A quantitative indirect immunofluorescence technique was developed utilizing a monoclonal antibody (6A10) recognizing the imidazole ring-opened form of the major N-7 guanine adduct of aflatoxin B1 (AFB1). This method was used to investigate adduct formation in woodchuck hepatocytes treated in culture and in liver tissue of rats treated i.p. with AFB1. Fluorescein isothiocyanate-labeled secondary antiserum was used for adduct localization in conjunction with 4′,6-diamidino-2-phenylindole dihydrochloride staining to localize nuclei. Quantitation of AFB1-DNA adducts was carried out by densitometric analysis of photographic slides. Specific nuclear staining was observed in both woodchuck hepatocytes and rat liver tissue. There was a dose-response relationship between fluorescence intensity and AFB1 dose in treated animals. Turnover of adducts could also be followed in animals over 48 h with this method. DNA was isolated from liver tissue of treated animals and adduct levels were quantitated by competitive enzyme-linked immunosorbent assay with antibody 6A10 and by fluorescence spectroscopy. There was a significant correlation of the quantitative immunofluorescence intensity with levels of AFB1 adducts detected by enzyme-linked immunosorbent assay (r = 0.61, P < 0.05) and spectrofluorescence (r = 0.78, P < 0.01). This immunohistochemical method should be applicable to the detection of adducts in liver tissues of humans exposed to high levels of dietary AFB1.
This study was supported by NIH Grants ES05116 and CA21111 and by a gift from the Lucille P. Markey Charitable Trust. Dr. C-J. Chen is the recipient of an NIH Fogarty International Research Fellowship (1 F05 TW04161-01).