[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.

Paraquat-induced membrane dysfunction in pulmonary microvascular endothelial cells.

Membrane dysfunction monitored by lactate dehydrogenase release from cultured pulmonary microvascular endothelial cells of pigs, which were exposed to paraquat at different concentrations (0.1-2 mM), was examined. Paraquat caused a time-dependent increase in lactate dehydrogenase release. Lactate dehydrogenase releases after 72 hr, 32, 58, and 84% by 0.1, 0.5, and 2 mM paraquat, respectively, were well correlated with cell viability measured by cell adherence. In contrast, reductions of two tetrazolium compounds were depleted profoundly by 72 hr after exposure to 0.5 mM paraquat, suggesting depletion of intracellular reductive substances. Extracellular hydrogen peroxide began to significantly increase 56 hr or 32 hr after exposure to 0.5 mM or 1.5 mM paraquat, respectively, preceding the initial increase of lactate dehydrogenase release (64 hr by 0.5 mM or 48 hr by 1.5 mM). Lactate dehydrogenase release 72 hr after exposure to 0.5 mM paraquat was prevented strongly by catalase (1000 units/ml), but weakly by superoxide dismutase (1000 units/ml). These enzymes failed to restore the reduced acid phosphatase activity. Also, 0.1 mM desferal or alpha,alpha'-dipyridyl protected lactate dehydrogenase release. Similarly, 1 mM thiourea or dimethylthiourea, and 0.5 mM alpha-tocopherol or trolox, were effective, but diethylenetriaminepentaacetic acid (0.1 mM) and probucol (5 or 10 microM) were ineffective. Exposure of 0.5 or 1.5 mM paraquat suppressed levels of lipid peroxidation. These results indicate that membrane dysfunction by paraquat is ascribed to an iron-catalyzed reaction of extracellularly increased hydrogen peroxide. A deleterious species for the membrane dysfunction is discussed.

Superoxide production from paraquat evoked by exogenous NADPH in pulmonary endothelial cells.

Superoxide production from paraquat in a pulmonary microvascular endothelial cell (PMEC) suspension was demonstrated using 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one (MCLA), a chemiluminescence probe, to detect superoxide anions. Increased rates of superoxide production from paraquat, which were sensitive to superoxide dismutase (SOD), required the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the reaction medium, and occurred instantaneously after the addition of NADPH, which is impermeable to cell membranes. NADH as an electron donor was not as effective, and xanthine or succinate had no influence. Paraquat was anaerobically reduced in the presence of NADPH and PMECs to yield a one-electron reduced radical, and the reduction was inhibited by NADP+. Diphenyleneiodonium, an inhibitor of flavoprotein reductases, also markedly inhibited both paraquat reduction and superoxide production. These results indicate that NADPH-dependent superoxide production from paraquat probably occurs by a flavoprotein with NADPH-dependent reductase activity in cell membranes. NADPH-dependent superoxide production from paraquat was also reproduced using adherent PMECs on wells. Under these conditions, superoxide production was enhanced with agonists, including interleukin-1beta, A23187, and phorbol 12-myristate 13-acetate. The effect of the former two was blocked with staurosporine, while the latter's effect was suppressed with calyculin A.

The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyra z in-3-one (MCLA), a chemiluminescence probe to detect superoxide anions.

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O2.-, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2'-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe2+-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.

Lucigenin reduction by NADPH-cytochrome P450 reductase and the effect of phospholipids and albumin on chemiluminescence.

To assess lucigenin, a chemilumigenic probe, as a detector of superoxide anion in microsomes, NADPH oxidation, lucigenin disappearance, and chemiluminescence in a system including purified NADPH-cytochrome P450 reductase were examined. NADPH oxidation was increased by adding lucigenin, and concurrently, its disappearance and oxygen consumption were also stimulated. Chemiluminescence, which is negligibly emitted in the presence of the reductase alone, was remarkably amplified with phospholipids and albumin. Menadione inhibited lucigenin disappearance resulting in suppression of chemiluminescence. Lucigenin chemiluminescence measured in microsomes appears not to reflect direct superoxide anion production from microsomal components and from quinones, such as menadione.

Enzymatic and molecular aspects of the antioxidant effect of menadione in hepatic microsomes.

The enzymatic features and molecular species of the inhibitory action of menadione on lipid peroxidation in rat liver microsomes were examined. In an ascorbate-supported system or a NADH-supported reconstituted system containing NADH-cytochrome b5 reductase and cytochrome b5, menadione was not an inhibitor of lipid peroxidation at pH 7.5, while some antioxidant ability was observed at lower pH ranges. Lipid peroxidation in the presence of menadione in the NADH-supported reconstituted system at pH 7.5 was markedly inhibited by adding lipoamide dehydrogenase. NAD(P)H-supported lipid peroxidation in microsomes with increased DT-diaphorase activity from 3-methylcholanthrene-treated rats was highly susceptible to menadione. These inhibitions were abolished by dicoumarol, an inhibitor of DT-diaphorase. Cumene hydroperoxide-dependent lipid peroxidation in microsomes, with desferal and NADP+ to prevent nonheme iron-dependent reactions and oxygen radical generation, was inhibited by menadione in the presence of NADPH, and the inhibition was also more effective in the microsomes with increased DT-diaphorase activity. Menadiol reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH) in ethanol at a molar ratio of DPPH/menadiol at 1.9. In an iron-supported reconstituted enzymatic or a nonenzymatic system at pH 7.5, menadiol showed an antioxidant effect at an early stage, followed by a prooxidant effect, which was prevented by SOD, probably by protecting menadiol autooxidation. These results show that menadione exerts an antioxidant effect through participation of microsomal DT-diaphorase by generating menadiol with a radical scavenging ability, while menadiol also has a prooxidant property.

Effects of membrane charges and hydroperoxides on Fe(II)-supported lipid peroxidation in liposomes.

The processes in producing a lag phase in Fe2+-supported lipid peroxidation in liposomes were investigated. Incorporation of phosphatidylserine (PS) or dicetyl phosphate (DCP) into phosphatidylcholine [PC(A)] liposomes, which have arachidonic acid, produced a marked lag phase in Fe(2+)-supported peroxidation, where PS was more effective than DCP. Phosphatidylcholine dipalmitoyl [PC(DP)] with a net-neutral charge was still effective in producing a lag phase, though weak. Increasing concentrations of PS, DCP, and PC(DP) prolonged the lag period. Initially after adding Fe2+, slight oxygen consumption occurred in PC(A)/PS liposomes including hydroperoxides, followed by a lag phase. An increase in the hydroperoxide resulted in a shortening of the lag period. The initial events of Fe2+ oxidation accompanied by oxygen consumption were dependent on the hydroperoxide content, but significant changes in diene conjugation and hydroperoxide levels at this stage were not found. The molar ratios of both disappeared Fe2+ and consumed O2 to preformed hydroperoxide in liposomes with or without tert-butylhydroxytoluene were constant, regardless of the different amounts of lipid hydroperoxides. The antioxidant completely inhibited the propagation of lipid peroxidation in the lipid phase, following a lag phase. In a model system containing 2,2'-azobis (2-amidinopropane) dihydrochloride, Fe2+ were consumed. We suggest that Fe2+ retained at a high level on membrane surfaces play a role in producing a lag phase following the terminating behavior of a sequence of free radical reactions initiated by hydroperoxide decomposition, probably by intercepting peroxyl radicals.

The reducing ability of iron chelates by NADH-cytochrome B5 reductase or cytochrome B5 responsible for NADH-supported lipid peroxidation.

We studied iron reduction, NADH oxidation, and lipid peroxidation in the presence of iron chelates with a chelator, such as nitrilotriacetate, ADP, citrate, and pyrophosphate, in NADH-supported reconstituted system. The results showed the selectivity of NADH-cytochrome b5 reductase or cytochrome b5 towards iron chelates and the subsequent ability of this system to promote peroxidation. The lipid peroxidation was partially inhibited by superoxide dismutase. In the presence of any iron chelate, hydrogen peroxide was produced in the systems including the reductase, and the production was accompanied with an increase in NADH oxidation.

A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation.

This study was conducted to determine whether a factor responsible for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in rat liver microsomes is involved in iron reduction by cooperation with NADPH-cytochrome P450 reductase. Under anaerobic conditions, NADPH-dependent reduction of ferric pyrophosphate in microsomes was not dependent on cytochrome P450 levels and was not inhibited by carbon monoxide (CO). All of the iron complexes with chelators such as adenosine 5'-diphosphate, pyrophosphate, nitrilotriacetate, oxalate or citrate were reduced in microsomes, although in the reconstituted system containing purified NADPH-cytochrome P450 reductase little or no iron reduction was found. A cytochrome P450-free fraction from a cholate-solubilized preparation of microsomes after passage through a laurate sepharose column was required for reduction of iron pyrophosphate in the reconstituted system leading to lipid peroxidation. The iron reduction was not inhibited by CO and was destroyed by heat treatment or trypsin digestion of the fraction. All iron complexes were reduced in the presence of the fraction, using a reducing equivalent of NADPH via NADPH-cytochrome P450 reductase. The results indicate that a heat-labile component, which is probably a protein distinct from cytochrome P450, is associated with iron reduction responsible for lipid peroxidation in microsomes.

Mechanism of the biphasic effect of ethylenediaminetetraacetate on lipid peroxidation in iron-supported and reconstituted enzymatic system.

The biphasic action of ethylenediaminetetraacetate (EDTA), depending on its concentration, on lipid peroxidation was examined in an iron-supported and reconstituted enzymatic system. In the presence of NADPH-cytochrome P450 reductase and NADPH, Fe(3+)-PPi or Fe(3+)-ADP, though not reducible in the absence of EDTA, was markedly reduced with increasing concentration of EDTA. Lipid peroxidation, in the reconstituted system containing negatively charged liposomes, showed the maximal rate at 0.5 molar ratio of EDTA/iron, but no peroxidation occurred in positively charged liposomes, suggesting production of a positively charged iron complex as the prooxidant. Isotachophoresis indicated production of net-negative charge, EDTA-Fe(3+)-PPi complex, from Fe(3+)-PPi and EDTA at 1.1 ratio of EDTA/iron. The complex quenched Fe(2+)-PPi-supported lipid peroxidation. We suggest that EDTA-iron complexes of different charges are generated, depending on the amount of EDTA in the enzymatic system and, consequently, there is a switch between prooxidant and inhibitory effect at some critical ratio of EDTA/iron.

Antioxidant mechanism of Mn(II) in phospholipid peroxidation.

Antioxidant action of Mn2+ on radical-mediated lipid peroxidation without added iron in microsomal lipid liposomes and on iron-supported lipid peroxidation in phospholipid liposomes or in microsomes was investigated. High concentrations of Mn2+ above 50 microM inhibited 2,2'-azobis (2-amidinopropane) (ABAP)-supported lipid peroxidation without added iron at the early stage, while upon prolonged incubation, malondialdehyde production was rather enhanced as compared with the control in the absence of Mn2+. However, in a lipid-soluble radical initiator, 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN)-supported lipid peroxidation of methyl linoleate in methanol Mn2+ apparently did not scavenge lipid radicals and lipid peroxyl radicals, contrary to a previous report. At concentrations lower than 5 microM, Mn2+ competitively inhibited Fe(2+)-pyrophosphate-supported lipid peroxidation in liposomes consisting of phosphatidylcholine with arachidonic acid at the beta-position and phosphatidylserine dipalmitoyl, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in the presence of iron complex in microsomes. Iron reduction responsible for lipid peroxidation in microsomes was not influenced by Mn2+.

Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation.

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation.

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.

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.

Bromosulfophthalein abolishes glutathione-dependent protection against lipid peroxidation in rat liver mitochondria.

The effect of bromosulfophthalein (BSP) on GSH-dependent protection against lipid peroxidation in rat liver mitochondria was examined. Mitochondrial lipid peroxidation induced by ascorbate-Fe2+ was prevented by GSH, and addition of BSP abolished the protective effect of GSH. The effect of BSP was apparently not due to causing disappearance of GSH from the reaction mixture by interacting directly with GSH. BSP strongly inhibited the mitochondrial GSH S-transferase activity rather than the GSH peroxidase activity. Ascorbate-Fe2+-induced lipid peroxidation in mitochondria without addition of GSH was also stimulated to some extent by BSP, and the stimulation seems likely to be due to abolition of the inhibitory effect of endogenous GSH. GSH could not be replaced as an inhibitor of lipid peroxidation by cysteine, beta-mercaptoethanol, or dithiothreitol. The inhibitory effect of GSH on lipid peroxidation was not observed in vitamin E-deficient mitochondria. No inhibitory effect of exogenous vitamin E was demonstrated either in vitamin E-deficient mitochondria or in vitamin E-sufficient mitochondria in the presence of BSP, whether GSH was added or not. These results indicate that a mitochondrial GSH-dependent factor which inhibits lipid peroxidation requires vitamin E to exert its function. It is suggested that mitochondrial GSH S-transferase(s) may be responsible for GSH-dependent inhibition of lipid peroxidation in mitochondria, probably by scavenging lipid radicals.