DIFFERENCES AMONG THE ACTION OF , METHYLCHOLANTHRENE AND MALE SEX HORMONE ON MICROSOMAL -METABOLIZING ENZYME SYSTEMS OF RAT *

RYUICHI KATO AND MICHIKO TAKAYANAGHI Department of and Department of Toxicology, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo

Received for publication May 30, 1966

The administration of phenobarbital, , , chlorcyclizine, or other different kinds of 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 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 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), chlo ride (25 umoles), (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, , , meperidine, aniline, neoprotosil ; 4 pmoles of hexobarbital ; 3 pmoles of zoxazolamine , p-nitroanisole, p-nitrobenzoic acid; 2 amoles of , 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 . 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 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 amino pyrine, butynamine, diphenhydramine, meperidine, N-methylbarbital and morphine, but it slightly increased the activities of N-methylaniline, N,N'-dimethylnaphthylamine. Moreover, the administration of methylcholanthrene to male rats markedly depressed the activities of N-demethylation of aminopyrine, butynamine, diphenhydramine, meperi dine, N-methylbarbital and morphine, but it slightly increased the activities of N-de methylation of N-methylaniline, N,N'-dimethylaniline and N,N'-dimethylnaphthylamine.

2. Effect of administration of Phenobarbital or methylcholanthrene on the activities of microsomal hydroxylases and 0-dealkylase of female and male rats Figs. 4 and 5 show that there were marked sex difference in the activities of hydro xylation of carisoprodol, pentobarbital and hexobarbital, while there were no clear sex difference in the activities of hydroxylation of aniline and zoxazolamine, moreover, there were slight sex difference in the activities of O-dealkylation of p-nitroanisole. The activities of hydroxylation of carisoprodol, pentobarbital and hexobarbital were markedly increased by the phenobarbital treatment in both female and male rats, while they were not changed in female and markedly depressed in male by the treatment with methylcholanthrene. In contrast, the activities of hydroxylation of aniline and zoxazolamine and moreover O-dealkylation of p-nitroanisole were increased by the treat ment with phenobarbital as well as methylcholanthrene in both male and female rats.

FIG. 4. Effect of administration of phenobarbital or methylcholanthrene on the oxidation of cari sprodol and pentobarbital and on hydroxylation of hexobarbital. Adult female and rats were treated with phenobarbital (80 mg/kg, i.p.) or methylcholan threne (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 : Carisoprodol oxidation (187 mpmole/g/30 min), pentobarbital oxidation (147 mpmole/g/30 min) and hexobarbital hydro xylation (783 mpmole/g/30 min). These results consistently indicated that the effect of methylcholanthrene on the microsomal drug-metabolizing systems of female rats is clearly different from that of male rats.

FiG. 5. Effect of administration of phenobarbital or methylcholanthrene on the 0-nitroanisole and hydroxylations of aniline and zoxazolamine. 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 : p-Nitroanisole O-demethylation (148 mpmole/g/30 min), aniline hydroxylation (298 mpmole/g/30 min) and zoxazolamine hydroxylation (315 mpmole/g/30 min).

FIG. 6. Effect of administration of phenobarbital or metylcholanthrene on the nitro-reduction of p-nitrobenzoic acid, azo-reduction neoprontsil, NADPH-oxidase, and NADPH-cytochrome c reductase. 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 average obtained from at least six animals and as percentages of control female rats. The average enzyme activities of female controls are as follows : p-Nitrobenzoic acid nitro-reduc tion (253 mpmole/g/30 min), neoprontosil azo-reduction (741 mpmole/g/30 min), NADPH oxidase (1.09 Iamole/g/5 min) and NADPH-cytochrome c reductase (11.3 pmole/g/5 min). 3. Effect of administration of Phenobarbital or methylcholanthrene on the activities of NADPH dependent enzymes of liver microsomes in female and male rats Effect of administration of phenobarbital or methylcholanthrene on the activities of NADPH-dependent enzymes of liver microsomes in female and male rats was given in Fig. 6. The activities of nitro-reduction of p-nitrobenzoic acid and azo-reduction of neo prontosil were clearly different between female and male rats, but they were stimulated by phenobarbital as well as methylcholanthrene in both female and male rats. As reported in previous papers (7, 23), phenobarbital stimulated the activities of NADPH oxidase and NADPH-cytochrome c reductase in both female and male rats, while methyl cholanthrene did not significantly alter the activities. Moreover, phenobarbital stimulated the activities of NADPH-neotetrazolium diaphorase, NADPH-dichlorindophenol diapho rase and NADPH-ferricyanide reductase in both female and male rats, while methyl cholanthrene did not significantly or only slightly increased the activities. 4. Different effects of Phenobarbital and methylcholanthrene on the activities of microsomal enzymes in methylteststerone treated castrated female rats Effect of phenobarbital or methylcholanthrene on the activities of N-demethylation of aminopyrine, butynamine and N-methylaniline and hydroxylation of zoxazolamine in the methylteststerone treated castrated female rats is given in Fig . 7. The treatment

FIG. 7. Effect of administration of phenobarbital or methylcholanthrene on the N-dernethylations of aminopyrine, butynamine and N-methylaniline and on the hydroxylation of zoxazolamine in castrated female rats. Female rats were castrated 20 days before sacrifice and treated with methylteststerone (MT, 10 mg/kg, s.c.) every other days for 18 days or treated with phenobarbital (PB, 80 mg/kg, i.p.) or methylcholanthrene (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 castrated female controls. The average enzyme activities of the castrated female rats are as follows : Aminopyrine N-demethylation (85 m,umole/g/30 min), butynamine N-demethylation (155 m,umole/g/30 min), N-methylaniline N-demethylation (481 mumole,/g/30 min) and zoxa zolamine hydroxylation (288 mumole/g/30min) . with methylteststerone markedly stimulated the activities of N-demethylation of amino

pyrine and butynamine, while it slightly stimulated the activities of N-methylaniline N-demethylation and it did not sign significantly alter the activities of zoxazolamine hydro xylation. The administration of Phenobarbital additionally increased the activities of all four enzymes. In contrast, the administration of methylcholanthrene antagonistically

decreased N-demethylation of aminopyrine and butynamine , while it additionally increase N-demethylation of N-methylaniline and hydroxylation of zoxazolamine.

5. Different effect of methylteststerone , Phenobarbital or methylcholanthrene on the acti~nitieso f micro somal enzymes in fasted castrated female rats Effect of administration of methylteststerone, phenobarbital or methylcholanthrene on the activities of microsomal enzymes in the fasted castrated female rats was given in Fig. 8. As reported in a previous paper (11), starvation markedly increased the activities of aminopyrine N-demethylation, aniline hydroxylation and nitro-reduction of p-nitrobenzoic acid in castrated female rats as well as in female rats (135%, 105% and 123% respectively). The effect of methylteststerone in the fasted rats was antagonistic in the N-demethylation of aminopyrine, but it was not significant in the hydroxylation of aniline and the nitro-reduction of p-nitrobenzoic acid . Moreover, the effect of methyl cholanthrene in the fasted rats was depressive in the N-demethylation of aminopyrine ,

Fir 8. Effect of administration of methylteststerone, phenobarbital or methylcholanthrene on the N-demethylation of aminopyrine, hydroxylation of aniline and nitro-reduction of p-nitroben zoic acid in fasted castrated female rats. Female rats were castrated 20 days before sacrifice and treated with methylteststerone (10 mg/kg, s.c.) every other days for 18 days or with phenobarbital (80 mg/kg , i.p.) or methyl cholanthrene (40 mg/kg, i.p.) 48 hours and 72 hours before sacrifice. Half of rats in each group were fasted for 72 hours before sacrifice. The results are expressed as averages obtain ed from at least six animals and as percentages of the castrated rats. The average enzyme activities of female controls are as follows : Aminopyrine N-demethylation (75 m,umole/g/30 min), aniline hydroxylation (319 mlimole/g/30 min) and p-nitrobenzoic acid nitro-reduction (219 m,umole/g/30 min). but it was not significant in the hydroxylation of aniline and the nitro-reduction of p nitrobenzoic acid. In contrast, the effect of phenobarbital in the fasted female rats was clearly increased synergistically in the activities of all three enzymes.

DISCUSSION

It was demonstrated that the administration of phenobarbital, methylcholanthrene and methyltesterone increased the activities of microsomal enzyme systems of rat liver. The administration of phenobarbital increased the activities of all enzymes tested in both female and male rats, although the activities in female rats were generally more markedly stimulated than in male rats. Moreover, the administration of phenobarbital in castrated female rats showed additional action on the effect of methylteststerone and also showed synergistic action on the effect of the starvation. On the other hand, the administration of methylcholanthrene increased the activities of N-demethylation of methylaniline, N,N'-dimethylaniline, N,N'-dimethylnaphthylamine and hydroxylation of aniline and zoxazolamine and O-demethylation of p-nitroanisole in both female and male rats. In contrast, the administration of methylcholanthrene de creased the activities of N-demethylation of aminopyrine, butynamine, diphenhydramine, meperidine, N-methylbarbital and morphine and oxidation of carisoprodol, pentobarbital and hydroxylation of hexobarbital in male rats, but it did not significantly alter these activities in female rats. It was clearly observed that the action of methylcholanthrene on the activities of microsomal enzyme systems is related to the sex difference of the enzyme activity. There was clear tendency that the administration of methylcholanthrene to female rats did not alter the activities of the enzymes having clear sex difference and it increased the activities of the enzymes having slight or no clear sex difference. And moreover, the administration of methylcholanthrene to male rats markedly decreased the activities of the enzymes having clear sex difference and it increased the activities of the enzymes having slight or no clear sex difference. These results as well as the results from Fig. 7 indicate that the administration of phenobarbital act additionally to the effect of anabolic action of male sex hormone and, in contrast, the administration of methylcholanthrene act antagonistically. Moreover, the administration of phenobarbital to castrated fasted female rats synergistically in creased the activities of microsomal enzymes, but the administration of methylcholan threne antagonistically decreased the activities. Although the nitro-reduction of p-nitrobenzoic acid and azo-reduction of neoprontosil showed clear sex difference, the administration of methylcholanthrene increased these activities in both female and male rats. Moreover, NADPH oxidase and NADPH-cyto chrome c reductase showed no clear sex difference and administration of methylcholan threne did not increase those activities. The detailed mechanism of the different effects observed between phenobarbital and methylcholanthrene is not clear, but it is of interest that the differences are likely related to the anabolic action of male sex hormones on the microsomal enzymes. It was reported that the stimulating process and the increased activity of the microsomal enzyme by male sex hormone-like substances are likely unstable and they may easily lose their activity under non-physiological condition, such as adrenalectomy, fasting, hyperthyroi dism and alloxan diabetes, and moreover, administration of morphine or high doses of adrenaline (12). Thus, it is reasonable to assume that the administration of methyl cholanthrene acts in dual mode on the microsomal enzyme system, that is, it acts as inducer of the microsomal enzymes and, on the other hand, it acts as depresser. There fore, it is probable that the activities of the enzyme systems of relatively stable and of being unable to stimulate by anabolic action are increased by the administration of methylcholanthrene and the activities of the enzymes of unstable and of being able to stimulate by anabolic action are decreased. It is of interest that the administration of teststerone and methylteststerone increases the activity of only some enzymes and these increases are observed only in rats, but not in other species of animals (2). However, detailed mechanisms are still in obscure. The relationships between the action of methyl teststerone and methylcholanthrene may offer some insights for the mechanism of stimu lation enzymes by various compounds in further investigations.

SUMMARY

Differences among the actions of phenobarbital, methylcholanthrene and male sex hormone on the activities of drug-metabolizing enzymes of liver microsomes in female and male rats were investigated. The administration of phenobarbital markedly increased the activities of all the enzymes studied more clearly in female rats than in male rats. On the other hand, the administration of methylcholanthrene to female rats increased the activities of the enzymes having no sex difference, but it did not alter the activities of the enzymes having clear sex difference. Moreover, the administration of methylcholanthrene to male rats increased the acti vities of the enzymes having no sex difference, but it markedly decreased the activities of the enzymes having clear sex difference. The administration of methylteststerone markedly increased N-demethylation of aminopyrine and butynamine, while it increased only slightly N-demethylation of N methylaniline and hydroxylation of zoxazolamine in castrated female rats. The admi nistration of phenobarbital to the methyl test sterone-treated castrated female rats resulted in further increases in the activities of all enzymes. In contrast, the administration of methylcholanthrene to the methylteststerone-treated castrated female rats resulted in further increases in the activities of N-methylaniline N-demethylase and zoxazolamine hydroxylase, but it decreased the activities of aminopyrine and butynamine N-methylases. The starvation of methylteststerone treated rats markedly depressed the effect of methyl teststerone on the enzymes having clear sex difference, such as aminopyrine, and the starvation of phenobarbital treated rats markedly increased the effect of phenobarbital, and more the starvation of methylcholanthrene treated rats did not significantly alter the effect of methylcholanthrene. There results indicate that the effect of methylcholanthrene on the activities of drug metabolizing enzyme of liver microsomes related to that of male sex hormone.

REFERENCES

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