Genotoxicity screening via the γH2AX by flow assay

https://doi.org/10.1016/j.mrfmmm.2011.07.001Get rights and content

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.

Highlights

► Development of “γH2AX by flow” genotoxicity screening assay that incorporates bioactivation capability, providing scope for detection of both pro- and proximate genotoxins. ► High predictivity (sensitivity, specificity and concordance) of the γH2AX by flow assay with respect to the in vitro genetic toxicology test battery, i.e. Ames and mammalian assays such as mouse lymphoma and chromosome aberrations assays. ► High content nature of assay provides capability to determine atypical genotoxic effects as illustrated by γH2AX response to simple aniline chemicals.

Introduction

Genetic toxicology assessment represents a front-line safety tool in chemical and pharmaceutical development. Positive results in one or more of the core tests in the standard battery, typically bacterial mutagenesis, in vitro mammalian genotoxicity assays and in vivo bone marrow cytogenetic assessment can have significant implications for development. In addition, many chemical synthesis intermediates, degradation products and impurities may also require testing and therefore the development of new technologies for the reliable and accurate assessment of genotoxic potential is required [1], [2], [3]. To this end, several rapid and high-throughput assays have been developed that claim to be predictive of the core genetic toxicology test battery [4], [5].

Measurement of serine-139-phosphorylated histone H2AX (defined as γH2AX) using flow cytometry technology is an emerging assay for the detection of genotoxic potential. The assay is mechanistically-underpinned by the knowledge that γH2AX facilitates the repair of clastogenic DNA double-strand breaks (DSBs) and is an integral component in the DNA damage response machinery of mammalian cells [6]. Indeed, h2ax gene “knock-out” mice exhibit both radiosensitivity and broad genomic instability, thus illustrating the physiological importance of H2AX protein as a tumour suppressor [7]. Flow cytometry assessment of γH2AX is particularly appealing because of the high-content nature of the technology; genotoxic “signatures” consisting of DNA damage response and cell cycle information are readily elucidated, whilst compound requirements are relatively low and data acquisition is both rapid and of higher-throughput compared to standard mammalian genotoxicity assays, such as the mouse lymphoma assay (MLA) and chromosome aberration (CA) assay [8], [9].

The role of γH2AX was first elucidated around a decade ago and since then researchers have endeavoured to use it as a biological tool to study in situ DNA damage, specifically DSBs, in a number of contexts. γH2AX expression has been utilised as a biodosimeter for ionising radiation exposure, a surrogate of cell killing and also as a means to monitor both clinical progress and inform on disease prognosis in an oncology setting [10], [11], [12]. This expanding body of data appears to reflect the promise of γH2AX as a biomarker of DSBs and potential genotoxic stress.

In the present pilot study, γH2AX levels were used as a surrogate for DNA damage and evaluated in L5178Y cells exposed to a variety of chemicals with well-characterised genotoxic potential in standard Ames bacterial mutagenesis and in vitro mammalian genotoxicity tests, i.e. MLA and CA assay (see Table 1), mainly from the European Centre for the Validation of Alternative Methods (ECVAM) list [13]. Two assay “arms” were conducted; 3h +S9 and 24h −S9 treatment regimens were used in this study to mimic the in vitro mammalian assay screening strategy used at GlaxoSmithKline, where experience to date indicates that 3h −S9 unique positives are extremely rare (<0.5%; data not shown). To define evaluation criteria for this novel approach, a validation set of chemicals consisting of 6 prototypical genotoxins (+S9: DMBA, 2-AAF and B[a]P; −S9: MMS, MNU and 4-NQO) and 3 non-genotoxic cytotoxins (±S9: phthalic anhydride, n-butyl chloride and hexachloroethane) were analysed initially, followed by the remaining 25 chemicals. Results of the γH2AX by flow assay are presented and discussed in terms of their concordance with standard in vitro genotoxicity assays (Ames and in vitro mammalian genotoxicity tests).

Section snippets

Cell culture and treatment

Mouse lymphoma L5178Y cells (tk+/−) were maintained in exponential growth phase in “complete” RPMI 1640 medium (GIBCO, UK), i.e. supplemented with heat-inactivated donor horse serum (10% v/v; Biosera, UK), penicillin (50 U/ml; GIBCO), streptomycin (50 μg/ml; GIBCO), l-glutamine (2 mM; GIBCO), sodium pyruvate (0.5 mM; GIBCO) and pluronic F68 (0.05% v/v; GIBCO), at 37 °C in a humidified atmosphere (5% CO2).

All test compounds were of the highest purity available and purchased from Sigma–Aldrich, UK

Defining assay evaluation criteria using the validation set

The pro-genotoxins DMBA, 2-AAF and B[a]P, which require P450-mediated bioactivation to proximate genotoxins, the proximate genotoxins MMS, MNU and 4-NQO, and the non-genotoxic cytotoxins phthalic anhydride, n-butyl chloride and hexachloroethane were used to define assay evaluation criteria for the γH2AX by flow assay. Selection was based on recommended lists of genotoxic and non-genotoxic chemicals for performance assessment of new or improved genotoxicity tests [13].

As expected, all pro- and

Discussion

The first objective of the current study was to define robust positive evaluation criteria for the γH2AX by flow assay that would enable reliable discrimination of chemicals with genotoxic potential from non-genotoxins. As with any genetic toxicology assay, the consideration of confounding factors for assay interpretation, such as cytotoxicity, was central to the definition of the evaluation criteria of this assay [29]. To this end, a set of validation chemicals consisting of the

Conflict of interest statement

None declared.

Competing interests

The authors are working at GlaxoSmithKline.

Funding

GlaxoSmithKline was responsible for funding of this work.

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