Different effects of paraquat on microsomal lipid peroxidation in mouse brain, lung and liver

Paraquat stimulates NADPH-Fe(2+)-dependent microsomal lipid peroxidation in mouse brain and strongly inhibits it in the liver. In lung microsomes, the lipid peroxidation was stimulated by paraquat at 10(-4) M, but not at higher doses. An antioxidant action of paraquat seemed to account, at least in part, for the lack of stimulation in lung microsomes, but it was inappropriate to explain the result in hepatic microsomes. There was no apparent correlation between the effects of paraquat on the lipid peroxidation and on the activity of NADPH-cytochrome P-450 reductase, the enzyme which initiates redox cycling of paraquat, resulting in generation of active oxygen species. In fact, the effect of paraquat on the lipid peroxidation was independent of paraquat radical production, an intermediate in the cycle. However, the inhibitory potency of N-ethylmaleimide on NADPH-cytochrome P-450 reductase activity paralleled that on the lipid peroxidation stimulated by paraquat in brain and lung. These findings indicate that the effect of paraquat on microsomal lipid peroxidation differs among the organs and that other factors, besides NADPH-cytochrome P-450 reductase, might be involved in the stimulation of lipid peroxidation by paraquat.     

Stimulation by adriamycin of rat heart and liver microsomal NADPH-dependent lipid peroxidation

Rat liver and heart microsomes catalyze the transfer of single electrons from NADPH to adriamycin forming semiquinone radicals which, in turn, activate molecular oxygen. This process stimulated lipid peroxidation 5- to 7-fold as measured by malonaldehyde formation. Adriamycinaugmented lipid peroxidation was linear with time to 60 min, optimal at 1.0 mg of microsomal protein/ml and pH 7.5, and was proportional to the adriamycin concentration up to 100 μM. An NADPH-generating system was superior to NADPH, and an oxygen atmosphere tripled the rate of peroxidation as compared to air. Nitrogen abolished adriamycin-stimulated peroxidation. Superoxide dismutase, reduced glutathione, α-tocopherol, EDTA, dioxopiperazinylpropane (ICRF-187), and dimethylurea were effective inhibitors of lipid peroxidation. This suggests that Superoxide anion and possibly hydroxyl radical may be formed by the oxidation of the adriamycin semiquinone radical and thus stimulate the peroxidation of microsomal unsaturated fatty acids. Although adriamycin failed to stimulate lipid peroxidation in heart microsomes from control animals, peroxidation was dramatically increased when adriamycin was added to cardiac microsomes from α-tocopherol-deficient rats. Lipid peroxidation in α-tocopheroldeficient liver microsomes was four times greater than in control microsomes with the NADPH-generating system, and adriamycin did not further increase that high rate of peroxidation; however, when NADPH was used as the source of electrons in place of the NADPH-generating system, adriamycin stimulated peroxidation more than 2-fold. These results suggest that microsomal lipid peroxidation may play a role in the cytotoxicity and cardiotoxicity of adriamycin.     

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.     

Lipid peroxidation and paraquat toxicity.

Paraquat, added in vitro, stimulated lipid peroxidation in lung microsomes obtained from mouse lung but not from rat lung. Pretreatment of mice with N, N'-diphenyl-p-phenylene diamine, an antioxidant, or a high carbohydrate diet prevented the stimulatory effects of paraquat on lipid peroxidation but did not protect the animals against the lethal effects of paraquat. Conjugated dienes were not elevated in vivo after a dose of paraquat in mice equivalent to twice the ld₅₀. Plasma and lung concentrations and edematogenic activities of paraquat and diquat were measured in rats after subcutaneous injections of these chemicals. At equimolar doses, diquat was less edematogenic and found at lower concentrations in lung than was paraquat, but, considering the greater efficacy of diquat in stimulating lipid peroxidation in vitro (R. Talcott, H. Shu and E. Wei, [24], these results indicate that the effects of bipyridinium herbicides in vitro may not be related to their mechanisms of toxicity in vivo.     

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).     

Mechanism of paraquat-stimulated lipid peroxidation in mouse brain and pulmonary microsomes.

Paraquat-stimulated NADPH-dependent lipid peroxidation in mouse brain and pulmonary microsomes was inhibited by superoxide dismutase and singlet oxygen quenchers, but not by catalase or hydroxyl radical scavengers. MnCl2, which might form a salt with unsaturated lipid, inhibited the lipid peroxidation in brain microsomes, but not that in pulmonary microsomes. These findings suggest that activated oxygen species, especially superoxide and singlet oxygen, may play a major role in the stimulation of microsomal lipid peroxidation by paraquat in both brain and lung, and that the nature of the lipids exposed to peroxidative attack may be different in microsomes of the two organs.     

Interactions between paraquat and ferric complexes in the microsomal generation of oxygen radicals

Transition metals may play a central role in the toxicity associated with paraquat. Studies were carried out to evaluate the interaction of paraquat with several ferric complexes in the promotion of oxygen radical generation by rat liver microsomes. In the absence of added iron, paraquat produced some increase in low level chemiluminescence by microsomes; there was a synergistic increase in light emission in the presence of paraquat plus ferric-ATP or ferric-citrate, but not paraquat plus either ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (ferric-DETAPAC). Synergistic interactions could be observed at a paraquat concentration of 100 microM and a ferric-ATP concentration of 3 microM. In the absence or presence of paraquat, microsomal light emission was not affected by catalase or dimethyl sulfoxide (DMSO), indicating no significant role for hydroxyl radicals. Superoxide dismutase (SOD) did not affect chemiluminescence in the absence of paraquat but produced some inhibition in the presence of paraquat; this inhibition by SOD was most prominent in the absence of added iron and less pronounced in the presence of ferric-ATP or ferric-citrate. Although microsomal chemiluminescence is closely associated with lipid peroxidation, paraquat did not increase malondialdehyde production as reflected by production of thiobarbituric acid-reactive components. However, lipid peroxidation was sensitive to inhibition by SOD in the presence, but not in the absence, of paraquat, analogous to results with chemiluminescence. Paraquat synergistically increased microsomal hydroxyl radical production as measured by the production of ethylene from 2-keto-4-thiomethylbutyrate in the presence of ferric-EDTA or ferric-citrate. The interaction of paraquat with microsomes and ferric complexes resulted in an increase in oxygen radical generation. Various ferric complexes can increase the catalytic effectiveness of paraquat in promoting microsomal generation of oxygen radicals, although, depending on the reaction being investigated, the nature of the ferric complex is important.     

Comparison of one-electron reduction activity against the bipyridylium herbicides, paraquat and diquat, in microsomal and mitochondrial fractions of liver, lung and kidney (in vitro).

The first one-electron reduction steps of paraquat and diquat were compared using microsomal and mitochondrial fractions of rat liver, lung and kidney. Both fractions reduced each herbicide effectively, with the order of the Vmax values in microsomes and mitochondria being liver greater than lung greater than kidney and kidney greater than liver greater than lung, respectively. Although similar Vmax values were obtained from the liver and lung with the two subcellular fractions, the affinity of mitochondrial enzymes was lower, suggesting that the reduction of both herbicides in a microsomal site would be dominant in these two organs. The Vmax values for radical formation of paraquat were higher than those of diquat in all the endogenous one-electron reducing systems. The apparent Km values for diquat, however, were lower than those for paraquat in both subcellular fractions from the three tissues, indicating the superiority of the reduction for diquat to that for paraquat at low concentrations. This difference in the Km values supported the finding that the reduction velocity for diquat was significantly higher than that for paraquat at 1 mM concentration. Thus, at low concentrations, diquat would be reduced more easily than paraquat. In addition, tissue enzymatic specificity for paraquat was not obtained. From these data, it seems reasonable to conclude that the tissue-selective accumulation of paraquat previously proposed determines its toxicity.     

Stimulation of cyclophosphamide-induced pulmonary microsomal lipid peroxidation by oxygen

Cyclophosphamide (CP) causes lung toxicity in a wide variety of animals including humans. Recent reports suggest that CP increases lipid peroxide formation in the lung, and that oxygen (O2) potentiates CP-induced lung toxicity. We hypothesized that CP, or one of its toxic metabolites, acrolein, stimulates lung lipid peroxide formation in the presence of high O2 tensions. To test this, rat lung microsomes were treated in vitro with CP or acrolein in the presence of NADPH and 0-100% O2 with and without superoxide dismutase (SOD), glutathione (GSH), dithiothreitol (DTT), and EDTA (agents which scavenge reactive O2 species and/or detoxify reactive metabolites). Lipid peroxide formation in untreated microsomes was increased 40, 39, and 37% in 60, 80 and 100% O2 respectively (P less than 0.02 vs. 21% O2 air). Lipid peroxide formation in microsomes treated with CP increased 2-3-fold under 21% O2 (P less than 0.05 vs. untreated under 21% O2). However, increases in lipid peroxide formation were 3-4 fold in CP treated microsomes under 40-100% O2 (P less than 0.001 vs. untreated at same % O2). CP and acrolein-stimulated lipid peroxidation with and without O2 exposure was significantly (P less than 0.05) reduced by prior addition of SOD, GSH, DTT, or EDTA to the lung microsomal suspension. These results indicate that lipid peroxide formation increases in CP and acrolein-treated lung microsomes, and high O2 tensions stimulate CP-induced lipid peroxidation. Stimulation of CP-induced microsomal lipid peroxidation appears to be mediated by reactive O2 species or metabolites.     

Bleomycin toxicity: alterations in oxidative metabolism in lung and liver microsomal fractions

Separate groups of male rats received low doses (5 units) or high doses (15 units) of bleomycin i.p. twice weekly for 1.5, 3 or 6 weeks. The susceptibility of tissue lipid to peroxidation and the activities of mixed function oxidations were examined in microsomal fractions prepared from lung and liver. ADP-Fe (III)-initiated lipid peroxidation was stimulated in lung microsomal fractions only in animals treated with high-dose bleomycin for 1.5 weeks, whereas a 2- to 4-fold enhancement was observed in liver preparations from all bleomycin-treated animals. Microsomal ADP-Fe (III)-initiated lipid peroxidation was inhibited, however, by the in vitro addition of bleomycin in both lung and liver preparations, but this inhibition was an artifact resulting from the chelation of Fe (III) by bleomycin. Soybean lipoxygenase I-initiated microsomal lipid peroxidation, which does not require added iron, was unaffected by bleomycin in lung preparations but was inhibited in liver. Following in vivo treatment, lung microsomal hydrogen peroxide generation was inhibited by 1.5 weeks of high-dose bleomycin treatments, whereas benzphetamine N-demethylation was unchanged. These cytochrome P-450-dependent reactions were both suppressed, however, in liver microsomal fractions. In vitro, both reactions were also inhibited by bleomycin in liver but not in lung microsomal fractions. The lack of effect of in vitro bleomycin treatments on Superoxide generation in lung or liver preparations suggests that the NADPH cytochrome P-450 reductase component of the mixed function oxidase system was not affected. Minimal alterations in lipid peroxidizability and mixed function oxidase activities in lung microsomal fractions of bleomycin-treated animals suggest that the insensitivity could be due to: (1) the site of toxicity not being at the level of the endoplasmic reticulum; or (2) the target of bleomycin toxicity being limited to a small population of pulmonary cell types. Even though the liver is not susceptible to bleomycin toxicity, the hepatic microsomal mixed function oxidase system is highly sensitive to this chemical.     

Prevention of microsomal production of hydroxyl radicals, but not lipid peroxidation, by the glutathione-glutathione peroxidase system

The glutathione-glutathione peroxidase system is an important defense against oxidative stress. The ability of this system to protect against iron-catalyzed microsomal production of hydroxyl radicals [oxidation of 4-methylmercapto-2-oxo-butyrate (KMBA)] and lipid peroxidation was evaluated. When rat liver cytosol was added to microsomes, strong inhibition against KMBA oxidation was observed. No protection was found when the cytosol was boiled or dialyzed. In the latter case, the addition of 0.5 mM glutathione restored almost complete protection, whereas in the former case protection could be restored by the addition of both glutathione and glutathione peroxidase. Cysteine could not replace glutathione, nor could glutathione S-transferase replace glutathione peroxidase. The glutathione-glutathione peroxidase system was also very effective in decreasing production of hydroxyl radicals stimulated by the addition of menadione or paraquat to microsomes. In the absence of cytosol, the addition of glutathione plus glutathione peroxidase was also effective; however, 5 mM glutathione was necessary to protect against KMBA oxidation. The effective concentration of glutathione required for protection was lowered when glutathione reductase was added to the system, to regenerate reduced glutathione. These results indicate that low concentrations of glutathione in conjunction with glutathione peroxidase plus reductase can be very effective in preventing microsomal formation of hydroxyl radicals catalyzed by iron and other toxic compounds. Microsomal lipid peroxidation was decreased 40% by glutathione alone, and this decrease was potentiated in the presence of glutathione reductase. In contrast to KMBA oxidation, the combination of glutathione plus glutathione peroxidase was not any more effective than glutathione alone in preventing lipid peroxidation. The differences in sensitivities of microsomal lipid peroxidation and KMBA oxidation to glutathione peroxidase suggest that these two processes can be distinguished from each other, and that free H2O2 and hydroxyl radicals are involved in KMBA oxidation, but not lipid peroxidation.     

Generation of superoxide anion and lipid peroxidation in different cell types and subcellular fractions from rat testis

Mitochondria and microsomes from whole rat testis, seminiferous tubules and Leydig cells were investigated with respect to their capacity to generate superoxide anion. In addition, lipid peroxidation by whole testis mitochondria and microsomes was measured. In the presence of NADH and various respiratory inhibitors all three mitochondrial preparations catalyzed the formation of superoxide anion at a rate of 0.27-1.67 nmol/min.mg. This formation was concluded to be confined mainly to the NADH dehydrogenase region of the respiratory chain. Addition of NADPH to whole testis or Leydig cell mitochondria, but not tubule mitochondria, caused an additional formation of superoxide anion, which was unrelated to the respiratory chain, accelerated several-fold by menadione, and presumably catalyzed by NADPH-cytochrome c reductase and cytochrome P-450. Microsomes isolated from whole testis, seminiferous tubules, and Leydig cells generated superoxide anion at rates between 0.19 and 0.44 nmol/min.mg. These rates were also strongly stimulated by menadione. It is likely that both NADPH-cytochrome c reductase and cytochrome P-450 were involved in the microsomal generation of superoxide. Free radical scavengers of various types inhibited both the mitochondrial and microsomal formation of superoxide anion. Lipid peroxidation in whole testis essentially paralleled superoxide anion generation. However, the rate of mitochondrial lipid peroxidation was twice that of the microsomal rate. It is concluded that seminiferous tubules and Leydig cells generate superoxide anion at different rates and by different mechanisms. Together with cytochrome P-450-dependent hydroxylases, e.g., BP and DMBA hydroxylases, this superoxide generation may reflect a potential for cell-specific peroxidative damage in the testis.     

Studies on the in vitro interaction of mitomycin C,nitrofurantoin and paraquat with pulmonary microsomes: Stimulation of reactive oxygen-dependent lipid peroxidation

In vitro experiments were performed to evaluate the capacity of the redox cycling compounds mitomycin C (MC), nitrofurantoin (NF) and paraquat (PQ) to stimulate pulmonary microsomal lipid peroxidation. It was observed that the interaction of MC, NF or PQ with rat or mouse lung microsomes in the presence of an NADPH-generating system and an O₂ atmosphere resulted in significant lipid peroxidation. All three compounds demonstrated similar concentration dependency, similar time courses and the ability to generate lipid peroxidation-associated chemiluminescence. The stimulation of lipid peroxidation by MC, NF or PQ was inhibited significantly by Superoxide dismutase, glutathione, ascorbic acid, catalase and EDTA, agents which either scavenge reactive oxygen and/or prevent the generation of secondary reactive oxygen metabolites. In addition, the ability of MC or NF, but not PQ, to stimulate lipid peroxidation was reduced significantly following preincubation with microsomes and NADPH under N₂ (15–20 min) prior to incubation under O₂. During this period under N₂, MC and NF underwent reductive metabolism of their quinone and nitro moieties respectively. Thus, it appears that under aerobic conditions the pulmonary microsomal-mediated redox cycling of MC, NF and PQ is accompanied by the stimulation of reactive oxygen-dependent lipid peroxidation.     

Lipid peroxidation and generations of oxygen radicals induced by cephaloridine in renal cortical microsomes of rats

To investigate whether oxygen radicals would be generated by cephaloridine (CER) in the renal cortical microsomes obtained from rats and whether the microsomal lipid peroxidation would be promoted by CER, the microsomes were incubated under a pure oxygen atmosphere in a medium containing the reduced nicotinamide adenine dinucleotide phosphate regenerating system, under various conditions. Generations of superoxide anion and hydrogen peroxide and malondialdehyde formation were all dependent on microsomal protein concentrations, incubation periods and CER concentrations. Scavengers of the microsomal lipid peroxidation induced by CER, (+)-cyanidanol-3, mannitol, sodium benzoate and N-acetyl tryptophan, which are scavengers of hydroxyl free radicals, inhibited the CER-stimulated lipid peroxidation in the microsomes. Histidine, a scavenger of hydroxyl free radicals and singlet oxygen, and alpha-tocopherol, reduced-glutathione and NN'-diphenyl-p-phenylenediamine, the three of which are non-specific antioxidants, also inhibited the CER-stimulated lipid peroxidation in the microsomes. Accordingly, our findings may strongly support that CER generates not only superoxide anions and hydrogen peroxide but also hydroxyl free radicals in the kidney, and these generated oxygen radicals react with the membrane lipids to induce peroxidation and nephrotoxicity.     

Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin

Mitochondrial degeneration is a consistently prominent morphological alteration associated with adriamycin toxicity which may be the consequence of adriamycin-enhanced peroxidative damage to unsaturated mitochondrial membrane lipids. Using isolated rat liver mitochondria as an in vitro model system to study the effects of the anticancer drug adriamycin on lipid peroxidation, we found that NADH-dependent mitochondrial peroxidation--measured by the 2-thiobarbituric acid method--was stimulated by adriamycin as much as 4-fold. Marker enzyme analysis indicated that the mitochondria were substantially free of contaminating microsomes (less than 5%). Lipid peroxidation in mitochondria incubated in KCl-Tris-HCl buffer (pH 7.4) under an oxygen atmosphere was optimal at 1-2 mg of mitochondrial protein/ml and with NADH at 2.5 mM. Malonaldehyde production was linear with time to beyond 60 min, and the maximum enhancement of peroxidation was observed with adriamycin at 50-100 microM. Interestingly, in contrast to its stimulatory effect on NADH-supported mitochondrial peroxidation, adriamycin markedly diminished ascorbate-promoted lipid peroxidation in mitochondria. Superoxide dismutase, catalase, 1,3-dimethylurea, reduced glutathione, alpha-tocopherol and EDTA added to incubation mixtures inhibited endogenous and adriamycin-augmented NADH-dependent peroxidation of mitochondrial lipids, indicating that multiple species of reactive oxygen (superoxide anion radical, hydrogen peroxide and hydroxyl radical) and possibly trace amounts of endogenous ferric iron participated in the peroxidation reactions. In submitochondrial particles freed of endogenous defenses against oxyradicals, lipid peroxidation was increased 7-fold by adriamycin. These observations suggest that some of the effects of adriamycin on mitochondrial morphology and biochemical function may be mediated by adriamycin-enhanced reactive oxygen-dependent mitochondrial lipid peroxidation.     

Paraquat-stimulated binding of dopa to liver and lung microsomal protein

1. ³H-Dopa is converted by lung and liver microsomes to a reactive intermediate which binds covalently to lung and liver microsomal protein.

2. Binding of radioactivity from ³H-dopa to lung and liver microsomes was decreased by superoxide dismutase to the level observed with boiled microsomes.

3. Paraquat (5 mM) caused a 133% increase in binding of radioactivity from ³H-dopa with lung microsomes compared with 224% increase with liver microsomes. However, superoxide dismutase decreased the binding by 38% and 50% with lung and liver microsomes respectively.

4. Benzoate and 1, 4-diazobicyclo[2, 2, 2]octane in the absence and presence of paraquat had no effect on the binding of radioactivity from ³H-dopa.

5. Glutathione and ascorbic acid in the absence and presence of paraquat decreased the binding to below the level obtained with boiled microsomes.

6. The soluble fraction when present in the physiological ratio to microsomal protein decreased the binding, in the presence of paraquat, to the level of the control.     

A possible mechanism of naproxen-induced lipid peroxidation in rat liver microsomes

Previous papers from our laboratory report that naproxen and salicylic acid induced lipid peroxidation in rat liver microsomes, however, the mechanism is still unclear. In the present paper, ferrous iron release, nicotinamide-adenine dinucleotide phosphate reduced form (NADPH) oxidation and hydrogen peroxide (H2O2) formation have been measured to find out which mechanisms are involved in naproxen- and salicylic acid-induced lipid peroxidation. While the increase of ferrous iron release was observed with high concentrations of naproxen, salicylic acid did not stimulate ferrous iron release. Neither of these drugs stimulated NADPH oxidation and H2O2 formation. However hexobarbital and perfluorohexane, known as uncouplers of cytochrome P450, stimulated microsomal NADPH oxidation, O2 consumption, H2O2 formation and water (H2O) formation involving four-electron oxidase reaction. These results suggest that ferrous iron release contributes to naproxen-induced microsomal lipid peroxidation and that naproxen and salicylic acid are not uncouplers of cytochrome P450. Apparently H2O2 does not play an important role in naproxen- and salicylic acid-induced microsomal lipid peroxidation.     

Effect of hydrogen peroxide on the initiation of microsomal lipid peroxidation

Hydrogen peroxide reacts with reduced transition metals to generate the highly reactive hydroxyl radical (·OH), most often proposed as the predominant species for initiating microsomal lipid peroxidation. To assess the potential involvement of ·OH, generated from hydrogen peroxide, in microsomal lipid peroxidation, we have altered the concentration of microsomal hydrogen peroxide and measured the resulting rates of malondialdehyde production. Hydrogen peroxide concentration in microsomes was changed by adding exogenous catalase, by washing to reduce both endogenous catalase activity and hydrogen peroxide-dependent glutathione oxidase activity, and by inhibiting endogenous catalase activity with azide in either the presence or absence of exogenous hydrogen peroxide. In only one instance was the rate of lipid peroxidation affected; exogenous hydrogen peroxide added to microsomes, previously incubated with azide, inhibited lipid peroxidation, the opposite effect from that predicted if ·OH, generated from hydrogen peroxide, is actually the major initiating species. Neither these results, nor the inability of known ·OH traps to inhibit microsomal lipid peroxidation, support the role of free hydrogen peroxide in the initiation of microsomal lipid peroxidation.     

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by beta-lapachone and related naphthoquinones

The lipophilic o-naphthoquinones beta-lapachone, 3,4-dihydro-2-methyl-2-ethyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 8-935), 3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 9-442), and 3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione (CG 10-248) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P-450 destruction; (c) inhibited microsomal aniline 4-hydroxylase, aminopyrine N-demethylase and 7-ethoxycoumarin deethylase; (d) did not inhibit the ascorbate- and tert-butyl hydroperoxide-dependent lipid peroxidation and the cumenyl hydroperoxide-linked aniline 4-hydroxylase reaction; and (e) stimulated NADPH oxidation, superoxide anion radical generation and Fe(III)ADP reduction by NADPH-supplemented microsomes. In the presence of ascorbate, the same o-naphthoquinones stimulated oxygen uptake and semiquinone formation, as detected by ESR measurements. The p-naphthoquinones alpha-lapachone and menadione were relatively less effective than the o-naphthoquinones. These observations support the hypothesis that, in the micromolar concentration range, o-naphthoquinones inhibit microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions, by diverting reducing equivalents from NADPH to dioxygen.     

The effect of diethyldithiocarbamate on the lipid peroxidation of rat-liver microsomes and intact hepatocytes

The role of the oxygen radicals in lipid peroxidation, induced by ADP/Fe³⁺ or cumene hydroperoxide was investigated by administering diethyldithiocarbamate, an inhibitor of Superoxide dismutase, to hepatocytes or rats.Intact rat-liver hepatocytes perform a delayed ADP/Fe³⁺-induced lipid peroxidation after pretreatment with diethyldithiocarbamate. The cumene hydroperoxide-induced lipid peroxidation is unchanged. Hepatocytes, isolated from a rat administered with diethyldithiocarbamate in vivo, exhibit the same pattern, a delayed iron-induced lipid peroxidation and an unchanged cumene hydroperoxide-induced lipid peroxidation.Liver microsomes isolated from liver of a rat administered with diethyldithiocarbamate do not perform lipid peroxidation with NADPH/ADP/Fe³⁺, but do undergo lipid peroxidation with cumene hydroperoxide.It can be concluded that besides the inhibition of Superoxide dimutase, diethyldithiocarbamate inhibits directly the microsomal lipid peroxidation. Although this inhibition hampers the conclusion, evidence is obtained that Superoxide dismutase is probably involved in the protection against lipid peroxidation of the mitochondria, but not of the microsomes.