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Capillary Electrophoretic Separation and Laser-Induced Fluorescence Detection of the Major DNA Adducts of Cisplatin and Carboplatin

✍ Scribed by M. Sharma; R. Jain; E. Ionescu; H.K. Slocum


Publisher
Elsevier Science
Year
1995
Tongue
English
Weight
478 KB
Volume
228
Category
Article
ISSN
0003-2697

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✦ Synopsis


Micellar electrokinetic capillary chromatographic separation of a dansylated mixture of normal nucleotides and platinated cross-link adducts of (\mathrm{d}(\mathrm{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/mg DNA by fluorescence postlabeling assay. The enrichment of the adduct, prior to dansylation, enhanced the detection limit to 1 adduct (/ 10^{7}) normal nucleotides (/ \mathrm{mg}) 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 (\mathrm{mM}) cisplatin for (2 \mathrm{~h}). The cells were incubated with drug-free medium for (3 \mathrm{~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. 1995 Academic Press, Inc.


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