O6-alkyldeoxyguanosine detection by 32P-postlabeling and nucleotide chromatographic analysis.

ABSTRACT

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-methl- and O6-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O6-alkyl-2'-deoxyguanosine residue per 10(4) and 10(7) 2'-deoxyguanosines. The limit of detection was less than one O6-alkyl-2'-deoxyguanosine in 10(7) 2'-deoxyguanosine residues in a sample size of 100 micrograms 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.