Detection of carcinogen-DNA adducts in exfoliated urothelial cells of cigarette smokers: association with smoking, hemoglobin adducts, and urinary mutagenicity.

Abstract

The presence of covalent modifications in DNA obtained from exfoliated urothelial cells of smokers and nonsmokers was determined using 32P postlabeling methods. Urine and blood samples were procured from 73 persons. Cells were removed from the urine by filtration. DNA was isolated using an enzyme-solvent extraction method and then coprecipitated with glycogen. Sufficient DNA to detect 1 carcinogen-DNA adduct/10(9) normal nucleotides was obtained from 40 of the 73 samples. DNA was hydrolyzed to 3'-phosphodeoxynucleotides and then 32P postlabeled under conditions of excess [32P]ATP. Carcinogen-DNA adducts were resolved using anion-exchange thin-layer chromatography and visualized by autoradiography; film exposures lasted as long as 7 days. Twelve different carcinogen-DNA adducts and a diagonal zone of radioactivity could be found, but no sample contained all adducts. At least four adducts appeared to be cigarette smoking related. These adducts were from 2 to 20 times higher in the smokers than the nonsmokers. Two carcinogen-DNA adducts were qualitatively very similar to adducts described earlier in a study of human bladder biopsies. One of these corresponded to N-(deoxyguanosin-8-yl)-4-aminobiphenyl. Adducts were correlated significantly with the levels of 4-aminobiphenyl hemoglobin adducts and number of cigarettes smoked. In addition, levels of the putative N-(deoxyguanosin-8-yl)-4-aminobiphenyl adduct and a measure of total adducts were correlated with the mutagenic activity of the individual's urine. These data suggest that noninvasive, biological monitoring techniques can be applied to the study of carcinogen-DNA adducts in humans at high risk for bladder cancer.