Lipid peroxidation activity mediated by NADPH-cytochrome C reductase purified from rabbit liver microsomes.

Purified NADPH-cytochrome c reductase of rabbit liver microsomes was examined to determine whether or not the reported low lipid peroxidation activity of rabbit liver microsomes is due to the enzyme, NADPH-cytochrome c reductase. NADPH-cytochrome c reductase was purified from phenobarbital-treated rabbit liver microsomes to a specific activity of 14.9 to 21.4 unit per mg of protein with a yield of 15.2 to 16.4%. The purified sample (21.4 unit/mg of protein) was almost homogenous as determined by sodium dodecylsulfate gel electrophoresis. This sample was used for determining lipid peroxidation activity. EDTA and ferrous ion but not ADP were essential requirements for the activity. FMN enhanced the activity when low concentrations of the NADPH-cytochrome c reductase were used for the assay. NADP and 2'-AMP, which are inhibitors of NADPH-cytochrome c reductase, inhibited the lipid peroxidation activity. a-Tocopherol and p-chloromercuribenzoate (PCMB) also inhibited the activity. From these results, we confirmed the rabbit liver microsomal enzyme NADPH-cytochrome c reductase plays a role in lipid peroxidation activity. The reported low lipid peroxidation activity in rabbit liver microsomes does not appear to be caused by the NADPH-cytochrome c reductase.     

Inhibition of rat liver microsomal lipid peroxidation by boldine.

The alkaloid boldine, found in the leaves and bark of boldo, was an effective inhibitor of rat liver microsomal lipid peroxidation under a variety of conditions. The following systems all displayed a similar sensitivity to boldine: non-enzymatic peroxidation initiated by ferrous ammonium sulfate; iron-dependent peroxidation produced by ferric-ATP with either NADPH or NADH as cofactor; organic hydroperoxide-catalyzed peroxidation; and carbon tetrachloride plus NADPH-dependent peroxidation. Boldine inhibited the excess oxygen uptake associated with microsomal lipid peroxidation. Thus, boldine was effective in inhibiting iron-dependent and iron-independent microsomal lipid peroxidation, with 50% inhibition occurring at a concentration of about 0.015 mM. Boldine did not appear to react efficiently with superoxide radical or hydrogen peroxide, but was effective in competing for hydroxyl radicals with chemical scavengers. Concentrations of boldine which produced nearly total inhibition of lipid peroxidation had no effect on microsomal mixed-function oxidase activity nor did boldine appear to direct electrons from NADPH-cytochrome P450 reductase away from cytochrome P450. Boldine completely protected microsomal mixed-function oxidase activity against inactivation produced by lipid peroxidation. The effectiveness of boldine as an anti-oxidant under various conditions, and its low toxicity, suggest that this alkaloid may be an attractive agent for further evaluation as a clinically useful anti-oxidant.     

Activation of misonidazole by rat liver microsomes and purified NADPH-cytochrome c reductase

Rat liver microsomes and purified NADPH-cytochrome c reductase metabolized [14C]misonidazole anaerobically to a reactive intermediate that covalently binds to tissue macromolecules. Air strongly inhibited the binding whereas carbon monoxide had no effect, indicating that misonidazole is activated via reduction and not by cytochrome P-450-dependent oxidation. Both systems showed an absolute requirement for NADPH and were stimulated by flavine (FAD) and paraquat. The apparent Km for misonidazole binding to microsomal protein was 0.74 mM the apparent Vmax was 0.64 nmole 14C bound . mg-1 . min-1. At a single substrate concentration, nitrofurantoin, nitrofurazone and desmethylmisonidazole inhibited the covalent binding of misonidazole to microsomal protein by 47, 26, and 38% respectively. The effect of nitrofurantoin on the kinetics of misonidazole binding gave a complex interaction indicative of uncompetitive inhibition. Glutathione reduced the binding of misonidazole to microsomal protein below the level observed for boiled microsomes while ascorbic acid had no effect. Compared to nitrofurantoin and paraquat, misonidazole was a poor stimulator of superoxide production as measured by adrenochrome formation.     

Selective inactivation of rat lung and liver microsomal NADPH-cytochrome c reductase by acrolein

Addition of acrolein to rat lung or liver microsomal suspensions resulted in total inactivation of NADPH-cytochrome c reductase and partial conversion of cytochrome P-450 to P-420 in a concentration- and time-dependent fashion. Acrolein also caused total loss of nonprotein sulfhydryl content in both preparations, whereas protein sulfhydryl content was decreased by 40% and 28% in lung and liver preparations, respectively. Maxima of about 60% of the total lung cytochrome P-450 and 50% of the liver cytochrome P-450 in acrolein-treated microsomes did not support the N-demethylation of benzphetamine or ethylmorphine or hydroxylation of aniline because of the total loss of NADPH-cytochrome c reductase. Addition of purified NADPH-cytochrome c reductase to the acrolein-treated lung or liver microsomal suspension largely restored these monooxygenase activities. Addition of glutathione or dithiothreitol to the lung or liver microsomal suspension prior to the addition of acrolein significantly protected cytochrome P-450 from conversion to cytochrome P-420 as well as NADPH-cytochrome c reductase from inactivation. Thus, selective conjugation of acrolein with lung and liver NADPH-cytochrome c reductase but not cytochrome P-450 was responsible for total loss of these lung and liver monooxygenase activities.     

Effects of lipid peroxidation on neurotransmitters uptake by rat synaptosomes

This paper described the effect of in vitro peroxidation achieved by 60 s or 5 min exposure to 60 microM Fe2+ with 200 microM ascorbic acid, on selected properties of rat brain synaptosomes reflecting some steps of chemical neurotransmission. The studies have revealed dramatic differences between dopamine, GABA and choline high affinity uptake systems in response to peroxidation. The uptake of calcium by synaptosomes submitted to free radical oxidation, mostly its K+-depolarization-dependent portion, was significantly suppressed. In contrast, peroxidation appeared not to influence the transmembrane synaptosomal potential. It is concluded, that peroxidation of synaptic endings modifies the lipid content of synaptoplasmatic membranes and consequently leads to severe disturbances in the function of neurotransmitter uptake systems and depolarization-dependent calcium channels.     

Enhancement of ethanol-induced lipid peroxidation in rat liver by lowered carbohydrate intake.

In order to investigate the effect of carbohydrate intake on ethanol-induced lipid peroxidation and cytotoxicity, rats were maintained on four different test diets, a medium-carbohydrate (carbohydrate intake, 8.4 g/day/rat on average), a low-carbohydrate (carbohydrate intake, 2.8 g/day/rat on average), an ethanol-containing medium-carbohydrate (carbohydrate and an ethanol intake, 8.4 and 2.9 g/day/rat on average, respectively), and an ethanol-containing low-carbohydrate diet (2.8 and 2.9 g/day/rat on average, respectively). Ethanol and the low-carbohydrate diet each increased the liver malondialdehyde content, but the combined effect of both (ethanol-containing low-carbohydrate diet) was much more prominent than either alone. The degree of increase in malondialdehyde content almost paralleled the activity of the microsomal ethanol oxidizing system. Both the low-carbohydrate and the ethanol-containing low-carbohydrate diets decreased the liver glutathione content, but ethanol combined with the medium-carbohydrate diet had no effect on the content. Ethanol treatment increased the liver triglyceride content only when combined with the low-carbohydrate diet. The rate of NADPH-dependent microsomal malondialdehyde formation was much higher in microsomes from rats maintained on the ethanol-containing low-carbohydrate diet than in those from rats on the ethanol-containing medium-carbohydrate diet, indicating that lowered carbohydrate intake augments ethanol-induced malondialdehyde accumulation in the liver by enhancing the rate of lipid peroxidation. In addition, when incubated with red blood cells in the presence of NADPH, microsomes from rats fed the ethanol-containing low-carbohydrate diet caused marked hemolysis, which was prevented by the addition of 5 mM glutathione to the incubation system. Furthermore, addition of 50 mM ethanol to the reaction system greatly accentuated the hemolysis. These results suggest that lowered carbohydrate intake at the time of ethanol consumption potentiates ethanol cytotoxicity by enhancing ethanol-induced lipid peroxidation.     

Effect of delta9-tetrahydrocannabinol on rat liver microsomal lipid peroxidation.

In vivo ip administration of saline-Tween 80 suspensions of pure Δ⁹-tetrahydrocannabino (Δ⁹-THC) under both acute (10 and 50 mg/kg) and chronic (10 mg/kg/day for 21 days) conditions, to adult male albino rats inhibited liver microsomal lipid peroxidation. In vitroΔ⁹-THC (0.5–8 μg/mg protein) also markedly lowered NADPH- and ascorbate-induced microsomal lipid peroxidation. Δ⁹-THC was also effective in lowering CCl₄-induced lipid peroxidation in vitro. These results suggest that Δ⁹-THC exerts a stabilizing effect on hepatic microsomal membrane.     

Inhibition of rat heart and liver microsomal lipid peroxidation by nifedipine

Lipid peroxidation in rat heart and liver microsomes was induced by an NADPH-generating system or by ascorbate in the presence of an ADP-iron complex. Microsomal lipid peroxidation, as measured by malonaldehyde formation, was inhibited by nifedipine over a wide range of concentrations (47 microM to 6 mM). Nifedipine also decreased the oxygen consumption of cardiac and hepatic microsomes in a concentration-dependent manner. These results indicate that nifedipine may perturb microsomal electron transport systems. Nifedipine may have the potential to alter the sensitivity of cardiac and hepatic membranes to peroxidative damage.     

Carbon tetrachloride-induced lipid peroxidation of rat liver microsomes in vitro.

Characteristics of carbon tetrachloride-induced lipid peroxidation of rat liver microsomes and effect on microsomal enzymes were studies in vitro. Microsomes isolated from well-perfused livers and washed with EDTA-containing medium exhibited low endogenous lipid peroxidation when incubated in a phosphate buffer (> 0.1 M) in the presence of NADPH, whereas carbon tetrachloride stimulated to a great extent the peroxidation under these conditions. The stimulation was dependent on the concentration of NADPH, neither NADH nor ascorbic acid being replaced. The stimulatory action by bromotrichloromethane was more marked than that by carbon tetrachloride, however chloroform had no stimulatory action. N,N-Diphenyl-p-phenylene diamine, diethyldithiocarbamate and disulfiram inhibited carbon tetrachloride-induced lipid peroxidation in low concentrations. Inhibitions by thiol compounds and EDTA were weaker. Ferricyanide, cytochrome c and vitamine K₃ inhibited the stimulation by carbon tetrachloride while no inhibition was seen with carbon monoxide. An increase in the degree of carbon tetrachloride-induced lipid peroxidation resulted in a coincidental decrease in microsomal cytochrome P-450 content accompanying a parallel loss in aminopyrine demethylase activity, while NADH-ferricyanide dehydrogenase and NAD(P)H-eytochrome c reductase activities, and cytochrome b₅ content remained unaffected. Similar results were obtained when microsomes were peroxidized with NADPH in combination with ferric chloride and pyrophosphate. Regarding the mechanism of hepatotoxic action of carbon tetrachloride, these results support the hypothesis of lipid peroxidation.     

Suppressive effect of bifemelane on lipid peroxidation in rat liver

The effects of Bifemelane (BF) on lipid peroxidation, the activities of hepatic drug metabolizing enzymes, and the function of cell membranes were examined in rats. In the liver ischemia-reperfusion model, BF suppressed the elevation of the lipid peroxidation level during the period of reperfusion. BF did not exhibit a radical-trapping action using a stable free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), which was estimated by electron spin resonance (ESR). BF remarkably inhibited NADPH-dependent lipid peroxidation in vitro. BF had no effect on the contents of cytochrome P-450 and b5 and the activities of NADPH cytochrome P-450 reductase and Cu,Zn-superoxide dismutase (SOD). BF suppressed phorbol myristate acetate (PMA)-induced superoxide formation of polymorphonuclear leukocytes (PMNs), protected hypotonic hemolysis of erythrocyte and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and serum phospholipase A activity. These results suggest that BF has neither radical-trapping activity nor any influence on the drug metabolizing enzymes, but BF has a membrane-stabilizing action and it attributes to the suppressive effect of lipid peroxidation.     

Effect of sex hormones on lipid peroxidation in rat liver

The role of sex hormones in hepatic lipid peroxidation, and in hepatic aldehyde oxidase and xanthine oxidase activities were investigated using rat liver homogenates. It was observed that male rat had a significantly greater content of malondialdehyde in liver than female. Among the sex hormones tested, estradiol, one of female hormones, markedly inhibited the formation of lipid peroxides in liver tissues in vitro. Especially, the inhibitory effect of estradiol appeared more remarkably in Fe+2-induced lipid peroxidation. The hepatic xanthine oxidase activity was decreased about 15% by 10(-6) M estradiol, whereas, the aldehyde oxidase activity was almost completely disappeared at the same concentration of estradiol. It implies that sex differences in lipid peroxidation is attributed to the suppression of free radical generating system by estradiol.     

异莲心碱对大鼠肝匀浆脂质过氧化的影响

目的:研究异莲心碱(isoliensinine,IL)对大鼠肝匀浆脂质过氧化的影响.方法:用硫代巴比妥酸(TBA)法测定异莲心碱对大鼠肝匀浆自氧化及Vit C-Fe2 系统诱导引起的脂质过氧化产物丙二醛的含量.结果:异莲心碱能显著抑制大鼠肝匀浆自氧化及Vit C-Fe2 系统诱导所产生MDA的含量,且呈现出一定的量效关系,达到50%抑制率所需药物浓度IC50分别为0.67 g·L-1和1.05 g·L-1.结论:异莲心碱具有显著的抗脂质过氧化作用.     

Inhibitory effect of eugenol on non-enzymatic lipid peroxidation in rat liver mitochondria.

The anti-peroxidative activity of eugenol on Fe(2+)-ascorbate- and Fe(2+)-H2O2-induced lipid peroxidation was studied using rat liver mitochondria. Eugenol inhibited thiobarbituric acid reactive substance (TBARS) formation induced by both the systems in addition to oxygen uptake and mitochondrial swelling induced by Fe(2+)-ascorbate. Time course studies on TBARS formation indicated the ability of eugenol to inhibit initiation and propagation reactions. There was no measurable chemical modification of eugenol during the course of mitochondrial peroxidation by both the systems. Mitochondrial peroxidation by Fe(2+)-H2O2 was inhibited by hydroxyl radical (OH) scavengers like mannitol, benzoate, formate and dimethyl sulfoxide apart from eugenol. The OH scavenging ability of eugenol was evident from its inhibitory effect on OH-mediated deoxyribose degradation. The second-order rate constant for the reaction of OH with eugenol was about 4.8 x 10(10) M-1 sec-1. Eugenol reduced Fe3+ ions and Fe3+ chelated to citrate or ADP but it did not exhibit pro-oxidant activity in OH-mediated deoxyribose degradation. Incubation of mitochondria with eugenol resulted in the uptake of small but significant quantities of eugenol which inhibited subsequent lipid peroxidation by acting as a chain breaking antioxidant.     

Nuclear lipid peroxidation induced in rat liver by T-2 mycotoxin

Oral administration to rats of T-2 mycotoxin (1.25 mg/kg) for five days causes an increase in lipid peroxidation (ascorbate-induced as well as NADPH-dependent) in hepatic nuclei, while the activity of liver glutathione-S-transferase (EC. 2.5.1.18) is decreased. The hepatotoxicity could be due to lipid peroxidation induced by depletion of hepatic reduced glutathione and/or production of free radicals.     

NADPH-cytochrome P-450 reductase: Preferential inhibition by ellipticine and other type II compounds having little effect on NADPH-cytochrome c reductase

Ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b]carbazole) binds with an affinity greater than most other compounds known to interact with P-450. Control and 3-methylcholanthrene-induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) and acetanilide 4-hydroxylase and control and phenobarbital-induced ethylmorphine N-demethylase activities are all markedly inhibited by ellipticine to about the same extent. Ellipticine and other Type II compounds (metyrapone, octylamine-1, pyridine and aniline) preferentially inhibit NADPH-cytochrome P-450 reductase activity, while affecting NADPH-cytochrome c reductase activity very little. Butanol-1, a compound having pure Reverse Type I character, does not block P-450 reductase activity like these Type II compounds. These data suggest that Type II compounds bind to P-450 ferric iron in the sixth coordinate position in such a way as to impede transfer of the first electron from the hydrophobic binding site of the reductase to the P-450-substrate complex, while leaving unencumbered any transfer of electrons from the hydrophilic binding site of the reductase to soluble electron acceptors such as cytochrome c. These data indicate that ellipticine may be very useful in attempting to understand the mechanism by which electrons are transferred from the reductase to the cytochrome(s) P-450.     

Effects of antioxidant vitamins on glutathione depletion and lipid peroxidation induced by restraint stress in the rat liver

BACKGROUND AND AIM: Stress as a cofactor has been reported to affect the progression and severity of several diseases. The influence of stress on the liver is of interest from the clinical point of view because stress plays a potential role in aggravating liver diseases in general and hepatic inflammation in particular, probably through generation of reactive oxygen species. The present study was undertaken to investigate the potential of the antioxidant vitamins A (retinol), E (tocopherol) and C (ascorbic acid) individually and in combination (vitamin E + C) to modulate restraint stress-induced oxidative changes. These effects were determined by measuring changes in hepatic levels of free radical scavenging enzymes such as superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase, as well as levels of total glutathione (GSH), malondialdehyde (MDA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). METHODS: Immobilisation was achieved by placing the animals in wire mesh cages of their size. The rats were orally administered vitamins A, E and C individually and in combination (E + C) prior to and after 6 hours of immobilisation stress exposure. The hepatic levels of SOD, GST, catalase, GSH and MDA were determined by spectrophotometric methods. Liver SOD activity was assayed by monitoring the amount of enzyme required to inhibit autoxidation of pyrogallol by 50%. Hepatic GST was monitored by following the increase in absorbance at 340 nm of CDNB-GSH conjugate generated due to GST catalysis between GSH and CDNB. Catalase activity in liver tissues was determined using peroxidase as the substrate. Lipid peroxidation was measured by determining the level of thiobarbituric acid reactive substances. ALT and AST were determined by commercial kits. RESULTS: Six hours of immobilisation stress caused a decrease in liver levels of SOD (p = 0.001), catalase (p = 0.031), GST (p = 0.021) and GSH (0.013), while levels of MDA (p = 0.0015), AST (p = 0.05) and ALT (p = 0.046) were increased compared with non-stressed control rats. Both pre-vitamin stress and post-vitamin stress treatments either alone or in combination were associated with increased normalisation of these parameters towards control values, with post-vitamin treatment being the more effective of the two. Vitamins E and C individually were found to be more effective in restoring the endogenous antioxidant system than vitamin A. The combined vitamin (E + C) post-stress treatment was found to be effective but not additive in combating hepatic oxidative stress. The beneficial effects of these vitamin treatments were also reflected in reversions of altered AST and ALT levels towards their control values. CONCLUSION: Vitamins E or C alone or in combination can be given as prophylactic/therapeutic supplements for combating scavenging free radicals generated in liver tissue. This approach may reduce oxidative stress caused by diseases such as cirrhosis.     

Investigation of lipid peroxidation induced by hydrazine compounds in vivo in the rat

Phenylhydrazine caused lipid peroxidation in rats in vivo as detected by expiration of ethane but this was not due to lipid peroxidation in liver, as there was no associated MDA production in this tissue. Hydrazine did not cause either ethane expiration or MDA formation. Both hydrazine and phenylhydrazine caused a significant increase in propane expiration. Phenylhydrazine significantly decreased packed cell volume and haemoglobin levels but hydrazine had no effect on these parameters. These data indicate that the early toxicity of hydrazine does not involve peroxidation of lipids whereas phenylhydrazine causes lipid peroxidation possibly within the erythrocyte, perhaps by interaction with red cell haemoglobin.     

Stimulation by adriamycin of rat heart and liver microsomal NADPH-dependent lipid peroxidation

Rat liver and heart microsomes catalyze the transfer of single electrons from NADPH to adriamycin forming semiquinone radicals which, in turn, activate molecular oxygen. This process stimulated lipid peroxidation 5- to 7-fold as measured by malonaldehyde formation. Adriamycinaugmented lipid peroxidation was linear with time to 60 min, optimal at 1.0 mg of microsomal protein/ml and pH 7.5, and was proportional to the adriamycin concentration up to 100 μM. An NADPH-generating system was superior to NADPH, and an oxygen atmosphere tripled the rate of peroxidation as compared to air. Nitrogen abolished adriamycin-stimulated peroxidation. Superoxide dismutase, reduced glutathione, α-tocopherol, EDTA, dioxopiperazinylpropane (ICRF-187), and dimethylurea were effective inhibitors of lipid peroxidation. This suggests that Superoxide anion and possibly hydroxyl radical may be formed by the oxidation of the adriamycin semiquinone radical and thus stimulate the peroxidation of microsomal unsaturated fatty acids. Although adriamycin failed to stimulate lipid peroxidation in heart microsomes from control animals, peroxidation was dramatically increased when adriamycin was added to cardiac microsomes from α-tocopherol-deficient rats. Lipid peroxidation in α-tocopheroldeficient liver microsomes was four times greater than in control microsomes with the NADPH-generating system, and adriamycin did not further increase that high rate of peroxidation; however, when NADPH was used as the source of electrons in place of the NADPH-generating system, adriamycin stimulated peroxidation more than 2-fold. These results suggest that microsomal lipid peroxidation may play a role in the cytotoxicity and cardiotoxicity of adriamycin.