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PubMed2001

Antioxidative defence mechanisms against reactive oxygen species in bovine retained and not-retained placenta: activity of glutathione peroxidase, glutathione transferase, catalase and superoxide dismutase.

Glutathione peroxidase (GSH-Px), glutathione transferase (GSH-Tr), catalase (CAT) and superoxide dismutase (SOD)-the members of enzymatic antioxidative defence mechanisms against reactive oxygen species-may play an important role in proper or improper release of bovine fetal membranes. The aim of the following study was the determination of GSH-Px, GSH-Tr, CAT and SOD activity in order to define antioxidative status of bovine placenta during retention of fetal membranes (RFM) in cows. Placental samples were collected immediately after spontaneous parturition or during caesarean section before term and at term and divided into six groups as follows: A: caesarean section before term without RFM; B: caesarean section before term with RFM; C: caesarean section at term without RFM; D: caesarean section at term with RFM; E: spontaneous delivery at term without RFM; F: spontaneous delivery at term with RFM. The enzyme activities in placental homogenates were measured spectrophotometrically. GSH-Px activity was statistically significantly higher in fetal than in maternal placenta in all examined groups, increased towards parturition and was higher in caesarean section groups than spontaneous delivery groups. Statistically significantly higher activities were noticed in retained than not-retained placentae. GSH-Tr activity was significantly lower in fetal than in maternal placenta. In preterm groups, the activity was statistically significantly higher in retained than not retained placenta. In term groups, the opposite relationship was observed, higher values in caesarean section groups than spontaneous delivery were noticed. CAT activity was statistically significantly higher in fetal than in maternal part of placenta in all groups examined. The highest values in C and D groups and the differences between retained and not-retained placenta were observed. SOD exhibited the highest values in preterm placenta and alterations between retained and not-retained fetal membranes. In conclusion, the activities of GSH-Px, GSH-Tr, CAT and SOD are altered in cases of retained fetal membranes which may suggest the activation of antioxidative mechanisms caused by the imbalance between production and neutralization of reactive oxygen species.

Animals↗
PubMed1999

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles.

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.

Alamethicin↗
PubMed2000

The enhanced affinity for thiolate anion and activation of enzyme-bound glutathione is governed by an arginine residue of human Mu class glutathione S-transferases.

A series of chimeric human Mu class glutathione S-transferases were designed to determine mechanisms by which they activate enzyme-bound glutathione (GSH) for reaction with electrophilic substrates. In view of evidence that the His(107) residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y. V., Patskovska, L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-1202), the cognate Arg(107) residue of the hGSTM2 subunit was replaced (R107N or R107H) and arginine residues were also incorporated into position 107 of hGSTM1 (H107R) and hGSTM4 (S107R) subunits. The major distinguishing kinetic properties invariably associated with enzymes containing an Arg(107) residue include an inverse dependence of k(cat) on viscosity and lower K(m(GSH values relative to enzymes with other residues at that position. Moreover, affinities for GSH thiolate anion binding are greater for enzymes containing Arg(107))), with K(d) values of 20-50 microM that are consistent with the K(m(GSH values (10-25 microM) obtained by steady-state kinetic analyses. Both thermodynamic and kinetic and data indicate that the Arg(107))) residue is specifically involved in enhancing the binding affinity of GSH thiolate anion relative to that of the protonated form. These enzymes therefore, can be more effective at lower GSH concentrations. Combined mutations indicate that both Arg(107) and Tyr(6) residues are required for thiolate anion formation and stabilization. The three-dimensional structure of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that Arg(107) maintains an electrostatic interaction with the Asp(161) side chain (3 A apart), but is distant from the GSH-binding site. However, an alternative energetically favorable model places the guanidino group 4 A from the sulfur atom of bound GSH. It is suggested therefore, that in solution, motion of the positively charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the observed greater affinity of enzymes containing Arg(107) for binding of thiolate anion.

Arginine↗
PubMed2000

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases.

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(a) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M(-1) s(-1), k(off) = 36 +/- 9 s(-1), at 37 degrees C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK(a) value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.

Arginine↗
PubMed2000

Role of multidrug resistance protein 1 (MRP1) and glutathione S-transferase A1-1 in alkylating agent resistance. Kinetics of glutathione conjugate formation and efflux govern differential cellular sensitivity to chlorambucil versus melphalan toxicity.

We investigated the role of phase II (conjugation) and phase III (efflux) detoxification of the anticancer drugs melphalan (MLP) and chlorambucil (CHB). Although both drugs are substrates of Alpha-class glutathione S-transferases (GST) and the monoglutathionyl conjugates formed in these enzymatic reactions are transported by MRP1, we found that GSTA1-1 and MRP1 acted in synergy to confer resistance to CHB but not to MLP (Morrow, C. S., Smitherman, P. K., Diah, S. K., Schneider, E., and Townsend, A. J. (1998) J. Biol. Chem. 273, 20114-20120). To explain this selectivity of MRP1/GST-mediated resistance, we report results of side-by-side experiments comparing the kinetics of MLP- versus CHB-glutathione conjugate: formation, product inhibition of GSTA1-1 catalysis, and transport by MRP1. The monoglutathionyl conjugate of CHB, CHB-SG, is a very strong competitive inhibitor of GSTA1-1 (K(i) 0.14 microM) that is >30-fold more potent than that of the corresponding conjugate of MLP, MLP-SG (K(i) 4.7 microM). The efficiency of GSTA1-1-mediated monoglutathionyl conjugate formation is more than 4-fold higher for CHB than MLP. Lastly, both CHB-SG and MLP-SG are efficiently transported by MRP1 with similar V(max) although the K(m) for CHB-SG (0.37 microm) is significantly lower than for MLP-SG (1.1 microM). These results indicate that MRP1 is required for GSTA1-1-mediated resistance to CHB in order to relieve potent product inhibition of the enzyme by intracellular CHB-SG formed. The kinetic properties of MRP1 are well suited to eliminate CHB-SG at pharmacologically relevant concentrations. For MLP detoxification, where product inhibition of GSTA1-1 is less important, GSTA1-1 does not confer resistance because of the relatively poorer catalytic efficiency of MLP-SG formation. Similar analyses can be useful for predicting the pharmacological and toxicological consequences of MRP and GST expression on cellular sensitivity to various other electrophilic xenobiotics.

ATP-Binding Cassette Transporters↗
PubMed2005

Glutathione and glutathione peroxidase activities in blood of patients in early stages following kidney transplantation.

This study focuses on glutathione (GSH) level in red blood cells, as well as on glutathione peroxidases (GSH-Px) activities in red blood cells and in plasma of chronic renal failure (CRF) patients following renal transplantation. We want to focus our main attention on plasma GSH-Px, the selenoenzyme that is synthesized primarily in the kidney. In CRF patients, activity of this enzyme is significantly reduced, and the reduction decreases with the progress of the disease, reaching in the end-stage 20% to 30% of the activity of healthy patients. We have shown that following renal transplantation the activity of plasma GSH-Px is restored very rapidly, and 2 weeks after surgery it reached the value of the control group. Red blood cell GSH level is significantly higher in CRF patients, and following transplantation, no significant changes were observed. Red blood cell GSH-Px activity before transplantation was the same as in healthy patients and did not change significantly after surgery.

Adult↗
PubMed1990

The importance of glutathione and glutathione transferase for somatic mutations in Drosophila melanogaster induced in vivo by 1,2-dichloroethane.

Two principal pathways of metabolism of the carcinogenic compound 1,2-dichloroethane (DCE) have been proposed. One is a mixed function oxidase dependent pathway requiring oxygen and NADPH. The other pathway depends on the presence of glutathione (GSH) and glutathione transferase (GST). The aim of this study was to investigate the role of the latter pathway for the in vivo mutagenicity of DCE in the somatic wing spot test in Drosophila melanogaster. DCE caused a dose-dependent increase of wing spots. In order to investigate the role of cellular GSH for the mutagenicity, the level of GSH was decreased by 24 h pretreatment with buthionine sulfoximine (BSO), an efficient inhibitor of GSH synthesis. This pretreatment decreased the GSH level to approximately 6% as compared to the control. The pretreatment also resulted in a significant decrease of the mutagenicity of DCE. Treatment of the larvae with phenobarbiturate (PB) resulted in approximately 200% induction of cytosolic GST, and a corresponding increase in the DCE mutagenicity. These results indicate that the important pathway in vivo for the mutagenicity of DCE is dependent on GSH and GST. A similar experimental protocol was used to study interactions between aflatoxin B1 (AFB) and GSH and GST. No effect of the treatment with BSO on the mutagenicity of AFB was observed, while pretreatment with PB caused a decrease of the mutagenicity of AFB.

Aflatoxins↗
PubMed1991

Assessment of the antioxidant/prooxidant status of murine skin following topical treatment with 12-O-tetradecanoylphorbol-13-acetate and throughout the ontogeny of skin cancer. Part II: Quantitation of glutathione and glutathione disulfide.

Tissue glutathione (GSH) and glutathione disulfide (GSSG) contents were quantitated in the skins of female SENCAR mice following the topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), and in the skin tumors generated by an initiation-promotion protocol. Total epidermal GSHt (GSH + GSSG) and GSSG contents were not reproducibly and significantly altered 0.5, 4 or 24 h after one or four topical applications of 1 microgram TPA, relative to the values obtained in age-matched, solvent-treated mice. Similar findings held for dermal GSHt at all times of analyses, and for dermal GSSG contents 0.5 and 4 h after TPA application. However, dermal GSSG contents were slightly elevated 24 h after TPA application. The GSHt and GSSG contents of skins initiated with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and harvested 17, 29 and 37 days after the cessation of chronic treatment with acetone (14 weeks, twice a week) were comparable to the values measured in age-matched, non-treated skins. In contrast, GSHt contents of papillomas harvested 17, 29 and 37 days after the cessation of chronic treatment with 1 microgram TPA (14 weeks, twice a week) were 2- to 4-fold greater than the values measured in non-treated mice, and DMBA-initiated, acetone-promoted mice, and the non-tumorous tissue adjacent to the papillomas. Comparable changes did not occur in papilloma GSSG contents. GSHt contents in squamous cell carcinomas (SCC) were twice the values measured in papillomas and 5- to 8-fold greater than the values measured in non-treated skins, and the non-tumorous tissue adjacent to SCC. Similarly, GSSG contents in SCC were elevated multifold relative to papillomas, non-treated skin and the non-tumorous tissue adjacent to SCC. Epidermal cell suspensions prepared by the trypsin-flotation procedure retained less than 2% of their original GSHt content and had reduced GSHt/GSSG ratios. Collectively these studies suggest that (i) if promoting doses of TPA induce oxidative stress in murine epidermis, it cannot be detected by measurements of GSH/GSSG; (ii) the antioxidant capacity of epidermal cells prepared by the trypsin-flotation procedure is severely compromised; and (iii) GSHt contents progressively increase during skin tumor ontogeny.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Modulation of glutathione S-transferases and glutathione peroxidase by the anticarcinogen butylated hydroxyanisole in murine extrahepatic organs.

Induction of glutathione S-transferases (GST) by the anticarcinogen butylated hydroxyanisole (BHA) has been examined in lung, kidney and small intestine of male and female BALB/c mice. BHA produced maximal induction of GST in the gut and although it increased GST levels in the kidney, it had little effect on pulmonary GST. Dietary BHA induced Alpha (Ya and Yk), Mu (Yb) and Pi (Yf) class GST subunits at least 10-fold in the small intestine but, by contrast, selenium-dependent glutathione peroxidase activity was reduced by approximately 4-fold in this organ following BHA treatment. In the kidney, all of the GST subunits, apart from Yk in males, showed modest levels of induction by BHA. However, a pronounced sex difference in the expression of renal alpha class subunits in both control and BHA-treated mice was observed, with female mice expressing approximately 4-fold greater levels of Ya and Yk than male mice. All renal GST were localized primarily in the proximal tubules. Dietary BHA was found to have the least inductive effect in the lung, where the GST were localized solely in the bronchi. The pulmonary Mu class GST subunits were induced approximately 2-fold by BHA; the expression of other GST was marginally increased by this inducer. Alpha class GST was also subject to sexual differentiation in the lung with female mice possessing higher levels of Yc and Yk than males. The Ya-type subunit was not detected in the lung nor was it induced by BHA.

Animals↗
PubMed1992

Glutathione conjugation of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene isomers by human glutathione transferases.

Each of the four stereoisomers of trans-3,4-dihydroxy 1,2-epoxy 1,2,3,4-tetrahydrobenzo[c]phenanthrene [(+)- and (-)-anti-BPhDE and (+)- and (-)-syn-BPhDE] has been incubated with the human glutathione transferase (GST) isoenzymes GST A1-1, GST M1-1 and GST P1-1, representing class alpha, mu and pi respectively, and glutathione (GSH). The conjugates formed were analyzed by HPLC and the results demonstrate that all GST isoenzymes catalyze the formation of GSH conjugates of all BPhDE isomers. However, a marked variation in catalytic efficiencies was observed (0.122-1.28/mM/s). These values are considerably lower than those previously estimated for the bay-region diol epoxides of benzo[a]pyrene (B[a]P) and human GSTs. The (+)-syn and (-)-anti-BPhDE (1R,2S-epoxide absolute configuration) were in general better substrates than the corresponding 1S,2R-epoxides. In accordance with previous observations with the diolepoxides of B[a]P, GST P1-1 was highly selective towards the BPhDE isomer with 4R,3S-diol 2S,1R-epoxide absolute configuration, i.e. (-)-anti-BPhDE, whereas GST A1-1 and M1-1 preferentially catalyzed the conjugation of (+)-syn-BPhDE (4R,3S-diol 2R,1S-epoxide absolute configuration). Overall, the most active isoenzyme was GST A1-1. Analysis by NMR spectroscopy of the GSH conjugates of BPhDE demonstrate that the reaction with GSH generally takes place by trans-addition of the thiol group at the benzylic C-1 carbon. The low catalytic efficiencies of human GSTs with BPhDE as compared to diolepoxides of B[a]P may be explained in part by the more crowded bay-region and substantially lower chemical reactivity (e.g. delta Edeloc/beta) of the former compounds.

Carcinogens↗
PubMed1994

Quantification of induction of rat oesophageal, gastric and pancreatic glutathione and glutathione S-transferases by dietary anticarcinogens.

Four dietary, naturally occurring anticarcinogens (flavone, coumarin, alpha-angelicalactone and ellagic acid) were studied with respect to their effects on oesophageal, gastric and pancreatic (i) glutathione S-transferase (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content in male Wistar rats. GST enzyme activity was significantly increased in the oesophagus by flavone, coumarin and alpha-angelicalactone (125, 240 and 155% respectively) and in the stomach by coumarin and alpha-angelicalactone (140 and 230%). No change in pancreatic GST activity was observed. In addition, class- and tissue-specific changes in GST isozyme levels occurred. Class alpha GSTs were induced in the oesophagus by flavone, coumarin and alpha-angelicalactone (570, 1580 and 570%), but did not change in the stomach. GST-alpha was undetectable in the pancreas. GST-mu was expressed at high levels in all three tissues investigated, but only pancreatic GST-mu levels of ellagic acid-fed rats were increased (160%). GST-pi was induced in the stomach by coumarin and alpha-angelicalactone (470 and 1120%) and in the pancreas by flavone (200%). GST-pi was detectable at low levels in rat oesophageal epithelium of coumarin-fed animals only. GSH concentrations were uninfluenced by the dietary anticarcinogens in all tissues. These results suggest that dietary ellagic acid and, more especially, flavone, coumarin and alpha-angelicalactone may exert strong chemoprotective effects by selective enhancement of members of the GST detoxification system in the oesophagus or stomach and, to a lesser extent, in the pancreas.

Animals↗
PubMed1995

Enhancement of rat hepatic and gastrointestinal glutathione and glutathione S-transferases by alpha-angelicalactone and flavone.

The naturally occurring anticarcinogens flavone and alpha-angelicalactone incorporated separately and simultaneously in the diet at 0.5, 0.1, 0.05 and 0.01% w/w, were studied with respect to their effects on oesophageal, gastric, intestinal, colonic and hepatic (i) glutathione S-transferase (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content in male Wistar rats. GST enzyme activity was significantly increased in the three treatment groups at one or more sites. The most substantial inductions were seen in oesophagus and stomach by 0.5% alpha-angelicalactone (1.9- and 2.3-fold respectively); and in small intestine, colon and liver by 0.5% combination diet (2.5-, 1.4- and 4.0-fold respectively). The inducing capacities declined with decreasing anticarcinogen concentrations. GST enzyme activity was induced in liver and to a lesser extent in small intestine and stomach. In general, in combination groups similar effects were seen as after treatment with alpha-angelicalactone or flavone separately. However, colonic GST enzyme activity was increased in the 0.5% combination group (1.4-fold), whereas in the corresponding flavone or alpha-angelicalactone groups no induction was observed. Concomitant changes in GST isozyme levels occurred. The involvement was the highest for GST-alpha (75%), followed by GST-mu (58%) and GST-pi (33%). Increased GSH levels were obtained in stomach and liver in all three treatment groups at various concentrations. These data demonstrate that dietary administration of flavone or alpha-angelicalactone, even at relatively low concentrations, may exert chemopreventive effects in stomach, small intestine, liver and to a lesser extent in oesophagus by enhancing the GST detoxification system, mainly by induction of GST-alpha and GST-mu isozymes. In addition, simultaneous administration of flavone and alpha-angelicalactone may result in anticarcinogenic effects in the colon by the same principle.

4-Butyrolactone↗
PubMed1995

Glutathione S-transferases and glutathione in human head and neck cancer.

Glutathione S-transferase (GST) enzyme activity, GST isoenzyme composition and glutathione (GSH) concentration were assessed in normal and squamous cell carcinoma specimens of 14 patients with oral or oropharyngeal cancer and 11 patients with laryngeal cancer. Comparing malignant with normal oral/oropharyngeal tissues, no significant differences in GSH content, GST enzyme activity or isoenzyme composition were found. However, some tumours had up to 3-fold increased GST enzyme activities and 11 malignant samples over-expressed GST-pi. GST-pi was present in all normal and malignant oral/oropharyngeal specimens investigated, whereas class alpha and class mu were detected in only a few samples. GST-mu was present in 28% of the patients with oral/oropharyngeal tumors as compared with approximately 60% in the normal population. GST-alpha, -mu and -pi were detected in 91, 64 and 100% of the normal laryngeal tissues respectively. In laryngeal tumours significantly higher levels of GST-pi and GSH but significantly lower amounts of GST-alpha were detected. Levels of class mu GST were generally lower in cancerous tissues, but differences were not significant. In comparison with normal oral/oropharyngeal tissues, normal laryngeal tissues contained almost twice the amount of GST enzyme activity due to higher class alpha enzyme levels. It is concluded that GST-pi is elevated in 11 out of 14 tumours of the oral cavity and values are significantly increased in tumours of the larynx, which may contribute to the inherent anti-cancer drug resistance of these malignancies. In laryngeal tumours the increased GSH levels may confer additional resistance to radiation therapy.

Aged↗
PubMed1995

The high non-enzymatic conjugation rates of some glutathione S-transferase (GST) substrates at high glutathione concentrations.

Enzymatic properties of five human glutathione S-transferases (GSTs) and non-enzymatic conjugation rates of GST substrates were examined as a function of glutathione (GSH) concentration and pH. GSTP1-1 showed a broad substrate specificity with both low and high GSH (10 mM) concentrations at pH 7.0, and the inhibitor insensitivity was then prominent. Among the GST substrates tested, ethacrynic acid, p-nitrophenylacetate, 1-chloro-2,4-dinitrobenzene and trans-4-phenyl-3-buten-2-one conjugated nonenzymatically with half-times of 0.39, 3.1, 9.4 and 10.0 min respectively at 10 mM GSH, pH 7.0 and 35 degrees C. The half-time for acrolein estimated by extrapolation was approximately 0.5 s. While the enzymatic reaction rates were independent of GSH at concentrations larger than Km values according to Michaelis-Menten kinetics and relatively insensitive to a pH change from 6.5 to 7.0, the non-enzymatic ones were linearly proportional to the GSH concentration and sensitive to pH. The enhanced non-enzymatic conjugation of various electrophiles at high GSH concentrations may in part account for the physiological significance of GSH elevation in preneoplastic and neoplastic cells.

Female↗
PubMed1996

Glutathione S-transferase A1-1-catalysed conjugation of bay and fjord region diol epoxides or polycyclic aromatic hydrocarbons with glutathione.

Mammalian metabolism of polycyclic aromatic hydrocarbons results in the formation of vicinal diol epoxides (existing as enantiomeric pairs of two diastereomers) considered as important ultimate carcinogens if the oxirane ring is located in a bay or fjord region of the parent hydrocarbon. In the present study, individual stereoisomers of the bay region diol epoxides of chrysene, dibenz[a,h]-anthracene and benzo[a]pyrene, as well as of the fjord region diol epoxides of benzo[c]phenanthrene, benzo[c]chrysene and benzo[g]chrysene, have been incubated with glutathione (GSH) in the presence or absence of human glutathione S-transferase isoenzyme GST A1-1, a class Alpha enzyme. The formation of GSH conjugates was determined and quantified by HPLC. The results demonstrate that the GST A1-1 isoenzyme catalyzes the formation of GSH conjugates of all diol epoxides tested, although a marked variation in catalytic efficiency (>20-fold) was observed. With both bay and fjord region anti-diol epoxides a significant preference for conjugation of the enantiomer with the R configuration at the benzylic position of the oxirane ring was noted. Among the syn diastereomers of the fjord region diol epoxides a similar substrate enantioselectivity was noted, i.e. the enantiomer with the corresponding R configuration was again preferentially conjugated. In contrast, for the bay region syn-diol epoxides this substrate selectivity was reversed, resulting in a preference for the enantiomer with the S configuration. The chemically more reactive syn diastereomers were in general better substrates for GST A1-1 than the corresponding anti diastereomers. However, a comparison between different diol epoxide diastereomers revealed no obvious correlation between chemical reactivity of the compounds and catalytic efficiencies. Furthermore, no significant correlation between diol epoxide lipophilicity and catalytic efficiency was observed. It is suggested that stereochemical factors, including the size and the geometry of the aromatic ring system and the preferred conformation of the diol epoxide, are involved as the major determinant for the rate of catalysis by GST A1-1.

Chromatography, High Pressure Liquid↗
PubMed1998

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.

Adult↗
PubMed2002

Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronosyl transferase (UGT1A1) and glutathione transferase (GSTA1) in cultured cells.

Glucoraphanin in Brassica vegetables breaks down to either sulforaphane or sulforaphane nitrile depending on the conditions, and sulforaphane can be further conjugated with glutathione. Using a high-throughput microtitre plate assay and TaqMan real time quantitative RT-PCR to measure mRNA, we show that sulforaphane and its glutathione conjugate, but not the nitrile, increased significantly (P < 0.05) both UGT1A1 and GSTA1 mRNA levels in HepG2 and HT29 cells. These changes were accompanied by an increase in UGT1A1 protein, as assessed by immunoblotting, and a 2-8-fold increase in bilirubin glucuronidation. When treated together, the nitrile derivative did not affect sulforaphane induction. The induction of UGT1A1 and GSTA1 mRNA by sulforaphane was time and concentration dependent. The results show a functional induction of glucuronidation by sulforaphane but not sulforaphane nitrile, and show that the pathway of metabolism of glucosinolates in Brassica vegetables is important in determining the resulting biological and anticarcinogenic activities.

Base Sequence↗
PubMed1991

Changes in Cu,Zn-superoxide dismutase, cytochrome c oxidase, glutathione peroxidase and glutathione transferase activities in copper-deficient mice and rats.

Dietary copper deficiency was produced in Swiss albino mice and Sprague Dawley rats to compare changes in selected antioxidant enzymes. A 5-wk dietary treatment was employed, starting approximately 1 wk after birth for mice (initially via dams) and 3 wk after birth for rats. An additional confirmatory experiment was conducted with mice using the postweanling paradigm. Mouse offspring (6 wk of age) and rats (8 wk of age) maintained on a Cu-deficient treatment were compared with Cu-adequate controls. Compared with Cu-adequate animals, Cu-deficient mice and rats were anemic, had lower ceruloplasmin activities and liver copper levels, and had higher relative weights of heart and small intestine. Activity of cytochrome c oxidase (mice) and Cu,Zn-superoxide dismutase (mice and rats) was lower in all seven organs examined from Cu-deficient animals compared with Cu-adequate animals, although there were organ and species differences. Compared with Cu-adequate controls, glutathione peroxidase activity was lower in liver and plasma of Cu-deficient mice and rats. Hepatic glutathione transferase activity was markedly lower in those Cu-deficient mice started on treatment at 1 wk of age but not in those mice or rats subjected to postweanling copper deficiency.

Animals↗