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 hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (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 Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. 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 H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.     

Cause of decrease of ethylmorphine N-demethylase activity of lipid peroxidation in microsomes from the rat, guinea pig and rabbit.

There were marked differences among animal species between NADPH-dependent and ascorbic acid-Fe++-dependent lipid peroxidation. In NADPH-dependent lipid peroxidation, this activity occurred to the greatest extent in rats followed by guinea pigs and rabbits and such was much lower in rabbits than in guinea pigs. On the other hand, rabbit microsomes exhibited higher lipid peroxidation activity than guinea pigs in ascorbic acid plus Fe++ or Fe++-dependent lipid peroxidation although the activity was still lower than in rats. The ascorbic acid plus Fe++-stimulated lipid peroxidation produced a decrease in ethylmorphine N-demethylase activity which was closely related to ethylmorphine-enhanced NADPH-cytochrome P-450 reductase activity but was not related to the change of the apparent content of cytochrome P-450 in all animal species. These results indicate that decrease of NADPH-cytochrome P-450 reductase activity induces a decrease in ethylmorphine N-demethylase activity by lipid peroxidation.     

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.     

Protective role of ascorbic acid against asbestos induced toxicity in rat lung: in vitro study

Asbestos fibers adsorb cytochrome P-450 and P-448 proteins from rat lung micosomal fractions and liberate heme from cytochrome P-448 on prolonged incubation in vitro. further, fibers, decrease the activities of benzo(a)pyrene hydroxylase and glutathione-S-transferase in microsomal and cytosolic fractions respectively. Mineral fibers also stimulate both the enzymatic (NADPH-induced) and non-enzymatic (Fe2(+)-induced) lipid peroxidation in microsomal fractions. Preincubation of microsomal and cytosolic fractions with a physiological concentration of ascorbic acid ameliorates, to a large extent, the changes induced by asbestos fibers.     

Effects of mansonones on lipid peroxidation, P450 monooxygenase activity, and superoxide anion generation by rat liver microsomes

Several structurally related ortho-naphthoquinones isolated from Mansonia altissima Chev (mansonones C, E and F) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P450 destruction; (c) inhibited NADPH-supported aniline 4-hydroxylase activity; (d) inhibited Fe(III)ADP reduction by NADPH-supplemented microsomes; (e) stimulated superoxide anion generation by NADPH-supplemented microsomes; and (f) stimulated ascorbate oxidation. ESR investigation of ascorbate-reduced mansonone F demonstrated semiquinone formation. Mansonone C had a greater effect than mansonones E and F on NADPH-dependent lipid peroxidation, O2- production and ascorbate oxidation, whereas mansonone E was more effective than mansonones C and F on aniline 4-hydroxylase activity. Mansonones E and F did not inhibit hydroperoxide-dependent lipid peroxidation, cytochrome P450 destruction or microsomal aniline 4-hydroxylase activity. Mansonone C inhibited to a limited degree tert-butyl hydroperoxide-dependent lipid peroxidation, this inhibition being increased by NADPH. Mansonone A, a tetrahydro orthonapthoquinone derivative, was in all respects relatively less effective than mansonones C, E and F. It is postulated that mansonones C, E and F inhibited microsomal lipid peroxidation and cytochrome P450 catalyzed reactions by diverting reducing equivalents from NADPH to dioxygen, but mansonone C (including its reduced form) may also exert direct antioxidant activity.     

Stimulation of the conjugation of lipid dienes in hepatic microsomes by 3,3'-dichlorobenzidine

Pretreatment of male rats with 3,3'-dichlorobenzidine (DCB) resulted in the accumulation of conjugated dienes in lipids from hepatic microsomes. In vitro, these microsomes had 2-fold the NADPH-dependent malondialdehyde (MDA)-forming capacity of microsomes from untreated rats. To determine the mechanisms of the DCB-induced accumulation of diene conjugation, the effects of added DCB on NADPH- or iron + ascorbic acid- (Fe2+-ascorbate-) dependent diene conjugation, oxygen uptake and MDA formation were examined in microsomes from untreated rats in vitro. In the presence of NADPH, added DCB stimulated diene conjugation in microsomal lipids as did in vivo DCB pretreatment but inhibited the uptake of oxygen and the formation of MDA. When Fe2+-ascorbate was substituted for NADPH, the formation of diene conjugation, oxygen uptake, and MDA formation were inhibited by added DCB. The DCB-induced stimulation of diene conjugation, in addition to being strictly NADPH dependent, was carbon monoxide sensitive and was concomitant with the binding of added DCB to microsomal lipids. It is postulated that a metabolite of DCB generated by cytochrome P-450 reacts with membrane lipids both in vivo and in vitro in a manner analogous to the initiation of lipid peroxidation but at the same time prevents the autocatalytic decomposition of the lipids. The DCB-induced diene conjugation is interpreted as predisposing to deleterious changes in microsomes.     

Sex-related differences in NADPH-dependent lipid peroxidation induced by cadmium

Male and female rats were dosed once a day for 2 days with injections of 1.5 mg Cd/kg. Formation of thiobarbituric acid reactive substances (TBA-RS) was significantly increased in male rat liver but not in the females. NADPH-dependent lipid peroxidation in vitro in microsomes derived from untreated rat liver was greater in males than in females. Furthermore, addition of cadmium (Cd) to microsomes isolated from male rat liver produced a dose-dependent potentiation of NADPH-dependent lipid peroxidation from low concentrations of Cd. In microsomes derived from females a significant increase in lipid peroxidation was observed only at high Cd concentrations. NADPH-dependent lipid peroxidation enhanced by Cd was greater in the males than in the females. These data suggest that a sex-related difference in the ability of Cd to induce lipid peroxidation in vivo in rat liver appears to be mediated partly through differences in hepatic microsomal NADPH-dependent lipid peroxidation.     

Kinetics of dextromethorphan-O-demethylase activity and distribution of CYP2D in four commonly-used subcellular fractions of rat brain

Abstract

The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions.

Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis–Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot.

Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (～50-fold) affinity but a higher (～six-fold) capacity. The apparent CYP2D affinity and capacity of the crude mitochondria were in between those of the microsomes and purified mitochondria. Additionally, the CYP2D activity in the whole homogenate was much higher than that in the total membranes at higher DXM concentrations. A CYP2D immune-reactive band in the brain mitochondria appeared at a lower MW but had a much higher intensity than that in the microsomes.

Mitochondrial brain CYP2D has a much higher capacity than its microsomal counterpart. Additionally, brain homogenate is more representative of the overall CYP2D activity than the widely-used total membrane fraction.     

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.     

Dissociation of microsomal oxygen reduction and lipid peroxidation with the electron acceptors, paraquat and menadione.

Paraquat, diquat and menadione, electron acceptors which interact with the microsomal electron transport chain, were used to investigate the relationship between microsomal lipid peroxidation and microsomal oxygen reduction. All three compounds stimulated hydrogen peroxide production and the rate of superoxide production by mouse liver microsomes. However, while paraquat and diquat stimulated microsomal lipid peroxidation (2-fold in liver microsomes and 6- to 10-fold in lung microsomes), menadione was a potent inhibitor. Superoxide dismutase and catalase had no effect on paraquat-stimulated lipid peroxidation. Diquat, at concentrations sufficient to stimulate Superoxide production, was unable to stimulate lipid peroxidation. Based on the above observations, a mechanism of paraquat- and diquat-initiated lipid peroxidation independent of superoxide and peroxide generation is proposed. The stimulatory effects of paraquat and diquat on lung microsomal lipid peroxidation are also discussed in relation to the lipid peroxidation hypothesis of paraquat lung toxicity.     

Generation of superoxide anion and lipid peroxidation in different cell types and subcellular fractions from rat testis

Mitochondria and microsomes from whole rat testis, seminiferous tubules and Leydig cells were investigated with respect to their capacity to generate superoxide anion. In addition, lipid peroxidation by whole testis mitochondria and microsomes was measured. In the presence of NADH and various respiratory inhibitors all three mitochondrial preparations catalyzed the formation of superoxide anion at a rate of 0.27-1.67 nmol/min.mg. This formation was concluded to be confined mainly to the NADH dehydrogenase region of the respiratory chain. Addition of NADPH to whole testis or Leydig cell mitochondria, but not tubule mitochondria, caused an additional formation of superoxide anion, which was unrelated to the respiratory chain, accelerated several-fold by menadione, and presumably catalyzed by NADPH-cytochrome c reductase and cytochrome P-450. Microsomes isolated from whole testis, seminiferous tubules, and Leydig cells generated superoxide anion at rates between 0.19 and 0.44 nmol/min.mg. These rates were also strongly stimulated by menadione. It is likely that both NADPH-cytochrome c reductase and cytochrome P-450 were involved in the microsomal generation of superoxide. Free radical scavengers of various types inhibited both the mitochondrial and microsomal formation of superoxide anion. Lipid peroxidation in whole testis essentially paralleled superoxide anion generation. However, the rate of mitochondrial lipid peroxidation was twice that of the microsomal rate. It is concluded that seminiferous tubules and Leydig cells generate superoxide anion at different rates and by different mechanisms. Together with cytochrome P-450-dependent hydroxylases, e.g., BP and DMBA hydroxylases, this superoxide generation may reflect a potential for cell-specific peroxidative damage in the testis.     

A possible role for membrane lipid peroxidation in anthracycline nephrotoxicity

Adriamycin causes both glomerular and tubular lesions in kidney, which can be severe enough to progress to irreversible renal failure. This drug-caused nephrotoxicity may result from the metabolic reductive activation of Adriamycin to a semiquinone free radical intermediate by oxidoreductive enzymes such as NADPH-cytochrome P-450 reductase and NADH-dehydrogenase. The drug semiquinone, in turn, autoxidizes and efficiently generates highly reactive and toxic oxyradicals. We report here that the reductive activation of Adriamycin markedly enhanced both NADPH- and NADH-dependent kidney microsomal membrane lipid peroxidation, measured as malonaldehyde by the thiobarbituric acid method. Adriamycin-enhanced kidney microsomal lipid peroxidation was diminished by the inclusion of the oxyradical scavengers, superoxide dismutase and 1,3-dimethylurea, and by the chelating agents, EDTA and diethylenetriamine-pentaacetic acid (DETPAC), implicating an obligatory role for reactive oxygen species and metal ions in the peroxidation mechanism. Furthermore, the inclusion of exogenous ferric and ferrous iron salts more than doubled Adriamycin-stimulated peroxidation. Lipid peroxidation was prevented by the sulfhydryl-reacting agent, p-chloromercuribenzenesulfonic acid, by omitting NAD(P)H, or by heat-inactivating the kidney microsomes, indicating the requirement for active pyridine-nucleotide linked enzymes. Several analogs of Adriamycin as well as mitomycin C, drugs which are capable of oxidation-reduction cycling, greatly increased NADPH-dependent kidney microsomal peroxidation. Carminomycin and 4-demethoxydaunorubicin were noteworthy in this respect because they were three to four times as potent as Adriamycin. In isolated kidney mitochondria, Adriamycin promoted a 12-fold increase in NADH-supported (NADH-dehydrogenase-dependent) peroxidation. These observations clearly indicate that anthracyclines enhance oxyradical-mediated membrane lipid peroxidation in vitro, and suggest that peroxidation-caused damage to kidney endoplasmic reticulum and mitochondrial membranes in vivo could contribute to the development of anthracycline-caused nephrotoxicity.     

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.     

Mechanism of paraquat-stimulated lipid peroxidation in mouse brain and pulmonary microsomes.

Paraquat-stimulated NADPH-dependent lipid peroxidation in mouse brain and pulmonary microsomes was inhibited by superoxide dismutase and singlet oxygen quenchers, but not by catalase or hydroxyl radical scavengers. MnCl2, which might form a salt with unsaturated lipid, inhibited the lipid peroxidation in brain microsomes, but not that in pulmonary microsomes. These findings suggest that activated oxygen species, especially superoxide and singlet oxygen, may play a major role in the stimulation of microsomal lipid peroxidation by paraquat in both brain and lung, and that the nature of the lipids exposed to peroxidative attack may be different in microsomes of the two organs.     

Protective action of a new benzofuran derivative on lipid peroxidation and sulphydryl groups oxidation

The antioxidant properties of a novel water-soluble antioxidant of the benzofuran family (5-hydroxy-4,6,7-trimethyl-2,3-dihydrobenzofuran-2-acetic acid, BFA) were studied. In rat liver mitochondria BFA increases the lag-time and decreases the extent of lipid peroxidation induced by ascorbate/Fe2+; an IC50 value of about 12 microM was observed. In rat liver microsomes it inhibits the lipid peroxidation induced both by NADPH/Fe2+/ADP (iron-dependent) and by cumene hydroperoxide (iron-independent), showing IC50 values of 25 and 30 microM respectively. The antioxidant efficiency of BFA is slightly higher than that of the congener compound Trolox C. BFA is also able to inhibit the oxidation of protein sulphydryl groups consequent to microsomal lipid peroxidation induced by NADPH/Fe2+/ADP. The antioxidant properties of BFA are discussed considering its hydrophilic character and pharmacological features.     

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by beta-lapachone and related naphthoquinones

The lipophilic o-naphthoquinones beta-lapachone, 3,4-dihydro-2-methyl-2-ethyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 8-935), 3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 9-442), and 3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione (CG 10-248) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P-450 destruction; (c) inhibited microsomal aniline 4-hydroxylase, aminopyrine N-demethylase and 7-ethoxycoumarin deethylase; (d) did not inhibit the ascorbate- and tert-butyl hydroperoxide-dependent lipid peroxidation and the cumenyl hydroperoxide-linked aniline 4-hydroxylase reaction; and (e) stimulated NADPH oxidation, superoxide anion radical generation and Fe(III)ADP reduction by NADPH-supplemented microsomes. In the presence of ascorbate, the same o-naphthoquinones stimulated oxygen uptake and semiquinone formation, as detected by ESR measurements. The p-naphthoquinones alpha-lapachone and menadione were relatively less effective than the o-naphthoquinones. These observations support the hypothesis that, in the micromolar concentration range, o-naphthoquinones inhibit microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions, by diverting reducing equivalents from NADPH to dioxygen.     

Antioxidant effects of albumin-bound sulfur in lipid peroxidation of rat liver microsomes.

The antioxidant potential of albumin-bound sulfur (SBA) was investigated in rat liver microsomes using lipid peroxidation systems in vitro. Sulfur bound to protein is a reduced metabolite which is produced from cystine by gamma-cystathionase. Lipid peroxidation was induced either chemically by ferrous ions and ascorbate or enzymatically by carbon tetrachloride or tert-butyl hydroperoxide as indicated by the increase in thiobarbituric acid reactive substances (TBA-RS) and oxygen consumption. Although the antioxidant effect of SBA was weak on the non-enzymatic lipid peroxidation system, the addition of SBA significantly inhibited TBS-RS formation and oxygen consumption compared with non-treated bovine serum alubumin (BSA) in a microsomal lipid peroxidation system induced enzymatically. The sulfur bound to albumin disappeared during incubation with liver microsomes. However, slight differences in the disappearance were observed depending on whether or not lipid peroxidation was induced in the enzymatic systems. In the CCl4-induced lipid peroxidation system, the cytochrome P-450 level was significantly decreased by the addition of SBA. Therefore, in cytochrome P-450 dependent lipid peroxidation system, the potential effects of sulfur bound to albumin are due to an inhibition of cytochrome P-450 rather than by the oxidation itself caused by radical trapping.     

NADH-dependent generation of reactive oxygen species by microsomes in the presence of iron and redox cycling agents.

NADH was found previously to catalyze the reduction of various ferric complexes and to promote the generation of reactive oxygen species by rat liver microsomes. Experiments were conducted to evaluate the ability of NADH to interact with ferric complexes and redox cycling agents to catalyze microsomal generation of potent oxidizing species. In the presence of iron, the addition of menadione increased NADPH- and NADH-dependent oxidation of hydroxyl radical (.OH) scavenging agents; effective iron complexes included ferric-EDTA, -diethylenetriamine pentaacetic acid, -ATP, -citrate, and ferric ammonium sulfate. The stimulation produced by menadione was sensitive to catalase and to competitive .OH scavengers but not to superoxide dismutase. Paraquat, irrespective of the iron catalyst, did not increase significantly the NADH-dependent oxidation of .OH scavengers under conditions in which the NADPH-dependent reaction was increased. Menadione promoted H2O2 production with either NADH or NADPH; paraquat was stimulatory only with NADPH. Stimulation of H2O2 generation appears to play a major role in the increased production of .OH-like species. Menadione inhibited NADH-dependent microsomal lipid peroxidation, whereas paraquat produced a 2-fold increase. Neither the control nor the paraquat-enhanced rates of lipid peroxidation were sensitive to catalase, superoxide dismutase, or dimethyl sulfoxide. Although the NADPH-dependent microsomal system shows greater reactivity and affinity for interacting with redox cycling agents, the capability of NADH to promote menadione-catalyzed generation of .OH-like species and H2O2 or paraquat-mediated lipid peroxidation may also contribute to the overall toxicity of these agents in biological systems. This may be especially significant under conditions in which the production of NADH is increased, e.g. during ethanol oxidation by the liver.     

The role of lipid peroxidation in the nephrotoxicity of cisplatin.

The possible role of lipid peroxidation in the nephrotoxicity of the antitumour drug cisplatin was studied in vitro. In contrast to Adriamycin, cisplatin did not induce lipid peroxidation in rat kidney microsomes containing a NADPH-generating system. Pretreatment of rat kidney microsomes with cisplatin did not reduce the activity of a microsomal glutathione (GSH)-dependent protective factor against lipid peroxidation induced by Fe(2+)-ascorbate. However, pretreatment of rat kidney microsomes with 0.1 mM N-ethyl maleimide (NEM) did reduce this GSH-dependent protection. Cisplatin also did not reduce the activity of a cytosolic GSH-dependent protective factor against Fe(2+)-ascorbate-induced lipid peroxidation. The results of our experiments indicate that, in contrast to Adriamycin, cisplatin does not induce lipid peroxidation in vitro in various test systems. It also does not destroy microsomal and cytosolic GSH-dependent protective factors against lipid peroxidation.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced oxidative stress in female rats

Oxidative stress may play a role in the toxic manifestations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Therefore, the time-dependent effects of 100 micrograms TCDD/kg on various indices of oxidative stress including lipid peroxidation. DNA damage, membrane fluidity, calcium homeostasis, nonprotein sulfhydryl content, and NADPH content of hepatic subcellular fractions of female rats were followed for 12 days. Increases in lipid peroxidation of 400-500% occurred in mitochondrial and microsomal membranes and nuclei, with maximum increases occurring 5-6 days post-treatment. Decreases in the nonprotein sulfhydryl content of mitochondrial and microsomal fractions of approximately 80% were observed by Day 12 posttreatment. Membrane fluidity gradually decreased following administration of TCDD, with decreases of 30-40% being observed in mitochondria, microsomes, and plasma membranes. A sharp increase in the incidence of hepatic nuclear DNA single strand breaks was observed 3 days after treatment with an increase of approximately 600% by Day 9. Following the administration of TCDD, increases of 70-80% occurred in the calcium content of mitochondria and microsomes. An 18% increase in cytosolic calcium was present 12 days after the administration of TCDD. Cytosol and mitochondria both exhibited an initial increase in NADPH content following administration of TCDD, but by Day 12 both had decreased to approximately two-thirds of control values. The results clearly demonstrate that TCDD administration induces an oxidative stress in rat liver. The most pronounced effects were observed in membrane lipid peroxidation and DNA damage with gradual changes being observed in calcium and nonprotein sulfhydryl contents and membrane fluidity.