Quantification of Tamoxifen DNA Adducts Using On-Line Sample Preparation and HPLC-Electrospray Ionization Tandem Mass Spectrometry
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- 作者：Gonç ; alo Gamboa da Costa ; M. Matilde Marques ; Frederick A. Beland ; James P. Freeman ; Mona I. Churchwell ; and Daniel R. Doerge
- 刊名：Chemical Research in Toxicology
- 出版年：2003
- 出版时间：March 2003
- 年：2003
- 卷：16
- 期：3
- 页码：357 - 366
- 全文大小：151K
- 年卷期：v.16,no.3(March 2003)
- ISSN：1520-5010
文摘
The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agentfor the treatment of all stages of hormone-dependent breast cancer and more recently as achemopreventive agent in women with elevated risk of developing the disease. While clearlybeneficial for the treatment of breast cancer, tamoxifen has been reported to increase the riskof endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic inrats. Tamoxifen is clearly genotoxic in rat liver, as indicated by the formation of DNA adducts;the occurrence of tamoxifen DNA adducts in human endometrial tissue is more controversial.The detection and quantitation of tamoxifen DNA adducts have relied primarily upon 32P-postlabeling, with other techniques, such as immunoassays and accelerator mass spectrometry,being used to a much lesser extent. To expand the range of available analytical methodologiesfor quantifying tamoxifen DNA adducts, we have developed an assay using on-line samplepreparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-MS/MS). 
chars/alpha.gif" BORDER=0>-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ngand 1 mg chars/alpha.gif" BORDER=0>-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, themost highly modified DNA samples were analyzed by HPLC-UV, which indicated the presenceof two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorouslycharacterized as (E)-chars/alpha.gif" BORDER=0>-(deoxyguanosin-N2-yl)tamoxifen, and quantified on the basis of its molarextinction coefficient. A similar reaction was conducted with [N(CD3)2]-chars/alpha.gif" BORDER=0>-acetoxytamoxifen toprepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. Thelimit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/109 nucleotides, with an intra- and interassay precision of 3% relative standard deviation. The methodwas validated over the range of 8-1 520 000 adducts/108 nucleotides using 100 
g samples ofDNA modified in vitro. Analysis of liver DNA from female Sprague-Dawley rats treated bygavage with seven daily doses of 20 mg tamoxifen/kg body weight gave a value of 496 ± 16adducts/108 nucleotides for (E)-chars/alpha.gif" BORDER=0>-(deoxyguanosin-N2-yl)tamoxifen and 626 ± 18 adducts/108nucleotides for (E)-chars/alpha.gif" BORDER=0>-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen. These data indicate thatthe HPLC-ES-MS/MS methodology has sufficient sensitivity and precision to be useful in theanalysis of tamoxifen DNA adducts formed in vivo in experimental models and may be able todetect tamoxifen DNA adduct formation in human tissue samples.
chars/alpha.gif" BORDER=0>-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ngand 1 mg chars/alpha.gif" BORDER=0>-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, themost highly modified DNA samples were analyzed by HPLC-UV, which indicated the presenceof two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorouslycharacterized as (E)-chars/alpha.gif" BORDER=0>-(deoxyguanosin-N2-yl)tamoxifen, and quantified on the basis of its molarextinction coefficient. A similar reaction was conducted with [N(CD3)2]-chars/alpha.gif" BORDER=0>-acetoxytamoxifen toprepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. Thelimit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/109 nucleotides, with an intra- and interassay precision of 3% relative standard deviation. The methodwas validated over the range of 8-1 520 000 adducts/108 nucleotides using 100 g samples ofDNA modified in vitro. Analysis of liver DNA from female Sprague-Dawley rats treated bygavage with seven daily doses of 20 mg tamoxifen/kg body weight gave a value of 496 ± 16adducts/108 nucleotides for (E)-chars/alpha.gif" BORDER=0>-(deoxyguanosin-N2-yl)tamoxifen and 626 ± 18 adducts/108nucleotides for (E)-chars/alpha.gif" BORDER=0>-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen. These data indicate thatthe HPLC-ES-MS/MS methodology has sufficient sensitivity and precision to be useful in theanalysis of tamoxifen DNA adducts formed in vivo in experimental models and may be able todetect tamoxifen DNA adduct formation in human tissue samples.