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.     

Induction of drug metabolism. VI. Effects of phenobarbital and 3-methylcholanthrene administration on N-demethylating enzyme systems of rough and smooth hepatic microsomes

The induction of the microsomal hemoproteins, cytochromes P-450 and Pi-450, and of N-demethylase activities in hepatic microsomal subfractions from rats were studied at various times after administration of phenobarbital or 3-methylcholanthrene. After a single dose of phenobarbital, N-demethylase activity and cytochrome P-450 levels increased initially only in rough microsomes (RER) whereas a single dose of 3-methylcholanthrene caused almost simultaneous increases of the two enzymes in both RER and smooth microsomes (SER). The increases in N-demethylase activities during this early period of induction by 3-methylcholanthrene were paralleled by a change in P-450 hemoprotein from cytochrome P-450 to cytochrome P₁-450 in both microsomal subfractions, but the total amount of P-450 hemoprotein remained essentially unchanged. These results add to existing evidence that phenobarbital and 3-methylcholanthrene produce their inductive effects by different mechanisms and raise the possibility that cytochrome P₁-450 may be synthesized in both RER and SER.     

Stimulation of lipid peroxidation and heme oxygenase activity with inhibition of cytochrome P-450 monooxygenase in the liver of rats repeatedly exposed to cadmium

Exposure of rats to 0.1 and 0.5 mg Cd/kg subcutaneously (s.c.) thrice weekly for 5 weeks resulted in an accumulation of cadmium in the liver in concentrations of 40 and 95 micrograms/g tissue, respectively, and a microsomal burden of Cd amounting to approx. 2-3% of the retained cadmium. The cytoplasm contained about 80% of the cadmium. At an exposure dose of 0.1 mg Cd/kg, stimulation of lipid peroxidation by 22% and inhibition of ALA synthetase by 16% in the liver were observed. The higher exposure of 0.5 mg Cd/kg caused an inhibition of microsomal monooxygenase with depression of cytochrome P-450 and cytochrome b5 by 20% (over 2-fold prolongation of hexobarbital sleeping time and statistically significant decrease of activity of aniline p-hydroxylase). The loss of cytochrome P-450 probably was due to an intensified lipid peroxidation and induction of heme oxygenase (30% and 60% over control, respectively). Sequestration of cadmium by cytoplasm (metallothionein) does not protect microsomes against cadmium accumulation and specific biochemical disturbances.     

Effects of motorcycle exhaust inhalation exposure on cytochrome P-450 2B1, antioxidant enzymes, and lipid peroxidation in rat liver and lung

The effects of motorcycle exhaust (ME) on metabolic and antioxidant enzymes and lipid peroxidation were determined using male rats exposed to 1:10 diluted ME by inhalation 2 h daily for 4 wk. For microsomal cytochrome P-450 enzymes, ME resulted in threefold increases of 7-ethoxyresorufin and pentoxyresorufin O-deethylase activities in liver and a sixfold increase of 7-ethoxyresorufin O-deethylase activity and an 80% decrease of pentoxyresorufin O-dealkylase activity in lung. The results of immunoblot analysis of microsomal proteins revealed that ME increased liver and lung cytochrome P-450 1A1 with minimal effects on cytochrome P-450 2E1. ME increased cytochrome P-450 2B1/2 proteins in liver but decreased cytochrome P-450 2B1 in lung. ME did not change microsomal cytochrome P-450 enzyme activity or protein level in kidney. For phase II enzymes, ME resulted in 53% and twofold increases of cytosolic NAD(P)H:quinone oxidoreductase activities in liver and lung, respectively, and no effect on microsomal UDP-glucuronosyltransferase activities. For antioxidant enzymes, ME produced 23% and 35% decreases of superoxide dismutase, 9% and 27% decreases of catalase, and no changes of glutathione peroxidase activities in liver and lung cytosols, respectively. For lipid peroxidation, the results of thiobarbituric acid assay showed that ME resulted in a twofold increase of formation of malondialdehyde by liver microsomes incubated with FeCl(3) -ADP. ME produced a threefold increase of malondialdehyde formation by lung microsomes. The present study demonstrates that ME inhalation exposure differentially modulates cytochrome P-450 2B1 and antioxidant enzymes and increases susceptibility to lipid peroxidation in rat liver and lung.     

Ethanol metabolism in vivo by the microsomal ethanol-oxidizing system in deermice lacking alcohol dehydrogenase (ADH)

To assess the importance of non-ADH ethanol metabolism, ADH-negative and ADH-positive deermice were fed liquid diets containing ethanol or isocaloric carbohydrate for 2-4 weeks. Blood ethanol disappearance rate increased significantly after chronic ethanol feeding in both strains. Although at low ethanol concentrations (between 5 and 10 mM) there was no significant difference between ethanol-fed and pair-fed control animals, at high ethanol concentrations (between 40 and 70 mM) blood ethanol elimination rates were increased significantly after chronic ethanol feeding in both ADH-positive and ADH-negative animals. There was no significant effect of the catalase inhibitor 3-amino-1,2,4-triazole on the ethanol elimination/rates in both strains. Whereas catalase and ADH activities were not altered after chronic ethanol treatment, the activity of the microsomal ethanol-oxidizing system (MEOS) was enhanced three to four times in both strains, and microsomal cytochrome P-450 content was also increased significantly. When MEOS activity was expressed per cytochrome P-450 content, it was higher in ADH-negative than in ADH-positive animals, and it increased after ethanol administration. When microsomal proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ethanol-fed animals had a distinct band which reflected the increase in microsomal cytochrome P-450 content and seemed to reflect a unique form of cytochrome P-450 induced by ethanol. Thus, despite the absence of the ADH pathway, a large amount of ethanol was metabolized by MEOS in ADH-negative deermice; this was associated with increased blood ethanol elimination rates, enhanced MEOS activity, and quantitative and qualitative changes of cytochrome P-450.     

Halomethane hepatotoxicity: induction of lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under low oxygen partial pressures

Halomethane-induced lipid peroxidation and inactivation of cytochrome P-450 were studied in liver microsomes from phenobarbital-pretreated rats in the presence of NADPH at steady-state O2 partial pressures (PO2). As indicated by the formation of thiobarbituric acid-reactive material and the stimulation of O2 uptake, significant lipid peroxidation was induced by those halomethanes containing more than two Cl, Br, or I atoms. Lipid peroxidation decisively depended on the PO2 present, showing distinct maxima at PO2 between 1 and 10 mm Hg. Those halomethanes inducing lipid peroxidation also led to inactivation of microsomal cytochrome P-450, as indicated by a loss of cytochrome P-450 detectable as ferrous CO complex and an equimolar loss of microsomal heme. Under anaerobic conditions inactivation of cytochrome P-450 presumably resulted solely from an attack of halomethane radicals on its heme moiety. Under aerobic conditions lipid peroxidation made an additional contribution to the inactivation of cytochrome P-450. These results suggest that the reductive activation to free radicals, catalyzed by cytochrome P-450, and thus the induction of lipid peroxidation at low but physiological PO2 are characteristic not only of CCl4 but also of other polyhalogenated methanes, especially CBrCl3, CBr4, CHI3, CHBr3, and CHBr2Cl.     

Inhibition of hepatic microsomal drug-metabolizing enzymes by habu snake (trimeresurus flavoviridis) venom fractions

An inhibitor of hepatic microsomal drug-metabolizing enzyme activity was isolated from the venom of the Habu snake (Trimeresurus flavoviridis) by gel filtration through Sephadex G-100 and Amberlite CG-50 column chromatography. The inhibitor, designated R-CG-50-2, gave one band on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis and caused high hemorrhagic activity when administered i.c. to rabbits. R-CG-50-2 inhibited the drug-metabolizing enzyme system even after being heated at 70° for 5 min, in spite of a complete loss of hemorrhagic activity. Cytochrome P-450 content and NADPH-cytochrome c reductase activity of rat hepatic microsomes were decreased by administration in vivo either R-CG-50-2 or heated R-CG-50-2. The extent of the decrease was greater with unheated R-CG-50-2 than with heated R-CG-50-2. In both cases, cytochrome P-420, the inactive form of cytochrome P-450, was not detected. Lipid peroxidation in hepatic microsomes was also decreased by administration of unheated R-CG-50-2 but the decrease was not significant. In an in vitro experiment, both heated and unheated R-CG-50-2 decreased the cytochrome P-450 content and the NADPH-cytochrome c reductase activity of microsomes, but, unlike the results in vivo, cytochrome P-450 was converted to cytochrome P-420. Microsomal lipid peroxidation was greatly inhibited by either heated or unheated R-CG-50-2 in vitro. It was concluded that the inhibition of the hepatic microsomal drug-metabolizing enzyme system by either heated or unheated R-CG-50-2 may have been due to the decrease in the cytochrome P-450 content and the NADPH-cytochrome c reductase activity, and that lipid peroxidation may not have had an effect on the inhibition.     

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.     

Modulatory influence of noscapine on the ethanol-altered hepatic biotransformation system enzymes, glutathione content and lipid peroxidation in vivo in rats.

The modulatory potential of noscapine, an opium alkaloid was assessed on the ethanol-induced changes in hepatic drug metabolizing enzyme systems, glutathione content and microsomal lipid peroxidation. Noscapine was administered orally to male Wistar rats at a dose level of 200 mg/kg bw alone as well as in combination with 50% ethanol (v/v) for 5 days. Noscapine administration was associated with a approximately 91% decrease in hepatic microsomal cytochrome P-450 content. A decline of approximately 36% was observed in the NADPH-cytochrome c reductase activity on noscapine administration. The lowering of cytochrome P-450 levels on noscapine administration was accompanied by a concomitant increase in heme oxygenase activity as well as serum bilirubin levels. Our results indicate that the combination dosage of noscapine and ethanol antagonised the ethanol-induced elevation of cytochrome P-450 levels. Noscapine fed rats had decreased glutathione (GSH) content and enhanced lipid peroxidation compared to control rats as indexed by MDA method. Further, noscapine and ethanol coexposure produced a more pronounced elevation in lipid peroxidation and the glutathione levels also decreased significantly. We speculate on the basis of our results that the significant enhancement of lipid peroxidation on combination dosage of noscapine and ethanol is a consequence of depletion of glutathione to certain critical levels. The inhibition of glutathione-S-transferase (GST) as well as lowering of cytochrome P-450 suggests that the biotransformation of noscapine and ethanol is significantly altered following acute coexposures.     

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats was examined with respect to the mixed function oxidase system, anaerobic dehalogenation activity of halothane, and the antioxidant system. The levels of cytochrome P-450 and NADPH-cytochrome P-450 reductase were significantly higher in guinea pigs than in rats. There was no difference between the two animals in anaerobic dehalogenation activity of halothane per cytochrome P-450 in microsomes. Microsomal alpha-tocopherol was significantly lower in guinea pigs than in rats, and was increased by multiple exposure to halothane in guinea pigs but remained lower than in rats. Microsomal alpha-tocopherol was decreased in rats by multiple exposure. The concentration of reduced glutathione and ascorbic acid was decreased significantly by multiple exposure to halothane in guinea pigs but not in rats. These results suggest that the higher level of halothane-induced hepatic microsomal lipid peroxidation in guinea pigs is due to the large production of radical metabolites resulting from the large amounts of cytochrome P-450, the high activity of NADPH-cytochrome P-450 reductase, and the low concentration of microsomal alpha-tocopherol.     

Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase

The role of flavin-containing monooxygenase (FMO) in the decrease in cytochrome P-450 content during the microsomal metabolism of methimazole (N-methyl-2-mercaptoimidazole) was investigated by heat inactivation of FMO. Incubation of liver microsomes from untreated Fischer 344 rats with NADPH and methimazole resulted in a 25% loss of cytochrome P-450 detectable as its ferrous-carbon monoxide complex. The same extent of cytochrome P-450 loss was observed with 1 and 20 mM methimazole, suggesting saturation of the process. There was no significant loss of cytochrome P-450 when microsomal FMO was heat-inactivated prior to incubation with NADPH and methimazole. Heat pretreatment of the microsomes did not affect cytochrome P-450 concentrations and cytochrome P-420 was not observed. These results indicate that FMO-catalyzed metabolism of methimazole is necessary for the loss of cytochrome P-450 in microsomes from untreated rats. Sulfite and N-methylimidazole, the ultimate products of methimazole metabolism, did not cause a significant loss of cytochrome P-450. There was no loss of cytochrome P-450 when glutathione was included in the incubation with methimazole, suggesting that cytochrome P-450 loss was due to an interaction with oxygenated metabolites of methimazole formed by FMO. Losses of cytochrome P-450 were also observed after incubation of microsomes from phenobarbital- (31%) of beta-naphthoflavone-pretreated rats (44%) with NADPH and methimazole. In contrast to microsomes from untreated rats, heat inactivation of FMO did not prevent the loss of cytochrome P-450 in microsomes from the pretreated rats. These results indicate that both phenobarbital and beta-naphthoflavone induce isozymes of cytochrome P-450 capable of directly activating methimazole.     

Inhibition of hepatic microsomal lipid peroxidation by endogenous glycogen in the rat

The effect of endogenous glycogen on lipid peroxidation was examined in hepatic microsomes from rats. Microsomes were prepared to retain endogenous hepatic glycogen (Pg+) or to minimize it (Pg-). The indices of lipid peroxidation examined included the rate of NADPH-dependent formation of malondialdehyde (MDA) and the concomitant destruction of cytochrome P-450 and decline in the linearity of benzphetamine N-demethylase activity in microsomes. Cytochrome P-450 was destroyed during benzphetamine N-demethylation in microsomes with the loss being more extensive in Pg- than in Pg+. The destruction of cytochrome P-450 and the concomitant loss in linearity of benzphetamine N-demethylation in Pg- were prevented by added EDTA. Added linoleic acid hydroperoxide (LAHP) also caused a time-dependent loss of cytochrome P-450 in microsomes with the rate being greater in Pg- than in Pg+. The results show that glycogen inhibits hepatic microsomal lipid peroxidation and suggest that variations in glycogen content may contribute to disparities in in vitro oxidative activities between different microsomal samples. Such disparities may be minimized by the removal of glycogen during the preparation of microsomes and then supplementing the incubation mixtures with EDTA. The in vivo relevance of the observed antioxidant effect of glycogen is discussed in terms of the possible modulation by the polysaccharide of hepatotoxicity by agents whose effects may be mediated by lipid peroxidation.     

Covalent binding in vitro of polychlorinated biphenyls to microsomal macromolecules. Involvement of metabolic activation by a cytochrome P-450-linked mono-oxygenase system.

Incubation of ¹⁴C-labeled polychlorinated biphenyls (PCBs) with rat, mouse or rabbit liver microsomes in the presence of an NADPH-generating system and molecular oxygen caused covalent binding of radioactive metabolites of PCBs to microsomal macromolecules. The binding was more pronounced with liver microsomes from animals pretreated with inducers of the microsomal mono-oxygenase system. The order of induction effect of the inducers used was KC-500 (a PCB preparation containing 55% chlorine) ≥ phenobarbital (PB) ? 3-methylcholanthrene (3-MC) in rats, PB > KC-500 > 3-MC in mice, and PB > KC-500 in rabbits. [¹⁴C]KC-300 (a PCB preparation containing 42% chlorine) was more effective than [¹⁴C]KC-500 as substrate for all the microsomal preparations. The binding reaction was dependent on both NADPH and oxygen, sensitive to carbon monoxide, glutathione, cysteine, hexobarbital, and aniline, and enhanced by EDTA, which inhibits lipid peroxidation. The addition of NADH, which was by itself a very poor electron donor, caused a synergistic increase of the NADPH-dependent binding of PCBs. It is concluded that the conversion of PCBs to active metabolites by the cytochrome P-450-linked mono-oxygenase system is prerequisite to the binding reaction. A survey of the effects of various inducers suggested that a cytochrome P-450 having a high aminopyrine N-demethylation activity is mainly responsible for the metabolic activation of PCBs in liver microsomes. Kidney and lung microsomes from untreated rats were virtually devoid of the PCB-binding capacity, but in kidney microsomes this capacity could be induced by pretreatment with 3-MC or KC-500, though not with PB.     

Sulphoxidation of albendazole by the FAD-containing and cytochrome P-450 dependent mono-oxygenases from pig liver microsomes

1. Two distinct microsomal pathways involved in the metabolism of albendazole (ABZ) to albendazole-sulphoxide (SO.ABZ) by pig liver microsomes have been identified and quantified. 2. The binding of ABZ to microsomal cytochrome P-450 (Type I spectrum, Ks = 25.5 microM), the decrease of the rate of sulphoxidation by antibody against NADPH cytochrome c reductase, and the use of purified cytochrome P-450 A demonstrated the contribution of a cytochrome P-450-dependent mono-oxygenase to the metabolism of ABZ. 3. The involvement of FAD-containing mono-oxygenase (FMO) was shown by thermal pretreatment of microsomes, n-octylamine activation of the reaction, and by using purified pig liver FMO. 4. From Km and Vmax values, it would appear that the relative contributions of the two systems depend on the concentration of ABZ.     

Effect of phenobarbital and 3-methylcholanthrene on the early oxidative stress component induced by lindane in rat liver

1. Lindane administered to untreated rats or rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) increased liver lipid peroxidation, of the same magnitude in all groups.

2. PB pretreatment produced a 50% increase in lipid peroxidation (TBAR) by liver homogenates and microsomes, an effect accompanied by increases in cytochrome P-450, NADPH-cytochrome P-450 reductase, NADPH oxidase and microsomal superoxide anion production, MC pretreatment resulted in increases in liver cytochrome P-450 and NADPH oxidase only.

3. Pretreatment of rats with PB, but not MC or lindane, gave increases in glutathione peroxidase and reductase.

4. Pretreatment with PB, but not MC, increased liver GSH. Lindane decreased liver GSH to the same extent as PB plus lindane.

5. Biliary GSH, GSSG and bile flow were decreased by lindane to similar extents in all groups.

6. Lindane induced periportal necrosis with haemorrhagic foci in all groups.

7. Data presented indicate that the early lipid peroxidative response of liver to lindane was unchanged by PB- or MC-stimulated hepatic microsomal enzyme induction.     

Immunohistochemical localization and quantitation of NADPH-cytochrome P-450 reductase in human liver

An antibody raised in a goat against the human liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4.) enzyme has been used to: 1) immunoquantify the level of this enzyme in human liver microsomes, and 2) study the distribution of the reductase across the human liver acinus. Employing the Western blot procedure, anti-human reductase IgG recognized a single band in human liver microsomes which corresponded in molecular weight to the purified reductase. The content of the NADPH-cytochrome P-450 reductase in six normal human livers varied from 87 to 121 pmol/mg of microsomal protein. NADPH-cytochrome P-450 reductase activity of the same microsomes ranged from 107 to 222 nmol of cytochrome c reduced per min per mg of protein. The correlation between reductase content and activity (r = 0.54) was not statistically significant (p greater than 0.1). The total cytochrome P-450 content (cytochrome P-450 and P-420) of the same microsomes varied from 423 to 1413 pmol/mg of microsomal protein. The average ratio of cytochrome P-450 to NADPH-cytochrome P-450 reductase was 7.1:1 +/- 3.1 (mean +/- SD) in the human liver microsomal preparations studied. The reductase was found to be nonuniformly distributed across the human liver acinus. Although all hepatocytes stained positively for NADPH-cytochrome P-450 reductase, the staining intensity was highest in zone 3 and in some cases also in zone 1 hepatocytes. These results show that human liver contains a gross excess of cytochrome P-450 molecules to NADPH-cytochrome P-450 reductase molecules. Furthermore, the differential distribution of the reductase within the human liver acinus may lead to a better understanding of the mechanism underlining site-specific drug hepatotoxicity.     

Effect of phenobarbital and 3-methylcholanthrene on the early oxidative stress component induced by lindane in rat liver.

1. Lindane administered to untreated rats or rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) increased liver lipid peroxidation, of the same magnitude in all groups. 2. PB pretreatment produced a 50% increase in lipid peroxidation (TBAR) by liver homogenates and microsomes, an effect accompanied by increases in cytochrome P-450, NADPH-cytochrome P-450 reductase, NADPH oxidase and microsomal superoxide anion production, MC pretreatment resulted in increases in liver cytochrome P-450 and NADPH oxidase only. 3. Pretreatment of rats with PB, but not MC or lindane, gave increases in glutathione peroxidase and reductase. 4. Pretreatment with PB, but not MC, increased liver GSH. Lindane decreased liver GSH to the same extent as PB plus lindane. 5. Biliary GSH, GSSG and bile flow were decreased by lindane to similar extents in all groups. 6. Lindane induced periportal necrosis with haemorrhagic foci in all groups. 7. Data presented indicate that the early lipid peroxidative response of liver to lindane was unchanged by PB- or MC-stimulated hepatic microsomal enzyme induction.     

Comparison of effects of acetaminophen on liver microsomal drug metabolism and lipid peroxidation in rats and mice.

Studies were conducted to determine the in vivo effect of acetaminophen (AAP) on the lipid peroxidation, drug metabolizing enzyme activity and microsomal electron transfer system of rat and mouse liver. AAP was found to inhibit ethylmorphine N-demethylase activity in the presence of NADPH and this inhibition of the enzyme was due to decrease in cytochrome P-450 content, but not due to change in lipid peroxidation in liver microsomes. Kinetical data showed that AAP administration had no effect on Km values of ethylmorphine N-demethylase, however, a decrease in the Vmax values was seen in rats and mice. There was no significant effect of AAP on both NADPH-cytochrome c reductase and the content of cytochrome b5 3 hours after this administration to rats and mice. On the other hand, AAP induced a significant decrease in NADH-ferricyanide reductase in mice, but not in rats. The greatest decrease in cytochrome P-450 observed among the components of the liver microsomal electron transfer system of rats and mice.     

Cytochrome P-450 oxidation of alkanes originating as scission products during lipid peroxidation

Alkanes of low molecular weight, as well as malondialdehyde, originate during lipid peroxidation. Ethane and pentane are the most prominent and are probably scission products of omega-3 and omega-6 unsaturated fatty acids, respectively. Measurement of exhaled alkanes is the most reliable procedure for determining lipid peroxidation in vivo. Alkanes appear in the breath of rats 15 min after administration of CBrCl3 i.p., and are also formed in small amounts endogenously. Alkanes exhaled from untreated rats in a closed system, in which CO2 is absorbed and O2 supplied, reached steady-state levels after different times, indicating that these volatile gases are metabolized at variable rates. Metabolism was verified by injecting alkanes into the closed system. Pentane was metabolized 5-10 times faster than ethane, and was species- and strain-dependent. Administration of drugs which inhibit or induce cytochrome P-450 indicated that a particular isoenzyme might be involved in the oxidation of small alkanes. SKF 525-A or benzoflavone did not inhibit, but tetrahydrofuran and ethanol were effective inhibitors. Inducing effects of phenobarbital, methylcholanthrene or ethanol were insignificant. Incubation of microsomes with NADPH and O2, either with or without Fe-ADP, to elicit lipid peroxidation confirmed the findings in vivo. Ethane and pentane were formed in similar quantities. Inhibition of alkane oxidation with CO or ethanol increased the amount of pentane three- to four-fold, indicating that inhibition of metabolism enhances alkane release. The ratio of unmetabolized pentane to ethane reflects the membrane ratio of omega-6 to omega-3 unsaturated acids. Different types of alkane release were observed following administration of paracetamol or CCl4 to mice, indicating differences in the peroxidative attack. CCl4 destroys cytochrome P-450 dose dependently, so that it loses its capacity to oxidize pentane, whereas paracetamol does not inactivate the mono-oxygenase. Monitoring the elimination rate of injected pentane is recommended as a reliable non-invasive procedure for testing the functional state of hepatic cytochrome P-450.     

Triphenyltin acetate-mediated in vitro inactivation of rat liver cytochrome P-450

The in vitro effects of the organotin (OT) compound triphenyltin acetate (TPTA) on cytochrome P-450 content and functions were investigated in liver microsomes from untreated, phenobarbital (PB)- or beta-naphthoflavone- (betaNAF) pretreated rats. At a concentration of 0.5 mM, TPTA caused a marked loss in the spectrally detectable content of cytochrome P-450 up to 27% of its original value, along with an increase in the inactive form cytochrome P-420. Both effects were most pronounced in betaNAF-treated microsomes, which showed a shift in the hemoprotein absorption maximum from 448 nm to 451 nm, but in all cases TPTA failed to affect either cytochrome b5 or total heme content, or to increase the production of malondialdehyde. These results suggest that lipid peroxidation of microsomal membranes or damage to the heme moiety should be excluded as contributing factors in the hemoprotein loss. TPTA also produced a concentration-related functional inactivation of cytochrome P-450 that was most pronounced in betaNAF-exposed microsomal preparations, as denoted by a striking reduction in the ethoxyresorufin O-deethylase (EROD) activity (IC50 = 0.088 mM). In contrast, the activities of cytochrome P-450-independent microsomal enzymes such as NADPH cytochrome c reductase and indophenyl acetate esterase (IPA-EST) were not markedly affected even by 0.5 mM TPTA (-30%). As assessed by Lineweaver-Burk plots, the mechanism of inhibition appeared to be noncompetitive for IPA-EST and of mixed type (competitive-noncompetitive) for EROD. Among sulfhydryl-containing compounds, dithiothreitol was considerably more effective than albumin and reduced glutathione in preventing cytochrome P-450 inactivation and even was able to partially reverse the hemoprotein damage when added after TPTA; glycerol, which is known to protect the hydrophobic environment of cytochrome P-450, was as effective as albumin. This study indicates that TPTA behaves as an almost specific and powerful in vitro inhibitor of cytochrome P-450-dependent monooxygenases, apparently through the interaction with critical sulfhydryl groups of the hemoprotein.