Increased GST Activity Research Articles

Mechanisms of primary cancer prevention by butyrate and other products formed during gut flora-mediated fermentation of dietary fibre

Dietary fibres are indigestible food ingredients that reach the colon and are then fermented by colonic bacteria, resulting mainly in the formation of short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate. Those SCFA, especially butyrate, are recognised for their potential to act on secondary chemoprevention by slowing growth and activating apoptosis in colon cancer cells. Additionally, SCFA can also act on primary prevention by activation of different drug metabolising enzymes. This can reduce the burden of carcinogens and, therefore, decrease the number of mutations, reducing cancer risk. Activation of GSTs by butyrate has been studied on mRNA, protein, and enzyme activity level by real-time RT-PCR, cDNA microarrays, Western blotting, or photometrical approaches, respectively. Butyrate had differential effects in colon cells of different stages of cancer development. In HT29 tumour cells, e.g., mRNA GSTA4, GSTP1, GSTM2, and GSTT2 were induced. In LT97 adenoma cells, GSTM3, GSTT2, and MGST3 were induced, whereas GSTA2, GSTT2, and catalase (CAT) were elevated in primary colon cells. Colon cells of different stages of carcinogenesis differed in post-transcriptional regulatory mechanisms because butyrate increased protein levels of different GST isoforms and total GST enzyme activity in HT29 cells, whereas in LT97 cells, GST protein levels and activity were slightly reduced. Because butyrate increased histone acetylation and phosphorylation of ERK in HT29 cells, inhibition of histone deacetylases and the influence on MAPK signalling are possible mechanisms of GST activation by butyrate. Functional consequences of this activation include a reduction of DNA damage caused by carcinogens like hydrogen peroxide or 4-hydroxynonenal (HNE) in butyrate-treated colon cells. Treatment of colon cells with the supernatant from an in vitro fermentation of inulin increased GST activity and decreased HNE-induced DNA damage in HT29 cells. Additional animal and human studies are needed to define the exact role of dietary fibre and butyrate in inducing GST activity and reducing the risk of colon cancer.

Role of microsomal glutathione transferase 1 in the mechanism-based biotransformation of glyceryl trinitrate in LLC-PK1 cells

Although glyceryl trinitrate (GTN) has been used in the treatment of angina for many years, details of its conversion to the proximal activator (presumed to be NO or an NO congener) of soluble guanylyl cyclase (sGC) are still unclear. We reported previously that purified microsomal glutathione transferase 1 (MGST1) mediates the denitration of GTN. In the current study, we investigated in intact cells whether this enzyme also converts GTN to species that activate sGC (mechanism-based biotransformation). We utilized LLC-PK1 cells, a cell line with an intact NO/sGC/cGMP system, and generated a stable cell line that overexpressed MGST1. MGST1 in the stably transfected cells was localized to the endoplasmic reticulum, and microsomes from these cells exhibited markedly increased GST activity. Although incubation of these cells with GTN resulted in a 3–4-fold increase in GTN biotransformation, attributed primarily to an increase in formation of the 1,3-glyceryl dinitrate metabolite, GTN-induced cGMP accumulation in cells overexpressing MGST1 was not different than that observed in wild type cells or in cells stably transfected with empty vector. To determine whether overexpression of NADPH cytochrome P450 reductase might act in concert with MGST1 to generate activators of sGC, we assessed GTN-induced cGMP accumulation in MGST1-overexpressing cells that had been transiently transfected with CPR. In this case, GTN-induced cGMP accumulation was also not different than that observed in wild type cells. We conclude that although MGST1 mediates the biotransformation of GTN in intact cells, this biotransformation does not contribute to the formation of activators of sGC.

Activity of Antioxidative Enzymes in Erythrocytes after a Single Dose Administration of Doxorubicin in Rats Pretreated with Fullerenol C60(OH)24

In earlier in vitro investigations, fullerenol was shown to have a strong antioxidative capability. The present study examined the role of fullerenol as a potential antioxidative protector for doxorubicin-induced oxidative stress in the blood of rats through an investigation of the activity of glutathione-dependent enzymes (glutathione-S-transferase and glutathione peroxidase). It also assessed the influence of fullerenol on the number of blood cells (leukocytes and erythrocytes) as well as on the content of hemoglobin after a single dose administration of doxorubicin. Experiments were performed on six groups of adult male Wistar rats, each group containing eight individuals. Doxorubicin was administrated i.v. (tail vein) in a single dose of 10 mg/kg. Fullerenol C60(OH)24 was administrated to the treated animals i.p. (in doses 50, 100, 200 mg/kg) 30 min before the dosing with doxorubicin. The control group animals were given saline (1 ml/kg; i.p.). One group of animals was treated only with fullerenol (100 mg/kg i.p.). The animals were sacrificed 2 and 14 days after the treatment. Each experiment was repeated twice. The results may indicate that fullerenol induces a decrease in the antioxidative capacity of erythrocytes in oxidative stress conditions, whereas, without doxorubicin, the application of fullerenol did not induce any changes in the enzyme activity of erythrocytes. The results of GST activity might indicate that 50 mg/kg are not sufficient to protect from doxorubicin toxicity, while 200 mg/kg might be toxic for animals, judging from the increase in GST activity.

Oxidative injury and enzymic antioxidant misbalance in schizophrenics with positive, negative and cognitive symptoms

With its hallucinations, delusions, thought disorder, and cognitive deficits, schizophrenia affects the most basic human processes of perception, emotion, and judgment. Evidence increasingly suggests that schizophrenia is a subtle disorder of brain development and plasticity and oxidative injury contributes largely to pathophysiology of schizophrenia, indicated by the increased lipid peroxidation products in plasma and altered levels of enzymatic antioxidants in schizophrenic patients. However, the status of antioxidants and the extent of lipid peroxidation in erythrocytes have not been investigated so far in schizophrenia patients with different symptoms. In the present study, in order to examine the antioxidant status and lipid peroxidation in the schizophrenics with positive, negative and cognitive symptoms, the activities of three free radical scavenging enzymes glutathione transferase (GST), glucose-6-phosphate dehydrogenase (G6PD), ceruloplasmin ferroxidase (Cp) and the level of thiobarbituric acid-reactive substances (TBARS) as an index of lipid peroxidation were analyzed. Results showed that there was a significant increase in GST activity in all the schizophrenics when compared to normal and it was observed that there was a significant decrease in erythrocyte, G6PD and ceruloplasmin ferroxidase levels in patients with schizophrenia, when compared to controls. Schizophrenics with positive symptomology were found to have pronounced decrease in the activities of Cp ferroxidase and statistically more significant decrease in G6PD levels were found in patients with negative symptoms. Further a significant rise in oxidative stress and decreased secondary enzymic antioxidant status was observed in the chronic stage of schizophrenics as compared to those in acute condition. The study showed that the level of malondialdehyde was increased in schizophrenics with positive (163%), negative (137%) and cognitive (132%) symptoms compared to control groups .The increased activities of antioxidant enzymes may be a compensatory regulation in response to increased oxidative stress. The decreased concentrations of the antioxidants status support the hypothesis that lipid peroxidation is an important causative factor in the pathogenesis of schizophrenia. These data reveal that antioxidant defense mechanisms might be impaired in schizophrenic patients. Understanding these basic pathologic processes may yield novel targets for the development of more effective treatments. Keywords: Schizophrenia, symptoms, secondary antioxidant enzymes, malondialdehyde (MDA), oxidative stress

Transcriptome analysis provides new insights into liver changes induced in the rat upon dietary administration of the food additives butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole

Transcriptomics was performed to gain insight into mechanisms of food additives butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG), and thiabendazole (TB), additives for which interactions in the liver can not be excluded. Additives were administered in diets for 28 days to Sprague–Dawley rats and cDNA microarray experiments were performed on hepatic RNA. BHT induced changes in the expression of 10 genes, including phase I (CYP2B1/2; CYP3A9; CYP2C6) and phase II metabolism (GST μ2). The CYP2B1/2 and GST expression findings were confirmed by real time RT-PCR, western blotting, and increased GST activity towards DCNB. CC altered the expression of 12 genes. Three out of these were related to peroxisomes (phytanoyl-CoA dioxygenase, enoyl-CoA hydratase; CYP4A3). Increased cyanide insensitive palmitoyl-CoA oxidation was observed, suggesting that CC is a weak peroxisome proliferator. TB changed the expression of 12 genes, including CYP1A2. In line, CYP1A2 protein expression was increased. The expression level of five genes, associated with p53 was found to change upon TB treatment, including p53 itself, GADD45α, DN-7, protein kinase C β and serum albumin. These array experiments led to the novel finding that TB is capable of inducing p53 at the protein level, at least at the highest dose levels employed above the current NOAEL. The expression of eight genes changed upon PG administration. This study shows the value of gene expression profiling in food toxicology in terms of generating novel hypotheses on the mechanisms of action of food additives in relation to pathology.

Effects of Aroclor 1254 on oxidative stress in developing Xenopus laevis tadpoles

Over the last decades, amphibians decline has been reported worldwide. Exposure to polychlorinated biphenyls (PCBs) is one of the possible causes in addition to climate changes, UV-radiation or habitat destruction. In the present study, we tested the hypothesis that PCBs could induce oxidative stress in young tadpoles. Developing Xenopus laevis were exposed from 2- to 5-d postfertilization (pf) to 0.1 or 1 mg/l of Aroclor 1254. Lipid peroxidation and antioxidant systems (SOD, CAT, GST, GPx, GR activities and t-GSH level) were investigated in whole organisms. Exposure to both concentrations did not impact on the survival and development whereas the average body weight decreased. Exposure to 1 mg/l of Aroclor 1254 induced a significant ( p<0.05) increase of GST activity when compared to controls 0 and DMSO. The other antioxidant enzymes and LPO evaluation remained unchanged. Our results demonstrate that exposure of X. laevis tadpoles to environmental concentrations of Aroclor 1254 interfere with normal growth. They also highlight that very young X. laevis tadpoles express antioxidant systems.

Dynamics of glutathione- S-transferases in Mytilus galloprovincialis exposed to toxic Microcystis aeruginosa cells, extracts and pure toxins

Molluscs and especially bivalves are able to accumulate dinoflagelates, diatoms and cyanobacteria toxins, and, being vectors for these toxins, transfer them along food chains. The data obtained from laboratory experiments showed that bivalve molluscs are resistant to cyanobacteria toxins. In this work, we wanted to test if Mytilus galloprovincialis organs react to microcystins and other cyanobacteria compounds by inducing or decreasing its GST activity. Acclimated mussels M. galloprovincialis were exposed to the toxic Microcystis aeruginosa M13 strain. Exposure of mussels to toxins was done in three ways: living Microcystis cells, crude Microcystis extracts and pure toxins. The measurement of soluble and microsomal GST activity in the different mussel organs was done by using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichloro-1-nitrobenzene (DCNB). Analysis of the GST activity of the control mussels using CDNB as a substrate showed that cytosolic activity is much more significant than microsomal. Intact M. aeruginosa cells did not induce any significant response from the mussels, showing that these animals are quite resistant to the cyanobacteria if they are intact. On the other hand, cell extracts caused an important effect in the gut, in the gills and in the labial palps, although in different ways. There was an increase in GST activity in the gut and gills of mussels exposed to Microcystis extracts, showing a response of this detoxication pathway, but in the labial palps a severe reduction in GST activity occurred. Pure MC LR+YR induced an increase in GST activity in all organs but the labial palps. The results showed that other substances apart from microcystins may cause stress to mussels and affect detoxication enzymes such as GST.

Salt and paraquat stress tolerance results from co-expression of the Suaeda salsa glutathione S-transferase and catalase in transgenic rice

GST (Glutathione S-transferase, EC 2.5.1.18) and CAT (Catalase, EC 1.11.1.6) play important roles in oxidative stress resistance. In this study, we transferred both GST and CAT1 of Suaeda salsa into rice (Oryza sativa cv. Zhonghua No.11) by Agrobacterium tumefaciens-mediated transformation under the control of cauliflower mosaic virus (CaMV) 35S promoter, and investigated whether co-expressing the GST and CAT1 in transgenic rice could reduce oxidative damage. Salt and paraquat stresses were applied. The data showed that co-expression of the GST and CAT1 resulted in greater increase of CAT and SOD (Superoxide Dismutase, EC 1.15.1.1) activity in the transgenics compared to non-transgenics following both stress imposition. Whereas the significant increase of GST activity in transgenics only occurred in paraquat stressed plants. While the generation of H2O2, Malon dialdehyde and plasma membrane relative electrolyte leakage decreased in the transgenics than in non-transgenics under the same conditions. Moreover, the transgenic rice seedlings showed markedly enhanced tolerance to salt stress compared with non-transgenics upon 200 mM NaCl treatment in greenhouse. The enhancement of the active oxygen-scavenging system that led to increased oxidative stress protection in GST + CAT1-transgenic rice plants could result not only from increased GST and CAT activity but also from the combined increase in SOD activity.

The role of aryl hydrocarbon receptor and the reactive oxygen species in the modulation of glutathione transferase by heavy metals in murine hepatoma cell lines

Glutathione transferase (GST) is a phase II detoxifying enzyme that plays a protective mechanism against oxidizing substances and toxic contaminants. Among these contaminants, heavy metals and polycyclic and halogenated aromatic hydrocarbons (PHAHs) have been shown to exert their toxic effects through the modulation of detoxifying enzymes, including the GSTs. Recently, we showed that heavy metals particularly Hg 2+, Pb 2+, and Cu 2+ modulate the expression of phase II detoxifying enzymes such as NAD(P)H:quinone oxidoreductase 1 and Gsta1 in a concentration- and time-dependent manner. However, the effect of heavy metals and their potential interactions with aryl hydrocarbon receptor (AhR) ligands, PHAHs, on total Gst activity is still unknown. In the current study, we have investigated the effects of Hg 2+, Pb 2+, and Cu 2+ in the absence and presence of four AhR ligands on the total Gst activity and reactive oxygen species (ROS) production in wild-type and AhR-deficient Hepa 1c1c7 cells. Our results showed that Hg 2+ and Cu 2+, but not Pb 2+, significantly induced Gst activity in wild-type cells, whereas all metals induced the Gst activity in AhR-deficient cells. The induction of Gst activity by heavy metals was strongly correlated with an increase in the ROS production in wild-type, but not in AhR-deficient cells. Co-administration of heavy metals with AhR ligands differentially modulated Gst activity, in that co-exposure to Hg 2+ plus AhR ligands could be beneficial in protecting against cytotoxicity as demonstrated by the increase in Gst activity with a proportional decrease in ROS production. Whereas co-exposure to Cu 2+ plus AhR ligands was more toxic in that a decrease in Gst activity and an increase in oxidative stress of the cell were observed. We concluded that heavy metals differentially modulate the Gst activity through oxidative stress- and AhR-mediated mechanisms.

Chemosensitizing multiple drug resistance of human carcinoma by bicyclol involves attenuated P-glycoprotein, GST-P and Bcl-2

Bicyclol, a second generation of synthetic hepatoprotectant being used in China for anti hepatitis therapy, shows chemosensitizing effect on reverting multiple drug resistance (MDR) of cytostatic agents in two established MDR carcinoma cell lines, vincristine resistant human stomatic epidermoid carcinoma VinRKB and adriamycin resistant human breast carcinoma AdrRMCF-7. The reversal rate of drug resistance was calculated from the changes of the IC50 of cell growth inhibition. Bicyclol at the concentration of 25, 50, 100 mM induced 2.8 7.3 and 20.7 fold, respectively, reversal of vincristine resistance in VinRKB cell. Bicyclol also reversed the cross-resistance of VinRKB cell to taxol and AdrRMCF-7 cell resistance to adriamycin at the similar range of potency. Further, Bicyclol recovered the reduced accumulation of adriamycin in AdrRMCF-7 cell partially to the level in drug-sensitive MCF-7 cell, indicate the inhibition of MDR related membrane efflux pump system. Overexpression of membrane p-glycoprotein coded by Mdr-1 genes, the most common efflux pump correlated to MDR, was found in both VinRKB and AdrRMCF-7 cells by Western blot and immunocytochemistry as compared with drug-sensitive cells. The p-glycoprotein was decreased to the levels in drug-sensitive cells when VinRKB and AdrRMCF-7 cells were treated with Bicyclol for 12-72 hours. Both VinRKB and AdrRMCF-7 cells showed increased GSH contents, and AdrRMCF-7 cell showed increased GST activity and the overexpression of Bcl-2 protein, by which molecules are tightly related to the MDR formation besides Mdr-1 p-glycoprotein. Bicyclol reduced the GSH contents, GST activities and Bcl-2 expression. All these data demonstrate that, by modifying the expressions of Mdr-1, GSH/GST and Bcl-2, Bicyclol increases the intracellular drug concentration and sensitizes the resistant cells to the anti-carcinoma agents.

Glutathione and GSH-dependent enzymes in patients with liver cirrhosis and hepatocellular carcinoma.

We investigated glutathione level, activities of selenium independent GSH peroxidase, selenium dependent GSH peroxidase, GSH S-transferase, GSH reductase and the rate of lipid peroxidation expressed as the level of malondialdehyde in liver tissues obtained from patients diagnosed with cirrhosis or hepatocellular carcinoma. GSH level was found to be lower in malignant tissues compared to adjacent normal tissues and it was higher in cancer than in cirrhotic tissue. Non-Se-GSH-Px activity was lower in cancer tissue compared with adjacent normal liver or cirrhotic tissue, while Se-GSH-Px activity in cancer was found to be similar to its activity in cirrhotic tissue and lower compared to control tissue. An increase in GST activity was observed in cirrhotic tissue compared with cancer tissue, whereas the GST activity in cancer was lower than in adjacent normal tissue. The activity of GSH-R was similar in cirrhotic and cancer tissues, but higher in cancer tissue compared to control liver tissue. An increased level of MDA was found in cancer tissue in comparison with control tissue, besides its level was higher in cancer tissue than in cirrhotic tissue. Our results show that the antioxidant system of cirrhosis and hepatocellular carcinoma is severely impaired. This is associated with changes of glutathione level and activities of GSH-dependent enzymes in liver tissue. GSH and enzymes cooperating with it are important factors in the process of liver diseases development.

The Bipyridyl Herbicide Paraquat Produces Oxidative Stress-Mediated Toxicity in Human Neuroblastoma SH-SY5Y Cells: Relevance to the Dopaminergic Pathogenesis

Paraquat (PQ) is a cationic nonselective bipyridyl herbicide widely used to control weeds and grasses in agriculture. Epidemiologic studies indicate that exposure to pesticides can be a risk factor in the incidence of Parkinson's disease (PD). A strong correlation has been reported between exposure to paraquat and PD incidence in Canada, Taiwan, and the United States. This correlation is supported by animal studies showing that paraquat produces toxicity in dopaminergic neurons of the rat and mouse brain. However, it is unclear how paraquat triggers toxicity in dopaminergic neurons. Based on the prooxidant properties of paraquat, it was hypothesized that paraquat may induce oxidative stress-mediated toxicity in dopaminergic neurons. To explore this possibility, dopaminergic SH-SY5Y cells were treated with paraquat, and several biomarkers of oxidativestress were measured. First, a specific dopamine transporter inhibitor GBR12909 significantly protected SY5Y cells against the toxicity of paraquat, indicating that paraquat exerts its toxicity by a mechanism involving the dopamine transporter (DAT). Second, paraquat increased intracellular levels of reactive oxygen species (ROS), but decreased the levels of glutathione. Third, paraquat inhibited glutathione peroxidase activity, but did not affect glutathione reductase activity. On the other hand, paraquat increased GST activity by 24 h, after which GST activity returned to the control value at 48 h. Fourth, paraquat dissipated mitochondrial transmembrane potential (MTP). Fifth, paraquat produced increases of malondialdehyde (MDA) and protein carbonyls, as well as DNA fragmentation, indicating oxidative damage to major cellular components. Sixth, paraquat increased the protein level of heme oxygenase-1 (HO-1). Taken together, these findings verify our hypothesis that paraquat produces oxidative stress-mediated toxicity in SH-SY5Y cells. Thus, current findings suggest that paraquat may induce the pathogenesis of dopaminergic neurons through oxidative stress.

Garlic Organosulfur Compounds Upregulate the Expression of the π Class of Glutathione S-Transferase in Rat Primary Hepatocytes

The chemopreventive property of garlic is related in part to its induction of phase II detoxification enzymes. In the present study, we investigated the modulatory effect of 3 garlic organosulfur compounds, i.e., diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), which differ in their number of sulfur atoms, on the gene expression of the pi class of glutathione S-transferase (GSTP). Hepatocytes isolated from male Sprague-Dawley rats were cultured with 50-200 micromol/L of DAS, DADS, or DATS for 24 h. DADS and DATS increased GST activity toward ethacrynic acid by 40 and 66%, respectively (P < 0.05). Moreover, both garlic allyl sulfides dose dependently induced GSTP mRNA and protein expression. DATS increased the protein level more than DADS (P < 0.05). In contrast, DAS did not affect the activity or the protein or mRNA levels of this phase II drug-metabolizing enzyme. In Clone 9 liver cells, the pTA-luciferase reporter assay showed that luciferase activity in DADS- and DATS-treated cells was 2.8- and 3.9-fold higher than that in control cells, respectively (P < 0.05). Again, luciferase activity was not affected by treatment with DAS. Deletion of -2.7 to -2.6 kb in the GSTP promoter region, which contains the GSTP enhancer (GPE) I element, abolished the upregulation of GSTP transcription by DADS and DATS. Deletion of GPE II, however, did not affect the induction of reporter activity. In conclusion, the effectiveness of 3 garlic allyl sulfides on GSTP expression was related to the number of sulfur atoms in the molecules, and GPE I was responsible for this upregulation.

In vitro effect of low concentrations of oltipraz on the antioxidant defence of mouse hepatocytes and Schistosoma mansoni worms

The direct effects of oltipraz (OPZ) on mouse hepatocytes and Schistosoma mansoni worms are studied in vitro at a concentration range of 5–25 µmol/L following one- and three-hour incubations. Oxidative stress is reflected by increases in malondialdehyde (MDA), representing the end products of lipid peroxidation, and depletion of glutathione (GSH), representing protective thiol groups. Activities of glutathione peroxidase isoenzymes, GST and GR as components of antioxidative defence are also determined. The opposite effects of low concentrations of OPZ on mammalian hepatocytes and S. mansoni worms were confirmed. In incubation with S. mansoni, addition of OPZ resulted in significantly increased production of MDA, together with depletion of GSH, both of which were time- and OPZ concentration-dependent. In incubation with mouse hepatocytes, however, there was little change in MDA concentrations, and a gradual increase in GSH levels, both of which were time- and concentration-dependent. Addition of OPZ to the incubation media also affected the activities of antioxidant enzymes. Although total GPx activity increased in both mammalian hepatocyte and S. mansoni experiments, the opposite was noted with the selenium-dependent isoenzyme. While there was a gradual increase in sGPx in hepatocytes, there was a timeand concentration-dependent inhibition in the worm isoenzyme. Contrasting results were also obtained with GR. While increased activity was obtained with the enzyme from mouse hepatocytes, the worm enzyme was inhibited, especially at the upper end of the OPZ concentration range and also following longer periods of incubation. The increase in GST activity followed the same qualitative pattern in both hepatocytes and schistosomes. Therefore, OPZ given in doses that maintain a serum concentration in the range 5–25 µmol/L induces biochemical changes in mouse hepatocytes that could be utilised for chemo-preventive purposes and prevention of oxidative damage. However, progressive oxidative damage to S. mansoni worms occurred despite some protective biochemical changes.

An investigation into the detoxification of microcystin-LR by the glutathione pathway in Balb/c mice

Toxin-producing cyanobacteria pose a world-wide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. The predominant cyanotoxin, microcystin-LR (MCLR), targets the liver and its toxicity depends on the uptake and removal rates in the liver. The role of the glutathione detoxification pathway in protecting the liver from the effects of MCLR was investigated. Mice exposed to a single 75% LD 50 dose of pure MCLR were sacrificed at 8, 16, 24 and 32 h post-exposure (pe). Toxin induced liver damage was observed 8 and 16 h pe as evidenced by raised serum ALT and LDH levels, reduced glycogen levels and liver histology. A significant increase in lipid peroxidation was seen at 16 h pe that decreased after 24 and 32 h pe, the time-points which showed significant increases in GPX activity. An increase in soluble GST activity was noted between 8 and 16 h pe, levels of total GSH increased at 24 h while oxidised glutathione increased throughout the investigation. The increase in activity of both GPX and GST corresponded with increased transcription of these enzymes, as well as the rate-limiting enzyme in GSH synthesis, γ-glutamyl transferase. In conclusion, this study confirms that an increase in GST activity is critical for the detoxification of MCLR, that this is regulated at the transcriptional level, and that exposure to MCLR induces the de novo synthesis of GSH. Finally, we report the involvement of GPX in the removal of MCLR-induced lipid hydroperoxides.

Chronic Arsenic-Exposed Human Prostate Epithelial Cells Exhibit Stable Arsenic Tolerance: Mechanistic Implications of Altered Cellular Glutathione and Glutathione S-transferase

Acquisition of stable arsenic tolerance in human cells following chronic arsenic exposure has not been previously reported. In the present work, we describe acquisition of stable arsenic tolerance in the human prostate epithelial cell line RWPE-1 following chronic arsenic exposure in vitro. RWPE-1 cells continuously exposed to 5 μM sodium arsenite for ≥18 weeks exhibited dramatic resistance to acute arsenite toxicity. The LC50 for acute arsenite exposure in these chronic arsenic-exposed prostate epithelial (CAsE-PE) cells was 43.8 μM versus 17.6 μM in control cells. Similar results were obtained using the antineoplastic agent arsenic trioxide. This tolerance was stable, as CAsE-PE cells grown in arsenic-free medium for 5 weeks retained their resistant phenotype. Compared to control cells, CAsE-PE cells showed a 90% reduction in arsenic accumulation over 24 h coupled with a 2.6-fold increase in the rate of arsenic efflux. CAsE-PE cells had increased basal GSH levels (4.9-fold) and increased GST activity (2.4-fold) and both GSH depletion and inhibition of GST activity abolished arsenic tolerance. Arsenic tolerance was also abolished by treatment with inhibitors of the Mdr1 and Mrp1 transporters, although no increases in mdr1 or mrp1 gene expression were observed. Our results indicate that this tolerance in human cells involves increases in GSH levels and GST activity that allow for more efficient arsenic efflux by MRP1 and MDR1. This study represents the first report of stable acquired arsenic tolerance in human cells, which could have important implications for both the toxicology and the pharmacology of arsenic.

Chronic Arsenic-Exposed Human Prostate Epithelial Cells Exhibit Stable Arsenic Tolerance: Mechanistic Implications of Altered Cellular Glutathione and Glutathione S-transferase

Acquisition of stable arsenic tolerance in human cells following chronic arsenic exposure has not been previously reported. In the present work, we describe acquisition of stable arsenic tolerance in the human prostate epithelial cell line RWPE-1 following chronic arsenic exposure in vitro. RWPE-1 cells continuously exposed to 5 μM sodium arsenite for ≥18 weeks exhibited dramatic resistance to acute arsenite toxicity. The LC50 for acute arsenite exposure in these chronic arsenic-exposed prostate epithelial (CAsE-PE) cells was 43.8 μM versus 17.6 μM in control cells. Similar results were obtained using the antineoplastic agent arsenic trioxide. This tolerance was stable, as CAsE-PE cells grown in arsenic-free medium for 5 weeks retained their resistant phenotype. Compared to control cells, CAsE-PE cells showed a 90% reduction in arsenic accumulation over 24 h coupled with a 2.6-fold increase in the rate of arsenic efflux. CAsE-PE cells had increased basal GSH levels (4.9-fold) and increased GST activity (2.4-fold) and both GSH depletion and inhibition of GST activity abolished arsenic tolerance. Arsenic tolerance was also abolished by treatment with inhibitors of the Mdr1 and Mrp1 transporters, although no increases in mdr1 or mrp1 gene expression were observed. Our results indicate that this tolerance in human cells involves increases in GSH levels and GST activity that allow for more efficient arsenic efflux by MRP1 and MDR1. This study represents the first report of stable acquired arsenic tolerance in human cells, which could have important implications for both the toxicology and the pharmacology of arsenic.

Characterization and Safener Induction of Multiple Glutathione S-Transferases in Three Genetic Lines of Rice

Multiple glutathione S-transferase (GST; EC 2.5.1.18) activity peaks were characterized by fast protein liquid chromatography (FPLC) in three genetic lines of rice (Oryza sativa L.). Root-applied pretilachlor (0.15 μM) inhibited root growth of 5-day-old etiolated seedlings by 67% in Teqing (indica type), 54% in Koshihikari (japonica type), and 34% in Lemont (indica x japonica type). The safener fenclorim (1 μM) prevented the injury caused by pretilachlor in all genotypes. GST activity from 5-day-old untreated or fenclorim-treated seedlings of the three genetic lines was assayed using 1-chloro-2,4-dinitrobenzene (CDNB), radiolabeled pretilachlor, and radiolabeled fenclorim as substrates. GST activity with each substrate was greater in roots than in shoots of the three genotypes. Treatment with fenclorim increased GST activity in roots and shoots by 20 to 90%. GST activity in all genotypes was greater with CDNB, followed by pretilachlor, and then fenclorim. GST(CDNB) and GST(pretilachlor) activities were greater in Lemont, followed by Koshihikari, and then Teqing. In contrast, GST(fenclorim) activity was greater in Koshihikari and least in Lemont. FPLC elution patterns (MonoQ column) of partially purified GST extracts from untreated and fenclorim-treated seedlings of the three rice genotypes revealed the presence of multiple GST activities. Teqing shoots had six GST activity peaks with fenclorim. Four of them were also active with CDNB and pretilachlor. Koshihikari shoots had six GST activity peaks with pretilachlor. Five of them were also active with fenclorim, but only three were active with CDNB. Lemont shoots exhibited three GST activity peaks with fenclorim, five with CDNB, and seven with pretilachlor. Multiple GST activity peaks using CDNB and pretilachlor as substrates were also present in the roots of Teqing, Koshihikari, and Lemont seedlings. Fenclorim increased the activity of GST peaks conjugating pretilachlor and CDNB more than those active with fenclorim. Induction of GST peaks was more evident in Lemont, followed by Koshihikari, and then Teqing. The differential patterns of GST peaks active toward pretilachlor show that rice japonica lines may be more tolerant to pretilachlor than indica lines.

Dietary Fat and Garlic Oil Independently Regulate Hepatic Cytochrome P450 2B1 and the Placental Form of Glutathione S-Transferase Expression in Rats

The individual and combined effects of dietary fat and garlic oil on two drug-metabolizing enzymes, cytochrome P(450) 2B1 and the placental form of glutathione (GSH) S-transferase (PGST), in rat liver were examined in this study. Rats were fed a low corn oil, high corn oil or high fish oil diet and received various amount of garlic oil (0, 30, 80, 200 mg/kg body) orally three times per week for 6 wk. The fat energy in the low and high fat diets accounted for 11.6 and 45.7% of total energy, respectively. Final body weights did not differ among the three dietary fat groups and were not affected by garlic oil treatment. The fatty acid profile in hepatic phospholipids revealed higher eicosapentaenoic acid [20:5(n-3)] and docosahexaenoic acid [22:6(n-3)] levels in the fish oil-fed group than in the low and high corn oil-fed groups (P < 0.05). In contrast, the corn oil-fed groups had greater hepatic phospholipid arachidonic acid [20:4(n-6)] levels (P < 0.05). Both dietary fat and garlic oil significantly affected hepatic cytochrome 7-pentoxyresorufin O-dealkylase (PROD) activity and GST activity toward ethacrynic acid. Rats fed the high fish oil diet had 85 and 51% higher PROD activity compared with those fed the low or the high corn oil diet, respectively (P < 0.05). The GST activity in the high fish oil and the high corn oil groups was 33 and 18% higher than that in the low corn oil group (P < 0.05), respectively, and the GST activity in rats fed the high fish oil diet was higher than in those fed the high corn oil diet (P < 0.05). Garlic oil dose-dependently increased GST activity. No interaction between dietary fat and garlic oil on PROD or GST activity was noted. Northern and Western blot analysis revealed that dietary fish oil increased both cytochrome P(450) 2B1 and PGST mRNA and protein levels. Cytochrome P(450) 2B1 and PGST mRNA and protein levels were also dose-dependently increased by garlic oil treatment. The effects of garlic oil and dietary fat on P(450) 2B1 and PGST mRNA and protein expression were independent. These results indicate that dietary fat and garlic oil independently modulate P(450) 2B1 and PGST expression at transcriptional and/or post-transcriptional stages.

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.