Our goal was to develop an in vitro MN test coupled with a metabolically well-equipped RGHep+ cell line, derived from HepaRG®, by monitoring the production of chromosome damage in real time. Therefore, two fluorescent biotracers were expressed into non-metabolically active cells (HT1080 and HCT116) and hepatic metabolically active RGHep+ cells. The fluorescent chromatibody biotracer, directed against the H2A-H2B histones, labels nuclei allowing the observation in real-time of the chromatin dynamics and the localization of a centromeric protein. A positive MN test associated with either a positive or negative centromeric signal will give insights regarding the MN origin, induced by clastogenic or aneugenic mechanisms respectively.
The developed in vitro MN assay with HepaRP cells validated for toxicity studies is being adapted to a middle- high throughput readable assay, thanks to the implementation of high-content screening imaging protocols and the development of an image analysis and classification-based pipeline. This may bring new capacity to classic MN test and lead to progress in the characterization of the mechanism of DNA damaging event by chemical agents.