Increased Glutathione S-transferase Activity Research Articles

Mechanisms of resistance to organophosphorus insecticides in populations of the obliquebanded leafroller Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae) from southern Ontario.

Populations of Choristoneura rosaceana (Harris) from orchards in Ontario were shown to be resistant to azinphos-methyl and to other types of organophosphorus insecticides. Resistance extended to methyl carbamates and to methomyl. The laboratory population used for these assays and selected with azinphosmethyl was also resistant to the pyrethroid, cypermethrin. Resistance was associated with increased esterase activity and was reduced by the addition of the synergist DEF. IEF studies of esterases also indicated increased activity in resistant populations, but did not identify any unique esterases associated with the resistance. Resistance was highly correlated (r = 0.78) with elevated esterases but not with increased glutathione-S transferase activity (r = 0.13). Other mechanisms did not appear to be related.

Differential influences of various arsenic compounds on glutathione redox status and antioxidative enzymes in porcine endothelial cells.

The cellular response and detoxification mechanisms in porcine endothelial cells (PAECs) to arsenic trioxide (As2O3), sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4) were investigated. NaAsO2 at 20 microM for 72 h increased Cu/Zn superoxide dismutase activity resulting in elevated intracellular hydrogen peroxide levels, but As2O3 and Na2HAsO4 did not. Trivalent arsenic compounds increased intracellular oxidized glutathione (GSSG) and total glutathione (GSH) and cellular glutathione peroxidase (cGPX) and glutathione S-transferase (GST) activity, but not glutathione reductase activity. The increased cGPX activity resulted in an elevated cellular GSSG content. Na2HAsO4 increased the cellular GSSG level at 72 h compared to controls. These results imply that the increased GSH content responding to the oxidative stress by trivalent arsenic compounds may be mainly related to the regulation of GSH turnover. The increased GST activity implies that the elevated intracellular GSH level responding to the oxidative stress may be used to conjugate arsenic in PAECs and facilitate arsenic efflux.

Comparative study of the xenobiotic metabolising system in the digestive gland of the bivalve molluscs in different aquatic ecosystems and in aquaria experiments

A comparative study has been performed on populations of Unionidae from the Lake Suszek and Brda river situated in the centre of Tucholski Landscape Park, around which there are no factories and the Pilica river—affected by the influence of the nearby town agglomeration. Mussels collected from Suszek were also treated (72 h) with various concentrations of dichlorophenol (DCP; 0.1, 0.15, 0.2 ppm) and paraquat (PQ; 1, 5, 10 ppm) in laboratory conditions (aquarium). The activities of glutathione S-transferase (GST) and cytochrome P450 monooxygenase system (NAD(P)H ferricyanide reductase, NAD(P)H cytochrome c reductase), cytochrome P450 content and b 5 in microsomal and cytosolic fractions of digestive gland were investigated. The differences in enzyme activities between groups of mussels, which were exposed to various concentrations of chemical pollutants, as well as the dependence on geographical distribution in Poland, were observed. In experiments with DCP the dose-dependent increase in GST activity was found, but no changes after PQ treatment were observed. Results, in experiments with DCP and PQ, have varied from no change to increase or decrease in the measured monooxygenase activities and cytochrome P450 content. Increases have been recorded in two cases (NADPH ferricyanide reductase and cytochrome P450) after exposure to DCP and in the case of NADH ferricyanide reductase following the exposure to PQ. NAD(P)H cytochrome c reductase activity and content of P450 decreased considerably in 5 and 10 ppm PQ-treated mussels. Thus, the treatment with DCP and PQ in water changed the properties of the mussels digestive gland cytochrome P450 monooxygenase system. These changes may be used as a bioindicator, at the molecular level, of exposure to those xenobiotics not only in controlled experiments (aquaria) but also in the natural environment.

New, quick tests for herbicide resistance in black-grass (Alopecurus myosuroides Huds) based on increased glutathione S-transferase activity and abundance

New, quick tests for herbicide resistance in black-grass (Alopecurus myosuroides Huds) based on increased glutathione S-transferase activity and abundance

Effects of Mannich bases on cellular glutathione and related enzymes of Jurkat cells in culture conditions

Jurkat cells were exposed to representative acetophenone-derived mono Mannich bases 2 and 3 and also cyclic Mannich base C1 in culture conditions to see the alterations in the most abundant cellular thiol, glutathione and also some of the enzymes in its metabolic pathway. Jurkat cells were exposed to the compounds for 24 h in cell culture medium with fetal bovine serum (1%) at 37 °C under a humidified atmosphere of 95% air and 5% CO 2. Mannich bases generally increased total glutathione level (123–151% of control). Glutathione S-transferase (GST) activity also increased (150–363% of control), while glutathione disulfide reductase (GRD) activity was not affected. The increase in cellular glutathione level may possibly result from de novo glutathione synthesis. The consumption of the glutathione due to alkylation by Mannich bases might have stimulated the enzymes in the γ-glutamyl cycle in our experimental design, where the cells had nutrients and time to react with their feedback mechanisms. A remarkable increase in GST activity might be a compensatory up-regulation to detoxify Mannich bases by conjugating them with cellular thiols.

Response of antioxidant enzymes in freshwater fish populations ( Leuciscus alburnoides complex) to inorganic pollutants exposure

Evidence is accumulating indicating the importance of antioxidant enzyme activity measurements in eco-toxicological studies, as they may constitute markers for exposure to a large variety of pollutants. Variation of antioxidant enzymes, superoxide dismutase (SOD) and glutathione S-transferases (GST) and the effect of heavy metals and selenium exposure on these enzymes were investigated in the livers of Iberian endemic minnows ( Leuciscus alburnoides complex) captured in a copper (Cu) mining area. Higher hepatic levels of copper and selenium were always observed in fish captured at the polluted site relative to the reference area population, reflecting the environmental monitoring results. A seasonal fluctuation in zinc and selenium levels for both populations was also observed which could be related to gonad maturation. The activity of SOD did not show significant regional alterations, but a seasonal variation occurred presumably associated with the Leuciscus life cycle. The GST activity was higher in the fish population from the polluted area (except in spring) and GST variability was associated with selenium and copper levels when both regions were compared. The increased GST activity was probably a metabolic adaptation to the continuous exposure to higher levels of those elements.

Glutathione-S-transferase-pi expression regulates sensitivity to glutathione-doxorubicin conjugate.

We have reported that glutathione-doxorubicin conjugate (GSH-DXR) exhibited potent cytotoxicity against tumor cells and inhibited glutathione-S-transferase (GST) enzyme activity. In order to determine whether or not the expression of GST-pi lowered the cytotoxicity of GSH-DXR, cytocidal activity of the conjugate was examined using tumor cells in which the level of GST-pi expression was regulated by transfecting GST-pi cDNA in the correct or reverse direction and comparing with that of DXR. Enhancement of GST-pi expression by transfecting GST-pi sense cDNA into human hepatoblastoma HepG2 cells in which GST-pi expression was extremely low caused an increase in GST activity from 0.26 to 55.0 nmol/mg/min and a marked reduction in transfectant sensitivity to GSH-DXR to 1/120 (0.15-18 nM IC50) although the sensitivity to DXR was slightly decreased to 1/2.6 (380-990 nM IC50). By contrast, a high GST-pi-expressing human colon cancer cell line, HT29, showed a decrease in GST enzyme activity from 72.0 to 45.9 nmol/mg/min after transfecting GST-pi antisense cDNA and a marked improvement in transfectant sensitivity to GSH-DXR was observed (28-2.9 nM IC50) compared with the transfectant sensitivity to DXR (1020-320 nM IC50). Additionally, the expression of GST-pi in HepG2 cells caused a decrease in GSH-DXR-induced activation of caspase-3, which was an apoptotic marker, whereas the suppression of GST-pi in HT29 cells showed an increase in caspase-3 activation. These results suggested that the cytocidal efficacy of GSH-DXR, but not that of DXR, was controlled by the level of GST-pi expression in the cells.

Glyphosate-Induced Increase in Glutathione S-Transferase Activity and Glutathione Content in Groundnut (Arachis hypogaea L.)

Abstract The obvious sensitivity of most plant species to glyphosate suggests that this phosphonate is poorly metabolized in plants. The effect of glyphosate on glutathione S -transferase (GST, EC 2.5.1.18) and glutathione (GSH) levels was examined in groundnut ( Arachis hypogaea L.), in view of the adaptive significance of elevated thiol metabolism in catalyzing metabolism of several herbicides in vivo . Glyphosate treatment resulted in a significant and concentration-dependent increase in the activity of GST and GSH levels in the three groundnut cultivars, JL24, CO2, and TMV2, tested. The glyphosate-induced increase in the enzyme activity was maximum in the foliar explants of all three cultivars, but was not correlated to their sensitivity to the herbicide. Glyphosate-tolerant cell lines of groundnut ( A. hypogaea cv. JL24) selected in vitro showed an elevated basal level of GST activity and nonprotein thiol content, as opposed to the glyphosate-sensitive cell line. The glyphosate-induced increase in GST activity could be prevented by cycloheximide and actinomycin D.

Oxidative stress in the latissimus dorsi muscle of diabetic rats.

The purpose of the present study was to investigate the effects of experimental diabetes on the oxidant and antioxidant status of latissimus dorsi (LD) muscles of male Wistar rats (220 +/- 5 g, N = 11). Short-term (5 days) diabetes was induced by a single injection of streptozotocin (STZ, 50 mg/kg, iv; glycemia >300 mg/dl). LD muscle of STZ-diabetic rats presented higher levels of thiobarbituric acid reactive substances (TBARS) and chemiluminescence (0.36 +/- 0.02 nmol/mg protein and 14706 +/- 1581 cps/mg protein) than LD muscle of normal rats (0.23 +/- 0.04 nmol/mg protein and 7389 +/- 1355 cps/mg protein). Diabetes induced a 92% increase in catalase and a 27% increase in glutathione S-transferase activities in LD muscle. Glutathione peroxidase activity was reduced (58%) in STZ-diabetic rats and superoxide dismutase activity was similar in LD muscle of both groups. A positive correlation was obtained between catalase activity and the oxidative stress of LD, as evaluated in terms of TBARS (r = 0.78) and by chemiluminescence (r = 0.89). Catalase activity also correlated inversely with glutathione peroxidase activity (r = 0.79). These data suggest that an increased oxidative stress in LD muscle of diabetic rats may be related to skeletal muscle myopathy.

Biochemical responses in farmed mussel Perna perna transplanted to contaminated sites on Santa Catarina Island, SC, Brazil

The effects of contaminants on the biochemical parameters of the intensively farmed mussel Perna perna, are unknown. The aim of this study was to compare biochemical responses in mussels held in clean and contaminated sites in Santa Catarina Island, Brazil. Mussels were transplanted from a farming area, Ratones Grande Island (RGI), to two contaminated sites, Itacorubi (ITAC) and Hercı́lio Luz Bridge (HLB). A reference group was kept at RGI. After 150 and 180 days of exposure, the digestive glands of the mussels were analyzed for catalase, glucose 6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST) activities. No changes were observed in the catalase activity, in both periods. Low G6PDH activity was observed in mussels transplanted for 150 days at the ITAC site. Increased GST activity was observed in mussels from ITAC and HLG sites after 180 days. These responses are probably related to the augmented discharges of domestic effluents associated with elevated rainfall index.

Effects on Detoxification Enzymes in Different Life Stages of Honey Bees (Apis mellifera L., Hymenoptera: Apidae) Treated with a Synthetic Pyrethroid (Flumethrin)

The activities of two detoxification enzymes, glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, and glutathione peroxidase with tertbutyl hydroperoxide as substrate (GSH-Px[TBH]), were measured in the larvae, pupae and adults of honey bees (Apis mellifera ligustica Spinola) originating from two colonies, one untreated acting as a control group and the other treated with flumethrin. The treatment with flumethrin led to increased GST activity in the larval instars, pupae and nurse bees compared with the control group. In particular, the late fifth larval instars (LS) and the pupal instars showed increased GST activities. GSH-Px[TBH] activities were highest in the early larval instars (L1-L2). Treatment with flumethrin resulted in the induction of GSH-Px[TBH] activity in the L4-L5 instars and LS. In the forager bees, the enzyme activity was lower in the group treated with flumethrin than in the control group. This could have been due to unknown extrinsic environmental factors. In general, the average protein contents were lower in the flumethrin-treated group than in the control group. In the LS and pupal stage, and in the adult worker bees, the differences were significantly lower. This inhibition of growth could be due to metabolic costs resulting from exposure to toxicants.

Enhancement of glutathione S-transferase using diallyl sulfide from garlic in Hepa-1c1c7 cells

Modification of the expression of xenobiotic metabolizing enzymes, such as glutathione S-transferase (GST), is thought to be one of the mechanisms of the anticarcinogenic effect of garlic. To obtain further insight into the induction of the GST enzyme and on the anticarcinogenic action of garlic, we examined the effect of daillyl sulfide (DAS), an organosulfur compound derived from garlic, on the induction of GST in the murine hepatoma cells (Hepa-1c1c7), which is sensitive to the xenobiotics. We observed that the GST enzyme activity was enhanced after treatment with 5 mM DAS for 16, 24 and 48 hours. Dose-response experiments demonstrated that the GST enzyme activity increased with increasing concentrations of DAS. Furthermore, Western blotting analysis revealed that three GST isoenzyme proteins, GST-α, GST-μ and GST-π, tended to increase with increasing DAS concentrations. In summary, the results suggest that GST enzyme activity can be enhanced by DAS in Hepa-1c1c7 cells, and the increased expression of the GST isoenzyme proteins contributed to the augmentation of the corresponding enzyme activity. The increased GST activity may explain, at lease in part, the anticarcinogenic effect of garlic.

Reactive oxygen species-specific mechanisms of drug resistance in paraquat-resistant acute myelogenous leukemia sublines.

Reactive oxygen species (ROS)-specific mechanisms of drug resistance were explored in paraquat (PQ)-resistant acute myelogenous leukemia cell (OCI/AML-2) sublines. For this, PQ-resistant AML sublines, AML-2/PQ100 and AML-2/PQ400, were selected in the presence of PQ concentrations of 100 microg/ml and 400 microg/ml, respectively. They showed a moderate level of cross resistance to cisplatin and doxorubicin. They were also slightly more resistant than the parental cell (AML-2/WT) to etoposide, camptothecin and daunorubicin. The resistance of PQ-resistant AML-2 sublines to cisplatin seemed to be due to increased amounts of metallothionein, which was not only supported by reversal of resistance to cisplatin by propargylglycin (an inhibitor of metallothionein synthesis) but also confirmed by Western blot analysis and reverse transcription-PCR assay. In addition, both AML-PQ100 and /PQ400 sublines showed increased activities of Cu-, Zn-containing superoxide dismutase (Cu,Zn-SOD) and Mn-containing superoxide dismutase (Mn-SOD), whereas AML-2/PQ400, but not AML-2/PQ100, showed increased glutathione S-transferase activity as compared to that of AML-2/WT. However, there was no difference in other ROS-related cellular antioxidants between AML-2/WT and its PQ-resistant sublines. Taken together, these results strongly suggest that increases in levels of metallothionein, glutathione S-transferase, Cu,Zn-SOD and Mn-SOD play important roles in protective mechanisms against toxicity of PQ or ROS in AML cells.

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Modulation of hepatic and intestinal Glutathione S-transferases and other antioxidant enzymes by dietary lipids in streptozotocin diabetic rats

Antioxidant enzymes in liver and small intestine were investigated using control and streptozotocin diabetic rats fed diets with 5% olive, sunflower or fish oil for five weeks. In liver, Glutathione Peroxidase and Superoxide Dismutase decreased and in intestine Glutathione-S-transferase (GST) increased by diabetes. In isolated jejunum and ileum, this increase in GST activity was due to an increase in GST-α and -μ isoenzymes in jejunum and GST-α, μ and -π in ileum. Since GST plays an important role in protecting tissues from oxidative damage, our results highlight the role of the intestine against free radicals in physiological or pathological situations.

Induction of New GlutathioneS-Transferase Isozymes by Allelochemicals in the Fall Armyworm

The induction of glutathioneS-transferases in different tissues of fall armyworm (Spodoptera frugiperda) larvae was studied using the allelochemical xanthotoxin as a model inducer. Newly molted sixth instars were fed an artificial diet containing 0.01% xanthotoxin for 2 days prior to enzyme preparation and characterization. Xanthotoxin treatment resulted in significant increases in glutathioneS-transferase activity (DCNB) in midguts (76-fold), fat bodies (59-fold), and Malpighian tubules (32-fold) compared with the respective controls. Nondenaturing gel electrophoresis of glutathione–agarose affinity-purified preparations from control larvae showed that the midgut possessed six heterodimeric isozymes with subunit molecular weights of 26,700 to 30,000. The fat body contained three homodimeric isozymes with subunit molecular weights of 20,100 to 29,000. The Malpighian tubules had five heterodimeric isozymes with subunit molecular weights of 26,700 to 30,000. Induction of glutathioneS-transferases by xanthotoxin in the larvae resulted in production of two new isozymes in fat bodies but had no effect on isozyme composition in midguts and Malpighian tubules. Both induced isozymes were homodimers with a subunit molecular weight of 28,000. The allelochemicals, indole 3-acetonitrile and flavone, also induced these new isozymes in fat bodies but, unlike xanthotoxin, both inducers decreased levels of the constitutive isozymes. Using isozyme-specific antisera as probes, one of the induced isozymes was immunologically related to two midgut isozymes. Actinomycin D at 0.1 μg/larva completely blocked the xanthotoxin induction of new isozymes in fat bodies.

Effects of Toxic and Deterrent Terpenoids on Digestive Protease and Detoxication Enzyme Activities of Colorado Potato Beetle Larvae

Larvae of Colorado potato beetle,Leptinotarsa decemlineata, were fed on potato leaf disks treated with the toxic limonoids azadirone and a mixture of 1,7-di-O-acetylhavanensin and 3,7-di-O-acetylhavanensin (F18) fromTrichilia havanensis(Meliaceae) and a deterrentneo-clerodane (scutalpin-B) fromScutellaria alpina(Labiatae). Azadirone, a toxicant with low mortality action, decreased esterase activity and increased glutathioneS-transferase activity during the treatment period, but these detoxication enzymes reached normal levels of activity when the larvae were shifted to untreated leaf disks during the post-treatment period. The mixture of limonoids F18, a strong toxicant that produces high levels of larval mortality, significantly reduced digestive protease and esterase activities and increased glutathioneS-transferase and polysubstrate monooxygenases during the treatment period. All enzymatic activities but glutathioneS-transferase remained altered during the post-treatment period. Scutalpin-B, with a deterrent mode of action, did not have any significant effect on any of the enzymatic processes. We concluded that the effects of these compounds on digestive proteases and detoxication enzymes in the larval midgut ofL. decemlineatalarvae reflect their postulated mode of action.

α,β-Unsaturated Aldehydes Increase GlutathioneS-Transferase mRNA and Protein: Correlation with Activation of the Antioxidant Response Element

A series of α,β-unsaturated aldehydes was evaluated to determine if these compounds could mediate inducible expression of glutathioneS-transferase (GST) through the 5′-flanking antioxidant response element (ARE). The ARE from rGST A1 was subcloned into a luciferase reporter construct and used to transiently transfect rat Clone 9 hepatoma cells. Transfected cells were treated with 4-hydroxy-trans-2-nonenal (4-HNE),trans-2-hexenal (t-2-HE), 2-propenal (acrolein, 2-PE), and ethacrynic acid (EA), a control compound also containing an α,β-unsaturated carbonyl moiety. Each compound was evaluated for cytotoxicity to construct dosing regimens in transfection studies. IC50values for growth inhibition were measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. IC50values in Clone 9 cells were: 4-HNE, 6.3 ± 0.7 μM;t-2-HE, 16.0 ± 0.7 μM; 2-PE, 2.2 ± 0.4 μM; and EA, 38.0 ± 1.6 μM. A dose-dependent increase in luciferase activity was observed in transfected cells with all four compounds tested, indicating that α,β-unsaturated aldehydes function as direct activators of the ARE. To determine whether or not the observed promoter activation led to increased transcriptional and translational induction of GST, cells were treated with the various compounds and assayed for increases in GST mRNA, protein, and enzyme activity. Studies in Clone 9 cells revealed increased steady-state message for GST A1 and A4, increased GST A4-4 protein by Western blotting, and increased GST activity toward 1-chloro-2,4-dinitrobenzene in response to treatment with all four compounds evaluated. Collectively, these studies demonstrate that EA and certain α,β-unsaturated aldehydes produced as a result of cellular membrane lipid peroxidation are activators of the ARE and efficient inducers of GST A1-1 and A4-4.

Glutathione and glutathione S-transferase in the rainbow trout olfactory mucosa during retrograde degeneration and regeneration of the olfactory nerve.

In the peripheral olfactory organ, continual olfactory receptor neuron (ORN) turnover exposes neighboring cells to potentially damaging cellular debris such as free radicals. These, in turn, may be inactivated by binding directly onto glutathione (GSH) or by enzymatic conjugation with glutathione S-transferase (GST). In this study, we have investigated GSH and GST during retrograde degeneration and regeneration of the olfactory nerve in rainbow trout. In these fish, prolonged ORN physiological activity and structural integrity following transection of the olfactory nerve may be mediated by GSH and GST. In the olfactory mucosa, early changes following nerve lesion and prior to ORN degeneration included a shift of intense GSH labeling from the dendrites and perikarya of a subpopulation of ORN, and from melanophores, to olfactory nerve fascicles. GSH levels were unchanged, but GST activity decreased by 33% and GST-immunoreactivity (GST-IR) in nerve fascicles diminished slightly. When the process of massive degeneration terminated and ORN were largely absent, GSH levels and GST activity decreased further, GSH labeling was confined to melanophores, and GST-IR was absent. As ORN repopulated the olfactory mucosa, GST-IR was widespread. The combination of increased GST activity (92% of preoperative values) and low GSH levels suggests GSH utilization for GST conjugation reactions. These changes imply that GSH provides protection from cellular debris associated with ORN degeneration. Recovery of GST activity and widespread GST-IR during regeneration indicates modulation of neuroprotective, developmental, and/or physiological processes by GST.

Preferential Overexpression of a Class MU GlutathioneS-Transferase Subunit in Mouse Liver by Myristicin

The present studies were undertaken to elucidate the mechanism of induction of glutathione S-transferase (GST) in mouse liver by myristicin, an active constituent of parsley leaf. A/J albino mice, given 5 to 50 mg doses of myristicin, showed 4- to 14-fold increase in liver GST specific activity over the control. GST purified from equal amounts of control and myristicin-treated livers indicated a marked increase in the GST activity. A relatively higher increase in GST activity towards 2,4-dichloronitrobenzene and a profound increase in the levels of GST μ on Western blot analysis of the myristicin-treated mouse liver suggest a preferential induction of GST μ. Results of the study also indicate that out of the two μ class GST subunits (Mr. 26,500 and Mr. 25,000) expressed in liver only one (Mr. 26,500) is significantly elevated. Myristicin treatment caused a slight change in the GST π levels while the levels of GST α showed a modest increase. These results suggest that myristicin could be an effective chemopreventive agent, particularly for carcinogens that are detoxified by the μ class GST.