Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome P450 enzymes

Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs fo rmed in the mercapturic acid pathway in mammals. They are effective chemopr eventive agents in mouse lung tumor bioassays and in other models. Thiol-IT Cs are inhibitors of P450s, but it has not been determined if P450 inhibiti on is due to conjugates themselves or to parent ITCs released by deconjugat ion reactions. In studies of mechanism of chemopreventive action of thiol-I TCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conj ugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6 -phenylhexyl isothiocyanate, (PHITC), and sulforaphane (SFN), expressed as the first-order rate constant k(1) and the half-life of decomposition Dt(1/ 2), were measured in aqueous solutions at pH 7.4 and 37 degrees. The Dt(1/2 )S for the Cys conjugates were severalfold shorter than the Dt(1/2)s for re spective GSH conjugates, while the Dt(1/2)S for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was ro ughly parallel to the extent of decomposition of the conjugate that had occ urred, indicating that both potency of the respective parent ITC and the ra te of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 muM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD w as also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechani sms probably contribute to their chemopreventive activity.