Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a 32P-postlabeling/HPLC method for detection of (i) two DHR−3‘-dGMP and four DHR−3‘-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR−3‘-dGMP, DHR−3‘-dAMP, and DHR−3‘,5‘-dG-bisphosphate standards and characterization of their structures by mass and 1H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and 32P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR−3‘,5‘-dG-bisphosphate adducts both in vitro and in vivo.
