Paraquat and nitrofurantoin inhibit growth of Escherichia coli by inducing stringency

The herbicide paraquat and the antibiotic nitrofurantoin (redox-active compounds that can transfer electrons singly to oxygen) induced intracellular accumulation of the regulatory inhibitor guanosine tetraphosphate (stringency) in Escherichia coli. This mechanism is sufficient to account for the rapid bacteristasis produced in minimal medium by these agents. The growth inhibition and stringency induction were prevented by inclusion of specific amino acids in the medium. Stringency was first reported to result from amino acid starvation, with unloaded transfer ribonucleic acids (tRNAs) acting as the trigger. Previously, inhibition of growth of E. coli by paraquat and hyperbaric oxygen were shown to be prevented by inclusion in the medium of a nearly identical profile of specific amino acids, including branched-chain amino acids, which were required because of poisoning of their biosynthesis at the dihydroxyacid dehydratase site, and stringency has been induced by hyperbaric oxygen poisoning. Thus, stringency induction via a common poisoned site in branched-chain amino acid biosynthesis appears to be a shared mechanism of toxicity for these agents and hyperbaric oxygen, which also share the propensity for one-electron-transfer, free-radical reactions in cells.     

An evaluation of the redox cycling potencies of paraquat and nitrofurantoin in microsomal and lung slice systems

The redox cycling abilities of the pulmonary toxins paraquat and nitrofurantoin have been compared with those of the potent redox cyclers, diquat and menadione in lung and liver microsomes by using the oxidation of NADPH and consumption of oxygen. The relative potencies of these compounds to undergo redox cycling were in the order: diquat approximately menadione much greater than paraquat congruent to nitrofurantoin. This was partly attributed to the much lower affinity (Km) of lung and liver microsomes for paraquat and nitrofurantoin than for diquat and menadione. The potential to redox cycle was assessed in an intact cellular system by determining the oxygen consumption of rat lung slices in the presence (10(-6), 10(-5) and 10(-4) M) or absence of each of the four substrates. At concentrations of paraquat (10(-5) M) known to be accumulated by lung slices, a small but significant stimulation of lung slice oxygen uptake was observed. Nitrofurantoin (10(-4)-10(-6) M) did not affect lung slice oxygen uptake in lung slices, an observation consistent with its being a poor redox cycling compound, which is not actively accumulated into lung cells. This data has important implications in assessing the risk of exposure to paraquat. Low levels of paraquat would not be expected to cause lung damage because insufficient compound is present in the lung to exert its toxicity by redox cycling (due to the high Km observed).     

A mechanism of paraquat toxicity in mice and rats.

The purpose of this study was to investigate the hypothesis that paraquat toxicity results from cyclic reduction-oxidation of paraquat in vivo, with subsequent generation of superoxide radicals and initiation of lipid peroxidation. Phenobarbital pretreatment (0.1% in drinking water for 10 days) significantly increased the paraquat LD50 in mice, but only when the mice were continued on phenobarbital after paraquat administration. Phenobarbital, through its own metabolism, may be competing for electrons which might otherwise be utilized in paraquat reduction and thus decrease paraquat toxicity. Paraquat, given at 30 mg/kg ip to mice, significantly decreased liver concentrations of the water-soluble antioxidant, reduced glutathione, and lung concentrations of lipid-soluble antioxidants. The decrease in tissue antioxidants may reflect the initiation of lipid peroxidation by paraquat. Oxygen-tolerant rats (exposure to 85% oxygen for 7 days) have increased activities of pulmonary enzymes which combat lipid peroxidation. The paraquat Lt50 (median time to death) after 45 mg/kg of paraquat ip was significantly increased in oxygentolerant rats compared to room-air-exposed controls. Rats exposed to 100 ppm paraquat in the drinking water for 3 weeks had significantly elevated activities of lung glucose-6-phosphate dehydrogenase and glutathione reductase. The cross-tolerance of oxygen and paraquat and the induction by paraquat of pulmonary enzymes that supply reducing equivalents to combat oxidative damage support the proposal that paraquat may initiate lipid peroxidation in vivo.     

Postnatal toxicity of chronically administered paraquat in mice and interactions with oxygen and bromobenzene.

The purpose of this study was to investigate the effect of chronic paraquat administration on developing mice and to examine the interaction of chronic paraquat exposure with 100% oxygen and the hepatotoxin bromobenzene. Paraquat was administered at 50 and 100 ppm in the drinking water to pregnant mice beginning at Day 8 of gestation, with continued exposure to the newborns up to 42 days after birth. Neither paraquat treatment altered the postnatal growth rate; however, 100 ppm but not 50 ppm paraquat significantly increased the postnatal mortality. Both 50- and 100-ppm paraquat-treated mice were sensitized to the onset of oxygen toxicity, determined by a significant reduction in the LT50 at 42 days after birth. An enhanced sensitivity to oxygen toxicity was also detectable in 100 ppm but not 50 ppm mice at Days 1 and 28 after birth. In 42-day-old mice, 50 and 100 ppm paraquat treatment also significantly reduced the LT50 after 3100 mg/kg ip (LD85) bromobenzene. These observations suggest that the toxicity of paraquat may be mediated through an interaction with oxygen and describe possible interactions that could occur with the environmental use of paraquat.     

Protection by selective amino acid solutions against doxorubicin induced growth inhibition of Escherichia coli

• 1.1. Incubation of Escherichia coli with 0.7 mM doxorubicin in MBS-glucose medium resulted in complete growth inhibition, an inhibition that was blocked by placing specific amino acids (AA) in the medium.
• 2.2. The mechanism of protection by AA was similar to that reported previously for cells poisoned by hyperoxia and by paraquat, e.g. of 20 common AA, ten protect, ten do not and the branched-chair AA are among those required for inhibition.
• 3.3. Unlike hyperoxia and paraquot stringency which caused elevation of intracellular concentrations of guanosine tetraphosphate (ppGpp), doxorubicin inhibition did not elevate ppGpp.
• 4.4. Concentrations of ppGpp were increased by isoleucine starvation as expected, and the subsequent addition of doxorubicin did not abolish that increase; however, pretreatment with doxorubicin prevented the induction of stringency by isoleucine starvation.
• 5.5. This suggests that doxorubicin directly inhibits ppGpp synthesis or protein biosynthesis to leave tRNA loaded as is the case with chloramphenicol.

     

Synergistic interactions between NADPH-cytochrome P-450 reductase, paraquat, and iron in the generation of active oxygen radicals

The toxicity associated with paraquat is believed to involve the generation of active oxygen radicals and the production of oxidative stress. Paraquat can be reduced by NADPH-cytochrome P-450 reductase to the paraquat radical; this results in consumption of NADPH. A variety of ferric complexes, including ferric-ATP, -citrate, -EDTA, ferric diethylenetriamine pentaacetic acid and ferric ammonium sulfate, produced a synergistic increase in the paraquat-mediated oxidation of NADPH. This synergism could be observed with very low concentrations of iron, e.g. 0.25 microM ferric-ATP. Very low rates of hydroxyl radical were generated by the reductase with paraquat alone, or with ferric-citrate or -ATP or ferric ammonium sulfate in the absence of paraquat; however, synergistic increases in the rate of hydroxyl radical generation occurred when these ferric complexes were added together with paraquat. Ferric-EDTA and -DTPA catalyzed some production of hydroxyl radicals, which was also synergistically elevated in the presence of paraquat. Ferric desferrioxamine was essentially inert in the absence or presence of paraquat. This enhancement of hydroxyl radical generation was sensitive to catalase and competitive scavengers but not to superoxide dismutase. The interaction of paraquat with NADPH-cytochrome P-450 reductase and ferric complexes resulted in an increase in oxygen radical generation, and various ferric complexes increased the catalytic effectiveness and potentiated significantly the toxicity of paraquat via this synergistic increase in oxygen radical generation by the reductase.     

Metabolic activation of oxygen by nitrofurantoin in the young chick

Anaerobic incubations containing nitrofurantoin, and NADPH-generating system, and chick hepatic microsomes produced an electron spin resonance spectrum identified as the nitro anion free radical. Aerobically, nitrofurantoin markedly stimulated oxygen consumption, superoxide formation, and NADPH oxidation in hepatic microsomal preparations from control and selenium-deficient chicks. The nitrofurantoin-stimulated oxidation of NADPH was inhibited by superoxide dismutase (SOD). The superoxide-dependent oxidation of NADPH did not appear to be mediated by an NADP^? radical, as has been shown for lactate dehydrogenase. Further, the aerobic metabolism of the nitro drug was also affected by SOD, suggesting the existence of a previously unreported metabolic pathway for nitrofurantoin. These studies support the growing body of evidence which suggests that nitrofurantoin toxicity is mediated, at least in part, by the metabolic activation of oxygen by the nitro aromatic anion radical. Further, these data suggest that superoxide may be involved in the oxidative metabolism of the aromatic nitro compounds.     

Induction of phenotypes resembling CuZn-superoxide dismutase deletion in wild-type yeast cells: an in vivo assay for the role of superoxide in the toxicity of redox-cycling compounds

Yeast (Saccharomyces cerevisiae) lacking the enzyme CuZn-superoxide dismutase (sod1delta) display a large number of dioxygen sensitive phenotypes, such as amino acid auxotrophies, sensitivity to elevated temperatures, and sensitivity to 100% dioxygen, which are attributed to superoxide stress. Such cells are exquisitely sensitive to small amounts of the herbicide paraquat (methyl viologen), which is known to produce high fluxes of superoxide in vivo via a redox-cycling mechanism. We report that dioxygen sensitive phenotypes similar to those seen in sod1delta cells can be induced in wild-type cells by treatment with moderate concentrations of paraquat or diquat, another bipyridyl herbicide, providing strong evidence that the mechanism of toxicity for both of these compounds is attributable to superoxide stress. Certain redox-cycling quinone compounds (e.g., menadione and plumbagin) are also far more toxic toward sod1delta than to wild type. However, treatment of wild-type yeast with menadione or plumbagin did not induce sod1delta-like phenotypes, although toxicity was evident. Thus, their toxicity in wild type cells is predominantly, but not exclusively, due to mechanisms unrelated to superoxide production. Further evidence for a different basis of toxicity toward wild-type yeast in these two classes of redox-cycling compounds includes the observations that (i) growth in low oxygen alleviated the effects of paraquat and diquat but not those of menadione or plumbagin and (ii) activity of the superoxide sensitive enzyme aconitase is affected by very low concentrations of paraquat but only by higher, growth inhibitory concentrations of menadione. These results provide the basis for an easy qualitative assay of the contribution of redox-cycling to the toxicity of a test compound. Using this method, we analyzed the Parkinsonism-inducing compound 1-methyl-4-phenylpyridinium and found that redox cycling and superoxide toxicity are not the predominant factor in its toxic mechanism.     

The bipyridyl herbicide paraquat produces oxidative stress-mediated toxicity in human neuroblastoma SH-SY5Y cells: relevance to the dopaminergic pathogenesis

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

Inhibition of c-Src protects paraquat induced microvascular endothelial injury by modulating caveolin-1 phosphorylation and caveolae mediated transcellular permeability

The mechanisms underlying paraquat induced acute lung injury (ALI) is still not clear. C-Src plays an important role in the regulation of microvascular endothelial barrier function and the pathogenesis of ALI. In the present study, we found that paraquat induced cell toxicity and an increase of reactive oxygen species (ROS) in endothelium. Paraquat exposure also induced significant increase of caveolin-1 phosphorylation, caveolae trafficking and albumin permeability in endothelial monolayers. C-Src depletion by siRNA significantly attenuate paraquat induced cell toxicity, caveolin-1 phosphorylation, caveolae formation and endothelial hyperpermeability. N-acetylcysteine (NAC) failed to protect endothelial monolayers against paraquat induced toxicity. Thus, our findings suggest that paraquat exposure increases paracellular endothelial permeability by increasing caveolin-1 phosphorylation in a c-Src dependant manner. The depletion of c-Src might protect microvascular endothelial function by regulating caveolin-1 phosphorylation and caveolae trafficking during paraquat exposure, and might have potential therapeutic effects on paraquat induced ALI.     

Uptake and efflux of 14C-paraquat by rat lung slices: the effect of imipramine and other drugs.

Paraquat accumulation by rat lung slices incubated at 10 or 100 μm concentration was linear with time and the accumulated paraquat was “noneffluxable.” Imipramine (100 or 500 μm) inhibited paraquat (10 μm) uptake by 38 and 85%, respectively, and 500 μm imipramine enhanced paraquat efflux by 40%. The combination of impaired uptake and enhanced efflux suggested the possibility that imipramine might reduce the toxicity of paraquat in intact animals. However, at the doses used, imipramine did not alter paraquat toxicity in vivo. Eleven other drugs were shown to inhibit uptake, but only five enhanced paraquat efflux.     

Pulmonary Effects to Repeated Exposures to Paraquat Aerosol in Guinea Pigs

Pulmonary Effects of Repeated Exposures to Paraquat Aerosolin Guinea Pigs. BURLEIGH-FLAYER, H. AND ALARIE, Y. (1988). Fundam.Appl. Toxicol 10, 717–729. Exposure to paraquat, a widelyused herbicide, has been shown to produce a concentration dependentrapid, shallow breathing pattern in guinea pigs 18 hr followingexposure (H. Burleigh-Flayer and Y. Alarie, 1987, Arch Toxicol.59(6), 391–396). To further explore the pulmonary effectsfollowing exposure to paraquat, two experiments were carriedout. The first experiment consisted of exposing a group of guineapigs for a period of 4 hr to 0.7 mg/m³ paraquat aerosol andmonitoring respiratory variables for 2 weeks following the exposure.In the second experiment, three groups of guinea pigs were repeatedlyexposed to three concentrations of paraquat aerosol (0.1,0.4,and 0.8 mg/m³) for 6 hr a day, 5 days a week for 3 weeks. Respiratoryvariables were measured each day of these 3-week experiments.The respiratory variables evaluated in both experiments weretidal volume (VT) and respiratory frequency (/). These variableswere monitored during air breathing and upon challenge with10% CO₂ in 20% O₂ and 70% N₂ in order to evaluate the pulmonaryeffects of exposure to paraquat. Following a single exposureto 0.7 mg/m³ paraquat aerosol, a decrease in VT and increasein f were seen during air and 10% CO₂ challenge which reacheda maximum several days following exposure. After reaching maximalchanges, the respiratory variables returned to control values.With repeated 6-hr exposures to paraquat aerosol, guinea pigsexposed to 0.4 and 0.8 mg/m³ also displayed a rapid, shallowbreathing pattern. Adaptation to the exposures for these twoconcentration groups was evidenced by a return of the respiratoryvariables toward control levels. This adaptation typically occurredduring the first 7 days of exposures. A cumulative effect wastherefore not detected with repeated exposures to paraquat aerosols.     

Superoxide independence of paraquat toxicity in Escherichia coli

Hyperbaric oxygen failed to enhance the lethal effect of the herbicide paraquat towards Escherichia coli B. Variations induced in the intracellular concentrations of either of two endogenous superoxide dismutases had no effect on bacterial susceptibility to paraquat, even in the presence of hyperbaric oxygen. It is concluded that paraquat toxicity is not mediated by superoxide in this organism.     

Role of antioxidants in paraquat toxicity

Paraquat, a quarternary nitrogen herbicide, is a highly toxic compound for humans and animals and many cases of acute poisoning and death have been reported over the past few decades. The mechanisms of paraquat toxicity involve: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species, such as hydrogen peroxide and hydroxyl radical; and the oxidation of the cellular NADPH, the major source of reducing equivalents for the intracellular reduction of paraquat, which results in the disruption of important NADPH-requiring biochemical processes. The major cause of death in paraquat poisoning is respiratory failure due to an oxidative insult to the alveolar epithelium with subsequent obliterating fibrosis. Management of paraquat poisoning has remained mostly supportive and has been directed towards the modification of the toxicokinetics of the poison. Currently, there are no true pharmacological antagonists for paraquat and there are no chelating agents capable of binding the poison in the blood or other tissues. Recognizing the fact that paraquat induces its toxic effects via oxidative stress-mediated mechanisms, innovations in the management of paraquat poisoning are directed towards the use of antioxidants. In this review, the status of antioxidants in ameliorating or treating the toxic effects produced by paraquat is presented.     

Testicular Toxicity of N-Methyltetrazolethiol Cephalosporin Analogs in the Juvenile Rat

Testicular Toxicity of N-Methyltetrazolethiol CephalosporinAnalogs in the Juvenile Rat. COMERESKI, C. R., BREGMAN, C. L.,and BUROKER, R. A. (1987). Fundam. Appl. Toxicol. 8, 280–289.The testicular toxicity of 10 antibiotics was evaluated in juvenilerats. Three of the antibiotics, cefbuperazone, cefamandole,and cefoperazone, contain the N-methyltetrazolethiol group asthe 3-substituent; ampicillin, cefazolin, cefotaxime, cephalothin,cefoxitin, piperacillin, and ceforanide do not contain thismoiety. Testicular degeneration, partially irreversible in nature,was observed with those antibiotics which contain the N-methyltetrazolethiolsubstituent. Further, free N-methyltetrazolethiol also producedtesticular degeneration in the juvenile rat. This substituentis most likely responsible for the testicular toxicity observedwith cefbuperazone, cefamandole, and cefoperazone in the juvenilerat. The implications of these findings to man are undetermined.     

Effect of proline analogs on oxygen toxicity-induced pulmonary fibrosis in the rat

Proline analogs inhibit collagen biosynthesis and prevent accumulation of collagen in tissues. The antifibrotic effects of three proline analogs, cis-hydroxyproline, L-azetidine-2-carboxylic acid, and L-3,4-dehydroproline, were compared in a rat oxygen toxicity model. The specificity of these agents for collagen was examined by measuring their effects on noncollagen protein and elastin accumulation in the lung. Increased lung collagen was produced by exposing rats to 95% O2 for 60 hr followed by a 2-week recovery period. Animals were treated with the proline analogs for the 2-week period. Oxygen exposure in untreated animals increased lung collagen 26% above air-breathing controls, and this increase was prevented by all three analogs. Increased noncollagen protein was also prevented by these agents, suggesting they were not entirely specific for collagen. Elastin accumulation, however, was not inhibited by cis-hydroxyproline. It was concluded that proline analogs were antifibrotic, but affected the metabolism of noncollagen protein.     

Effects of glutathione depletion using buthionine sulphoximine on the cytotoxicity of nitroaromatic compounds in mammalian cells in vitro

The inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO) has been used to deplete endogenous thiols in mammalian cells in vitro. The effect of such depletion on the toxicity of nitroaromatic compounds has been investigated. Substantial enhancement of both aerobic and hypoxic toxicity of the 2-nitroimidazole, misonidazole is observed in thiol-depleted cells; the hypoxic toxicities of metronidazole, nitrofurantoin and nimorazole are also increased by thiol depletion. These data of significance for the potential combined use of BSO with nitroaromatic radiosensitizers to increase their radiosensitizing efficiency in radiotherapy, and as a potential method for enhancing the efficiency of anti-protozoal nitroaromatic drugs.     

Nitrofurantoin-mediated oxidative stress cytotoxicity in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism(s) of toxicity of the antimicrobial drug nitrofurantoin. This 5-nitrofuran derivative stimulated hepatocyte oxygen uptake in the presence of the mitochondrial respiration inhibitors KCN or antimycin A. This could indicate the formation of O2- and H2O2, following intracellular nitrofurantoin reduction. Addition of nitrofurantoin to suspensions of isolated rat hepatocytes produced a dose- and time-dependent decrease of cell viability. H2O2 probably plays a significant role in the cytotoxic effects of nitrofurantoin as the catalase inhibitors azide or aminotriazole markedly enhanced cytotoxicity. The loss of cell viability was preceded by glutathione (GSH) depletion and a concomitant and nearly stoichiometric formation of oxidised glutathione (GSSG) that did not occur in hepatocytes lacking glutathione peroxidase activity isolated from rats fed a low-selenium diet. This indicates that H2O2 and the seleno-enzyme glutathione peroxidase are responsible for GSH oxidation. Furthermore, addition of nitrofurantoin to isolated rat hepatocytes produced a reversible inactivation of hepatocyte glutathione reductase activity and explains the maintenance of high GSSG levels. The compromised hepatocytes were also highly susceptible to H2O2. The hepatocyte toxicity of nitrofurantoin may, therefore, be attributed to oxidative stress caused by redox-cycling mediated oxygen activation.     

Nitrofurantoin-induced pulmonary toxicity. In vivo evidence for oxidative stress-mediated mechanisms.

The present study was carried out to examine whether nitrofurantoin-induced pulmonary toxicity in normal rats was mediated via oxidant stress mechanisms. The relative importance of the cellular antioxidant enzymes in nitrofurantoin toxicity was also assessed. For this, the pulmonary toxicity induced by nitrofurantoin in rats was evaluated at various time intervals after a single subcutaneous injection. Data from this study showed that nitrofurantoin (200 mg/kg, s.c.) resulted in transient but measurable lung damage as evidenced by the increases in wet lung weight/body weight ratio and decreases in lung angiotensin converting enzyme activity. A transient decrease in GSH concentrations with a concurrent increase in GSSG concentrations as well as an increase in lipid peroxidation levels (measured by the formation of diene conjugates and thiobarbituric acid reactants) were also evident in lungs of nitrofurantoin-treated rats. In addition, nitrofurantoin did not alter the pulmonary superoxide dismutase and glutathione peroxidase activities, but it did produce transient decreases in catalase and glutathione reductase activities. These data indicate that impairment of the ability of the lung to detoxify reactive oxygen species may play an important role in the development of nitrofurantoin-induced pulmonary toxicity. The results of the present study suggest that nitrofurantoin can damage the lungs of rats, probably through oxidative stress-mediated mechanisms. Also, our data have provided in vivo evidence for substantiating lipid peroxidation as a possible cause of lung damage.     

NADH-dependent generation of reactive oxygen species by microsomes in the presence of iron and redox cycling agents.

NADH was found previously to catalyze the reduction of various ferric complexes and to promote the generation of reactive oxygen species by rat liver microsomes. Experiments were conducted to evaluate the ability of NADH to interact with ferric complexes and redox cycling agents to catalyze microsomal generation of potent oxidizing species. In the presence of iron, the addition of menadione increased NADPH- and NADH-dependent oxidation of hydroxyl radical (.OH) scavenging agents; effective iron complexes included ferric-EDTA, -diethylenetriamine pentaacetic acid, -ATP, -citrate, and ferric ammonium sulfate. The stimulation produced by menadione was sensitive to catalase and to competitive .OH scavengers but not to superoxide dismutase. Paraquat, irrespective of the iron catalyst, did not increase significantly the NADH-dependent oxidation of .OH scavengers under conditions in which the NADPH-dependent reaction was increased. Menadione promoted H2O2 production with either NADH or NADPH; paraquat was stimulatory only with NADPH. Stimulation of H2O2 generation appears to play a major role in the increased production of .OH-like species. Menadione inhibited NADH-dependent microsomal lipid peroxidation, whereas paraquat produced a 2-fold increase. Neither the control nor the paraquat-enhanced rates of lipid peroxidation were sensitive to catalase, superoxide dismutase, or dimethyl sulfoxide. Although the NADPH-dependent microsomal system shows greater reactivity and affinity for interacting with redox cycling agents, the capability of NADH to promote menadione-catalyzed generation of .OH-like species and H2O2 or paraquat-mediated lipid peroxidation may also contribute to the overall toxicity of these agents in biological systems. This may be especially significant under conditions in which the production of NADH is increased, e.g. during ethanol oxidation by the liver.