HPLC-32P-postlabelling analysis of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine

A 32P-postlabelling procedure coupled with HPLC has been developed to detect and measure the cyclic nucleic acid adducts 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in DNA. Chloroacetaldehyde-modified DNA containing these adducts was enzymatically digested to 3'-monophosphates and adducts were separated by ion-pair reverse-phase HPLC on PL RP-S prior to 32P-postlabelling with carrier free [gamma-32P]ATP. Following 3'-dephosphorylation with nuclease P1 the resulting [5'-32P]monophosphate adducts were finally resolved by HPLC on PL RP-S and assayed by liquid scintillation counting.