The deuterium isotope effect for the α-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D₅-ethyl]tamoxifen

Abstract

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.

Tamoxifen Induction of Aryl Sulfotransferase and Hydroxysteroid Sulfotransferase in Male and Female Rat Liver and Intestine

“…TAM-induced hepatogenotoxicity in rats and mice but hepatocarcinogenicity only in rats has been shown (Carthew et al, 1995;Martin et al, 1998;Firozi et al, 2000). Metabolic activation of TAM is believed to be a prerequisite to form an electrophile leading to the formation of DNA adduct, resulting in carcinogenicity (Jarman et al, 1995;Poon et al, 1995;Moorthy et al, 1996). Some reports reveal that, apart from its anticancer and chemoprotective effect (Heel et al, 1978), TAM may be involved in influencing some of the drugmetabolizing enzymes (Hellriegel et al, 1996;Kasahara et al, 2002).…”

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

“…In humans (49-53) and rats (46,47,54), the major metabolic pathway for tamoxifen is through N-demethylation to give N-desmethyltamoxifen (Figure 1, pathway C). For example, when assessed in breast cancer patients after daily dosing for up to 2.5 years, N-desmethyltamoxifen was found in plasma at approximately twice the concentration of tamoxifen (51).…”

Toremifene metabolism in rat, mouse and human liver microsomes: Identification of α‐hydroxytoremifene by LC‐MS

“…There have been many structures proposed as the reactive tamoxifen metabolites responsible for gentoxicity (Smith et al, 2000). Among these, a-hydroxytamoxifen has attracted much interest in this respect and has been widely postulated as the most important reactive species derived from the metabolic activation of tamoxifen (Potter et al, 1994;Jarman et al, 1995;Phillips et al, 1995). The arguments put forward in support of this hypothesis include the substantial increased in DNAadduct formation when synthetic acetoxy-or sulphatederivative of a-hydroxytamoxifen was incubated with DNA (Dasaradhi and Shibutani, 1997;Osborne et al, 1997;Davis et al, 1998;Shibutani et al, 1998) and decreased when D5-ethyl tamoxifen was activated (Jarman et al, 1995).…”