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.     

A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes

The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine). The labile sulfur contained in trisulfide disappeared during incubation with liver microsomes. In the CCl₄-induced lipid peroxidation system, the cytochrome P-450 level and NAD(P)H-cytochrome P-450 reductase activity were significantly decreased by the addition of trisulfide derivatives. Therefore, in cytochrome P-450-dependent lipid peroxidation system, the potential effects of trisulfide appear to be mediated via enzyme inhibition. These results suggest that pretreatment of the trisulfide derivatives may affect the toxic function of exogenous xenobiotics or drugs, which are reduced by the liver enzyme cytochrome P-450 to radical species.     

A possible mechanism of naproxen-induced lipid peroxidation in rat liver microsomes

Previous papers from our laboratory report that naproxen and salicylic acid induced lipid peroxidation in rat liver microsomes, however, the mechanism is still unclear. In the present paper, ferrous iron release, nicotinamide-adenine dinucleotide phosphate reduced form (NADPH) oxidation and hydrogen peroxide (H2O2) formation have been measured to find out which mechanisms are involved in naproxen- and salicylic acid-induced lipid peroxidation. While the increase of ferrous iron release was observed with high concentrations of naproxen, salicylic acid did not stimulate ferrous iron release. Neither of these drugs stimulated NADPH oxidation and H2O2 formation. However hexobarbital and perfluorohexane, known as uncouplers of cytochrome P450, stimulated microsomal NADPH oxidation, O2 consumption, H2O2 formation and water (H2O) formation involving four-electron oxidase reaction. These results suggest that ferrous iron release contributes to naproxen-induced microsomal lipid peroxidation and that naproxen and salicylic acid are not uncouplers of cytochrome P450. Apparently H2O2 does not play an important role in naproxen- and salicylic acid-induced microsomal lipid peroxidation.     

Inhibitory action of Oren-gedoku-to extract on enzymatic lipid peroxidation in rat liver microsomes

We examined the inhibitory action of the extract of Oren-gedoku-to, a traditional herbal medicine known to act as an antioxidant, on enzymatic lipid peroxidation in rat liver microsomes. Simultaneous addition of a spray-dried preparation of Oren-gedoku-to extract (Tsumura TJ-15) inhibited enzymatic lipid peroxidation induced by reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH) and ADP/Fe3+ complex in liver microsomes in a dose-dependent manner. When the inhibition by TJ-15 of enzymatic lipid peroxidation in liver microsomes was kinetically analyzed, this medicine showed a competitive inhibition against NADPH or ADP/Fe3+ complex. TJ-15 inhibited the NADPH-driven enzymatic reduction of ADP/Fe3+ complex or cytochrome c in liver microsomes competitively. TJ-15 enhanced NADPH consumption by liver microsomes with ADP/Fe3+ complex. Treatment with TJ-15 after the onset of enzymatic lipid peroxidation in liver microsomes inhibited the progression of lipid peroxidation in a dose-dependent manner. The present results indicate that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes in the initiation and propagation steps in a dose-dependent manner. These results also suggest that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes not only through its antioxidant action but also through reduction of the supply of electrons derived from NADPH to ADP/Fe3+ complex in liver microsomes both in a competitive manner and through stimulation of NADPH oxidation.     

The role of iron in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced lipid peroxidation by rat liver microsomes

Treatment of female Sprague-Dawley rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances hepatic lipid peroxidation, and the role of iron in TCDD-induced lipid peroxidation was examined. Ferrous and ferric ions, and the chelators adenosine diphosphate (ADP), ethylenediaminetetraacetic acid (EDTA) and desferrioxamine (DFX) were added to an in vitro microsomal lipid peroxidation system using microsomes from control and TCDD-treated animals. Both ferrous and ferric ions enhanced microsomal lipid peroxidation, with the greatest effect being produced by the combination of ferrous and ferric ions. The addition of ADP and EDTA produced modest increases in microsomal lipid peroxidation, suggesting that these chelators facilitated formation of reactive oxygen species by iron. The addition of DFX markedly inhibited microsomal lipid peroxidation, with greatest inhibition occurring with microsomes from TCD-treated animals. The results indicate that iron is involved in TCDD-induced lipid peroxidation.     

Stimulative effects of chelating agents, 2,2'-bipyridine and 1,10-phenanthroline, on lipid peroxidation in rat liver microsomes.

Well known lipid peroxidation inhibitors, 1,10-phenanthroline and 2,2'-bipyridine, stimulated microsomal NADPH- and ascorbic acid-dependent lipid peroxidation when low concentrations of these chelating agents were added to incubation mixture. The stimulatory effects of the chelating agents on lipid peroxidation were enhanced when ferrous ion was added together with the chelating agents to the mixture at a molar ratio of 1:1. Ethylenediaminetetraacetic acid (EDTA) had no stimulatory effect on lipid peroxidation. Ferrous ion-EDTA complex increased lipid peroxidation by only 20-30%, which was lower than that obtained by addition of the same concentration of ferrous ion alone. On the other hand, manganese and calcium ions, which are also inhibitors of lipid peroxidation, had no ability to stimulate lipid peroxidation even in the presence of extra ferrous ions. Changes in the lipid peroxidation by chelating agents affected the apparent activity of ethylmorphine N-demethylation.     

Effect of multiple administration of calcium antagonists on lipid peroxidation in rat liver microsomes

The effects of three calcium antagonists, nifedipine (NF), verapamil (VP), and diltiazem (DT), on the lipid peroxidation (LPO) in rat liver microsomes were studied. The drugs were administered in oral doses of 50, 40, and 30 mg/kg daily for 21 days in male Wistar rats. Nonstimulated LPO was significantly decreased by NF and was not changed by VP and DT. There was a correlation between the extent of the previously found enzyme-inducing action and the potency of the antioxidant effects of calcium antagonists. Fe²⁺/NADPH- and Fe²⁺/ascorbate-stimulated microsomal LPO was increased by the calcium antagonists studied in the following order: VP>DT>NF (the increase caused by NF was insignificant in Fe²⁺/NADPH stimulation).     

Protection by gsh against lipid peroxidation induced by ascorbate and iron in rat liver microsomes

GSH is considered to be a potent inhibitor of 1ipid peroxidation, but the mechanisms by which it carries out this function are not clear. GSH-dependent factors which inhibit 1ipid peroxidation in the NADPH and in the ascorbate-iron microsomal 1ipid peroxidation systems have been demonstrated in rat liver 105,000 $\text{g}$ supernatant (1,2). This communication describes a GSH-dependent factor in the microsomal fraction of rat liver which inhibits ascorbate and iron-induced microsomal 1ipid peroxidation.     

Effect of the flavanolignans of Silybum marianum L. on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes.

The effect of several flavanolignans (silicristin, silidianin, silybin and isosilybin) present in silymarin, the extract of Silybum marianum fruits, was tested on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes. In microsomes lipid peroxidation was generated by ADP/Fe2+ and NADPH. All flavanolignans inhibited peroxidation in a concentration dependent manner. In hepatocytes lipid peroxidation was induced by ADP/Fe3+ complex and cell damage was evaluated as LDH activity released in the medium. The inhibition of the peroxidative process by flavanolignans was also evident in this model, even if with a potency order different from that found in microsomes. In contrast, the effect on LDH release was significant only for silybin and isosilybin, the other compounds being inactive on this parameter.     

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.     

Lipid peroxidation in rat liver microsomes: influence of carcinogenic N-nitrosocarbaryl

The reactivities of carbaryl, N-methyl 1-naphthylcarbamate insecticide and its N-nitrosated derivative carcinogenic, N-nitrosocarbaryl, were investigated on the microsomal hepatic lipid peroxidation and NADPH-dependent reductase activities. The in vivo treatment by N-nitrosocarbaryl produced a reduction in lipoperoxidative degradation induced in vitro by NADPH with regard to the formation of malonaldehyde and conjugated dienes. Carbaryl, its precursor did not affect lipid peroxidation under the same in vivo conditions. Moreover, following administration of the 2 compounds, the activities of NADPH-cytochrome c reductase as well as NADPH-neotetrazolium reductase were significantly decreased by N-nitrosocarbaryl but not influenced by carbaryl. Correspondingly, in vitro studies were performed; different action patterns of the 2 tested xenobiotics were also noted after treatment of rat liver microsomes in vitro by carbaryl and N-nitrosocarbaryl especially in their ability to cope with microsomal oxygen activation. N-Nitrosocarbaryl proved to have a potent inhibitor concentration effect on NADPH-dependent chemiluminescence response in vitro; carbaryl was virtually ineffective on this parameter. No significant difference appeared in the affinity of N-nitrosocarbaryl and carbaryl for the microsomal phospholipids. From the in vitro explorations, it was suggested that carcinogenic N-nitrosocarbaryl may be involved in the inhibition mechanism of microsomal lipid peroxidation through decreases in both NADPH-dependent reductase activities and superoxide generation.     

Alterations in lipid peroxidation, antioxidant enzymes, and carcinogen metabolism in liver microsomes of vitamin E-deficient trout and rat.

Feeding rainbow trout for 16 weeks a diet in which the levels of vitamin E were reduced 70-fold resulted in marked depletion (18-fold) of vitamin E levels in liver microsomes from these fish. The susceptibility of hepatic microsomes to lipid peroxidation in vitro and the levels of plasma and liver microsomal lipid hydroperoxides generated in vivo were markedly elevated in vitamin E-depleted trout. No appreciable alterations were observed in the liver microsomal cytochrome P450-dependent mixed-function oxidase system or in the fatty acid composition of trout liver microsomal membranes. Livers from rats fed a vitamin E-deficient diet for 10 weeks also had significantly lower levels of microsomal vitamin E. In addition, total cytochrome P450 levels were depressed (15%) and cytosolic glutathione was enhanced (40%) in livers from rats fed the vitamin E-depleted diet. Covalent binding of [3H]-(+)-benzo[a]pyrene-7,8-dihydrodiol to exogenous DNA in vitro was enhanced with liver microsomes from vitamin E-deficient trout and these fish were much more sensitive to the acute toxicity of this carcinogenic polycyclic aromatic hydrocarbon. These results indicate that trout may be a useful model for studying the significance of peroxidative pathways in carcinogenesis and their manipulation by dietary antioxidants.     

Adriamycin-induced lipid peroxidation in mitochondria and microsomes

The effect of the anti-neoplastic agent adriamycin on the peroxidation of lipids from rat liver and heart mitochondria and rat liver microsomes was investigated. The extent of total lipid peroxidation was determined by assaying for malondialdehyde (MDA), while the degradation of unsaturated fatty acids was monitored using gas chromatography. For liver mitochondria and microsomes, the formation of MDA was dependent on the concentrations of adriamycin, Fe3+, and protein, as well as time. In the presence of 50 microM adriamycin and saturating amounts of NADH, 1.5 +/- 0.2 nmol MDA/mg protein/60 min was produced with liver mitochondria. Upon addition of 25 microM Fe3+, the amount of MDA generated was increased to 6.5 +/- 0.1 nmol/mg protein/60 min. Liver microsomes produced amounts which were approximately 2-fold higher under all conditions. No MDA formation could be detected in rat heart mitochondria. The addition of 50 microM chlorpromazine completely inhibited peroxidation, whereas 0.5 to 1.0 mM p-bromophenacyl bromide blocked MDA formation by 50%. Analysis of fatty acids by gas chromatography showed that there was about a 50% decrease in arachidonic and docosahexaenoic acids in liver mitochondria and microsomes, but no change in the fatty acid content of heart mitochondria when incubated with both 50 microM adriamycin and 25 microM Fe3+ for 1 hr. These results suggest that (1) therapeutic concentrations of adriamycin enhance the peroxidation of lipids in liver mitochondria and microsomes through an enzymatic mechanism, especially in the presence of Fe3+; and (2) toxicity of this drug may be related to the degradation of membrane lipids.     

Effects of lipid peroxidation on activities of drug-metabolizing enzymes in liver microsomes of rats

A relationship was found between the formation of lipid peroxides and the activities of drug-metabolizing enzymes in liver microsomes of rats. Induction of lipid peroxidation by incubation with ferrous ion led to a sharp decline in the ability of the microsomal enzyme system to demethylate ethylmorphine. Inhibition of lipid peroxidation by EDTA increased the enzyme activity about two-fold. Addition of EDTA (0.1 mM) to the incubation mixture produced marked changes in the Michaelis constants of drug-metabolizing enzymes and in inhibition constants of SKF 525-A for drug-metabolizing enzymes.     

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.