Inhibition Of Microsomal Lipid Peroxidation Research Articles

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by nitrofuran compounds.

(5-Nitro-2-furfurylidene)amino compounds bearing triazol-4-yl, benzimidazol-1-yl, pyrazol-1-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-1-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-1-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-1-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-1-yl and substituted triazol-4-yl derivatives.

Inhibition of microsomal lipid peroxidation by α-tocopherol and α-tocopherol acetate

1. The antioxidant effects of alpha-tocopherol and alpha-tocopherol acetate were assayed for the (a) oxygen uptake, (b) chemiluminescence and (c) malondialdehyde formation, of tert-butyl hydroperoxide-supplemented rat liver microsomes. 2. Oxygen uptake was inhibited 60% by both alpha-tocopherol and alpha-tocopherol acetate with the half-maximal effect at 5 nmol tocopherol/mg protein. Chemiluminescence and malondialdehyde formation were equally inhibited 35% by both tocopherols with half-maximal effects at 2 nmol tocopherol/mg protein. 3. The rate of O2 uptake by tocopherol-supplemented microsomes was dependent on O2 concentration. A 60% inhibition by 5 nmol tocopherol/mg protein at 0.2 mM O2 is decreased to 5% inhibition at 0.6 mM O2. 4. The inhibition of O2 uptake, chemiluminescence and malondialdehyde formation indicate that both alpha-tocopherol and alpha-tocopherol acetate have similar effects as free radical traps in the hydrophobic domain of biomembranes. The different inhibition observed at different O2 concentrations indicate competition between vitamin E and O2 by unoxygenated lipid radicals.

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

The lipophilic o-naphthoquinones β-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 α-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.

Inhibition of Lipid Peroxidation by Spin Labels: Relationships between structure and function

Inhibition of lipid peroxidation by nitroxide radicals and their corresponding hydroxylamines was investigated. The nitroxides were either oxazolidines or piperidines, differing in substitution of the backbone of the molecule (a five or six-membered ring structure, respectively). Concentration requirements for 50% inhibition of microsomal lipid peroxidation varied from 340 to 6 microM for the nitroxides, and from 120 to 3 microM for the hydroxylamines, correlating with lipophilicity and chemical structure. Intramembrane concentrations required for 50% inhibition was independent of lipophilicity when peroxidation was initiated with ADP-Fe2+ but increased with lipophilicity when peroxidation was initiated with t-butylhydroperoxide. During studies of the kinetics of the inhibition, two modes were seen: a delay or a decreased rate of the process. The former mode was seen with the more lipophilic inhibitors. The mechanism of inhibition was similar for all nitroxides and consisted of the following three major components: blocking of primary initiation, prevention of secondary (peroxide-dependent) initiation, and scavenging of various lipoid radicals in the membrane, the major mode of action of the hydroxylamines. Inhibitory efficiency was interpreted in terms of steric hindrance, diffusibility, regeneration of inhibitor, and ability to interact with hydrophilic sites in a hydrophobic environment.

Halothane-Induced Liver Injury as a Consequence of Enhanced Microsomal Lipid Peroxidation in Guinea Pigs

We investigated the role of microsomal lipid peroxidation in halothane hepatotoxicity in guinea pigs. Animals were exposed to halothane, isoflurane or enflurane. Enhancement of microsomal lipid peroxidation was specific to halothane. The time-course of lipid peroxidation and hepatic damage following a single exposure to halothane was investigated by measuring the thiobarbituric acid (TBA)reactive products, serum transaminase activity, reduced glutathione (GSH) concentration and histopathological examination. Microsomal lipid peroxidation was enhanced most rapidly and preceded GSH depletion and hepatic injury. Metyrapone, an inhibitor of cytochrome P-450, and N-tert-butyl-alpha-phenylnitrone (BPN), a radical trapping agent, inhibited halothane-induced lipid peroxidation and the incidence and severity of liver injury. The metabolism of halothane was considered to be inhibited by metyrapone and the reactivity of radical intermediates was considered to be decreased by BPN. Microsomal lipid peroxidation was initiated by radical metabolites of halothane but did not result from GSH depletion. Inhibition of microsomal lipid peroxidation reduced the halothane hepatotoxicity. These results demonstrated that the halothane-induced liver injury was caused by microsomal lipid peroxidation in guinea pigs.

Effects of aurothioglucose on iron-induced rat liver microsomal lipid peroxidation.

Aurothioglucose (ATG), an inhibitor of selenium-dependent glutathione peroxidase activity, at a concentration of 100 microM, strongly increases lipid peroxidation of rat liver microsomes exposed to either ferrous ion (10 microM) or the combination of ferric ion (10 microM) and ascorbic acid (500 microM), in the presence of reduced glutathione (GSH, 800 microM). This effect was not achieved using heat-inactivated microsomes and was dependent on the presence of GSH. ATG did not affect the lag period associated with ascorbic acid/ferric ion-induced microsomal lipid peroxidation (previously attributed to an undefined GSH-dependent microsomal agent), but did increase the rate of peroxidation subsequent to the lag period. The potent GSH-dependent inhibition of microsomal lipid peroxidation by cytosol (10% of total volume) was completely reversed by ATG (100 microM). ATG similarly reversed an inhibition of phosphatidylcholine hydroperoxide-dependent liposomal peroxidation that has been attributed to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme distinct from the classical glutathione that cannot utilize intact phospholipids. ATG inhibited, in addition to the classical selenium-dependent glutathione peroxidase, both cytosolic and microsomal (basal and N-ethyl maleimide-stimulated) glutathione S-transferase activities with greater than 80% inhibition achieved at 100 microM ATG. ATG, at concentrations up to 250 microM, did not inhibit PHGPX activity measured by the coupled-enzyme method in the presence of Triton X-100 (0.1%).(ABSTRACT TRUNCATED AT 250 WORDS)

PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE

Abstract “Phospholipid Hydroperoxide Glutathione Peroxidase” (PHGPX) is a new selenoenzyme recently identified and purified from mammalian tissues that reduces membrane hydroperoxides. This monomeric enzyme (M.W. 21,000), different from all previously known glutathione peroxidases and glutathione transferases, has been purified from different mammalian tissues and accounts for the cytosolic inhibition of microsomal lipid peroxidation induced by NADPH or ascorbate and iron. A selenocysteine is present at the active site, as suggested by the kinetics of the inhibition in the presence of iodoacetate. The kinetics, as in the case of GPX, is compatible with a ter-uni ping pong mechanism. The kinetic comparison between PHGPX and GPX in the presence of different substrates and detergents, indicates that, while GPX is more active on soluble hydrophilic substrates, PHGPX is more active on the hydroperoxides in the membranes.

Inhibition of microsomal lipid peroxidation by naphthoquinones: Structure-activity relationships and possible mechanisms of action

Menadione (2-methyl-1,4-naphthoquinone) is a remarkably potent inhibitor of microsomal lipid peroxidation, effective at submicromolar concentrations. Its possible mechanism of action and the relationship between naphthoquinone structure and antioxidant activity were the topics of this investigation. In the microsomal lipid-peroxidizing system dependent on NADPH and ferric pyrophosphate, menadione, at concentrations of 50 μ m or higher virtually eliminated the accumulation of malondialdehyde and lipid hydroperoxides. In the NADPH-independent, cumene hydroperoxide dependent system, menadione was also an effective antioxidant, but only in the presence of reducing equivalents. These and other observations indicate that a reduced form of menadione, either the hydroquinone or semiquinone, is the active antioxidant, and suggest that it may trap hydroperoxy radicals, alkoxy radicals, or other free radicals involved in propagating lipid peroxidation. Moreover, these results show that electron diversion per se cannot account for the antioxidant effects of menadione. A comparison of the antioxidant activities of eight 1,4-naphthoquinones indicated that methyl substitution of C-2, lack of steric hindrance at C-3 or C-5, and (in the case of weak acids) a relatively high p K a are favorable structural features associated with strong antioxidant activity.

Inhibition of in vitro microsomal lipid peroxidation by isoflavonoids

In a comparative study the inhibition of microsomal lipid peroxidation induced by an Fe 2+-ADP-complex and NADPH by naturally occurring isoflavones and their reduced derivatives (isoflavonones and isoflavans) has been examined. It is found that the isoflavanones are more active than the parent isoflavones and the isoflavans are by far the most potent inhibitors. In our in vitro test system 6,7,4′-trihydroxy- and 6,7-dihydroxy-4′-methoxyisoflavans ( ic 50 values 1.3×10 −6 and 1.1×10 −6mol/l respectively) surpass the inhibitory effect of α-tocopherol, (+)-cyanidanol-3 and BHA (butylated hydroxyanisole). In order to establish a structure-activity relationship, a few more isoflavans have been included in the investigation.

Inhibition of microsomal lipid peroxidation by cytosolic protein in presence of ADP and high concentration of Fe 2+

Microsomal lipid peroxidation induced by NADPH, but not by ascorbate, was found to be inhibited by liver cytosol. This inhibition was not dependent on glutathione and was enhanced by ADP in presence of Fe 2+ at a concentration of 50 μM or higher. ATP was also effective, but not AMP or cyclic AMP. The cytosolic factor appeared to be a protein as it was heat-labile (> 70°C), was non-dialyzable and was precipitated by ammonium sulfate and acetone. It was stable for several months in frozen state and also when heated at 50°C for 10 min. The inhibition by the cytosolic protein was obtained by producing a lag in the activity of lipid peroxidation and was reversed by ceruloplasmin but not by catalase, cytochrome c, hemoglobin or superoxide dismutase. This inhibitory effect by cytosol was limited to formation of lipid peroxides whereas oxygen uptake and NADPH oxidation remained unaffected. Regulation of lipid peroxidation by nucleotide-Fe complexes and cytosolic proteins is indicated by these studies.

Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2.

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.

Protection against lipid peroxidation by a microsomal glutathione-dependent labile factor

Glutathione (GSH) protects rat liver microsomes against ascorbic acid (0.2 mM)/ferrous iron (10 μM)-induced lipid peroxidation for some time. The inhibitory effect of GSH is concentration-dependent (0.1–1.0 mM). Our data suggest that GSH acts by preventing initial radical formation rather than via radical scavenging or GSH—peroxidase activity. A labile GSH-dependent factor is involved in the inhibition of microsomal lipid peroxidation by GSH, inasmuch as heating the microsomes abolishes the GSH effect. We found that besides heating, lipid peroxidation also destroys the GSH-dependent factor. Consequently, continuous radical stress will produce lipid peroxidation, despite the presence of GSH. Moreover, a detrimental effect of in vivo-induced lipid peroxidation (CCl 4-treatment) on the GSH-dependent factor was observed. The implications of the present data for the genesis of and the protection against peroxidative damage are discussed.

Levamisole inhibition of microsomal lipid peroxidation as related to its sulfhydryl metabolite dl-2-oxo-3-(2-mercaptoethyl)-5-phenylimadazolidine.

Levamisole was previously shown to protect rat liver microsomes from lipid peroxidation induced by ADP-Fe and either NADPH, ascorbate, or X irradiation. The present experiments provide information about the mechanism of protection. Incubation of levamisole with a microsomal system containing ADP-Fe and NADPH resulted in protection of sulfhydryl groups, whereas reaction of levamisole with ascorbate (nonenzymatic system) indicated generation of a sulfhydryl metabolite. Production of a sulfhydryl metabolite of levamisole, dl-2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI), in either the enzymatic or nonenzymatic system was demonstrated by gas chromatography-mass spectrometry. While levamisole acts as an antioxidant at concentrations of 1.0 and 2.0 mM, OMPI had an enigmatic effect on microsomal lipid peroxidation induced enzymatically or nonenzymatically. OMPI exhibited a biphasic effect: at concentrations below 25 microM a prooxidant effect was observed, and at concentrations exceeding 50 microM an antioxidant effect was observed. The data suggest that the inhibition of microsomal lipid peroxidation by levamisole is due to the generation of a sulfhydryl metabolite and that the active intermediate is probably OMPI.

Effect of dietary antioxidants and phenobarbital pretreatment on microsomal lipid peroxidation and activation by carbon tetrachloride

The effect of the dietary antioxidants, vitamin E and selenium, and the effect of phenobarbital pretreatment on in vitro NADPH-dependent microsomal lipid peroxidation and the activation of microsomal lipid peroxidation by CCl 4 were studied. The rate of microsomal lipid peroxidation decreased as a function of dietary anti-oxidant, while the degree of CCl 4 activation increased. Phenobarbital pretreatment diminished the antioxidant inhibition of microsomal lipid peroxidation found with microsomes from rats fed high levels of antioxidant. Phenobarbital pretreatment lowered the extent of lipid peroxidation as measured by malonaldehyde production but had little effect on the rate of lipid peroxidation as measured by oxygen uptake. The kinetics of lipid peroxidation and the stoichiometry of the reaction were assessed as a function of dietary antioxidant. The findings suggest that at low microsomal antioxidant concentrations, the lipid peroxidation reaction occurs at a maximal rate dependent upon some rate-limiting step, such as the reduction of Fe +3, which is unaffected by CCl 4 addition. Conversely, at high microsomal antioxidant concentrations, the antioxidant termination reactions appear to determine the overall reaction rate.