Identification and Quantification of Tamoxifen-DNA Adducts in the Liver of Rats and Mice

Abstract

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.

Cytotoxicity of tamoxifen in normal and tumoral cell lines and its ability to induce cellular transformation in vitro

“…These findings agree with previous studies indicating that DMBA and TAM can lead to DNA adduct formation. This injury is responsible for the activation of metabolic pathways that contribute for DNA fragmentation and the induction of cell death by apoptosis or necrosis, depending on the dose and the duration of exposure to the drug (Bursch et al, 1997;Umemoto et al, 2001;Barbisan et al, 1999).…”

Antiestrogens and the Formation of DNA Damage in Rats: A Comparison

“…formed through O-sulfonation of α-hydroxylated TAM metabolites (8); α-(N 2 -deoxyguanosinyl)tamoxifen (dG-N 2 -TAM) and α-(N 2 -deoxyguanosinyl)-Ndesmethyltamoxifen (dG-N 2 -N-desTAM) are detected as major hepatic DNA adducts using mass-spectroscopic and 32 P-postlabeling/HPLC analyses (12,15). There is a controversy regarding the detection of TAM-DNA adducts in the human tissues (16,17).…”

Inefficient Repair of Tamoxifen-Dna Adducts in Rats and Mice

“…When mice were treated with TAM (20 mg/ kg/day for 7 days) at the dose equivalent to that used for rat study, only a trace of TAM-DNA adduct (Ͻ1 adduct/10 8 nucleotides) was observed in the liver (data not shown). Therefore, mice were treated with a 6-fold higher dose (120 mg/kg/day) of TAM, as reported previously (Umemoto et al, 2001). A high level of TAM-DNA adducts was observed in the liver of all Xpc knockout mice (Fig.…”

“…1) (Osborne et al, 1996;Dasaradhi and Shibutani, 1997). dG-N 2 -TAM and ␣-(N 2 -deoxyguanosinyl)-N-desmethyltamoxifen (dG-N 2 -N-desTAM) were major adducts in the liver of rats and mice treated with TAM (Rajaniemi et al, 1999;Umemoto et al, 2001) and in several tissues including reproductive organs of monkeys treated with TAM Shibutani et al, 2003). ␣-(N 2 -Deoxyguanosinyl)tamoxifen N-oxide (dG-N 2 -TAM N-oxide) was detected as a minor adduct in mouse liver.…”