Evidence for phosphate adducts in DNA from mice treated with 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The weakly alkylating capacity of phosphotriesters (PTE) has been used for the determination of adducts to phosphate groups in DNA by specific transfer to the strongly nucleophilic compound cob(I)alamin [Cbl(I)]. When enzymatically degraded liver DNA from mice treated with 1-(N-methyl-N-nitrosamino)-4-(3-[H-3]pyridyl)-4-oxobutane ([H-3]NNK) was added to Cbl(I), a 4-(3-[H-3]pyridyl)-4-hydroxy-1-butyl-cobalamin ([H-3]PHB-Cbl) complex was formed and determined by HPLC and liquid scintillation counting. The PHB-Cbl formed was compared with a synthetic standard verified by LC/MS and H-1 NMR and corresponds to phosphate adducts formed from the pyridyloxobutylating species from NNK and from the pyridylhydroxybutylating species from NNAL, NNK being to a large extent converted to NNAL in vivo. It was concluded that about 22% of the total level of pyridyl (oxo or hydroxy) butyl adducts to DNA was bound to phosphate groups.