Effect of antioxidants on adriamycin-induced microsomal lipid peroxidation

Adriamycin (25 μM) stimulated NADPH-dependent microsomal lipid peroxidation about fourfold over control values. The tested antioxidants, zinc, superoxide dismutase, vitamin E, and desferrioxamine (Desferal) inhibited Adriamycin-enhanced lipid peroxidation to varying degrees. Others antioxidants, e.g., glutathione, catalase, and selenium, were found to have no effects. Our in vitro studies suggest that adriamycin effect is mediated by a complex oxyradical cascade involving superoxide, hydroxyl radical, and small amounts of iron.     

Effect of lecithin on liver microsomal lipid peroxidation]

The effect of exogeneous (egg) lecithin on peroxidation of microsomal lipids was studied with the view of elucidating the role of various components of lipid substrate in the overall oxidation rate of the lipids. The following processes were studied a) NADPH-dependent microsomal lipid peroxidation in the presence of lecithin; b) ascorbate-dependent microsomal lipid peroxidation in the presence of lecithin; c) oxidation of lipid mixture, isolated from the microsomes, and that of lecithin in the presence of the Fe2+ + ascorbate system; 4) oxidation of lecithin induced by the Fe2+ + ascorbate system. It was found that in the presence of exogeneous lecithin the oxidation of microsomal lipids in inhibited, which is probably due to the peculiarities of lecithin oxidation. It was shown that the specific rate of lecithin oxidation is decreased with an increase in lecithin concentration. Possible mechanisms of lecithin effect on microsomal lipid peroxidation are discussed.     

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 $\text{in}$$\text{vitro}$ NADPH-dependent microsomal lipid peroxidation and the activation of microsomal lipid peroxidation by CCl₄ were studied. The rate of microsomal lipid peroxidation decreased as a function of dietary anti-oxidant, while the degree of CCl₄ 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 CCl₄ addition. Conversely, at high microsomal antioxidant concentrations, the antioxidant termination reactions appear to determine the overall reaction rate.     

Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.     

Nitrofuran inhibition of microsomal lipid peroxidation

Two nitrofuran compounds, nifurtimox and nitrofurantoin, inhibited in a concentration-dependent manner the NADPH-, iron-induced lipid peroxidation in rat liver microsomes, as shown by the decreased rate of MDA accumulation. Other nitro compounds (benznidazole and chloramphenicol) were relatively inactive. Nifurtimox inhibition affected polyenoic fatty acids and cytochrome P-450 degradation that follows lipid peroxidation. The ascorbate- or tert-butyl hydroperoxide-dependent lipid peroxidations were much less inhibited than the NADPH-dependent one. Nifurtimox and nitrofurantoin, but not benznidazole and chloramphenicol, strongly stimulated the microsomal NADPH-oxidase activity, thus supporting electron diversion, as the main cause of the inhibition of peroxidation initiation.     

A new and suitable reconstructed system for NADPH-dependent microsomal lipid peroxidation

In order to evaluate the O-2 participation in NADPH-dependent microsomal lipid peroxidation, we used reconstructed system which contained detergent-solubilized NADPH-dependent cytochrome P-450 reductase, cytochrome P-450, phospholipid liposomes, NADPH and Fe3+-ADP. Lipid peroxidation, monitored by the formation of thiobarbituric acid-reactive substance, was increased with increasing concentration of detergent-solubilized NADPH cytochrome P-450 reductase, cytochrome P-450 or Fe3+-ADP. Cytochrome P-450-dependent lipid peroxidation was parallel to O-2 generation monitored by chemiluminescence probe with 2-methyl-6-(p-methoxyphenol)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one. Lipid peroxidation was significantly inhibited by superoxide dismutase, but not by catalase or sodium benzoate. The reconstructed system herein described is considered to be very close to NADPH-dependent microsomal lipid peroxidation system.     

Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.     

Effects of lipid peroxidation on adrenal microsomal monooxygenases

Incubation of guinea pig adrenal microsomes with 10^?6 M ferrous (Fe²⁺) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe²⁺ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe²⁺ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[α]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.     

Antioxidant effect of caeruloplasmin on microsomal lipid peroxidation

Microsomal NADPH-dependent lipid peroxidation catalyzed by ADP-Fe³⁺ was inhibited by the addition of caeruloplasmin. The antioxidant effect of caeruloplasmin was independent of the superoxide anion (O^?₂ scavenging activity. Since caeruloplasmin enhanced the function of ADP-Fe³⁺ acting as electron acceptor for microsomal electron transport system, the antioxidant effect of caeruloplasmin is considered to depend on the ferroxidase activity.     

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.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.     

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 ¹Δg type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with β-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.     

Lactoperoxidase-catalyzed lipid peroxidation of microsomal and artificial membranes

Lactoperoxidase, in the presence of H₂O₂, I^?, and rat liver microsomes, will peroxidize membrane lipids, as evidence by malondialdehyde formation. Fe³⁺ assists in the formation of malondialdehyde. Fe³⁺ can be added at the end of the reaction period as well as at the beginning with equal effectiveness, suggesting that it only acts to assist in the conversion of lipid peroxides, previously formed by lactoperoxidase, to malondialdehyde. The addition of EDTA to the microsomal reaction mixture results in a 40% decrease in malondialdehyde formation. The antioxidant butylated hydroxytoluene will completely block the formation of malondialdehyde. Malondialdehyde formation is not dependent upon the production of superoxide, singlet oxygen, or hydroxyl radicals. Peroxidation of membrane lipids by this system is equally effective in both intact microsomes and in liposomes, indicating that iodination of microsomal protein is not required for lipid peroxidation to occur.     

Spectroscopic evidence for the generation of singlet oxygen in the reduced nicotinamide adenine dinucleotide phosphate-dependent microsomal lipid peroxidation system.

In the presence of Fe3+-ADP and NADPH, washed rat liver microsomes at 37 degrees and at pH 7.5 emitted ultraweak light with the peroxidative cleavage of endogenous lipid. The main light-emitting species of this system was analyzed spectroscopically in the visible region and estimated to be singlet oxygen. The addition of superoxide dismutase or catalase did not inhibit the light emission significantly. It is unlikely therefore that superoxide anion and OH are involved in the generation of singlet oxygen in the present system.     

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.     

Development of the cytosolic defence system against microsomal lipid peroxidation in rat liver

Ascorbate-induced lipid peroxidation in rat liver microsomes reaches the adult level in 2-3 days. NADPH-induced peroxidation develops more gradually, in parallel with the activity of NADPH-cytochrome P-450 reductase, attaining adult levels by 10-12 days. The glutathione-dependent cytosolic enzyme activity which inhibits peroxidation is inhibited by bromosulphophthalein. The development of this system lags behind the development of microsomal lipid peroxidation between the ages of 2 and 20 days, allowing peroxidation to proceed.     

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.     

Ferritin, a physiological iron donor for microsomal lipid peroxidation

In the process of lipid peroxidation of microsomes induced either by oxygen radicals generated by xanthine oxidase or by NADPH, ferritin is able to donate the necessary iron. The amount of ferritin necessary to catalyze the process of lipid peroxidation is in the physiological range. In contrast to the finding with phospholipid liposomes, catalase hardly stimulates the lipid peroxidation of microsomes.     

Effect of alkylresorcin on biological membranes during activation of lipid peroxidation

The effect of alkyl resorcin isolated from the cells of Azotobacter chroococcum and of its structural analog devoid of the alkyl chain (resorcin) on liver microsomes and brain synaptosomes of the rat as well as on rabbit skeletal muscle sarcoplasmic reticulum fragments during activation of lipid peroxidation was studied. Alkyl resorcin was shown to produce a much more potent antioxidant effect as compared with resorcin, since it inhibited lipid peroxidation in all the three types of membranes under study at much lower concentrations. Both alkyl resorcin and resorcin which inhibit lipid peroxidation prevented lipid peroxidation-induced structural-functional damages of synaptosomal and sarcoplasmic reticulum fragment membranes. Unlike resorcin, alkyl resorcin exerted an additional effect on brain synaptosomal membranes which consisted in the stabilization of barrier functions of membranes during incomplete inhibition of lipid peroxidation. The cumulative data suggest that stabilization necessitates the presence of both resorcin radical and alkyl chain in the alkyl resorcin molecule.