Analysis of tamoxifen-DNA adducts by high-performance liquid chromatography using postcolumn online photochemical activation.
Tamoxifen, a widely used nonsteroidal antiestrogen in the treatment of breast cancer, forms several metabolites. 4-Hydroxytamoxifen (4-OHTam), a metabolite found in the bloodstream, has much higher affinity for the estrogen receptor than tamoxifen itself. Oxidative activation of 4-OHTam induces DNA damage. DNA isolated from HL-60 cells exposed to 10 microM 4-OHTam in the presence of 1 microM hydrogen peroxide was digested enzymatically to release both normal and modified nucleosides. The modified nucleosides were enriched by butanol extraction. Using UV detection, HPLC analysis of the butanol extract from 200 microg DNA digest detected approximately 4 4-OHTam-dG adducts per 10(7) nucleotides (n = 3). Online postcolumn UV irradiation in HPLC and fluorescence detection improved the detection sensitivity by 3 x 10(2) times. Using 4-OHTam as an example, this report demonstrated for the first time the power of the technique to assay tamoxifen-DNA adducts directly in the DNA digest without relying on postlabeling.
Sharma
M
M
Department of Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. minoti.sharma@roswellpark.org
D000818:Animals; D000440:Butanols / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D003619:Dansyl Compounds / Q000378:metabolism; D005453:Fluorescence; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008297:Male; D010616:Phenanthrenes / Q000378:metabolism; D010777:Photochemistry; D015203:Reproducibility of Results; D012474:Salmon; D012680:Sensitivity and Specificity; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013737:Testis; D014466:Ultraviolet Rays
D000440:Butanols; D018736:DNA Adducts; D003619:Dansyl Compounds; D010616:Phenanthrenes; D013629:Tamoxifen; C016601:afimoxifene; D006861:Hydrogen Peroxide; C029038:dansyl chloride