Min microsomes prepared from control and 3-methylcholanthrene-pre- treated animals. Of the plant phenols studied tannic acid was found to be the most potent ...
[CANCER RESEARCH 47, 760-766, February 1, 1987)
Inhibition of Epidermal Xenobiotic Metabolism in SENCAR Mice by Naturally Occurring Plant Phenols1 Mukul Das,2 Hasan Mukhtar,3 Daniel P. Bik, and David R. Bickers Departments of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, and Veterans Administration Medical Center, Cleveland, Ohio 44106
ABSTRACT 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 10~5M to 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 them-
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
Received 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. 4 The 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.
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
760
INHIBITION
OF EPIDERMAL
MONOOXYGENASES
in vivo effects of plant phenols on epidermal monooxygenases, animals were treated topically with different doses of the compound (50-400 ¿tmol/kgbody weight) in 0.1 ml of acetone and/or dimethyl sulfoxide. Controls were treated with identical volumes of the vehicle. Preparation of Microsomal Fraction. The mice were killed 24 h after treatment by decapitation with surgical scissors. The skin was excised and immediately placed in an ice-cold solution of KC1 (0.15 M). Each skin sample was placed epidermal side down on a covered glass Petri dish containing crushed ice. The skin was scraped with a sharp scalpel blade (Bard-Parker No. 20) to remove dermis, subcutaneous fat, and muscle and epidermal microsomes were prepared as described earlier (13). The microsomal pellets were overlaid with buffer A [100 mM potassium phosphate, pH 7.4, containing 10 mM dithiothreitol, 10 mM EDTA, and 20% (v/v) glycerol] and frozen at -170'C under nitrogen. For the determination of microsomal enzyme activities the frozen pellets were slowly thawed (within 3-5 days of tissue preparation) in an ice bucket, suspended in buffer A, and used as the enzyme source. Enzyme activities were stable under these storage conditions for at least 3 weeks. Enzyme Assays. AHH activity was determined by a modification of the method of Nebert and Gelboin (14), the details of which have been described earlier (13). The quantitation of phenolic BP metabolites was based on comparison of fluorescence with a 3-hydroxy-BP standard. ECD activity was determined according to a slight modification of the procedure of Greenlee and Poland (15), the adaptations of which have been described earlier (13). ERD activity was determined according to the procedure of Pohl and Fouts (16). The reaction was initiated by the addition of 1.5 pM ethoxyresorufin in 5 pi of dimethyl sulfoxide and was incubated aerobically at 37*C for 30 min in a Dubnoff metabolic
OH
HOOC
DIGALLIC
ACID CH20-R /Ho V \0-R/
0-R \J
ov-r R
TANNIC ( R - Digol
0-R
ACID I ic acid
)
ANTHRAFLAVIC OH
0
Fig. 1. Structure of the plant phenols.
ACID
aqueous phase ranged from 0.3 to 0.4% of the total radioactivity and was proportional to the extent of BP metabolized. The organic phase was dried under a stream of nitrogen and dissolved in 100 ¿il of methanol for HPLC analysis as described earlier (8). All operations were per formed under subdued yellow light. HPLC Analysis of Formation of Metabolites. A Waters Associates model 204 liquid Chromatograph, fitted with a Waters Associate ( ',KfiBondapak column (4.6 mm x 25 cm) was used for the analysis of radiolabeled metabolite mixtures of BP. Identification of the metabo lites was based on reference standards. The column was eluted at ambient temperature with a 20min linear gradient of methanol:water (65:35, v/v) to methanol at a solvent flow rate of 0.6 ml/min. The eluates were monitored at 254 nm, fractions of 0.2 ml were collected dropwise, and the radioactivity of each fraction was determined on a Packard Tri-Carb 460 CD liquid scintillation spectrometer.
RESULTS
shaker. The reaction was terminated by the addition of 2.0 ml of methanol. Fluorescence of the deethylated metabolite was measured at excitation and emission wavelengths of 550 and 585 nm, respectively. The quantitation of the deethylated metabolite was based on compari son of its fluorescence with resorufin as standard. Protein was deter mined, after precipitation with trichloroacetic acid, by the procedure of Lowry et al. (17) using bovine serum albumin as reference standard. in Vitro System of BP Metabolism. The incubation mixture in a final volume of 1.0 ml contained 1.0 mg of microsomal protein, 0.10 mmol of phosphate buffer (pH 7.4), 3 / quercetin > myricetin > anthraflavic acid (Fig. 2). It is impor tant to point out that digallic acid, which occurs on five different positions of the glucose moiety in the structure of tannic acid, had no inhibitory effect on epidermal AHH activity (data not shown). Table 1 shows the I-,,,for these plant phenols against AHH activity in epidermal microsomal prepared from control and MCA-pretreated animals. Quercetin was the most effective inhibitor with an I5o of 5.4 xlO~5 M for AHH activity in epidermal microsomes prepared from control animals followed in decreasing order by myricetin (5.6 x 10~5M), tannic acid (6.0 x Ifr5 M), and anthraflavic acid (12.4 x 10~5 M) (Table 1). In contrast, tannic acid was the most effective inhibitor of AHH activity in microsomes prepared from MCA-treated animals showing a Iso of 4.4 x 10~5 M followed by quercetin (5.4 x 10~5 M), myricetin (5.8 x 10~5 M), anthraflavic acid (11.8 x 10~5 M),
and digallic acid (no inhibition). The kinetics of inhibition of these plant phenols on epidermal AHH activity is depicted in Fig. 3. Tannic acid, quercetin, myricetin, and anthraflavic acid inhibited epidermal AHH activity with K, of 81, 63, 135, and 165 MM,respectively (Fig. 3). Lineweaver-Burk plots of epider mal AHH activity in the presence and absence of fixed concen trations of the plant phenols are shown in Fig. 4. The Kmvalue for AHH activity in epidermal microsomes prepared from control animals was found to be 7.9 MMwhile in the presence of 240 MMtannic acid, quercetin, myricetin, or anthraflavic acid it was 12.2, 14.5, 16.4, and 18.5 MM,respectively. The ym,f was also altered in the presence of each of the plant phenols sug gesting that the inhibitory effect is of a mixed type. The effect of these plant phenols on epidermal AHH, ECD, and ERD activities is shown in Table 2. Tannic acid, quercetin, myricetin, and anthraflavic acid at a concentration of 240 MMresulted in 57-92% inhibition of AHH activity in epidermal microsomes prepared from control and MCA-induced animals (Table 2). These plant phenols were also found to inhibit ERD activity (33-85%) and ECD activity (19-59%) (Table 2) in epidermal microsomes of control as well as MCA-induced animals. Each of the plant phenols consistently exerted a greater inhibitory effect on ERD and AHH activities than on ECD activity. 761
INHIBITION
OF EPIDERMAL
MONOOXYGENASES
BY PLANT PHENOLS
•Tannic acid » Quercetin •Myricetin •Anthraflavic
acid
180
120
CONCENTRATION
240
300
((jM)
•Tannic acid «Quercetin »Myricetin •Anthraflavic
acid
120
240
180
CONCENTRATION
300
( \¡M )
Fig. 2. In vitro effect of plant phenols on epidermal AHH activity in SENCAR mice. Epidermal microsomes were prepared from control (A) and MCA-induced l /( I SENCAR mice. Plant phenols (30-300 />M)were added to the incubation mixtures and AHH activity was determined as described in "Materials and Methods." Data represent means of four individual values.
Table 1 Plant phenol inhibition of AHH activity in epidermal microsomes prepared from control and MCA-pretreated SENCAR mice 10-5M)Control of AHH (x MCA-induced phenolsTannic Plant acid Quercetin Myricetin Anthraflavic acidI»
microsomes microsomes6.0 5.4 5.6 12.4
4.4 5.4 5.8 11.8
In Vitro Inhibitory Effect of Plant Phenols on Epidermal BP Metabolism. The effect of tannic acid, quercetin, myricetin, and anthraflavic acid on epidermal microsomal BP metabolism is shown in Fig. S. Each of the plant phenols exhibited a signifi cant inhibitory effect on the formation of BP metabolites and tannic acid was most effective in this regard. The addition of these plant phenols (60 /AI) decreased the formation of BP diols and BP-phenols by 23-67 and 29-57%, respectively, whereas they had little or no effect on the formation of BPquinones. Tannic acid, quercetin, myricetin, and anthraflavic acid diminished BP-7,8-diol formation by epidermal micro somes. Their inhibitory effect on the formation of BP-phenols correlates well with their effect on epidermal AHH activity. In Vivo Inhibitory Effect of Topically Applied Plant Phenols on Epidermal Monooxygenases. The effect of a single topical application of tannic acid, quercetin, and myricetin to the skin of SENCAR mice on epidermal monooxygenase activities is shown in Fig. 6. The administered dose was limited by the maximum achievable solubility of the test compound in the vehicle. Topical application of tannic acid, quercetin, and my ricetin caused a dose-dependent inhibition of epidermal AHH, ECD, and ERD activities. Topical application of 400 /
