Control Microsomes Research Articles

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, O 2 −-formation, H 2O 2-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 H 2O 2-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.

Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the ω-1 and ω-2 hydroxylation of prostaglandins

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the ω-2 hydroxylation of prostaglandin E 2 (PGE 2) by liver microsomes ( K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477–489 ). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the ω-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1,1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450 57K and P450 56K (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450 57K; and 1-36-1 binds solely P450 57K. MAb 1-7-1 inhibited ω-2 and ω-1 PGE 2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE 2 ω-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the ω-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the ω-2 hydroxylation and to a lesser extent the ω-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the ω-2 hydroxylating activity in intact MC-microsomes supported by H 2O 2, by demonstrating a much higher activity when H 2O 2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE 2 at the ω-2 and ω-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the ω-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some ω-2 hydroxylating activity is due to P450d was not ruled out. Certain purified P450s (RLM 5 and 5a) from untreated rat liver catalyze the ω-2 hydroxylation of PGE 2 (D. Kupfer et al. (1988) Arch. Biochem. Biophys. 261, 186–195). The current findings demonstrate that all the ω-2 hydroxylation in control microsomes and about 25% of the activity in MC-microsomes is by P450s which are not inhibited by MAb 1-7-1. It is not known whether these resistant P450s are RLM 5, 5a, or different constitutive P450s.

Temperature dependence of the microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent reactions

The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 ± 2.5 °C with control microsomes. Energies of activation (about 15–17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (.OH)-dependent pathway. Arrhenius plots of the .OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450-dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the .OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by .OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions.

High affinity diethylnitrosamine-deethylase in liver microsomes from acetone-induced rats

The effects of acetone treatment on microsomal cytochrome P-450-dependent mono-oxygenases of the rat liver have been investigated to elucidate the role of this system in the metabolism of diethylnitrosamine (DEN). Acetone markedly enhanced the hepatic P-450 content and the activities of p-nitrophenol hydroxylase, acetone hydroxylase, ethoxycoumarin deethylase and DEN deethylase (DENd), whereas activities of pentoxy-resorufin O-deethylase and ethoxy-resorufin O-deethylase were not affected. Two distinct apparent Km values (0.43 and 9.1 mM), dependent on the substrate concentration, were observed for the DENd of acetone-induced microsomes. Only one Km value (8.4 mM) was observed for the DENd of control microsomes. In control microsomes at a DEN concentration of 1 mM, the N-deethylation of DEN was undetectable whereas in acetone-induced microsomes the N-deethylation rate was approximately 2.3 nmol/mg protein per min. The results suggest that acetone-induced microsomes of rat liver contain a high affinity form of DEN-deethylase which should be the P-450j isozyme (known to catalyze the oxidation of dimethylnitrosamine at low Km). P-450j is strongly enhanced by acetone treatment as indicated by the increase of the specific acetone hydroxylase. The treatment also enhanced the metabolism of DEN at substrate concentrations higher than 1 mM, suggesting that other P-450(s) catalyse DEN-deethylation although with lower substrate affinity. The low Km form of DENd is a P-450-dependent mono-oxygenase. It requires NADPH and O2, is inhibited by CO, but not by mannitol, superoxide dismutase, catalase or desferrioxamine. Its action therefore appears not to be mediated by oxygen radical species. Many solvents such as dimethylsulfoxide, dioxolane, chloroform and butanol when present at 10 mM in the incubation mixture inhibited the low Km form of DENd. However, pyrazole and piperonylbutoxide were found to be the strongest inhibitors. These results establish that acetone affects the metabolism of DEN, particularly at low concentrations, in a fashion somewhat similar to dimethylnitrosamine.

Pulegone mediated hepatotoxicity: Evidence for covalent binding of R(+)-[ 14C]pulegone to microsomal proteins in vitro

Incubation of R(+)-[ 14C]pulegone with rat liver microsomes in the presence of NADPH resulted in covalent binding of radioactive material to macromolecules. Covalent binding was much higher in phenobarbital-treated microsomes as compared to 3-methylcholanthrene treated or control microsomes. The K m and V max of covalent binding was 0.4 mM and 1.7 nmol min −1 mg −1, respectively. Covalent binding was drastically inhibited (93%) in the presence of piperonyl butoxide. Antibodies to phenobarbital-induced cytochrome P-450 and NADPH-cytochrome P-450 reductase inhibited covalent binding to an extent of 72% and 47%, respectively. Cysteine and semicarbazide also inhibited NADPH dependent binding of radiolabel from R(+)-[ 14C]pulegone to microsomal proteins. The results suggest the involvement of liver microsomal cytochrome P-450 in the bioactivation of R(+)-pulegone to reactive metabolite(s) which might be responsible for covalent binding to macromolecules resulting in toxicity.

Iron overload and the predisposition of cells to antioxidant consumption and peroxidative damage.

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

Increased oxygen radical-dependent inactivation of metabolic enzymes by liver microsomes after chronic ethanol consumption.

Enzymatic and nonenzymatic mixed-function oxidase systems have been shown to generate an oxidant that catalyzes the inactivation of glutamine synthetase and other metabolic enzymes. Recent studies have shown that microsomes isolated from rats chronically fed ethanol generate reactive oxygen intermediates at elevated rates compared with controls. Microsomes from rats fed ethanol were found to be more effective than control microsomes in catalyzing the inactivation of enzymes added to the incubation system. The enzymes studied were alcohol dehydrogenase, lactic dehydrogenase, and pyruvate kinase. The inactivation process by both types of microsomal preparations was sensitive to catalase and glutathione plus glutathione peroxidase, but was not affected by superoxide dismutase or hydroxyl radical scavengers. Iron was required for the inactivation of the added enzymes; microsomes from the rats fed ethanol remained more effective than control microsomes in catalyzing the inactivation of enzymes in the absence or presence of several ferric complexes. The inactivation of enzymes was enhanced by the addition of menadione or paraquat to the microsomes, and rates of inactivation were higher with the microsomes from the ethanol-fed rats. The enhanced generation of reactive oxygen intermediates and increased inactivation of enzymes by microsomes may contribute toward the hepatotoxic effects associated with ethanol consumption.

Alteration of liver microsomal monooxygenases and substrate competition with aniline hydroxylase from rats chronically fed low-fat and high-fat-containing alcohol diets.

Male Sprague-Dawley rats fed ethanol (EtOH) 36% of total calories for four weeks in a liquid diet containing either 34% (HF) or 12% (LF) of calories as fat were studied with respect to induction of microsomal monooxygenases (MFO) and substrate competition with EtOH-inducible aniline hydroxylase. The specific activity and turnover of aniline hydroxylase were induced to similar extents by HF-EtOH and LF-EtOH diets. Whereas, both LF-EtOH and HF-EtOH caused a decrease in the turnover of arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and aldrin epoxidase compared to pair-fed (PF) controls, LF-EtOH but not HF-EtOH increased the turnover of ethoxycoumarin and ethoxyresorufin O-deethylase (ECOD and EROD). The increase in ECOD and EROD and the decrease in AHH by EtOH is contrary to the parallel induction of these activities by 3-methylcholanthrene (3-MC) and Aroclor 1254 (Aroclor). Benzo(a)pyrene (BaP) stimulated aniline hydroxylase in the HF-EtOH and PF systems, whereas with LF diet, stimulation was seen only in the EtOH group. Ethoxycoumarin (EC) inhibited aniline hydroxylase by microsomes from EtOH- and pyrazole-treated rats, whereas it stimulated aniline hydroxylase by control microsomes, suggesting that the EC effects were associated with EtOH-inducible cytochrome P-450. Ethoxyresorufin (ER) inhibited aniline hydroxylase in EtOH and PF groups, thus the differential effects of EC were not nonspecific O-deethylase effects. The effects of EtOH feeding on ECOD, EROD, and AHH (ie, substrates for 3-MC-inducible cytochrome P-450) displayed a greater differential between the experimental and control group with the LF- than with the HF-containing diet. The findings suggest that the alteration of certain MFO activities by chronic EtOH ingestion can be modified by the content of dietary fat. Moreover, the competition dynamics of MFO substrates toward EtOH-inducible aniline hydroxylase are altered by EtOH feeding and, in turn, modified by dietary fat.

Biochemical toxicology of argemone alkaloids. III. Effect on lipid peroxidation in different subcellular fractions of the liver

Consumption of edible oils contaminated with Argemone mexicana seed oil causes various toxic manifestations. In this investigation the in vivo effect of argemone oil on NADPH-dependent enzymatic and Fe 2+-, Fe 2+/ADP- or ascorbic acid-dependent non-enzymatic hepato-subcellular lipid peroxidation was studied. Parenteral administration of argemone oil (5 ml/kg body weight) daily for 3 days produced a significant increase in both non-enzymatic and NADPH-supported enzymatic lipid peroxidation in whole homogenate, mitochondria, and microsomes. Lipid peroxidation aided by various pro-oxidants, namely Fe 2+, Fe 2+/ADP and ascorbic acid also revealed a significant enhancement in the whole homogenate, mitochondria and microsomes of argemone oil-treated rats. Further, when compared with whole homogenate, the hepatic mitochondria and microsomes of either control or argemone oil-treated rats showed a 4- and 6-fold increase in non-enzymatic, and a 5- and 18- fold increase in NADPH- dependent enzymatic lipid peroxidation, respectively. Similarly, both mitochondrial and microsomal fractions showed a 5- and 7-fold increase in Fe 2+-, and a 12- and 15-fold increase in either Fe 2+/ADP- or ascorbic acid-aided lipid peroxidation, respectively. These results suggest that the hepatic microsomal as well as the mitochondrial membrane is vulnerable to the peroxidative attack of argemone oil and may be instrumental in leading to the hepatotoxicity symptoms noted in argemone poisoning victims.

Identification of the hepatic cytochrome P-450 isozymes induced and decreased by picloram

Microsomes from male rats treated with plcloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16α-hydroxylase activity when incubated with (4- 14C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P 450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450d in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to plcloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbitalindudble forms of cytochrome P-450. Third, hepatic microsomes from plcloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98' revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from plcloramtreated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.

Hepatic microsomal metabolism of androst-4-ene-3,17-dione: Relative importance of ring hydroxylation and aromatization in control and induced rat liver

The purpose of these studies was to determine whether oestrogen production is a quantitatively important pathway in the hepatic microsomal metabolism of androst-4-ene-3,17-dione. The effects of the enzyme inducing agents phenobarbitone and β-naphthoflavone on microsomal cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation and aromatization was investigated in the rat in vitro. In microsomal fractions from untreated rats the ratio of hydroxylated products to aromatized (oestrogenic) metabolites was 33:1. Phenobarbitone pretreatment of rats increased total hydroxylation by about 20% but did not change the ratio of hydroxylated to aromatized products (27:1). In contrast, β-naphthoflavone induction decreased total hydroxylation to about 35% of control but did not affect total aromatization. Thus the ratio of hydroxylation to aromatization was significantly lower than in control microsomes (17:1). The principal aromatized products were oestriol and 2-hydroxyoestradiol-17β, with oestradiol-17β and its 4-hydroxy metabolite as minor products; no oestrone was observed. In further studies of the microsomal metabolism of oestrone, the major product was oestradiol-17β whereas hydroxylated metabolites were only minor products. Oestradiol-17β, in contrast, was hydroxylated to a considerable extent. These findings suggest that oestrone is a better substrate for the microsomal 17β-oxidoreductase than it is for cytochrome P-450. It therefore appears likely that any oestrone formed from the aromatization of androst-4-ene-3,17-dione would be readily converted to oestradiol-17β which, in turn, is subject to cytochrome P-450-mediated hydroxylation. Although the liver is a site of C 19-steroid aromatization, it appears unlikely that this organ could contribute significantly to serum oestrogen levels since microsomal hydroxylases are readily able to convert aromatized products to biologically inactive metabolites.

Alterations in hepatic microsomal protein levels in rainbow trout fed cyclopropenoid fatty acids analyzed by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis was used to analyze the effect of dietary cyclopropenoid fatty acids on hepatic microsomal polypeptide distribution patterns. Antibodies against rainbow trout type-LM2 cytochrome P-450 were employed to localize the corresponding polypeptide(s) by immunochemical staining. The LM2 antigen was purified from trout that had been fed beta-naphthoflavone. Microsomes from trout fed beta-naphthoflavone showed a decrease in a cytochrome P-450 polypeptide, detected with antibody against LM2. In contrast, microsomes from control fish contained two distinct cytochrome P-450 polypeptides, differing in their isoelectric points. Cyclopropenoid fatty acid treatment caused a preferential decrease in the additional isozyme seen in control samples. The distribution of concanavalin-A-binding glycopolypeptides was also assessed. Surprisingly, the two P-450 isozymes localized from control microsomes were found to bind concanavalin A. In addition to this, the cyclopropenoid fatty acid treatment generated a shift in a closely related group of microsomal glycopolypeptides, labeled gp80, gp82, gp80(1), and gp82(1). A decrease in the levels of gp80 and gp82 and a corresponding increase in gp80(1) and gp82(1) was observed.

Inducing effect of albendazole on rat liver drug-metabolizing enzymes and metabolite pharmacokinetics

Albendazole (ABZ), methyl (5-(propylthio)-1H-benzimidazol-2-yl)carbamate, is a broad spectrum anthelmintic drug. S-oxidation to the sulfoxide (SO-ABZ) and the sulfone (SO 2-ABZ) are the first steps of its bioconversion. SO-ABZ is pharmacologically active and embryotoxic in rats. In the present study, rat liver microsomal drug-metabolizing enzymes were assayed after 10 days oral administration with 40 μmol ABZ/kg per day. The activities of 4-nitroanisole O-demethylase, benzo[ a]pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase increased 6-, 7-, 8-, and 30-fold, respectively. By immunoblotting an increase in cytochrome P-448 was observed. UDP-glucuronosyltransferase (GT) type 1 activities (1-naphthol, 7-hydroxycoumarin, 4-nitrophenol, and 4-methylumbelliferone) were significantly higher than in control microsomes (3- to 4-fold), while GT type 2 activities and bilirubin-GT remained unchanged. Microsomal epoxide hydrolase (benzo[ a]pyrene oxide) increased 2-fold. Microsomal γ-glutamyltransferase activity was unchanged. The in vivo SO-ABZ plasma level was decreased when the SO 2-ABZ plasma level was increased. In vitro sulfoxidation and sulfonation were, however, unchanged. Although a range of imidazole derivatives, including benzimidazole itself, were commonly reported as inhibitors of monooxygenase activities, ABZ behaved as an inducer of cytochrome P-448, GT1, and epoxide hydrolase.

Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites.

1. The metabolism and covalent binding of [3H/14C]bromobenzene has been investigated using liver microsomes from untreated and phenobarbital (PB)-pretreated rats. A model has been developed to relate the observed 3H/14C ratios in the covalently bound residues to the type of metabolite (epoxide versus quinone) responsible for their formation. 2. With control microsomes metabolism was linear for 60 minutes, but with PB microsomes the time course showed a short-lived burst of rapid metabolism followed by a long phase with an overall rate comparable to control. With both types of microsomes covalent binding was synchronous with metabolism. 3. The normalized 3H/14C ratios of recovered substrate and water-soluble metabolites was 1.0, whereas that of the covalently bound material was only 0.5. Such extensive loss of tritium implies that a considerable portion of the covalent binding arises from bromobenzene metabolites more highly oxidized than an epoxide (e.g. quinones). 4. The normalized 3H/14C ratios for bromobenzene metabolites covalently bound to liver proteins in vivo (total and microsomal) was the same as with microsomes in vitro (0.5). However, for the lung and kidney the 3H/14C ratios were considerably higher (0.71 and 0.62), indicating that differences between tissues in vivo may be greater than between liver microsomes in vitro and in vivo.

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats.

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.

Evidence for cytochrome P-450 multiplicity in the midgut of the cluster caterpillar, Spodoptera litura (F.)

Treatment of Spodoptera litura (F.) (the cluster caterpillar) larvae with geraniol (0.4%), pentamethylbenzene (0.2%), β-naphthoflavone (0.2%), and phenobarbital (0.1%) increased cytochrome P-450 content, with geraniol causing a four-fold induction (no differential induction of cytochrome P-450 monooxygenase activity was observed). All treatments, except phenobarbital, increased aldrin epoxidase, ethoxyresorufin-, and ethoxycoumarin- O-deethylase activities. Only geraniol treatment induced epoxide hydrolase activity. Ethoxyresorufin and ethoxycoumarin- O-deethylase activities were insensitive to carbon monoxide and metyrapone inhibition. In contrast, aldrin epoxidase was sensitive to both inhibitors. Differential inhibition was also seen with α-naphthoflavone with the O-deethylase activities being more inhibited than aldrin epoxidase at equal inhibitor concentration. Subsequent studies with isosafrole (0.05%) resulted in qualitative changes in midgut microsomal cytochrome P-450 parameters compared to geraniol (0.4%) and control microsomes. Isosafrole treatment greatly induced ethoxyresorufin- (∼20-fold) and ethoxycoumarin- O-deethylase activity while only slightly inducing aldrin epoxidase activity (∼2-fold). Isosafrole treatments altered the apparent K m for ethoxyresourufin and ethoxycourmarin and increased the proportion of low-spin cytochrome P-450 present in midgut microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isosafrole-treated microsomes revealed the presence of two protein staining bands of molecular weights 53,500 and 50,500 not observed in control or geraniol-induced microsomes. The results suggest that like S. eridania multiple forms of cytochrome P-450 are present in S. litura midgut microsomes.

Impaired androgen 16α-hydroxylation in hepatic microsomes from carbon tetrachloride-cirrhotic male rats

Hepatic cirrhosis produced by repeated inhalation of carbon tetrachloride is associated with reduced levels of microsomal cytochrome P450. In this study the C19-steroids androstenedione and testosterone were used as specific probes of the functional activity of several forms of cytochrome P450 in microsomal fractions from control and cirrhotic rat liver. The principal finding, that androstenedione 16α-hydroxylation and testosterone 2α-, 16α-, and 17α-hydroxylation were reduced to 14%–38% of control activity, strongly suggests that levels of the male sexually differentiated cytochrome P450 (P45016α) are decreased in hepatic cirrhosis. The activity of other cytochrome P450-mediated C19-steroid hydroxylases, with the exception of androstenedione 6β-hydroxylase, appeared essentially unaltered in microsomes from cirrhotic rats. Cirrhosis induced by carbon tetrachloride was also associated with greatly decreased activity of the microsomal cytochrome P450-independent 17β-oxidoreductase, an enzyme that catalyzes the conversion of androstenedione to testosterone. Consequently, and in view of the impaired activity of cytochrome P450-mediated testosterone 17α-hydroxylation, the capacity of cirrhotic microsomes to catalyze the interconversion of androstenedione and testosterone was much lower than that of control microsomes. The present data confirm and extend earlier observations that selective impairment of drug oxidation pathways occurs in hepatic cirrhosis. These changes are unrelated to the acute toxicity produced by carbon tetrachloride exposure. The available evidence supports the assertion that specific forms of cytochrome P450 are subject to altered regulation in cirrhosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced lipid peroxidation in hepatic and extrahepatic tissues of male and female rats.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)2 administration to rats on lipid peroxidation (microsomal malondialdehyde formation and hepatic malondialdehyde content) was determined by the thiobarbituric acid method. The results demonstrate that dose and time-dependent lipid peroxidation occurs in hepatic as well as extrahepatic tissues. A 3–4 fold increase in hepatic lipid peroxidation was observed following the oral administration of TCDD. Lipid peroxidation also increases in other tissues as kidney and thymus, which are target tissues of TCDD. Sex differences exist with respect to the induction of lipid peroxidation by TCDD. A greater and more rapid induction of hepatic malondialdehyde (MDA) formation in response to TCDD is observed in female rats. When using arachidonic acid as an oxidizable substrate, more MDA was formed with hepatic, kidney and testes microsomes from TCDD-treated rats as compared to microsomes from control animals. No difference was found in the iron content of whole liver following TCDD administration. However, a 47% increase occurred in hepatic mitochondrial iron content, while a 52% decrease was observed in microsomal iron content per mg protein. The addition of ferrous ion to microsomes from control and treated animals resulted in a proportionally greater increase in MDA formation by control microsomes. Desferrioxamine addition to microsomes from control and TCDD-treated rats resulted in a decrease in MDA formation to the same basal level. The increase in MDA production by microsomes from TCDD-treated rats may be due, in part, to an increase in free ferrous ion.

Decrease in the levels of a constitutive cytochrome P-450 (RLM5) in hepatic microsomes of diabetic rats

Cytochrome P-450 dependent hydroxylation of testosterone has been measured in hepatic microsomes of control, diabetic and insulin-treated diabetic rats. The observed decrease in testosterone 16α-hydroxylase activity in diabetes, an activity previously shown to be largely due to RLM5, was accompanied by a dramatic decrease in immunodetectable RLM5. Diabetic rats which received insulin had elevated testosterone 16α-hydroxylase activity relative to the diabetic animals, which was accompanied by a corresponding increase in the levels of RLM5. These results provide evidence that specific constitutive cytochrome P-450 enzymes are altered in the diabetic state and that these changes are not permanent since they can be overcome, at least partially, by insulin replacement therapy.

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole.

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from beta-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation. 2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex. 3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c. 4. Both ethoxyresorufin and benzo[a]pyrene inhibited the development of the type I optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[a]pyrene was noncompetitive in nature. 5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type I optical difference spectrum non-competitively. 6. The results indicate that ethoxyresorufin and benzo[a]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.