ANALYSIS OF ALKYLATED SITES AT N-3 AND N-7 POSITIONS OF PURINES AS AN INDICATOR FOR CHEMICAL CARCINOGENS

  • 1 January 1981
    • journal article
    • research article
    • Vol. 41  (1) , 223-229
Abstract
A rapid assay to distinguish depurination from other forms of alkaline-labile lesions induced in DNA by alkylating agents is reported. Covalently closed circular duplex [bacteriophage] PM2 DNA was treated with various alkylating agents such as N-methyl-N-nitrosourea, dimethyl sulfate, methyl methanesulfonate, N-ethyl-N-nitrosourea, diethyl sulfate and ethyl methanesulfonate at pH 6.5. Apurinic sites and subsequent strand breaks were introduced by the hydrolysis of the alkylated purines under nondenaturing conditions by heating alkylated DNA at 70.degree. for 1.5 h with 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid:KOH (pH 7.4), 0.1 M KCl, 0.01 M MgCl2, 0.0005 M ethylenediaminetetraacetate, 0.05 M glycine and 0.01 M putrescine. The number of strand breaks produced, representing the alkylated sites at N-3 and N-7 positions of purines, were quantitated by electrophoresis in 1% neutral agarose slab gels. These results were compared with previously reported carcinogenic and mutagenic effects of these compounds and a correlation between the apurinic sites, the total alkylated sites and the biological effect of the alkylating agent was determined.