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.     

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.     

Dissociation of microsomal oxygen reduction and lipid peroxidation with the electron acceptors, paraquat and menadione.

Paraquat, diquat and menadione, electron acceptors which interact with the microsomal electron transport chain, were used to investigate the relationship between microsomal lipid peroxidation and microsomal oxygen reduction. All three compounds stimulated hydrogen peroxide production and the rate of superoxide production by mouse liver microsomes. However, while paraquat and diquat stimulated microsomal lipid peroxidation (2-fold in liver microsomes and 6- to 10-fold in lung microsomes), menadione was a potent inhibitor. Superoxide dismutase and catalase had no effect on paraquat-stimulated lipid peroxidation. Diquat, at concentrations sufficient to stimulate Superoxide production, was unable to stimulate lipid peroxidation. Based on the above observations, a mechanism of paraquat- and diquat-initiated lipid peroxidation independent of superoxide and peroxide generation is proposed. The stimulatory effects of paraquat and diquat on lung microsomal lipid peroxidation are also discussed in relation to the lipid peroxidation hypothesis of paraquat lung toxicity.     

A possible role for membrane lipid peroxidation in anthracycline nephrotoxicity

Adriamycin causes both glomerular and tubular lesions in kidney, which can be severe enough to progress to irreversible renal failure. This drug-caused nephrotoxicity may result from the metabolic reductive activation of Adriamycin to a semiquinone free radical intermediate by oxidoreductive enzymes such as NADPH-cytochrome P-450 reductase and NADH-dehydrogenase. The drug semiquinone, in turn, autoxidizes and efficiently generates highly reactive and toxic oxyradicals. We report here that the reductive activation of Adriamycin markedly enhanced both NADPH- and NADH-dependent kidney microsomal membrane lipid peroxidation, measured as malonaldehyde by the thiobarbituric acid method. Adriamycin-enhanced kidney microsomal lipid peroxidation was diminished by the inclusion of the oxyradical scavengers, superoxide dismutase and 1,3-dimethylurea, and by the chelating agents, EDTA and diethylenetriamine-pentaacetic acid (DETPAC), implicating an obligatory role for reactive oxygen species and metal ions in the peroxidation mechanism. Furthermore, the inclusion of exogenous ferric and ferrous iron salts more than doubled Adriamycin-stimulated peroxidation. Lipid peroxidation was prevented by the sulfhydryl-reacting agent, p-chloromercuribenzenesulfonic acid, by omitting NAD(P)H, or by heat-inactivating the kidney microsomes, indicating the requirement for active pyridine-nucleotide linked enzymes. Several analogs of Adriamycin as well as mitomycin C, drugs which are capable of oxidation-reduction cycling, greatly increased NADPH-dependent kidney microsomal peroxidation. Carminomycin and 4-demethoxydaunorubicin were noteworthy in this respect because they were three to four times as potent as Adriamycin. In isolated kidney mitochondria, Adriamycin promoted a 12-fold increase in NADH-supported (NADH-dehydrogenase-dependent) peroxidation. These observations clearly indicate that anthracyclines enhance oxyradical-mediated membrane lipid peroxidation in vitro, and suggest that peroxidation-caused damage to kidney endoplasmic reticulum and mitochondrial membranes in vivo could contribute to the development of anthracycline-caused nephrotoxicity.     

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.     

Protection of cells from menadione-induced apoptosis by inhibition of lipid peroxidation

Menadione is a commonly used compound that causes oxidative stress. We investigated the influence of lipid peroxidation on the apoptotic response of mouse myogenic C2C12 cells following menadione-induced oxidative stress. The presence of hypodiploid cells and phosphatidylserine translocation were assayed to detect apoptotic cells. Menadione at 10-40 micro M induced cell apoptosis. Menadione at dose of 80 micro M induced both apoptosis and necrosis. At a 160 micro M dosage, menadione induced cell necrosis. Caspase 3 activation is required for menadione-induced apoptosis. Incubation of cells with 40 micro M menadione resulted in the depletion of cellular glutathione and increased lipid peroxidation. Pre-treatment of cells with cysteine suppressed the menadione-induced apoptosis and prevented changes in reactive oxygen species levels, glutathione levels and lipid peroxidation. Pre-treatment of cells with deferoxamine mesylate, an iron chelator, also reduced both menadione-induced apoptosis and lipid peroxidation. However, this did not prevent menadione-induced glutathione depletion. Thus, the inhibition of lipid peroxidation by deferoxamine mesylate prevented apoptosis even though cellular glutathione remained depleted. Our data suggest that menadione-induced apoptosis is directly linked to iron-dependent lipid peroxidation.     

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.     

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.     

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.     

The role of iron in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced lipid peroxidation by rat liver microsomes

Treatment of female Sprague-Dawley rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances hepatic lipid peroxidation, and the role of iron in TCDD-induced lipid peroxidation was examined. Ferrous and ferric ions, and the chelators adenosine diphosphate (ADP), ethylenediaminetetraacetic acid (EDTA) and desferrioxamine (DFX) were added to an in vitro microsomal lipid peroxidation system using microsomes from control and TCDD-treated animals. Both ferrous and ferric ions enhanced microsomal lipid peroxidation, with the greatest effect being produced by the combination of ferrous and ferric ions. The addition of ADP and EDTA produced modest increases in microsomal lipid peroxidation, suggesting that these chelators facilitated formation of reactive oxygen species by iron. The addition of DFX markedly inhibited microsomal lipid peroxidation, with greatest inhibition occurring with microsomes from TCD-treated animals. The results indicate that iron is involved in TCDD-induced lipid peroxidation.     

Lipid peroxidation by bovine heart submitochondrial particles stimulated by 1,1′-dimethyl-4,4′-bipyridylium dichloride (paraquat)

Paraquat enhanced the NADH-dependent lipid peroxidation of bovine heart submitochondrial particles in the presence of ADE-Fe³⁺ chelate. The enhancement at physiological pH was about 3-fold. The pH optimum of the lipid peroxidation was shifted from pH 6.5 by paraquat. The submitochondrial particles catalyzed the reduction of paraquat when incubated anaerobically with NADH, whereas they did not reduce paraquat with succinate. The reduction was inhibited by phydroxymercuribenzoate or amytal, but it was not inhibited by rotenone, antimycin A or cyanide. The respiratory-chain inhibitors similarly affected the NADH-dependent O₂ consumption stimulated by paraquat, indicating that the NADH-dehydrogenase is involved in the reduction of paraquat at a region between the mercurial-sensitive site and the rotenone-sensitive site. The NADH-dependent reduction of ADP-Fe³⁺ chelate, a key step in lipid peroxidation, was stimulated by paraquat about 5-fold at physiological pH. The stimulation could mainly be ascribed to the direct electron transfer from a paraquat radical to the chelate and partially to the electron transfer from O₂^? produced by the reoxidation of the paraquat radical. ADP-Fe²⁺ produced lipid hydroperoxide in liposomes and decomposed cumene hydroperoxide. These reactions, the initiation reaction and the propagation reaction of peroxidation, were stimulated by paraquat. These results suggest that paraquat enhanced lipid peroxidation by stimulating (1) the reduction of ADP-Fe³⁺ chelate, and (2) the ADP-Fe²⁺-dependent initiation and propagation reactions of the peroxidation.     

Inhibition of rat liver microsomal lipid peroxidation by boldine.

The alkaloid boldine, found in the leaves and bark of boldo, was an effective inhibitor of rat liver microsomal lipid peroxidation under a variety of conditions. The following systems all displayed a similar sensitivity to boldine: non-enzymatic peroxidation initiated by ferrous ammonium sulfate; iron-dependent peroxidation produced by ferric-ATP with either NADPH or NADH as cofactor; organic hydroperoxide-catalyzed peroxidation; and carbon tetrachloride plus NADPH-dependent peroxidation. Boldine inhibited the excess oxygen uptake associated with microsomal lipid peroxidation. Thus, boldine was effective in inhibiting iron-dependent and iron-independent microsomal lipid peroxidation, with 50% inhibition occurring at a concentration of about 0.015 mM. Boldine did not appear to react efficiently with superoxide radical or hydrogen peroxide, but was effective in competing for hydroxyl radicals with chemical scavengers. Concentrations of boldine which produced nearly total inhibition of lipid peroxidation had no effect on microsomal mixed-function oxidase activity nor did boldine appear to direct electrons from NADPH-cytochrome P450 reductase away from cytochrome P450. Boldine completely protected microsomal mixed-function oxidase activity against inactivation produced by lipid peroxidation. The effectiveness of boldine as an anti-oxidant under various conditions, and its low toxicity, suggest that this alkaloid may be an attractive agent for further evaluation as a clinically useful anti-oxidant.     

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.     

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.     

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.     

Paraquat-induced alterations of phospholipids and GSSG-release in the isolated perfused rat liver,and the effect of SOD-active copper complexes

The possibility that generation of active oxygen species and lipid peroxidation are involved in paraquat toxicity was examined through the use of the Superoxide dismutase-active, low molecular weight copper complexes Cu(tyr)₂ and Cu-penicillamine. In addition, it was investigated whether the oxidation of glutathione is directly associated with paraquat-induced lipid peroxidation. An increase in diene absorption and a decrease in phospholipids of mitochondria and microsomes isolated after perfusion of rat livers with paraquat could be observed. These effects may be explained by lipid peroxidation. Cu(tyr)₂ abolished these effects of paraquat. In contrast, the increase of GSSG-release into the perfusate upon paraquat-treatment could not be influenced by Cu-penicillamine and was only partially inhibited by Cu(tyr)₂. Therefore the GSSG-release cannot be the result of paraquat-induced generation of O₂^? or lipid peroxidation and seems more likely to be caused by the NADPH depleting action of paraquat. It is proposed that the alterations suggesting lipid peroxidation may only be observed if hepatic glutathione content decreased below a critical value.     

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.     

Evaluation of microsomal pathways of oxidation of alcohols and hydroxyl radical scavenging agents with carbon monoxide and cobalt protoporphyrin IX

Rat liver microsomes catalyze the oxidation of hydroxyl radical scavenging agents by an iron-dependent process, and can oxidize alcohols by pathways dependent on, as well as independent of, .OH. Experiments were carried out to evaluate which microsomal components participate in the production of .OH, and in the two pathways of oxidation of alcohols. Cobalt protoporphyrin IX treatment of rats resulted in a decrease in microsomal oxidation of aminopyrine, .OH scavengers, and alcohols. However, this treatment not only lowered the content of cytochrome P-450, but also decreased the activity of NADPH-cytochrome P-450 reductase. Carbon monoxide, metyrapone and SKF-525A also inhibited the oxidation of aminopyrine but did not affect oxidation of .OH scavengers. Desferrioxamine, a potent iron chelator, inhibited the oxidation of .OH scavengers but not aminopyrine. The oxidation of alcohols was partly sensitive to desferrioxamine and partly sensitive to carbon monoxide, thus showing similarities to the oxidation of .OH scavengers and drugs. These results suggest that the desferrioxamine-sensitive, .OH-dependent pathway of alcohol oxidation is mediated by the reductase, in analogy to results with .OH scavengers, whereas the desferrioxamine-resistant pathway of alcohol oxidation is mediated by cytochrome P-450, in analogy to results with aminopyrine. By the use of desferrioxamine or carbon monoxide, either of the two alcohol-oxidizing pathways can be inhibited independently of each other.     

Antioxidant activity of probucol. Short communication

To evaluate a possible antioxidant activity of probucol on NADPH-dependent microsomal lipid peroxidation and on iron-doxorubicin (adriamycin) complex-induced phospholipid peroxidation, the effects were compared with another powerful antioxidant, 2-OH-estrone. Antioxidant activity of 10 mumol/l probucol in NADPH-dependent microsomal lipid peroxidation corresponded to 4 mumol/l 2-OH-estrone. Together with the results obtained from iron-doxorubicin complex-induced phospholipid peroxidation, it was concluded that probucol is a potent inhibitor of lipid peroxidation and has one-quarter to one-half the antioxidant activity of 2-OH-estrone in vitro.     

Free radicals mediate cardiac toxicity induced by adriamycin

Anthracycline antibiotics, including adriamycin (ADM), are widely used to treat various human cancers, but their clinical use has been limited because of their cardiotoxicity. ADM is especially toxic to heart tissue. The mechanisms responsible for the cardiotoxic effect of ADM have been very/extremely controversial. This review focuses on the participation of free radicals generated by ADM in the cardiotoxic effect. ADM is reduced to a semiquinone radical species by microsomal NADPH-P450 reductase and mitochondrial NADH dehydrogenase. In the presence of oxygen, the reductive semiquinone radical species produces superoxide and hydroxyl radicals. Generally, lipid peroxidation proceeds by mediating the redox of iron. ADM extracts iron from ferritin to form ADM-Fe3+, which causes lipid peroxidation of membranes. These events may lead to disturbance of the membrane structure and dysfunction of mitochondria. However, superoxide dismutase and hydroxyl radical scavengers have little effect on lipid peroxidation induced by ADM-Fe3+. Alternatively, ADM is oxidatively activated by peroxidases to convert to an oxidative semiquinone radical, which participates in inactivation of mitochondrial enzymes or including succinate dehydrogenase and creatine kinase. Here, we discuss the activation of ADM and the role of reductive and oxidative ADM semiquinone radicals in the cardiotoxic effect of this antibiotic.