P-Adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures

A 32P-adduct assay for the measurement of low levels (1 adduct per 107 nucleotides) of binding of carcinogens to DNA has been reported previously. In this procedure, DNA is enzymatkally hydrolyzed to 3-monophosphates of normal nucleosides and adducts, which are S'-^P-labeled by T4 polynucleotide kinase and [7-32P]ATP. Labeled adducts are resolved by TLC. Enrichment of adducts by extraction in 1-butanol [Gupta,R.C. (1985) Cancer Res., 45, 5656] or diges-tion with nuclease PI [Reddy,M.V. and Randerath,K. (1986) Carcinogenesis, 7, 1543] prior to ^P-labeling, however, increased the sensitivity of detection for many adducts to a level of 1 per 109"10 nucleotides, although adduct recovery particularly in the latter assay depended on the chemical nature of adducts. We have now compared recoveries for>70, different carcinogen-DNA adducts of known and unknown chemical nature in the two enrichment procedures as well as in a new procedure in which polynucleotide kinase is substituted for nuclease PI. When compared with the butanol extraction procedure, arylamines (such as 2-aminofluorene, 2-aminophenanthrene, 2-naphthylamine