Immunoperoxidase quantitation of 4-aminobiphenyl- and polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral and urothelial cells of smokers and nonsmokers.

Abstract

Immunoperoxidase methods using two antibodies were developed for detection and quantitation of DNA damage in single cells. A monoclonal antibody that recognizes 4-aminobiphenyl (4-ABP)-DNA adducts was initially tested on liver tissues of BALB/c mice treated with 4-ABP, then applied to the detection of adducts in oral mucosa and exfoliated urothelial cells of smokers and nonsmokers. Levels of 4-ABP-DNA in exfoliated urothelial cells were elevated in each of 20 smokers (mean relative staining intensity, 517 +/- 137) compared with age-, race-, and sex-matched nonsmokers (313 +/- 79; P < 0.0005). Significantly higher damage levels were also observed in oral mucosa cells of smokers compared with nonsmokers (552 +/- 157 versus 326 +/- 101; P < 0.0005). A polyclonal antiserum that recognizes benzo(a)pyrene and structurally related polycyclic aromatic hydrocarbon (PAH) diol epoxide-DNA adducts was also applied to the same study samples after validation by staining of 10T1/2 cells treated with (+/-)-trans-anti-benzo(a)pyrene diol epoxide. Smokers had higher levels of PAH-DNA damage in oral mucosa and exfoliated urothelial cells than nonsmokers (oral mucosa cells, 684 +/- 107 versus 370 +/- 83; P < 0.0005; urothelial cells, 689 +/- 72 versus 495 +/- 57; P < 0.0005). A similar 2-3-fold range in relative staining was found in smokers and nonsmokers for both 4-ABP- and PAH-DNA, suggesting the importance of individual differences in capacity to metabolize the carcinogens and/or repair damaged DNA. Significant correlations were found among the biomarkers in both cell types. This noninvasive method, requiring small numbers of cells and with a relatively low cost, will be useful for monitoring DNA damage in large-scale molecular epidemiology studies.