Hepatic Microsomal Hydroxylation Research Articles

Effect of ethanol on CHCl3 metabolism in hepatic microsomes from Osborne-Mendel rats.

The treatment of Osborne-Mendel rats with ethanol in drinking water for 2 weeks resulted in a 3-fold increase of hepatic microsomal hydroxylation of both p-nitrophenol and aniline, two substrates considered highly selective for P4502E1. No other forms of P450 seemed to be affected. These results, confirmed by the immunoblot analysis of microsomal protein, showed an induction of P4502E1. The levels of total covalent binding to microsomal phospholipid due to 14CHCl3 reactive intermediates in ethanol-pretreated microsomes were identical to those measured in microsomes from untreated rats at any pO2. The distribution of radioactivity obtained after transmethylation of the adducts of 14CHCl3 intermediates with microsomal phospholipids (PL) indicated that binding to fatty acyl chains (due to .CHCl2 radicals) increased with decreasing pO2. On the contrary, the binding to polar heads due to phosgene decreased. The ethanol treatment did not affect binding to either PL moieties. These results indicated that, in our experimental conditions, the in vitro production of both oxidative and reductive intermediates of CHCl3 in the liver of Osborne-Mendel rats were not influenced by ethanol consumption.

A novel rat hepatic clofibrate-inducible cytochrome P450 that is not a lauric acid hydroxylase

2-Methoxy-6-[1-methylethyl]naphthalene (MMEN) was hydroxylated in an NADPH-dependent manner to the (ω-1)-alcohol and the ( R)-ω- and ( S)- ω-alcohols by rat hepatic microsomes. ( S)- ω-Hydroxylation was selectively induced 7-fold by clofibrate treatment. Phenobarbital, 3-methyl-cholanthrene, dexamethasone, cholestyramine, and MMEN did not induce this activity to the same extent. Incubation of the racemic ω-alcohols with microsomes isolated from rats resulted in a greater rate of degradation of the ( S)- than the ( R)- ω-alcohol confirming ( S)- ω-hydroxylation to be an initial catalytic event. MMEN and lauric acid were not competitive inhibitors of each other in microsomes from clofibrate-treated rats, indicating the ( S)- ω-MMEN hydroxylase to be a different enzyme from the characterized clofibrate-inducible lauric acid hydroxylases, CYP4A1 and CYP4A3. This was confirmed by the observations that (1) lauric acid hydroxylation was inhibited by 0.02% Tween 20 or Tween 80 and 25 μM capric or myristic acids, whereas ω-MMEN hydroxylation was not, (2) ω-MMEN hydroxylation was inhibited by ketoconazole, cholesterol and acetone, whereas lauric acid hydroxylation was not, and (3) CYP4A1 and CYP4A3 expressed in Hep G2 cells did not catalyze MMEN hydroxylation. Microsomes from the lungs of rabbits treated with progesterone and kidney of untreated rats did not support selective ( S)- ω-MMEN hydroxylation, indicating that this activity is not associated with CYP4A4 or CYP4A2, respectively. Leukotriene B 4(LTB 4) hepatic microsomal hydroxylation was not inhibited by MMEN and microsomes from human neutrophils did not support the reaction. These data identify a hitherto uncharacterized cytochrome P450 which is selectively induced by clofibrate and does not catalyze the ω-hydroxylation of the fatty acids or prostaglandins investigated. It is proposed that the enzyme catalyzing the selective ( S)- ω-hydroxylation of MMEN is a novel rat P450 and that it is either a new member of the CYP4 family or a clofibrate-inducible P450 from another gene family.

Effect of inhibitors on ω‐and (ω‐1)‐hydroxylation of lauric acid by frog liver microsomes

To investigate the involvement of different cytochrome P-450 monooxygenases in fatty acid hydroxylation in frog liver microsomes, the effect of various inhibitors of cytochrome P-450 monooxygenases on the omega- and (omega-1)-hydroxylation of laurate was examined. The omega/omega-1-hydroxylation ratios were changed significantly by various levels of carbon monoxide (CO) inhibition; the formation of omega-hydroxylaurate was more sharply inhibited by various levels of CO than was the formation of (omega-1)-hydroxylaurate. On the contrary, metyrapone inhibited only the formation of (omega-1)-hydroxylaurate and stimulated the formation of omega-hydroxylaurate, 7,8-Benzoflavone as well as CO was more inhibitory to the omega-hydroxylation of laurate. At low concentrations of KCN (0.2 and 0.1 mM), the (omega-1)-hydroxylase activity was stimulated, but both the omega- and (omega-1)-hydroxylase activities were inhibited at the higher concentrations (5-10 mM). The effect of drugs and hydroxylaurate isomers on the omega- and (omega-1)-hydroxylation was also examined. Aminopyrine showed a stimulative effect on omega-hydroxylase activity and no effect on the (omega-1)-hydroxylase activity, while p-nitroanisole inhibited the (omega-1)-hydroxylase activity and showed almost no effect on the omega-hydroxylase activity. 12-Hydroxylaurate inhibited both the omega- and (omega-1)-hydroxylase activities, but the omega-hydroxylase activity was inhibited to a much greater extent. 11-Hydroxylaurate had no effect on either hydroxylation. These findings strongly support the hypothesis that different cytochrome P-450 species are involved in the hepatic microsomal hydroxylation of laurate at omega- and (omega-1)-positions in the frog.

Characterization of the activation of hepatic microsomal hydroxylation by betamethasone and α naphthoflavone

Biphenyl 2-hydroxylation is selectively activated in vitro by incubation of betamethasone or α naphthoflavone with control male rat liver microsomes. Biphenyl 3- and 4-hydroxylation activities are unchanged or marginally inhibited. The nature of the enzymes involved in the activation has been investigated. Metyrapone (1 mM) completely inhibited the expression of the activation but had a lesser effect on the basal 2-, 3- and 4-hydroxylation activities. SKF525A (1 mM) † inhibited both basal and betamethasone-activated enzyme activities by 25–35 per cent. Of other drug metabolizing enzymes investigated, only benzo[ a]pyrene hydroxylation activity was increased by betamethasone and α naphthoflavone. Acetone (0.6M) caused a small activation (40 per cent) of biphenyl 2-hydroxylation but inhibited 4-hydroxylation. The non-ionic detergent Brij 35 inhibited biphenyl 2-, 3- and 4-hydroxylation. It was concluded that activation of biphenyl 2-hydroxylation differs from activation of aromatic amine hydroxylation and glucuronyl transferase but may be related to activation of benzo[ a]pyrene hydroxylation by naphthoflavones.

Antipyrine as an in vitro probe of mixed function oxidase activity [proceeding

British Journal of Clinical PharmacologyVolume 9, Issue 3 p. 284P-283P Free Access Antipyrine as an in vitro probe of mixed function oxidase activity [proceeding] GC Kahn, GC KahnSearch for more papers by this authorAR Boobis, AR BoobisSearch for more papers by this authorI Blair, I BlairSearch for more papers by this authorMJ Brodie, MJ BrodieSearch for more papers by this authorDS Davies, DS DaviesSearch for more papers by this author GC Kahn, GC KahnSearch for more papers by this authorAR Boobis, AR BoobisSearch for more papers by this authorI Blair, I BlairSearch for more papers by this authorMJ Brodie, MJ BrodieSearch for more papers by this authorDS Davies, DS DaviesSearch for more papers by this author First published: March 1980 https://doi.org/10.1111/j.1365-2125.1980.tb04844.xCitations: 12AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References Boobis, A.R., Brodie, M.J., Kahn, G.C., Fletcher, D.R., Saunders, J.H. & Davies, D.S. (1980). Monooxygenase activity of human liver in microsomal fractions of needle biopsy specimens. Br. J. clin. Pharmac., 9, 11– 19. Danhof, M., Krom, D. & Breimer, D.D. (1979). Studies on the different metabolic pathways of antipyrine in rats: the influence of phenobarbital and 3-methylcholanthrene treatment. Xenobiotica, (In press). Tredger, J.M., McPherson, F.J., Chakraborty, J., Bridges, J.W. & Parke, D.V. (1976). The effect of some glucocorticoids on hepatic microsomal hydroxylation in the rat and hamster. Naunyn-Schmiedebergs Arch. Pharmac., 292, 267– 270. Citing Literature Volume9, Issue3March 1980Pages 284P-283P ReferencesRelatedInformation

Inhibition of thiabendazole metabolism in the rat.

1. A single oral dose of desmethylimipramine (80 mg/kg) administered to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (45%), aniline (30%), biphenyl (30%) and ethylmorphine (15%) in vitro at 5 h after dosage; there was no decrease in cytochrome P-450 or b5. 2. A single oral dose of ethoxyquin (200 mg/kg) to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (65%), aniline (40%) and biphenyl (40%) in vitro at 1 h after dosage; inhibition was less at 5 h. There were no changes in the contents of cytochromes P-450 and b5. 3. The max. plasma concn. of thiabendazole occurred 2--4 h after oral dosing (50--200 mg/kg) to rats. Thiabendazole (100 mg/kg) administered orally 30 min after oral ethoxyquin (400 mg/kg) or thiabendazole (200 mg/kg) administered orally 30 min after oral desmethylimipramine (80 mg/kg) delayed absorption of the thiabendazole and resulted in markedly markedly decreased plasma concentration of the anthelmintic. 4. Simultaneous administration of ethoxyquin (300 mg/kg) potentiated the anthelmintic effect of thiabendazole (750 mg/kg) on the helminth parasite, Nematospiroides dubius, in the mouse. Desmethylimipramine showed no similar potentiation.

The effect of some glucocorticoids on hepatic microsomal hydroxylation in the rat and hamster.

The effect of some glucocorticoids on hepatic microsomal biphenyl and aniline hydroxylations were investigated in rat and hamster. Steroids were either added to the standard incubation mixture or given as a single dose intraperitoneally before preparation of the liver microsomes for enzyme determination. The addition of steroids in vitro enhanced biphenyl-2-hydroxylation activity in hepatic microsomes of rat but not of hamster and the most pronounced effect was obtained with betamethasone. A similar species difference in the effects of betamethasone on this enzyme was also observed after administration of a single dose of the steroid in vivo.

The absence of an inhibitory effect of metyrapone (2-methyl-1,2-di (3-pyridyl) propan-1-one) on hepatic microsomal hydroxylation in scurvy.

Microsomes from livers of scorbutic guinea-pigs showed a reduced rate of acetanilide hydroxylation. The response of "scorbutic" liver microsomes to the inhibitor Metyrapone (2-methyl-1,2 di (3-pyridyl) propan-1-one) was different from that of liver microsomes from non-scorbutic guinea-pigs.

The effects of the non-ionic detergent Tween 80 on hepatic microsomal hydroxylation.

1. Effects of the non-ionic detergent Tween 80 on hamster liver microsomal components and on various reactions, including biphenyl hydroxylation, have been studied. Tween 80 competitively inhibited both 2- and 4-hydroxylation of biphenyl, with Ki values of 4-9 and 4-1 mM. The apparent kinetic constants corrected for the inhibitory effect of 2-9 mM Tween 80 (the concn. routinely used) were for 2-hydroxylation: Km = 0-47 mM, Vmax = 2-2 nmol/min/mg; and for 4-hydroxylation: Km = 0-14 mM, Vmax = 3-5 nmol/min/mg. 2. The type I spectrally apparent interaction of biphenyl with liver microsomes (Ks = 0-23 mM; deltaEmax = 8-2 E/2 mg protein) was also competitively inhibited by Tween 80 (Ki = 1-1 mM), itself an apparently type I substrate. 3. Tween 80 (9-5 mM) activated aniline 4-hydroxylation. The type II spectrally obvious interaction between aniline and microsomes was not affected by Tween 80. 4. Tween 80 and biphenyl together enhanced NADPH-cytochrome c reduction whereas Tween 80 and aniline together had no effect. Tween 80 did not affect the stability of cytochrome P-450 in microsomal suspensions over a 5 min period. 5. Results are discussed in terms of differences between biphenyl 2- and 4-hydroxylation systems, between type I and II substrate-enzyme complexes, and in terms of the relevance of spectral interactions to aryl hydroxylations.