2-Ethynylnaphthalene as a mechanism-based inactivator of the cytochrome P-450 catalyzed N-oxidation of 2-naphthylamine

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Abstract
Since the N-oxidation of several carcinogenic arylamines has been shown to be catalyzed preferentially by cytochrome P-450IA2 in several species, homologous ethynyl-substituted aromatic hydrocarbons, 2-ethynylnaphthalene, 1-ethynylnaphthalene, and 2-ethynylfluorene, were synthesized and examined as potential mechanism-based inactivators of this monooxygenase. By use of 2-naphthylamine, whose-N-oxidation was known to be selectively catalyzed by rat cytochrome P-450ISF-G (P450IA2), and hepatic microsomes from isosafrole-treated rats, each of these ethynyl derivatives was found to be strongly inhibitory at concentrations of 1 and 10 .mu.M. However, only inhibition by 2-ethynylnaphthalene was significantly enhanced by prior incubation with the microsomal system. The inactivation of 2-naphthylamine N-oxidation was found to be NADPH- and time-dependent and to follow pseduo-first-order kinetics, demonstrating that 2-ethynylnaphthalene is a potent mechanism-based inactivator of the enzymatic activity. The extrapolated kinactivation and KI were 0.23 min-1 and 8 .mu.M, respectively. By use of 2-aminofluorene, whose N-oxidation was known to be catalyzed by both cytochromes P-450ISF-G and P-450.beta.NF-B (P-450IA1), and the purified enzymes in a reconstituted system, both 2-ethynylnaphthalene and 1-ethynylnaphthalene were found to be strongly inhibitory. However, 2-ethynylnaphthalene was a more potent inhibitor of the purified P-450ISF-G than of P-450.beta.NF-B; and it was also found to be a more potent inhibitor of P-450ISF-G than was 1-ethynylnaphthalene. With radiolabeled 2-ethynylnaphthalene and purified P-450ISF-G, the metabolic formation of [3H]-2-naphthylacetic acid was measured and used to calculate a partition ratio of 64 (molecules of 2-ethynylnaphthalene metabolized per molecule of P-450 inactivated). Covalent binding of 2-ethynylnaphthalene to the purified P-450s was also demonstrated, resulting in 0.62-0.77 mol of [3H]-2-ethynylnaphthalene bound per mole of P-450ISF-G and 0.72 mol of [3H]-2-ethynylnaphthalene bound per mole of P-450.beta.NF-B. These studies suggest that 2-ethynylnaphthalene may be useful as a inhibitor of arylamine N-oxidation and as an active site probe for cytochrome P-450IA2.

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