Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies.
Conventional radiochromatographic procedures for the quantitation of carcinogen/mutagen-induced structural DNA modifications have a number of limitations. Thus, these techniques for the most part require application of radioactively labeled carcinogens and the use of relatively large amounts of DNA for analysis at low levels of DNA modification. Radiochromatographic methods also preclude analyses at the level of single cells and DNA molecules. Recently developed immunoanalytical methods have improved this situation considerably. Monoclonal antibodies (Mab) characterized by a high substrate specificity and affinity, in combination with radio- and enzyme-immunoassays, or with "immuno-slot-blot" techniques, now permit the detection of femtomole to subfemtomole amounts of, e.g., alkyldeoxynucleosides in small samples of DNA isolated from tissues or cultured cells previously exposed to nonradioactive N-nitroso compounds. Furthermore, selected Mab can be used to quantitate by direct immunofluorescence (with the aid of computer-based image analysis of electronically intensified fluorescence signals), specific alkyldeoxynucleosides in the nuclear DNA of single cells. With this method, the detection limit for the alkylation product O6-ethyldeoxyguanosine (O6-EtdGuo) is presently of the order of 10(2) -10(3) O6-EtdGuo residues per diploid mammalian genome. Individual cells can thus be monitored for the presence of specific carcinogen-DNA adducts, and with respect to their capacity for enzymatic removal of such modified structures from DNA (as exemplified here by the kinetics of the enzymatic elimination of O6-EtdGuo from the DNA of malignant neurogenic rat cell lines). In combination with transmission electron microscopy, Mab also permit direct visualization (via Mab binding sites) of specific carcinogen-modified structures in individual DNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)
D000477:Alkylating Agents / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D003853:Deoxyribonucleosides / Q000032:analysis*; D007118:Immunoassay / Q000379:methods; D008854:Microscopy, Electron; D011863:Radioimmunoassay / Q000379:methods
D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D003853:Deoxyribonucleosides; D004247:DNA