Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice.
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.
Umemoto
A
A
Second Department of Surgery, School of Medicine, University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan. umemoto@clin.med.tokushima-u.ac.jp
Komaki
K
K
Monden
Y
Y
Suwa
M
M
Kanno
Y
Y
Kitagawa
M
M
Suzuki
M
M
Lin
C X
CX
Ueyama
Y
Y
Momen
M A
MA
Ravindernath
A
A
Shibutani
S
S
D000818:Animals; D002273:Carcinogens / Q000032:analysis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018736:DNA Adducts / Q000032:analysis*; D006900:Hydroxylation; D008099:Liver / Q000473:pathology; D051379:Mice; D010761:Phosphorus Radioisotopes; D051381:Rats; D011916:Rats, Inbred F344; D012680:Sensitivity and Specificity; D013629:Tamoxifen / Q000032:analysis / Q000737:chemistry*
D002273:Carcinogens; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D013629:Tamoxifen