The autoradiographic test for unscheduled DNA synthesis: a sensitive assay for the detection of DNA repair in the HepG2 cell line

• Published in: Mutation research Vol. 559; no. 1-2; pp. 211 - 217
• Main Authors: Valentin-Severin, Isabelle, Thybaud, Véronique, Le Bon, Anne-Marie, Lhuguenot, Jean-Claude, Chagnon, Marie-Christine
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 11.04.2004; Elsevier
• Subjects: Analysis of Variance; Autoradiography; Autoradiography - methods; Biological and medical sciences; Direct mutagens; DNA Repair - drug effects; DNA Repair - genetics; DNA Replication - drug effects; DNA Replication - genetics; Endpoint Determination - methods; Fundamental and applied biological sciences. Psychology; Genetics of eukaryotes. Biological and molecular evolution; HepG2 cell line; Humans; Life Sciences; Medical sciences; Mutagens - toxicity; Mutagens requiring metabolic activation; Regression Analysis; Toxicology; Toxicology and food chain; Tumor Cells, Cultured; Unscheduled DNA synthesis; Direct mutagens; Autoradiography; Unscheduled DNA synthesis; HepG2 cell line; Mutagens requiring metabolic activation; Exploration; Mutagen; Toxicology; Cell line; Test; DNA; Metabolic activation; Genetics; Detection; Repair
• ISSN: 1383-5718; 0027-5107; 1879-3592
• DOI: 10.1016/j.mrgentox.2003.12.007

We assessed the DNA-repair capacity of HepG2 cells, which were derived from a human hepatoma, by the unscheduled DNA synthesis assay, using the autoradiography protocol (UDS-AR). We evaluated DNA repair following exposure to direct mutagens (4-nitroquinoline-N-oxide (4-NQO), methyl methanesulfonate (MMS)), to mutagens requiring metabolic activation (benzo[a]pyrene (B[a]P), 2-acetylaminofluorene (2-AAF), N-dimethylnitrosoamine (NDMA)) or to structurally related non-mutagens such as pyrene and 4-acetylaminofluorene (4-AAF). All positive compounds tested induced UDS in HepG2 cells. With 4-NQO and MMS, a concentration-dependent increase in net nuclear grains per cell was observed, with 73 and 90% of cells, respectively, in repair at the highest concentration. B[a]P, 2-AAF and NDMA displayed similar dose-dependent UDS responses, but the percentage of cells in repair was lower (about 45%) than that for 4-NQO and MMS. We assessed the genotoxicity of the compounds tested by determining IC5NNG: the concentration required to induce 5NNG. The compounds studied were ranked in order of IC5NNG as follows: 4-NQO=B[a]P>2-AAF>MMS>NDMA. The UDS assay discriminated between mutagens and non-mutagens, as pyrene and 4-AAF failed to induce DNA repair. The present study demonstrates that UDS can be used as an endpoint for the detection of DNA damage in HepG2 cells.