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Enhanced Sensitivity of ³²P-Postlabeling Analysis of Aromatic Carcinogen:DNA Adducts¹

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

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 kinasecatalyzed ³²P transfer from adenosine [-³²P]triphosphate. Labeled adducts are purified and resolved by four-directional thin-layer chromatography. This procedure can detect one adduct in 10⁷–10⁸ nucleotides but quantitation of adduct concentrations of one adduct in >5 x 10⁶ 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 [-³²P]triphosphate (100–200 µCi), thus enabling quantitative analysis of a single adduct in 10⁹–10¹⁰ nucleotides when 1–10 µg 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 10¹⁰ nucleotides.

¹ This work was supported by USPHS Grant CA 30606.

Received 4/ 2/85. Revised 6/13/85. Accepted 6/20/85.

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