Detection of N7-methyldeoxyguanosine adducts in human pulmonary alveolar cells.
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.
Petruzzelli
S
S
U.O. Fisiopatologia Respiratoria, Clinica Medica 2a, Università di Pisa, Italy.
Tavanti
L M
LM
Celi
A
A
Giuntini
C
C
D000328:Adult; D000368:Aged; D000477:Alkylating Agents; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects / Q000528:radiation effects; D018736:DNA Adducts / Q000032:analysis; D003848:Deoxyguanine Nucleotides / Q000032:analysis*; D004307:Dose-Response Relationship, Radiation; D015180:Electrophoresis, Gel, Two-Dimensional; D004848:Epithelium / Q000737:chemistry; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010761:Phosphorus Radioisotopes; D011650:Pulmonary Alveoli / Q000737:chemistry / Q000166:cytology*; D012907:Smoking / Q000009:adverse effects
D000477:Alkylating Agents; D018736:DNA Adducts; D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; C060943:7-methyl-2'-deoxyguanosine 3'-monophosphate; D004247:DNA