Biodistribution of, Antimutagenic Efficacies in Salmonella typhimurium

Quebec City, Canada G1K 7P4, and Department of Physiology and Pharmacology, School of. Medicine, Loma Linda University, Loma Linda, California 92350...
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Chem. Res. Toxicol. 1998, 11, 1258-1264

Articles Biodistribution of, Antimutagenic Efficacies in Salmonella typhimurium of, and Inhibition of P450 Activities by Ellagic Acid and One Analogue Andre Castonguay,*,† Mohamed Boukharta,† and Robert Teel‡ Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Quebec City, Canada G1K 7P4, and Department of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, California 92350 Received February 25, 1998

Ellagic acid (EA) is generated by hydrolysis of ellagitannins present in fruit berries and edible nuts and grapes. Large doses of EA prevent lung tumorigenesis induced by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. In this study, we document the efficacies of the EA structural analogue (3,4,7,8-tetrahydroxy-6H-benzo[b,d]pyran-6-one) (analogue 1) to inhibit specific P450 activities, pulmonary metabolism of NNK in A/J mice, and NNK-induced mutations in Salmonella typhimurium. Mouse lung microsomes metabolized benzyloxyresorufin, a marker of cytochrome P450 2B1 activity, more extensively than methoxyresorufin or ethoxyresorufin. The EA analogue was more effective than EA in inhibiting dealkylation of the three alkoxyresorufins, suggesting that it is a nonspecific inhibitor of P450s. Mouse lung microsomes hydroxylate testosterone in the 7R and 6β positions, suggesting contributions of P450 2A1 and P450 3A2 isozymes, respectively. Inhibition of both pathways was more effective with the EA analogue than with EA. Mouse lung explants metabolized NNK by R-carbon hydroxylation (activation) and pyridine N-oxidation (deactivation). Both pathways were inhibited when 100 µM EA was added to the culture medium. The EA analogue was a better inhibitor of the activation of NNK to electrophilic species than EA. Mouse lung microsomes activate NNK to intermediates mutagenic to S. typhimurium. Inhibition of NNK mutagenicity by EA or the EA analogue was 20 or 65%, respectively. The distribution of the EA analogue in lung and liver was determined following gavage with 1.7 mmol of the EA analogue. In the lung, a maximal level of EA analogue corresponding to 105 nmol was observed 30 min after administration of the analogue. The level in liver tissues was 4-fold lower than in the lung. Results of this study demonstrate that the EA analogue is more effective than EA in inhibiting the pulmonary activation of NNK and suggest that the EA analogue could be effective in preventing lung tumorigenesis.

Introduction Ellagic acid (EA)1 is generated by hydrolysis of complex polyphenolic compounds named ellagitannins. Ellagitannins are esters of glucose with hexahydroxydiphenic acid. Lactonization of hexahydroxydiphenic acid yields EA. The total annual per capita consumption of fruit berries and edible nuts containing ellagitannins is estimated to be 2.5 kg corresponding to 343 mg of EA per year (1, 2). Ellagitannins are also present in grapes (3). According to our laboratory studies, this consumption does not provide a level of EA intake sufficiently high to prevent lung carcinogenesis in humans (4). Evidence that ellagitannins inhibit cancer development is limited. Antitumor activities of monomeric and mac†

Laval University. Loma Linda University. Abbreviations: EA, ellagic acid; NNK, 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone; AROD, alkoxyresorufin dealkylase; MROD, methoxyresorufin dealkylase; EROD, ethoxyresorufin dealkylase; BROD, benzyloxyresorufin dealkylase; analogue 1, 3,4,7,8-tetrahydroxy-6Hbenzo[b,d]pyran-6-one; CYP, cytochrome P450 monooxygenase. ‡

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rocyclic ellagitannins have been reported in mice inoculated with the sarcoma-180 cell line (5). Pomegranate extract containing the ellagitannin punicalagin does not inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis, suggesting that EA is not liberated effectively from punicalagin (6). This observation is consistent with a previous study showing that less than 5% of the dose of pomegranate ellagitannins is excreted as EA in feces (7). Daniel et al. (8) demonstrated that hydrolysis of ellagitannins to EA was limited to the intestine. Modifications of the chemical structure of EA for improving its preventive efficacy have not been fully explored. The relatively low toxicity of EA and the ubiquitous occurrence of ellagitannins have led to many investigations of EA as an inhibitor of cancer development. EA has a wide range of preventing activities, inhibiting chemically induced carcinogenesis in the esophagus, tongue, liver, colon, and skin (9-13). This preventing activity is protocol-dependent and organ-specific. EA

10.1021/tx980033g CCC: $15.00 © 1998 American Chemical Society Published on Web 10/07/1998

Inhibition by Ellagic Acid and One Analogue

injected ip is not effective against benzo[a]pyrene-induced lung tumorigenesis (14). In contrast, Lesca observed a 90% inhibition of lung tumorigenesis induced by benzo[a]pyrene in mice fed a diet containing 400 mg of EA/kg (15). Boukharta et al. (4) showed that prevention of lung tumorigenesis induced by the tobacco-specific carcinogen NNK was proportional to the log of the dose of EA. EA at a dose of 4 g/kg inhibited lung tumor multiplicity by54% (4). The preventive efficacy of EA is limited by its poor absorption from the gastrointestinal tract, its rapid elimination in urine and feces, and its low bioavailibilty in internal organs (16, 17). A lipophilic derivative, 3-O-decylellagic acid, synthesized by Smart et al. (18), improved the lung bioavailibility 20-fold but did not inhibit the binding of benzo[a]pyrene to lung DNA. Boukhata et al. (4) doubled the lung bioavailibility of EA in mice by complexing EA with cyclodextrin but did not test the preventive efficacy of this complex. Glucopyranosyl derivatives of EA are more soluble but less effective preventive agents than EA (19). Modification of the phenolic bislactone structure of EA leading perhaps to a more effective chemopreventive agent remains an avenue to be explored. The analogue (3,4,7,8tetrahydroxy-6H-benzo[b,d]pyran-6-one) used in this study was synthesized by Alo et al. (20) and was found to be the most effective inhibitor of benzo[a]pyrene dihydrodiolepoxide-induced mutgenesis among the 13 analogues tested (21). Many observations have led to hypotheses regarding the mechanism of prevention by EA. Dietary EA inhibits P450-mediated carcinogen activations (22, 23) and induces phase II enzymes involved in the detoxification of carcinogens (24). Inhibition of ornithine decarboxylase and oxidative damage suggest that EA could inhibit tumor promotion (6, 25). There is evidence that EA can trap electrophilic intermediates derived from polyaromatic hydrocarbons and methylating N-nitrosamines (26, 27). Inhibition of tumor growth by EA is also documented (28). EA has multiple mechanisms of action. The aims of this study were (1) to compare the inhibition of P450 activities, the inhibition of pulmonary metabolism of NNK, and the inhibition of NNK-induced mutations by EA and analogue 1 and (2) to determine the biodistribution of analogue 1 given orally to A/J mice.

Experimental Procedures Caution: NNK should be handled as a potential human carcinogen. Chemicals. NNK was synthesized as previously described (29) and was 98.5% pure as determined by reversed phase HPLC (30). [5-3H]NNK (1.84 Ci/mmol) was purchased from Chemsyn Science Laboratory (Lenexa, KS). EA, ethoxyresorufin, and benzyloxyresorufin were purchased from Sigma Chemical Co. (St. Louis, MO). EA was crystallized from pyridine and dried at 55 °C for 3 days. Methoxyresorufin was purchased from Molecular Probes (Eugene, OR). EA analogue 1 was synthesized and characterized by V. Snieckus (University of Waterloo) as described previously (20) and was 97% pure as determined by reversed phase HPLC. EA and analogue 1 were dissolved in DMSO shortly before being used. Tissue Distribution. Female A/J mice (6-8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME). Six groups of three female mice were fasted for 24 h. They were gavaged with a 0.5 mL of a suspension containing 1.7 mmol of analogue 1/kg of body weight in distilled water. One group was sacrificed at intervals of 10, 15, 30, 45, 60, and 120 min after administration.

Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1259

Figure 1. Chemical structures of ellagic acid (EA) and analogue 1. Levels of the analogue 1 in lung and liver tissues were determined as follows. Liver or lung tissues were homogenized in 1 M sodium phosphate buffer (pH 3), and the analogue 1 was extracted with a mixture of acetone and ethyl acetate (1/2 v/v). A 5 mL aliquot of the organic layer was evaporated to dryness under nitrogen at