Mechanism-Based Inactivation of Hepatic Ethoxyresorufin O-Dealkylation Activity by Naturally Occurring Coumarins

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Abstract
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 [14C]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 [14C]coriandrin to microsomal protein. In addition, the covalent binding of [14C]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 [14C]coriandrin-bound microsomal protein from MC-pretreated mice showed that [14C]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 [14C]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.

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