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.     

Diquat-induced oxidative damage in hepatic microsomes: effects of antioxidants.

The ability of the redox cycling compound, diquat, to induce lipid peroxidation and oxidative damage was investigated using hepatic microsomes. Antioxidants, with demonstrated efficacy in physical models of oxidative stress, were examined in a diquat model. Diquat (10 microM-3 mM) induced lipid peroxidation (TBARS) in hepatic microsomes prepared from Fischer 344 rats. Diquat (1 mM) also increased protein carbonyl formation, NADPH oxidation and superoxide anion radical production (acetylated cytochrome c reduction). The novel antioxidants U-74,006F, U-78,517G and the known antioxidant, DPPD, decreased diquat-induced lipid peroxidation to levels below that of the control. These antioxidants also decreased protein carbonyl formation caused by diquat. U-74,006F and U-78,517G reduced NADPH oxidation slightly; although this inhibition was statistically significant, the biological significance is questionable. DPPD had no effect on this parameter. U-78,517G inhibited the reduction of acetylated cytochrome c slightly, whereas the other antioxidants had little effect. Thus overall, the increase in NADPH oxidation and the production of superoxide anion by redox cycling of diquat were not substantially affected by antioxidants. Neither did the test compounds show evidence of activity as iron chelators. This leads to the suggestion that antioxidants are preventing diquat-induced oxidative damage by scavenging lipid peroxyl radicals and preventing the propagation of the lipid peroxidation process.     

The role of iron in the paracetamol- and CCl4-induced lipid peroxidation and hepatotoxicity

Treatment of non-induced or phenobarbital-induced, glutathione-depleted mice with 400 mg/kg paracetamol led to a marked ethane exhalation as an index of in vivo lipid peroxidation (LPO) and to a significant elevation of liver-specific serum enzyme activities. Similar effects were seen with rats treated with 0.5 ml/kg CCl4. Pretreatment with the iron-chelating agent desferrioxamine (DFO) clearly suppressed lipid peroxidation in all cases, but inhibited only the CCl4-induced hepatotoxicity. Treatment of mice with desferrioxamine alone showed no hepatotoxicity at all, nor did it influence liver GSH-levels. In addition, DFO had no effect on hepatic microsomal enzyme activities responsible for the bioactivation of both paracetamol and CCl4. These findings are consistent with the theories which indicate that lipid peroxidation requires the presence of Fe2+-ions, regardless of the initiating agent, and that LPO is involved in CCl4-toxicity, but most probably not in paracetamol-induced liver damage. Furthermore, Fe2+-ions might play a role as mediators of CCl4-hepatotoxicity.     

NADPH- and NADH-dependent oxygen radical generation by rat liver nuclei in the presence of redox cycling agents and iron

Redox cycling agents such as paraquat and menadione increase the generation of reactive oxygen species in biological systems. The ability of NADPH and NADH to catalyze the generation of oxygen radicals from the metabolism of these redox cycling agents by rat liver nuclei was determined. The oxidation of hydroxyl radical scavenging agents by the nuclei was increased in the presence of menadione or paraquat, especially with NADPH as the reductant. Paraquat, even at high concentrations, was relatively ineffective with NADH. The highest rates of generation of .OH-like species occurred with ferric-EDTA as the iron catalyst. Certain ferric complexes such as ferric-ATP, ferric-citrate, or ferric ammonium sulfate, which were ineffective catalysts for .OH generation in the absence of paraquat or menadione, were reactive in the presence of the redox cycling agents. Oxidation of .OH scavengers was sensitive to catalase and competitive .OH-scavenging agents under all conditions. The redox cycling agents increased NADPH-dependent nuclear generation of H2O2; stimulation of H2O2 production may play a role in the increase in .OH generation by menadione and paraquat. Menadione inhibited nuclear lipid peroxidation, whereas paraquat and adriamycin were stimulatory. The nuclear lipid peroxidation with either NADPH or NADH plus the redox cycling agents was not sensitive to catalase or .OH scavengers. These results indicate that the interaction of rat liver nuclei with redox cycling agents and iron leads to the production of potent oxidants which initiate lipid peroxidation or oxidize .OH scavengers. Although NADPH is more effective, NADH can also participate in catalyzing the production of reactive oxygen intermediates from the interaction of quinone redox cycling agents with nuclei. The ability of redox cycling agents to interact with various ferric complexes to catalyze nuclear generation of potent oxidizing species with either NADPH or NADH as reductants may contribute to the oxidative stress, toxicity, and mutagenicity of these agents in biological systems.     

Effects of Oxidative Stress Caused by Hyperoxia and Diquat. A Study in Isolated Hepatocytes

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated.

The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not ' to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyI)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below ≈ 6 nmol/10⁶ cells.

The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant a-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxy! radical scavenger α-keto-γ-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation.

The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase – glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H₂O₂ and lipid hydroperoxides.     

Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: involvement of hydrogen peroxide and transition metals

Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O2-.), hydrogen peroxide (H2O2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H2O2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of O2-. remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that O2-. is unlikely to significantly contribute to the reduction of Fe3+ to Fe2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H2O2/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.     

Inhibition of ethanol induced ethane exhalation by carcinogenic pretreatment of rats 12 months earlier

The exhalation of ethane was used as a measure of in vivo lipid peroxidation in rats treated with the hepatocarcinogen diethylnitrosamine followed by 2-acetylaminofluorene (AAF), and carbon tetrachloride, and subsequently fed a liquid diet containing 7% ethanol for 12 months. The other groups consisted of animals treated with methylbenzylnitrosamine (MBN), an esophageal carcinogen, or non-carcinogen pretreated animals with or without 7% ethanol feeding. Ethane production was increased in rats consuming ethanol irrespective of their pretreatment with MBN. In sharp contrast, the ethanol-induced increase of ethane production was absent in rats given the hepatocarcinogenic regime. Our results strengthen recent observations indicating decreased susceptibility of tumor cells to lipid peroxidation. In addition, they confirm the debated concept that there is increased lipid peroxidation following ethanol consumption.     

Bacillus subtilis paraquat resistance is directed by sigmaM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC

A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function sigma(M) protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate sigma(M)-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.     

A new potent inhibitor of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione

The drug anisyldithiolthione (ADT) acted as a good inhibitor of lipid peroxidation induced in rat liver microsomes either chemically by FeSO4 and reducing agents (cysteine or ascorbate) or enzymatically by NADPH and CC14. ADT was found as potent as propylgallate with IC50 around 2 microM and much more potent than vitamin E and levamisole. ADT was also found as a good inhibitor of ethane exhalation by rats treated by CCI4 (ID50 approximately 5mg per kg) and by mice intoxicated by acetaminophen (ID50 approximately 0.7 mg per kg). At doses as low as 5 mg per kg it completely suppressed ethane exhalation by acetaminophen-intoxicated mice and also protected them very efficiently against mortality caused by acetaminophen overdose. The inhibitory effect of ADT toward lipid peroxidation seems to be linked to the presence of its dithiolthione function.     

Lipid peroxidation and tissue injury by ferric citrate in paraquat-intoxicated mice

To determine whether iron toxicity is caused by iron-catalyzed radical production, the in vivo effect of ferric citrate was studied in paraquat-intoxicated mice. Intraperitoneally injected Fe3+-citrate complex was distributed mainly in the liver and kidney, and promoted lipid peroxidation, as measured by expiratory ethane in both normal and paraquat-intoxicated mice. Plasma glutamic-oxaloacetic transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) activity increased significantly only in paraquat and Fe3+-citrate-injected mice (PFe group). The rate of ethane production increased prior to the elevation of plasma glutamic-oxaloacetic transaminase levels, and was greater in the PFe group than in the mice, that were injected Fe3+-citrate alone. Pretreatment of animals with desferrioxamine mesylate inhibited both ethane production and elevation of plasma glutamic-oxaloacetic transaminase levels in the PFe group. Administration of 100% oxygen or glucose, which is expected to increase cellular NADPH, to the PFe group further elevated the plasma glutamic-oxaloacetic transaminase level, but had little effect on ethane production, indicating that tissue injury occurs independently of lipid peroxidation. These results suggest that iron toxicity is due to radical production and that, although iron stimulated lipid peroxidation, it might not be the only cause of tissue injury.     

Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side

There was reason from bacterial and algal systems to expect that pretreatments with a paraquat analog might confer tolerance against a subsequent paraquat treatment. Thus, a series of compounds were tested for protective activity against bipyridinium herbicides. These included other bipyridinium compounds and derivatives, as well as compounds having similar or more positive redox potentials than paraquat and compounds known to increase or maintain high superoxide dismutase activity levels in plants.

Only treatments with benzyl viologen, a benzyl analog of paraquat, protected Spirodela oligorrhiza (Kurz) Hegelm. colonies from otherwise damaging levels of diquat.

NADP photoreduction by isolated thylakoids was inhibited by the same concentrations of paraquat, diquat, and benzyl viologen given separately. Thus, the benzyl viologen-mediated tolerance against the bipyridinium herbicides is probably not due to a direct interaction at the thylakoid level.

Superoxide dismutase activity was about 50% higher in broken plastids of benzyl viologen-treated plants compared to controls, which may partly explain the observed tolerance.

     

Microsomal reduction of low-molecular-weight Fe3+ chelates and ferritin: enhancement by adriamycin, paraquat, menadione, and anthraquinone 2-sulfonate and inhibition by oxygen

Reduction of iron is important in promoting xenobiotic-enhanced, microsomal lipid peroxidation, yet there is little evidence that Fe3+ chelates that promote lipid peroxidation can be reduced by the microsomal system. We have shown that rat liver microsomes catalyse NADPH-dependent reduction of Fe3+ without chelator, as well as Fe3+(ADP), Fe3+(ATP), Fe3+(citrate), Fe3+(EDTA), and ferrioxamine in N2. The NADPH oxidation that accompanied Fe3+ reduction was inhibited by CO for all chelates, except Fe3+ (EDTA). This implies that, except for Fe3+ (EDTA), cytochrome P450 was involved in reduction of the complexes. Adriamycin, paraquat, and anthraquinone 2-sulfonate (AQS) enhanced reduction of all the Fe3+ chelates, whereas menadione enhanced reduction only of Fe3+(ADP) and Fe3+(citrate). All the compounds enhanced oxidation of NADPH in the presence or absence of iron. This was not inhibited by CO, and the results are compatible with Fe3+ reduction occurring via the xenobiotic radicals produced by cytochrome P450 reductase. Microsomal reduction of the xenobiotics, except menadione, enabled the reduction and release of iron from ferritin. Fe3+ chelate reduction, both with and without xenobiotic, was inhibited by O2, although it still proceeded in air at 10-20% of the rate in N2. Iron-dependent lipid peroxidation was promoted by ADP and ATP, inhibited 50% by citrate, and completely inhibited by EDTA and desferrioxamine. Of the xenobiotics, only Adriamycin enhanced microsomal lipid peroxidation. These results indicate that the effects of chelators and xenobiotics on Fe3+ reduction do not correlate with lipid peroxidation and, although reduction is necessary, there must be other factors involved.     

In vivo oxidation of reduced nicotinamide-adenine dinucleotide phosphate by paraquat and diquat in rat lung.

Intravenous injection of rats with 156 mumol/kg of paraquat or 140 mumol/kg of diquat produced, within 60 min, a sharp drop in the ratios of NADPH to NADP in lung. The effect persisted for a time period of at least 24 h. Exposure to 100% oxygen enhanced the toxicity of both compounds without substantially amplifying changes in the NADPH/NADP ratio. Lungs retained the capability to synthesize adenine nucleotides de novo. Electron microscopic studies showed that both paraquat and diquat damage type I alveolar cells, but only paraquat produces type II cell lesions. Although bipyridylium herbicides produce acute oxidation of NADPH in vivo, there seems not to exist a straightforward relationship between this event and cell damage.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

Glutathione depletion in a liver microsomal assay as an in vitro biomarker for reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleophile trap for reactive species derived from xenobiotics. This has led to the development of an assay for the detection of reactive species generated by liver microsomal metabolism of xenobiotics. This assay has been used extensively to study reactive metabolites which initiate toxicity through a direct (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen formation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displayed GSH reactivity that was either dependent or independent (amodiaquine) on metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquinone, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoin and diquat, two other redox cycling xenobiotics. Induction of the mixed function oxidase system with Aroclor afforded little advantage when using rat liver microsomes, whilst there was considerable inter-individual variation in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived reactive species.     

Glutathione depletion in a liver microsomal assay as an in vitro biomarker for reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleophile trap for reactive species derived from xenobiotics. This has led to the development of an assay for the detection of reactive species generated by liver microsomal metabolism of xenobiotics. This assay has been used extensively to study reactive metabolites which initiate toxicity through a direct (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen formation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displayed GSH reactivity that was either dependent or independent (amodiaquine) on metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquinone, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoin and diquat, two other redox cycling xenobiotics. Induction of the mixed function oxidase system with Aroclor afforded little advantage when using rat liver microsomes, whilst there was considerable inter-individual variation in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived reactive species.     

Effect of BCNU pretreatment on diquat-induced oxidant stress and hepatotoxicity

Diquat administration produces hepatic necrosis in male Fischer-344 rats, and minimally in male Sprague-Dawley rats, with massive oxidant stress observable in both strains as evidenced by increased biliary efflux of glutathione disulfide (GSSG). Pretreatment of both strains of rats with 80 mg/kg of 1,3-bis(2-chloroethyl)-N-nitrosourea (BCNU) inhibited hepatic glutathione reductase by 75 percent and increased dramatically the biliary efflux of GSSG produced by administration of diquat. BCNU pretreatment markedly potentiated diquat hepatotoxicity in the Fischer rats and modestly in Sprague-Dawley rats. BCNU-pretreated Fischer rats did not show an enhanced depletion of nonprotein sulfhydryls in response to diquat, in spite of the dramatic potentiation of the hepatic necrosis produced, nor were protein thiols depleted. The effects of BCNU on diquat hepatotoxicity in the Fischer rat are consistent with a critical role for reactive oxygen species in the pathogenesis of the observed hepatic necrosis and for the protective role of the glutathione peroxidase/reductase system. The data suggest that shifts in thiol-disulfide equilibria are not responsible for the cell death produced by oxidant stress in vivo, but are consistent with a role for lipid peroxidation in the pathogenesis of the lesion.     

Lipid Peroxidation Induced ByN-Nitrosodimethylamine (Ndma) In Rats In Vivo and in Isolated Hepatocytes

To investigate the role of carcinogenic chemicals as a possible cause for oxidative damage, rats were treated with jV-nitrosodimethylamine (NDMA) and various measures of lipid peroxidation were followed. As an indication of enhanced peroxidative processes in vivo NMDA treatment produced rapidly an increase in the rate of ethane exhalation. A single i.p. or p.o. injection of lOmg/kg b.w. elevated ethane exhalation by 13-14 fold; a single dose of 0.5mg/kg of NDMA (the smallest dose tested) increased 5-fold the amount of ethane exhaled. Similarly, lipid peroxidation in the liver of NDMA-treated rats (measured by diene conjugation, chemiluminescence, the production of fluorescent and TBA-reactive material) was found to be increased rapidly showing a peak already 20min after dosing. Simultaneously, NDMA-treatment slightly decreased antioxidant enzyme activities and GSH contents in the liver. In isolated rat hepatocytes the lucigenin-dependent chemiluminescence, as well as H,0₂ release, were increased by micromolar concentrations of NDMA. Finally, it was shown that the rate of NADPH-stimulated ethane production by hepatic microsomes, prepared from untreated rats, was increased in the presence of NDMA. Thus, our results demonstrate that the alkylating NDMA can induce oxidative stress in rodents. Whether the same is true for other classes of carcinogens and processes known to affect tumor initiation/progression is presently under investigation.