[CANCER RESEARCH 44, 4233-4240. October 1984] Clotrimazoie, an Inhibitor of Epidermal Benzo(a)pyrene Metabolism and DMA Binding and Carcinogenicity of the Hydrocarbon1 Hasan Mukhtar,2 Benjamin J. Del Tito, Jr., Mukul Das, Evan P. Cherniack, Andrew D. Chemiack, and David R. Bickers Department of Dermatology, Case Western Reserve University [H. M,, B. J. D., M. D., E. P. C., A. D. C., D. R. B.¡,and the Veterans Administration Medical Center [H. M., B. J. D.. M. D., £.P. C., A. D. C., D. R. B.], Cleveland, Ohio 44106 ABSTRACT Clotrimazoie, a topically applied imidazole antifungal agent widely used in dermatological practice, was shown to be a potent inhibitor of the epidermal metabolism of benzo(a)pyrene (BP) and its microsomal enzyme-mediated binding both to neonatal rat epidermal DMA in vivo and to calf thymus DNA in vitro. Varying concentrations of Clotrimazoie added to in vitro incubation sys tems resulted in a dose-dependent inhibition of cytochrome P- 450-dependent microsomal aryl hydrocarbon hydroxylase (AHH) in control animals as well as in animals pretreated with topical application of known inducers of the enzyme. Inhibition of epi dermal AHH by topically applied Clotrimazoie was time and dose dependent. The 50% inhibition of Clotrimazoie for epidermal AHH ranged from 0.12 to 0.25 ^M. which suggests that Clotrimazoie is among the most potent inhibitors of epidermal AHH yet iden tified. Clotrimazoie was also found to be a potent inhibitor of epoxide hydrolase activity in vitro with a 50% inhibition at 0.1 row. High-pressure liquid Chromatographie analysis of the metab olism of BP in rat epidermal microsomes revealed substantial inhibition of metabolite formation by Clotrimazoie. This occurred in microsomes prepared from untreated as well as animals pretreated with inducers of the enzyme. Furthermore, a single topical application of Clotrimazoie resulted in 80 and 30% induc tion of epidermal and hepatic glutathione S-transferase activity, respectively. Topical application of Clotrimazoie to the skin of BALB/c mice substantially increased the latent period for the development of skin tumors by 3-methylcholanthrene. These studies indicate that Clotrimazoie is an extremely potent inhibitor of epidermal BP metabolism and of the DMA-binding of polycyclic aromatic hydrocarbon (PAH) carcinogens, and is an enhancer of enzymes necessary for detoxification of the PAH. Clotrimazoie also reduces the formation of carcinogenic and mutagenic metabolites of BP in vitro and in vivo and inhibits induction of skin tumors by the PAH. These data indicate that the imidazole antifungal Clotrimazoie offers promise as an agent useful for the modulation of PAH cancer risk in the skin. INTRODUCTION Many of the PAHs,3 some of which are common environmental pollutants, are known to have carcinogenic and mutagenic ef- 1 Supported in part by NIH Grant ES-1900 and research funds from the Veterans Administration. * To whom requests for reprints should be addressed, at Veterans Administration Medical Center, 10701 East Boulevard, Cleveland. OH 44106. 3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; BP, benzo(a)pyrene; BP 4,5-oxide, benzo(a)pyrene 4,5-oxide; AHH, aryl hydrocarbon hydroxylase; EH, epoxide hydrolase; GSH transferase, glutathione S-transferase; HPUC, high-pressure liquid chromatography; 3-MC, 3-methylcholanthrene; PCB, Aroctor 1254; ln, 50% inhibition. Received December 7,1983; accepted June 28,1984. fects (9). The PAHs themselves are relatively inert biologically and essentially act as precarcinogens that must first undergo metabolic activation by mammalian enzymes to their biologically active ultimate carcinogenic metabolites (10, 20, 23, 34). These metabolites are highly reactive unstable moieties that can bind covalently to cellular RNA, DNA, and proteins, as well as to synthetic copolymers of nucleic acids (7, 27, 55). It is generally agreed that the covalent interaction of reactive metabolites of the PAHs with DNA may represent an essential initial step in tumor induction (7, 20, 27, 55). Studies during the past decade have identified one of the isomerie metabolites of BP known as (+)-7/3,8«-dihydroxy-9a-10a-epoxy-7,8,9,10-tetrahydrobenzo (a)pyrene, the ultimate carcinogenic species of this PAH (45, 51). The formation of this metabolite is catalyzed by 3 successive enzymatic steps: first, turnover by AHH, a cytochrome P-450- dependent mixed-function oxidase, followed by EH and again by AHH (10,14, 20, 23, 34). The reactive epoxide intermediates are detoxified by conjugation with glutathione, a reaction catalyzed by GSH transferases (10, 19, 35). These enzymatic pathways for the metabolic activation and inactivation of PAHs have been identified in the skin (3, 35). A number of investigators have shown that imidazole-contain- ing compounds, including certain widely used drugs (e.g., cime- tidine, benzimidazole, antifungal agents) can alter hepatic xeno- biotic metabolism by inhibiting microsomal cytochrome P-450- dependent monooxygenase activities (6, 22, 25, 54). In addition to inhibiting certain of these cytochrome P-450-dependent activ ities, imidazole derivatives have also been shown to modify EH activity (22, 25). Kahl et al. (25) previously reported the inhibition of hepatic microsomal BP metabolism and its DNA adduct for mation in vitro by Clotrimazoie. Cancer of the skin is the most common form of malignant neoplasm which occurs in the human population. Skin is a major interface between the body and its environment and is a potential portal of entry for numerous environmental chemicals, drugs, and other agents, some of which exhibit local toxic and/or tumorigenic effects in cutaneous tissue, whereas others have been shown to exert toxic effects in extracutaneous tissues (2, 33). In recent years, it has become quite clear that organ-specific toxicity including carcinogenic responses are dependent upon metabolic events that occur in the target tissue itself. It is reasonable to assume that the balance of activating and inacti vating enzymatic pathways in the skin would be a major deter minant of the amount of carcinogenic metabolites available for initiating oncogenic responses in cutaneous tissue. Clotrimazoie (for structure, see Chart 1) is currently used throughout the world in medical practice for the management of a wide variety of superficial dermatophyte infections (56). Be cause of its proven safety in clinical medicine, the potential effectiveness of Clotrimazoie as an inhibitor of the metabolism of OCTOBER 1984 4233