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

Abstract

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-(N:(2)-deoxyguano- sinyl)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 (32)P-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.

OXIDATION OF TAMOXIFEN BY HUMAN FLAVIN-CONTAINING MONOOXYGENASE (FMO) 1 AND FMO3 TO TAMOXIFEN-N-OXIDE AND ITS NOVEL REDUCTION BACK TO TAMOXIFEN BY HUMAN CYTOCHROMES P450 AND HEMOGLOBIN

“…Our current study demonstrates the participation of human P450 enzymes, among these CYP3A4 in the reduction of the N-oxide of TAM. These findings and the observations that TNO forms far less DNA and protein adducts than does TAM (Dehal and Kupfer, 1999;Umemoto et al, 2000) suggest that TNO could be an attractive candidate for a prodrug of TAM. Other tamoxifen analogs undergoing clinical trials for antibreast cancer treatment (e.g., iodoxifene, toremifene, and droloxifene) have also been shown to form the corresponding N-oxides (McCague et al, 1990;John et al, 2002;Jones and Lim, 2002).…”

The gene expression of hepatic proteins responsible for DNA repair and cell proliferation in tamoxifen‐induced hepatocarcinogenesis

“…[14][15][16][17][18][19][20][21] Although N-desmethyltamoxifen, tamoxifen N-oxide, and 4-hydroxytamoxifen are major metabolites of tamoxifen, DNA adducts of these metabolites are hardly detected in the liver, 19,22) suggesting that these metabolites might be considered primarily as detoxification forms.…”

“…[8][9][10][11][12][13] α-(N 2 -Deoxyguanosinyl)tamoxifen is detected as a major DNA adduct of tamoxifen in the rat liver, and the adduct has been shown to be formed through metabolic activations, α-hydroxylation and O-sulfation, of tamoxifen. [14][15][16][17][18][19][20][21] Although N-desmethyltamoxifen, tamoxifen N-oxide, and 4-hydroxytamoxifen are major metabolites of tamoxifen, DNA adducts of these metabolites are hardly detected in the liver, 19,22) suggesting that these metabolites might be considered primarily as detoxification forms.…”

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

“…Preparation of Tamoxifen-dG3′P Standards. dG3′P-N 2 -TAM, R-(N 2 -deoxyguanosinyl)-N-desmethyltamoxifen 3′-monophosphate (dG3′P-N 2 -N-desmethyl-TAM) and dG3′P-N 2 -TAM N-oxide were prepared by reacting dG3′P with R-acetoxytamoxifen, R-acetoxy-N-desmethyltamoxifen or R-acetoxytamoxifen N-oxide, as reported previously (9,(14)(15)(16) (Figure 1). The trans and cis stereoisomers of each nucleotide were isolated by HPLC.…”

“…In our previous study, two types of tamoxifen-DNA adducts, R-(N 2 -deoxyguanosinyl)tamoxifen 3′-monophosphate (dG 3′P -N 2 -TAM) 1 and R-(N 2 -deoxyguanosinyl)tamoxifen N-oxide 3′-monophosphate (dG 3′P -N 2 -TAM N-oxide) were prepared as the authentic standards. It was demonstrated that in mouse liver the epimers of trans-dG 3′P -N 2 -TAM and trans-dG 3′P -N 2 -TAM N-oxide were present as the first and the third major adducts, respectively (9). However, the second major adduct remains to be identified.…”