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.     

Role of cytochrome P-450 in ochratoxin A-stimulated lipid peroxidation.

The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe3+, ethylenediaminetetraacetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe3+. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.     

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

Reduced rates of lipid peroxidation have been observed in Yoshida hepatoma cells and microsomes when compared with appropriate control tissue (normal rat liver) under the same pro-oxidant conditions. The pro-oxidant conditions used were incubation with NADPH+ADP+iron or ascorbate+iron or exposure to gamma-irradiation. As previously shown with the Novikoff hepatoma, the relative concentrations of alpha-tocopherol and polyunsaturated fatty acids are important in conferring resistance to lipid peroxidation in the Yoshida hepatoma. Furthermore, NADPH-cytochrome c reductase and the NADPH-cytochrome P-450 electron transport chain, which are involved in the initiation and propagation of certain types of lipid peroxidation, are found at very much reduced levels in the Yoshida hepatoma. The relative importance of these aberrations are discussed.     

Role of cytochrome P-450 in ochratoxin a-stimulated lipid peroxidation

The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe³⁺, ethylenediaminetetra-acetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe³⁺. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro. Interaction sites in the endoplasmic reticulum

1. The actions of various inhibitors of the microsomal NADPH-cytochrome P-450 electron-transport chain have been studied on the stimulatory effect of carbon tetrachloride on malonaldehyde production. 2. Carbon monoxide, p-chloromercuribenzoate, beta-diethylaminoethyl-3,3'-diphenylpropyl acetate (SKF 525A) and nicotinamide did not decrease the stimulatory action of carbon tetrachloride on malonaldehyde production when present in concentrations shown to be capable of strongly inhibiting the demethylation of aminopyrine. 3. In contrast with the effects of the substances mentioned above, low concentrations of cytochrome c strongly depressed the stimulatory action of carbon tetrachloride on malonaldehyde production while increasing the endogenous rate of peroxidation. 4. Aging the microsomal suspensions at 0 degrees C caused a rapid decrease in aminopyrine demethylation activity and in lipid peroxidation catalysed by ADP and Fe(2+). The stimulation of malonaldehyde production by carbon tetrachloride was relatively unaffected, however, by aging the microsomes at 0 degrees C for 3 days; during this period cytochrome P-450 decreased by more than 30%. 5. The conclusion is reached that the interaction between carbon tetrachloride and the NADPH-cytochrome P-450 electron-transport chain necessary for the stimulation of malonaldehyde production involves a locus near to if not identical with the NADPH-cytochrome c reductase flavoprotein.     

Reconstitution studies on the involvement of radiation-induced lipid peroxidation in damage to membrane enzymes

The effect of radiation on the drug-metabolizing enzyme system of microsomes, reconstituted with liposomes of microsomal phospholipids, NADPH-cytochrome P-450 reductase and cytochrome P-450, was examined to elucidate the role of lipid peroxidation of membranes in radiation-induced damage to membrane-bound enzymes. The reconstituted system of non-irradiated enzymes with irradiated liposomes showed a low activity of hexobarbital hydroxylation, whereas irradiated enzymes combined with non-irradiated liposomes exhibited an activity equal to that of unirradiated controls. Irradiation of liposomes caused a decrease in cytochrome P-450 content by destruction of the haem of cytochrome P-450 and also inhibited the binding capacity of cytochrome P-450 for hexobarbital. The relationship between radiation-induced lipid peroxidation and membrane-bound enzymes is discussed.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.     

Subfractionation of rat liver microsomes by immunoprecipitation and immunoadsorption methods.

Rabbit antisera were prepared against cytochrome b5 and NADPH-cytochrome c reductase [EC 1.6.2.4] purified from rat liver microsomes, and utilized in examining the distribution of these and other membrane-bound enzymes among the vesicles of rat liver microsomal preparations by immunoprecipitation and immunoadsorption methods. Smooth microsomes with an average vesicular size of 200 nm (diameter) and sonicated smooth microsomes with an average diameter of 40-60 nm were used in subfractionation experiments. Immunoprecipitation of microsomal vesicles with anti-cytochrome b5 immunoglobulin failed to show any separation of the microsomes into fractions having different enzyme compositions. Cytochrome b5 was apparently distributed among all vesicles even when sonicated microsomes were used. When the antibody against NADPH-cytochrome c reductase was used, however, immunoadsorption of microsomes on Sepharose-bound antibody produced some separation of NADPH-cytochrome c reductase and cytochrome P-450 from NADH-cytochrome b5 reductase and cytochrome b5. The separation was more pronounced when sonicated microsomes were used. These results indicate microheterogeneity of the microsomal membrane, and suggest the clustering of NADPH-cytochrome c reductase and cytochrome P-450 molecules in the membrane.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Collagenolytic activity of rabbit V2-carcinoma growing at multiple sites.

Interaction between lanosterol and cytochrome P-450 purified from microsomes of anaerobically-grown Saccharomyces cerevisiae was studied. Lanosterol (4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol) stimulated the oxidation of NADPH by molecular oxygen in the presence of cytochrome P-450 and NADPH-cytochrome P-450 reductase both purified from S. cerevisiae microsomes. Lanosterol stimulated the reduction of cytochrome P-450 by NADPH with the cytochrome P-450 reductase, and induced Type I spectral change of cytochrome P-450. These observations suggest that lanosterol interacts to the substrate region of cytochrome P-450 of S. cerevisiae. Based on these facts, possible role of cytochrome P-450 in lanosterol metabolism in yeast cell is discussed.     

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.     

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.     

Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.

Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes.

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

Changes in the ratio of microsomal electron carriers in reconstituted microsomal membranes

The reconstitution of microsomal membrane monooxygenase system with variable contents of the hydroxylating chain enzymatic components was carried out. It was found that during self-assembly of microsomal membranes solubilized with 4% sodium cholate and gel filtration through Sephadex LH-20 in the presence of isolated microsomal enzymes, two forms of cytochrome P-450, i. e. phenobarbital- and 3-methylcholantrene-induced ones, and NADPH-cytochrome P-450 reductase, the exogenous enzymes are incorporated into the microsomal membrane matrices of control and methyl-cholantrene-treated animals. In the membranes reconstituted from the microsomes of the methylcholantrene-induced animals the catalytic activity of cytochrome P-448 in the metabolism of benz(a)pyrene at varying cytochrome P-448 and NADPH-cytochrome P-450 reductase contents were investigated.     

The consequences of lipid peroxidation in isolated hepatocytes.

Lipid peroxidation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to isolated rat hepatocytes and the resultant biochemical and morphological alterations investigated. As previously observed with microsomes, malonaldehyde formation was associated with the inactivation of glucose-6-phosphatase. Inhibition of microsomal oxidative drug metabolism was correlated with the release and subsequent inactivation of NADPH-cytochrome c reductase, whereas cytochrome P-450 destruction occurred only in the presence of high concentrations of the organic hydroperoxide which were associated with extensive malonaldehyde formation. Under these conditions there were also marked ultrastructural alterations in the hepatocytes which were not apparent after incubation in the presence of iron (less than or equal to 187 muM Fe3+). The latter treatment was, however, associated with moderate biochemical effects such as glucose-6-phosphatase inactivation and increased membrane permeability. The cellular defence system against lipid peroxidation is discussed and it is concluded that the isolated liver cell system provides a valuable tool for the study of lipid peroxidation and its pathological implications.