The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

Mechanisms of the initiation of lipid peroxidation in the synaptosomes of marine teleosts

The initial rates of NAD- and NADPH-dependent enzymic and Fe+-ascorbic acid-dependent nonenzymic lipid peroxidation have been measured in synaptosomes from the brain of 4 teleost species. The rates of peroxidation were compared with lipid composition and fatty acid composition of total lipids in order to reveal factors accounting for the intensity of peroxidation in the excitable membranes from the brain of ectotherms. The data obtained indicate that the rates of enzymic lipid peroxidation do not correlate with lipid and fatty acid compositions, depending on the efficiency of production of oxygen in the active form by pyridine nucleotide-dependent enzymic systems. Activation of lipid peroxidation during adaptation of animals to the environment may be considered as one of the mechanisms which account for compensatory changes in fatty acid composition of the membrane lipids.     

The mechanism of liver microsomal lipid peroxidation

In the presence of Fe³⁺ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1,3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. These results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe³⁺ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe²⁺ by oxygen.     

Effect of aminazin on the enzymatic oxidation of lipids]

An inhibitory effect of chlorpromazine on the enzymatic NADPH-dependent lipid peroxidation in rat liver microsomal fraction was found. This inhibition was caused by the 1) antioxidative effect of hydroxy-derivatives appearing during the oxidative metabolism of chlorpromazine with NADPH-dependent microsomal oxygenases, and by the 2) competition for reduced components of electron-carriers between the NADPH-dependent processes: chlorpromazine metabolism and lipids peroxidation.     

The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria

Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.     

Participation of oxygen activated species in enzymatic lipid peroxidation in biomembranes]

The participation of oxygen activated species in the induction of lipid peroxidation (LPO) in the membrane systems containing cytochrome P-450 (liver microsomes) and in the membrane fragments devoid of this hemoprotein (brain and skeletal muscle microsomes) was studied. It was shown that the rate of NADH-dependent LPO does not depend on the presence of hemoproteins and the activity of NADH-specific flavoprotein in the membranes. On the other hand, the microsomal membranes of the liver with high specific contents of b5 and P-450 cytochromes and NADPH-specific flavoprotein, had the highest rates of NADPH-dependent LPO. It was found that the most effective inhibitors of free oxygen activated species in the case of NADPH- and NADH-dependent LPO in the microsomal fractions of liver, brain and skeletal muscles are the superoxide (O ./2) anion radical inhibitors. The singlet oxygen (1O2) quenchers inhibit only NADPH-dependent LPO in the liver, however, in a far lesser degree. The hydroxyl radical (OH) scavengers had no effect on enzymatic LPO in all systems studied.     

Selective effects of some anesthetics and detergents on lipid peroxidation of mouse heart homogenates.

1. The effects of some anesthetics and detergents on the Fe2+/ascorbate-stimulated non-enzymatic lipid peroxidation potential and on the NADPH-dependent enzymatic lipid peroxidation capacity were characterized in mouse heart homogenates. 2. Chlorpromazine turned out to be the most efficient inhibitor, causing a 50% inhibition at a concentration of 0.03 mM in the non-enzymatic assay, and at a concentration of 0.02 mM in the enzymatic assay. 3. Tetracaine was about a 10-times weaker inhibitor with IC50-values of 0.25 mM. High concentration of dibucaine (1 mM) exerted a 60% inhibition in the non-enzymatic assay, but lidocaine and procaine had no prominent effect with the concentrations used. 4. In the non-enzymatic, Fe(2+)-stimulated system, a 50% inhibition was obtained by using SDS, Triton X-100, and deoxycholic acid at concentrations of 0.004, 0.03, and 0.15%, respectively. 5. In the NADPH-dependent enzymatic lipid peroxidation system, corresponding concentrations were 0.02, 0.04 and 0.1%. Deoxycholate and Triton X-100 even stimulated (10-20%) the enzymatic lipid peroxidation at the lowest concentrations (0.005-0.01%). Saponin was the least effective of these detergents. 6. It is suggested that anesthetics and detergents induce structural rearrangements in the myocardiac membranes which result in the unavailability of phospholipid substrates to lipid peroxidation.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-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-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-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-l-yl and substituted triazol-4-yl derivitives.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-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-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-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-l-yl and substituted triazol-4-yl derivitives.     

Cytochrome P-450 involvement in the NADPH-dependent lipid peroxidation in human placental mitochondria

The NADPH-dependent lipid peroxidation in human placental mitochondria has been found to be inhibited strongly by amphenone B, aminoglutethimide and carbon monoxide, inhibitors of cytochrome P-450-mediated reactions, but was hardly affected by respiratory chain inhibitors. Cytochrome c, an exogenous electron acceptor which is known to compete with cytochrome P-450 for the reducing equivalents, showed an inhibitory effect on NADPH-dependent lipid peroxidation. The observed NADPH-dependent superoxide generation was also strongly inhibited by amphenone B and aminoglutethimide. Moreover, the lipid peroxidation in placental mitochondria was demonstrated to be stimulated by xanthine/xanthine oxidase added as superoxide generating system. This peroxidation was not affected by amphenone B and aminoglutethimide. On the other hand, the superoxide dismutase was found to inhibit both the xanthine oxidase- and NADPH-dependent lipid peroxidation. These data provide evidence that cytochrome P-450 is involved in NADPH-dependent mitochondrial lipid peroxidation. It is suggested that superoxide liberated from cytochrome P-450, in combination with iron, may be responsible for initiation of NADPH-dependent lipid peroxidation in human placental mitochondria.     

A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation.

A simplified system, consisting of NADPH, Fe3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris - HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves 1deltag type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with beta-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9,10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals.     

The influence of NADPH-dependent lipid peroxidation on the progesterone biosynthesis in human placental mitochondria.

In an in vitro system consisting of human term placental mitochondria and an NADPH-generating system plus Fe2+, significant lipid peroxidation was observed along with a concomitant inhibition of progesterone biosynthesis. This inhibition could be markedly blocked by Mn2+, superoxide dismutase and dimethylfuran, inhibitors of NADPH-dependent lipid peroxidation. In addition, it has been found that malondialdehyde formation is accompanied by a corresponding decrease in placental mitochondrial cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in cell-free systems. These measurements provide the first evidence that the inhibition of progesterone biosynthesis by a NADPH-dependent lipid peroxidation in placental mitochondria is a consequence of cytochrome P-450 degradation due to lipid peroxidation.     

Antiarrhythmic agents. Scavengers of hydroxyl radicals and inhibitors of NADPH-dependent lipid peroxidation in bovine lung microsomes.

Antiarrhythmic drugs, e.g. lidocaine, quinidine, and procainamide have been suggested as a means of reducing myocardial damage. The mode of action of these drugs have been attributed to their "membrane-stabilizing" properties. However, as tissue ischemia reperfusion is reported to generate toxic species of oxygen, we investigated the oxygen radical scavenging properties of these drugs and their effect on NADPH-dependent lipid peroxidation. These antiarrhythmic drugs are found to be ineffective as superoxide radical scavengers but are potent scavengers of hydroxyl radical with rate constants of 1.8 x 10(10) M-1 s-1, 1.61 x 10(10) M-1 s-1, and 1.45 x 10(10) M-1 s-1 for quinidine, lidocaine and procainamide, respectively, as determined by deoxyribose assay. In EPR study, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, lidocaine, quinidine, and procainamide caused a dose-dependent inhibition of DMPO-OH adduct formation. These drugs also caused a dose-dependent inhibition of NADPH-dependent lipid peroxidation when lung microsomes were incubated with NADPH in presence of Fe(3+)-ADP. We propose that the antiarrhythmic agents exert their beneficial effects, in part, by their ability to scavenge toxic species of oxygen and by reducing membrane lipid peroxidation.     

The role of lipid radicals in electron transport and energy transformation

Data given propose two regimes of lipid radicals and oxygen utilization realized in microsomal and mitochondrial membranes. The first one, lipid peroxidation, i.e. interaction of lipid radicals and oxygen is an empty step. In converting this regime to the functional one NADPH-dependent lipid peroxidation is inhibited. A change of this regime to the functional one in microsome demand the presence of hydroxylation substrates. Setting lipid radical-dependent coupling apparatus on phosphorylation in mitochondria occur in the presence of ADP and Pi-phosphorylation substrates.     

Immunochemical study on the pathway of electron flow in reduced nicotinamide adenine dinucleotide-dependent microsomal lipid peroxidation.

NADH could support the lipid peroxidation of rat liver microsomes in the presence of ferric ions chelated by ADP(ADP-Fe). The reaction had a broad pH optimum (pH 5.8--7.4) and was more active in the acidic pH range. Antibodies to NADH-cytochrome b5 reductase [EC 1.6.2.2] and cytochrome b5 inhibited NADH-dependent lipid peroxidation in the presence of ADP-Fe, whereas the antibody against NADPH-cytochrome c reductase [EC 1.6.2.4] showed no inhibition. These oberservations suggest that the electron from NADH was supplied to the lipid peroxidation reaction via NADH-cytochrome b5 reductase and cytochrome b5. On the other hand, NADPH-supported lipid peroxidation was strongly inhibited by the antibody against NADPH-cytochrome c reductase, confirming the participation of this this flavoprotein in the NADPH-dependent reaction. In the presence of both ADP-Fe and ferric ions chelated by EDTA(EDTA-Fe), NADH-dependent lipid peroxidation was highly stimulated up to the level of the NADPH-dependent reaction. In this case, the antibody against cytochrome b5 could not inhibit the reaction, while the antibody against NADH-cytochrome b5 reductase did inhibit it, suggesting the direct transfer of electrons from NADH-cytochrome b5 reductase to EDTA-Fe complex.     

Attenuation of daunorubicin-augmented microsomal lipid peroxidation and oxygen consumption by calcium channel antagonists.

Daunorubicin (20 microM) stimulated NADPH-dependent microsomal lipid peroxidation about 2-fold over control values and enhanced the rate of oxygen utilization by microsomes. The calcium channel blockers tested inhibited daunorubicin-augmented lipid peroxidation and O2 consumption to varying degrees. Inhibition of daunorubicin-stimulated lipid peroxidation was found to be dose dependent; the IC50 (drug concentration producing 50% inhibition of lipid peroxidation) values for verapamil, nifedipine and diltiazem were approximately 150 microM, 200 microM, and 600 microM respectively. Our in vitro studies suggest that calcium channel antagonists may modulate the free radical-mediated, cardiotoxic effects of daunorubicin.     

Changes in peroxidation under conditions of 3,4-benzypyrene-induced carcinogenesis

The activities of enzymatic systems generating and destroying peroxides and the lipid peroxide content in neoplastic rat liver and 3,4-benzpyrene-induced sarcoma were studied. The tumour was characterized by high activity of glutathione peroxidase and low activity of catalase. No urate- and glycolate oxidases or ascorbat dependent peroxidation of lipids and lipid peroxides were found in the tumour. In the liver of neoplastic animals the activities of glutathione peroxidase and NADPH-dependent system of microsomal phospholipid peroxidation and the lipd peroxides content were increased, whereas the activities of catalase and urate oxidase were decreased.     

NADPH-dependent lipid peroxidation and its effects on aminopyrine N-demethylation in subcellular fractions of human neonatal liver.

NADPH-dependent lipid peroxidation was determined in humans, using subcellular fractions of livers obtained from newborn infants. As reported for other species, activity was concentrated in the microsomal fraction and was similar to that found in the rat. High activity of lipid peroxidation induced by iron decreased aminopyrine N-demethylation and slightly reduced linearity time for the reaction. Compared with the rat, however, human microsomes were more resistant to the effects of lipid peroxidation. If liped peroxidation occurs in vivo it is unlikely to affect drug oxidation to any great degree in human infants.