phosphorus-radioisotopes and 7-methyl-2--deoxyguanosine-3--monophosphate

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

As N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) is the major, persistent DNA lesion generated by methylating agents, a combined HPLC/32P-postlabeling assay has been developed to quantitate this adduct in human DNA. N7-MedGp was purified from normal nucleotides by anion-exchange chromatography followed by reverse-phase HPLC procedures. The adduct was then 32P-postlabeled and resolved by two-dimensional TLC for detection and quantitation by storage-phosphor imaging. The effect of conditions used for DNA purification and digestion on the recovery of N7-MedGp has been investigated. Extended, raised temperature incubations normally employed during DNA purification were demonstrated to result in considerable loss of adduct through depurination after 22 h at 65 and 37 degrees C (82% and 20% loss, respectively), but depurination was reduced to 5% if the incubation was performed at either 4 or 22 degrees C. Similarly, close to optical recovery (83%) of N7-MedGp was achieved after DNA digestion by incubating at 4 degrees C, pH 7.4, for 18 h in the presence of micrococcal nuclease and calf spleen phosphodiesterase from Sigma and Boehringer Mannheim, respectively. Overall, the recovery of N7-MedGp was 40%, resulting in a detection limit of 1.3 fmol which is equivalent to 0.16 mumol of adduct/mol of 2'-deoxyguanosine-3'-monophosphate (dGp) when analyzing 10 micrograms of DNA. The N7-MedGp content of DNA that had been methylated in vitro using 0, 16, and 80 microM N-methyl-N-nitrosourea (NMU) was determined by 32P-postlabeling to be 12, 112, and 671 mumol of N7-MedGp/mol of dGp. Electrochemical detection of N7-methylguanine (N7-MeG) after HPLC purification measured approximately 2-fold higher levels, i.e., 25, 225, and 1080 mumol of N7-MeG/mol of Gua, at each NMU concentration, respectively. The levels of N7-MedGp in the white blood cell (WBC) DNA of patients receiving a single dose of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) chemotherapy were determined by 32P-postlabeling. Maximum levels were found 4-6 h after treatment, and in two out of four individuals adduct levels were decreased by 21 h. Prior to treatment, N7-MedGp was detectable in WBC DNA in two out of the four individuals indicating that nontherapeutic exposure to methylating agents had occurred.

Topics: Animals; Autoradiography; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; DNA; DNA Adducts; Humans; Image Processing, Computer-Assisted; Isotope Labeling; Phosphorus Radioisotopes; Radiographic Image Enhancement; Reproducibility of Results; Sensitivity and Specificity

Alkylating agents may cause DNA damage in different human cells and tissues, including lungs. For instance, tobacco-specific N-nitrosamines are known to produce methyl-DNA adducts, such as N7-methyldeoxyguanosine, and to induce lung tumors. We applied a combined high-performance liquid chromatography (HPLC)/32P-postlabeling technique for measurement of N7-methyldeoxyguanosine in human pulmonary alveolar cells (HPAC). Thirty patients (13 males, 17 females; mean age 51 +/- 17 yr) undergoing bronchoalveolar lavage for diagnosis of nonmalignant lung diseases were studied. DNA was extracted from HPAC, digested to 2'-deoxyribonucleotide 3'-monophosphates and HPLC separated to obtain deoxyguanosine (dGp) and N7-methyldeoxyguanosine (N7-MedGp) monophosphates. Fractions corresponding to normal (1:10,000) and N7-methylated dGp were subsequently 32P-postlabeled by T4 polynucleotide kinase with high specific activity 32P-ATP, resolved by two-dimensional thin-layer chromatography (TLC) and autoradiographed after 3 to 18 h exposure. Spots corresponding to dGp and N7-MedGp were scraped off the plates and quantitated by liquid scintillation counting to calculate direct molar ratios. Recovered HPAC (14.4 +/- 10.0 x 10(6)) were predominantly macrophages (73.8 +/- 16.4%) and lymphocytes (9.8 +/- 11.6%). N7-MedGp was detected in 11 patients, the level ranging from 0.10 to 48.03 fmol/micrograms DNA which corresponded to 0.31-79.00 x 10(-6) N7-MedGp/dGp ratios. Detection of N7-MedGp in HPAC was associated with the smoking habit of patients: N7-MedGp was present in 7 of 10 smokers, 2 of 10 ex-smokers, and 2 of 10 nonsmokers (P < 0.05). These results show that HPAC may be used for molecular dosimetry of DNA damage by alkylating agents, including tobacco-specific N-nitrosamines, in cigarette smokers and thus used for cancer risk assessment.

Topics: Adult; Aged; Alkylating Agents; Bronchoalveolar Lavage Fluid; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; DNA; DNA Adducts; Dose-Response Relationship, Radiation; Electrophoresis, Gel, Two-Dimensional; Epithelium; Female; Humans; Male; Middle Aged; Phosphorus Radioisotopes; Pulmonary Alveoli; Smoking

7-Alkyldeoxyguanosine DNA adducts may be a marker for some N-nitroso compound exposures and subsequent human cancer risk. A sensitive and highly specific assay for the detection of 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-methyldGp) and 7-ethyl-2'-deoxyguanosine-3'-monophosphate (7-ethyldGp) has been developed by combining two different HPLC purification steps with the 32P-postlabeling assay. We previously reported that ion-pair reverse-phase (IP) chromatography coupled with the 32P-postlabeling assay detects 7-methyldGp in human lung, but have found that other nucleotides and unknown adducts co-elute. Thus, weak anion exchange (AE) HPLC was added in tandem with IP HPLC prior to the 32P-postlabeling assay. 2'-Deoxyguanosine-3'-monophosphate (dGp) is incorporated into the assay as an internal standard for the assessment of enzyme labeling efficiency and adduct recovery. The methodology was validated using radiolabeled DNA and liquid scintillation counting, which accounts for adduct loss from enzymatic digestion to detection. Levels of 7-ethyldGp also were correlated with accelerator mass spectrometry. The overall adduct recovery with this method was 58% for 7-methyldGp and 98% for 7-ethyldGp. The detection limit for both assays using 100 micrograms of DNA was one adduct in 10(8) unmodified dGp. 7-MethyldGp and 7-ethyldGp levels were determined in ten human lung samples at levels of 1.4-5.4 and 0.6-3.1 adducts per 10(7) dGp respectively, and in five human lymphocyte samples at levels of 5.0-8.3 and 0.3-1.4 adducts per 10(7) dGp respectively. Combining the two HPLC purification steps and the 32P-postlabeling assay attains chemical specificity, retains sufficient quantitative sensitivity and should be useful in human biomonitoring studies.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; DNA; Ethylnitrosourea; Guanosine Monophosphate; Humans; Methylnitrosourea; Phosphorus Radioisotopes

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

The 32P-postlabelling technique introduced by Randerath and coworkers was used to investigate the efficiency of the phosphorylation reaction by T4 polynucleotide kinase using three synthesized adducts: 7-methyl-dGMP, ring-opened 7-methyl-dGMP and platinated dGpdG. The methylated substrates were detected at sub-fmol sensitivities. 7-Methyl-dGMP was quantitatively phosphorylated at these low concentrations. The efficiency of phosphorylation of the ring-opened product was less (about one order of magnitude) and that of Pt(dGpdG) about three orders of magnitude less. These results show that T4 polynucleotide kinase phosphorylation is an efficient reaction with 7-methyl-dGMP and with ring-opened 7-methyl-dGMP, even though in the latter case longer incubation times may have to be used to boost the reaction towards completion. By contrast, the low level of phosphorylation with Pt(dGpdG) does not appear encouraging for quantitative determination requiring a high sensitivity.

Topics: Deoxyguanine Nucleotides; Dinucleoside Phosphates; Isotope Labeling; Kinetics; Molecular Conformation; Molecular Structure; Organoplatinum Compounds; Phosphorus Radioisotopes; Phosphorylation; Polynucleotide 5'-Hydroxyl-Kinase; T-Phages; X-Ray Diffraction