INHIBITION OF N-NITROSODIMETHYLAMINE DEMETHYLASE IN RAT AND HUMAN LIVER-MICROSOMES BY ISOTHIOCYANATES AND THEIR GLUTATHIONE, L-CYSTEINE, AND N-ACETYL-L-CYSTEINE CONJUGATES

Natural and synthetic isothiocyanates and their conjugates were examin ed for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometri c NDMAd assay. Substrate concentrations of 30 and 60 mu M were used to probe the activity of cytochrome P4502E1 isozyme through the a-hydrox ylation of NDMA. It was found that alkyl isothiocyanates such as sulfo raphane and allyl isothiocyanate displayed very weak inhibition, where as the arylalkyl isothiocyanates such as benzyl and phenethyl isothioc yanate showed significant inhibition toward rat liver NDMAd activity w ith IC50 values of 9.0 and 8.3,mu M, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothi ocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differ ences in their inhibitory activity; i.e., its conjugates with L-cystei ne (IC50 = 4.3 mu M) and with glutathione (IC50 = 4.0 mu M) are more p otent than its conjugate of N-acetylcysteine (IC50 = 24.0 mu M). The s ame trend was also observed for the human liver microsomal NDMAd activ ity. The half-lives of these conjugates were determined in the presenc e of other free thiols from L-cysteine or glutathione using an HPLC sy stem. It was shown that isothiocyanates are released from their conjug ates and react with the free thiols present in the solution. The longe r half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhib itory activity. The inhibition of NDMAd, and therefore cytochrome P450 2E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrom e P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemoprev entive agents.