Mechanism-Based Inactivation of Hepatic Ethoxyresorufin O-Dealkylation Activity by Naturally Occurring Coumarins Yingna Cai, † Wanda Baer-Dubowska, † Mike J. Ashwood-Smith, ‡ Oluna Ceska, ‡ Sanro Tachibana, § and John DiGiovanni* ,† The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Department of Carcinogenesis, P.O. Box 389, Smithville, Texas 78957, and Department of Biology and Department of Botany, University of Victoria, Victoria, British Columbia V8W 2YZ, Canada Received December 11, 1995 X Several naturally occurring coumarins contained in the human diet have been found to be effective inhibitors and inactivators of murine hepatic ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase in vitro [Cai, Y., Bennett, D., Nair, R. V., Ceska, O., Ashwood-Smith, M., and DiGiovanni, J. (1993) Chem. Res. Toxicol. 6, 872-879]. In the present study, these same coumarins decreased the content of cytochrome P450 (P450) in either 3-methylcholanthrene (MC)- or phenobarbital-induced murine hepatic microsomes but did not have a major effect on heme content. Detailed in vitro studies with [ 14 C]coriandrin, which selectively inhibits and inactivates P450 1A1-mediated EROD activity, demonstrated that it covalently bound, in a preferential manner, to hepatic microsomal protein from MC-pretreated mice. A linear relationship was observed between covalent binding and loss of EROD activity. The inclusion of electrophile trapping agents in the incubations significantly inhibited the covalent binding of [ 14 C]coriandrin to microsomal protein. In addition, the covalent binding of [ 14 C]coriandrin was decreased 46% by 7,8-benzoflavone (7,8-BF), 58% by a monoclonal antibody with specificity toward MC-induced form(s) of P450, and 60% by ethoxyresorufin, implicating the bioactivation of coriandrin by P450 1A1. Analysis by sodium dodecyl sulfate- polyacrylamide gel electrophoresis of [ 14 C]coriandrin-bound microsomal protein from MC- pretreated mice showed that [ 14 C]coriandrin bound covalently to a protein with an approximate molecular mass of 49 kDa. Again, addition of 7,8-BF or polyclonal antibody against P450 1A1 reduced the covalent binding of [ 14 C]coriandrin to this specific protein band. Interestingly, coriandrin was also found to be a potent inhibitor and inactivator of purified human P450 1A1. These results demonstrate that certain coumarins to which humans are exposed in the diet are bioactivated by P450 1A1 to reactive intermediates that subsequently form covalent adducts with the apoprotein, effectively destroying enzyme activity. Thus, certain naturally occurring coumarins may have a significant effect on human health. Introduction The cytochrome P450 (P450) 1 superfamily consists of many enzymes that play a central role in both metabolic activation and detoxication of a variety of xenobiotics, including drugs, chemical toxicants, and carcinogens. To date, 12 P450 gene families comprising over 100 enzymes have been identified in mammalian species (1). Although there is some overlap in substrate specificity among the enzymes, individual enzymes may prefer a specific substrate (2). Many synthetic as well as naturally occurring compounds can inhibit P450-mediated reac- tions (reviewed in refs 3 and 4), including polycyclic aromatic acetylenes (5) and flavonoids (6, 7). These inhibitors may be classified as either reversible or ir- reversible inhibitors. Mechanism-based inactivators of P450 are a category of irreversible inhibitors that require metabolic activation by P450 to form reactive intermedi- ates, leading to the formation of covalent adducts (8). Without exception, this covalent binding ultimately leads to the loss of the catalytic function of the P450 molecule (8). Furthermore, some inactivators appear to selectively inactivate specific P450 enzymes (reviewed in ref 9). For example, as reported by Hopkins et al., 1-ethynylpyrene (1-EP) effectively inactivated P450 1A1 but not P450 2B1 (5). Mechanism-based inactivators of P450 provide valu- able probes to study the catalytic site of the enzyme molecule. Interestingly, inactivators such as 1-EP have also displayed anticarcinogenic effects in experimental carcinogenesis systems (10). A number of naturally occurring coumarins have been found to both inhibit and inactivate P450-mediated enzyme activity. Earlier studies (11, 12) indicated that xanthotoxin (7, Chart 1) and several linear furanocou- marins were irreversible inhibitors of P450 enzymes both in vitro and in vivo. Recently, we reported that certain naturally occurring coumarins, including the linear fura- nocoumarins imperatorin (6, Chart 1) and isopimpinellin * Author to whom correspondence and reprint requests should be addressed. † The University of Texas M. D. Anderson Cancer Center. ‡ Department of Biology, University of Victoria. § Department of Botany, University of Victoria. X Abstract published in Advance ACS Abstracts, May 1, 1996. 1 Abbreviations: P450, cytochrome P450; 1-EP, 1-ethynylpyrene; EROD, ethoxyresorufin O-dealkylase; MC, 3-methylcholanthrene; PROD, pentoxyresorufin O-dealkylase; PB, phenobarbital; -NF, -naph- thoflavone; NBS, hybrid cells formed from RGNS-1 myeloma cells and spleen cells from unimmunized mice; G-6-P, glucose 6-phosphate; G-6- PD, glucose-6-phosphate dehydrogenase; -ME, -mercaptoethanol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophore- sis; DLPC, dilauroylphosphatidylcholine; 7,8-BF, 7,8-benzoflavone; EH, epoxide hydrolase; ER, ethoxyresorufin; SCZ, sodium semicarbazide. 729 Chem. Res. Toxicol. 1996, 9, 729-736 S0893-228x(95)00208-6 CCC: $12.00 © 1996 American Chemical Society + +