06-Alkyldeoxyguanosine Detection by 32P-Postlabeling and Nucleotide Chromatographie Analysis

The "P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quantitation of O'-alkyl-2'-deoxyguanosine residues in DNA. Chromato graphie techniques were developed to resolve individually the normal deoxyribonucleotide-3'-monophosphates and the O'-alkyldeoxyguanosine-3'-monophosphates by high-pressure liquid chromatography. Selec tive deoxyribonucleotide-3/-monophosphates (e.g., O'-alkyldeoxyguanosine-3'-monophosphates) were then converted to labeled deoxyribonucleotide-(5'-"P]monophosphates by 32P-postlabeling and nuclease PI treat ment and separated by two-dimensional thin layer chromatography. The 0*-methyland 0'-ethyl-2'-deoxyguanosine-3'-monophosphate nucleolides. and the respective 5'-monophosphates, were chemically synthe sized for standardization of these quantitative procedures. The quantitation of O'-methyland O6-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O'-aIkyl-2'-deoxyguanosine residue per 10' and 1112'-deoxyguanosines. The limit of detection was less than one 0*-alkyl-2'-deoxyguanosine in III 2'-deoxyguanosine residues in a sample size of 100 Mgof DNA, i.e., approximately 10 pg of adduct. The guaniitaiiim of O'-methyl-2'-deoxyguanosine in the liver DNAs of rats treated with ["C-MeyV-nitrosodimethylamine compared well with values obtained by both I4C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these "P-postlabeling and nucleotide Chromatographie procedures should be useful in monitoring human ex posure to methylating and ethylating carcinogens.