A simple and sensitive method for in vitro quantitation of abasic sites inDNA

A novel method for the quantitation of abasic sites (AP sites) in DNA is de scribed. As abasic sites can be generated by controlled thermal treatment o f base-modified DNA, this method can be used for estimation of the extent o f DNA damage resulting from exposure to genotoxic agents. The method involv es use of probe molecules 1 and 2 that contain a fluorescent label linked t o an aminooxy group which reacts specifically with the aldehydic function o f the ring-opened form of abasic sites. The two fluorescent probes 1 and 2 were found to react with 2-deoxyribose, a model substrate, at the optimum o f PH 4.0. As spontaneous depurination occurs at low PH, the reactions with abasic DNA were carried out at neutral PH with an excess concentration of t he probes. Studies with alkylated, depurinated calf thymus DNA showed that the method is selective and quantitative. Good correlations were found betw een the level of 7-methylguanine (7-MeGua), generated in vitro in DNA by th e methylating agent dimethyl sulfate, and the amount of AP sites as determi ned by the method presented here. In addition, similar correlations were fo und when the assay was used to detect abasic sites in DNA isolated from rat s treated with carcinogenic alkylating agents. In each case, the level of a basic sites, as expected, is slightly higher than the level of 7-MeGua whic h is known to represent about 70% of the total modifications of DNA followi ng exposure to the methylating agent. This method may be useful not only in experimental settings but also in studies of DNA damage in humans resultin g from chemotherapy or exposure to environmental agents.