Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice.
Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice.
复制标题
大鼠和小鼠肝脏中他莫昔芬-DNA 加合物的鉴定和定量。
DOI：
--
复制
发表时间：
2001
影响因子：
4.1
通讯作者：
S. Shibutani
中科院分区：
文献类型：
--
作者：
A. Umemoto;K. Komaki;Y. Monden;M. Suwa;Y. Kanno;M. Kitagawa;M. Suzuki;C. X. Lin;Y. Ueyama;M. A. Momen;Ravinder Nath ANISETTI;S. Shibutani
建立了一种新的HPLC梯度系统，用于（32）P-后标记分析，以鉴定和定量用他莫昔芬处理的大鼠和小鼠的肝他莫昔芬-DNA加合物。α-（N（2）-脱氧鸟苷基）他莫昔芬（dG（3 ′）（P）-N（2）-TAM）、α-（N（2）-脱氧鸟苷基）-N-去甲基他莫昔芬（dG（3 ′）（P）-N（2）-N-去甲基-TAM）和α-（N（2）-脱氧鸟苷基）他莫昔芬N-氧化物（dG（3 ′）（P）-N（2）-TAM N-氧化物）的四种立体异构体通过使α-乙酰氧基他莫昔芬，α-乙酰氧基-N-去甲基他莫昔芬或α-乙酰氧基他莫昔芬N-氧化物与2 ′-脱氧鸟苷3 ′-单磷酸，并用作（32）P-后标记/HPLC分析的标准标记物。我们的HPLC梯度系统可以将上述12种核苷酸异构体分离为9个峰; 6个峰代表dG（3 '）（P）-N（2）-TAM、dG（3'）（P）-N（2）-N-去甲基-TAM和dG（3 '）（P）-N（2）-TAM N-氧化物的各两种反式差向异构体（fr-1和fr-2），3个峰代表核苷酸的两种顺式差向异构体（fr-3和fr-4）的混合物。雌性F344大鼠和DBA/2小鼠分别以45 mg/kg/天和120 mg/kg/天剂量灌胃给予他莫昔芬7天。在大鼠和小鼠中分别检测到15和17种他莫昔芬-DNA加合物，其中13种在大鼠和小鼠中均观察到。trans-dG-N（2）-TAM（fr-2）和trans-dG（3 '）（P）-N（2）-N-去甲基-TAM（fr-2）是两种主要的加合物。除这两种加合物外，反式-dG-N（2）-TAM N-氧化物（fr-2）是第三丰富的加合物，在小鼠中占总加合物的6.4%，而在大鼠中仅占0.3%。除大鼠中dG-N（2）-TAM N-氧化物的顺式形式外，在两只动物中也检测到dG（3 '）（P）-N（2）-TAM、dG（3'）（P）-N（2）-N-去甲基-TAM和dG（3 '）（P）-N（2）-TAM N-氧化物的反式异构体（fr-1）和顺式异构体（fr-3和fr-4）作为次要加合物。尽管大鼠的给药剂量比小鼠少2.7倍，但大鼠的总加合物水平（216个加合物/10（8）个核苷酸）比小鼠（56.2个加合物/10（8）个核苷酸）高3.8倍。因此，这三种类型的他莫昔芬加合物分别占大鼠和小鼠总DNA加合物的95.0%和92.5%。他莫昔芬加合物的形成主要由他莫昔芬的α-羟基化引起。
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.