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.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

Effect of thiols on lipid peroxidation in rat liver microsomes

The stimulatory or inhibitory effects of various thiol compounds on in vitro lipid peroxidation by iron-ascorbate in rat liver microsomes were determined. Glutathione had no measurable pro-oxidant capacity, in contrast, it protected against lipid peroxidation. N-Acetyl l-cysteine and S-methyl-glutathione had no effect on in vitro lipid peroxidation. l-Cysteine stimulated lipid peroxidation and also of d-penicillamine and dl-dithiothreitol the pre-oxidant capacity predominated the anti-oxidant capacity. Cysteamine afforded a pronounced protection against in vitro lipid peroxidation. In contrast to the labile character of the glutathione dependent protection, the protection by cysteamine was not affected by heat-pretreatment of the liver microsomes or alkylating protein sulfhydryl groups by N-ethyl maleimide. Again in contrast to glutathione, the protection against in vitro microsomal lipid peroxidation by cysteamine was not reduced after in vivo lipid peroxidation induced by CC14. This suggests that even after the process of lipid peroxidation has been started, administration of cysteamine might still be beneficial.     

The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation.

NADPH-dependent lipid peroxidation occurs in two distinct sequential radical steps. The first step, initiation, is the ADP-perferryl ion-catalyzed formation of low levels of lipid hydroperoxides. The second step, propagation, is the iron-catalyzed breakdown of lipid hydroperoxides formed during initiation generating reactive intermediates and products characteristic of lipid peroxidation. Propagation results in the rapid formation of thiobarbituric acid-reactive material and lipid hydroperoxides. Propagation can be catalyzed by ethylenediamine tetraacetate-chelated ferrous ion, diethylenetriamine pentaacetic acid-chelated ferrous ion, or by ferric cytochrome P-450. However, cytochrome P-450 is destroyed during propagation.     

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 electron transport chain in microsomes and lipid peroxidation catalyzed by metal ions.

Like iron ions copper ions are also able to stimulate the NADPH-dependent lipid peroxidation in rat liver microsomes. This effect is strongly dependent on the concentration of Cu2+ added. Initial concentrations of Cu2+ above 50 microM completely inhibit the formation of malonaldehyde. The activator and inhibitor functions may be interpreted by a simultaneous participation of Cu+ ions formed in the chain branching and termination reaction of the free radical lipid peroxidation process. Inhibition studies with pCMB and the His-reagent diethyl pyrocarbonate indicate an essential role of cysteine and histidine residues in the Cu+-NADPH-dependent lipid peroxidation process.     

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.     

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.     

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.     

NADPH-dependent drug redox cycling and lipid peroxidation in microsomes from human term placenta

1. NADPH-dependent iron and drug redox cycling, as well as lipid peroxidation process were investigated in microsomes isolated from human term placenta. 2. Paraquat and menadione were found to undergo redox cycling, catalyzed by NADPH:cytochrome P-450 reductase in placental microsomes. 3. The drug redox cycling was able to initiate microsomal lipid peroxidation in the presence of micromolar concentrations of iron and ethylenediaminetetraacetate (EDTA). 4. Superoxide was essential for the microsomal lipid peroxidation in the presence of iron and EDTA. 5. Drastic peroxidative conditions involving superoxide and prolonged incubation in the presence of iron were found to destroy flavin nucleotides, inhibit NADPH:cytochrome P-450 reductase and inhibit propagation step of lipid peroxidation. 6. Reactive oxo-complex formed between iron and superoxide is proposed as an ultimate species for the initiation of lipid peroxidation in microsomes from human term placenta as well as for the destruction of flavin nucleotides and inhibition of NADPH:cytochrome P-450 reductase as well as for impairment of promotion of lipid peroxidation under drastic peroxidative conditions.     

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.     

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.     

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.     

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes.

Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co²⁺, Mn²⁺, Hg²⁺, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional 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.     

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.