Abstract
Alkylation at the N-3 and N-7 sites of purines in rat mammary gland DNA induced in vivo by N-ethyl- N-nitrosourea (ENU) and N-methyl- N-nitrosourea (MNU) was quantitated using a newly developed, quantitative, and relatively rapid assay that does not require the use of radiolabeled DNA or radiolabeled alkylators. The assay was based on the selective depurination of the alkylated N-3 and N-7 purine sites in nonradiolabeled DNA followed by hydrolysis at neutral pH to form single-strand breaks in the DNA at each alkylated site. The number of single-strand breaks and thus alkyl- N-3 and alkyl- N-7-purines was determined from the number-average molecular weight of the nonradiolabeled DNA denatured by formamide and sedimented through 5 to 20% neutral sucrose gradients. It was found that MNU alkylates more effectively at the N-3 and N-7 positions of purines than ENU, as expected. These sites appeared to be lost from the rat mammary gland DNA within 24 h. These data are in agreement with those obtained using other approaches.
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