Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells

• Published in: Archives of toxicology 2014-05, Vol.88 (5), p.1141-1159
• Main Authors: Ireno, Ivanildce C., Baumann, Cindy, Stöber, Regina, Hengstler, Jan G., Wiesmüller, Lisa
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Biomedicine; Carcinogens; Carcinogens - analysis; Carcinogens - toxicity; Cell Line - drug effects; Cells; Cyclophosphamide - pharmacokinetics; Cyclophosphamide - toxicity; DNA Damage; Environmental Health; Fluorescence; Genomics; Genotoxicity and Carcinogenicity; Green Fluorescent Proteins - genetics; Humans; Metabolism; Mutagenicity Tests - methods; Mutagens - analysis; Occupational Medicine/Industrial Medicine; Pharmacology/Toxicology; Recombinant Proteins - genetics; Recombination, Genetic - drug effects; Sensitivity and Specificity; Toxicity
• ISSN: 0340-5761; 1432-0738
• DOI: 10.1007/s00204-014-1229-3

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens (“true positives”), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect (“true negatives”) and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays (“false positives”). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities 85 %, specificity of ≥90 % and detection of false-positive compounds