title: Optimisation of the comet genotoxicity assay in freshly isolated murine hepatocytes: detection of strong in vitro DNA damaging properties for styrene.
abstract: While the comet assay is used to detect DNA damage in isolated cells following exposure to chemicals in vitro, few publications report the use of the procedure in liver cells isolated from mice. Our initial efforts to use the assay to assess DNA damage in mouse hepatocytes maintained on collagen-coated dishes were hampered by high levels of baseline damage in controls, which appeared to result from mechanical damage sustained during the dislodgement of adherent cells in the early stages of the assay protocol. Here we describe an efficient version of the comet assay in cultured mouse hepatocytes that involves careful recovery of cells using a "scraping" buffer supplemented with 10% high purity grade DMSO. Use of this buffer strongly diminished the frequency of false positives. Using the industrial reagent styrene as a positive control in the optimised procedure, non-cytotoxic concentrations of this substance (2.5-10 mM) significantly increased mean comet tail length, area, and moment. Co-incubation with the CYP inhibitor SKF-525A strongly attenuated these effects of styrene. Collectively, these findings confirm this method is highly suitable for the detection of DNA damage by bioactivation-dependent compounds in freshly isolated mouse hepatocytes.
authors.affiliation: Molecular Toxicology Research Group, Department of Clinical and Experimental Pharmacology, University of Adelaide, Adelaide, SA 5005, Australia. frank.fontaine@adelaide.edu.au
authors.forename: F R
authors.initials: FR
authors.lastname: Fontaine
authors.forename: Y C
authors.initials: YC
authors.lastname: DeGraaf
authors.forename: R
authors.initials: R
authors.lastname: Ghaoui
authors.forename: B C
authors.initials: BC
authors.lastname: Sallustio
authors.forename: J
authors.initials: J
authors.lastname: Edwards
authors.forename: P C
authors.initials: PC
authors.lastname: Burcham
mesh_terms: D000328:Adult; D000818:Animals; D020552:Comet Assay / Q000379:methods; D004249:DNA Damage; D022781:Hepatocytes / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000187:drug effects; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D015203:Reproducibility of Results; D020058:Styrene / Q000633:toxicity*
chemical_list: D020058:Styrene