title: Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.
abstract: In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.
authors.affiliation: Department of Laboratory Sciences and Investigative Toxicology, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Gealy
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Wright-Bourque
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Kraynak
authors.forename: Troy W
authors.initials: TW
authors.lastname: McKelvey
authors.forename: John E
authors.initials: JE
authors.lastname: Barnum
authors.forename: Richard D
authors.initials: RD
authors.lastname: Storer
mesh_terms: D000818:Animals; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002733:Chlorophenols / Q000633:toxicity; D002744:Chlorpheniramine / Q000633:toxicity; D004247:DNA / Q000032:analysis; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009596:Nitrophenols / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018675:Toxicity Tests / Q000379:methods / Q000592:standards
chemical_list: D002733:Chlorophenols; D009153:Mutagens; D009596:Nitrophenols; D002744:Chlorpheniramine; D004247:DNA; C004762:2,4-dichlorophenol; C024836:4-nitrophenol