A proposed mechanism for the metabolic activation of tamoxifen to electrophilic species that form DNA adducts leading to liver cancer involves alpha-hydroxylation of the ethyl group in the critical first step. This mechanism predicts that tamoxifen deuterated at the alpha-position would be less genotoxic than the non-deuterated compound owing to an isotope effect that would reduce the rate of oxidative metabolism at this position. This hypothesis has now been tested with experiments conducted in rats in vivo and in human cells in vitro. It was found that the deuterated compound [D5-ethyl]-tamoxifen is significantly less genotoxic than tamoxifen. Administration of 0.06 and 0.12 mmol/kg [D5-ethyl]-tamoxifen to female Fischer rats by gavage resulted in a 2.5- and 1.7-fold reduction respectively in the levels of hepatic DNA adducts present 24 h after treatment, compared with the non-deuterated compound. Treatment of MCL-5 cells with [D5-ethyl]-tamoxifen resulted in a 2- to 3-fold decrease, compared with tamoxifen, in the number of micronuclei induced in cells arrested in cytokinesis by cytochalasin-B. Further evidence is provided by UV irradiation of the major hepatic DNA adducts isolated from tamoxifen-treated rats, which caused a large shift in the chromatographic mobility of the adducts, consistent with the presence of a 1,2-olefinic linkage in the tamoxifen residue of the adduct leading to cyclization to a phenanthrenylic compound, and consistent with this adduct having arisen from reaction with DNA at the alpha-position of tamoxifen. Taken together, these data strongly suggest that tamoxifen is metabolized to a liver carcinogen by alpha-hydroxylation of its ethyl group.

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 [D5-ethyl]-analogue. The ratio of ca 3:1 between unlabelled and D4-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 D5-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-D2-ethyl]- and [beta-D3-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.

Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via alpha-hydroxylation leading to modification of the N(2)-position of guanine in DNA and to a lesser extent the N(6)-position of adenine. Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifen-N(2)-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 alpha-hydroxytamoxifen gave rise to both. Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen, alpha-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the alpha position was confirmed in experiments in which [alpha-D(2)-ethyl]tamoxifen, but not [beta-D(3)-ethyl]tamoxifen, produced reduced levels of DNA adducts. Tamoxifen N-oxide and alpha-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 alpha-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 alpha-hydroxylation and N-demethylation, which is followed by Phase II activation at the alpha-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by (32)P-postlabelling is achieved with >90% labelling efficiency.

Tamoxifen [(E)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1, 2-diphenylbut-1-ene], a nonsteroidal antiestrogen, induces liver tumors in rats by a genotoxic mechanism. The mechanism of DNA adduct formation is believed to proceed via the formation of a reactive carbocation at the alpha-position from the alpha-hydroxylated metabolite. Molecular mechanics calculations [Kuramochi, H. (1996) J. Med. Chem. 39, 2877-2886] have predicted that 4-substitution will affect the stability of the carbocation and thus will alter its reactivity toward DNA. We have synthesized the putative alpha-hydroxylated metabolites of 4-hydroxytamoxifen [(E)-1-(4-(2-(N, N-dimethylamino)ethoxy)phenyl)-1-(4-hydroxyphenyl)-3-hydroxy-2-phenyl but-1-ene] and idoxifene [(Z)-1-(4-iodophenyl)-3-hydroxy-2-phenyl-1-(4-(2-(N-pyrrolidino) ethoxy)phenyl)but-1-ene] and compared their reactivities with DNA with that of alpha-hydroxytamoxifen [(E)-1-(4-(2-(N, N-dimethylamino)ethoxy)phenyl)-3-hydroxy-1,2-diphenylbut-1-ene]. As predicted, the bis-hydroxylated compound reacted with DNA in aqueous solution at pH 5 to give 12-fold greater levels of adducts than alpha-hydroxytamoxifen, whereas alpha-hydroxyidoxifene gave one-half the number of adducts. The results demonstrate that idoxifene presents a significantly lower genotoxic hazard than tamoxifen for the treatment and prophylaxis of breast cancer.

Tamoxifen which incorporates a fully deuterated ethyl group, [DS-ethyl]-tamoxifen, has been synthesised in order to probe the mechanism of tamoxifen induced hepatic DNA adduct formation. The pentadeuteroethyl group was introduced into the tamoxifen structure by treatment of the ketone precursor I-[4-(2-chloroethoxy)phenyl]-2-phenylethanone, as its sodium enolate, with [Ds]-iodoethane. Abbreviations: tamoxifen = (Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-l-butene; [D5-ethyll-tamoxifen = [3,3,4,4,4-*H5]-(2)-1-[4-[2-(dimethylamino)ethoxy]pheny~]-12-diphenyl-1butene.