GH2AX/pH3 - RE-Place

Pros, cons & Future potential

Advantages

• a) The gH2AX/pH3 method has been reported to be more predictive of genotoxicity (for specificity and sensitivity) than the commonly used assays (MNvit and Ames) and could detect efficiently different types of genotoxic compounds at low concentrations.
• b) This assay is faster than MNvit test because it did not require two cell cycles completion.
• c) This assay is the first one to permit to easily discriminate the genotoxic mode of action very powerfully (clastogens, aneugens and misleading cytotoxic chemicals) in any human cell type. The use of cell lines with different metabolism capacities enabled differentiation between directly from bioactivated genotoxins.
• d) The use of multi well plates allows high-throughput format for both cell treatment and parameter measurement.
• e) The biomarkers and the antibodies used for the activity measurement are not protected by any patent or license and the technologies are relatively inexpensive to perform.

Challenges

The gH2AX/pH3 genotoxic method is highly suitable for the detection of genotoxic properties of substances amenable to solution. Chemicals are routinely dissolved in aqueous solvent such as water or organic solvents such as DMSO. For hydrophobic substances like oil-derived chemicals, DMSO extracts can be tested. As with conventional in vitro genotoxicity assays, the gH2AX/pH3 biomarkers do not easily allow the evaluation of gasses. It is hoped that new advances in this area will enable such investigations. The method is able to detect genotoxins that require metabolic activation, providing that metabolically competent cells are used or external metabolic activation system is added (e.g., S9 fraction). Since the gH2AX signal is expected to in part result from DNA replication or transcription blocking lesions induced by genotoxins, cells in a proliferative state may be more appropriate than cells in a confluence state.

Modifications

The use of high content analysis platform permits to detect in parallel different biomarkers. So it is possible to detect other biomarker of interest than only phosphorylated histone H2AX and H3.

Future & Other applications

The gH2AX/pH3 test method is not aimed to replace or delete an existing method. The gH2AX/pH3 method has the unique possibility to provide mechanistic insights into the mechanism of genotoxicity by combining two relevant endpoints in a single assay. gH2AX/pH3 method can be particularly useful in an Adverse Outcome Pathway (AOP) and in the development of an Integrated Approach to Testing and Assessment (IATA). The mechanistic information that is provided by the gH2AX/pH3 method assay can be applied to translate the Molecular Initiating Events (MIE) and cellular responses that are activated upon chemical exposure to carcinogenicity hazards for humans. Discussion will begin this year at OECD for a official test guideline.