Detection of DNA adducts in N-acetoxy-2-acetylaminofluorene-treated human fibroblasts by means of immunofluorescence microscopy and quantitative immunoautoradiography.
An immunohistochemical procedure was developed for the detection of adducts in DNA of cultured cells exposed to N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) with the use of antibodies raised in rabbits against N-(guanosin-8-yl)-2-acetyl-aminofluorene (Guo-8-AAF) conjugated to bovine serum albumin. Binding of these antibodies to the nuclear DNA was visualized by means of immunofluorescence microscopy with fluorescein-conjugated anti-rabbit-Ig antibodies, or by means of immunoautoradiography with 125I-labeled protein A. The dose-response curve obtained when the number of autoradiographic grains developed over the nuclei was plotted as a function of the concentration of N-AcO-AAF used to treat the cells, indicated that the extent of specific antibody-binding is determined by the amount of adducts in the cells. DNA modification levels allowing for 20% survival of the cells could be detected with the immunofluorescence technique, while cells exposed to concentrations of N-AcO-AAF resulting in 60% survival were still positive with the immunoautoradiographic method.
D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives*; D000099:Acetoxyacetylaminofluorene / Q000378:metabolism / Q000633:toxicity*; D001345:Autoradiography; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera; D007457:Iodine Radioisotopes; D007700:Kinetics; D013205:Staphylococcal Protein A
D007106:Immune Sera; D007457:Iodine Radioisotopes; D013205:Staphylococcal Protein A; D000099:Acetoxyacetylaminofluorene; D004247:DNA; D015073:2-Acetylaminofluorene