title: Genotoxicity screening via the γH2AX by flow assay.
abstract: The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on γH2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the γH2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.
authors.affiliation: Safety Assessment, GlaxoSmithKline R&D, Park Road, Ware, Hertfordshire SG12 0DP, UK. Daniel.J.Smart@GSK.com
authors.forename: D J
authors.initials: DJ
authors.lastname: Smart
authors.forename: K P
authors.initials: KP
authors.lastname: Ahmedi
authors.forename: J S
authors.initials: JS
authors.lastname: Harvey
authors.forename: A M
authors.initials: AM
authors.lastname: Lynch
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis; D053903:DNA Breaks, Double-Stranded; D005434:Flow Cytometry / Q000379:methods*; D006657:Histones / Q000378:metabolism*; D007940:Leukemia L5178; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity
chemical_list: D015415:Biomarkers; D006657:Histones; D009153:Mutagens; C475961:gamma-H2AX protein, mouse