The assessment of genotoxicity upon exposure to chemical and environmental agents plays an important role in basic research as well as in pharmaceutical, chemical, cosmetic and food industry. Low sensitivity and lack of inter-laboratory comparability are considered problematic issues in genotoxicity testing. Moreover, commonly used mutagenicity assays lack information about early and specific genotoxic events. Previously, we developed an automated version of the “Fluorimetric detection of Alkaline DNA Unwinding” (FADU) assay as a high-throughput screening method for the detection of DNA strand breaks in living cells. Here, we report an enzyme-modified version of the cell-based FADU assay (emFADU) for the determination of oxidative and methylation lesions in cellular DNA. Our method is based on the use of formamidopyrimidine DNA glycosylase or human alkyladenine DNA glycosylase for the detection of chemically-induced nucleobase modifications in lysates of immortalized cell lines, growing in suspension or as adherent cells, and in peripheral blood mononuclear cells. We could show that upon treatment with sub-cytotoxic doses of known genotoxins, oxidative and methylation lesions are readily detectable. This fast, inexpensive, and convenient method could be useful as a high-content screening approach for the sensitive and specific assessment of genotoxicity in human cells. Thus, when implemented in the early compound development in an industrial setting, the emFADU assay could help reduce the number of animals used for toxicity testing. Furthermore, as we established the method for different cell types, this new assay may provide an opportunity for population studies and/or mechanistic research into DNA repair pathways.
Keywords: human cell lines; DNA alkylation; automation; genotoxicity; in-vitro method; oxidative DNA damage; peripheral blood mononuclear cells.