Glutathione-dependent enzymes alone can produce paraquat resistance

HL60 cells exposed to increasing paraquat concentrations were screened for clones without increased superoxide dismutase activities in an effort to examine cytotoxic events occurring after superoxide production. The resulting resistance to paraquat was not associated with alterations in paraquat uptake, catalase, or NADPH-P450 reductase activity, but to alterations in glutathione-dependent enzyme activities. While increases in glutathione-dependent enzymes upon exposure to paraquat have been reported, the increases were considered a secondary response to increases in superoxide dismutase activities. Our results demonstrate that glutathione-dependent enzymes alone provide protection against paraquat toxicity, and their increase upon exposure to paraquat can be independent of the response of superoxide dismutases. This supports a previous finding that cells resistant to Adriamycin, based on elevated glutathione peroxidase and transferase activities are also cross-resistant to paraquat. Unlike this previous report, the increase in glutathione peroxidase was not a persistent genetic event, as activities returned to normal upon removal of paraquat. An isolated increase in glutathione peroxidase without accompanying increases in superoxide dismutases was a rare event, as nearly all clones examined after exposure to paraquat had increased superoxide dismutase.     

Oxidative stress response of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> to hydrogen peroxide,paraquat and pressure

The aim of this work was to study the oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide (50 mM), paraquat (1 mM), an increase in air pressure (120 kPa, 600 kPa) and pure oxygen pressure (120-600 kPa) in a pressurized bioreactor. The effect of these oxidants on metabolism and on the induction of antioxidant enzymes was investigated. The exposure for 1 h of K. marxianus at exponential growth phase with either H(2)O(2) or paraquat, under air pressure of 120 kPa or 600 kPa, induced an increase in both superoxide dismutase (SOD) and glutathione reductase (GR) content. SOD induction by the chemical oxidants was independent of the air pressure values used. A 2-fold increase in SOD activity was observed after 1 h of exposure to H(2)O(2) and a 3-fold increase was obtained by the presence of paraquat, with both air pressures studied. In contrast, GR activity was raised 1.7-fold by the exposure to both chemicals with 120 kPa, but a 2.4-fold GR induction was obtained with 600 kPa. As opposed to Saccharomyces cerevisiae, catalase was not induced and was even lower than the normal basal levels. This antioxidant enzyme seemed to be inhibited under increasing oxygen partial pressure. The cells showed a significant increase in SOD and GR activity levels, 4.7-fold and 4.4-fold, when exposed for 24 h to 120 kPa pure oxygen pressure. This behaviour was even more patent with 400 kPa. However, whenever cells were previously exposed to low air pressures, low enzymatic activity levels were measured after subsequent exposure to pure oxygen pressure.     

Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase

Diquat and paraquat are nonspecific defoliants that induce toxicity in many organs including the lung, liver, kidney, and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In this study, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese hamster ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild-type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective at generating ROS compared to paraquat (K(M)=1.0 and 44.2μM, respectively, for H(2)O(2) generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5μM, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (V(max)≈6.0nmol H(2)O(2)/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than in CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than in CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity.     

Resistance of paraquat and adriamycin in human breast tumor cells: role of free radical formation

Generation and enhanced detoxification of toxic free radicals by glutathione peroxidase and glutathione transferase in human breast tumor cells have been suggested to play an important role in toxicity and in resistance to adriamycin. We have examined the biochemical basis of paraquat-induced free radical formation and the mechanism of resistance to this agent in human breast tumor cell lines. We have also compared the similarities and differences between adriamycin and paraquat in their mode of free radical formation and tumor cell kill. Anaerobic incubation of paraquat resulted in the formation of the paraquat cation radical in both the sensitive and resistant cells which increased with time and was enhanced by NADPH addition. Our studies show that while both adriamycin and paraquat form hydroxyl radicals (.OH) in these cell lines, adriamycin was 2-3 fold better at reducing oxygen. The formation of .OH was inhibited by exogenously added superoxide dismutase and catalase, indicating the involvement of both superoxide anion radical and hydrogen peroxide. In the adriamycin-resistant cell line, less .OH was formed by each of these drugs. While the .OH appeared to be formed outside by both adriamycin and paraquat in the drug-sensitive cells, experiments using chromium oxalate as a spin-broadening agent suggest that the drug-induced .OH formation in the resistant cells is an intracellular event. The adriamycin-resistant cell line was also cross-resistant to paraquat, suggesting a common mechanism of toxicity for both drugs. However, adriamycin was significantly more toxic (4000-times) to the sensitive cells suggesting that either other mechanisms or site-specific free radical formation are also important in biochemical mechanisms of adriamycin toxicity.     

Paraquat-generated oxidative stress in rat liver induces heme oxygenase-1 and aminolevulinic acid synthase

The in vivo effect of the known herbicide, paraquat, on both hepatic oxidative stress and heme metabolism was studied. A marked increase in lipid peroxidation and a decrease in reduced glutathione (GSH) content were observed 1 h after paraquat administration. The activity of liver antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase was decreased 3 h after paraquat injection. Heme oxygenase-1 induction started 9 h after treatment, peaking at 15 h. delta-aminolevulinic acid synthase induction occurred once heme oxygenase had been enhanced, reaching its maximum (1.5-fold of control) at 16 h. delta-aminolevulinic acid dehydratase activity was 40% inhibited at 3 h showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of alpha-tocopherol (35 mmol/kg body weight) 2 h before paraquat treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels as well as heme oxygenase-1 and delta-aminolevulinic acid synthase induction. This study shows that oxidative stress produced by paraquat leads to an increase in delta-aminolevulinic acid synthase and heme oxygenase-1 activities, indicating that the herbicide affects both heme biosynthesis and degradation.     

Prion protein protects against DNA damage induced by paraquat in cultured cells

Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.     

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).     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.     

Relationship of resistance to oxygen free radicals to CuZn-superoxide dismutase activity in transgenic, transfected, and trisomic cells.

Although CuZn-superoxide dismutase (CuZnSOD) has been shown to reduce oxidative damage in several systems, the quantitative relationship between the degree of protection and CuZnSOD activity has not been well investigated. Therefore, the ability of cells to tolerate superoxide toxicity was assessed as a function of endogenous CuZnSOD activity in several mouse and human cell lines with progressively higher levels of CuZnSOD activity. In five lines of fetal fibroblasts derived from SOD1-transgenic mice, with CuZnSOD activities of 1.7- to 7.1-fold the nontransgenic level and no changes in the cellular glutathione peroxidase (GSHPx) activity, a direct relationship (r = 0.97) between the LD50 to paraquat and enzyme activity was observed, suggesting that CuZnSOD activity is the single most important factor in determining the paraquat LD50. Mouse trisomy 16 fetal fibroblasts and human trisomy 21 lung fibroblasts, both expressing a 1.5-fold increase in CuZnSOD activity, were 1.5-fold more tolerant to paraquat than were their diploid counterparts. Furthermore, the protective effect of CuZnSOD at the DNA level, as shown by reduced thymine glycol generation, was demonstrated in paraquat-treated transgenic fibroblasts. A direct relationship (r = 0.78) of paraquat LD50 and CuZnSOD activity was also observed with a panel of six lines of SOD1- transfected HeLa cells with 1.6- to 7.3-fold the basal CuZnSOD activity. Moreover, there was no correlation between resistance to paraquat toxicity and the cellular GSHPx and/or catalase activity. Taken together, these results demonstrate a consistently protective effect of endogenous CuZnSOD against superoxide toxicity in both primary and transformed cell lines.     

The formation of mixed disulphides in rat lung following paraquat administration Correlation with changes in intermediary metabolism

We have investigated the hypothesis that the formation of mixed disulphides between protein sulphydryl and glutathione may be responsible for controlling the activity of the pentose phosphate pathway and fatty acid synthesis in rat lung. Using lung slices, taken from rats 2 h after dosing with a range of concentrations (5–80 mg/kg) of the pulmonary toxin paraquat, the pentose phosphate pathway was found to be stimulated in direct proportion to a reduction in fatty acid synthesis. These effects were also linearly related to an increase in mixed (total) disulphide levels in the lung. This was quantitatively similar to an increase in mixed (glutathione) disulphides, although non-protein sulphydryl and oxidised levels remained normal. Thus, an early biochemical event in the mechanism of paraquat toxicity in the lung involves an increased formation of mixed (glutathione) disulphides and simulatneous regulation of pentose phosphate pathway activity and fatty acid synthesis. These data support the concept that the formation of mixed disulphides of protein and glutathione is a mechanism for maintaining NADPH levels despite the ‘redox’ stress caused by the cyclical and NADPH dependent reduction and reoxidation of paraquat.     

Oxidative stress responses in transgenic tobacco containing altered levels of glutathione reductase activity

A pea glutathione reductase cDNA was expressed in tobacco. Three classes of construct were used which gave a range of elevated levels of glutathione reductase (GR) activity in the cytosol (GR32), chloroplasts (GR36), or in both chloroplasts and mitochondria (GR46). In some transgenic progeny (T₂) from self-fertilized GR32 and GR36 primary transformants, having approximately twofold elevation of GR activity as compared with recessive siblings, there was an amelioration of the effect on leaf discs of up to 15 µM paraquat. However, lines with similarly elevated levels of GR activity showed no decreased sensitivity to the herbicide. None of the GR32 and GR36 lines was less sensitive to ozone. Conversely, T₂ progeny of GR46 lines, with greater than 4.5-fold elevations of GR activity, showed no reduced sensitivity to paraquat but two out of four of these lines were less sensitive to ozone fumigation. The differential response to stress co-segregated with the presence of the transgene but there was no relationship between the degree of stress response and the level of GR activity. There was an elevation in the total glutathione pool in all lines showing increased GR activity but there was no change in the ratio of oxidized to reduced glutathione. These results demonstrate that the mechanisms of protection against ozone and paraquat are different although both can be mediated by elevated GR activity.     

Increase of Cu,Zn-superoxide dismutase activity during differentiation of human K562 cells involves activation by copper of a constantly expressed copper-deficient protein.

Cu,Zn-superoxide dismutase activity, expressed on the basis of cell number, increased by 50% during sodium butyrate-induced differentiation of human K562 erythroleukemia cells. The increased enzyme activity was found to be concomitant with constant Cu,Zn-superoxide dismutase mRNA and immunoreactive protein levels and was accompanied by a rise in intracellular copper and glutathione. Incubation of K562 cell homogenates with copper caused an increase of Cu,Zn-superoxide dismutase activity which reached the levels observed after differentiation in the presence of sodium butyrate. The same treatment led to no significant activity increase in homogenates derived from differentiated cells. Externally added ceruloplasmin increased both intracellular copper levels and Cu,Zn-superoxide dismutase activity in undifferentiated cells to a level comparable with that observed after induction of differentiation. Both increments were abolished by depletion of cell glutathione. Cu,Zn-superoxide dismutase purified from control cells had both a lower kcat and a lower copper content than the enzyme purified from differentiated cells. From these data we conclude that: 1) Cu,Zn-superoxide dismutase is present in K562 cells also under the form of a less active copper-deficient enzyme, 2) the extent of enzyme activation is regulated post-translationally by differential delivery of copper as a function of differentiation stage, and 3) glutathione is likely to play a role in delivering copper to the copper-deficient protein in intact K562 cells.     

Metabolic response to treatment with cold, paraquat, or 3-amino-1,2,4-triazole in leaves of winter wheat.

We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.     

Differential response to paraquat induced oxidative stress in two rice cultivars on antioxidants and chlorophyll a fluorescence

The responses of antioxidative system and photosystem II photochemistry of rice (Oryza sativa L.) to paraquat induced oxidative stress were investigated in a chilling-tolerant cultivar Xiangnuo no. 1, and a chilling-susceptible cultivar, IR-50. Electrolyte leakage and malondialdehyde (MDA) content of Xiangnuo no. 1 were little affected by paraquat, but they increased in IR-50. After paraquat treatment, superoxide dismutase (SOD) activity remained high in Xiangnuo no. 1, while it declined in IR-50. Activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) declined with oxidative stress in both cultivars, but Xiangnuo no. 1 had higher GR activity than IR-50. Under paraquat induced oxidative stress, ascorbic acid (AsA) and reduced glutathione (GSH) concentrations remained high in Xiangnuo no. 1, but decreased in IR-50. The results indicated that higher activities of SOD and GR and higher contents of AsA and GSH in Xiangnuo no. 1 under paraquat induced oxidative stress were associated with its tolerance to paraquat, while paraquat induced damage to IR-50 was related to decreased activities of SOD, APX and GR and contents of AsA and GSH. F _v/F _m, Φ _PSII, and qP remained high in Xiangnuo no. 1, while they decreased greatly in IR-50 under paraquat induced oxidative stress.     

Hypothermia enhances bcl-2 expression and protects against oxidative stress-induced cell death in Chinese hamster ovary cells.

Oxidative stress is one of the major causes of cellular injury. Various reactive oxygen (ROS) and nitrogen (RNS) species such as superoxide, hydroxyl radical, peroxynitrite, and nitric oxide are involved in the manifestations of different types of organ toxicity and the resultant syndromes, symptoms, or diseases. Hypothermic conditions have been reported to reduce the oxidative stress in various in vitro and in vivo studies. In the present study, we sought to determine the effect of lowered temperatures on oxidative stress-induced cell death in Chinese hamster ovary (CHO) cells. We also investigated the oxidative stress-induced alterations in the expression of anti-apoptotic protein, bcl-2, in CHO cells at lowered temperatures. CHO cells were incubated at four different temperatures of 30, 32, 35, and 37 degrees C (control temperature) from 1 to 4 d. In another set, the cells were incubated with 100 microM hydrogen peroxide (H(2)O(2)) for 30 min before harvesting at different time points. The cells were harvested at 1, 2, 3, and 4 d. Cell survival was significantly higher at 30 degrees C as compared to 37 degrees C over 4 d of incubation. In cells incubated with H(2)O(2), significantly higher cell viability was observed at lower temperatures as compared to the cells incubated at 37 degrees C. The activity of glutathione peroxidase (GSH-Px) also increased significantly at lower temperatures. Lowered temperature also provided a significant increase in the expression of anti-apoptotic protein, bcl-2 after 4 d of incubation. These data suggest that hypothermic conditions lowers the risk of oxidative stress-induced cellular damage and programmed cell death by increasing the activity of GSH-Px and by the induction in the expression of the anti-apoptotic protein, bcl-2.     

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.     

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.