Tamoxifen: evidence by 32P–postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4–hydroxytamoxifen as a proximate metabolite
Exposure to pentachlorophenol (PCP) strongly intensifies the formation of mouse hepatic DNA adducts elicited by oral administration of tamoxifen (TAM), as previously shown by 32P-postlabeling. To explain this effect, PCP was proposed to interfere with the detoxication by sulfate conjugation of an as yet unidentified hydroxylated proximate TAM metabolite. A comparison of the present and earlier results shows that the hepatic TAM adduct pattern in female ICR mice depended on the route of administration of TAM (120 μmol/kg), with oral administration primarily eliciting formation of more polar adducts (termed group I adducts), while after i.p. administration less polar adducts (group II) predominated over group I adducts by a factor of 17.5. All these adducts were also formed in female Sprague–Dawley rats after i.p. dosing with TAM, but total adduct levels were 3.5- to 5-fold higher than in mice. After four daily i.p. treatments, TAM adducts accumulated in mouse liver DNA in a non-linear fashion. Adduct levels were 30–50 times lower in mouse kidney and lung than in liver. The phenolic metabolite 4-hydroxy TAM (120 μimol/kg) exclusively led to formation of polar (group I) hepatic adducts, and this process was stimulated 8-fold by coadministration of PCP (75 μimol/kg). Co-administration of PCP with the parent compound led to an 11-fold enhancement of group I adduct formation; simultaneously, levels of group II adducts were suppressed 6-fold. Another inhibitor of sulfate conjugation, 2,6-dichloro-4-nitrophenol, unlike PCP, had no effect on group I adducts, but it reduced group II adduct formation 2.2-fold. The PCP metabolite 2,3,5,6-tetrachlorohydroquinone (75 μimol/kg) did not significantly affect any major TAM adduct, suggesting that PCP itself was the active compound. Similar to group II TAM adducts, the formation of hepatic safrole–DNA adducts was inhibited in female ICR mice by both sulfotransferase inhibitors, consistent with the proposal that metabolic α-hydroxylation of the ethyl group of TAM followed by sulfate conjugation represented a mechanism of TAM activation. On the other hand, the strong intensification of group I adducts by PCP and the lack of this effect by 2,6-dichloro-4-nitrophenol suggested that inhibition of sulfate conjugation may not have been the primary mechanism underlying the intensification of group I adducts formed from TAM or 4-hydroxy TAM. The results presented herein demonstrate conclusively that TAM was activated to DNA-reactive compounds along two distinct pathways which contrasted in their responses to metabolic inhibitors.