QUANTIFICATION OF SPECIFIC DNA O-ALKYLATION PRODUCTS IN INDIVIDUAL CELLS BY MONOCLONAL-ANTIBODIES AND DIGITAL IMAGING OF INTENSIFIED NUCLEAR FLUORESCENCE

We report the establishment of a standardized, monoclonal antibody (Ma b)-based immunocytological assay (quantitative ICA) for the visualizat ion and quantification of low levels of specific DNA O-alkylation prod ucts in individual cells by electronically intensified, indirect or di rect immunofluorescence. In terms of specific binding to alkali-denatu red nuclear DNA and low background noise, 10 Mabs from a collection of 154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-eth yl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-Bud Guo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity constants ranging between 1.0 x 10(-6)-3.0 x 10(10)/mol were found to be best suited for ICA. At present, greater-than-or-equal-to 200 O6-E tdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA of greater-than-or-equal-to 8.4 x 10(-8)), greater-than-or-equal-to 40 0 O6-BudGuo residues (O6-BudGuo/dGuo, greater-than-or-equal-to 1.7 x 1 0(-7)), greater-than-or-equal-to 1800 O4-EtdThd residues (O4-EtdThd/dT hd, greater-than-or-equal-to 7.5 x 10(-7)) and greater-than-or-equal-t o 4800 O6-MedGuo residues (O6-MedGuo/dGuo, greater-than-or-equal-to 2. 0 x 10(-6)), can be quantified per diploid genome. Using a SIT video c amera in combination with multiparameter image digital analysis, DNA a dduct-specific rhodamine fluorescence signals are measured relative to nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence recordings in three different rat cell lines (BT3Ca, Fao and NO) were in excellent agreement with the data obtained by competitive radioimm unoassay (RIA) for hydrolysates of DNA isolated from the respective ce lls exposed in parallel to the same alkylating carcinogens (N-methyl-, N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of O6-EtdGuo repair, as determined by ICA and RIA, respectively, were su perimposable. Cell-specific, quantitative ICA can, therefore, be used for the quantification of specific, stable DNA adducts induced by alky lating carcinogens or chemotherapeutic agents and for DNA repair measu rements in individual (e.g. human) cells. Work is currently underway t o extend the spectrum of carcinogen - DNA adduct-specific Mabs suited for quantitative ICA.