title: Usefulness of monitoring γ-H2AX and cell cycle arrest in HepG2 cells for estimating genotoxicity using a high-content analysis system.
abstract: Formation of the phosphorylated protein γ-H2AX is a well-established marker of DNA strand breakage induced by DNA-damaging compounds. Many of these genotoxic compounds also inhibit cell division, leading to arrest at specific points in the cell cycle. Detection of γ-H2AX in combination with cell cycle arrest may therefore be useful for estimating the genotoxicity of experimental compounds. In this study, we examined γ-H2AX formation and cell cycle arrest using high-content screening (HCS) as a method for determining genotoxicity. HepG2 cells were treated with a panel of compounds and then stained with Hoechst 33342 and anti-γ-H2AX, anti-phospho-histone H3, and anti-tubulin antibodies. In total, 19 genotoxic and 7 nongenotoxic compounds were tested in this study. γ-H2AX production was observed within 1 h posttreatment for the majority of Ames-positive compounds, topoisomerase inhibitors, and DNA polymerase inhibitors. Cell cycle arrest in either the S or G2 phase was detected for all DNA-damaging compounds 24 h posttreatment, whereas tubulin-targeting compounds were shown to induce cell cycle arrest in the mitotic phase. Together, these results show that HCS is a simple, rapid, and effective tool for estimating the genotoxicity of compounds through detection of γ-H2AX production and cell cycle arrest.
authors: Safety Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Chiba, Japan Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan ando.masamitsu@mm.mt-pharma.co.jp.
authors: Masamitsu
authors: M
authors: Ando
authors: Safety Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Chiba, Japan.
authors: Keisuke
authors: K
authors: Yoshikawa
authors: Safety Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Chiba, Japan.
authors: Yumiko
authors: Y
authors: Iwase
authors: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
authors: Shoichi
authors: S
authors: Ishiura
mesh_terms: D015415:Biomarkers; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D015415:Biomarkers; C468783:H2AX protein, human; D006657:Histones; D009153:Mutagens
keywords: cell cycle arrest; genotoxicity; high-content screening; γ-H2A.X