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 microM 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.
al-Atrash
J
J
Cancer Center/Division of Environmental Science, School of Public Health, Columbia University, New York, New York 10032, USA.
Zhang
Y J
YJ
Lin
D
D
Kadlubar
F F
FF
Santella
R M
RM
D016475:3T3 Cells; D000611:Aminobiphenyl Compounds / Q000378:metabolism*; D000818:Animals; D002273:Carcinogens / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D008401:Gas Chromatography-Mass Spectrometry; D008099:Liver / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D001743:Urinary Bladder / Q000737:chemistry
D000611:Aminobiphenyl Compounds; D002273:Carcinogens; D018736:DNA Adducts; C006757:4-biphenylamine