Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion (MPP+): reductive activation by NADPH cytochrome P-450 reductase

Formation of free radical intermediates from 1--methyl-4-phenylpyridinium ion(MPP+) has been studied using spin-trapping techniques. Incubation of MPP+ with purified NADPH cytochrome P-450 reductase and NADPH under anaerobic conditions failed to produce any detectable radical intermediates. However, in the presence of air and a spin-trap, a significant stimulation of superoxide and hydroxyl radicals was detected. Formation of these toxic radicals from MPP+ was inhibited by superoxide dismutase, catalase, and ethanol. Under identical conditions, however, considerably less of these radicals were formed with MPP+ in comparison to paraquat, a lung toxin containing two pyridinium moieties.     

Differential sensitivities of plant and animal mitochondria to the herbicide paraquat

Paraquat herbicide is toxic to animals, including humans, via putative toxicity mechanisms associated to microsomal and mitochondrial redox systems. It is also believed to act in plants by generating highly reactive oxygen free radicals from electrons of photosystem I on exposure to light. Paraquat also acts on non-chlorophyllous plant tissues, where mitochondria are candidate targets, as in animal tissues. Therefore, we compared the interaction of paraquat with the mitochondrial bioenergetics of potato tuber, using rat liver mitochondria as a reference. Paraquat depressed succinate-dependent mitochondrial Delta(psi), with simultaneous stimulation of state 4 O2 consumption. It also induced a slow time-dependent effect for respiration of succinate, exogenous NADH, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)/ascorbate, which was more pronounced in rat than in potato mitochondria. However, with potato tuber mitochondria, the Delta(psi) promoted by complex-I-dependent respiration is insensitive to this effect, indicating a protection against paraquat radical afforded by complex I redox activity, which was just the reverse of to the findings for rat liver mitochondria. The experimental set up with the tetraphenyl phosphonium (TPP+)-electrode also indicated production of the paraquat radical in mitochondria, also suggesting its accessibility to the outside space. The different activities of protective antioxidant agents can contribute to explain the different sensitivities of both kinds of mitochondria. Values of SOD activity and alpha-tocopherol detected in potato mitochondria were significantly higher than in rat mitochondria, which, in turn, revealed higher values of lipid peroxidation induced by paraquat.     

Paraquat toxicity

Paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride) is marketed as a contact herbicide. Although it has proved safe in use there have been a number of cases of poisoning after the intentional swallowing of the commercial product. The most characteristic feature of poisoning is lung damage, which causes severe anoxia and may lead to death. The specific toxicity to the lung can be explained in part by the accumulation of paraquat into the alveolar type I and type II epithelial cells by a process that has been shown to accumulate endogenous diamines and polyamines. When accumulated, paraquat undergoes an NADPH-dependent, one-electron reduction to form its free radical, which then reacts avidly with molecular oxygen to reform the cation and produce superoxide anion, which in turn will dismutate to form H2O2. This may lead to the formation of more reactive (and hence toxic) radicals which have the potential to cause lipid peroxidation and lead to cell death. Biochemical changes provoked by paraquat in the lung suggest that it causes a rapid, pronounced and prolonged oxidation of NADPH that initiates compensatory biochemical processes in the lung. NADPH may be further depleted as it is consumed in an attempt to detoxify H2O2 or lipid hydroperoxides. Thus it is possible that with toxic levels of paraquat in the cell, compensatory biochemical processes are insufficient to maintain levels of NADPH consistent either with cell survival or with the ability to detoxify H2O2 or prevent lipid peroxidation.     

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.     

Effects of the free radical generator paraquat on differentiation, superoxide dismutase, glutathione and inorganic peroxides in microplasmodia of Physarum polycephalum

The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. The responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. The addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. The white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. The results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Effects of the Free Radical Generator Paraquat On Differentiation, Superoxide Dismutase, Glutathione and Inorganic Peroxides In Microplasmodia of Physarum Polycephalum

Abstract. The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. the responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. the addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. the white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. the results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Reductive release of iron from ferritin by cation free radicals of paraquat and other bipyridyls

NADPH-cytochrome P-450 reductase-catalyzed reduction of paraquat promoted the release of iron from ferritin. Aerobically, iron release was inhibited approximately 60% by superoxide dismutase, whereas xanthine oxidase-dependent iron release was inhibited nearly 100%. This suggests that both superoxide and the paraquat cation radical can catalyze the release of iron from ferritin. Accordingly, under anaerobic conditions, the paraquat radical mediated a very rapid, complete release of iron from ferritin. Similarly, the cation free radicals of the closely related chemicals, diquat and benzyl viologen, also promoted iron release. ESR studies demonstrated that electron transfer from the paraquat cation radical to ferritin accounts for the reductive release of iron. The ferritin structure was not altered by exposure to the paraquat radical and also retained its ability to re-incorporate iron. These studies indicate that release of iron from ferritin may be a common feature contributing to free radical-mediated toxicities.     

Methylene blue competes with paraquat for reduction by flavo-enzymes resulting in decreased superoxide production in the presence of heme proteins

Methylene blue competes 100 to 600 times more effectively than paraquat for reduction by three different flavo-containing enzymes; xanthine oxidase, NADH cytochrome c reductase, and NADPH cytochrome c reductase. Paraquat and methylene blue both interact with deflavo xanthine oxidase, indicating that neither electron acceptor reacted at the FAD site of the enzyme where molecular oxygen is reduced to superoxide. As the paraquat radical also directly reduced acetylated cytochrome c the hemeprotein could not be utilized for measuring superoxide production in the presence of the herbicide. In the presence of cytochrome c the methylene blue caused a sharp decrease in both paraquat-induced superoxide and hydroxyl radical production.     

Complex I is the major site of mitochondrial superoxide production by paraquat

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. This superoxide production causes extensive mitochondrial oxidative damage, however, there is considerable uncertainty over the mitochondrial sites of paraquat reduction and superoxide formation. Here we show that in yeast and mammalian mitochondria, superoxide production by paraquat occurs in the mitochondrial matrix, as inferred from manganese superoxide dismutase-sensitive mitochondrial DNA damage, as well as from superoxide assays in isolated mitochondria, which were unaffected by exogenous superoxide dismutase. This paraquat-induced superoxide production in the mitochondrial matrix required a membrane potential that was essential for paraquat uptake into mitochondria. This uptake was of the paraquat dication, not the radical monocation, and was carrier-mediated. Experiments with disrupted mitochondria showed that once in the matrix paraquat was principally reduced by complex I (mammals) or by NADPH dehydrogenases (yeast) to form the paraquat radical cation that then reacted with oxygen to form superoxide. Together this membrane potential-dependent uptake across the mitochondrial inner membrane and the subsequent rapid reduction to the paraquat radical cation explain the toxicity of paraquat to mitochondria.     

The Role of Free Radicals in Paraquat-Induced Corneal Lesions

Paraquat is a synthetic bipyridylium salt widely used as herbicide and defoliant. Enzyme-catalyzed redox-cycling of paraquat generates oxygen radicals. The toxic, even lethal, effects of paraquat are due to free radical-mediated tissue injury. Ocular lesions, sometimes quite severe, have been observed following accidental splashing of paraquat solutions onto the eyes.

These studies were designed to document the generation of paraquat free radicals in corneal tissue, and to describe the histological nature of the corneal injuries in experimental animals (rabbits and monkeys). The EPR spectrum of rabbit corneas, 30 min. after intrastromal injection of paraquat, showed the signal of the free radical of paraquat. Ultrastructural studies of corneas 8 days after intrastromal injections (100μl) of paraquat solutions showed that the initial lesions occur at the epithelium/basement membrane interface. In rabbit cornea, dose dependent lesions were observed, i.e. whereas 50 mM paraquat caused only minimal damage to the epithelial basement membrane, 75 mM caused complete dissolution to the basement membrane with some damage to stromal collagen, and loss of epithelium with stromal ulceration and severe inflammatory response were observed with 150 mM paraquat. Monkey corneas were less susceptible than those of rabbits to the effects of paraquat. No lesions were observed following intrastromal injections of 50 mM or 75 mM paraquat. With higher concentrations of paraquat (100 mM and 150 mM) the primary injuries were to the proximal and lateral plasma membranes of basal epithelial cells; basement membrane alterations were detected only adjacent to areas of significant plasma membrane damage. The underlying Bowman's membrane and stroma were not affected. Anatomical differences between the corneas of rabbit and monkeys as well as possible biochemical differences may account for the species differences observed.     

The protective effect of desferrioxamine on paraquat-treated pea (Pisum sativum)

Paraquat, a widely used herbicide, is photoreduced by photosystem I to the monovalent cation radical, which in turn, can react quickly and efficiently with molecular oxygen to produce superoxide anion radicals. In the presence of redox-active iron (or copper) superoxide radicals can serve as a source for the more active species such as hydroxyl radicals. The present sludv investigated the possible mediatory role of iron in paraquat to xicity. The results demonstrate that desferrioxamme (0–150μM) a highiy specific iron chelator, reduces the loss of proteins (by 34–69%) and lipid peroxidation (by 31–96%) in paraquat treated leaf cuts. Dcsferrioxamine also protects malate dehydrogenase (61–70%) hydroxvpyruvate reductase (54–100%), and Ca²⁺-dependent ATPase (25–34%) against the paraquat-induced loss of their activity. It also induces an increase in glutathione reductase activity (by 188%). These results, together with those from other experiments concerning the effect of desferrioxamine on paraquat uptake by the leaf cuts, suggest that the protection by desferrioxamine arises from its specific iron chelanon properties, and lead to the conclusion that nan-protein-bound and redoxactive forms of iron pluy a role in the manifestation of paraquat toxicity in plants.     

An oxidative stress-specific bacterial cell array chip for toxicity analysis

An oxidative stress-specific bacterial cell array chip was fabricated and implemented in the analysis of various different chemicals. The chip consisted of twelve toxicity responsive strains that respond specifically to different oxidative toxicities such as the generation of the superoxide radical, except for strain EBMalK, which was included as a negative control. Each bioluminescent strain carried a fusion of a stress gene promoter (sodA, pqi-5, soxR, fumC, soxS, inaA, hmp, malK, katG, zwf, fpr or pgi) to the bacterial lux reporter genes. A total of nine chemicals were selected to exhibit the capabilities of this array when analyzing different oxidative toxicities. Each of the chemicals were categorized according to their structure and their ability to form radicals in vivo: (I) paraquat, an active radical producer, (II) structural analogs of paraquat that produce radicals, (III) chemicals that are distinct from paraquat but still produce radicals and (IV) chemicals having similar structures as paraquat but do not produce radicals. The results found that each strain was responsive to one or more of the compounds tested but, as a definitive factor, the responses from the chip were dependent upon the production of radicals, i.e., the strains were unresponsive to compounds that were similar in structure to paraquat but lacked the ability to generate radicals. The specificity of the strains used in the chip was also demonstrated by their ability to discriminate between the superoxide radical and hydrogen peroxide. Therefore, this cell array chip could be implemented in characterizing and understanding the toxic impacts of newly synthesized chemicals and drugs in terms of toxicity classification and the nature of oxidative damage experienced by cells.     

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.     

Paraquat increases cyanide-insensitive respiration in murine lung epithelial cells by activating an NAD(P)H:paraquat oxidoreductase: identification of the enzyme as thioredoxin reductase

Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity mediating this reaction in lung cells is not completely understood. Using self-referencing microsensors, we measured the effects of paraquat on oxygen flux into murine lung epithelial cells. Paraquat (10-100 microm) was found to cause a 2-4-fold increase in cellular oxygen flux. The mitochondrial poisons cyanide, rotenone, and antimycin A prevented mitochondrial- but not paraquat-mediated oxygen flux into cells. In contrast, diphenyleneiodonium (10 microm), an NADPH oxidase inhibitor, blocked the effects of paraquat without altering mitochondrial respiration. NADPH oxidases, enzymes that are highly expressed in lung epithelial cells, utilize molecular oxygen to generate superoxide anion. We discovered that lung epithelial cells possess a distinct cytoplasmic diphenyleneiodonium-sensitive NAD(P)H:paraquat oxidoreductase. This enzyme utilizes oxygen, requires NADH or NADPH, and readily generates the reduced paraquat radical. Purification and sequence analysis identified this enzyme activity as thioredoxin reductase. Purified paraquat reductase from the cells contained thioredoxin reductase activity, and purified rat liver thioredoxin reductase or recombinant enzyme possessed paraquat reductase activity. Reactive oxygen intermediates and subsequent oxidative stress generated from this enzyme are likely to contribute to paraquat-induced lung toxicity.     

Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid

Low density lipoprotein (LDL) has been reported to be injurious or toxic to cells in vitro. This injurious effect is, in some instances, due to oxidation of the lipid moiety of the lipoprotein. The objectives of this study were to determine if the oxidation rendering the lipoprotein toxic to human skin fibroblasts occurred by free radical mechanisms, and if so, which of the common free radical oxygen species were involved. The selective free radical blockers or scavengers employed included superoxide dismutase for superoxide, catalase for hydrogen peroxide, dimethylfuran for singlet molecular oxygen, and mannitol for hydroxyl radical. The presence during lipoprotein preparation of general free radical scavengers (vitamin E, butylated hydroxytoluene) or the divalent cation chelator ethylenediamine tetraacetic acid prevented the formation of cytotoxic low density lipoprotein, while the simultaneous presence of superoxide dismutase and catalase partially inhibited its formation. The results indicate that superoxide and/or hydrogen peroxide are involved in the formation of the toxic LDL lipid. The toxic action of oxidized LDL could not be prevented by inclusion of antioxidants in the culture medium, indicating that an oxidized lipid was responsible for cell injury rather than free radicals generated in culture by the action of oxidized LDL. Three separate assays for cell injury (enumeration of attached cells, cell loss of lactate dehydrogenase into the culture medium, and trypan blue uptake) indicated a sequence of events in which the fibroblasts are injured, die, and then detach.     

Mutagenicity of active oxygen species in bacteria and its enzymatic or chemical inhibition

The mono-electronic reduction of oxygen in the hypoxanthine-xanthine oxidase system led to the formation of active species eliciting an evident and highly reproducible mutagenic response in strain TA104 of S. typhimurium. Similar effects were observed by generating oxy radicals either extracellularly or inside bacterial cells. Mutagenicity was selectively detected in TA104 and not in other Salmonella strains, which points out the importance of the hisG428 mutation and of the deletion excising the uvrB gene, as far as sensitivity to oxy radicals is concerned. The mutagenicity of the system was further enhanced in the presence of superoxide dismutase. Catalase did not affect the mutagenicity of hypoxanthine plus xanthine oxidase, whereas it inhibited the mutagenicity induced by the mixture of hypoxanthine with xanthine oxidase and superoxide dismutase. This demonstrates that not only hydrogen peroxide but also the superoxide radical anion is positive in this system. Glutathione and 2 synthetic thiols, i.e., N-acetylcysteine and alpha-mercaptopropionylglycine, besides decreasing the high spontaneous mutagenicity of TA104, efficiently prevented the mutagenicity of active oxygen species.     

Cancer chemopreventive oltipraz generates superoxide anion radical

The cancer chemopreventive actions of oltipraz, a member of a class of 1,2-dithiolethiones, have been primarily associated with the induction of phase 2 enzymes mediated by a 41bp enhancer element known as the anti-oxidant response element in the promoter regions of many phase 2 genes. It has been suggested that oxygen radical formation by oltipraz may be a critical mechanism by which it exerts chemoprevention. Therefore, in the present work, studies were performed to directly determine if oltipraz generates oxygen free radicals. Electron paramagnetic resonance (EPR) spin trapping demonstrated that oltipraz slowly reacts in the presence of oxygen to generate the superoxide anion radical. This formation of superoxide by oltipraz was concentration- and time-dependent. EPR oximetry studies showed that oxygen was also slowly consumed paralleling the process of superoxide formation. Thus, oltipraz induced superoxide formation occurs and could be involved in the mechanism by which it exerts chemoprotection.     

Assessment of the genotoxic potential of riboflavin and lumiflavin. B. Effect of light.

On exposure to visible light, riboflavin and lumiflavin produced reactive oxygen species such as singlet oxygen and superoxide radicals. The reaction was found to be time- and concentration-dependent. Both riboflavin and lumiflavin, upon illumination, showed mutagenic response in the umu test as well as in the Ames/Salmonella assay with Salmonella typhimurium TA102. The mutagenic response was partially abolished by superoxide dismutase while sodium azide did not have any effect. No mutagenicity was observed if the compounds were not illuminated. The results suggested the involvement of superoxide radicals in light-induced mutagenicity of riboflavin as well as lumiflavin.     

Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity

Paraquat-induced nephrotoxicity involves severe renal cell damage caused by reactive oxygen species (ROS), specifically via increasing concentrations of superoxide anions in the kidney. Recently, superoxide dismutase (SOD) mimetics (SODm) have been developed that display safe SOD activities but which also possess additional antioxidant enzyme (e.g., catalase) or ROS-scavenging activities. The aim of this study was to compare the effects of two such SODm, specifically, EUK-134, a SODm with catalase activity, and tempol, a SODm with ROS-scavenging properties, on paraquat-induced nephrotoxicity of renal NRK-52E cells. Incubation with paraquat (1 mM) for 24 h reduced cell viability and increased necrosis significantly. Paraquat also generated significant quantities of superoxide anions and hydroxyl radicals. Both EUK-134 (10-300 microM) and tempol (0.3-1.0 mM) were able to improve cell viability and reduced paraquat-induced cell death significantly via dismutation or scavenging of superoxide anions and reduced hydroxyl radical generation. The data presented here suggest that SODm such as EUK-134 and tempol, which possess additional catalase and/or ROS-scavenging activities, can significantly reduce renal cell damage caused by paraquat. These effects were evident at concentrations which avoid the pro-oxidant activities associated with higher concentrations of SOD. Such SODm could therefore prove to be beneficial as therapies for paraquat nephrotoxicity.