NADPH-dependent Lipid Peroxidation Research Articles

Effects of p-tert-butylphenyl trans-4-guanidinomethylcyclohexane carboxylate hydrochloride (NCO-650) and its metabolite, p-tert-butylphenol(BP) on drug-metabolizing enzymes and fine structure in rat liver.

The effects of p-tert-butylphenyl trans-4-guanidinomethylcyclohexane carboxylate hydrochloride (NCO-650) and its metabolite, p-tert-butylphenol(BP), on the drug-metabolizing enzymes and fine structure in the rat liver were examined. Aminopyrine demethylase activity was inhibited by the administration of NCO-650 and BP at a dose of 2 and 10 mg/kg, p.o. The increases of microsomal cytochrome b5 and cytochrome P-450 contents were noticed at 1, 12 and 24 hr after NCO-650 and BP administration. Ascorbate-dependent lipid peroxidation of mitochondria and microsome increased by the administration of NCO-650 and BP, but NADPH-dependent lipid peroxidation decreased by these drugs. In the morphological observations of fine structure in the liver, NCO-650 and BP caused the swelling and decrease of rough endoplasmic reticulum and the increase of smooth endoplasmic reticulum. The morphological changes of liver fine structure were related to the changes of drug-metabolizing enzymes in the liver by the administration of NCO-650 and BP, which may be suggest the transitory and functional responses of these drugs in the liver. The effect of BP on the drug-metabolizing enzymes and fine structure in the liver was similar to that of NCO-650.

Stabilizing action of hydroxybenzimidazole and its derivatives on biological membranes during activation of lipid peroxidation

Inhibition of lipid peroxidation (LPO) by oxybenzimidazole (OBI) and its derivatives--alkyloxybenzimidazole (AOBI) and alkylethoxybenzimidazole (AEBI) was studied in liver microsomes and brain synaptosomes. It has been shown that both OBI and AOBI strongly inhibit LPO in microsomes and not synaptosomes. AEBI failed to inhibit LPO in microsomes. AOBI is more potent than OBI both in ascorbate- and NADPH-dependent LPO of microsomes. An antioxidant effect of both compounds is more marked in ascorbate-dependent LPO. The investigation of the possible use of AOBI for the protection of liver membranes in various pathological conditions associated with LPO activation seems promising.

NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes

Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoride (LAMP) were compared. In the absence of added Fe 2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe 2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloro-mercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation. the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe 2+, whereas LAHP-dependent lipid peroxidation is auto-catalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H 2O 2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.

Effect of prolonged administration of clofibric acid and di-(2-ethylhexyl)phthalate on hepatic enzyme activities and lipid peroxidation in the rat

Male Sprague-Dawley rats were fed diets containing either 0.5% clofibric acid (CA) or 2% di-(2-ethylhexyl)phthalate (DEHP) for 2 years. Both compounds produced liver enlargement which was accompanied by the formation of liver nodules. Hepatic peroxisomal and microsomal fatty acid oxidising enzyme activities were induced in both large nodules and host tissue (i.e. tissue remaining after removal of large nodules) preparations from CA and DEHP treated rats. In contrast, little change in catalase activity was observed and the activities of cytosolic GSH peroxidase and GSH S-transferases were markedly reduced. Increased lipid peroxidation was observed by measurement of conjugated dienes in host tissue homogenates from CA and DEHP treated rats. Microsomal NADPH-dependent lipid peroxidation was also stimulated. Histological examination revealed extensive lipofuscin deposition in non-nodular, but not in nodular, tissue sections from treated rats. These results demonstrate that prolonged peroxisome proliferation can result in lipid peroxidation and that certain enzymes which metabolise hydrogen peroxide and organic hydroperoxides are either little affected or markedly inhibited.

Application of spin-trapping study to rat ischemic brain homogenate incubated with NADPH and Fe-EDTA

A spin-trapping technique was applied to the detection of free radicals generated in nicotinamide-adenine denucleotide phosphate (NADPH)-dependent lipid peroxidation of rat ischemic brain homogenate. The spin adduct of phenyl-t-butylnitrone if(A N = 16.2−16.5 g, A β H = 3.6−3.8 g ) was observed, and it was thought to be derived from NADPH-dependent lipid peroxidation from its oxygen and NADPH dependency. Its intensity was increased in the recirculated condition following ischemic insult, indicating the susceptibility of brain tissue to lipid peroxidation in such a condition.

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.

Action of Carbon Tetrachloride in the Reconstituted Monooxygenase System Using Partially Purified Cytochrome P-450 and P-448

In the cytochrome P-450-reconstituted system, CCI4 stimulated NADPH-dependent lipid peroxidation of the system containing the P-450 form to a much greater extent than that of the system containing the P-448 form. When the P-450-reconstituted system was preincubated in the presence of both NADPH and CCI4, 7-ethoxycoumarin O-deethylase, aminopyrine N-demethylase and aniline hydroxylase activities were decreased by 40–60%, whereas, with P-448 form reconstituted system, no suppression was observed in these enzyme activities or in 7-ethoxyresorufin O-deethylase activity. These observations suggest that the P-450 form, but not the P-448 form, is active in metabolizing CCI4 to a reactive species that subsequently impairs the hemoprotein.

Action of carbon tetrachloride in the reconstituted monooxygenase system using partially purified cytochrome P-450 and P-448.

In the cytochrome P-450-reconstituted system, CCl4 stimulated NADPH-dependent lipid peroxidation of the system containing the P-450 form to a much greater extent than that of the system containing the P-448 form. When the P-450-reconstituted system was preincubated in the presence of both NADPH and CCl4, 7-ethoxycoumarin O-deethylase, aminopyrine N-demethylase and aniline hydroxylase activities were decreased by 40-60%, whereas, with P-448 form reconstituted system, no suppression was observed in these enzyme activities or in 7-ethoxyresorufin O-deethylase activity. These observations suggest that the P-450 form, but not the P-448 form, is active in metabolizing CCl4 to a reactive species that subsequently impairs the hemoprotein.

Application of the thiobarbiturate assay to the measurement of lipid peroxidation products in microsomes

By applying two different thiobarbiturate assay procedures in parallel to aliquots of a microsomal incubation mixture one can simultaneously monitor free malondialdehyde and malondialdehyde plus labile lipid peroxidation products. The levels of malondialdehyde increase continuously during the incubation of microsomes, NADPH and ferrous-ADP complex, while the lipid precursors of MDA stop forming when the system becomes depleted in DADPH. In contrast to systems in which lipids are undergoing autooxidation, NADPH-dependent lipid peroxidation does not appear to generate significant amounts of water-soluble malondialdehyde precursors. As a result, quantitative interpretation of results is straightforward in the microsomal system. In spite of the lack of specificity of the thiobarbiturate coupling reaction, interferences can be easily compensated for by using zero time controls.

Inactivation of transforming activity of plasmid DNA by lipid peroxidation

DNA damage due to NADPH-dependent lipid peroxidation of liposomes was examined using liposomes prepared from lipids, NADPH-cytochrome P-450 reductase and cytochrome P-450 isolated from rat liver microsomes. Plasmid pBR322 DNA was incubated in the reaction mixture for liposomal lipid peroxidation and introduced to Escherichia coli CSR603 ( uvrArecA). More of the transforming activity of the DNA was lost as the lipid peroxidation progressed, and this inactivation was dependent on the extent of lipid peroxidation. Single strand breaks occurred in the plasmid DNA. Hydroxyl radical scavengers could not prevent most of the strand breaks or the lipid peroxidation reaction. Chloroform extracts from the reaction mixture of peroxidized microsomes also inactivated the transforming activity of pBR322 DNA but did not cause strand breaks. The 105 000 × g supernatant of the reaction mixture, which contained more than 85% of the thiobarbituric acid-reactive substances, did not inactivate the plasmid DNA. The degradative products of [U- 14C]arachidonic acid in the liposomes did not bind to DNA. These results led to the conclusion that at least two types of DNA damaging agent are produced during NADPH-dependent microsomal lipid peroxidation. One induces single strand breaks of DNA and another inactivates the plasmid-transforming activity without inducing strand breaks.

Hydroxyl radicals are not involved in NADPH dependent microsomal lipid peroxidation.

NADPH dependent H2O2 formation in microsomes in the presence of chelated iron leads to formation of hydroxyl radicals. Enhancement of hydroxyl radical generation (via ferric-EDTA or sodium azide) did not result in a concomitant increase in lipid peroxidation; rather, a decrease was observed. Moreover, the hydroxyl radical scavenger DMSO did not inhibit lipid peroxidation. This comparison of hydroxyl radical formation with lipid peroxidation suggests that hydroxyl radicals do not play a part in NADPH-dependent lipid peroxidation.

Alterations in Hepatic Microsomal Mixed-Function Oxidase System during Different Levels of Food Restriction in Adult Male and Female Rats

Wistar strain adult male and female rats were given 25, 50 and 75% less food than an ad libitum-fed group of rats for 45 d and the effects of food restriction on hepatic drug metabolizing enzymes, microsomal electron transport components, NADPH-dependent lipid peroxidation and glutathione-S-transferase activities were studied. Compared to ad libitum-fed controls, the cytochrome P-450 levels were higher in food restricted male rats, while they were lower in food restricted females. The activities of NADPH cytochrome c reductase were lower in food restricted females than in ad libitum-fed controls. The activities of drug metabolizing enzymes, aminopyrine N-demethylase and acetanilide hydroxylase were higher in food restricted males, whereas in food restricted females these activities were lower than in respective groups fed ad libitum. Microsomal, NADPH-dependent lipid peroxidation was higher in 25 and 50% food restricted females while in 50 and 75% food restricted males it was lower than in ad libitum controls of the same sex. The cytosolic glutathione-S-transferase activities were lower in food restricted rats of both the sexes than in the same sexed controls. Another group of male and female rats were given 75% less food than the ad libitum-fed rats and refed for 3 d prior to killing. Here also, the effects of restriction were different between sexes. It is concluded that hepatic microsomal mixed-function oxidase system (MFOS) is altered due to feed restriction and food restriction followed by refeeding, in a sex-related manner.

Importance of the polyunsaturated fatty acid to vitamin E ratio in the resistance of rat lung microsomes to lipid peroxidation

Rat lung microsomes and liposomes made from isolated lung microsomal lipids were found to be much more resistant to lipid peroxidation than those from liver in both enzymatic and nonenzymatic systems. The polyunsaturated fatty acid (PUFA) content of isolated lung microsomal lipids was 28% of total fatty acids, while liver was 54%. The vitamin E (α-tocopherol) content of isolated lung microsomal lipids was 2.13 nmol/ μmol lipid phosphate and that of liver was 0.43. Individually, neither the lower PUFA content nor higher vitamin E levels could account for the resistance of lung microsomal lipids to peroxidation. Distearoyl-L-a-phosphatidylcholine and/or α-tocopherol were added to liver microsomal lipids to achieve different PUFA to vitamin E ratios at PUFA contents of 28% or 54%, and the resulting liposomes were subjected to an NADPH-dependent lipid peroxidation system utilizing cytochrome P450 reductase, EDTA-Fe +3, and ADP-Fe +3. Liposomes having PUFA to vitamin E ratios less than approximately 250 nmol PUFA/nmol vitamin E were resistant to peroxidation, whereas lipid peroxidation, as evidenced by malondialdehyde production, occurred in liposomes having higher ratios. When lipid peroxidation occurred, 40%–60% of the liposomal vitamin E was irreversibly oxidized. Irreversible oxidation did not occur in the absence of lipid peroxidation. These studies indicated that the low PUFA to vitamin E ratio in lung microsomes and isolated microsomal lipids was sufficient to account for the observed resistance to lipid peroxidation.

Mutation induction in Escherichia coli incubated in the reaction mixture of NADPH-dependent lipid peroxidation of rat-liver microsomes

Experiments were carried out to examine mutation induction in E. coli cells incubated in the reaction mixture of NADPH-dependent lipid peroxidation of microsomes isolated from rat liver. The results obtained were as follows: (1) Lipid peroxidation of microsomes occurred extensively on incubation with NADPH and Fe 2+. In the E. coli WP2 uvrA(pKM101) system, the mutation frequency to streptomycin resistance increased markedly when the cells were incuated in the reaction mixture of microsomal lipid peroxidation. The induced mutation frequencies were dependent on the extent of the lipid peroxidation. (2) It was also found that the mutations were induced at the same rate as in the case (1) when the cells added to the microsomal suspensions after the reactions due to short-lived free radicals had terminated.(3) The cytotoxicity of the lipid peroxidation products was larger in the DNA repair-defective mutant, E. coli SR18 ( uvrArecA) than the wild-type strain, SR749. From these results it is concluded that some DNA-damaging and mutagenic substances are indeed produced in the degradation process of peroxidized polyunsaturated fatty acids in liver microsomal lipids.

Effects of diethylhydroxylamine on hepatic microsomal lipid peroxidation and glutathione S-transferases.

The effects of diethylhydroxylamine (DEHA), a potent free-radical scavenger, on lipid peroxidation of rat liver microsomes were investigated in vitro. DEHA strongly inhibited ascorbate-dependent nonenzymatic microsomal lipid peroxidation. DEHA also completely inhibited nonenzymatic lipid peroxidation of heat-denatured microsomes, indicating that inhibition is protein-independent. DEHA only moderately inhibited NADPH-dependent enzymatic microsomal lipid peroxidation. DEHA has been shown to exhibit antitumorogenic properties. However, it had no significant effect on hepatic glutathione S-transferase, selenium-independent glutathione peroxidase, or selenium-dependent glutathione peroxidase activity in the DEHA-treated CD-1 (lCR) Br male mouse. This suggests that the mode of action of DEHA as an antitumorogenic agent may be different from that of butylated hydroxyanisole, whose antitumor function is attributed to induction of glutathione S-transferase activity.

Rat liver microsomal NADPH-dependent release of iron from ferritin and lipid peroxidation

Microsomes prepared by the usual method of differential centrifugation were found to contain ferritin, superoxide dismutase (SOD), and catalase which could be removed by chromatography on Sepharose CL-2B. Addition of purified rat liver ferritin to chromatographed microsomes resulted in a significant stimulation of NADPH-dependent lipid peroxidation which was inhibited by exogenously added SOD. Iron release from ferritin by these microsomes was also inhibited by SOD. Ferritin did not promote NADPH-dependent microsomal lipid peroxidation when added to microsomes isolated in the usual manner, presumably due to the endogenous SOD present in the microsomes. Accordingly, only very low rates of iron release from ferritin were observed with these microsomes. Paraquat (PQ), which generates superoxide O 2 ⨪ via redox cycling, greatly stimulated iron release from ferritin and lipid peroxidation in chromatographed microsomes. Paraquat had no effect on iron release from ferritin or lipid peroxidation in microsomes which were not chromatographed unless they were first treated with CN − to inhibit endogenous SOD. These studies indicate that the majority of microsomal iron is contained within ferritin and that following release by O 2 ⨪ this iron serves to promote the peroxidation of microsomal lipids

Effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in brain mitochondria from rats and dogs.

The effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in isolated brain mitochondria from rats and dogs were studied. CV-2619 was easily reduced by canine brain mitochondria in the presence of respiratory substrates. Reduced CV-2619 (2H-CV-2619) was rapidly oxidized through the cytochrome b chain, indicating that the compound functioned simply as an electron carrier of mitochondrial respiratory system. Both nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidations were examined in canine brain mitochondria in the presence of adenosine diphosphate (ADP) and Fe3+. NADH-cytochrome c reductase activity was sensitive to NADPH-dependent lipid peroxidation. CV-2619 (10(-5)M) strongly inhibited both types of the lipid peroxidation reactions and protected the resultant inactivation of the NADH-cytochrome c reductase activity. Activities of succinate oxidase in rat and canine brain mitochondria were virtually unaffected by CV-2619 and its metabolites (10(-5)-10(-6) M). On the other hand, CV-2619 markedly suppressed the state 3 respiration in glutamate oxidation in a dose dependent manner without any effect on the state 4 respiration and the ADP/O ratio in intact rat brain mitochondria. The inhibitory effect of CV-2619 was also observed in NADH-cytochrome c reductase, but not in NADH-2,6-dichlorophenolindophenol (DCIP) and NADH-ubiquinone reductases in canine brain mitochondria. These facts and results of inhibitor analysis suggest that the action site of CV-2619 is NADH-linked complex I in the mitochondrial respiratory chain and is different from that of inhibitors of oxidative phosphorylation such as rotenone, oligomycin and 2,4-dinitrophenol. Finally, the above findings suggest that CV-2619 acts as an electron carrier in respiratory chains and functions as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria. It appears likely that the inhibition of oxygen consumption caused by CV-2619 is related to the effect on non-respiratory systems such as lipid peroxidation which also consumes oxygen.

Acute 60Co-gamma irradiation of rats decreases the inhibitory effect of succinate on the lipid peroxidation of liver mitochondria.

Succinate inhibits NADPH-dependent lipid peroxidation of liver mitochondria. This effect of succinate decreased 12 h after whole-body 60Co-gamma irradiation, depending on the dose of irradiation.

Generation of low-level chemiluminescence during the metabolism of 1-naphthol by rat liver microsomes

The metabolism of 1-naphthol in rat liver microsomal fractions supplemented with NADPH is accompanied by low-level chemiluminescence which reflects the formation of molecular excited states. Photoemission consists of two phases which both are dependent on microsomal protein and 1-naphthol concentration. The involvement of cytochrome P-450 in the microsomal metabolism of 1-naphthol was indicated by an inhibition of chemiluminescence by aminopyrine or metyrapone. Oxygen is required for light emission. Whereas phase I is hardly influenced by superoxide dismutase, phase II is suppressed. Chemiluminescence was not associated with malondialdehyde accumulation, in contrast to NADPH-dependent lipid peroxidation in microsomal fractions in the absence of 1-naphthol. Phase I of chemiluminescence appears to directly reflect cytochrome P-450-dependent hydroxylation, and phase II is attributed to redox cycling of products arising from these reactions, e.g. the 1,4- and/or 1,2-naphthoquinones as oxidation products of the corresponding dihydroxynaphthalenes.

Stimulation of nadph-dependent lipid peroxidation by 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid and diquat in microsomes isolated from Pisum sativum

2,4-Dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and diquat (DQ) stimulate NADPH-dependent lipid peroxidation (in the presence of ADP-Fe2+ complex) in microsomes from green leaves and etiolated seedlings. The activity was not shown with 2,6-D and 2,4,6-T which are non-active as herbicides. The stimulation was not observed when NADPH was replaced with NADH or ascorbate. It is suggested that the increasing of dark lipid peroxidation in microsomes plays some role in the toxicity of the herbicides to plants.