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O⁶-Alkyldeoxyguanosine Detection by ³²P-Postlabeling and Nucleotide Chromatographic Analysis

Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [V. L. W., R. A. S., C. C. H.]; and Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02319 [A. K. B., J. M. E.]

The ³²P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quantitation of O⁶-alkyl-2'-deoxyguanosine residues in DNA. Chromatographic techniques were developed to resolve individually the normal deoxyribonucleotide-3'-monophosphates and the O⁶-alkyldeoxyguanosine-3'-monophosphates by high-pressure liquid chromatography. Selective deoxyribonucleotide-3'-monophosphates (e.g., O⁶-alkyldeoxyguanosine-3'-monophosphates) were then converted to labeled deoxyribonucleotide-[5'-³²P]monophosphates by ³²P-postlabeling and nuclease P1 treatment and separated by two-dimensional thin layer chromatography. The O⁶-methyl- and O⁶-ethyl-2'-deoxyguanosine-3'-monophosphate nucleotides, and the respective 5'-monophosphates, were chemically synthesized for standardization of these quantitative procedures. The quantitation of O⁶-methyl- and O⁶-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O⁶-alkyl-2'-deoxyguanosine residue per 10⁴ and 10⁷ 2'-deoxyguanosines. The limit of detection was less than one O⁶-alkyl-2'-deoxyguanosine in 10⁷ 2'-deoxyguanosine residues in a sample size of 100 µg of DNA, i.e., approximately 10 pg of adduct. The quantitation of O⁶-methyl-2'-deoxyguanosine in the liver DNAs of rats treated with [¹⁴C-Me]N-nitrosodimethylamine compared well with values obtained by both ¹⁴C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these ³²P-postlabeling and nucleotide chromatographic procedures should be useful in monitoring human exposure to methylating and ethylating carcinogens.

¹ To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, NCI/NIH, Building 37, Room 2C20, Bethesda, MD 20892.

² Supported by Grant CA-43066 from the NIH, Department of Health and Human Services.

Received 9/ 8/87. Revised 12/28/87. Accepted 1/19/88.