phosphorus-radioisotopes and 7-methylguanine

N7-Methylguanine (N7-MeG) DNA adducts are markers of human exposure to methylating agents including tobacco-specific nitrosamines (TSNAs). Repair of this adduct is poor, so levels in lung tissue should reflect variation in both intensity of exposure and in metabolism. N7-MeG adducts in lung DNA from bronchial lavage samples were measured to determine whether levels were higher in smokers than non-smokers, and if levels were modified by genetic variation in carcinogen-metabolising enzymes. Adducts were detected in 38 out of 44 DNA samples by 32P post-labelling of the N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) isolated from DNA digests by two-stage HPLC. N7-MeG adduct levels were higher in smokers than in never smokers ((9.99 +/-20.3)x10(-7) versus (0.58+/-0.50)x10(-7) N7-MedGp/deoxyguanosine-3'-monophosphate (dGp); P=0.02) and intermediate in ex-smokers ((5.59+/-15.6)x10(-7) N7-MedGp/dGp). Adduct levels tended to be higher in individuals with GSTM1 null, GSTT1 null or GSTP1 ile/ile genotypes. When genotypes were combined, N7-MedGp levels among GSTM1 null/GSTT1 null individuals (n=6) were higher than among those having at least one wild-type allele of these two genes ((26.1+/-38.0)x10(-7) versus (2.73+/-4.07)x10(-7) N7-MedGp/dGp), although the results were not statistically significant (P=0.13). Adduct levels were highest in individuals with three unfavourable genotypes (GSTM1 null/GSTT1 null and GSTP1 ile/ile) compared with others ((74.5+/-13.1)x10(-7) versus (2.64+/-3.89)x10(-7) N7-MedGp/dGp, P=0.02). N7-MeG adduct levels in DNA isolated from lung tissue thus reflect exposure to cigarette smoke, and genetic variation in carcinogen-metabolising enzymes may modify these levels.

Topics: Adult; Bronchoalveolar Lavage Fluid; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; DNA Adducts; Genetic Variation; Genotype; Glutathione Transferase; Guanine; Humans; Lung Neoplasms; Phosphorus Radioisotopes; Smoking

The application of a 32P-postlabeling assay for 7-methylguanines in DNA was studied either by labeling the imidazole ring-opened dinucleotide derivatives or by using strong-anion-exchange column chromatography for the adduct enrichment from normal nucleotides. Data showed that 7-methylguanines can be efficiently labeled as dinucleotides when in vitro methylated DNA was first imidazole ring-opened and then digested to the dinucleotide level with deoxyribonuclease I, snake venom phosphodiesterase, and prostatic acid phosphatase. When using ion exchange chromatography for the adduct enrichment, DNA was digested with micrococcal nuclease and spleen phosphodiesterase. Anion exchange chromatography was applied for 7-methylguanine measurements in white blood cell DNA of healthy nonsmokers (n = 17) and patients (n = 4) treated with the methylating drugs procarbazine and decarbazine. We found that the mean level of 7-methylguanine residues in nonsmokers was 2.5 per 10(7) nucleotides. The corresponding level in the patient samples immediately after the drug treatment was 57 per 10(7) nucleotides.

Topics: Adult; Aged; Chromatography, Ion Exchange; Dacarbazine; DNA; Dose-Response Relationship, Drug; Guanine; Humans; Leukocytes; Middle Aged; Neoplasms; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Procarbazine

7-methylguanine DNA adducts were determined in macroscopically normal bronchial specimens and peripheral blood lymphocytes of 20 patients undergoing pulmonary surgery. A recently developed 32P-postlabeling assay was applied with anion exchange chromatography as an adduct enrichment method. The material consisted of 13 smokers and 7 non-smokers. The mean bronchial 7-methylguanine levels of 11 smokers and 6 non-smokers were 17.3 and 4.7 adducts/10(7) nucleotides. In lymphocyte DNA, the respective mean levels were 11.5 and 2.3 adducts/10(7) nucleotides. The bronchial DNA adduct levels in smokers were statistically higher than those in non-smokers. Among 5 smokers, for whom both bronchial and lymphocyte DNA was available, 7-methylguanine levels correlated in the two tissues (r = 0.77).

Topics: Adult; Aged; Bronchi; Carcinogens; DNA; DNA Damage; Female; Guanine; Humans; Lymphocytes; Male; Middle Aged; Nitrosamines; Phosphorus Radioisotopes; Smoking

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.

Topics: Chromatography, Thin Layer; Dacarbazine; Deoxyguanine Nucleotides; DNA; DNA, Neoplasm; Guanine; Humans; Leukocytes; Neoplasms; Phosphorus Radioisotopes; Phosphorylation; Procarbazine