Effect of 2-mercaptopropionylglycine on lipid peroxidation and drug oxidation in rat liver microsomes

2-Mercaptopropionylglycine, a synthetic thiol, significantly stimulated NADPH-dependent lipid peroxidation by rat liver microsomes, while the thiol inhibited the microsomal aminopyrine N-demethylase activity with an increase in lipid peroxidation. But, a strong inhibition of lipid peroxidation by EDTA could not abolish the inhibition of the N-demethylase activity by the thiol. Besides, the thiol markedly increased not only the Km value for aminopyrine N-demethylase but also the apparent Ks value for aminopyrine binding to the microsomal oxidized cytochrome P-450 by interacting with the cytochrome P-450.     

Senescence-associated decrease of NADPH-induced lipid peroxidation in rat liver microsomes

Senescence is associated with decrease in the NADPH-induced lipid peroxidation in liver homogenate as well as rough and smooth microsomes of female rats. In the microsomal fractions, sensitivity to NADPH-induced lipid peroxidation is high in young adults (3-month-old), decreases in middle aged (12-month-old) and reaches lowest levels in senescent (30-month-old) rats. Increasing the concentration of co-factors or time of incubation does not alter this resistance observed in the senescent rats. Major factors responsible for this resistance in senescent rats seem to be low levels of substrate in the c reductase, cytochrome P-450 and high cholesterol:phospholipid ratios besides enhanced levels of superoxide dismutase, alpha-tocopherol and reduced glutathione.     

NADPH-dependent inhibition of lipid peroxidation in rat liver microsomes.

Microsomal NADPH-driven electron transport is known to initiate lipid peroxidation by activating oxygen in the presence of iron. This pro-oxidant effect can mask an antioxidant function of NADPH-driven electron transport in microsomes via vitamin E recycling from its phenoxyl radicals formed in the course of peroxidation. To test this hypothesis we studied the effects of NADPH on the endogenous vitamin E content and lipid peroxidation induced in liver microsomes by an oxidation system independent of iron: an azo-initiator of peroxyl radicals, 2,2'-azobis (2,4-dimethylvaleronitrile), (AMVN), in the presence of an iron chelator deferoxamine. We found that under conditions NADPH: (i) inhibited lipid peroxidation; (ii) this inhibitory effect was less pronounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats; (iii) protected vitamin E from oxidative destruction; (iv) reduced chromanoxyl radicals of vitamin E homologue with a 6-carbon side-chain, chromanol-alpha-C-6. Thus NADPH-driven electron transport may function both to initiate and/or inhibit lipid peroxidation in microsomes depending on the availability of transition metal catalysts.     

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes.

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes. This reaction was heat-insensitive contrary to the mitochondrial peroxidation reported in the previous paper, and was enhanced by p-chloromercuribenzoate. Additions of Fe²⁺ and Fe³⁺ stimulated both the lipid peroxidation and the disappearance of dihydroxyfumaric acid. On the other hand, addition of Mn²⁺ or Cu²⁺, which stimulated the disappearance of dihydroxyfumaric acid, inhibited the lipid peroxidation. Hydroxyl radical scavengers, superoxide dismutase and catalase had no effect on this lipid peroxidation and dihydroxyfumaric acid disappearance. The cytochrome p-450 content decreased about 70 % in parallel with the lipid peroxidation.     

Action of retinoic acid on lipid peroxidation in rat liver microsomes in vitro

It has been shown in experiments in vitro that preincubation of rat liver microsomes with an ethanol solution of all-trans-retinoic acid in the final concentration 7.0 X 10(-5) M results in a decrease of both NADPN-dependent and spontaneous lipid peroxidation (to 53 and 70% of control, respectively) but did not influence ascorbate-dependent lipid peroxidation. Retinol at the same concentration induces more pronounced inhibition of all types of microsomal lipid peroxidation. The rate of NADPN-dependent lipid peroxidation decreases linearly as the retinoic acid concentration in the incubation medium is raised, whereas the rate of ascorbate-dependent lipid peroxidation drastically lessens only after the retinoic acid concentration in the medium is increased to 1.4 X 10(-4) M. The data obtained provide evidence in favour of the concepts of a possible role of vitamin A in LPO regulation in the body and point to the necessity of taking into consideration the antioxidant properties of retinol and retinoic acid while analysing their pharmacological action.     

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.     

The stimulatory effects of asbestos on NADPH-dependent lipid peroxidation in rat liver microsomes.

Lipid peroxidation in rat liver microsomes induced by asbestos fibres, crocidolite and chrysotile, is greatly increased in the presence of NADPH, leading to malondialdehyde levels comparable with those induced by CCl4, a very strong inducer of lipid peroxidation. This synergic effect only occurs during the first minutes and could be explained by an increase or a regeneration of the ferrous active sites of asbestos by NADPH, which in turn could rapidly be prevented by the adsorption of microsomal proteins on the surface of the fibres. It is not inhibited by superoxide dismutase, catalase and mannitol, indicating that oxygen radicals are not involved in the reaction. It is also not inhibited by desferrioxamine, indicating that it is not due to a release of free iron ions in solution from the fibres. Lipid peroxidation in NADPH-supplemented microsomes is also greatly increased upon addition of magnetite. This could be linked to the presence of ferrous ions in this solid iron oxide, since the ferric oxides haematite and goethite are completely inactive.     

Stimulation of lipid peroxidation in rat liver microsomes by tetraphenylporphine and its metallocomplexes

It is shown that tetraphenylporphyrin (TPP) and its complexes with metals decrease the rate of the diene conjugate formation. The above compounds increase the malonic dialdehyde accumulation. The effect of TPP and its complexes with metals is connected with stimulation of lipid peroxidation in biomembranes.     

Glutathione disulfide enhances the reduced glutathione inhibition of lipid peroxidation in rat liver microsomes

Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver microsomes. Dependence on microsomal alpha-tocopherol was shown for the GSH inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 mM) were combined in the assay system, inhibition of lipid peroxidation was enhanced markedly over that with GSH alone in microsomes containing alpha-tocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in alpha-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal alpha-tocopherol and likely requires GSH for alpha-tocopherol regeneration from the alpha-tocopheroxyl radical during lipid peroxidation. The second factor appears to be independent of alpha-tocopherol and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety.     

The critical steady-state hypoxic conditions in carbon tetrachloride-induced lipid peroxidation in rat liver microsomes

Defined steady-state oxygen partial pressures (PO2) were maintained constant with an oxystat system to study carbon tetrachloride (CCl4)-induced lipid peroxidation and oxygen uptake in rat liver microsomes. The initial rates of oxygen uptake and malondialdehyde formation indicated drastically increasing lipid peroxidation by decreasing PO2, attaining a maximum between 1-10 mmHg (0.1-1.3 kPa). Under these conditions, at the hypoxic end of the physiological PO2 in liver, CCl4 caused a 5-fold increase in the oxygen uptake rate and a 20-fold increase in the malondialdehyde formation rate while, at 80 mmHg (10.7 kPa) the haloalkane caused only an increase of 2- and 4-fold, respectively; in comparison, there was only a slight increase in NADPH-induced lipid peroxidation with increasing PO2. These data clearly demonstrate the critical role of low steady-state PO2 in CCl4-induced lipid peroxidation and support lipid peroxidation as a key factor in CCl4 hepatotoxicity.     

Studies on spice principles as antioxidants in the inhibition of lipid peroxidation of rat liver microsomes

Polyunsaturated fatty acids (PUFA) are vulnerable to peroxidative attack. Protecting PUFA from peroxidation is essential to utilize their beneficial effects in health and in preventing disease. The antioxidants vitamin E, t-butylhydroxy toluene (BHT) and t-butylhydroxy anisole (BHA) inhibited ascorbate/Fe²⁺-induced lipid peroxidation in rat liver microsomes. In addition, a number of spice principles, for example, curcumin (5–50 µM) from turmeric, eugenol (25–150 µM) from cloves and capsaicin (25–150 µM) from red chillies inhibited lipid peroxidation in a dose-dependent manner. Zingerone from ginger inhibited lipid peroxidation at high concentrations (> 150 µM) whereas linalool (coriander), piperine (black pepper) and cuminaldehyde (cumin) had only marginal inhibitory effects even at high concentrations (600 µM). The inhibition of lipid peroxidation by curcumin and eugenol was reversed by adding high concentrations of Fe²⁺.     

NADPH-dependen lipid peroxidation catalyzed by purified NADPH-cytochrome C reductase from rat liver microsomes

A purified preparation of rat liver microsomal NADPH-cytochrome c reductase has been shown to catalyze the NADPH-dependent peroxidation of isolated microsomal lipid. In addition to ADP and ferric ion required for NADPH-dependent lipid peroxidation in whole microsomes, this system requires high ionic strength and a critical concentration of EDTA. The peroxidation activity can be inhibited by superoxide dismutase suggesting that the superoxide anion, produced by this flavoprotein, is involved in the lipid peroxidation reaction.     

Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes.

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.     

Effect of C27-steroids on lipid peroxidation in rat liver mitochondria

The influence of C27-steroids--vitamin D3 and 20-hydroxyecdysone in free-radical reactions on lipid peroxidation in the mitochondrial fraction of rat's liver has been studied. Antioxidant activity of superoxide dismutase and catalase and the content of lipid components--cholesterol and phospholipids has been determined. The assumption was put forward that antioxidant action of steroids was possible as a result of to the modification of mitochondrial membrane through its lipid content and changing of its cholesterol: phospholipids content.