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

Abstract

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 mumol/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 mumol/kg) exclusively led to formation of polar (group I) hepatic adducts, and this process was stimulated 8-fold by co-administration of PCP (75 mumol/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 mumol/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 alpha-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.

α-Hydroxytamoxifen, a genotoxic metabolite of tamoxifen in the rat: identification and quantification in vivo and in vitro

“…α-Hydroxylation, epoxidation and generation of a quinone methide have all been presented as likely candidates (22,(42)(43)(44) but α-hydroxylation is considered to be the major pathway in rat liver (34). The key step in the mechanism of DNA adduct formation from α-hydroxytamoxifen is thought to involve the reversible O-sulphonation of the hydroxyl moiety generating a highly reactive carbocation (30,45). In support of this, experiments using 32 P-post-labelling of DNA have shown that α-sulphate cis-tamoxifen is highly reactive to DNA, yielding up to 1600 times the number of DNA adducts obtained from α-hydroxytamoxifen (31).…”

“…Tamoxifen is metabolized variously via primary N-demethylation, 4-hydroxylation, α-hydroxylation and N-oxidation ( Figure 1) by hepatic microsomes and cell lines (19)(20)(21)(22)(23), and additionally via secondary O-glucuronidation in hepatocytes (24) and in vivo (25)(26)(27)(28)(29), Ndemethylation being the principal biotransformation in humans and human liver microsomes (20,23,28). Both aromatic hydroxylation and α-hydroxylation have been consistently implicated in the formation of reactive tamoxifen metabolites in vitro and in experimental animals, and they appear to represent the initial steps of distinct bioactivation pathways in vivo (16,30). Although α-hydroxytamoxifen reacts directly, if slowly, with isolated DNA (31), both it and the chemically unreactive 4-hydroxytamoxifen are regarded as precursors of DNA-binding derivatives that have yet to be identified as metabolites, namely α-esters such as the highly reactive α-sulphate tamoxifen (31,32) and 4-hydroxytamoxifen quinone methide (33), respectively.…”

“…Although α-hydroxytamoxifen reacts directly, if slowly, with isolated DNA (31), both it and the chemically unreactive 4-hydroxytamoxifen are regarded as precursors of DNA-binding derivatives that have yet to be identified as metabolites, namely α-esters such as the highly reactive α-sulphate tamoxifen (31,32) and 4-hydroxytamoxifen quinone methide (33), respectively. The balance of the published evidence suggests that α-hydroxytamoxifen is the precursor of the principal DNA adducts formed in rat liver (30,31,34). This would explain why toremifene (β-chlorotamoxifen) does not give rise to such DNA adducts (11,35).…”

Evaluation of tamoxifen and alpha-hydroxytamoxifen 32P-post-labelled DNA adducts by the development of a novel automated on-line solid-phase extraction HPLC method

“…DNA adducts are detected in mouse liver following oral (8)(9)(10) and i.p. (11) dosing, but are present at lower levels than similarly treated rats (10). With long-term dietary exposure (~40 mg tamoxifen/kg per day) adducts are present in mouse liver at 3 months (~67 adducts/ 10 8 nucleotides), but decrease with time and are not detectable in tamoxifen-treated mice by 2 years (10).…”

Implications for risk assessment of suggested nongenotoxic mechanisms of chemical carcinogenesis.

“…More important, the fact that several nonmutagenic carcinogens have been found to be carcinogenic in experimental animals as well as in humans (e.g., benzene, 2,3,7,8-tetrachlorodibenzo-p-dioxin [ (8). An extensive literature base shows that tamoxifen forms DNA adducts in the livers of rats, mice, and hamsters (9,10); it is activated to form DNA adducts by rat or human liver microsomes (11); it is dastogenic in human lymphoblastoid cells (12); and liver tumors in rats treated with tamoxifen have a high frequency of p53 mutations (13). These genotoxic activities demonstrate that tamoxifen does not act simply as tumor promoter.…”