title: Incorporation of metabolic activation potentiates cyclophosphamide-induced DNA damage response in isogenic DT40 mutant cells.
abstract: Elucidating the DNA repair pathways that are activated in the presence of genotoxic agents is critical to understand their modes of action. Although the DT40 cell-based DNA damage response (DDR) assay provides rapid and sensitive results, the assay cannot be used on genotoxic compounds that require metabolic activation to be reactive. Here, we applied the metabolic activation system to a DDR and micronucleus (MN) assays in DT40 cells. Cyclophosphamide (CP), a well-known cross-linking agent requiring metabolic activation, was preincubated with liver S9 fractions. When DT40 cells and mutant cells were exposed to the preactivated CP, CP caused increased cytotoxicity in FANC-, RAD9-, REV3- and RAD18-mutant cells compared to isogenic wild-type cells. We then performed a MN assay on DT40 cells treated with preactivated CP. An increase in the MN was observed in REV3- and FANC-mutant cells at lower concentrations of activated CP than in the parental DT40 cells. These results demonstrated that the incorporation of metabolic preactivation system using S9 fractions significantly potentiates DDR caused by CP in DT40 cells and their mutants. In addition, our data suggest that the metabolic preactivation system for DDR and MN assays has a potential to increase the relevance of this assay to screening various compounds for potential genotoxicity.
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA, Drug Safety Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.
authors.forename: Kiyohiro
authors.initials: K
authors.lastname: Hashimoto
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Swenberg
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA, ynakamur@email.unc.edu.
authors.forename: Jun
authors.initials: J
authors.lastname: Nakamura
mesh_terms: D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D003520:Cyclophosphamide / Q000633:toxicity*; D004249:DNA Damage / Q000187:drug effects*; D006801:Humans; D053858:Metabolic Networks and Pathways / Q000187:drug effects*; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced; D015162:Micronucleus Tests; D009154:Mutation
chemical_list: D003520:Cyclophosphamide