A 32P-postlabeling method for simultaneous detection and quantification of exocyclic etheno and propano adducts in DNA

Abstract

A ³²P-postlabeling method is described that specifically detects and quantifies the 1,N² adducts derived from acrolein (AdG) and crotonaldehyde (CdG) and 1,N²-ethenodexoxyguanosine (EdG) in DNA. These exocyclic adducts are potential DNA lesions caused by exposure to enals as environmental pollutants and as endogenous compounds. This method was developed with the use of the synthetic adduct standards of these exocyclic adducts. The assay relies on HPLC for adduct enrichment prior to labeling and for quantitation and identification after labeling. The labeling efficiencies of adducts at the 1 fmol level ranged from 74 to 96%, whereas they were only 49–60% at the 100 fmol level. This method can detect as low as 0.2 fmol of adduct and allows the detection and quantitative determination of stereolsomers of AdG and CdG. The method was validated by using a sample of enzyme digests of 180 μg calf thymus DNA spiked with 25 or 75 fmol of adducts, which is equivalent to 5 or 15 adducts in 10⁸ nucleotides. The recovery rates of these adducts in DNA ranged from 30 to 90% at the 25 fmol level and 21 to 55% at the 75 fmol level. Similar to the labeling efficiency, a greater recovery was observed with a lower amount of adduct in DNA. Overall, this method allows the simultaneous identification and quantification of exocycic adducts AdG, CdG and EdG in DNA. Therefore, it provides a potential tool for studies of the in vivo formation of exocyclic adducts.