title: The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.
abstract: The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.
authors.affiliation: Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany guenter.speit@uni-ulm.de.
authors.forename: Günter
authors.initials: G
authors.lastname: Speit
authors.affiliation: Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vesely
authors.affiliation: Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Schütz
authors.affiliation: Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Linsenmeyer
authors.affiliation: Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Bausinger
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D018053:Arsenites / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D020552:Comet Assay / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004058:Diffusion; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008970:Molecular Weight; D017670:Sodium Compounds / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
chemical_list: D018053:Arsenites; D017670:Sodium Compounds; D012967:Sodium Dodecyl Sulfate; C017947:sodium arsenite; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D008741:Methyl Methanesulfonate