Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

Induction of the ethanol-inducible form of rabbit liver microsomal cytochrome P-450 by inhibitors of alcohol dehydrogenase

Cytochrome P-450 LMeb was purified from liver microsomes obtained from rabbits treated with either benzene or imidazole and was shown to have identical N-terminal amino acid sequence as that of cytochrome P-450 LM3a. The amino acid compositions of the proteins were indistinguishable. Quantitation of P-450 LMeb in various types of microsomes using radial immunodiffusion, revealed that pyrazole- or imidazole-treatment of the animals caused a 2-3-fold induction of the enzyme, accompanied by 2-3-fold increases of the rates of ethanol and aniline oxidation.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

The role of lipid peroxidation in CCl4-induced damage to liver microsomal enzymes: comparative studies in vitro using microsomes and isolated liver cells

The question as to whether CCl4 decreases the activities of glucose-6-phosphatase and cytochrome P-450 in liver endoplasmic reticulum mainly through its action in stimulating lipid peroxidation has been investigated using Promethazine to block lipid peroxidation. The investigation, moreover, has compared the effects of CCl4, with and without Promethazine, on isolated rat hepatocytes with corresponding effects on rat liver microsomal suspensions. Our data give no support for the view that products of lipid peroxidation are the main cause of the decrease in cytochrome P-450 observed in CCl4-intoxication. However, our present results are consistent with lipid peroxidation being a major contributory factor to the decrease in glucose-6-phosphatase activity observed in CCl4-induced liver injury.     

The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 mumol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parallel with the decrease in microsomal enzyme activities.     

Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

Deuterium isotope effects [D(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled or [1,1-2H2]ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1-13C]- and [2H6]ethanol or [2,2,2-2H3]- and [1,1-2H2]ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The D(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM2 oxidized the alcohol with D(V/K) of about 2.8 and 1.8, respectively. Addition of FeIIIEDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect, which approached that of the xanthine-xanthine oxidase system (1.4), whereas desferrioxamine had no significant effect. Incubations of all cytochrome P-450 containing systems or the xanthine-xanthine oxidase systems with (1R)- and (1S)-[1-2H]ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Reduced glutathione protection against rat liver microsomal injury by carbon tetrachloride. Dependence on O2.

Rat liver microsomal membranes contain a reduced-glutathione-dependent protein(s) that inhibits lipid peroxidation in the ascorbate/iron microsomal lipid peroxidation system. It appears to exert its protective effect by scavenging free radicals. The present work was carried out to assess the effect of this reduced-glutathione-dependent mechanism on carbon tetrachloride-induced microsomal injury and on carbon tetrachloride metabolism because they are known to involve free radicals. Rat liver microsomes were incubated at 37 degrees C with NADPH, EDTA and carbon tetrachloride. The addition of 1 mM-reduced glutathione (GSH) markedly inhibited lipid peroxidation and glucose 6-phosphatase inactivation and, to a lesser extent, inhibited cytochrome P-450 destruction. GSH also inhibited covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. These results indicate that a GSH-dependent mechanism functions to protect the microsomal membrane against free-radical injury in the carbon tetrachloride system as well as in the iron-based systems. Under anaerobic conditions, GSH had no effect on chloroform formation, carbon tetrachloride-induced destruction of cytochrome P-450 or covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. Thus, the GSH protective mechanism appears to be O2-dependent. This suggests that it may be specific for O2-based free radicals. This O2-dependent GSH protective mechanism may partly underlie the observed protection of hyperbaric O2 against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity.     

Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats.

In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.     

Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.     

Protective effect of Aquilegia vulgaris (L.) on carbon tetrachloride-induced oxidative stress in rats

The ethyl ether extract of A. vulgaris inhibited in vitro microsomal lipid peroxidation (IC50 58.8 microg/ml) and showed moderate ability to scavenge superoxide radicals and to chelate iron ions. The extract (100 mg/kg body weight, po) decreased uninduced and enzymatic microsomal lipid peroxidation in the liver of male rats pretreated with CCl4 (1 ml/kg body weight) by 27 and 40%, respectively. Activity of antioxidant and related enzymes (catalase and glucose-6-phosphate dehydrogenase) inhibited by CCl4 was significantly restored after administration of the extract. The extract itself significantly enhanced superoxide dismutase activity. There was no effect of the extract on hepatic glutathione level and cytochrome P450 content, both were decreased by CCl4. Neither CCl4 nor the tested extract affected activities of NADPH-cytochrome P450 reductase and two monooxygenases, aniline hydroxylase and aminopyrine n-demethylase. It can be concluded that the protective effect of the A. vulgaris extract in CCl4-induced liver injury is mediated by inhibition of microsomal lipid peroxidation and restoring activity of some antioxidant and related enzymes.     

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.     

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl(4) to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl(4) supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.     

CCl4-induced alterations in Ca++ homeostasis in chlordecone and phenobarbital pretreated animals

Male S-D rats were maintained on normal powdered diet or on the same diet containing 10 ppm chlordecone or 225 ppm phenobarbital for 15 days. On day 15, all the animals received a single ip injection of either corn oil or a subtoxic dose of CCl4 (25-200 microliter/kg) in corn oil vehicle (1 ml/kg). The animals were sacrificed 12 hrs later. Liver microsomal cytochrome P-450 and Ca++ levels in whole liver, mitochondria, microsomes and cytosol were determined. Cytochrome P-450 induction was greater with phenobarbital pretreatment than with chlordecone but the CCl4 induced destruction of cytochrome P-450 was almost similar in both groups and progressive with the dose of CCl4. CCl4 given to animals on normal diet in a dose range of 25-200 microliter/kg did not significantly alter the cytochrome P-450 levels. These findings are consistent with greater bioactivation of CCl4 after the above two pretreatments. There was a massive accumulation of Ca++ in chlordecone and phenobarbital pretreated animals after CCl4 administration. Cytosolic Ca++ levels remained high despite the mitochondrial and microsomal sequestration. This perturbation of hepatocellular Ca++ homeostasis might lead to hepatic lesion and hepatic failure. Chlordecone or phenobarbital alone do not alter hepatic Ca++ levels. These findings suggest that excessive accumulation of Ca++ may be causally related to the progression of hepatotoxic response due to CCl4 in chlordecone treated animals.     

The mechanism of hydroperoxide-dependent reactions with participation of cytochrome P-450

Cytochrome P-450 destruction kinetics by cumene hydroperoxide (CHP) has been studied at 25 degrees C in phosphate buffer, pH 7.25-7.50, in various systems: intact and induced rat or rabbit microsomes, highly purified LM2- and LM2- and LM4-forms of cytochrome P-450 from rabbit liver microsomes. The destruction kinetics is characterized by three phases in all systems. The CHP-influenced cytochrome P-450 destruction is a radical chain process with linear termination of the chains. The acidic phospholipids, phosphatidylserine and phosphatidylinositol and total microsomal phospholipids containing the acidic lipid components activate cytochrome P-450 in the hydroxylation of aniline and naphthalene by CHP. Phosphatidylcholine and sphingomyelin have no effect upon the cytochrome P-450 activity in the type I and II substrates oxidation by CHP. The phase transitions of the microsomal phospholipids influence the interaction of cytochrome P-450 with its reductase, altering the activation energy of type I substrates oxidation. The type II substrate oxidation is not affected by phase transitions in the full microsomal hydroxylating system.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.