Comparative Investigations of Deoxyribonucleic Acid Interstrand Crosslinks Induced by Semustine Using Fluorescence and High Performance Liquid Chromatography-Mass Spectrometry

Semustine(Me-CCNU) is an important alkylating agent used in the clinical treatment of cancer.The anticancer mechanism of Me-CCNU is associated with the formation of DNA interstrand crosslinks(ICLs).In this study,the ICLs induced by Me-CCNU were investigated using fluorescent assay and high performance liquid chromatography-electronspray ionization-tandem mass spectrometry(HPLC-ESI-MS/MS).The results showed that the maximum of the crosslinking factor(CF) obtained from fluorescent assay was 8.81%,11.17%,17.61% and 23.39%,respectively,with the concentration of Me-CCNU of 2,4,8 and 16 mmol/L;the maximum of the CF obtained from HPLC-ESI-MS/MS was 138,256,378 and 433 crosslinked dG-dCs per 107 base pairs,respectively,with the concentration of Me-CCNU of 2,4,8 and 16 mmol/L.By comparing these two methods for the determination of DNA interstrand crosslinks,the CF obtained from fluorescent assay characterized the crosslinks of the DNA double strands;on the other hand,the CF obtained from HPLC-ESI-MS/MS characterized the content of the crosslinked dG-dC.The latter gave a more essential quantification of the ICLs than the former.An "induction phase" was found by both of the two methods,in which the CF increased slowly during the beginning period of the reaction.This result provided evidences for the clarifing of the mechanism of the formation of ICLs.