Alteration of endogenous glutathione peroxidase, manganese superoxide dismutase, and glutathione transferase activity in cells transfected with a copper-zinc superoxide dismutase expression vector. Explanation for variations in paraquat resistance

Transfection of a human pSV2 (copper-zinc) superoxide dismutase expression vector into murine fibroblasts resulted in stable clones producing increased amounts of copper-zinc superoxide dismutase. A marked increase in endogenous glutathione peroxidase activity (up to 285%) and a smaller increase in glutathione transferase activity (up to 16%) also occurred. Manganese superoxide dismutase activity was decreased in all clones, whereas catalase and NADPH reductase activities were not affected. Alterations in glutathione peroxidase and manganese superoxide dismutase activities correlated with increases in copper-zinc superoxide dismutase activity. Whereas all clones were resistant to paraquat, a direct correlation between copper-zinc superoxide dismutase activity and resistance to paraquat did not exist. In agreement with previous reports clones expressing the highest copper-zinc superoxide dismutase activity did not display the highest resistance to paraquat. However, there was a direct correlation between the increase in glutathione peroxidase activity and paraquat resistance (p less than 0.002).     

The effect of paraquat on the peroxide metabolism enzymes in erythrocytes of freshwater fish species

The toxic effects of 10 ppm paraquat in vivo on the enzymes superoxide dismutase (SOD), catalase (C), peroxidase (P), glutathione peroxidase (GSH-Px) and on lipid peroxidation (LP) were estimated in erythrocytes of the carp, the tench and the crucian carp. Paraquat caused activity enhancement of the peroxide metabolism enzymes and increase of the lipid peroxidation in the carp and the crucian carp. The enzyme activities and lipid peroxidation were dependent on the species and on the length of the exposure to paraquat.     

A high-throughput reporter gene assay to prove the ability of natural compounds to modulate glutathione peroxidase,superoxide dismutase and catalase gene promoters in V79 cells

The aim of the study was to establish a 96-well microtiter plate-based reporter gene assay to test the influence of natural compounds on the promoter activities of rat catalase, human glutathione peroxidase and human superoxide dismutase expressed in V79 cells. Luciferase expression vectors with the promoter regions of the genes coding for the three above-mentioned enzymes were constructed and transfected into V79 cells. Thereafter the ability of sodium ascorbate, L-carnitine, catechin, epigallocatechin gallate, genistein, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate and Trolox to enhance the promoter activities was evaluated. Genistein, paraquat and quercetin led to a statistically significant increase in the glutathione peroxidase and superoxide dismutase gene promoter activities. None of the compounds tested enhanced the catalase gene promoter activity. The reporter gene assay described in this report is easy to perform, fast and allows one to test a high number of compounds and different concentrations of a single compound at the same time.     

A high-throughput reporter gene assay to prove the ability of natural compounds to modulate glutathione peroxidase, superoxide dismutase and catalase gene promoters in V79 cells

The aim of the study was to establish a 96-well microtiter plate-based reporter gene assay to test the influence of natural compounds on the promoter activities of rat catalase, human glutathione peroxidase and human superoxide dismutase expressed in V79 cells. Luciferase expression vectors with the promoter regions of the genes coding for the three above-mentioned enzymes were constructed and transfected into V79 cells. Thereafter the ability of sodium ascorbate, L-carnitine, catechin, epigallocatechin gallate, genistein, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate and Trolox to enhance the promoter activities was evaluated. Genistein, paraquat and quercetin led to a statistically significant increase in the glutathione peroxidase and superoxide dismutase gene promoter activities. None of the compounds tested enhanced the catalase gene promoter activity. The reporter gene assay described in this report is easy to perform, fast and allows one to test a high number of compounds and different concentrations of a single compound at the same time.     

Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury

The effect of ischemia-reperfusion on activity, protein and m-RNA levels of catalase, copper-zinc and manganese containing superoxide dismutases and glutathione peroxidase, the enzymes that are involved in free radical detoxification was studied in rat kidney. Ischemia alone did not alter either the activities or protein levels of superoxide dismutase and glutathione peroxidase. However, catalase activity was found to be inhibited to 82% of control. The inhibition of catalase was due to the inactivation of the enzyme as there was no significant change in enzyme protein level. Reperfusion following ischemia, however, led to a significant decrease in both the activities as well as the protein levels of all the antioxidant enzymes. The observed overall decrease in total superoxide dismutase activity was the net effect of a decrease in copper-zinc superoxide dismutase while manganese superoxide dismutase activity was found to be increased following reperfusion. This observed increased manganese superoxide dismutase activity was the result of its increased protein level. The mRNA levels for catalase, superoxide dismutases, and glutathione peroxidase were observed to be increased (100–145% of controls) following ischemia; reperfusion of ischemic kidneys, however, resulted in a significant decrease in the levels of mRNAs coding for all the enzymes except manganese superoxide dismutase which remained high. These results suggest that in tissue, the down regulation of the antioxidant enzyme system could be responsible for the pathophysiology of ischemia-reperfusion injury.     

Role of Glutathione-Dependent Peroxidase in Regulation of Lipoperoxide Utilization in Malignant Tumors

Glutathione content, the activity of glutathione-dependent enzymes (glutathione reductase, glutathione peroxidase, and glutathione S-transferase), and also SOD (superoxide dismutase) and catalase were studied in human malignant tumors (uterus, breast, and ovaries) and normal tissues. Glutathione level and the activity of glutathione-dependent enzymes were 2-3 times higher in the malignant tumors than in normal tissues. A negative correlation between the level of glutathione and glutathione-dependent enzymes (glutathione peroxidase and glutathione S-transferase) in tumors and the efficacy of postoperative chemotherapy may characterize the degree of tumor resistance to chemotherapy and therefore may have prognostic value. Low SOD and catalase activity and high activity of glutathione-dependent enzymes in tumors suggest that glutathione peroxidase and glutathione S-transferase play a major role in peroxide utilization in malignant tumors.     

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.     

Expression of antioxidant enzymes in rat lungs after inhalation of asbestos or silica.

Several studies indicate that active oxygen species play an important role in the development of pulmonary disease (asbestosis and silicosis) after exposure to mineral dust. The present study was conducted to determine if inhaled fibrogenic minerals induced changes in gene expression and activities of antioxidant enzymes (AOE) in rat lung. Two different fibrogenic minerals were compared, crocidolite, an amphibole asbestos fiber, and cristobalite, a crystalline silicon dioxide particle. Steady-state mRNA levels, immunoreactive protein, and activities of selected AOE were measured in lungs 1-10 days after initiation of exposure and at 14 days after cessation of a 10-day exposure period. Exposure to asbestos resulted in significant increases in steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) at 3 and 9 days and of glutathione peroxidase at 6 and 9 days. An increase in steady-state mRNA levels of copper, zinc-containing superoxide dismutase (CuZnSOD), was observed at 6 days. Exposure to asbestos also resulted in overall increased enzyme activities of catalase, glutathione peroxidase and total superoxide dismutase in lung. In contrast, silica caused a dramatic increase in steady-state levels of MnSOD mRNA at all time periods and an increase in glutathione peroxidase mRNA levels at 9 days. Activities of AOE remained unchanged in silica-exposed lungs. In both models, increases in gene expression of MnSOD correlated with increased amounts of MnSOD immunoreactive protein in lung and the pattern and extent of inflammation. These data indicate that the profiles of AOE are dissimilar during the development of experimental asbestosis or silicosis and suggest different mechanisms of lung defense in response to these minerals.     

Paraquat-resistant cell lines derived from Chinese hamster ovary cells

Two paraquat-resistant clones, PR-1 and PR-2, were selected from CHO K1 cells pretreated with ethyl methanesulfonate. PR-1 and PR-2, routinely cultured in a normal medium without paraquat, were six fold more resistant to paraquat than the parental CHO K1 cells. There was no difference in the uptake of [3H]paraquat among PR-1, PR-2, and CHO K1 cells. Both PR-1 and PR-2 cells showed no cross resistance to free radical generating agents and no increase in total activity of superoxide dismutase. The activities of paraquat-dependent NADPH oxidase and glucose-6-phosphate dehydrogenase were significantly reduced in PR-1 and PR-2 cells, hence the rate of paraquat radical formation will be limited. In addition, an elevation of glutathione levels in PR-1 cells or an increase in glutathione S-transferase activity in PR-2 cells may also play a certain role in protective mechanisms against the toxicity of paraquat.     

Imbalance of the antioxidant network of mouse small intestinal mucosa after radiation exposure

The aim of this study was to investigate acute variations in antioxidant defense systems in the intestinal mucosa after abdominal radiation exposure and the role played by radiation-induced inflammation in these variations. Antioxidant defense systems of mouse small intestinal mucosa were studied at 6 h and 4 days after abdominal radiation exposure. Superoxide dismutases, glutathione peroxidases, catalase, metallothioneins and thioredoxins were followed in terms of mRNA expression, protein expression and enzyme activities. Dexamethasone was administered to investigate the relationship between variations in mucosal antioxidant capacity and radiation-induced inflammation. Six hours after exposure, only mitochondrial-associated antioxidant systems were induced (the superoxide dismutase and thioredoxin 2). Four days after exposure, during the inflammatory phase, superoxide dismutases were decreased and modulations of the second line of the antioxidant network were also observed: Catalase was decreased and glutathione peroxidases and metallothioneins were induced. Dexamethasone treatment modulated only glutathione peroxidase expression and did not influence either metallothionein or superoxide dismutase expression. Our findings provide direct in vivo evidence that antioxidant mechanisms of the small intestinal mucosa were not markedly mobilized during the very acute tissue radiation response. During the radiation-induced acute inflammatory response, the antioxidant capacity appeared to be dependent on inflammatory status to a certain extent.     

Assay of free-radical toxicity and antioxidant effect on the Hep 3B cell line: a test survey using lindane

The content of reduced glutathione and of glutathione disulfide as well as the activities of glutathione reductase, glutathione peroxidase, glutathione S-transferases, catalase and superoxide dismutases were determined in human hepatoma Hep 3B cells in relation to free-radical toxicity in order to appreciate the defense capacities of these cells compared to data on normal hepatocytes. When Hep 3B cells were exposed to lindane, a known inducer of free-radical production, superoxide dismutase activity appeared as the best-adapted cellular parameter for early detection of the resulting free-radical toxicity.Abbreviations AAS atomic absorption spectrometry - CDNB 1-chloro-2,4-dinitrobenzene - DMEM Dulbecco's modified Eagle medium - GPx glutathione peroxidase - G.Red glutathione reductase - GSH reduced glutathione - GSSG glutathione disulfide - GST glutathione S-transferases - Prot proteins - SOD superoxide dismutase     

Overexpression of superoxide dismutase or glutathione peroxidase protects against the paraquat + maneb-induced Parkinson disease phenotype

Oxidative stress has been implicated in the pathogenesis of Parkinson disease based on its role in the cascade of biochemical changes that lead to dopaminergic neuronal death. This study analyzed the role of oxidative stress as a mechanism of the dopaminergic neurotoxicity produced by the combined paraquat and maneb model of the Parkinson disease phenotype. Transgenic mice overexpressing either Cu,Zn superoxide dismutase or intracellular glutathione peroxidase and non-transgenic mice were exposed to saline, paraquat, or the combination of paraquat + maneb twice a week for 9 weeks. Non-transgenic mice chronically exposed to paraquat + maneb exhibited significant reductions in locomotor activity, levels of striatal dopamine and metabolites, and dopaminergic neurons in the substantia nigra pars compacta. In contrast, no corresponding effects were observed in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. Similarly, the increase in levels of lipid hydroperoxides in the midbrain and striatum of paraquat + maneb-treated non-transgenic mice was not detected in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. To begin to determine critical pathways of paraquat + maneb neurotoxicity, the functions of cell death-inducing and protective mechanisms were analyzed. Even a single injection of paraquat + maneb in the non-transgenic treated group modulated several key pro- and anti-apoptotic proteins, including Bax, Bad, Bcl-xL, and upstream stress-induced cascade. Collectively, these findings support the assertion that protective mechanisms against paraquat + maneb-induced neurodegeneration could involve modulation of the level of reactive oxygen species and alterations of the functions of specific signaling cascades.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Biochemical Studies of Paraquat-Tolerant Mutants of the Fern Ceratopteris richardii

Enzymes and metabolites associated with mitigation of paraquat toxicity were compared in two paraquat-tolerant mutants and a sensitive wild-type strain of the fern Ceratopteris richardii Brongn. In 21-day-old gametophytes, the specific activities of superoxide dismutase, catalase, peroxidase, glutathione reductase, dehydroascorbate reductase, and ascorbate peroxidase showed no differences that would explain mutant tolerance. Constitutive levels of ascorbate and glutathione also did not differ significantly in the three strains. An experiment testing the inducibility of paraquat tolerance revealed no change in the dose response of mutant or wild type gametophytes after exposure to sublethal concentrations of the herbicide. Uptake of paraquat by whole gametophytes was also equivalent in mutants and wild type. These data suggest that the physiological basis for tolerance in these mutants, unlike several other tolerant biotypes reported, does not lie in the oxygen radical scavenging system, in an inducible stress response, or in a block to whole-plant uptake.     

Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice

Andrographis paniculata (AP) treatment prevents BHC induced increase in the activities of enzymes y-Glutamyl transpeptidase, glutathione-S-transferase and lipid peroxidation. The activities of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and the levels of glutathione were decreased following BHC effect. Administration of AP showed protective effects in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase as well the level of glutathione. The activity of lipid peroxidase was also decreased. The result indicate antioxidant and hepatoprotective action of A. paniculata.     

Exogenous treatment with salicylic acid leads to increased antioxidant capacity in leaves of barley plants exposed to paraquat

Our previous study suggests that salicylic acid mediates tolerance in barley plants to paraquat (Ananieva et al. 2002). To further define the role of SA in paraquat induced responses, we analysed the capacity of the antioxidative defence system by measuring the activities of several antioxidative enzymes: superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), dehydroascorbate reductase (DHAR, EC 1.8.5.1), catalase (CAT, EC 1.11.1.6), and guaiacol peroxidase (POX, EC 1.11.1.7). Twelve-day-old barley seedlings were supplied with 500 micromol/L SA or 10 micromol/L Pq via the transpiration stream and kept in the dark for 24 h. Then they were exposed to 100 micromol m(-2) s(-1) PAR and samples were taken 6 h after the light exposure. Treatment of seedlings with 10 micromol/L Pq reduced the activity of APX and GR, did not affect the activity of POX and DHAR but caused over a 40% increase in the activity of CAT. Pre-treatment with 500 micromol/L SA for 24 h in the dark before Pq application increased the activities of the studied enzymes in both the chloroplasts (SOD activity) and the other compartments of the cell (POX, CAT activity). The effect of SA pre-treatment was highly expressed on DHAR and POX activity. The data suggest that SA antagonizes Pq effects, via elicitation of an antioxidative response in barley plants.