Enzymatic lipid peroxidation and oxidative metabolism of aminazine in brain microsomal fractions]

NAD (P) H-dependent enzymic systems, both of lipid peroxidation and chlorpromazine oxidative metabolism are shown to be localized in the microsomal fractions from human and rat brain. Hydroxy-derivatives of chlorpromazine (e.g. 7-OH-chlorpromazine) formed in the course of enzymic NADPH-dependent metabolism possess antioxidant activity and inhibit lipid peroxidation in the brain microsomes. The properties of enzymic NAD (P) H-dependent oxigenase systems in the membranes of the microsomal reticulum of the liver and brain are compared.     

Non-Enzymic Lipid Peroxidation in Microsomes and Microsomal Phospholipids Induced by Anthracyclines

The stimulation of non-enzymic lipid peroxidation by doxorubicin, daunorubicin and 7 derivatives was investigated in extracted microsomal phospholipids and in intact microsomes.

Evidence was obtained for the necessity of a free amino-sugar moiety for a stimulative effect on lipid peroxidation. Binding of anthracyclines to RNA (which is present in microsomes) was inhibitory towards stimulation.

Drugs that stimulated lipid peroxidation in a non-enzymic system with extracted phospholipids also were stimulative in an enzymic, NADPH-dependent, microsomal system. They were not always effective in intact microsomes without the enzymic system.

The role of the enzymic system in the stimulation of anthracycline induced lipid peroxidation is thought to be the reduction of iron ions rather than the stimulation of oxygen radical production via the anthracyclines.     

Non-Enzymic Lipid Peroxidation in Microsomes and Microsomal Phospholipids Induced by Anthracyclines

The stimulation of non-enzymic lipid peroxidation by doxorubicin, daunorubicin and 7 derivatives was investigated in extracted microsomal phospholipids and in intact microsomes.

Evidence was obtained for the necessity of a free amino-sugar moiety for a stimulative effect on lipid peroxidation. Binding of anthracyclines to RNA (which is present in microsomes) was inhibitory towards stimulation.

Drugs that stimulated lipid peroxidation in a non-enzymic system with extracted phospholipids also were stimulative in an enzymic, NADPH-dependent, microsomal system. They were not always effective in intact microsomes without the enzymic system.

The role of the enzymic system in the stimulation of anthracycline induced lipid peroxidation is thought to be the reduction of iron ions rather than the stimulation of oxygen radical production via the anthracyclines.     

LIPID PEROXIDE FORMATION IN RAT BRAIN

Abstract— Lipid peroxide formation as measured by the thiobarbituric acid reaction was demonstrated in subcellular fractions of rat brain. The ascorbic acid induced nonenzymic lipid peroxidation was distributed in all the subcellular fractions with a maximum in microsomes. The NADPH dependent enzymic lipid peroxidation occurred mainly in microsomes and to a smaller extent in synaptosomes; NADH could replace NADPH for the enzymic lipid peroxidation under the assay conditions employed. Fe²⁺ but not Fe³⁺ stimulated the NADPH or NADH dependent lipid peroxide formation. The optimum conditions with respect to pH, ascorbic acid or NADPH concentration, time of incubation and protein concentration were studied. Heating the microsomes at 100^oCdid not influence the ascorbate-induced lipid peroxidation but completely abolished the NADPH linked peroxidation. Several heavy metal ions, surface active agents and EDTA were inhibitory to lipid peroxidation. The effect of thiol agents indicated that -SH groups were involved in the enzymic lipid peroxidation. Studies on subcellular fractions of developing rat brain showed an increasing trend in lipid peroxidation with the advancing age of the animal. No significant difference in lipid peroxidation was observed between brains from normal rats and those from rats affected by experimental allergic encephalomyelitis.     

Lipid peroxidation in rat uterus

Lipid peroxidation in rat uterus has been studied using NADPH- and ascorbate-induced systems. Lipid peroxidation in rat uterus is low as compared to rat liver. Uterus is more sensitive to ascorbate-induced lipid peroxidation than that induced by NADPH. Uterus contains lower amounts of phospholipids and has a lesser degree of unsaturation in lipids. Co-factor studies show that Fe2+ is more important for ascorbate-induced lipid peroxidation. Endometrium is more sensitive to ascorbate-induced lipid peroxidation than myometrium. It also contains more total lipids and phospholipids besides having a higher degree of unsaturation in the lipids as compared to myometrium. Among the subcellular fractions, mitochondria are more prone to ascorbate-induced lipid peroxidation, whereas microsomes are more sensitive to NADPH-induced lipid peroxidation. Uteri from old rats (24 months) and pregnant rats are more resistant to lipid peroxidation than those from 3-month-old control rats. Uterus of pregnant rats contains more factors which inhibit lipid peroxidation and also has a lesser degree of unsaturation in lipids compared with uterus of control rats. The possible consequences of the resistance of uterus to lipid peroxidation, especially during pregnancy and senescence, are discussed.     

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.     

Iron Overload and the Predisposition of Cells to Antioxidant Consumption and Peroxidative Damage

We have investigated the effects of iron overload in vivo on the tocopherol levels and the extent of lipid peroxidation in rat liver microsomes and their response to subsequent oxidative stress in vitro. The results demonstrate a direct correlation between consumption of antioxidant defences and the induction and extent of malondialdehyde production in microsomes prepared from iron-loaded rats. The data are consistent with the requirement for iron (II)/iron (III) ratios in lipid peroxidation in control microsomes.     

Effect of seminal plasma antioxidant on lipid peroxidation in spermatozoa, mitochondria and microsomes

Seminal plasma antioxidant inhibited ascorbate/iron-induced lipid peroxidation in spermatozoa, brain and liver mitochondria. The concentration required to produce inhibition in brain and liver mitochondria was high. Denaturation of spermatozoa resulted in complete loss of antioxidant action. Maintenance of native structure was essential for action of seminal plasma antioxidant in spermatozoal lipid peroxidation. The antioxidant inhibited NADPH, Fe3+-ADP induced lipid peroxidation in microsomes and consequences of lipid peroxidation such as glucose-6-phosphatase inactivation were prevented by presence of antioxidant. It did not inhibit microsomal lipid peroxidation induced by ascorbate and iron and xanthine-xanthine oxidase.     

Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine

Four aqueous extracts from different parts of medicinal plants used in Ayurveda (an ancient Indian Medicine) viz., Momardica charantia Linn (AP1), Glycyrrhiza glabra (AP2), Acacia catechu (AP3), and Terminalia chebula (AP4) were examined for their potential as antioxidants. The antioxidant activity of these extracts was tested by studying the inhibition of radiation induced lipid peroxidation in rat liver microsomes at different doses in the range of 100-600 Gy as estimated by thiobarbituric acid reactive substances (TBARS). Of all these extracts, AP4 showed maximum inhibition in the TBARS formation and hence is considered the best antioxidant among these four extracts. The extracts were found to restore antioxidant enzyme superoxide dismutase (SOD) from the radiation induced damage. The antioxidant capacities were also evaluated in terms of ascorbate equivalents by different methods such as cyclic voltammetry, decay of ABTS(.-) radical by pulse radiolysis and decrease in the absorbance of DPPH radicals. The results were found to be in agreement with the lipid peroxidation data and AP4 showed maximum value of ascorbate equivalents. Therefore AP4, with high antioxidant activity, is considered as the best among these four extracts.     

Effects of gallic acid on brain lipid peroxide and lipid metabolism in streptozotocin-induced diabetic Wistar rats

This study evaluated the protective effects of gallic acid on brain lipid peroxidation products, antioxidant system, and lipids in streptozotocin-induced type II diabetes mellitus. Streptozotocin-induced diabetic rats showed a significant increase in the levels of blood glucose, brain lipid peroxidation products, and lipids and a significant decrease in the activities of brain enzymic antioxidants. Oral treatment with gallic acid (10 mg and 20 mg/kg) for 21 days significantly decreased the levels of blood glucose, brain lipid peroxidation products, and lipids and significantly increased the activities of brain enzymic antioxidants in diabetic rats. Histopathology of brain confirmed the protective effects of gallic acid. Furthermore, in vitro study revealed the free radical scavenging action of gallic acid. Thus, our study shows the beneficial effects of gallic acid on brain metabolism in streptozotocin-induced type II diabetic rats. A diet containing gallic acid may be beneficial to type II diabetic patients.     

Antioxidant role of Umbelliferone in STZ-diabetic rats

The objective of the study was to investigate the role of Umbelliferone (UMB) on lipid peroxidation, nonenzymic and enzymic antioxidants in the plasma and liver of streptozotocin (STZ)-induced diabetic rats. Adult male albino rats of Wistar strain, weighing 180-200 g, were induced diabetes by administration of STZ (40 mg/kg b.wt.) intraperitoneally. The normal and diabetic rats were treated with UMB (30 mg/kg b.wt.) dissolved in 10% dimethyl sulfoxide (DMSO) for 45 days. Diabetic rats had an elevation in the levels of lipid peroxidation markers (thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (HP) and conjugated dienes (CD)), and a reduction in nonenzymic antioxidants (vitamin C and reduced glutathione (GSH) except vitamin E in the plasma and liver, and enzymic antioxidants (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in the liver. Decreased level of beta-carotene and increased level of ceruloplasmin (Cp) were observed in the plasma of diabetic rats. Treatment with UMB and glibenclamide brought back lipid peroxidation markers, nonenzymic and enzymic antioxidants to near normalcy. Since UMB treatment decreases lipid peroxidation markers and enhances antioxidants' status it can be considered as a potent antioxidant.     

Morin augments anticarcinogenic and antiproliferative efficacy against 7,12-dimethylbenz(a)-anthracene induced experimental mammary carcinogenesis

In general, oxidative stress resulting from an imbalance between prooxidant and antioxidant systems plays an important role in the pathogenesis of cancer. Morin (3,5,7,2',4'-pentahydroxyflavone), a member of the flavanol group, has been shown to possess chemopreventive potential against hepatocellular and colon cancer in experimental animals. Given the demonstrated importance of morin, aim of the present study was to evaluate the effect of morin on antiproliferative and anticarcinogenic effect against DMBA-induced experimental mammary carcinogenesis. Oral administration of 7,12-dimethylbenz(a)-anthracene (25 mg/kg body weight) to rats resulted in significant reduction of body weight, enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase), and nonenzymic antioxidants (reduced glutathione, vitamin C, and vitamin E). The levels of lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides) and tumor markers such as CA 15-3, AFP and CEA in serum were increased significantly in cancer-induced animals as compared to control rats. Oral supplementation of morin at a dose of 50 mg/kg body weight significantly improved the body weight, enzymic, and nonenzymic antioxidants and considerably decreased the lipid peroxidation marker and tumor markers levels. Histological observations also correlated with the biochemical parameters. Tumor bearing animals showed marked increase in proliferating cell nuclear antigen-positive cells and also the number of AgNOR/nuclei compared with control rats while this expression levels were significantly reduced upon morin treatment. Thus, this study reveals the possible beneficial effect of morin as chemopreventive agent against the oxidative stress induced during mammary carcinogenesis.     

Selective promotion of ferrous ion-dependent lipid peroxidation in Ehrlich ascites tumor cells by histidine as compared with other amino acids

Among tumors in general, Ehrlich ascites tumor cells are particularly resistant to lipid peroxidation. In this study lipid peroxidation was measured in terms of the formation of malondialdehyde-equivalent material in Ehrlich tumor cells during incubation in vitro. It was shown that the high antioxidant potential of these cells could be overcome by a strong radical-promoting agent like ferrous ion. Various amino acids were tested for their capability to augment the effect of Fe(II). Histidine and its 3-methyl-derivative turned out to be the most effective pro-oxidants, whose action could be ascribed to the presence of the imidazole group. From studies with homogenized and denatured cells it was concluded that lipid peroxidation stimulated by Fe(II)-histidinate is an autoxidation process and that no carrier effect of iron by histidine is predominating. The stimulatory action of Fe(II)-histidinate could be completely suppressed by vitamin C, which was shown to be a potent anti-oxidant under the conditions used. The combined application of Fe(II)-histidinate and vitamin C may offer a means to study lipid peroxidation of Ehrlich tumor cells in a controlled manner.     

Prevention of Carbon Tetrachloride-Induced Lipid Peroxidation in Liver Microsomes from Dehydroepiandrosterone-Pretreated Rats

Dehydroepiandrosterone (DHEA), a lipid soluble steroid, administered to rats (100 mg/kg b.wt) by a single intraperitoneal injection, increases to twice its normal level in the liver microsomes. Microsomes so enriched become resistant to lipid peroxidation induced by incubation with carbon tetrachloride in the presence of a NADPH-regenerating system: also the lipid peroxidation-dependent inactivation of glucose-6-phosphatase and gamma-glutamyl transpetidase due to the haloalkane are prevented. Noteworthy, the liver microsomal drug-metabolizing enzymes and in particular the catalytic activity of cytochrome P₄₅₀IIE1, responsible for the CCl₄-activation, are not impaired by the supplementation with the steroid. Consistently, in DHEA-pretreated microsomes the protein covalent binding of the trichloromethyl radical (CCl₃°), is similar to that of not supplemented microsomes treated with CCl₄. It thus seems likely that DHEA protects liver microsomes from oxidative damage induced by carbon tetrachloride through its own antioxidant properties rather than inhibiting the metabolism of the toxin.     

Protective Effect of Dehydroepiandrosterone Against Copper-Induced Lipid Peroxidation in the Rat

This study investigates the effectiveness and multitargeted activity of dehydroepiandrosterone (DHEA) as antioxidant in vivo. A single dose of DHEA was given IP to male rats. Liver and brain microsomes, and plasma low density lipoprotein (LDL), were isolated from rats sacrified 17 h later. Liver and brain microsomes were challenged with CuSO₄ and, as index of lipid peroxidation, the production of thiobarbituric acid reactive substances (TBARS) was measaured. Also, plasma low-density lipoprotein (LDL) were challenged with copper and the time course of lipid peroxidation was evaluated following the formation of conjugated dienes. The onset of TBARS generation induced by copper was marked delayed in both liver and brain microsomes from DHEA-treated animals. Also, the resistance of LDL to oxidation, expressed by the duration of the lag-phase of the kinetic curve, was significantly enhanced in DHEA-treated rats. Results indicate that in vivo DHEA supplementation makes subcellular fractions isolated from different tissues and plasma constituents (LDL) more resistant to lipid peroxidation triggered by copper. The antioxidant effect on plasma LDL might be of special relevance to the proposed antiatherogenic activity of DHEA. Moreover, multitargeted antioxidant activity of DHEA might protect tissues from oxygen radicals damage. © 1997 Elsevier Science Inc.     

Lack of melatonin effect on hydrogen peroxide induced bronchoconstriction in isolated and perfused rat lung

The effect of melatonin on hydrogen peroxide- induced broncho-and vasoconstriction was examined in vivo in the model of the isolated, perfused and ventilated lung. The administration of hydrogen peroxide (500 microM) to the perfusate caused a marked decrease in lung compliance, conductance and flow rate. The administration of melatonin (500 microM) to the perfusate 20 min before and during the hydroperoxide exposure did not cause any change in lung function. Exposure of lung microsomes to hydrogen peroxide (1-100 microM) did not induce any significant increase in malonaldehyde (MDA), an index of lipid peroxidation, and it was not affected by treatment with melatonin (500 microM). On the other hand, brain microsomes exposed to hydrogen peroxide (1-100 microM) give rise to increased levels of MDA, which were decreased by pre-treatment with melatonin (500 microM). The results suggest that melatonin may exert an antioxidant effect in conditions were lipid peroxidation is occurring. Its use may not be relevant in conditions where the mechanisms of the reactive oxygen species damage appears to be lipid peroxidation independent, such as the case of hydrogen peroxide induced broncho- and vasoconstriction.     

Protective effect of <Emphasis Type="Italic">Spirulina</Emphasis> against 4-nitroquinoline-1-oxide induced toxicity

In the present study, we focused on the protective effect of Spirulina against 4-nitroquinoline-1-oxide (4NQO) induced hepato and nephrotoxicity in the experimental rats. The 4NQO administration resulted in increased levels of hepatic and renal markers [Alanine Transaminase (ALT), Aspartate Transaminase (AST), Lactate Dehydrogenase (LDH), urea, creatinine and uric acid] in the serum of experimental animals. It also increased the oxidative stress resulting in increased levels of the lipid peroxidation with a concomitant decline in the levels of non enzymic [reduced glutathione (GSH)] and enzymic antioxidants [(Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), and Glutathione-S-transferase (GST)] in both liver and kidney. Oral pretreatment with aqueous extract of Spirulina prevented 4NQO induced changes in the levels of hepatic and kidney diagnostic marker enzymes in the serum of experimental rats. It counteracted the 4NQO induced lipid peroxidation and maintained the hepatic and kidney antioxidant defense system at near normal in both liver and kidney. The antioxidant responsiveness mediated by Spirulina may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect normal cell functioning and provide a scientific rationale for the use of Spirulina.     

Melatonin and N-acetyl serotonin inhibit selectively enzymatic and non-enzymatic lipid peroxidation of rat liver microsomes

Melatonin (N-acetyl-5-methoxytryptamine) and its immediate precursor N-acetyl serotonin in the metabolism of tryptophan are free radical scavengers that have been found to protect against non-enzymatic lipid peroxidation in many experimental models. By contrast, little is known about the antioxidant ability of these indoleamines against NADPH enzymatic lipid peroxidation. The light emission produced by rat-liver microsomes, expressed as total cpm during 180 min of incubation at 37 degrees C, was two-fold greater in the presence of ascorbate (0.4mM) when compared with NADPH (0.2 mM). Maximal peaks of light emission produced by microsomes lipid peroxidized with ascorbic-Fe(2+) or NADPH and expressed as cpm were 354,208 (at 60 min) and 135,800 (at 15 min), respectively. During non-enzymatic lipid peroxidation a decrease of total chemiluminescence (inhibition of lipid peroxidation) was observed when increasing concentrations of melatonin were added to liver microsomes. The protective effect was concentration-dependent. The inhibition observed in light emission was coincident with the protection of the most PUFAs. Preincubation of microsomes with N-acetyl serotonin reduced these changes very dramatically. Thus, in the presence of both antioxidants (0.36, 0.75, 1.5 mM), light emission percent inhibition during non-enzymatic (ascorbate-Fe(2+)) lipid peroxidation of rat liver microsomes was for melatonin: 6.12, 16.20, 34.88 and for N-acetyl serotonin: 85.10, 88.48, 84.4 respectively. The incubation of rat liver microsomes in the presence of NADPH (0.36, 0.75, 1.5 mM) produce a sudden increase of chemiluminescence that gradually increased and reached a maximal value at about 15 min; however, N-acetyl serotonin reduced these changes very efficiently.     

Vitamin E derivatives as new potent inhibitors of microsomal lipid peroxidation

Several synthetic Vitamin E derivatives are strong inhibitors of lipid peroxidation induced in rat liver microsomes either chemically by ferrous ions and ascorbate or enzymatically by NADPH and carbon tetrachloride. The relative activities of these inhibitors are consistent with their intrinsic antioxidant properties, as peroxyl radicals scavengers. Among them, a 3,4-dihydro-6-hydroxy-2H-1-naphtopyran with IC50 around 0.08 microM is one of the most potent yet known inhibitor of lipid peroxidation.     

Prevention of Carbon Tetrachloride-Induced Lipid Peroxidation in Liver Microsomes from Dehydroepiandrosterone-Pretreated Rats

Dehydroepiandrosterone (DHEA), a lipid soluble steroid, administered to rats (100 mg/kg b.wt) by a single intraperitoneal injection, increases to twice its normal level in the liver microsomes. Microsomes so enriched become resistant to lipid peroxidation induced by incubation with carbon tetrachloride in the presence of a NADPH-regenerating system: also the lipid peroxidation-dependent inactivation of glucose-6-phosphatase and gamma-glutamyl transpetidase due to the haloalkane are prevented. Noteworthy, the liver microsomal drug-metabolizing enzymes and in particular the catalytic activity of cytochrome P₄₅₀IIE1, responsible for the CCl₄-activation, are not impaired by the supplementation with the steroid. Consistently, in DHEA-pretreated microsomes the protein covalent binding of the trichloromethyl radical (CCl₃°), is similar to that of not supplemented microsomes treated with CCl₄. It thus seems likely that DHEA protects liver microsomes from oxidative damage induced by carbon tetrachloride through its own antioxidant properties rather than inhibiting the metabolism of the toxin.