Determination of DNA Damage in F344 Rats Induced by Geometric Isomers of Tamoxifen and Analogues

Abstract

To investigate the activation mechanisms involved in tamoxifen carcinogenicity, analogues of tamoxifen isomers modified at the ethyl group were synthesized and assessed for their ability to induce hepatic DNA damage following their administration to female F344 rats. The cis isomer was prepared by acid-catalyzed isomerization of tamoxifen and isolated by preparative HPLC. The active metabolite alpha-hydroxytamoxifen and geometric isomers of bromotamoxifen and C-desmethylenetamoxifen, analogues in which the ethyl group has been replaced by a bromine atom and methyl group, respectively, were synthesized according to published procedures. The levels of hepatic DNA adducts induced were determined by 32P-postlabeling. Bromotamoxifen and tamoxifen 1,2-epoxide caused no detectable DNA damage relative to controls. Trans isomers of tamoxifen, C-desmethylenetamoxifen, and alpha-hydroxytamoxifen all produced DNA adducts at a 5-90-fold higher level than the corresponding cis isomers. In contrast, both the cis and trans isomers of alpha-hydroxytamoxifen showed similar reactivity toward calf thymus DNA in vitro. Molecular models of alpha-hydroxytamoxifen isomers suggest this difference in DNA adduct-forming ability is due to steric hindrance of the enzymes involved in the activation of this metabolite. There were high adduct levels in the liver, but no uterine DNA adducts were detected in rats treated with alpha-hydroxytamoxifen. This suggests that in contrast to the liver, alpha-hydroxytamoxifen is not further activated in rat uterus. This may help to explain the absence of uterine tumors in rats following long-term tamoxifen treatment.

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

“…As reported by others (26,67), an adduct resulting from α-hydroxytamoxifen is clearly present. This was accompanied by a second major adduct, the identity of which is currently unknown.…”

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

“…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). It has also been shown that the number of DNA adducts in the livers of rats treated with α-hydroxytamoxifen is Ͼ50 times that found in rats treated with the corresponding cis isomer (36).…”

“…α-Hydroxytamoxifen is a metabolite of tamoxifen in human plasma (22), in rat (12), mouse (12) and human (12,22) hepatocytes, in human liver homogenate (22) and in rat liver microsomes (36). Only the Z-isomer of tamoxifen undergoes α-hydroxylation by rat liver microsomes (36).…”

“…Only the Z-isomer of tamoxifen undergoes α-hydroxylation by rat liver microsomes (36). α-Hydroxytamoxifen N-oxide is a metabolite of the N-oxide in rat liver microsomes (19,21) and N-desmethyl-α-hydroxytamoxifen has been identified in human plasma (22), as an O-glucuronide in human urine (28) and in incubations of rat liver microsomes (21), but their contributions to adduct formation are unknown.…”

Alterations of the K-ras and p53 genes in Tamoxifen-associated endometrial carcinoma

“…Shibutani et al (9) and Schild et al (10) observed similar TAM-DNA adducts in the brain, ovary, and uterus, in addition to the liver, in monkeys treated with TAM. Further, Shibutani et al have reported finding TAM-DNA adducts in human endometrial samples, using a combination of 32 P-postlabeling and HPLC analysis (11), and they have suggested that the organ-specific formation of TAM-DNA adducts may be species-specific, referring to other reports (9,12). This may suggest that the tissue-specific repair efficiency of TAM-DNA adducts may be a key factor in TAM-induced carcinogenicity.…”