Oxidation of ethylene glycol to formaldehyde by rat liver microsomes. Role of cytochrome P-450 and reactive oxygen species.

Rat liver microsomes oxidized ethylene glycol to formaldehyde in a NADPH-dependent, carbon monoxide-sensitive manner. Formaldehyde production was inhibited by substrates and ligands for cytochrome P-450 such as aniline, p-nitrophenol, pyrazole, and 4-methylpyrazole, and inhibitors such as tryptamine, cimetidine, and miconazole. The apparent Km for ethylene glycol was about 25 mM and the apparent Vmax was about 6 nmol/min/mg protein. Microsomes isolated from rats treated with pyrazole or 4-methylpyrazole to induce cytochrome P-450IIE1 oxidized ethylene glycol at rates which were about twice those found with control microsomes or microsomes isolated from rats treated with phenobarbital or 3-methylcholanthrene, although significant rates were found with all microsomal preparations. Antibody raised against the pyrazole-induced P-450IIE1 inhibited formaldehyde production from ethylene glycol in microsomes from pyrazole-treated rats. H2O2 itself did not oxidize ethylene glycol to formaldehyde; however, the microsomal reaction was inhibited by catalase or glutathione plus glutathione peroxidase and was stimulated by added H2O2 in the presence of NADPH. Nonheme iron also appeared to be required for ethylene glycol oxidation in view of the inhibition of formaldehyde production by desferrioxamine, EDTA, and DTPA. Microsomal oxidation of ethylene glycol was not sensitive to superoxide dismutase, hydroxyl radical scavengers, or Trolox, suggesting that the oxidant derived from H2O2 and iron and responsible for the production of formaldehyde from ethylene glycol was not superoxide, hydroxyl radical, or lipid hydroperoxide. These results suggest that ethylene glycol is oxidized to formaldehyde by an oxidant derived from H2O2 and nonheme iron, and that cytochrome P-450 may function to generate the H2O2 and to catalyze reduction of the nonheme iron.     

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

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

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

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

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.     

Cause of decrease of ethylmorphine N-demethylase activity of lipid peroxidation in microsomes from the rat, guinea pig and rabbit.

There were marked differences among animal species between NADPH-dependent and ascorbic acid-Fe++-dependent lipid peroxidation. In NADPH-dependent lipid peroxidation, this activity occurred to the greatest extent in rats followed by guinea pigs and rabbits and such was much lower in rabbits than in guinea pigs. On the other hand, rabbit microsomes exhibited higher lipid peroxidation activity than guinea pigs in ascorbic acid plus Fe++ or Fe++-dependent lipid peroxidation although the activity was still lower than in rats. The ascorbic acid plus Fe++-stimulated lipid peroxidation produced a decrease in ethylmorphine N-demethylase activity which was closely related to ethylmorphine-enhanced NADPH-cytochrome P-450 reductase activity but was not related to the change of the apparent content of cytochrome P-450 in all animal species. These results indicate that decrease of NADPH-cytochrome P-450 reductase activity induces a decrease in ethylmorphine N-demethylase activity by lipid peroxidation.     

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.     

Lipid peroxidation of rat erythrocyte membrane induced by adriamycin-Fe3+.

Adriamycin-Fe3+ caused lipid peroxidation of erythrocyte membrane in relation to its concentration. Adriamycin-Fe3+ had a high affinity for membrane and the adriamycin-Fe(3+)-binding membranes membranes was also found to cause lipid peroxidation. Under aerobic conditions, adriamycin-Fe3+ caused a reduction of cytochrome c and ferrous iron formed spontaneously. Superoxide dismutase (EC 1.15.1.1) (SOD) strongly inhibited the reduction of cytochrome c; however, the enzyme promoted formation of ferrous iron independent of enzymatic action. These results suggest that cytochrome c was reduced by superoxide radical (O2-) or an adriamycin-iron-O2 complex such as adriamycin-Fe(3+)-O2-, but not by adriamycin-Fe2+. The ferrous iron chelator bathophenanthroline sulfonate (BPS) completely inhibited oxygen consumption caused by adriamycin-Fe3+, indicating that ferrous iron is absolutely required for the lipid peroxidation. SOD and hydroxyl radical scavengers did not inhibit the lipid peroxidation, indicating that O2- and hydroxyl radical were not involved in membrane peroxidation. The peroxidation reaction was dramatically inhibited by Tris buffer (2-amino-2-hydroxymethyl-1,3-propanediol). However, hydroxyl radical generation and lipid peroxidation in Tris buffer were not related obviously, indicating that Tris did not act as a hydroxyl radical scavenger. The initial rate of TBARS (thiobarbituric acid reactive substances) formation induced by a mixture of adriamycin-Fe3+ and adriamycin-Fe2+ was much faster than that induced by adriamycin-Fe2+ or adriamycin-Fe3+ alone. These results made it became possible to speculate that the lipid peroxidation might be initiated by an adriamycin-Fe(3+)-oxygen-adriamycin-Fe2+ complex.     

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.     

The involvement of cytochrome P-488 and P-450 in NADH-dependent O-demethylation of p-nitroanisole in rat liver microsomes.

These studies have shown that addition of p-nitroanisole to a reaction mixture containing rat liver microsomes resulted in an increase the reoxidation rate of NADH-reduced cytochrome b5. Fortification of rat liver microsomes with partially purified cytochrome b5 produces an increase in both NADPH-dependent and NADH-dependent p-nitroanisole O-demethylation activity. Antiserum to cytochrome P-450 isolated from phenobarbital-treated rat liver microsomes inhibited the NADH-dependent O-demethylation activity as well as the NADPH-dependent O-demethylation activity seen in rat liver microsomes. Addition of either purified cytochrome P-450 or cytochrome P-448 to an incubation mixture containing phenobarbital-treated rat liver microsomes enhanced the NADH-dependent p-nitroanisole O-demethylation activity. These results suggest that NADH-dependent and, in part, NADPH-dependent O-demethylations are catalyzed by cytochrome P-448 and cytochrome P-450 receiving electrons from cytochrome b5.     

Stimulation of the conjugation of lipid dienes in hepatic microsomes by 3,3'-dichlorobenzidine

Pretreatment of male rats with 3,3'-dichlorobenzidine (DCB) resulted in the accumulation of conjugated dienes in lipids from hepatic microsomes. In vitro, these microsomes had 2-fold the NADPH-dependent malondialdehyde (MDA)-forming capacity of microsomes from untreated rats. To determine the mechanisms of the DCB-induced accumulation of diene conjugation, the effects of added DCB on NADPH- or iron + ascorbic acid- (Fe2+-ascorbate-) dependent diene conjugation, oxygen uptake and MDA formation were examined in microsomes from untreated rats in vitro. In the presence of NADPH, added DCB stimulated diene conjugation in microsomal lipids as did in vivo DCB pretreatment but inhibited the uptake of oxygen and the formation of MDA. When Fe2+-ascorbate was substituted for NADPH, the formation of diene conjugation, oxygen uptake, and MDA formation were inhibited by added DCB. The DCB-induced stimulation of diene conjugation, in addition to being strictly NADPH dependent, was carbon monoxide sensitive and was concomitant with the binding of added DCB to microsomal lipids. It is postulated that a metabolite of DCB generated by cytochrome P-450 reacts with membrane lipids both in vivo and in vitro in a manner analogous to the initiation of lipid peroxidation but at the same time prevents the autocatalytic decomposition of the lipids. The DCB-induced diene conjugation is interpreted as predisposing to deleterious changes in microsomes.     

Evaluation of iron binding and peroxide-mediated toxicity in rat hepatocytes.

A novel assay was developed to determine subnanomolar amounts of Fenton-reactive iron (FRI) in biological tissues. FRI represents that pool of iron that is redox active and capable of participating in a model Fenton reaction. The FRI was used to identify a kinetically-defined cellular iron binding site. This site displays positive cooperativity, with apparent kinetic constants of Kd = 10.6 microM, Bmax = 20.7 nmol/mg protein, and the Hill coefficient = 1.4. After addition of exogenous ferrous ammonium sulfate to hepatocytes, binding occurred within a few seconds and was stable for at least an hour. Free extracellular iron, but not bound iron, stimulated lipid peroxidation in hepatocytes. In contrast, bound but not free iron produced a concentration-dependent increase in tert-butyl hydroperoxide (TBH)-mediated toxicity, suggesting the toxicological relevance of bound, rather than free iron. Furthermore, the hydroxyl radical scavengers mannitol and 2-deoxyribose inhibited Fe2/TBH-mediated lipid peroxidation, but not cell killing, suggesting that hydroxyl radical may not be involved in the critical toxic event. The divalent cations Mn2+ and Co2+ inhibited iron-mediated hepatocyte killing in the presence of TBH, but only if added prior to Fe2+. Mn2+, but not Co2+, inhibited Fe(2+)-mediated lipid peroxidation regardless of the order of addition. These results indicate the existence of a specific, kinetically-defined cellular iron binding site. Such binding is involved in peroxide-mediated toxicity, but independent of lipid peroxidation. The specific nature of this site and involvement with other forms of chemical intoxication or cellular iron homeostasis are unknown.     

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.     

Antioxidant effects of albumin-bound sulfur in lipid peroxidation of rat liver microsomes.

The antioxidant potential of albumin-bound sulfur (SBA) was investigated in rat liver microsomes using lipid peroxidation systems in vitro. Sulfur bound to protein is a reduced metabolite which is produced from cystine by gamma-cystathionase. Lipid peroxidation was induced either chemically by ferrous ions and ascorbate or enzymatically by carbon tetrachloride or tert-butyl hydroperoxide as indicated by the increase in thiobarbituric acid reactive substances (TBA-RS) and oxygen consumption. Although the antioxidant effect of SBA was weak on the non-enzymatic lipid peroxidation system, the addition of SBA significantly inhibited TBS-RS formation and oxygen consumption compared with non-treated bovine serum alubumin (BSA) in a microsomal lipid peroxidation system induced enzymatically. The sulfur bound to albumin disappeared during incubation with liver microsomes. However, slight differences in the disappearance were observed depending on whether or not lipid peroxidation was induced in the enzymatic systems. In the CCl4-induced lipid peroxidation system, the cytochrome P-450 level was significantly decreased by the addition of SBA. Therefore, in cytochrome P-450 dependent lipid peroxidation system, the potential effects of sulfur bound to albumin are due to an inhibition of cytochrome P-450 rather than by the oxidation itself caused by radical trapping.     

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.     

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.     

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.     

Protective role of ascorbic acid against asbestos induced toxicity in rat lung: in vitro study

Asbestos fibers adsorb cytochrome P-450 and P-448 proteins from rat lung micosomal fractions and liberate heme from cytochrome P-448 on prolonged incubation in vitro. further, fibers, decrease the activities of benzo(a)pyrene hydroxylase and glutathione-S-transferase in microsomal and cytosolic fractions respectively. Mineral fibers also stimulate both the enzymatic (NADPH-induced) and non-enzymatic (Fe2(+)-induced) lipid peroxidation in microsomal fractions. Preincubation of microsomal and cytosolic fractions with a physiological concentration of ascorbic acid ameliorates, to a large extent, the changes induced by asbestos fibers.     

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.     

Suppressive effect of bifemelane on lipid peroxidation in rat liver

The effects of Bifemelane (BF) on lipid peroxidation, the activities of hepatic drug metabolizing enzymes, and the function of cell membranes were examined in rats. In the liver ischemia-reperfusion model, BF suppressed the elevation of the lipid peroxidation level during the period of reperfusion. BF did not exhibit a radical-trapping action using a stable free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), which was estimated by electron spin resonance (ESR). BF remarkably inhibited NADPH-dependent lipid peroxidation in vitro. BF had no effect on the contents of cytochrome P-450 and b5 and the activities of NADPH cytochrome P-450 reductase and Cu,Zn-superoxide dismutase (SOD). BF suppressed phorbol myristate acetate (PMA)-induced superoxide formation of polymorphonuclear leukocytes (PMNs), protected hypotonic hemolysis of erythrocyte and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and serum phospholipase A activity. These results suggest that BF has neither radical-trapping activity nor any influence on the drug metabolizing enzymes, but BF has a membrane-stabilizing action and it attributes to the suppressive effect of lipid peroxidation.     

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.