QUANTITATIVE IMMUNOHISTOCHEMICAL ANALYSIS OF 4-AMINOBIPHENYL-DNA IN CULTURED-CELLS AND MICE - COMPARISON TO GAS-CHROMATOGRAPHY MASS-SPECTROSCOPY ANALYSIS

Two monoclonal antisera, 4C11 and 3C8, recognizing 4-aminobiphenyl (4-ABP)-DNA adducts were developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Both antisera are highly specific for 4-ABP-DNA and, at the highest concentration tested, do not recognize the DNA adducts of several other aromatic amines tested including 1-aminopyrene, 8-nitro-1-aminopyrene, and 6-nitro-1-aminopyrene. An immunohistochemical method for detecting adducts was developed in R52 cells, a mouse NIH3T3 cell line expressing high levels of cytochrome P450 1A2. Quantitation of fluorescence labeling indicated a dose-related increase in staining in cells treated with 0, 6, 30, 60, and 300 mu M 4-ABP. To apply the method to tissue samples, Balb/c mice were treated with 0, 4, 10, 20, 40, and 80 mg/kg 4-ABP and liver, bladder, and lung tissue analyzed by immunohistochemical staining of tissue sections. There was a dose-related increase in specific nuclear staining in liver and bladder tissues with no detectable staining in lung tissue. DNA from liver tissue was also analyzed by alkaline hydrolysis of 4-ABP, derivatization with pentafluoropropionic anhydride, and gas chromatography/mass spectroscopy analysis. A good correlation (r = 0.98, p < 0.0001) was found between DNA damage levels determined by the two methods. Based on adduct levels determined by GC/MS in both R52 cells and liver tissue, the immunohistochemical method has a limit of sensitivity of approximately 1 adduct/10(7-8) nucleotides. Immunohistochemistry should be useful for analysis of 4-ABP-DNA adducts in human tissue biopsies as well as exfoliated cells from the oral mucosa and urinary bladder.