A P-32 POSTLABELING METHOD FOR DETECTING UNSTABLE N-7-SUBSTITUTED DEOXYGUANOSINE ADDUCTS IN DNA

Many antitumor agents, including the mustards, form N-7 deoxyguanosine adducts in DNA that are difficult to quantitate by the P-32-postlabeling procedure because of their instability. We have developed a method that is successful for the analysis of such adducts using, as a prototype mustard, C-14-labeled bis(2-chloroethyl)sulfide. This agent forms the unstable product 7-hydroxyethylthioethyldeoxyguanosine in DNA. By performing enzymatic digestions to 3'-deoxynucleotides at 10 degrees C, including a second N-7-substituted guanine deoxynucleotide as an internal standard, removing most of the unmodified nucleotides and [P-32]ATP on disposable anion columns, and measuring the labeled products after separation on a Ca column, we are able to detect 1 unstable N-7 deoxyguanosine adduct in 10(7) normal nucleotides with good precision.