Identification of hepatic tamoxifen-DNA adducts in mice:: α-(N2-deoxyguanosinyl)tamoxifen and α-(N2-deoxyguanosinyl)tamoxifen N-oxide

Tamoxifen-DNA adducts detected in the liver of mice treated with tamoxifen have not yet been identified. In the present study a new type of tamoxifen-DNA adduct, four stereoisomers of alpha-(N-2-deoxyguanosinyl)tamoxifen N-oxide 3'-monophosphate (dG(3'P)-N-2-TAM N-oxide) were prepared as standard DNA adducts by reacting 2'-deoxyguanosine 3'-monophosphate with trans-alpha-acetoxytamoxifen N-oxide in addition to four stereoisomers of alpha-(N2-deoxyguanosinyl)tamoxifen 3'-monophosphate (dG(3'P)-N-2-TAM) that was reported previously, Liquid chromatography-electrospray ionization-mass spectrometry of the reaction products gave the most abundant ion at m/z 731 ([M - H](-)), which corresponded to dG(3'P)-N-2-TAM N-oxide, The modified products digested by alkaline phosphatase corresponded to the isomers of dG-N-2-TAM N-oxide whose structures were identified previously by mass spectrometry and nuclear magnetic resonance. Using these standard markers, we analyzed the hepatic DNA adducts of female DBA/2 mice treated with tamoxifen at a dosage of 120 mg/ kg/day for 7 days by P-32-post-labeling coupled with an HPLC/radioactive detector. Mixtures of eight isomers of dG(3'P)-N-2-TAM and dG(3'P)-N-2-TAM N-oxide were separated into six peaks, since each of the cis epimers were not separated under the present HPLC conditions. Nine adducts were detected in all liver samples of mice. An epimer of trans-dG(3'P)-N-2-TAM was detected as the principal DNA adduct at a level of 29.0 adducts/10(8) nucleotides, which accounted for 53.3% of the total tamoxifen-DNA adducts, Lesser amounts of cis-dG(3'P)-N-2-TAM (2.8%) were also observed. An epimer of the trans-dG(3'P)-N-2-TAM N-oxide (3.9 adducts/10(8) nucleotides) was detected as the third biggest adduct (7.2% of the total). The Cis-dG(3'P)-N-2-TAM N-oxide (0.4 adducts/10(8) nucleotides) accounted for 0.7% of the total. Thus, dG(3'P)-N-2-TAM and dG(3'P)-N-2-TAM N-oxide were identified in tamoxifen-treated mouse liver.