Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts

Abstract

We have previously described a 32P assay for the detection and quantitation of aromatic carcinogen:DNA adducts (R. C. Gupta et al., Carcinogenesis (Lond.), 3: 1081-1092, 1982). The method entails enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates which are then converted to deoxynucleoside 3',5'-[5'-32P]diphosphates by T4 polynucleotide kinase-catalyzed 32P transfer from adenosine [gamma-32P]triphosphate. Labeled adducts are purified and resolved by four-directional thin-layer chromatography. This procedure can detect one adduct in 10(7)-10(8) nucleotides but quantitation of adduct concentrations of one adduct in greater than 5 X 10(6) nucleotides becomes exceedingly difficult. I have now found that isolation of DNA adducts by extraction with 1-butanol in the presence of the phase-transfer agent tetrabutylammonium chloride prior to the labeling allows one to use excess carrier-free adenosine [gamma-32P]triphosphate (100-200 microCi), thus enabling quantitative analysis of a single adduct in 10(9)-10(10) nucleotides when 1-10 micrograms of the DNA are used. Further increase in the sensitivity of the assay requires higher amount of DNA. The four-directional thin-layer chromatography system has been modified so as to analyze simultaneously as many as 35-40 DNA samples. The new protocol, as applied to a number of carcinogenic aromatic amines and polycyclic aromatic hydrocarbons of diverse structure, is capable of detecting and quantitating adducts at the level of one adduct per 10(10) nucleotides.