Decreased Glutathione S-transferase Activity Research Articles

Glutathione S-transferase (GST) activity as a biomarker in ecological risk assessment of pesticide contaminated environment

The behaviour and fate of pesticides in the environment will determine their impact on both humans and non-target organisms. Biochemical biomarkers are increasingly used in ecological risk assessment to identify the incidence of exposure to and effects caused by xenobiotics. This study was undertaken to investigate the potential toxic effect of a locally produced insect powder called “Rambo” (which contain 0.6% permethrin) on non-target organisms exemplified with albino rats. The results obtained showed that glutathione S-transferase (GST) activity in the newly-weaned rats (NWR) and middle-aged rats (MAR) groups were found to increase significantly (p homogenates, a decrease in GST activity was observed, this decrease was significant (p 0.05). However, the highest GST activity (398.44 ± 23.44) U/L was recorded in the liver homogenates while the least activity (9.07 ± 3.44) U/L was obtained in the plasma sample. The significance of such a decrease in intracellular GST is that, protection against reactive intermediates may be lost and thus affect vital metabolic processes that may result to death. This shows that GST can be used as a biomarker in ecological risk assessment of pesticide contaminated environment.

Effect of chronic treatments with GH, melatonin, estrogens, and phytoestrogens on oxidative stress parameters in liver from aged female rats

The aging theory postulates that this process may be due to the accumulation of oxidative damage in cells and molecules. The present study has investigated the effect of castration in old female rats on various parameters related to the antioxidant properties of several cellular fractions obtained from the liver, and the influence of several chronic treatments on it, both in intact and castrated animals. Sixty-one 22-month-old Wistar female rats, were used. About 21 intact animals were divided into three groups and treated for 10 weeks with GH, melatonin or saline, and 40 ovariectomized (at 12 months of age) animals were divided into five groups and treated for the same time with GH, melatonin, estrogens (Eos), phytoestrogens (Phyt) or saline. All animals were sacrificed at 24 months of age by decapitation. The activity of glutathione peroxidase (GPx) in cytosolic fraction, glutathione-S-transferase (GST) in cytosol and microsomal fractions, and the levels of nitric oxide (NO) and cytochrome C in mitochondrial and cytosol fractions of liver were determined. A decrease in GST activity was detected in cytosol and in the microsomal fraction in ovariectomized animals as compared to intact rats. The activity of GPx was also decreased in ovariectomized as compared with the intact group. NO level was increased and cytochrome C decreased in the mitochondrial fraction of the liver in ovariectomized females as compared with the intact group, respectively. No significant changes after melatonin or GH treatments were found in GPx, GST activity and NO level in mitochondrial fraction in the intact group. Administration of GH, melatonin, Eos and Phyt in the ovariectomized groups significantly increased the GPx, and GST activity in the cytosol and microsomal fraction and decreased the level of NO in the mitochondrial fraction as compared with the untreated rats. A significant increase in the level of cytochrome C in the mitochondrial fraction and a decrease in the cytosol fraction were also found with all treatments. The administration of GH, melatonin, Eos and Phyt to castrated females seem to reduce oxidative changes in the liver from old ovariectomized rats.

Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M. aeruginosa

The aim of this study was to examine the effects of chemical nonylphenols (NPs) on the antioxidant system of Microcystis aeruginosa strains. The degradation and sorption of NPs by M. aeruginosa were also evaluated. High concentrations of NPs (1 and 2 mg/l) were found to cause increases in superoxidase dismutase (SOD) and glutathione-S-transferase (GST) activities and in glutathione (GSH) levels. These results suggest that toxic stress manifested by elevated SOD and GST levels and GSH contents may be responsible for the toxicity of NPs to M. aeruginosa and that the algal cells could improve their antioxidant and detoxification ability through the enhancement of enzymatic and nonenzymatic prevention substances. The observed elevations in GSH levels and GST activities were relatively higher than those in SOD activities, indicating that GSH and GST contributed more in eliminating toxic effects than SOD. Low concentrations of NPs (0.05-0.2 mg/l) enhanced cell growth and decreased GST activity in algal cells of M. aeruginosa, suggesting that NPs may have acted as a protecting factor, such as an antioxidant. The larger portion of the NPs (>60%) disappeared after 12 days of incubation, indicating the strong ability of M. aeruginosa to degrade the moderate persistent NP compounds. The sorption ratio of M. aeruginosa after a 12-day exposure to low nominal concentrations of NPs (0.02-0.5 mg/l) was relatively high (>30%). The fact that M. aeruginosa effectively resisted the toxic effects of NPs and strongly degraded these pollutants indicate that M. aeruginosa cells have a strong ability to adapt to variations in environmental conditions and that low and moderate concentrations of organic compounds may favor its survival. Further studies are needed to provide detailed information on the fate of persistent organic pollutants and the survival of algae and to determine the possible role of organic pollutants in the occurrence of water blooms in eutrophic lakes.

Erythrocyte antioxidant defense response against cigarette smoking in humans—the glutathione S‐transferase vulnerability

Cigarette smoking leads to uptake of a multitude of reactive chemicals including many electrophiles and may also give rise to oxidative stress. Human red blood cells are important targets for electrophilic and oxidant foreign compounds. We investigated the oxidative stress in erythrocytes upon cigarette smoking, and the response of antioxidant defense system against it. With this aim, simultaneous determination of erythrocyte superoxide dismutase (SOD), selenium dependent glutathione peroxidase (Se-GPx), catalase (CAT), glutathione S-transferase (GST) activities and plasma levels of thiobarbituric acid reactive substances (TBARS), and the degree of erythrocyte membrane lipid peroxidation (EMLP) were carried out in blood samples of smokers and their controls. Plasma TBARS levels and EMLP in smokers were significantly higher than the control levels (p < 0.01 and p < 0.005, respectively). SOD activity was diminished in smokers compared to nonsmoker controls (p < 0.005). Erythrocyte Se-GPx activity was also found significantly diminished in smokers (p < 0.005), while plasma Se-GPx activity was not changed. We observed that erythrocyte CAT activity was not different in smokers compared to nonsmoker controls. We found that the erythrocyte GST activity is significantly lower in young adult smokers (3.03 +/- 0.18 U/mg protein; mean +/- SEM; n = 46) than in nonsmoking contemporaries (3.98 +/- 0.26 U/mg protein; mean +/- SEM; n = 41). Together with previously reported data, it can be concluded that the decrease in GST activity leads to extra GST synthesis during erythrocyte proliferation. The same data were also analyzed for the sex differences. The statistically significant differences remained the same between nonsmoker and smoker females. Only EMLP degree and SOD activity were significantly different between nonsmoker and smoker males; however, when compared the parameters between male and female nonsmokers, GST activity was found to be significantly higher in females than that of males.

Effect of curcumin and 4-n-propil curcumin on glutation s-transferase activity in rat liver

Curcumin, a plant phenol in rhizome Curcuma longa L. has been reported to be a potent inhibitor of glutathione S-transferase (GST) in rat liver with in vitro with substrate 1,2-dikloro-4-nitrobenzen (DCNB). By using DCNB, a specific substrate for GST mu, This work was aimed to know whether curcumin derivatives: 4-n-propyl curcumin can also inhibit GST activity in vitro. Glutathione activity can be measured by conjugating GSH and DCNB catalized with GST. The product can be measured spectrofotometrically. Curcumin and its derivatives decreased GST activities with IC50 values 6,86 μM for curcumin and 8,41 μM for 4-n-propyl curcumin. This means that 4-n-propyl curcumin has a weaker inhibition potency than curcumin. Curcumin (consentration 10μM) inhibited GST activity by mixed with the Ki dan Ki’ values 2,77 μM dan 7,33 μM, whereas 4-n-propyl curcumin (consentration 10μM) inhibited GST activity by competitive with the Ki values 6,28 μM. Key words : Curcumin, 4-n-propyl curcumin, glutathione S-transferase, 1,2-dikloro-4-nitrobenzen.

An investigation of the role of vitamin E in the protection of mice against microcystin toxicity.

The presence of cyanobacterial toxins in drinking and recreational waters represents a potential public health risk. Microcystin-LR (MC-LR) is a potent cyclic heptapeptide hepatotoxin produced by the blue-green alga Microcystis aeruginosa. Chemoprotectant studies have indicated that membrane-active antioxidants such as vitamin E may offer protection against microcystin toxicity. This study investigated the effect of vitamin E supplementation on microcystin toxicity in mouse liver. Groups of mice were fed vitamin E supplements (8.33 or 33.3 U/mouse/day) for 4 weeks, with intraperitoneal doses of MC-LR extract (70% LD(50)) every 3 days from day 8. The potential benefits of vitamin E were evaluated based on lipid peroxidation, alanine transaminase (ALT), and glutathione S-transferase (GST) levels. Vitamin E supplementation at 33.3 U/mouse/day offered some protection against lipid peroxidation induced by repeated exposure to MC-LR extract and limited both the toxin-induced increase in ALT leakage and decrease in GST activity. Vitamin E supplementation at 66.6 U/mouse/day significantly increased the time to death and reduced the increase in liver percentage body weight induced in mice given a lethal dose challenge of MC-LR extract. Therefore, vitamin E, taken as a dietary supplement, may have a protective effect against chronic exposure to MC-LR.

Sensitization of cells overexpressing multidrug-resistant proteins by pluronic P85.

This study evaluated the chemosensitizing effects of Pluronic P85 (P85) on cells expressing multidrug resistance-associated proteins, MRPI and MRP2. Cell models included MRP1- and MRP2-transfected MDCKII cells as well as doxorubicin-selected COR-L23/R cells overexpressing MRP1. Effects of P85 on cellular accumulation and cytotoxicity of vinblastine and doxorubicin were determined. Mechanistic studies characterized the effects of P85 on ATP and reduced glutathione (GSH) intracellular levels as well as MRP ATPase and glutathione-S-transferase (GST) activities in these cells. Considerable increases of vinblastine and doxorubicin accumulation in the cells overexpressing MRP1 and MRP2 in the presence of P85 were observed, although no statistically significant changes in drug accumulation in the parental cells were found. P85 treatment caused an inhibition of MRP ATPase activity. Furthermore, P85 induced ATP depletion in these cells similar to that previously reported for Pgp-overexpressing cells. In addition, reduction of GSH intracellular levels and decrease of GST activity were observed following P85 treatment. Finally, significant enhancement of cytotoxicity of vinblastine and doxorubicin by P85 in MRP-overexpressing cells was demonstrated. This study suggests that P85 can sensitize cells overexpressing MRP1 and MRP2, which could be useful for chemotherapy of cancers that display these resistant mechanisms.

Studies on some enzymes involved in insecticide resistance in fenitrothion-resistant and -susceptible strains of Musca domestica L. (Dipt, Muscidae)

Abstract: The toxicity of fenitrothion and fenitrothion plus synergists was determined by topical application to adults of fenitrothion-resistant (571ab) and -susceptible (Cooper) strains of Musca domestica L. The strain 571ab was 232-fold resistant to fenitrothion when compared with the Cooper strain. Co-administration of fenitrothion with three synergists, namely piperonyl butoxide (PBO), tributylphosphorotrithioate (DEF) and diethyl maleate (DEM) was investigated, respectively, at 1 : 5, 1 : 5 and 1 : 10 ratio. This co-administration of fenitrothion with PBO, DEF and DEM caused a decrease in the doses which produced 50% lethality (LD50s) in 571ab but had no synergistic effect on fenitrothion toxicity was observed in the Cooper strain. The effect of topical application of fenitrothion alone and in combination with PBO, DEF and DEM at the LD50 level on some enzyme activities in 571ab and Cooper strains was examined. The application of fenitrothion alone and in combination with DEF and DEM at LD50 level caused a significant decrease in activities of total esterases, acetylcholinesterase (AChE) and glutathione S-transferase (GST) in the 571ab strain. The decrease in GST activity was not significant in treated flies of the Cooper strain when compared with GST activity of control flies. A non-significant effect on total cytochrome P450 level was observed with fenitrothion alone and the fenitrothion + PBO treatment. No increase in activity level of total esterases, AChE and GST was found, which might suggest that changes in activity level of these enzymes are not related to fenitrothion resistance in the 571ab strain.

Basal Cellular Alterations of Esterase, Glutathione, Glutathione S-Transferase, Intracellular Calcium, and Membrane Potentials in Coumaphos-Resistant Boophilus microplus (Acari: Ixodidae) Cell Lines

Organophosphate (OP) acaricide resistance in Boophilus microplus ticks compromises the efficacy of tick control programs. Mechanisms that may contribute to acaricide resistance were evaluated by partial characterization of three OP-resistant B. microplus tick cell lines (C34, C44, and C54) compared to nonresistant control cells. The resistant cells showed an increase in glutathione coupled with a decrease in glutathione S-transferase activity, decreased plasma and mitochondrial membrane potentials, increased intracellular calcium concentration, and increased esterase levels. These results are consistent with resistance mechanisms found in other cell systems and with in vivo observations of increased esterase activity in organophosphate-resistant ticks. No significant differences were found in intracellular pH, drug efflux, or reactive oxygen species. The use of organophosphate-resistant cell lines offers a convenient vehicle for studying mechanisms of acaricide resistance development in vitro.

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.

In vitro effects of NSAIDS and paracetamolon oxidative stress-related parameters of human erythrocytes

In vitro effects of widely used nonsteroidal antiinflammatory drugs (NSAIDs) and paracetamol were studied on oxidative stress-related parameters of human red blood cells (RBC). Membrane lipid integrity, activity of erythrocyte antioxidant enzymes; i.e. glutathione S-transferase (GST), selenium dependent-glutathione peroxidase (Se-GPx), and catalase (CAT), and hemolytic/stabilizing action of the drugs on erythrocyte membrane were assessed. Diclofenac, indomethacin and paracetamol at the therapeutic and higher concentrations, and dipyrone at the high concentration exerted a statistically significant inhibition on H2O2 forced erythrocytic membrane lipid peroxidation (EMLP). Increased hemolysis was observed by Na-salicylate, naproxen and ketorolac at therapeutic and higher concentrations, and by diclofenac and tiaprofenic acid at high concentrations, while the others seemed to stabilize the membrane at the same conditions. Na-salicylate inhibited GST activity at the therapeutic dose, however activated the same enzyme at high concentrations. Naproxen, tiaprofenic acid and piroxicam caused a decrease in GST activity at therapeutic doses. Paracetamol caused an activation at a high dose. Tiaprofenic acid, ketorolac, naproxen and piroxicam caused a significant Se-GPx inhibition. Erythrocyte CAT activity was increased by Na-salicylate, acemetacin, and tenoxicam at the therapeutic, and by dipyrone at the high concentration. Our results suggest that NSAIDs and paracetamol may be involved in oxidative/antioxidative processes of human erythrocytes. Also, the in vitro EMLP method can be considered as a simple test for evaluating possible antioxidant potency of chemicals.

Evaluation of glutathione-sensitive fluorescent dyes in cortical culture

The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione-S-transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (CMAC), 7-amino-4-chloromethylcoumarin (CMAC-blue), monochlorobimane (MCB), and 5-chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3-naphthalenedicarboxaldehyde (NDA) and o-phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL-buthionine-(S, R)-sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC-blue, and MCB and partial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocytes between 5-6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC-blue and MCB. This decrease in staining was not due to a decrease in GST activity, as neurons stained with the GST-insensitive OPD also exhibited a decline in GSH-sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co-localized with GFAP-positive astrocytes, but not with MAP-2-positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve astrocytes in living mixed cultures co-loaded with the Ca(2+)-sensitive dye, calcium green 5N-AM. GLIA 30:329-341, 2000. Published 2000 Wiley-Liss, Inc.

The expression and regulation of drug metabolism in human placenta

The human placenta oxidizes several xenobiotics, although the spectrum of substrates and metabolic activities when compared with the liver appears restricted. Maternal cigarette smoking or PCB exposure increase the expression of CYP1A1. This induced activity is able to catalyze the activation of benzo(a)pyrene into DNA-bound adducts, both in vitro and in vivo. Studies with RT–PCR technique have demonstrated that first trimester placentae express at the mRNA level CYP1A1, 1A2, 2C, 2D6, 2E1, 2F1, 3A4, 3A5, 3A7 and 4B1 and at full term CYP1A1, 2E1, 2F1, 3A3/4, 3A5 and 3A7. However, more detailed studies on cDNA probes or with specific antibodies or ‘diagnostic’ substrates for other than CYP1A1, 2E1 and 3A gene products have yielded negative results. Studies on human placenta and a chorioncarcinoma cell line, JEG 3 cells, boulster the concept that placental CYP1A1 and 1B1 – although their expression is Ah receptor and ARNT mediated – is controlled by distinct mechanisms. Aromatase, CYP19, and cholesterol side-chain cleaving, CYP11B, genes, proteins and activities are catalytically active in human placentae throughout the pregnancy and those parameters do not seem to be affected by maternal cigarette smoking but rather maternal health status. However, the substrate binding pocket of aromatase accepts as its substrate several xenobiotics and is responsible for constitutive xenobiotic biotransformations. Functional placental glutathione S-transferase, N-acetyl transferase and epoxide hydrolase are expressed via one gene each and their function reflects the placenta as an endocrine organ rather than a xenobiotic-metabolizing unit. However, markers for oxidative stress can be detected in decreased glutathione S-transferase activities. Because human placenta has quite well defined metabolic characteristics, and obtaining placental samples will not meet any drastic ethical difficulties, it could be used more intensively as a source of metabolizing enzymes in in vitro studies during the course of a drug development program. The human placenta, or its subcellular organelles, could serve as a real alternative model for an extrahepatic tissue in replacing recombinant expression systems especially if CYP11, 19, 1A1 or potentially 2E1 are target enzymes for potential metabolic interactions.

Glutathione metabolism and glutathione S-conjugate export ATPase (MRP1/GS-X pump) activity in cancer. I. Differential expression in human cancer cell lines.

Mg(2+)-dependent vanadate-sensitive glutathione S-conjugate ATPase (GS-X pump) activity is a common feature of some ATP-binding cassette (ABC) transporters, such as the multidrug resistance-associated protein (MRP1) gene product, that exports biologically active electrophiles after their conjugation with intracellular glutathione (GSH) from normal and cancer cells. Antitumor electrophiles (e.g. naturally occurring cyclopentenone prostaglandins and anticancer chemicals) can be intracellularly conjugated with GSH via a glutathione S-transferase catalyzed reaction and be eliminated through GS-X pumps thus threatening cancer chemotherapeutics. Since different sensitivities to antitumor electrophiles are shown by different cell types, the ability of several human cancer cell lines to produce and export S-(2,4-dinitrophenyl)-glutathione (DNP-SG) conjugate through the GS-X pump, using whole cells and inside-out membrane vesicle preparations, is investigated. Different cancer cell lines exhibited characteristically different GS-X pump activity. In particular, HEp-2 larynx carcinoma cells possess an elevated DNP-SG export rate through the GS-X pump compared with HeLa, K562, U937 or HL-60 cells, which exhibit the lowest activity. The differences in DNP-SG export rates are not due to decreased glutathione S-transferase activity or impaired de novo synthesis of GSH. The findings suggest that the GS-X pump may be involved in the modulation of the biological activity of both naturally occurring electrophiles and anticancer drugs. The differential expression of GS-X pumps may lead to an improved understanding of multidrug resistance and may be exploited in the development of new therapeutic strategies for the treatment of cancer patients.

Glutathione S-transferases—biomarkers of cancer risk and chemopreventive response

The critical role of the glutathione S-transferase (GST) multigene family in cellular protection in combination with the large interindividual variability in the expression of these enzymes has prompted an investigation of their importance in cancer prevention and susceptibility. Previous preclinical and clinical studies from this laboratory have established an association between decreased GST activity and increased risk for colorectal cancer. Based upon the increased incidence of colon malignancies among patients with ulcerative colitis, GST activity has been examined in a mouse model of induced colitis. Significant decreases (50% of controls) in the GST activity of colon tissue were observed during the establishment and progression of colitis. These data suggested that depletion of cellular protection may be an important event in the carcinogenic progression of ulcerative colitis. The ability of the dithiolthione oltipraz to induce GST expression within the murine colon has been demonstrated. Use of chemopreventive regimens to induce phase 2 detoxication enzyme expression represents a promising strategy for the prevention of cancer. Clinical studies revealed that the GST activity of blood lymphocytes from individuals with either a personal or family history of colorectal cancer or a personal history of colon polyps was decreased significantly when compared to that of healthy controls. Phase 1 clinical evaluation of oltipraz has demonstrated its ability to induce GST activity as well as the level of transcripts encoding γ-glutamylcysteine synthetase ( γ-GCS) and DT-diaphorase in the colon mucosa of individuals at increased risk for colorectal cancer. The observed correlation between the posttreatment response in blood lymphocytes and colon mucosa suggested that blood lymphocytes may be used in future trials as a surrogate biomarker of the responsiveness of colon tissue to chemopreventive regimens.

Ethanol effects on lipid peroxidation and glutathione-mediated defense in rat small intestine: Role of dietary fats

The effect of ethanol feeding for 5 weeks on lipid peroxidation status of small intestine was studied in rats maintained on either a rat pellet (RP) or a semisynthetic diet containing coconut oil (CCO), corn oil (CO), or fish oil (FO). Highest rate of iron/ascorbate-induced lipid peroxidation was observed in intestinal mucosa of FO-fed rats, which was further elevated ( p < 0.05) upon ethanol administration. Purified brush borders from all the ethanol-treated dietary groups were more susceptible to iron-induced lipid peroxidation. Level of nonprotein thiols was increased by ethanol feeding to rats given CO or FO. FO-fed rats exhibited increased activities of glutathione reductase (GR), glutathione- S-transferase (GST), and catalase (Cat). Glutathione peroxidase (GPx) was the lowest in the CCO group. Ethanol-treated FO group exhibited increased GST and GPx activities compared to controls, whereas in rats fed the RP or CO diet, ethanol feeding significantly decreased GST activity. GR and Cat activities were not affected under these conditions. Thus, ethanol exposes the small intestinal mucosa to oxidative stress. The effects were more pronounced in rats fed n-3 fatty acid-rich (FO) diet. The corresponding rise in GPx and GST levels may reflect the adaptive changes in intestine.

Combined effects of ethanol and cigarette smoke on hepatic and pulmonary xenobiotic metabolizing enzymes in rats

The combined effects of ethanol (EtOH) and cigarette smoke (CS) on hepatic and pulmonary monooxygenase (MO) activities (aniline 4-hydroxylase (AH), aminopyrine N-demethylase (AMND), 7-ethoxyresorufin O-deethylase (EROD), p-nitroanisole O-demethylase ( p-NAOD)), lipid peroxidation (LP) and reduced glutathione (GSH) levels and glutathione S-transferase (GST) activities toward several substrates (1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), 1,2-epoxy-3-( p-nitrophenoxy)-propane (ENPP)) were determined and compared with those of EtOH or CS alone in rats. When the male adult rats (225–275 g) were treated with 10% EtOH (v/v) in their drinking for 21 days AH, AMND and EROD activities and LP and GSH levels increased significantly whereas GST activity for EAA decreased significantly in liver as compared to controls. EtOH did not change the hepatic p-NAOD and GST activities toward CDNB, DCNB and ENPP. In lung, EtOH increased GST activities toward CDNB and ENPP and LP level but decreased GST activity toward DCNB, significantly. No alterations were noted in pulmonary MO activities and GST activity toward EAA and GSH level by EtOH treatment. When the animals were exposed to CS five times a day, with 1 h intervals, for 3 days in a chamber where smoke and fresh air lead alternatively, AMND, EROD and p- NAOD activities, GST activity toward EAA and GSH level increased but LP level and GST activity for ENPP decreased significantly in liver. CS did not alter the hepatic AH and GST activities toward CDNB and DCNB. In lung, CS increased AH, EROD and p- NAOD activities and LP and GSH levels and decreased all the GST activities studied significantly. CS had no influence on pulmonary AMND activity. For the combined treatment, the animals were treated with 10% EtOH (v/v) in their drinking water for 21 days and during the last 3 days they were exposed to CS five times a day, with 1, h intervals, in a chamber where smoke and fresh air lead alternatively. In these animals, augmentation of elevations were noted in AH and p- NAOD activities and LP and GSH levels but not in EROD and AMND activities in liver. Combined treatment significantly decreased GST activity toward CDNB, ameliorated the alteration caused by either EtOH or CS treatment alone on GST activity toward EAA and potentiated the depression of GST activity toward ENPP to a greater degree. No change was observed in GST activity toward DCNB. In lung, combined treatment potentiated the elevations of AMND and p- NAOD activities and LP level and not those of AH and EROD activities. GST activities toward CDNB, DCNB and ENPP were highly elevated by the combined treatment. No changes were observed in pulmonary GSH level and GST activity for EAA by the combined treatment. These results reveal that the regulations of the hepatic and pulmonary MO and GST are differentially influenced by EtOH, CS and the combined treatment.

Induction of glutathione S-transferase in the freshwater bivalve Sphaerium corneum as a biomarker for short-term toxicity tests?

A study was carried out to examine the use of glutathione S-transferase (GST) in freshwater bivalves as a short-term biochemical marker for organic contaminants. In contrast with earlier reports, induction of GST activity towards 1-Chloro-2,4-dinitrobenzene (CDNB) was not found in any of the experiments performed. These included 10–12 days exposures to sediments spiked with 2,3′,4,4′,5-pentachlorobiphenyl, benzo[a]pyrene or lindane and 7–8 days exposures to dieldrin, lindane or benzo[a]pyrene in a water-only system. A nonsignificant decrease in GST activity (ca. − 20%) was found at the highest sediment concentration of lindane (10,000 μg/kg) and at the highest water concentrations of dieldrin (4.9 and 29 μg/l). Between experiments differences in GST activity were found that were ascribed to seasonal influences and maintenance in the laboratory. It is concluded that under the applied conditions glutathione S-transferase activity of Sphaerium corneum towards CDNB is not a suitable short-term biomarker with respect to the toxicants tested.

402 “Glutathion system” and chemoresponse to 5-fluorouracil and cisplatine in head and neck squamous cell carcinoma: A pilot study

The relationship between the glutathione system and the chemoresponse to nea-adjuvant chemotherapy (CT) commonly used (cisplatine/5-fluorouracil: CDDP/5-FU) in head and neck squamous carcinoma (HNSCC). We assayed in the total blood of 38 patients (pt) the glutathione system before treatment (BT) and after 3 courses of CT. Objective response based on clinical examination (CE) and CT-Scan was achieved in 19/38 patients. The regression stage (RS) based on tumors histology obtained surgically (n=28) after 3 courses of chemotherapy was achieved in 8/28 patients. Total glutathione (GSH), glutathione-S-transferase (GST) activity and GST-π content were assayed on cytosols of total blood. For each cycle and each pt, GST activity and GSH content were investigated by spectrophotometric assays, GST-π by Western blot. According to CE and RS, our results showed difference between non responders (NR) and objective responders pt (R) only for GST activity. The difference was significant after one course of CT [according to CE for NR (5±2nmoles/min/mg) and R (4±2nmoles/min/mg) (<i>P</i><0.05), according to RS for NR (5±2nmoles/min/rng) and R (3±1mmoles/min/mg) (<i>P</i><0.01)]. No difference were detected for GSH and for GST-π. These preliminary results suggest that GST activity plays a role in the chemoresponse of HNSCC pts. Patients with decreased GST activity may be more chemosensitive than others. Indeed, it is too early to detect some correlation between disease free and overall survival.

Role of calcium fluxes in the action of glucagon on cytosolic glutathione S-transferase activity in rat liver slices.

In a previous study we demonstrated that the administration of 20 micrograms/kg b.wt. of glucagon to rats caused a significant diminution of hepatic cytosolic glutathione S-transferase (GST) activity. This inhibition was non-competitive and reversible. We suggested that the effect would be mediated by cytosolic effectors. The present work was performed to characterize the mechanism involved in this inhibition. Liver tissue slices (170 to 200 mg) were incubated during different periods of time (0, 5, 10, 15, 20 and 30 min.) with several concentrations of glucagon (10(-5) M, 10(-8) M and 10(-10) M), dibutiryl cyclic AMP (10(-4) M, 10(-6) M and 10(-9) M), divalent cation ionophore A23187 (10(-4) M, 10(-6) M and 10(-9) M) or vasopressin (10(-7) M, 5 x 10(-7) M and 10(-8) M). The incubation was done with or without calcium in the medium. In all cases the cytosolic GST activity were determined in liver slices. The percentage of inhibition of GST activity was directly related to the increase of concentration of the test substances. An inhibition between 40% to 45% after 10 min. of incubation with the highest concentrations was observed (except vasopressin which caused 10% of inhibition). 10(-10) M glucagon did not produce a decrease of GST activity. The inhibition disappeared in calcium-free incubated slices, but direct relationship between plasma-membrane calcium influx and inhibition of GST activity (r = 0.950, P < 0.001, n = 24) could be obtained. By using calmodulin antagonists, we conclude that the inhibition process of the enzyme was mediated by calmodulin. In summary, we propose that plasma-membrane calcium influx induced by high concentrations of glucagon activates calmodulin, which promotes a modification (actually a methylation, according to other authors) on GST, thereby causing a decrease in its activity.