Measurement by 32P-postlabelling of 7-methylguanine levels in white blood cell DNA of healthy individuals and cancer patients treated with dacarbazine and procarbazine. Human data and method development for 7-alkylguanines.
A 32P-postlabelling method was developed to measure 7-methylguanine in human DNA. DNA was digested to nucleotides and 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-me-dGMP) was isolated from normal nucleotides using strong anion-exchange column chromatography. Overall the method gave 35-45% yield as measured with DNA methylated with tritiated dimethyl sulfate. Total white blood cell DNA from healthy non-smokers (n = 17) contained from 2.5 7-methylguanine residues/10(7) nucleotides, corrected for the losses in preparation. Among four patients sampled immediately after a total dose of 1050-2800 mg of dacarbazine or procarbazine, the mean adduct level was 57 7-methylguanine residues/10(7) nucleotides. As further method development, we also investigated the phosphorylation reaction by T4 polynucleotide kinase using dinucleotides containing 7-methylguanine and corresponding imidazole ring-opened products as substrates. We found that imidazole ring-opened dTpdG-Me is resistant to digestion with deoxyribonuclease I, snake venom phosphodiesterase and prostatic acid phosphatase. It is quantitatively phosphorylated at femtomolar levels. This method is shown to be suitable for the detection of 7-methylguanine in DNA, and is suggested to be the approach most suited to postlabelling large and labile 7-alkylguanines in DNA.
Mustonen
R
R
Institute of Occupational Health, Helsinki, Finland.
Försti
A
A
Hietanen
P
P
Hemminki
K
K
D002855:Chromatography, Thin Layer / Q000379:methods; D004247:DNA / Q000737:chemistry*; D004273:DNA, Neoplasm / Q000737:chemistry*; D003606:Dacarbazine / Q000627:therapeutic use; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000302:isolation & purification*; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D009369:Neoplasms / Q000097:blood / Q000188:drug therapy; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011344:Procarbazine / Q000627:therapeutic use
D004273:DNA, Neoplasm; D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; C060943:7-methyl-2'-deoxyguanosine 3'-monophosphate; D011344:Procarbazine; D006147:Guanine; C008450:7-methylguanine; D003606:Dacarbazine; D004247:DNA