Theoretical And Experimental Study On DNA Cross-Linking Induced By Two Nitrosoureas

Nitrosoureas act as an anticancer drug are widely used in cilinic, however,almost all the nitrosoureas may lead to second tumor in the process of treatment, this problem affects the application and further development of nitrosoureas seriously. The anticancer mechanism of nitrosoureas is very similar to the carcinogenic mechanism, both of them can alkylate DNA bases and form DNA cross-linking further. Therefore, it is significant to elucidate the similarities and differences of carcinogentic and anticancer mechanism of nitrosoureas. This thesis mainly investigated on DNA cross-linking induced by nitrosoureas by biochemistry and molecular biology methods, furthermore, studied theβ-position activity of nitrosoureas, three decomposition mechanisms of nitrosoureas and the stabilization of their guanine alkylation adducts by quantum chemistry calculation methods.The research on nitrosoureas by biochemistry and molecular biology methods mainly took two kinds of nitrosourea anticancer drugs semustine (Me-CCNU) and nimustine (ACNU) as research objects, Agarose gel electrophoresis method and fluorescence method were used to investigate DNA cross-linking induced by Me-CCNU and ACNU. Agarose gel electrophoresis method was mainly used to study the relationship between DNA cross-linking rate and reaction time , and the relationship between the degree of cross-linking and drug concentration; Fluorescence method studied the cross-linking of calf thymus DNA induced by Me-CCNU and ACNU, this method was on the basis of the characteristic of fluorescent dyes Hoechst 33258 which can excite fluorescence by binding double-stranded DNA but not single- stranded DNA. We established fluorescence reaction conditions after the hydrolysis of the two drugs in different solutions was studied. Then we worked on the change relationship between DNA cross-linking rate and reaction time at different drug concentrations and DNA concentrations.We found that there was an"induction period"in the initial stage of the reaction of drug and DNA, which the formation of cross-linking should undergo, and obtained the change trend of cross-linking in eight hours after the reaction of drug and DNA.In quantum chemistry calculation of nitrosoureas, SR870BH2 server and Gaussian A03 and GaussView software were used to calculate the reaction activity onβ-position, three decomposition mechanisms of chloroethylnitrosoureas and their six guanine alkylation adducts. All the reactants, fortransition states and intermediates were calculated by full geometric structure optimizations at RHF/6-311G(d,p) basis set using ab initio method, fortransition states were comfirmed by STQN method. Vibrational frequencies analysis was carried out on the molecular structures after optimization at the same basis set, and IRC was carried out to confirm the relationship between the related structures. By extracting and evaluating molecular structure parameters and energy parameters, we made certain that the processes of the anchimeric assistance ofβ-position of nitrosoureas played an important role in DNA cross-linking, the active energies of three decomposition mechanisms of nitrosoureas and the stability of guanine alkylation adducts. Theoretical calculation provided theoretical evidence for elucidating action manners of drug and DNA further.