DIFFERENCES AMONG THE ACTION OF PHENOBARBITAL, METHYLCHOLANTHRENE AND MALE SEX HORMONE ON MICROSOMAL DRUG-METABOLIZING ENZYME SYSTEMS OF RAT LIVER* RYUICHI KATO AND MICHIKO TAKAYANAGHI Department of Pharmacology and Department of Toxicology, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo Received for publication May 30, 1966 The administration of phenobarbital, phenaglycodol, glutethimide, chlorcyclizine, chlorpromazine or other different kinds of drugs induce an increase in the activities of drug-metabolizing enzyme system of liver microsome (1-3). Moreover, the administration of caricnogenic polycyclic hydrocarbons, such as 20 methylcholanthrene or 3,4-benzopyrene also induce an increase in the activities of drug metabolizing enzyme systems of liver microsome (4, 5), however, the enzyme systems stimulated by methylcholanthrene or benzopyrene are apparently different from which stimulated by phenobarbital (4, 6, 7). On the other hand, Brodie et al. demonstrated that the liver microsomes from male rats metabolized hexobarbital more rapidly than the liver microsomes from female rats did and the castration of male rats abolished the sex difference and the administration of teststerone restored the sex difference. More over, Kato et al. demonstrated that synthetic anabolic hormone, such as 4-chlorteststerone increased activities of strychnine on carisoprodol metabolizing enzymes of liver micro somes of castrated male and female rats (8, 9). However, Kato et al. observed that the single injection of phenobarbital or methylcholanthrene increased the activities of the drug-metabolizing enzyme system of liver microsomes already within 24 hours after the administration, while single injection of teststerone did not increase the activities of the enzyme systems and the activities were increased after successive injection of 4-5 days (10). Furthermore, Kato and Gilllette observed that the sex difference in the metabolism of drugs by liver microsomal enzymes was not observed in all of the enzyme systems, and some enzyme systems had no clear sex difference (11). These results indicate that the action of phenobarbital, methylcholanthrene and male sex hormone on microsomal drug-metabolizing enzyme systems of rat liver is likely dif ferent. The purpose of present communication is to elucidate these differences. 加 藤 隆 一 ・高 柳 美 智 子 MATERIALS AND METHODS Female (160 g) and male (180 g) rats of Sprague-Dawley strain were used and in some experiments female and male rats of Wistar strain were also used. The results were almost same in the both strains. Phenobarbital sodium (80 mg/kg) was dissolved in distilled water and methylcholanthrene (40 mg/kg) was dissolved in corn oil and both drugs were given intraperitoneally 72 hours and 48 hours before the sacrifice. Methyl teststerone (10 mg/kg) was dissolved in corn oil and given subcutaneously. Since the results of previous papers (11, 12) and preliminary works indicate that the enzyme systems having clear sex difference were stimulated by the administration of methyl teststerone and other anabolic hormones and in contrast, the enzyme systems having no clear sex difference were not stimulated, in most of experiments male rats were used as substitute for castrated female rats treated with methylteststerone. Preparation of microsomes: The rats were killed by decapitation and the liver was im mediately removed and homogenized in 4 volumes of ice cold 1.15% KC1 solution with a Teflon-glass homogenizer. The homogenate was centrifuged at 9,000 x g for 20 minutes and the supernatant fraction was then centrifuged at 144,000 x g for 1 hour and the microsomes were suspended in 1.15% KC1 solution. Enzyme assays: Drug-metabolizing enzymes; a typical incubation mixture consisted of 2.5 ml of microsomal solution equivalent to 1 g of liver, NADP (1.5 ,umoles), glucose 6-phosphate (50 ,umoles), glucose-6-phosphate dehydrogenase (0.5 units), magnesium chlo ride (25 umoles), nicotinamide (50 ,amoles), 1.4 ml of 0.2 M phosphate buffer (pH 7.4), various substrates (5 moles of aminopyrine, butynamine, N-methylbarbital, N-methyl aniline, N,N'-dimethylaniline, N,N'-dimethylnaphthylamine, diphenhydramine, morphine, meperidine, aniline, neoprotosil ; 4 pmoles of hexobarbital ; 3 pmoles of zoxazolamine , p-nitroanisole, p-nitrobenzoic acid; 2 amoles of pentobarbital, carisoprodol), 0.45 ml of 1.15% KC1 and water to a final volume of 5 ml. The mixtures were incubated for 30 minutes under air except the mixtures containing p-nitrobenzoic acid and neoprotosil which were incubated under an atomosphere of nitrogen. The amount of N-demethyla tion of aminopyrine was estimated by measuring the 4-aminoantipyrine formed accord ing to the method of La Du et al. (13). The amount of N-methylation of butynamine , N-methylbarbital, N-methylaniline, N,N'-dimethylaniline, N,N'-dimethylnaphthylamine, diphenhydramine, morphine, meperidine was estimated by measuring the amount of formaldehyde formed according to the method of Nash modified by Cochin and Axelrod (14). The aliphatic hydroxylation of hexobarbital, pentobarbital and carisoprodol was determined by measuring the disappearance of the substrates according to the methods of Cooper and Brodie, Brodie et al. and Kato et al., respectively (15-17). The aromatic hydroxylation of aniline and zoxazolamine was determined by measuring the disappear ance of the substrates according to the methods described in a previous paper and of Conney et al. (11, 18). O-Demethylation of p-nitroanisole was estimated by measuring the formation of p nitrophenol as described in a previous paper (11). Reduction of neoprontosil was deter mined by estimating the formation of sulfanilamide according to Fouts et al. (19). Reduc tion of p-nitrobenzoic acid was determined by estimating the formation of p-aminobenzoic acid CFouts and Brodie (20)j. Microsomal NADPH-oxidase was assayed spectrophotometrically as described by Gillette et al. (21). Microsomal NADPH-cytocrome c reductase, NADPH-neotetrazolium diaphorase, NADPH-dichlorphenolindophenol diaphorase and NADPH-ferricyanide re ductase were determined according to the methods of Williams and Kamin (22). RESULTS 1. Effect of administration of Phenobarbital or methylcholanthrene on the activities of microsomal N-demethylases of female and male rats Effect of administration of phenobarbital or methylcholanthrene on the activities of microsomal N-demethylases of female and male rats was comparatively investigated. As demonstrated in Figs. 1, 2 and 3, the activities of N-demetylation of aminopyrine, butynamine and diphenhydramine, meperidine, N-methylbarbital and morphine had very marked sex difference, while the activities of N-methylaniline, dimethylaniline and dimethylnaphthylamine had only slight sex difference. The administration of pheno barbital to female rats markedly increased the activities of N-demethylation of all the drugs studied. The rate of the increase was, however, markedly different for each sub strates, for example, the activities of N-demethylation of butynamine, N-methylbarbital FiG. 1. Effect of administration of phenobarbital or methylcholanthrene on the N-demethylations of aminopyrine, butynamine and diphenhydramine. Adult female and male rats treated with phenobarbital (PB, 80 mg/kg, i.p.) or methylcho lanthrene (MC, 40 mg/kg, i.p.) 48 hours and 72 hours before sacrifice. The results are expressed as averages obtained from at least six animals and as percentages of female control. The average enzyme activities of female controls are as follows : Amino pyrine N-demethylation (81 mpmole/g/30 min) butynamine N-demethylation (142 mItmole/g/ 30 min) and diphenhydramine N-demethylation (843 mpemole/g/30 min). FIG. 2. Effect of administration of phenobarbital or methylcholanthrene on the N-demethylations of meperidine, N-methylbarbital and morphine. Adult female and male rats were treated with phenobarbital (80 mg/kg, i.p.) or methyl cholanthrene (40 mg/kg, i.p.) 48 hours and 72 hours before sacrifice. The results are ex pressed as average obtained from at least six animals and as percentages of control female rats. The average enzyme activities of female controls are as follows : Meperidine N-deme thylation (798 mpmole/g/30 min), N-methylbarbital N-demethylation (83 mpmole/g/30 min) and morphine N-demethylation (342 m pmole/g/30 min). FIG. 3. Effect of administration of phenobarbital or methylcholanthrene on the N-demethylations of N-methylaniline, N,N'-dimethylaniline and N,N'-dimethylnaphthylamine. Adult female and male rats were treated with phenobarbital (80 mg/kg, i.p.) or methyl cholanthrene (40 mg/kg, i.p.) 48 hours and 72 hours before sacrifice. The results are expressed as averages obtained from at least six animals and as percentages of control female rats. The average enzyme activities of female controls are as follows : N-Methylaniline N-demetylation (553 mpmole/g/30 min), N,N'-dimethylaniline N-demethylation (425 momole/g/30 min) and N,N'-dimethylamine N-demethylation (716 mpmole/g/30 min). and aminopyrine were increased by 774%, 412% and 280%, respectively, while activities of N-demethylation of diphenhydramine, meperidine, morphine, N-methylaniline, N,N' dimethylaniline and N,N'-dimethylnaphthylamine were increased by 108%, 106%, 71%, 113%, 96% and 59%, respectively. The administration of phenobarbital to male rats also increased the activities of N demethylation less markedly than female rats. In contrast, the administration of methyl cholanthrene to female rats did not increase the activities of N-demethylation of