Pyrrolizidine alkaloid-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and humans.
Pyrrolizidine alkaloids are among the first chemical
carcinogens identified in plants. Previously, we determined that metabolism of
pyrrolizidine alkaloids in vivo and in vitro generated a common set of
DNA adducts that are responsible for
tumor induction. Using LC-ESI/MS/MS analysis, we previously determined that four
DNA adducts (DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4) were formed in rats dosed with
riddelliine, a tumorigenic
pyrrolizidine alkaloid. Because of the lack of an adequate amount of authentic standards, the structures of DHP-dA-3 and DHP-dA-4 were not elucidated, and the structural assignment for DHP-dG-4 warranted further validation. In this study, we developed an improved synthetic methodology for these
DNA adducts, enabling their full structural elucidation by mass spectrometry and NMR spectroscopy. We determined that DHP-dA-3 and DHP-dA-4 are a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N(6)-yl)
dehydrosupinidine, while DHP-dG-4 is 7-hydroxy-9-(deoxyguanosin-N(2)-yl)dehydrosupinidine, an epimer of DHP-dG-3. With the structures of these
DNA adducts unequivocally elucidated, we conclude that cellular
DNA preferentially binds dehydropyrrolizidine
alkaloid, for example, dehydroriddelliine, at the C9 position of the necine base, rather than at the C7 position. We also determined that DHP-dA-3 and DHP-dA-4, as well as DHP-dG-3 and DHP-dG-4, are interconvertible. This study represents the first report with detailed structural assignments of the
DNA adducts that are responsible for
pyrrolizidine alkaloid tumor induction on the molecular level. A mechanism of
tumor initiation by
pyrrolizidine alkaloids is consequently fully determined.