Short Communication Metabolism of dacarbazine by rat liver microsomes contribution of CYP1A enzymes to dacarbazine N-demethylation

The N-demethylation of dacarbazine in liver microsomes was significantly increased by treatment of rats with b-naphthoflavone, dexamethasone, or phenobarbital. However, the extent of increase in the N-demethylation observed in b-naphthoflavone-treated rats was much greater than that observed in dexamethasone-treated rats. A good correlation between N-demethylation of dacarbazine and O-deethylation of phenacetin was observed when a low concentration of phenacetin was used. Furthermore, the activity of dacarbazine N-demethylase in rat liver microsomes was highly correlated with the amounts of CYP protein immunochemically determined with anti-rat CYP1A2 antibodies. In addition, antibodies to rat CYP1A2, and furafylline and a-naphthoflavone, which are known inhibitors of CYP1A enzymes, exhibited inhibitory effects on dacarbazine N-demethylation. These results indicated that CYP1A enzymes may be responsible for N-demethylation of dacarbazine in rat liver microsomes. 5-(3,3-Dimethyl-1-triazeno) imidazole-4-carboxamide (dacarbazine) is an antineoplastic drug which is classified as an alkylating agent (Newell et al., 1987). It is used extensively as a single drug in the treatment of metastatic malignant melanoma and in combination with other drugs for treating renal adenocarcinoma, soft tissue sarcoma, solid tumor, and malignant lymphomas (Lee et al., 1992; Mitchell and Dolan, 1993). In addition, dacarbazine has also shown that 1-aryl-3,3,-dialkyltriazenes undergo oxidative metabolism to form 1-aryl-3-monomethyltriazenes, and that triazenes including dacarbazine require metabolic activation by the host for antitumor activity (Newell et al., 1987; Audette et al., 1973; Gescher et al., 1981). Aminoimidazole carboxamide, which is a major urinary metabolite of dacarbazine in humans, has been demonstrated to be produced from dacarbazine in liver microsomes (Breithaupt et al., 1982; Skibba and Bryan, 1970; Hill, 1975). Furthermore, it has been suggested that both 5-(3-hydroxymethyl-3-methyltriazen-1-yl)-imidazole-4-carboxamide and 5-(3-methyltriazene-2-yl) imidazole-4-carboxamide were dacarbazine metabolites in mice, rats, and humans and that 5-(3-hydroxymethyl-3-methyltriazen-2-yl) imidazole-4-carboxamide decomposes spontaneously to form aminoimidazole carboxamide, with concomitant alkylation of celluar DNA (1, 10–12Newell et al., 1987; Beal et al., 1975; Kolar et al., 1980; Nagasawa et al., 1974; Mizuno et al., 1976). Therefore, N-demethylation of dacarbazine has been considered to be an important metabolic pathway for both the antineoplastic and carcinogenic activities of dacarbazine (Beal et al., 1975; Spassova and Golvovinsky, 1985). Since several lines of evidence have demonstrated that liver microsomal enzyme(s) is responsible for N-demethylation of dacarbazine leading to the formation of monomethyl triazene imidazole carboxamide and that the P450 is responsible for the bioactivation of dacarbazine (Hill, 1975; Mudipalli et al., 1995), it is likely that the antineoplastic activity of dacarbazine may be affected by the activity of P450 responsible for N-demethylation of the drug. However, the form(s) of P450 that contribute to N-demethylation of dacarbazine are still undetermined. Therefore, this study was conducted to get better understanding of the P450 enzyme responsible for N-demethylation of dacarbazine in rat liver microsomes. Materials and Methods Materials. Dacarbazine was a generous gift from Kyowa Hakko KK. (Tokyo, Japan). CYP1A2 and CYP2B1/2 were purified from liver microsomes of rats treated with 3-methylcholanthrene and phenobarbital by the methods reported previously, respectively (Tamataki et al., 1983; Kitada et al., 1984). CYP3A4 and CYP2C9 were purified from human liver microsomes according to methods described elsewhere (Shimada et al., 1986; Komori et al., 1988). The antibodies for purified P450 raised to rabbit were carried out according to the method previously reported (Kamataki et al., 1976). NADP, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase were obtained from Oriental Yeast (Tokyo, Japan). Other chemicals used were of the highest grade commercially available. Animals, Pretreatment of Animals and Preparation of Microsomes. Male Sprague-Dawley rats (8 weeks old), obtained from Takasugi Experimental Animals Co. Ltd. (Saitama, Japan), were used throughout this study. Rats were pretreated intraperitoneally with phenobarbital-Na at doses of 80 mg/kg for 5 days, dexamethasone 50 mg/kg for 3 days, and b-naphthoflavone 40 mg/kg for 3 days. The agents for pretreatment were dissolved in corn oil except for phenobarbital which was dissolved in water. Ethanol was given orally in a solution at a 10% concentration. Rats were given free access to food and water and were killed 24 hr after the last injection. Liver microsomes were prepared by differential centrifugation as described elsewhere (20). Assays. A typical reaction mixture consisted of 100 mM potassium This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Send reprint requests to: Mitsukazu Kitada, Ph.D., Division of Pharmacy, Chiba University Hospital, 1–8-1 Inohana, Chuo-ku, Chiba 260, Japan. 0090-9556/98/2604-0379–382$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 26, No. 4 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 379 at A PE T Jornals on A uust 4, 2017 dm d.aspurnals.org D ow nladed from