Potentiation by carbon tetrachloride of NADPH-dependent lipid peroxidation in lung microsomes.

NADPH-dependent lipid peroxidation occurs in rat lung microsomes in vitro. Expressed per wet weight of tissue we found that lung had only $\text{1}\text{100}$ the activity of liver. However, examination of the rate of malonyl dialdehyde production with different concentrations of NADPH revealed that the kinetics of lipid peroxidation in lung microsomes was indistinguishable from that of NADPH-dependent lipid peroxidation in liver microsomes. With lung microsomes supplemented with NADPH, lipid peroxidation was potentiated by CCl₄ and inhibited by EDTA, Mn²⁺, and cytochrome c.     

Influence of ellagic acid on antioxidant defense system and lipid peroxidation in mice

Addition of ellagic acid (EA) to liver microsomes of mice resulted in a steady increase in inhibition of NADPH-dependent lipid peroxidation up to 2 mM concentration. The maximum of 70% inhibition of ascorbate-dependent lipid peroxidation was achieved at 1 mM concentration of EA. Feeding of EA significantly increased the levels of reduced glutathione and glutathione reductase in liver and lungs of male and female mice. However, there were no changes in the activities of catalase and superoxide dismutase. On the other hand, microsomes from liver and lungs of EA fed animals showed significantly suppressed NADPH- and ascorbate-dependent lipid peroxidation.     

Effects of diethylhydroxylamine on hepatic microsomal lipid peroxidation and glutathione S-transferases

The effects of diethylhydroxylamine (DEHA), a potent free-radical scavenger, on lipid peroxidation of rat liver microsomes were investigated in vitro. DEHA strongly inhibited ascorbate-dependent nonenzymatic microsomal lipid peroxidation. DEHA also completely inhibited nonenzymatic lipid peroxidation of heat-denatured microsomes, indicating that inhibition is protein-independent. DEHA only moderately inhibited NADPH-dependent enzymatic microsomal lipid peroxidation. DEHA has been shown to exhibit antitumorogenic properties. However, it had no significant effect on hepatic glutathione S-transferase, selenium-independent glutathione peroxidase, or selenium-dependent glutathione peroxidase activity in the DEHA-treated CD-1 (lCR) Br male mouse. This suggests that the mode of action of DEHA as an antitumorogenic agent may be different from that of butylated hydroxyanisole, whose antitumor function is attributed to induction of glutathione S-transferase activity.     

Antioxidant properties of novel benzimidazole retinoids

Our approach was to examine the antioxidant activity of novel retinoid derivatives containing the benzimidazole moiety. Their in vitro effects on rat liver microasomal, NADPH-dependent lipid peroxidation levels and superoxide anion formation were determined. Lipid peroxidation in rat liver microsomes was reduced by some of the compounds in a dose-dependent manner.     

Inhibitory action of Oren-gedoku-to extract on enzymatic lipid peroxidation in rat liver microsomes

We examined the inhibitory action of the extract of Oren-gedoku-to, a traditional herbal medicine known to act as an antioxidant, on enzymatic lipid peroxidation in rat liver microsomes. Simultaneous addition of a spray-dried preparation of Oren-gedoku-to extract (Tsumura TJ-15) inhibited enzymatic lipid peroxidation induced by reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH) and ADP/Fe3+ complex in liver microsomes in a dose-dependent manner. When the inhibition by TJ-15 of enzymatic lipid peroxidation in liver microsomes was kinetically analyzed, this medicine showed a competitive inhibition against NADPH or ADP/Fe3+ complex. TJ-15 inhibited the NADPH-driven enzymatic reduction of ADP/Fe3+ complex or cytochrome c in liver microsomes competitively. TJ-15 enhanced NADPH consumption by liver microsomes with ADP/Fe3+ complex. Treatment with TJ-15 after the onset of enzymatic lipid peroxidation in liver microsomes inhibited the progression of lipid peroxidation in a dose-dependent manner. The present results indicate that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes in the initiation and propagation steps in a dose-dependent manner. These results also suggest that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes not only through its antioxidant action but also through reduction of the supply of electrons derived from NADPH to ADP/Fe3+ complex in liver microsomes both in a competitive manner and through stimulation of NADPH oxidation.     

Perturbation of liver microsomal calcium homeostasis by ochratoxin A

The effect of ochratoxin A on hepatic microsomal calcium sequestration was studied both in vivo and in vitro. The rate of ATP-dependent calcium uptake was inhibited by 42-45% in ochratoxin A intoxicated rats as compared to controls. In the presence of NADPH, addition of ochratoxin A (2.5 to 100 microM) caused a concentration-dependent inhibition of calcium uptake (28-94%) by untreated rat liver microsomes. The rate of NADPH-dependent lipid peroxidation, measured as malondialdehyde formed, was also greatly enhanced by ochratoxin A. Various agents that inhibited ochratoxin A enhanced lipid peroxidation were also able to block the destruction of calcium uptake activity. Lipid peroxidation enhanced by ochratoxin A was also accompanied by leakage of calcium from calcium-loaded microsomes. These results suggest that ochratoxin A disrupts microsomal calcium homeostasis by an impairment of the endoplasmic reticulum membrane probably via enhanced lipid peroxidation.     

Effects of mansonones on lipid peroxidation, P450 monooxygenase activity, and superoxide anion generation by rat liver microsomes

Several structurally related ortho-naphthoquinones isolated from Mansonia altissima Chev (mansonones C, E and F) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P450 destruction; (c) inhibited NADPH-supported aniline 4-hydroxylase activity; (d) inhibited Fe(III)ADP reduction by NADPH-supplemented microsomes; (e) stimulated superoxide anion generation by NADPH-supplemented microsomes; and (f) stimulated ascorbate oxidation. ESR investigation of ascorbate-reduced mansonone F demonstrated semiquinone formation. Mansonone C had a greater effect than mansonones E and F on NADPH-dependent lipid peroxidation, O2- production and ascorbate oxidation, whereas mansonone E was more effective than mansonones C and F on aniline 4-hydroxylase activity. Mansonones E and F did not inhibit hydroperoxide-dependent lipid peroxidation, cytochrome P450 destruction or microsomal aniline 4-hydroxylase activity. Mansonone C inhibited to a limited degree tert-butyl hydroperoxide-dependent lipid peroxidation, this inhibition being increased by NADPH. Mansonone A, a tetrahydro orthonapthoquinone derivative, was in all respects relatively less effective than mansonones C, E and F. It is postulated that mansonones C, E and F inhibited microsomal lipid peroxidation and cytochrome P450 catalyzed reactions by diverting reducing equivalents from NADPH to dioxygen, but mansonone C (including its reduced form) may also exert direct antioxidant activity.     

NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes

Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation, the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.     

Effects of various compounds on lipid peroxidation mediated by detergent-solubilized rat liver NADPH-cytochrome C reductase.

A reconstituted lipid peroxidation system containing NADPH-cytochrome c reductase isolated from detergent-solubilized rat liver microsomes was used to determine the effects of several compounds, including drugs, on the lipid peroxidation activity. EDTA and ferrous ion were essential requirements for reconstitution of the activity. The addition of 1,10-phenanthroline to the system containing both EDTA and ferrous ion further enhanced the activity. Pyrocatecol, thymol, p-aminophenol, imipramine, p-chloromercuribenzoate (PCMB) and alpha-tocopherol exhibited strong inhibition, aniline, N-monomethylaniline, aminopyrine, benzphetamine, SKF 525-A and NADP exhibited moderate inhibition, and phenol, benzoic acid, acetanilide and nicotinamide exhibited less or no inhibition at the concentrations lower than 1000 micron M. Metal ions such as Hg+, Hg2+, Co2+, Cu2+, Mn2+ and U6+ inhibited lipid peroxidation strongly. In addition, Cd2+, St2+ and Ca2+ exhibited less potent to moderate inhibition, and Ba2+ and Mg2+ were without effects on the activity. Among sulfhydryl compounds tested, dithiothreitol inhibited lipid peroxidation to a greater extent than did the other three compounds, glutathione, cysteine and mercaptoethanol.     

Dietary α-tocopherol prevents dehydroepiandrosterone-induced lipid peroxidation in rat liver microsomes and mitochondria

Dehydroepiandrosterone (DHEA), an adrenal steroid, causes lipid peroxidation in rat liver microsomes and mitochondria and induces hepatocarcinogenesis. It was investigated whether α-tocopherol, a naturally occurring free radical chain terminator, could decrease lipid peroxidation. When DHEA-free diet supplemented with increasing concentrations of α-tocopherol (25, 50, 100, 200, 400 and 1000 mg/kg diet) was fed to rats for 7 days, a marked lipid peroxidation (measured as thiobarbituric acid reactive substances formation) was observed at concentrations 25 and 50 mg/kg in liver microsomes and mitochondria isolated from these animals. Lipid peroxidation was significantly reduced at concentrations ≥ 100 mg/kg. When DHEA (500 mg/kg diet) was fed to rats simultaneously with increasing concentrations of α-tocopherol, strong lipid peroxidation was observed at a-tocopherol concentrations ≤ 200 mg/kg diet. However, microsomes and mitochondria isolated from livers of rats fed a-tocopherol at doses of 400 and 1000 mg/kg diet produced only negligible amounts of thiobarbituric acid reactive substances. The data show that high concentrations of α-tocopherol in the diet decrease DHEA-induced microsomal and mitochondrial lipid peroxidation. Our results support the concept thatα-tocopherol can protect against DHEA-induced lipid peroxidation and consequently against steroid-induced liver cell damage and, perhaps, also tumour development.     

Adriamycin stimulates NADPH-dependent lipid peroxidation in liver microsomes not only by enhancing the production of O2∸ and H2O2, but also by potentiating the catalytic activity of ferrous ions

The antitumor drug, adriamycin, enhances NADPH-dependent lipid peroxidation in liver microsomes via the formation of Superoxide anion radicals (O₂^?) and hydrogen peroxide (H₂O₂). In the presence of metal ions additional reactive species are generated, causing stimulation of lipid peroxidation.However, in this study it was found that the stimulation of NADPH-dependent lipid peroxidation by adriamycin was not only affected by the production of O₂^? and H₂O₂. Adriamycin also enhances the catalysis by metal ions of the formation of those reactive oxygen species which initiate peroxidation. This was inferred from the fact that adriamycin stimulated malondialdehyde production at low ferrous ion concentrations, whereas at high ferrous ion concentrations no stimulation was found. Additional evidence was found in experiments in which the enzymic redox cycle of adriamycin in microsomes was abolished by heat-inactivation of the microsomes, and O₂^? and H₂O₂ were only produced with xanthine and xanthine oxidase. In this case in the presence of ferrous ions, adriamycin stimulated lipid peroxidation.     

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.     

Enhancement of ethanol-induced lipid peroxidation in rat liver by lowered carbohydrate intake.

In order to investigate the effect of carbohydrate intake on ethanol-induced lipid peroxidation and cytotoxicity, rats were maintained on four different test diets, a medium-carbohydrate (carbohydrate intake, 8.4 g/day/rat on average), a low-carbohydrate (carbohydrate intake, 2.8 g/day/rat on average), an ethanol-containing medium-carbohydrate (carbohydrate and an ethanol intake, 8.4 and 2.9 g/day/rat on average, respectively), and an ethanol-containing low-carbohydrate diet (2.8 and 2.9 g/day/rat on average, respectively). Ethanol and the low-carbohydrate diet each increased the liver malondialdehyde content, but the combined effect of both (ethanol-containing low-carbohydrate diet) was much more prominent than either alone. The degree of increase in malondialdehyde content almost paralleled the activity of the microsomal ethanol oxidizing system. Both the low-carbohydrate and the ethanol-containing low-carbohydrate diets decreased the liver glutathione content, but ethanol combined with the medium-carbohydrate diet had no effect on the content. Ethanol treatment increased the liver triglyceride content only when combined with the low-carbohydrate diet. The rate of NADPH-dependent microsomal malondialdehyde formation was much higher in microsomes from rats maintained on the ethanol-containing low-carbohydrate diet than in those from rats on the ethanol-containing medium-carbohydrate diet, indicating that lowered carbohydrate intake augments ethanol-induced malondialdehyde accumulation in the liver by enhancing the rate of lipid peroxidation. In addition, when incubated with red blood cells in the presence of NADPH, microsomes from rats fed the ethanol-containing low-carbohydrate diet caused marked hemolysis, which was prevented by the addition of 5 mM glutathione to the incubation system. Furthermore, addition of 50 mM ethanol to the reaction system greatly accentuated the hemolysis. These results suggest that lowered carbohydrate intake at the time of ethanol consumption potentiates ethanol cytotoxicity by enhancing ethanol-induced lipid peroxidation.     

Enhancement of NADPH-dependent lipid peroxidation activity by ethylene-diamineteraacetic acid (EDTA) in the presence of ferrous ion in rabbit liver microsomes.

The addition of EDTA to the incubation mixture containing rabbit liver microsomes and ferrous ion resulted in 2-fold increase of lipid peroxidation activity. Such an enhancement was not observed in rat liver microsomes. The maximum lipid peroxidation activity seen in rabbit microsomes in the presence of EDTA and ferrous ion was about 80% that seen in rat liver microsomes. From these results, it is likely that low lipid peroxidation activity in rabbit liver microsomes may account for the insufficiency of an EDTA-LIKE FACTORS(S) IN RABBIT LIVER MICROSOMES.     

Inhibition of hepatic microsomal lipid peroxidation by endogenous glycogen in the rat

The effect of endogenous glycogen on lipid peroxidation was examined in hepatic microsomes from rats. Microsomes were prepared to retain endogenous hepatic glycogen (Pg+) or to minimize it (Pg-). The indices of lipid peroxidation examined included the rate of NADPH-dependent formation of malondialdehyde (MDA) and the concomitant destruction of cytochrome P-450 and decline in the linearity of benzphetamine N-demethylase activity in microsomes. Cytochrome P-450 was destroyed during benzphetamine N-demethylation in microsomes with the loss being more extensive in Pg- than in Pg+. The destruction of cytochrome P-450 and the concomitant loss in linearity of benzphetamine N-demethylation in Pg- were prevented by added EDTA. Added linoleic acid hydroperoxide (LAHP) also caused a time-dependent loss of cytochrome P-450 in microsomes with the rate being greater in Pg- than in Pg+. The results show that glycogen inhibits hepatic microsomal lipid peroxidation and suggest that variations in glycogen content may contribute to disparities in in vitro oxidative activities between different microsomal samples. Such disparities may be minimized by the removal of glycogen during the preparation of microsomes and then supplementing the incubation mixtures with EDTA. The in vivo relevance of the observed antioxidant effect of glycogen is discussed in terms of the possible modulation by the polysaccharide of hepatotoxicity by agents whose effects may be mediated by lipid peroxidation.     

Production of carbon monoxide by cytochrome P450 during iron-dependent lipid peroxidation.

Carbon monoxide (CO) formation was studied in the process of lipid peroxidation in phenobarbital-induced rabbit liver microsomes. The reaction was NADPH-dependent and required Fe(2+), which occurs in microsomes as being protein bound and is not a consequence of heme destruction. Zn-protoporphyrin IX, an inhibitor of the heme oxygenase activity, proved to have no effect on CO production, suggesting that heme oxygenase is not involved into the CO generation reaction. At the same time, the addition of cytochrome P450 typical inhibitors SKF 525A and metyrapone to the reaction mixture had an inhibitory effect on the CO formation rate. Antioxidants such as alpha-tocopherol and desferal inhibited lipid peroxidation in phenobarbital-induced rabbit liver microsomes, and in this case the CO production was not registered. Thus, on the basis of the results presented here it is possible to assert that the process of NADPH, Fe(2+)-dependent carbon monoxide formation in microsomes is a result of lipid peroxidation with cytochrome P450 2B4 participation.     

Mechanism of antihepatotoxic activity of atractylon, I: effect on free radical generation and lipid peroxidation1.

The mechanism of antihepatotoxic action of atractylon, a main sesquiterpenic constituent of ATRACTYLODES rhizomes, was studied. Atractylon inhibited carbon tetrachloride (CCl (4))-induced cytotoxicity in primary cultured rat hepatocytes and CCl (4)-induced lipid peroxidation by rat liver microsomes. However, atractylon increased the free radical generation by CCl (4) with rat liver microsomes in the presence of a radical trapping agent, phenyl T-butyl nitrone (PBN). In addition, atractylon generated free radical PER SE. Experiments using (13)CCl (4) instead of CCl (4) indicated that the increased free radicals consisted of those from (13)CCl (4) and from atractylon. Accumulated data support that although both CCl (4) and atractylon generate free radicals respectively by rat liver microsomes, free radical from CCl (4) conducts lipid peroxidation and produces liver lesion, while atractylon forms free radical which scavenges CCl (3) radical in the absence of PBN, inhibits lipid peroxidation by CCl (4) and suppresses CCl (4)-induced liver lesion.     

Lipid peroxidation in rat liver microsomes: influence of carcinogenic N-nitrosocarbaryl

The reactivities of carbaryl, N-methyl 1-naphthylcarbamate insecticide and its N-nitrosated derivative carcinogenic, N-nitrosocarbaryl, were investigated on the microsomal hepatic lipid peroxidation and NADPH-dependent reductase activities. The in vivo treatment by N-nitrosocarbaryl produced a reduction in lipoperoxidative degradation induced in vitro by NADPH with regard to the formation of malonaldehyde and conjugated dienes. Carbaryl, its precursor did not affect lipid peroxidation under the same in vivo conditions. Moreover, following administration of the 2 compounds, the activities of NADPH-cytochrome c reductase as well as NADPH-neotetrazolium reductase were significantly decreased by N-nitrosocarbaryl but not influenced by carbaryl. Correspondingly, in vitro studies were performed; different action patterns of the 2 tested xenobiotics were also noted after treatment of rat liver microsomes in vitro by carbaryl and N-nitrosocarbaryl especially in their ability to cope with microsomal oxygen activation. N-Nitrosocarbaryl proved to have a potent inhibitor concentration effect on NADPH-dependent chemiluminescence response in vitro; carbaryl was virtually ineffective on this parameter. No significant difference appeared in the affinity of N-nitrosocarbaryl and carbaryl for the microsomal phospholipids. From the in vitro explorations, it was suggested that carcinogenic N-nitrosocarbaryl may be involved in the inhibition mechanism of microsomal lipid peroxidation through decreases in both NADPH-dependent reductase activities and superoxide generation.     

Adriamycin-induced lipid peroxidation in mitochondria and microsomes

The effect of the anti-neoplastic agent adriamycin on the peroxidation of lipids from rat liver and heart mitochondria and rat liver microsomes was investigated. The extent of total lipid peroxidation was determined by assaying for malondialdehyde (MDA), while the degradation of unsaturated fatty acids was monitored using gas chromatography. For liver mitochondria and microsomes, the formation of MDA was dependent on the concentrations of adriamycin, Fe3+, and protein, as well as time. In the presence of 50 microM adriamycin and saturating amounts of NADH, 1.5 +/- 0.2 nmol MDA/mg protein/60 min was produced with liver mitochondria. Upon addition of 25 microM Fe3+, the amount of MDA generated was increased to 6.5 +/- 0.1 nmol/mg protein/60 min. Liver microsomes produced amounts which were approximately 2-fold higher under all conditions. No MDA formation could be detected in rat heart mitochondria. The addition of 50 microM chlorpromazine completely inhibited peroxidation, whereas 0.5 to 1.0 mM p-bromophenacyl bromide blocked MDA formation by 50%. Analysis of fatty acids by gas chromatography showed that there was about a 50% decrease in arachidonic and docosahexaenoic acids in liver mitochondria and microsomes, but no change in the fatty acid content of heart mitochondria when incubated with both 50 microM adriamycin and 25 microM Fe3+ for 1 hr. These results suggest that (1) therapeutic concentrations of adriamycin enhance the peroxidation of lipids in liver mitochondria and microsomes through an enzymatic mechanism, especially in the presence of Fe3+; and (2) toxicity of this drug may be related to the degradation of membrane lipids.     

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.