title: Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform.
abstract: DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 (-/-) /RAD54 (-/-) and REV3 (-/-) ) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity-2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether-were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA.
authors.affiliation: Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo, Kyoto 606-8501, Japan, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and.
authors.forename: Kana
authors.initials: K
authors.lastname: Nishihara
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and.
authors.forename: Ruili
authors.initials: R
authors.lastname: Huang
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Zhao
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and.
authors.forename: Sampada A
authors.initials: SA
authors.lastname: Shahane
authors.affiliation: Division of the National Toxicology Program, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Kristine L
authors.initials: KL
authors.lastname: Witt
authors.affiliation: Division of the National Toxicology Program, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Smith-Roe
authors.affiliation: Division of the National Toxicology Program, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tice
authors.affiliation: Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo, Kyoto 606-8501, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and mxia@mail.nih.gov.
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics; D001402:B-Lymphocytes / Q000187:drug effects; D002460:Cell Line; D002645:Chickens; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D000072200:Ku Autoantigen; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009153:Mutagens; D004259:DNA-Directed DNA Polymerase; D000072200:Ku Autoantigen