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Possible Role of P-450 in the Oxidation of Drugs in Liver Microsomes*

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Possible Role of P-450 in the Oxidation of Drugs in Liver Microsomes* The Journal of Biochemistry, Vol. 59, No. 6, 1966 Possible Role of P-450 in the Oxidation of Drugs in Liver Microsomes* By Ry RYUICHI KATO (From the Department of Pharmacology, National Institute of Hygienic Scienecs, Tamagawayoga-machi, Setagaya-ku, Tokyo) (Received for publication, November 17, 1965) B r o die and coworkers (1) demonstrated where A represents the reducible component. that many drugs and other unphysiological Gillette assumed that A is a microsomal materials with high lipid solubility are flavoprotein, NADPH-cytochrome c reductase oxidized by liver microsomes to more water- [EC 1.6.2.3], but gave no details on the soluble compounds. The microsomal systems nature of "active oxygen". On the other responsible for these reactions apparently hand, O m u r a and S a t o (7, 8) showed have a paradoxical requirement for NADPH that liver microsomes contain a hemoprotein, and molecular oxygen. The same require the reduced form of which combines with ments have also been demonstrated with the carbon monoxide to give a Soret peak at systems which hydroxylate phenylalanine and 45 mƒÊ. They have provisionally called this steroid hormones. It has been established hemoprotein as "P-450" and suggested its that atmospheric, but not aqueous, oxygen importance in electron transport in liver is in fact incorporated into the substrate microsomes. More recently, R e m m e r (9), molecules in the hydroxylation of phenyl Kato and Gillette (24)* and Orrenius alanine (2) and steroid hormones (3). and E r n s t e r (10) reported that admini Similarly, incorporation of atmospheric stration of phenobarbital to rats markedly oxygen into substrates has been demonstrated increase the content of P-450, the activity of in the oxidation of acetanilide (4) and NADPH-cytochrome c reductase and drug trimethylamine (5) by liver microsomes. metabolizing activity of liver microsomes. This suggests that in liver microsomes Furthermore, O r r e n i u s et al. (11) found NADPH reduces a component which in turn that carbon monoxide inhibits the oxidative reacts with molecular oxygen to form an N-demethylation of aminopyrine by liver "active oxygen" intermediate . The "active microsomes. These findings strongly suggest oxygen" seems to be involved in the oxida that P-450 may have some role in the oxida tion of a variety of substrates by a group of tion of drugs in liver microsomes. The non-specific enzymes in microsomes. present work was on the possible role of Gillette (6) formulated the following P-450 in the microsomal oxidation of drugs. coupled reactions for this : MATERIALS AND METHODS 1) NADPH+H++A - -.AH2+NADP+ Female rats of the Sprague-Dawly strain were used 2) AH2+02 ->" active oxygen" unless otherwise specified. Sodium phenobarbital, 3) " active oxygen" +drugoxidized drug chlorcyclizine hydrochloride and chlorpromazine * Preliminary reports of part of this work have hydrochloride were dissolved in water, while glutethi snide and carisoprodol were suspended in 0.5% been given at the Federation Meeting of the American carboxymethylcellulose. These materials were injected Society for Experimental Biology and Medicine into animals intraperitoneally. (Chicago, April, 1964). The annual Meeting of the Biochemical Society of Japan (Nagoya, October, 1964) * Reported in the Federation Meeting of the and the Kanto Branch Meeting of the Biochemical American Society for Experimental Biology and Society of Japan (Tokyo, November, 1964). Medicine (Chicago, April, 1964) 574 P-450 and Microsomal Oxidation of Drugs 575 Preparation of Liver Microsomes-Rats were killed c reductase was determined by the method of by decapitation, and the liver was removed, chopped W i l l i a m s and K a m i n (21). All activities were into small pieces, washed well, and homogenized in a expressed as the amount of the compounds metabolized Teflon glass homogenizer with 4 volumes of ice cold per one g. wet weight of liver. 1.15% KCI solution. Estimation of P-450-The amount of P-450 was The homogenate was centrifuged at 9,000•~g for determined by measuring the difference spectrum of 15 minutes and the supernatant fraction was then a microsomal preparation in a Beckman DK2 spectro centrifuged at 105,000•~g for I hour to sediment the photometer with cuvettes of 1 cm. optical path. One microsomes. In the first experiment, the microsomal ml. of microsomal preparation, equivalent to 250mg. pellet was resuspended in ice cold 1.15% KCI and of liver and 1.8 ml. of 0.1 M phosphate buffer (pH 7.4) recentrifuged to eliminate adsorbed hemoglobin. were placed in Thunberg-type cuvettes and chilled in However, this was omitted in later experiments, because an ice cold water bath. Then 0.2 ml. of NADPH recentrifugation decreased enzyme activities and did (2.0ƒÊmoles) was added and the gas-phase was not eliminate appreciable hemoglobin as judged by immediately replaced with oxygen-free nitrogen measuring its absorption in the form of CO-hemoglobin (reference cell) or oxygen-free carbon monoxide (sample at 419mƒÊ. It was also found that a perfusion of liver cell). The difference spectrum was measured at the in situ with isotonic KCI or NaCI significantly (about time of maximal absorbance. The P-450 content was 20%) decreased the recovery of microsomes. expressed as the difference between the optical densities The microsomal pellet was suspended in ice cold at 450 my and 490 my per 1 g. wet weight of liver. 1.15% KCI at a concentration such that 1.0ml. of suspension was equivalent to 400 mg. of liver. RESULTS Enzyme Assays-Microsomes (2.5ml.) equivalent to 1. Effect of Phenobarbital and Methyl 1.0g. of liver were mixed with 2.05 ml. of a solution cholanthreneon Drug Metabolism and the P-450 containing NADP (1.5lƒÊmoles), glucose-6-phosphate Contentof Microsomes-Administration of pheno (50 ƒÊmoles), glucose-6-phosphate dehydrogenase [EC 1. 1. 1.49] (0.5 ƒÊunit), magnesium chloride (25 ƒÊmoles), barbital, methylcholanthrene, benzopyrene, or nicotinamide (100 ƒÊmoles), potassium phosphate buffer various other drugs have been shown to (280 ƒÊmoles, pH 7.4) and various substrates (5 ƒÊmoles stimulate the oxidative metabolism of many of aminopyrine, methylaniline, aniline ; 4 ƒÊmoles of drugs in liver microsomes (22, 23). hexobarbital ; 3 ƒÊmoles of zoxazolamine ; or 2 ƒÊmoles As shown in Table I, it was in fact of pentobarbital) and 0.45m1. of 1.15% KCI. found that phenobarbital administration The mixtures were incubated in a Dubnoff significantly increased the P-450 content in metabolic shaker for 30 minutes at 37°C under air. liver microsomes even by 6 hours after its Demethylation of aminopyrine was determined by injection and the maximal increase in the mearsuring the formation of 4-aminoantipyrine, as P-450 content was attained after 24 to 36 described by Brodie and Axelrod (12). hours. Subsequently, the P-450 gradually Demethylation of N-methylaniline was estimated decreased, finally returning to the normal by measuring the amount of formaldehyde formed level after 120 hours. The microsomal hy according to the method of N a s h (13) as modified by droxylation of pentobarbital and the C o c h i n and A x e l r o d (14 ). Aromatic hydroxylation N-demethylation of aminopyrine followed of aniline was determined by measuring T-aminophenol similar patterns of increase and decrease in formation by the phenolindophenol method of B rod i e activity. However, 6 hours after administra and Axe 1 r o d (15) modified as reported previously tion when the P-450 content had increased (16). Aromatic hydroxylation of zoxazolamine was significantly these enzyme activates had not determined by measuring the disappearance of substrate yet increased. by the method of C o n n e y et al. (17). Aliphatic A similar discrepancy between the induc hydroxylations of hexobarbital and pentobarbital were tion of P-450 synthesis and drug-oxidizing determined by measuring the disappearance of the substrates according to the methods of Cooper and activity was demonstrated more clearly on Brodie (18) and Brodie et al. (19), respectively. administration of methylcholanthrene. As Microsomal NADPH dehydrogenase [EC 1.6. 99. 1] was shown in Table II, injection of this com- assayed spectrophotometrically as described by pound markedly increased the demethylation Gillette et al. (20). Microsomal NADPH-cytochrome of N-methylaniline and hydroxylation of 576 R. KATO TABLE I Effect of Phenobarbital Treatment on Aminopyrine N-Demethylation and Pentobarbital Hydroxylation Activities and Amount of P-450 Male rats weighing about 70g. were treated with phenobarbital (70 mg. /kg.) and killed 6, 12, 24, 48, 72 and 96 hours later. The livers from 2 rats were combined for each microsomal preparation. The determination of the enzyme activity and amount of P-450 were carried out as described in the methods. The amount of P-450 is expressed by the difference between in optical density at 450mƒÊ and 490mƒÊ. The results are expressed as averages±S.D. (standard deviation) of the values of 5 preparations. The figures in brackets indicate percentage differences from the control. TABLE II Effect of Methylcholanthrene Treatment on N-Methylaniline N-Demethylation and Zoxazolamine Hydroxylation Activities and Amount of P-450 Male rats weighing about 70g. were treated with methylcholanthrene (40mg./kg.) and killed 6, 12, 24, 36, 48, 72 and 96 hours later. P-450 and Microsomal Oxidation of Drugs 577 TABLE I I I Effect of Phenobarbital Treatment on Aminopyrine N-Demethylation, Pentobarbital Hydroxylation and NADPH Dehydrogenase Activities and Amount of P-450 of Rats Fed on Chow or Sucrose or Fasted Female rats weighing about 160g. were divided into three groups 72 hours before sacrifice. The first group was fed on standard chow diet, the second group was fasted and the third group was fed on sucrose only.
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