Lipid Peroxide Formation In Microsomes Research Articles

Stimulation of cyclophosphamide-induced pulmonary microsomal lipid peroxidation by oxygen

Cyclophosphamide (CP) causes lung toxicity in a wide variety of animals including humans. Recent reports suggest that CP increases lipid peroxide formation in the lung, and that oxygen (O 2) potentiates CP-induced lung toxicity. We hypothesized that CP, or one of its toxic metabolites, acrolein, stimulates lung lipid peroxide formation in the presence of high O 2 tensions. To test this, rat lung microsomes were treated in vitro with CP or acrolein in the presence of NADPH and 0–100% O 2 with and without superoxide dismutase (SOD), glutathione (GSH), dithiothreitol (DTT), and EDTA (agents which scavenge reactive O 2 species and/or detoxify reactive metabolites). Lipid peroxide formation in untreated microsomes was increased 40, 39, and 37% in 60, 80 and 100% O 2 respectively ( P < 0.02 vs. 21% O 2 air). Lipid peroxide formation in microsomes treated with CP increased 2–3-fold under 21% O 2 ( P < 0.05 vs. untreated under 21% O 2). However, increases in lipid peroxide formation were 3–4 fold in CP treated microsomes under 40–100% O 2 ( P < 0.001 vs. untreated at same % O 2). CP and acrolein-stimulated lipid peroxidation with and without O 2 exposure was significantly ( P < 0.05) reduced by prior addition of SOD, GSH, DTT, or EDTA to the lung microsomal suspension. These results indicate that lipid peroxide formation increases in CP and acrolein-treated lung microsomes, and high O 2 tensions stimulate CP-induced lipid peroxidation. Stimulation of CP-induced microsomal lipid peroxidation appears to be mediated by reactive O 2 species or metabolites.

Effects of trivalent and hexavalent chromium on lipid peroxidation in rat liver microsomes.

The effects of heavy metals, especially chromium, on lipid peroxidation in rat liver microsomes were studied. Lipid peroxidation was determined as thiobarbituric acidreacting materials. At lower concentrations in the range of 1-100μM, both hexavalent and trivalent chromium inhibited lipid peroxidation induced by ascorbate or NADPH in microsomes. In the presence of ascorbate, the inhibitions decreased on treatment with sulfhydryl reagents and were partly overcome by the addition of tartaric acid. It is suggested that chromium, presumably in the trivalent form, may bind with sulfhydryl groups of protein, resulting in the inhibition of lipid peroxidation. In the presence of NADPH, hexavalent chromium showed very potent inhibition. Hexavalent and trivalent chromium did not inhibit the electron-transport system in microsomes. The hexavalent form may act as a radical scavenger, since it strongly inhibited carbon tetrachloride-stimulated lipid peroxidation, could reduce 2, 2'-diphenyl-β-picrylhydrazyl to some extent in the presence of a reducing agent such as ethanol, and inhibited NADPH-induced peroxidation markedly in the presence of sulfhydryl compounds. Above 1 mM, hexavalent chromium caused lipid peroxide formation in microsomes, apparently not associated with iron, while trivalent chromium showed an inhibitory effect.

Effect of NADPH on the microsomal lipid peroxidation in the presence of the soluble fraction of rat liver (author's transl)

Effect of NADPH on lipid peroxidation in microsomes with the soluble fraction of rat liver was investigated. NADPH was found to inhibit lipid peroxidation in 9000×g supernatant fraction when over a certain amount of the fraction was added to the reaction mixture, even in the presence of Fe2+ or Fe2+-ADP which is known to activate the microsomallipid peroxidation induced by NADPH. In addition, the soluble fraction-stimulated lipid peroxidation in microsomes was found to be effectively inhibited by NADPH in the presence of a certain amount of the soluble fraction. NADPH extended the duration of depressing the lipid peroxidation in 9000×g supernatant fraction followed by a rise in the formation of lipid peroxides. When Zn2+, an inhibitor of glutathione reductase, was added to 9000×g supernatant fraction or the soluble fraction-microsomes system, lipid peroxide formation was not depressed by the addition of NADPH. Inversely, the addition of GSSG to the soluble fraction-microsome system was found to enhance the antiperoxidative effect of NADPH. These results indicate that a factor, which participates in the inhibitory action of NADPH on the microsomal lipid peroxidation in the presence of the soluble fraction, may be a glutathione reductase system present in the soluble fraction. Therefore, it is thought that NADPH does not only stimulate the microsomal lipid peroxidation, but also acts as an inhibitory factor on the lipid peroxide formation in microsomes together with the soluble fraction.

Effect of the soluble fraction of rat liver on the microsomal lipid peroxidation (author's transl)

Effect of the soluble fraction of rat liver on lipid peroxidation in hepatic microsomes was investigated. The soluble fraction markedly accelerated the lipid peroxidation in microsomes, even in the presence of 40μM Fe2+ and/or 10μM L-ascorbic acid. However, the soluble fraction effectively inhibited the NADPH-dependent lipid peroxidation in microsomes, especially in the presence of both glucose-6-phosphate and glucose-6-phosphate dehydrogenase. The inhibitory effect on the microsomal lipid peroxidation induced by NADPH-regenerating system was reduced by heating the soluble fraction. The addition of Zn2+, an inhibitor of glutathione reductase, to the reaction mixture weakened the anti-peroxidative action of the soluble fraction on the lipid peroxide formation in microsomes together with NADPH-regenerating system and, inversely, oxidized glutathione enhanced the action of this fraction. In addition, presence of oxidized glutathione and glutathione reductase preparation was as effective as reduced glutathione in depressing lipid peroxidation in microsomes stimulated by NADPH-regenerating system. These findings suggest that both the activation of glutathione reductase system by NADPH and a heat-stable factor (s) take part in the inhibitory effect of the soluble fraction on the NADPH-linked lipid peroxidation in microsomes. This soluble fraction also inhibited the microsomal lipid peroxidation stimulated by L-ascorbic acid at the concentration of 50μM or above, and the inhibitory effect was slightly increased by heating of this fraction. It is assumed that the inhibitory effect of the soluble fraction on L-ascorbic acid-dependent lipid peroxidation might be due to the mechanism different from that acts on the NADPH-dependent lipid peroxidation.

Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation.

1. Aminopyrine strongly inhibits NADPH-induced lipid peroxide formation in rat liver microsomes, but ascorbate-induced peroxidation is inhibited to a smaller extent. 2. Aminopyrine oxidation is stimulated by Mg(2+) but inhibited by Ca(2+). Concentrated solutions (10mm) of iron-chelating agents inhibit aminopyrine oxidation, but the more dilute solutions (0.5mm) of chelators that block lipid peroxide formation do not inhibit aminopyrine oxidation. Microsomes prepared from sucrose-EDTA homogenates rapidly oxidize aminopyrine, but do not form lipid peroxide when incubated with ascorbate or NADPH. 3. Aminopyrine oxidation is strongly inhibited by p-chloromercuribenzoate, less by iodoacetamide and weakly by N-ethylmaleimide. The site of action of these compounds is considered to be a ferredoxin-type protein. GSH and cysteine also inhibit. 4. Other drugs oxidized by microsomes such as caffeine, phenobarbitone and hexobarbitone had either no or little effect on lipid peroxide formation, but codeine inhibited. 5. Most aliphatic hydrocarbons, alcohols, ketones and aldehydes did not affect lipid peroxide formation, but chloroform and carbon tetrachloride inhibited. 6. Many aromatic compounds inhibited lipid peroxide formation. Only aromatic acids were without any effect and phenols and amines were very strong inhibitors. 7. Induction of lipid peroxide formation in microsomes by incubation with ascorbate or NADPH or by treatment with ionizing radiation leads to a sharp decline in the ability of microsomes to oxidize aminopyrine or hydroxylate aniline. 8. It is considered that the two processes of hydroxylation and lipid peroxide formation are closely linked in microsomes. They probably depend on the same electron-transport chain, and peroxide formation, which involves membrane disintegration, may be part of the normal membrane remodelling process.

Lipid peroxide formation in microsomes. The role of non-haem iron.

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe(2+) or Fe(3+)) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe(2+) is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.

Lipid peroxide formation in microsomes. General considerations.

1. Liver microsomes form lipid peroxide when incubated with ascorbate or NADPH, but not with NADH. Increasing the concentration of ascorbate beyond the optimum (0.5mm) decreases the rate of lipid peroxide formation, but this effect does not occur with NADPH. Other reducing agents such as p-phenylenediamine or ferricyanide were not able to replace ascorbate and induce lipid peroxide formation. 2. The rate of ascorbate-induced peroxidation is optimum at pH6.0 whereas the rate of the NADPH system is optimum at pH7.0. Both systems require phosphate for maximum activity. 3. Lipid peroxide formation occurs at the maximum specific rate in very dilute microsome suspensions (0.15mg. of protein/ml.). 4. Treatment of microsomes with deoxycholate and other detergents causes membrane disintegration and inhibits lipid peroxide formation. 5. Lipid peroxide formation is accompanied by a rapid uptake of oxygen and there is a large excess of oxygen utilized for each molecule of malonaldehyde measured in the peroxide method. 6. Boiled microsomes form lipid peroxide in the presence of ascorbate, but not if NADPH is added. 7. Lipid peroxide formation induced by NADPH is strongly inhibited by p-chloromercuribenzoate, weakly inhibited by N-ethylmaleimide and unaffected by iodoacetamide. Ascorbate-induced peroxidation in untreated microsomes is unaffected by p-chloromercuribenzoate, but inhibited if boiled microsomes are used. These experiments may be interpreted on the basis that a ferredoxin-type protein forms part of the system in which NADPH induces lipid peroxide formation. 8. Most heavy-metal ions, with the exception of inorganic iron (Fe(2+) or Fe(3+)), which activates, inhibit both ascorbate-induced and NADPH-induced peroxidation. Mg(2+) increases the rate of peroxidation whereas Ca(2+) inhibits it. 9. Lipid peroxide formation is inhibited strongly by GSH and weakly by cysteine. Ascorbate-induced peroxidation is much more sensitive than NADPH-induced peroxidation. 10. Peroxidation is strongly inhibited by addition of low concentrations (0.01-0.1mm) of cytochrome c or of haemoglobin. 11. It is considered that lipid peroxide formation occurs as a result of the operation of the microsomal electron-transport chain switching from hydroxylation to oxidize unsaturated lipids of the endoplasmic reticulum.