| Autor: |
Costa, G. Gamboa da, Marques, M. M., Beland, F. A., Freeman, J. P., Churchwell, M. I., Doerge, D. R. |
| Zdroj: |
Chemical Research in Toxicology; March 2003, Vol. 16 Issue: 3 p357-366, 10p |
| Abstrakt: |
The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent for the treatment of all stages of hormone-dependent breast cancer and more recently as a chemopreventive agent in women with elevated risk of developing the disease. While clearly beneficial for the treatment of breast cancer, tamoxifen has been reported to increase the risk of endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic in rats. 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 methodologies for quantifying tamoxifen DNA adducts, we have developed an assay using on-line sample preparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-MS/MS). α-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ng and 1 mg α-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, the most highly modified DNA samples were analyzed by HPLC−UV, which indicated the presence of two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorously characterized as (E)-α-(deoxyguanosin-N2-yl)tamoxifen, and quantified on the basis of its molar extinction coefficient. A similar reaction was conducted with [N(CD3)2]-α-acetoxytamoxifen to prepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. The limit 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 method was validated over the range of 8−1 520 000 adducts/108 nucleotides using 100 μg samples of DNA modified in vitro. Analysis of liver DNA from female Sprague−Dawley rats treated by gavage with seven daily doses of 20 mg tamoxifen/kg body weight gave a value of 496 ± 16 adducts/108 nucleotides for (E)-α-(deoxyguanosin-N2-yl)tamoxifen and 626 ± 18 adducts/108 nucleotides for (E)-α-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen. These data indicate that the HPLC-ES-MS/MS methodology has sufficient sensitivity and precision to be useful in the analysis of tamoxifen DNA adducts formed in vivo in experimental models and may be able to detect tamoxifen DNA adduct formation in human tissue samples. |
| Databáze: |
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