THE DEUTERIUM-ISOTOPE EFFECT FOR THE ALPHA-HYDROXYLATION OF TAMOXIFEN BY RAT-LIVER MICROSOMES ACCOUNTS FOR THE REDUCED GENOTOXICITY OF [D-5-ETHYL]TAMOXIFEN

This study describes the application of on line HPLC-electrospray ionization MS in the structural determination of the metabolites formed following incubation with rat liver microsomes of an equimolar mixture of the anticancer drug tamoxifen and its [D-5-ethyl]-analogue. The ratio of Ca 3:1 between unlabelled and D-4-labelled alpha-hydroxytamoxifen, indicating a large isotope effect for this metabolic process, accounted for the previously observed lower yield of DNA adducts formed in the livers of rats treated with D-5-tamoxifen compared with unlabelled drug, The loss of one deuterium atom on metabolism discriminated hydroxyethylated metabolites from others and enabled two further such metabolites to be detected, namely alpha-hydroxytamoxifen N-oxide and alpha-hydroxy-N-desmethyltamoxifen of which the latter is novel, Furthermore, the use of [alpha-D-2-ethyl]- and [beta-D-3-ethyl] tamoxifens discriminated alpha- from beta-hydroxylated metabolites and proved that the metabolites described here were alpha-hydroxylated. In contrast to the alpha-hydroxylated metabolites, the other metabolites identified, namely tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen and their deuterated counterparts were not depleted in the deuterated components, The use of on line HPLC-electrospray ionization MS combined with isotopic labelling is a powerful technique for probing the structures of metabolites, and, applied to tamoxifen, has provided further evidence that alpha-hydroxylation is an important pathway for the conversion of the drug into a DNA-reactive metabolite.