate by rat aortic glutathione S-transferases

The objective of this study was to purify and characterize rat aortic glutathione S-transferases (GSTs) and to assess their role in the biotransformation of the nitrovasodilator, glyceryl trinitrate (GTN). Two class alpha GSTs (Ya and Yc) a class mu GST (Yb2) and a class pi GST (Yp) were identified in rat aortic cytosol. Partial purification of three of these (Yb2, Yc and Yp) was achieved by affinity chromatography with S-hexylglutathione agarose. Further purification by cation- and anion-exchange chromatography resulted in the purification of GST Yc and GST Yb2/Yp to apparent homogeneity, a purification of 200- and 110-fold, respectively. Purified GST Yc and Yb2/Yp mediated GTN biotransformation with similar rates. GST activity and GTN biotransformation by rat aortic cytosol and affinity-purified GSTs were highly sensitive to inhibition by the class mu selective inhibitors Basilen Blue and bromosulfophthalein. Removal of GST Yb2 from rat aortic cytosol by immunoprecipitation resulted in marked inhibition of GST activity and GTN biotransformation. We conclude that the GSTs account for the major portion of GTN biotransformation in rat aortic cytosol, and that this is primarily attributable to the GST Yb2 isoform.     

An increase in cytosolic protease activity during liver preservation. Inhibition by glutathione and glycine

Degradative cytosolic proteolysis contributes to cell injury following ATP depletion. Although ATP depletion is a salient feature of ischemic liver storage for transplantation, information regarding cytosolic protease activity during liver storage is lacking. Thus our aim was to measure liver cytosolic protease activity following ischemic storage. A progressive increase in total cytosolic protease activity was observed over time at both 37 degrees C and 4 degrees C, but the increase was greater at 37 degrees C. Total cellular proteolysis was also temperature-dependent during anoxia (37 degrees C > 4 degrees C), demonstrating a physiologic correlation between cellular proteolysis and measurements of cytosolic protease activity. The stimulation of total cytosolic protease activity was due to an increase in metallo- and aspartate protease activity. Of particular interest, glutathione (GSH) inhibited both metalloprotease and aspartate protease activity from cytosol of stored livers. Glycine, the carboxyl-terminal amino acid of GSH, also inhibited both metalloprotease and aspartate protease activity. In addition to being an antioxidant, GSH may exert its protective effects during organ preservation by inhibiting cytosolic proteases--perhaps via its glycine moiety. These experiments support the hypothesis that degradative proteolysis contributes to liver injury during organ preservation.     

Inhibition of embryonic retinoic acid synthesis by aldehydes of lipid peroxidation and prevention of inhibition by reduced glutathione and glutathione S-transferases

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.     

Localization of glutathione S-transferases alpha and pi in human embryonic tissues at 8 weeks gestational age

Glutathione S-transferases (GST) are a family of enzymes involved in the detoxification of xenobiotics. In humans, GST are divided into four different classes, alpha, mu, pi and theta, with partly overlapping substrate specificity and a tissue-specific expression pattern. We studied the cellular distribution of GST alpha and pi in a variety of human embryonic organs obtained from an extra-uterine monozygotic twin pregnancy at 8 weeks' gestational age. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Three 4 microm thick sections were used, one for routine haematein and eosin staining, the others for immunohistochemical determination using monoclonal and polyclonal antibodies against GST alpha and pi, respectively. Both GST alpha and pi were present in hepatocytes, gastrointestinal epithelium, adrenal gland medulla, and tela chorioidea in the telencephalon. GST pi, but not alpha, was found in the epithelium of pancreatic and pulmonary glands, trachea, nephrons and urinary collecting ducts, as well as in the pia mater of the telencephalon and in developing nerve tissue in the gastrointestinal muscularis mucosae. In summary, we have demonstrated that immunoreactive protein for both GST alpha and pi is expressed in the human embryo at 8 weeks' gestational age. The early expression of GST alpha and pi in the epithelia of the urinary and digestive tracts and the respiratory system supports the importance of GST in the detoxification of potentially toxic or carcinogenic compounds. Our results suggest that the embryo itself is capable of detoxifying noxious compounds that are generated intracellularly or that cross the trophoblastic tissue.     

Menadione toxicity in Saccharomyces cerevisiae cells: activation by conjugation with glutathione

Menadione (2-methyl-1,4-naphthoquinone) has been used extensively as an oxidant stressor at the cellular level. However, the mechanism of cytotoxicity of this compound still remains controversial. This study deals with the role of intracellular glutathione in the resistance of the yeast Saccharomyces cerevisiae to menadione. Incubation with 0.5 mM menadione resulted in a decrease of total glutathione concentration in yeast cells, intracellular formation of menadione S-glutathione conjugate and export of the conjugate from cells. GSH-deficient mutants showed lower stimulation of superoxide and hydrogen peroxide production upon exposure to menadione and were more resistant to menadione than wild-type isogenic strains. These results indicate that in yeast cells the formation of S-glutathione conjugate is a major pathway of menadione metabolism and that this reaction leads to redox activation of menadione but permits its removal from the cells.     

Abnormal glutathione conjugation in patients with tyrosinaemia type I

Previous studies have suggested that tyrosinaemia type I may be associated with reduced glutathione availability due to conjugation of tyrosinaemia-associated reactive intermediates with glutathione. In the present study, the glutathione/ glutathione S-transferase system of two tyrosinaemia patients and three healthy controls were characterized by administering the racemic sedative drug bromisoval, a probe drug for assessing glutathione conjugation activity in vivo. Furthermore, concentrations of glutathione and glutathione S-transferase class alpha (GSTA) isoenzymes as well as the glutathione S-transferase class mu phenotype were assessed in the blood of six tyrosinaemia patients. The excretion of bromisoval mercapturates in healthy children was comparable to that observed in healthy adults. Tyrosinaemia patients were found to have a very high urinary recovery of bromisoval mercapturates (> or = 60% of the dose compared to about 30% for healthy, age-matched children and adults), which could be attributed mainly to a higher urinary excretion of the mercapturate derived from S-bromisoval. Healthy children and adults predominantly excrete the (R)-bromisoval mercapturate. The differences in amount excreted as well as in stereoselectivity of the urinary excretion of bromisoval mercapturates in tyrosinaemia patients are possibly related to an increased activity of specific glutathione S-transferase isoenzymes. Plasma glutathione and blood cell glutathione disulphide concentrations in tyrosinaemia patients were normal. Low blood cell glutathione concentrations were in general found only in two patients with a poor clinical condition. These results indicate that, in contrast to previous suggestions, reduced glutathione availability is not a generalized problem in (stabilized) tyrosinaemia patients.     

Thiol ester hydrolysis catalyzed by glutathione S-transferase A1-1

rGSTA1-1 has been shown to catalyze the hydrolysis of the thiol ester glutathionyl ethacrynate (E-SG). In contrast, neither the retro-Michael addition with the substrate EA-SG, to yield GSH and ethacrynic acid (EA), nor the conjugation reaction between GSH and EA to yield the thiol ester E-SG was catalyzed to any measurable extent under similar conditions. The steady state kcat and KM for hydrolysis of E-SG by wild type rGSTA1-1 were 0.11 +/- 0.009 min-1 and 15.7 +/- 1.6 mM, respectively. The site-directed mutant, Y9F, in which the catalytic Tyr-9 is substituted with Phe, was completely inactive in this reaction. To uncover a mechanistic signature that would distinguish between direct hydrolysis and covalent catalysis involving acylation of Tyr-9, solvent isotope exchange and mass spectrometry experiments were performed. No 18O incorporation into the starting thiol ester was detected with initial velocity solvent isotope exchange experiments. However, covalent adducts corresponding to acylated protein also were not observed by electrospray ionization mass spectrometry, even with an assay that minimized the experimental dead time and which allowed for detection of N-acetyltyrosine acylated with EA in a chemical model system. The kon and koff rate constants for association and dissociation of E-SG were determined, by stopped flow fluorescence, to be 5 x 10(5) s-1 M-1 and 6.7 s-1, respectively. Together with the isotope partitioning results, these rate constants were used to construct partial free energy profiles for the GST-catalyzed hydrolysis of E-SG, assuming that Tyr-9 acts as a general acid-base catalyst. The "one-way flux" of the thiol esterase reaction results directly from the thermodynamic stability of the products after rate-limiting attack of the thiol ester by H2O or Tyr-9, and is sufficient to drive the hydrolysis to completion, in contrast to GST-catalyzed breakdown of other GSH conjugates.     

Effect of ifosfamide treatment on glutathione and glutathione conjugation activity in patients with advanced cancers

Several studies have suggested that the glutathione/glutathione S-transferase (GSH/GST) system is involved in resistance of tumors toward ifosfamide and other cytostatic agents. Besides, ifosfamide metabolites (in vitro) as well as ifosfamide treatment (in vivo) have been shown to decrease cellular GSH availability. In the present study, the in vivo effects of three different ifosfamide treatment schedules on the GSH/GST system were studied in patients with advanced cancers (n = 24): continuous i.v. infusions of 1300 mg/m2 daily for 10 days and 5000 mg/m2/day for 24 h, as well as a 4-h infusion of 3000 mg/m2 daily for 3 days. The GSH/GST system was characterized by administering bromisoval, a probe drug to assess GSH conjugation activity in vivo, as well as by daily monitoring of GSH concentrations in blood cells and plasma. Bromisoval pharmacokinetics was assessed before and at the end of the ifosfamide treatment. Blood cell GSH levels decreased significantly (P < 0.05) during the 3- and 10-day ifosfamide treatment schedules; the 24-h treatment had no effect. The ifosfamide treatment schedules had only minimal effects on bromisoval pharmacokinetics. Assuming that the kinetics of the probe drug provide an accurate reflection of enzyme activity, this suggests that GST activity remains unchanged. Because GSH conjugation of bromisoval enantiomers requires both GST activity and GSH availability, these results also indicate that, despite the 35% decrease in GSH in blood cells of two patient groups, the GSH availability of the cancer patients was not rate-limiting for GSH conjugation of bromisoval enantiomers. If GSH levels in blood cells reflect those in tumors/other tissues, the present results indicate that ifosfamide may be used clinically to decrease GSH levels. However, whether a 35% decrease is sufficient to increase tumor sensitivity toward (other) cytostatics remains uncertain.     

Effects of chronic ethacrynic acid exposure on glutathione conjugation and MRP expression in human colon tumor cells

This study describes the presence of nitric oxide synthase (NOS-ir) immunoreactive neurons in the thalamic ventrobasal complex of the cat. NOS-ir is co-localized with gamma-aminobutyric acid (GABA-ir) in a subset of small neurons identified as local circuit neurons in previous studies. The double labeled neurons are further identified by a larger soma diameter when compared to GABA-ir only neurons. All NOS-ir somata exhibit GABA-ir but none exhibit immunoreactivity to calbindin.     

Sulfation of minoxidil by multiple human cytosolic sulfotransferases

Minoxidil is an antihypertensive agent and hair growth promoter that is metabolized by sulfation to the active compound, minoxidil sulfate. Thermostable phenol sulfotransferase (TS PST or P-PST) was initially thought to catalyze the reaction, and the enzyme was designated minoxidil sulfotransferase (MNX-ST). Information about human ST activities toward minoxidil would be useful in developing the capacity to predict individual responses to minoxidil based on tissue levels of STs. Therefore, human STs were studied from platelet homogenates, partially purified platelets, scalp skin high speed supernatants and COS-1 cell cDNA expressed preparations using a radiochemical enzymatic assay with minoxidil as the substrate. Studies showed the presence of TS PST, TL (thermolabile) PST and MNX-ST activities in human scalp skin. Biochemical properties and correlation studies suggested that in addition to TS PST, the TL PST activity, another ST activity or both were involved in the reaction. Partially purified human platelet TL PST tested with minoxidil and dopamine showed identical thermal stabilities and similar responses to the inhibitors 2,6-dichloro-4-nitrophenol (DCNP) and NaCl. To characterize the activity of TL PST toward minoxidil, several biochemical properties of the enzyme expressed from a human liver cDNA clone were investigated. When assayed with minoxidil and dopamine, thermal stabilities of the expressed enzyme were identical and IC50 values for the inhibitors DCNP and NaCl were similar. It was also demonstrated that cDNA encoded human liver dehydroepiandrosterone sulfotransferase and estrogen sulfotransferase contributed to the sulfation of minoxidil. The results confirm that at least four human STs contribute to minoxidil sulfation. MNX-ST activity represents a combination of ST activities. The data indicate that multiple ST activities should be taken into account in attempts to predict the regulation of minoxidil sulfation and individual responses to minoxidil.     

Glutathione S-transferases in carcinogenesis and diagnosis of liver cancer

We studied the relations between glutathione S-transferases (GSTs) from human liver and hepatic cancer and preneoplastic lesions in high risk area of Qidong city and Beijing. A biotin-avidin enzyme-linked immunoassay (BA-ELISA) for serum level of GSTs was developed. The results showed that the GSTs level of normal controls in Qidong city (0.66 +/- 0.54 ng/ml) was higher than that in Beijing (0.37 +/- 0.27 ng/ml). The members of high cancer families, HBVsAg carries and patients having a low level fluctuating pattern of serum AFP were the population with high risk of liver cancer; their serum level of GSTs was 1.25 +/- 1.46, 1.43 +/- 1.44 and 2.81 +/- 1.76 ng/ml respectively, and it was significantly higher than in the normal controls. The content of GSTs of hepatic cancer patients in Qidong city and Beijing was 3.03 +/- 3.35 and 3.60 +/- 3.70 ng/ml respectively, but the positive rate (97%) of GSTs in Qidong city was higher than that (82%) in Beijing. The results suggested that serum expression of GSTs can be used as an enzyme marker for hepatic cancer and preneoplastic lesions. Possible roles of these forms in hepatic carcinogenesis induced by chemical carcinogenesis were discussed.