Capillary electrophoretic separation and laser-induced fluorescence detection of the major DNA adducts of cisplatin and carboplatin.
Micellar electrokinetic capillary chromatographic separation of a dansylated mixture of normal nucleotides and platinated cross-link adducts of d(pGpG) and d(pApG) was optimized with baseline resolution. The introduction of a laser-induced fluorescence (LIF) detector overcame the lack of sensitivity characteristic of capillary electrophoresis (CE) due to the small injection volume and the short optical path length. CE/LIF was able to detect 1 adduct/10(4) normal nucleotides/micrograms DNA by fluorescence postlabeling assay. The enrichment of the adduct, prior to dansylation, enhanced the detection limit to 1 adduct/10(7) normal nucleotides/micrograms DNA. Calf thymus DNA was reacted in vitro with cisplatin and carboplatin with total input drug/nucleotide ratios of 0.05 and 0.5, respectively. A2780 human ovarian carcinoma cells were exposed in culture to 25 microM cisplatin for 2 h. The cells were incubated with drug-free medium for 3 h before harvesting. The identification of the cross-link adducts in modified DNA was confirmed by cochromatography with authentic markers. The same 1,2-intrastrand cross-link adducts were induced by both cisplatin and its second-generation drug carboplatin. This report has demonstrated, for the first time, the utility of CE/LIF as an analytical tool for assaying DNA damage [corrected].
Sharma
M
M
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Jain
R
R
Ionescu
E
E
Slocum
H K
HK
D000818:Animals; D016190:Carboplatin / Q000627:therapeutic use*; D002417:Cattle; D002945:Cisplatin / Q000627:therapeutic use*; D004247:DNA / Q000032:analysis / Q000187:drug effects*; D004586:Electrophoresis; D005260:Female; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D010051:Ovarian Neoplasms / Q000188:drug therapy*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D004247:DNA; D016190:Carboplatin; D002945:Cisplatin