Enhancement of lipid peroxidation in rat liver on acute exposure to styrene and acrylamide a consequence of glutathione depletion

Lipid peroxidation, glutathione level and activity of glutathione-S-transferase were studied in liver and brain of rats 4 and 3 h after a single i.p. administration of 0, 25, 75, 100 mg/kg acrylamide or 0, 50, 100, 200, 600 mg/kg styrene, respectively. In liver both acrylamide and styrene caused an increase in lipid peroxidation and decrease in glutathione contents and activity of glutathione-S-transferase in a dose dependent manner, while in brain only acrylamide produced a decrease in glutathione content. The decrease in glutathione content was not always associated with increase of lipid peroxidation. The enhancement of lipid peroxidation occurred only when glutathione contents were depleted to certain critical levels. No effect of acrylamide or styrene was seen on lipid peroxidation under in vitro conditions. The addition of glutathione in the incubation mixture significantly inhibited the rate of lipid peroxidation of liver homogenates of acrylamide and styrene treated animals.The results suggest that enhancement of lipid peroxidation in liver on exposure to acrylamide or styrene is a consequence of depletion of glutathione to certain critical levels. The inhibition of glutathione-S-transferase activity by acrylamide and styrene suggests that detoxication of these neurotoxic compounds could be suppressed following acute exposure.     

The effect of phenobarbital and 3′-methyl-N,N-dimethyl-4-aminoazobenzene administration on catalytic,binding and immunological properties of ligandin subunits in rat liver

Following administration of phenobarbital to rats, liver ligandin content, bilirubin binding, glutathione-S-transferase and steriod isomerase activities by 150% and the 22 000-dalton subunit was selectively increased. Following adminstration of 3′-methyl-N,N-dimethyl-4-aminoazobenzene, rat liver ligandin content and steroid isomerase decrased by 65%, glutathione-S-transferase incrased by 100%, bilirubin binding was abolished, and the relative proportion of the 22 000- and 25 000-dalton subunits remained unchanged.     

Age Dependent Damage and Glutathione Metabolism in Ozone Fumigated Barley: A Leaf Section Approach

First, second, and third leaves of barley (Hordeum vulgare L.cv. Atem) seedlings fumigated with 200 nl 1^–1 ozone for5 d were divided into equal length quarter sections before analysisof glutathione metabolism. Since the first leaf is the oldestand the third leaf is the youngest and since monocotyledonousleaves grow from the base, sections were of a widely differingage. Lipid peroxidation rose moderately in all sections in all leaves.However, severe damage, detected visibly as chlorosis and analyticallyas loss of soluble protein, occurred only in the oldest tissue. The specific activity of glutathione reductase was slightlyelevated in old and middle-aged tissue whilst the specific activityof glutathione-S-transferase rose markedly in old, middle-aged,and young tissue following fumigation. This may reflect theimportance of glutathione-S-transferase in the protection ofplant cells from oxidative stress products formed in membranes. Fumigation with ozone also caused a loss of total glutathionein the tip sections of all leaves. Explanations for these results, including age dependent proteinloss, are discussed. Key words: Barley, ozone, glutathione, developmental age, glutathione-S-transferase     

A single protein research integrated advanced biochemistry laboratory course: design and general outline

An advanced biochemistry laboratory has been designed to focus on a detoxifying enzyme, glutathione-S-transferase, which is involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs), pesticides, herbicides, and other electrophilic xenobiotic compounds. The enzyme is known to catalyze conjugation of glutathione to xenobiotics, which makes them water-soluble so that they can be easily discarded through further metabolism and excretion. About two-thirds of the laboratory course incorporates nine advanced biochemical techniques, all focused to analyze various chemical characteristics of the glutathione-S-transferase. The remaining third of the semester time students work on a project that involves application of all the newly acquired techniques to solve a biochemical problem that encompasses the same detoxifying enzyme.     

The hypothalamic–hypophyseal–gonadal regulation of hepatic glutathione S-transferases in the rat

Hepatic glutathione S-transferase activities were determined with the substrates 1,2-dichloro-4-nitrobenzene and 1-chloro-2,4-dinitrobenzene. Sexual differentiation of glutathione S-transferase activities is not evident during the prepubertal period, but glutathione conjugation with 1,2-dichloro-4-nitrobenzene is 2–3-fold greater in adult males than in females. Glutathione conjugation with 1-chloro-2,4-dinitrobenzene is slightly higher in adult males than adult females. No change in activity was observed after postpubertal gonadectomy of males or females. Neonatal castration of males results in a significant decrease in glutathione conjugation with 1,2-dichloro-4-nitrobenzene. Hypophysectomy, or hypophysectomy followed by gonadectomy did result in significantly higher glutathione S-transferase activities in both sexes. These increases can be reversed by implanting an adult male or female pituitary or four prepubertal pituitaries under the kidney capsule. Postpubertal sexual differentiation of glutathione S-transferase activities is neither dependent on pituitary sexual differentiation nor pituitary maturation. Prolactin concentrations are inversely related to glutathione S-transferase activities in hypophysectomized rats with or without ectopic pituitaries. Somatotropin exogenously administered to hypophysectomized rats results in decreased glutathione S-transferase activities, whereas prolactin has no effect. Adult male rats treated neonatally with monosodium l-glutamate to induce arcuate nucleus lesions of the hypothalamus have decreased glutathione S-transferase activities towards 1,2-dichloro-4-nitrobenzene and decreased somatotropin concentrations. Our experiments suggests that sexual differentiation of hepatic glutathione S-transferase is a result of a hypothalamic inhibiting factor in the male (absent in the female). This postpubertally expressed inhibiting factor acts on the pituitary to prevent secretion of a pituitary inhibiting factor (autonomously secreted by the female), resulting in higher glutathione S-transferase activities in the adult male than the adult female.     

Glutathione-S-transferase,a possible drug-metabolizing enzyme,in Haemonchus contortus: Comparative activity of a cambendazole-resistant and a susceptible strain

Kawalek J. C., Rew R. S. and Heavner J. 1984. Glutathione-S-transferase, a possible drug-metabolizing enzyme in Haemonchus contortus: comparative activity of a cambendazole-resistant and a susceptible strain. International Journal for Parasitology14: 173–175. A drug metabolizing enzyme (DME), glutathione-S-transferase, was detected in homogenates of a cambendazole-susceptible and a cambendazole-resistant strain of Haemonchus contortus. The activity was 1.5–1.8 times higher in the resistant strain. DME activation is a possible mechanism for anthelmintic resistance in H. contortus.     

Human glutathione-S-transferase: cloning and expression in Lactococcus Lactis

The glutathione-S-transferase A1 cDNA was amplified from the total RNAs of human liver by RT-PCR, and inserted into plasmid pMG36e. The hGSTA1 was expressed in Lactococcus lactis MG1363 and verified by SDS-PAGE and Western blot, purified by affinity chromatography and showed enzymatic activity.     

Probe molecule equipped with boronic acid moiety as a reversible cross-linking group improves its binding affinity

Syntheses of biotinylated probe molecules of l-glutathione (GSH) equipped with boronic acid moiety and evaluation of their binding affinities against glutathione-S-transferase (GST) were described. It revealed that the presence of boronic acid moiety in an appropriate position enhances binding affinity of GSH probe toward GST probably by forming a reversible cross-link. Among prepared, the boronate-containing probe 8b exhibited the highest recovering ability of GST from Escherichia coli cell lysate.     

A radioenzymatic ultramicro method applicable to the measurement of a wide range of metabolites

A procedure for the rapid identification of glutathione S-transferase isozymes from rat liver in polyacrylamide gels is described. The isozymes are separated by electrofocusing and then identified by bathing the gels in a solution containing substrates and scanning the gels at the appropriate wavelength for the appearance of product. Increase in absorbance as a function of time delineates areas containing enzyme from artifacts within the gel. This technique should be useful for the identification of isozymes of glutathione S-transferase in other tissues and also other species. Also, the technique provides for rapid confirmation of homogeneity of the isozymes of glutathione S-transferase.     

Semiquinone derivative isolated from Bacillus sp. INM-1 protects cellular antioxidant enzymes from γ-radiation-induced renal toxicity

This study was focused to evaluate protection of indigenous antioxidant system of mice against gamma radiation-induced oxidative stress using a semiquinone (SQGD)-rich fraction isolated from Bacillus sp. INM-1. Male C57bl/6 mice were administered SQGD (50 mg/kgb.w.i.p.) 2 h before irradiation (10 Gy) and modulation in antioxidant enzymes activities was estimated at different time intervals and compared with irradiated mice which were not pretreated by SQGD. Compared to untreated controls, SQGD pretreatment significantly (p < 0.05) accelerates superoxide dismutase, catalase, GSH, and glutathione-S-transferase activities. Similarly, significant (p < 0.05) increase in the expression of superoxide dismutase, catalase, GSH, and glutathione-S-transferase was observed in irradiated mice pretreated by SQGD, compared to only irradiated groups. Total antioxidant status equivalent to trolox was estimated in renal tissue of the mice after SQGD administration. Significant ABTS⁺ radical formation was observed in H₂O₂-treated kidney homogenate, due to oxidative stress in the tissue. However, significant decrease in the levels of ABTS⁺ radical was observed in kidney homogenate of the mice pretreated with SQGD. Therefore, it can be concluded that SQGD neutralizes oxidative stress by induction of antioxidant enzymes activities and thus improved total antioxidant status in cellular system and hence contributes to radioprotection.     

Dissection of glutathione conjugate turnover in yeast

Xenobiotics are widely used as pesticides. The detoxification of xenobiotics frequently involves conjugation to glutathione prior to compartmentalization and catabolism. In plants, degradation of glutathione-S-conjugates is initiated either by aminoterminal or carboxyterminal amino acid cleavage catalyzed by a γ-glutamyl transpeptidase and phytochelatin synthase, respectively. In order to establish yeast as a model system for the analysis of the plant pathway, we used monochlorobimane as a model xenobiotic in Saccharomyces cerevisiae and mutants thereof. The catabolism of monochlorobimane is initiated by conjugation to form glutathione-S-bimane, which is then turned over into a γ-GluCys-bimane conjugate by the vacuolar serine carboxypeptidases CPC and CPY. Alternatively, the glutathione-S-bimane conjugate is catabolized by the action of the γ-glutamyl transpeptidase Cis2p to a CysGly-conjugate. The turnover of glutathione-S-bimane was impaired in yeast cells deficient in Cis2p and completely abolished by the additional inactivation of CPC and CPY in the corresponding triple knockout. Inducible expression of the Arabidopsis phytochelatin synthase AtPCS1 in the triple knockout resulted in the turnover of glutathione-S-bimane to the γ-GluCys-bimane conjugate as observed in plants. Challenge of AtPCS1-expressing yeast cells with zinc, cadmium, and copper ions, which are known to activate AtPCS1, enhanced γ-GluCys-bimane accumulation. Thus, initial catabolism of glutathione-S-conjugates is similar in plants and yeast, and yeast is a suitable system for a study of enzymes of the plant pathway.     

Reduction of growth and acetyl-CoA carboxylase activity by expression of a chimeric streptavidin gene inEscherichia coli

The streptavidin gene fromStreptomyces avidinii was expressed inE. coli as a non-fusion protein and as a glutathioneS-transferase fusion protein. The streptavidin protein accumulated primarily in the inclusion bodies and did not alter cell growth. In contrast, the glutathione-S-transferase-streptavidin fusion protein was soluble. Nondenaturing polyacrylamide gel electrophoresis indicated that the chimeric glutathione-S-transferase-streptavidin protein was present mostly as a monomer, with some detectable polymeric forms. Cells grown in the presence of [³H]-biotin had label specifically associated with the expressed glutathione-S-transferase-streptavidin fusion protein, indicating this protein bound biotin in vivo. The mojority of the radiolabeled biotin was associated with polymetric forms of the gluthione-S-transferase-streptavin protein. The growth rates of biotin auxotrophs ofE. coli growing in biotin-deficient media were substantially decreased by the expression of the glutathione-S-transferase-streptavidin gene. The decreased growth rate correlated with a decrease in acetyl-CoA carboxylase activity.This work was supported in part by the Iowa State University Office of Biotechnology. Journal paper J16040 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2997     

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

Antihyperglycemic, hypolipidemic and antioxidant activities of ethanolic extract of Commiphora mukul gum resin in fructose-fed male Wistar rats

High fructose feeding (66?% of fructose) induces type-2 diabetes in rats, which is associated with the insulin resistance, hyperinsulinemia, hypertriglyceridemia and oxidative stress. The present study was undertaken to evaluate the effect of ethanol extract of Commiphora mukul gum resin (CMEE) on blood glucose, plasma insulin, lipid profiles, reduced glutathione, lipid peroxidation, protein oxidation and enzymatic antioxidants like superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione-S-transferase in fructose-induced type-2 diabetic rats. A significant gain in body weight, hyperglycemia, hyperinsulinemia, increased lipid profiles, lipid peroxidation, protein oxidation and decreased reduced glutathione, activities of enzymatic antioxidants and insulin sensitivity (increased homeostasis assessment assay) were observed in high-fructose-induced diabetic rats. The administration of CMEE (200?mg/kg/day) daily for 60?days in high-fructose-induced diabetic rats reversed the above parameters significantly. CMEE has the ability to improve insulin sensitivity and delay the development of insulin resistance, aggravate antioxidant status in diabetic rats and may be used as an adjuvant therapy for patients with insulin resistance.     

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. II. Enzyme induction and metabolism of benzo[a]pyrene

The effect of various pretreatments on the activities of several drug metabolizing enzymes was investigated in microsomes and postmicrosomal supernatant fractions isolated from whole body homogenates of Drosophila melanogaster larvae of different strains. Pretreatments of larvae with either phenobarbital (PB), β-naphthoflavone (BNF) or a mixture of polychlorinated biphenyls (Aroclor 1254, PCB) for 24 h increased microsomal benzo[a]pyrene (BP) monooxygenase activity 2- to 6-fold in all strains as compared to untreated larvae. A simultaneous increase in the contents of cytochrome P-450 occurred after pretreatment with PB and PCB. Comparison of the turnover rates of BP per molecule of cytochrome P-450 indicated that BP was a poor substrate for control cytochrome P-450 whereas BNF induced a most active hemoprotein for this metabolism. Marked differences in the qualitative pattern of BP metabolites were obtained between microsomes isolated from BNF-treated larvae or rat liver microsomes. 3-Hydroxy-BP (3-OH-BP) was the dominating metabolite with both preparations, while the BP dihydrodiols were formed in minor quantities in Drosophila as compared to rat liver. Metyrapone and SKF 525-A inhibited BP metabolism in microsomes isolated from untreated and BNF treated larvae of all strains. In contrast, α-naphthoflavone (ANF) stimulated the BP monooxygenase activity of microsomes isolated from untreated larvae approx. 3-fold but only slightly influenced the activity of microsomes from BNF treated larvae indicating that the latter species of cytochrome P-450 was less sensitive to ANF.In all strains, PCB and PB treatments approximately doubled microsomal epoxide hydrolase activity and increased cytosolic glutathione-S-transferase activity 25–60%, significant only in strain Berlin K after PB treatment. The activities of epoxide hydrolase and glutathione-S-transferase in control larvae were comparable in the different strains, whereas the content of cytochrome P-450 and BP monooxygenase activity was higher in the Hikone R strain. Variability in the induction response to the various pretreatment was observed among the three strains.     

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4–7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.     

Effect of D,L-buthionine-S,R-sulfoximine on the ratio of glutathione forms and the growth of Tatar buckwheat calli

We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase activity, glutathione-S-transferase, and ascorbate peroxidase in morphogenic and nonmorphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthase, the first enzyme of glutathione biosynthesis. We found that, during passaging, cultures only slightly differed in total glutathione content; however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG was higher in the nonmorphogenic culture. In the morphogenic callus, the glutathione-S-transferase activity was 10–20 times higher and the glutathione reductase activity was 2–2.5 times lower than in the nonmorphogenic callus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was temporary (on day 6–8 of passage), whereas that in the nonmorphogenic callus decreased within a day and remained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did not affect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. These differences are probably due to the fact that, in the BSO-containing medium, glutathione reductase is activated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSO caused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effect of BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on the differentiation of proembryonic cell complexes in the morphogenic callus. The role of the glutathione redox status in maintaining the embryogenic activity of cultured plant cells is discussed.     

The effect of n-3 long-chain polyunsaturated fatty acids and lipoic acid on the heart in the ovariectomized rat model of menopause

Menopause occurs as consequence of ovarian senescence that leads to a drop of oestrogen hormone. The decreased oestrogen levels combined with the impairment of the redox system may contribute to the increased risk of postmenopausal cardiovascular disease. Supplementation with antioxidants may be an alternative to reduce cardiovascular risk. The study evaluated the effect of dietary supplementation with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and α-lipoic acid (LA) for a period of 16 weeks on oxidative stress biomarkers in the hearts of ovariectomized 3-month-old rats. Ovariectomy did not increase the level of the damage markers malondialdehyde and carbonyl, and both were decreased by LA supplementation. Ovariectomy increased the levels of the endogenous antioxidants glutathione, vitamin C and H₂O₂ consumption, after restoration by DHA, EPA, and LA supplementation. Vitamin E, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase are not altered by ovariectomy. Lipid and protein damage are not increased after ovariectomy and a portion of the endogenous antioxidants concomitantly increased, suggesting that hearts may be protected by these antioxidants. DHA, EPA, and LA restored these endogenous antioxidants, showing that all evaluated supplements are effective in modulating the antioxidant redox system in the heart. LA showed additional effect on redox markers, decreasing lipid and protein damage markers.     

Glutathione-S-transferase selective release of metformin from its sulfonamide prodrug

In this study, three sulfonamide prodrugs of metformin were designed and synthesized. The bioconversion of the sulfonamide prodrugs by glutathione-S-transferase (GST) was evaluated in rat and human liver S9 fractions as well as with recombinant human GST forms. One of the prodrugs (3) was bioactivated by GST and released metformin in a quantitative manner, whereas the two others were enzymatically stable. Prodrug 3 had a much higher log D value relative to metformin and it was reasonably stable in both acidic buffer and rat small intestine homogenate, which indicates that this prodrug has the potential to increase the oral absorption of metformin.