Identification and quantification of tamoxifen-DNA adducts in the liver ofrats and mice

A new HPLC gradient system was developed for P-32-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treate d with tamoxifen. Four stereoisomers of alpha-(N-2-deoxyguanosinyl)tamoxife n (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-oxid e (dG(3 'P)-N-2-TAM N-oxide) were prepared by reacting either alpha -acetox ytamoxifen, alpha -acetoxy-N-desmethyltamoxifen or alpha -acetoxytamoxifen N-oxide with 2 ' -deoxyguanosine 3 ' -monophosphate, and used as standard m arkers for P-32-postlabeling/HPLC analysis. Our HPLC gradient system can se parate the above 12 nucleotide isomers as nine peaks; six peaks representin g 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-D NA adducts were detected in rats and mice, respectively; among them 13 addu cts 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 thir d abundant adduct that accounted for 6.4% of the total adducts in mice, whi le this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isom ers (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 e xcept 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.