N-Demethylation accompanies α-hydroxylation in the metabolic activation of tamoxifen in rat liver cells

Abstract

Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via α-hydroxylation leading to modification of the N2-position of guanine in DNA and to a lesser extent the N6-position of adenine. Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifen-N2-guanine adduct and the other the equivalent adduct in which the tamoxifen moiety has lost a methyl group. Treatment of rats or rat hepatocytes with N-desmethyltamoxifen gave rise to the second adduct, whereas treatment with tamoxifen or α-hydroxytamoxifen gave rise to both. Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen, α-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the a position was confirmed in experiments in which [α-D2-ethyl]tamoxifen, but not [β-D3-ethyl]tamoxifen, produced reduced levels of DNA adducts. Tamoxifen N-oxide and α-hydroxytamoxifen N-oxide also gave rise to DNA adducts in rat liver cells, but the adduct patterns were very similar to those formed by tamoxifen and α-hydroxytamoxifen, indicating that the N-oxygen is lost prior to DNA binding. These and earlier results demonstrate that in rat liver cells in vivo and in vitro, Phase I metabolic activation of tamoxifen involves both α-hydroxylation and N-demethylation, which is followed by Phase II activation at the α-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by 32P-postlabelling is achieved with > 90% labelling efficiency.