ABSTRACT
The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.
Subject(s)
Acetoxyacetylaminofluorene/analysis , Acetoxyacetylaminofluorene/immunology , Antibodies, Monoclonal , DNA Adducts/analysis , DNA Adducts/immunology , Animals , Cattle , Cell Line , DNA Damage , DNA Repair , Enzyme-Linked Immunosorbent Assay , Humans , Hybridomas/immunology , Mice , Microscopy, Fluorescence , Xeroderma Pigmentosum/metabolismABSTRACT
Sample stacking is used to improve the detection limits of capillary zone electrophoresis coupled to continuous flow fast atom bombardment mass spectrometry for the analysis of DNA adducts. It was found that, with stacking, the concentration detection limit of deoxynucleotide adducts could be improved by as much as 3 orders of magnitude, thereby bringing it into the 10(-8) M range. In addition, the mass spectrometric mass detection limits of a model acetylaminofluorene deoxyguanosine 5'-monophosphate adduct were found to be in the low picomole range for full scanning and the low femtomole range for multiple reaction monitoring of a selected fragmentation. The techniques have been applied to the analysis of adducts formed in the in vitro reaction of N-acetoxy-N-acetyl-2-aminofluorene with DNA.
Subject(s)
Acetoxyacetylaminofluorene/analysis , DNA Adducts/chemistry , DNA/analysis , Electrophoresis/methods , Spectrometry, Mass, Fast Atom Bombardment/methods , Acetoxyacetylaminofluorene/analogs & derivatives , Animals , Cattle , Deoxyguanine Nucleotides/chemistry , Hydrocarbons/chemistry , Molecular StructureABSTRACT
We have produced and characterized a murine monoclonal antibody that recognizes DNA modified with N-acetoxy-2-acetylaminofluorene. The effectiveness of competitive binding inhibitors in an ELISA indicates that this antibody binds most strongly to N-(guanin-8-yl)-2-acetylaminofluorene. It also binds to N-(guanin-8-yl)-2-aminofluorene, albeit some 20-fold less avidly. Thus the monoclonal antibody displays specificity generally similar to the polyclonal antisera elicited by other laboratories but having the advantage of stable and precisely defined specificities. We employed a biotin-streptavidin ELISA to demonstrate that the antibody can detect less than 10 fmol of N-(guanin-8-yl)-2-acetylaminofluorene. N-(guanin-8-yl)-2-acetylaminofluorene is a more effective competitive binding inhibitor of the antibody than is N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene or calf thymus DNA modified with N-acetoxy-2-acetylaminofluorene. Thus the antibody should be most useful in quantifying the persistence of N-acetoxy-2-acetylaminofluorene adducts in DNA hydrolyzed with trifluoroacetic acid.
Subject(s)
2-Acetylaminofluorene/analogs & derivatives , 2-Acetylaminofluorene/analysis , Acetoxyacetylaminofluorene/analysis , Antibodies, Monoclonal , Carcinogens/analysis , DNA/metabolism , Enzyme-Linked Immunosorbent Assay/methods , 2-Acetylaminofluorene/immunology , 2-Acetylaminofluorene/metabolism , Acetoxyacetylaminofluorene/immunology , Acetoxyacetylaminofluorene/metabolism , Animals , Antibody Specificity , Binding Sites, Antibody , Binding, Competitive , Carcinogens/metabolism , Hydrolysis , Mice , Trifluoroacetic Acid/pharmacologyABSTRACT
A method for the identification and quantitation of carcinogeniic O-acetates of fluorenylhydroxamic acids by high-pressure liquid chromatography is described. The adsorbent is Corasil II and a mixture of ethyl acetate-n-hexane (1:1) is used as the solvent. N-Acetoxy-2-fluorenylacetamide or N-acetoxy-3-fluorenylacetamide are separable as single peaks from N-acetoxy-4-fluorenylacetamide and from N-acetoxy-2-fluorenylbenzamide. The peak height traced by the recorder is linearly proportional to the amount of compound in the effluent. The method can be utilized for the detection of 0.1-1.0 mug of compound. The method has been used to identify the products of the decomposition of N-acetoxy-2-fluorenylacetamide in aqueous media.