Genotoxicity screening via the γh2aX by flow assay

Introduction:
This text presents a pilot study that aimed to define robust positive evaluation criteria for the /h9253h2aX by flow assay, a novel assay for the detection of genotoxic potential. The study also compared the performance of this assay with standard in vitro genetic toxicology test battery (ames and in vitro mammalian cell tests).

Key Points:

* The validation set of chemicals used to define assay evaluation criteria included pro-genotoxins, proximate genotoxins, and non-genotoxic cytotoxins.
* The assay evaluation criteria defined were a concentration-dependent response and a threshold of a 1.5-fold induction in h2aX phosphorylation together with a cytotoxicity limit of RCC 25%.
* The /h9253h2aX by flow assay identified all ames mutagens screened (100% sensitivity) and correctly identified most non-genotoxic chemicals (67% specificity).
* The assay also showed high sensitivity (91%) and specificity (89%) with respect to in vitro mammalian genotoxicity tests.
* The study identified some discordant findings, mainly due to chemicals that induced "biologically-irrelevant" genotoxicity via high cytotoxicity- or oxidative stress-driven mechanisms.
* In vitro mammalian genotoxicity tests are known to lack specificity with respect to in vivo endpoints, and a multi-endpoint approach is more appropriate for accurately determining whether a chemical possesses in vivo genotoxicity risk.
* The /h9253h2aX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.

Main Message:
The study demonstrates that the /h9253h2aX by flow assay is a promising tool for the detection of genotoxic potential, with high sensitivity and specificity compared to standard in vitro genetic toxicology tests. however, further validation is needed to fully characterize the performance of the assay, and it is important to consider the biological relevance of in vitro positivity and the potential impact of cytotoxicity on assay results. Overall, the /h9253h2aX by flow assay has the potential to be a valuable addition to the battery of tests used for genotoxicity screening in drug development.

Smart, D.J., K.P. ahmedi, J.S. harvey, and a.M. Lynch. “Genotoxicity Screening via the γh2aX by Flow assay.” Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 715, no. 1–2 (October 2011): 25–31. https://doi.org/10.1016/j.mrfmmm.2011.07.001.