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.     

Humic acid mediates iron release from ferritin and promotes lipid peroxidation in vitro: a possible mechanism for humic acid-induced cytotoxicity

Humic acid (HA) has been shown to be a toxic factor for many mammalian cells, however the specific mechanism of the cytotoxicity induced by HA remains unclear. From the assessment of its redox properties, HA has been shown to be capable of reducing iron(III) to iron(II) in aqueous conditions over a broad range of pH values (from 4.0 to 9.0). By using thiobarbituric acid-reactive substances as an index, the presence of HA was noted to increase the extent of lipid peroxidation both for linoleic acids and within rat liver microsomes. In addition, the increase in HA-induced lipid peroxidation is partially inhibited by sodium azide (a singlet oxygen scavenger) or disodium 4,5-dihydroxy-1,3-benzene-disulfonic acid (a superoxide scavenger), reflecting the involvement of singlet oxygen and superoxide in the process of lipid peroxidation. The addition of HA into a reaction system has been shown to generate superoxide in a dose-dependent manner by the superoxide dismutase-inhibitable cytochrome c reduction assay. In addition, HA is able to reduce and release iron from ferritin, but this process is partially inhibited by superoxide scavengers. Subsequently, the iron released from ferritin was shown to accelerate the HA-induced lipid peroxidation. From our results we conclude that HA has the ability to reduce and release iron from ferritin storage as well as to promote lipid peroxidation. Therefore, HA coupled with released iron can disturb the redox balance and elicit oxidative stress within a biological system. This may be one of the most important mechanisms for HA-induced cytotoxicity.     

Lipid peroxidation by bleomycin-iron complexes in vitro

Lipid peroxidation catalyzed by bleomycin (BLM)-metal complexes was studied in vitro using arachidonic acid as the substrate. Iron complexes of BLM caused extensive lipid peroxidation, but other metal complexes did not. The lipid peroxidation caused by the iron complexes was inhibited by antioxidants such as dl-alpha-tocopherol, ascorbic acid etc., but not by other scavengers of hydroxyl and superoxide radicals, and of singlet oxygen. Cyanide ion suppressed the lipid peroxidation caused by BLM-Fe(II), but did not suppress the peroxidation activity of BLM-Fe(III). The peroxidation activity of BLM-Fe(II) was lost instantly by pre-incubation of the complex at 37 degrees C before mixing with arachidonic acid, but that of BLM-Fe(III) was not. These results indicate that the active form for the lipid peroxidation derived from BLM-Fe(II) differs from that of BLM-Fe(III).     

Photosensitizing potential of benzanthrone

In skin-photosensitization studies on guinea-pigs benzanthrone induced significant erythema and oedema, depending upon the doses both of benzanthrone and of sunlight or artificially simulated UVA radiation. Maximum sensitization and delayed tanning response on the guinea-pig skin were observed 24-36 hr after irradiation. Photosensitized benzanthrone was found to produce significant amounts of singlet oxygen in vitro, as assessed by the bleaching of N,N-dimethyl-p-nitrosoaniline. As with haematoporphyrin and rose bengal, both of which are potent generators of singlet oxygen, the production of singlet oxygen by benzanthrone was dependent on both the concentration of the test chemical and the dose of solar radiation. Benzanthrone also produced a significant yield of superoxide anion radicals on exposure to simulated solar radiation or sunlight. Photosensitized benzanthrone induced photohaemolysis of rat erythrocytes and lipid peroxidation of erythrocyte ghosts, in vitro, probably largely through involvement of singlet oxygen (1O2). The rate of lipid peroxidation by photosensitized benzanthrone was inhibited by 64-80% with 1,4-diazabicyclo(2,2,2)octane and sodium azide, 15% with superoxide dismutase but was not affected by mannitol and sodium benzoate. Equimolar concentrations of haematoporphyrin and rose bengal also produced considerable photohaemolysis of erythrocytes and lipid peroxidation in erythrocyte ghosts; in both cases rose bengal was the most active and benzanthrone the least active of the three compounds.     

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.     

Lipid peroxidation induced by indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals

Some of the side-effects of using indomethacin (IM) involve damage to the gastric mucosa and liver mitochondria. On the other hand, neutrophils infiltrate inflammatory sites to damage the tissues through the generation of reactive oxygen species by myeloperoxidase. The stomach and intestine have large amounts of peroxidase. These findings suggest that peroxidases are involved in tissue damage induced by IM. To clarify the basis for the tissue damage induced by IM in the presence of horseradish peroxidase (HRP) and H2O2 (HRP-H2O2), lipid peroxidation was investigated. When IM was incubated with liver microsomes in the presence of HRP-H2O2 and ADP-Fe3+, lipid peroxidation was time-dependent. Catalase and desferrioxamine almost completely inhibited lipid peroxidation, indicating that H2O2 and iron are necessary for lipid peroxidation. Of interest, superoxide dismutase strongly inhibited lipid peroxidation, and it also inhibited the formation of bathophenanthroline-Fe2+, indicating that reduction of the ferric ion was due to superoxide (O2-). ESR signals of IM radicals were detected during the interaction of IM with HRP-H2O2. However, the IM radical by itself did not reduce the ferric ion. These results suggest that O2- may be generated during the interaction of IM radicals with H2O2. Ferryl species, which are formed during the reduction of iron by O2-, probably are involved in lipid peroxidation.     

Studies on membrane factors in iron-supported lipid peroxidation

Lipid peroxidation in biomembranes is mediated by free radical reactions. It leads to membrane damage and has been proposed to be associated with the pathogenesis to tissue injuries. Iron is known as a catalyst of lipid peroxidation. Microsomal lipid peroxidation by both NADPH and iron-chelate, such as Fe(3+)-ADP or Fe(3+)-PPi, is believed to be enzymatically associated with iron reduction. On the other hand, the addition of free Fe2+ to microsomes or liposomes produces a lag phase before the maximal rates of lipid peroxidation. We examined the interaction of iron with membrane in iron-supported lipid peroxidation and microsomal membrane components associated with iron reduction in NADPH-supported lipid peroxidation. Iron-supported lipid peroxidation was affected by the surface charges of liposomal membrane. Liposomes containing phosphatidylserine (PS) were most sensitive to iron-supported lipid peroxidation. The effect of PS on iron-supported lipid peroxidation indicates that iron participates in binding to membrane surface charges and also indicates that Fe2+ at high level bound to membranes plays a role in producing a lag phase. The mechanism producing a lag phase in Fe(2+)-PPi-supported lipid peroxidation is discussed. In NADPH-supported lipid peroxidation in microsomes, it seemed unlikely that superoxide may be involved in iron reduction. Alternatively, under anaerobic conditions, NADPH-supported iron reduction in microsomes was not dependent on cytochrome P450 content and not inhibited by CO. A cholate-solubilized fraction of microsomes was applied to a laurate-Sepharose column and an active fraction for lipid peroxidation was obtained. Involvement of a heat-labile component, distinct from cytochrome P450, responsible for iron reduction in microsomal lipid peroxidation was demonstrated.     

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.     

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.     

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.     

Photoirradiation of dehydropyrrolizidine alkaloids--formation of reactive oxygen species and induction of lipid peroxidation

Pyrrolizidine alkaloid (PA)-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and human. PAs require metabolic activation to generate pyrrolic metabolites (dehydro-PAs) that bind cellular protein and DNA, leading to hepatotoxicity and genotoxicity, including tumorigenicity. In this study we report that UVA photoirradiation of a series of dehydro-PAs, e.g., dehydromonocrotaline, dehydroriddelliine, dehydroretrorsine, dehydrosenecionine, dehydroseneciphylline, dehydrolasiocarpine, dehydroheliotrine, and dehydroretronecine (DHR) at 0-70 J/cm² in the presence of a lipid, methyl linoleate, resulted in lipid peroxidation in a light dose-responsive manner. When irradiated in the presence of sodium azide, the level of lipid peroxidation decreased; lipid peroxidation was enhanced when methanol was replaced by deuterated methanol. These results suggest that singlet oxygen is a photo-induced product. When irradiated in the presence of superoxide dismutase, the level of lipid peroxidation decreased, indicating that lipid peroxidation is also mediated by superoxide. Electron spin resonance (ESR) spin trapping studies confirmed that both singlet oxygen and superoxide anion radical were formed during photoirradiation. These results indicate that UVA photoirradiation of dehydro-PAs generates reactive oxygen species (ROS) that mediated the initiation of lipid peroxidation. UVA irradiation of the parent PAs and other PA metabolites, including PA N-oxides, under similar experimental conditions did not produce lipid peroxidation. It is known that PAs induce skin cancer and are secondary (hepatogenous) photosensitization agents. Our results suggest that dehydro-PAs are the active metabolites responsible for skin cancer formation and PA-induced secondary photosensitization.     

Anti-oxidative profile of lobenzarit disodium (CCA)

The effects of lobenzarit disodium (CCA) on various species of activated oxygen were investigated in chemiluminescence experiments. CCA showed a quenching effect against hydroxyl radicals generated by Fenton reaction. The inhibition of CCA was much more intense than that of mefenamic acid which is an anti-inflammatory drug and an analogous compound to CCA. CCA also showed a quenching effect against singlet oxygen generated in enzymatic systems. However, CCA had no effect against superoxide anion radicals generated in the xanthine oxidase-hypoxanthine system. As a model of lipid peroxidation and protein alteration induced by activated oxygen, we examined the auto-oxidation of linolenic acid and the UV irradiation of immunoglobulin G (IgG). CCA inhibited the production of lipid peroxide; however CCA did not show a direct quenching action against lipid radicals which had been previously generated. CCA also inhibited the IgG alteration induced by UV irradiation. These results indicate that CCA has anti-oxidative actions with specificity for activated oxygen species and that CCA protects against lipid and protein damage induced by activated oxygen.     

Effects of (+)-1,2-bis(3,5-dioxopiperazin-1-yl)propane (ADR-529) on iron-catalyzed lipid peroxidation

ADR-529 [(+)-1,2-bis(3,5-dioxopiperazin-1-yl)propane], a nonpolar, cyclic analogue of EDTA, protects against anthracycline cardiotoxicity in vivo. The protective mechanism presumably involves chelation of iron by a hydrolysis product of ADR-529, thus preventing the formation of reactive iron/oxygen species which can damage membrane lipids. We investigated the effects of ADR-529 and its hydrolysis products (the tetraacid and the diacid diamide) on NADPH- and ADP-Fe(3+)-dependent lipid peroxidation of rat liver microsomes and liposomes in the presence of cytochrome P-450 reductase. Hydrolyzed ADR-529 products caused inhibition of lipid peroxidation when in excess of the iron concentration. However, no inhibition of lipid peroxidation was detected by similar concentrations of nonhydrolyzed ADR-529. Microsomes did not affect the inhibition of lipid peroxidation, suggesting that rat liver microsomes do not hydrolyze ADR-529. Similarly, the diacid diamide hydrolysis product of ADR-529 inhibited ferritin- and adriamycin-iron-dependent liposomal lipid peroxidation in a concentration-dependent manner. No correlation between partially reduced oxygen species (O2.- and .OH; as measured by electron spin resonance) and lipid peroxidation (as assayed by malondialdehyde formation) was observed, suggesting that liposomal lipid peroxidation was strictly an iron-dependent phenomenon. These results suggest that inhibition of lipid peroxidation by iron chelation may be related to the protective effects of ADR-529 on in vivo anthracycline toxicity.     

Hydroxyl radical scavenging action of tinoridine

Radical scavenging action of tinoridine, a non-steroidal anti-inflammatory drug with a potent anti-peroxidative activity, was investigated. Tinoridine reduced a stable free radical, diphenyl-p-picryl-hydrazyl, in the molar ratio of about 1:2, indicating its free radical scavenging ability. Tinoridine inhibited the lipid peroxidation in rat liver microsomes induced by xanthine-xanthine oxidase system in the presence of ADP and Fe2+, in which hydroxyl radical (. OH) is formed. Tinoridine was demonstrated to be oxidized in the course of the lipid peroxidation by following the fluorescence derived from the oxidation product of tinoridine. It was also oxidized by the xanthine-xanthine oxidase system in the presence of Fe2+, but its oxidation was slow in the absence of Fe2+ and almost completely inhibited by catalase. Tinoridine was also oxidized by H2O2-Fe2+ system producing . OH (Fenton reaction), but it did not affect the reduction of cytochrome c caused by superoxide radical. These results indicate that tinoridine is able to scavenge . OH and the main active oxygen species responsible for the lipid peroxidation is . OH. The anti-peroxidative and . OH scavenging ability of tinoridine should contribute to its anti-inflammatory action.     

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.     

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.     

Nonenzymatic oxygen activation and stimulation of lipid peroxidation by doxorubicin-copper

Addition of cupric sulfate to neutral solutions of doxorubicin resulted in spectrophotometric, fluorometric, and chromatographic changes indicative of a direct chemical interaction. Associated with these changes was a copper-dependent consumption of dissolved oxygen and a superoxide dismutase-sensitive reduction of ferricytochrome c, suggesting the liberation of superoxide free radicals. Addition of equimolar ethylenediaminetetraacetic acid (EDTA) completely inhibited, but did not reverse, the effect of copper on the spectrophotometric, fluorometric, and chromatographic properties of the drug. EDTA also abolished the copper-stimulated consumption of oxygen and reduction of ferricytochrome regardless of the time of addition. Oxygen-free radical formation by the drug-copper complex was further implicated by the stimulation of lipid peroxidation, which was completely inhibited by adding EDTA. Inhibition by superoxide dismutase, catalase, and dimethyl urea implicates the involvement of assorted oxygen-free radicals in doxorubicin-copper stimulated lipid peroxidation. The data demonstrate that despite the implication of hydrogen peroxide and hydroxyl radicals in doxorubicin-copper stimulated lipid peroxidation, the immediate product of dioxygen reduction by the complex is superoxide-free radicals. The suggested occurrence of doxorubicin-copper complexes in vivo infers that nonenzymatic generation of oxygen-free radicals by the chelate may contribute to the mechanism of toxicity of doxorubicin and related anthraquinone anticancer agents observed clinically.     

Picroliv, picroside-I and kutkoside from Picrorhiza kurrooa are scavengers of superoxide anions.

Picroliv, the active principle of Picrorhiza kurrooa, and its main components which are a mixture of the iridoid glycosides, picroside-I and kutkoside, were studied in vitro as potential scavengers of oxygen free radicals. The superoxide (O2-) anions generated in a xanthine-xanthine oxidase system, as measured in terms of uric acid formed and the reduction of nitroblue tetrazolium were shown to be suppressed by picroliv, picroside-I and kutkoside. Picroliv as well as both glycosides inhibited the non-enzymic generation of O2- anions in a phenazine methosulphate NADH system. Malonaldehyde (MDA) generation in rat liver microsomes as stimulated by both the ascorbate-Fe2+ and NADPH-ADP-Fe2+ systems was shown to be inhibited by the Picroliv glycosides. Known antioxidants tocopherol (vitamin E) and butylated hydroxyanisole (BHA) were also compared with regard to their antioxidant actions in the above system. It was found that BHA afforded protection against ascorbate-Fe(2+)-induced MDA formation in microsomes but did not interfere with enzymic or non-enzymic O2- anion generation; and tocopherol inhibited lipid peroxidation in microsomes by both prooxidant systems and the generation of O2- anions in the non-enzymic system but did not interfere with xanthine oxidase activity. The present study shows that picroliv, picroside-I and kutkoside possess the properties of antioxidants which appear to be mediated through activity like that of superoxide dismutase, metal ion chelators and xanthine oxidase inhibitors.     

Vitamin E and glutathione are required for preservation of microsomal glutathione S-transferase from oxidative stress in microsomes

Glutathione (GSH) inhibited lipid peroxidation induced by NADPH-BrCCl3 in vitamin E sufficient microsomes, but did not in phenobarbital (PB)-treated microsomes (containing about 60% of normal vitamin E) or in vitamin E-deficient microsomes (containing about 30% of normal vitamin E). There was a good correlation between the increased formation of CHCl3 from BrCCl3 in the presence of GSH under anaerobic conditions and the vitamin E level in the microsomes. A normal level of vitamin E in microsomes was thus very important for GSH-dependent inhibition of lipid peroxidation and for the efficient formation of CHCl3 from BrCCl3. Bromosulfophthalein (BSP) eliminated the effects of GSH on lipid peroxidation and CHCl3 formation. The apparent Km and Vmax of substrates for GSH S-transferase were changed by in vivo depletion of vitamin E in microsomes, and the Vmax/Km values were significantly reduced. The enzyme activity in microsomes was inactivated following the loss of vitamin E during in vitro lipid peroxidation, and GSH prevented the loss of vitamin E and protected the enzyme from attack by free radicals. GSH inhibited lipid peroxidation induced by NADPH-Fe2+ and the loss of GSH S-transferase activity during the peroxidation in PB-treated microsomes, but did not in the case of induction by NADPH-BrCCl3. A possible relation between the microsomal GSH S-transferase activity and defense by GSH against lipid peroxidation in microsomes is discussed.     

Antioxidant effects of angiotensin-converting enzyme (ACE) inhibitors: free radical and oxidant scavenging are sulfhydryl dependent, but lipid peroxidation is inhibited by both sulfhydryl- and nonsulfhydryl-containing ACE inhibitors.

With an assay that generates free radicals (FR) through photooxidation of dianisidine sensitized by riboflavin, 4 x 10(-5) M captopril, epicaptopril (SQ 14,534, captopril's stereoisomer), zofenopril, and fentiapril [all sulfhydryl (-SH)-containing angiotensin-converting enzyme (ACE) inhibitors] were shown effective scavengers of nonsuperoxide free radicals whereas non-SH ACE inhibitors were not. Captopril was a more effective FR scavenger at pH 5.0 than at pH 7.5. Captopril (2 x 10(-5) M) also scavenged the other toxic oxygen species hydrogen peroxide and singlet oxygen and inhibited microsomal lipid peroxidation. Finally, captopril reduced the amount of superoxide anion-radical detected after neutrophils in whole blood were activated with zymosan, probably by inhibiting leukocyte superoxide production.