by Naturally Occurring Plant Phenols Inhibition of Epidermal Xenobiotic Metabolism in SENCAR Mice

Naturally occurring plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid have been shown to inhibit the mutagenicity of several bay-region diol-epoxides of polycyclic aromatic hydrocar bons including benzo(a)pyrene (BP). The present study was designed to determine whether these plant phenols can alter epidermal cytochrome P-450-dependent monooxygenases in SENCAR mice. In vitro addition of these plant phenols to epidermal microsomal preparations inhibited aryl hydrocarbon hydroxylase (AHH) activity in a concentration-depend ent manner. The 50% inhibitory concentrations for tannic acid, myricetin, quercetin, and anthraflavic acid ranged from 4.4 X 10~5Mto 12.4 x 10 ' M in microsomes prepared from control and 3-methylcholanthrene-pretreated animals. Of the plant phenols studied tannic acid was found to be the most potent inhibitor of epidermal AHH activity. Tannic acid, quer cetin, myricetin, and anthraflavic acid exhibited a mixed type of inhibitory effect with A, values of 81, 63, 135, and 165 /JM, respectively. In vitro addition of these plant phenols (240 MM) to the incubation mixture prepared from control and 3-methylcholanthrene-treated animals resulted in varying degrees of inhibition of epidermal microsomal AHH (5792%), ethoxycoumarin 0-deethylase (19-58%), and ethoxyresorufin Odeethylase (33-85%) activities. High pressure liquid Chromatographie analysis of the organic solvent-soluble metabolites of BP produced by epidermal microsomes indicated a substantial decrease in the formation of BP-diols (23-67%) and BP-phenols (29-57%) by each of the plant phenols. The formation of BP-7,8-diol was substantially inhibited (2952%) by each of the plant phenols. Further in vivo studies showed that a single topical application of tannic acid, quercetin, and myricetin greatly diminished epidermal AHH (53-65%), ethoxycoumarin 0-deethylase (30-68%), and ethoxyresorufin 0-deethylase (66-97%) activities whereas anthraflavic acid was ineffective in this regard even when re peatedly applied. Our results indicate that plant phenols have substantial though variable inhibitory effects on epidermal monooxygenase activities and BP metabolism suggesting that these compounds may be capable of inhibiting the carcinogenic effects of polycyclic aromatic hydrocarbons in the skin. INTRODUCTION The hemeprotein cytochrome P-450 is a heterogeneous mi crosomal enzyme system found in liver and many extrahepatic tissues including skin (1,2). This monooxygenase system func tions in the oxidative biotransformation of many drugs and foreign chemicals converting them to polar metabolites, thereby facilitating both their pharmacological inactivation and their elimination from the body (1-3). However, this enzyme system is also capable of transforming certain chemicals into highly reactive toxic metabolites that may produce a variety of path ological effects. For example, carcinogenic PAHs4 are themReceived 3/31/86; revised 9/11/86; accepted 10/21/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by NIH Grants ES-1900, CA-38028, and AM-34368; by American Institute for Cancer Research Grant 86A61; and by research funds from the Veterans Administration. 1 Recipient of Schering-Plough Foundation fellowship award from the Der matology Foundation. ' To whom requests for reprints should be addressed, at Veterans Administra tion Medical Center, 10701 East Boulevard, Cleveland, OH 44106. 4The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; AHH, aryl hydrocarbon hydroxylase; ECD, 7-ethoxycoumarin O-deethylase; ERD, 7ethoxyresorufin O-deethylase; BP, benzo(a)pyrene; BP-7,8-diol, (±)-/rani-7,8dihydro-7,8-dihydroxybenzo(a)pyrene; MCA, 3-methylcholanthrene; HPLC, high pressure liquid chromatography; I.,,. 50% inhibitory concentration. selves relatively inert compounds and essentially act as precarcinogens that must first undergo metabolic activation to biolog ically active metabolites that are the ultimate carcinogens (1, 4). The first step in the metabolic activation of PAHs is cata lyzed by the cytochrome P-450-dependent enzyme commonly known as AHH (1, 4). Knowledge of the importance of this enzyme in the metabolic activation of PAHs has led to the suggestion that inhibitors of AHH activity could diminish the carcinogenicity and mutagenicity of PAHs in target tissues such as the skin. For example a synthetic flavonoid, 7,8-benzoflavone, is an inhibitor of skin AHH activity, diminishes binding of 7,12-dimethylbenz(a)anthracene metabolites to cellular macromolecules, and reduces skin tumor initiation by 7,12-dimethylbenz(a)anthracene (5, 6). In previous studies our laboratory has shown that the antifungal imidazole, clotrimazole, inhibits epidermal AHH activity, decreases binding of BP metabolites to epidermal DNA, and protects against skin tumor induction by topically applied MCA (7). Recent attention has been focused on the identification of naturally occurring plant phenols as possible cancer chemopreventive agents. One promising approach to decreasing the risk of developing chemically induced tumors might be the modu lation of the activity of enzymes that participate in metabolic activation of carcinogens. Several recent studies have shown that a naturally occurring plant polyphenol, ellagic acid, inhibits the cytochrome P-450-dependent metabolism, the DNA bind ing, and the tumorigenicity of PAHs in skin and lung (8-12). In the present study we provide evidence for the in vitro and in vivo inhibition of cutaneous monooxygenases and BP metabo lism in SENCAR mice by a series of plant phenols including tannic acid, flavonoids, and anthraquinones (for structures see Fig. 1). MATERIALS AND METHODS Chemicals. Gold label BP, 7-ethoxycoumarin, resorufin, tannic acid, digallic acid, myricetin, and anthraflavic acid were obtained from Aidrich Chemical Co (Milwaukee, WI). 7-Ethoxyresonifin was purchased from Pierce Chemicals. Quercetin, MCA, NADPH, NADH, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). [7,10-I4C]BP (specific activity, 58.5 mCi/mmol) was purchased from Amersham Searle (Chicago, IL). Prior to use, radiolabeled BP was purified on a silica gel (l'ari¡siilO-jtm; Waters Associates) column with hexane as the eluting solvent and subsequently by reverse-phase HPLC using a Dupont Zorbax ODS column (6.2 mm x 25 cm) eluted with methanokwater (19:1 v/v). The purity of BP was greater than 99% as judged by HPLC. BP reference standards were provided by the Cancer Research Program, Division of Cancer Cause and Prevention, National Cancer Institute (Bethesda, MD). All other chemicals were obtained in the purest form commercially available. Treatment of Animals. Six-week-old female SENCAR mice, obtained from the NCI-Frederick Cancer Research Facility, Bethesda, MD, were used. The mice were shaved with electric clippers and Nair depilatory was applied 1 day prior to the beginning of the experiment. For evaluation of the in vitro effects of tannic acid, quercetin, myricetin, and anthraflavic acid, one group of animals received a single topical application of MCA (50 mg/kg) in 0.1 ml of acetone while control animals received an identical volume of the vehicle. For studies of the