ToxCast™ is a research initiative launched in 2007 by the United States Environmental Protection Agency (US EPA) to screen compounds and gain insight into their toxicological profiles. To date, more than 1800 chemicals have been assessed in high-throughput assays and the data catalogued and released to the public. Cyprotex’s US laboratory in Watertown, MA (previously named Apredica) have contributed to the High Content Imaging (HCI) research for the ToxCastTM project.
The US EPA recently published the results of this study with Apredica, which used Cyprotex’s HCI platform, CellCipr®, to elucidate cell damage caused by chemicals and at which point (either through concentration or exposure time) cells would no longer recover from the damage sustained. The study assessed phenotypic changes of 11 endpoints caused by 967 chemicals at a range of concentrations (up to 200 µM) over a 72 hour exposure period in HepG2 cells. The endpoints included p53, c-Jun, phospho-histone H3, alpha tubulin, mitochondrial membrane potential, mitochondrial mass, cell cycle arrest, nuclear size and cell number. To better understand the toxic properties of these test articles, the rate at which cells reached damage beyond repair, or a “tipping point”, was quantified. Unsurprisingly, compound concentration played a direct role in the speed with which this tipping point was reached. The results broke out quite neatly, with about one-third each showing either irreparable damage, resilience or need for further analysis.
However, there are some notable limitations to this study. First, HepG2 cells are not hepatocytes and as such, behave differently, in part because of their relatively lower metabolic activity. Furthermore, because this study was performed in a 2D monoculture, some of the nuances expressed in a multi cell-type organ may not have been expressed. Additionally, only two time points where examined – 24 and 72-hour. While a little more than one-third of the compounds reached or surpassed the phenotypic tipping point at 72 hours, one-third did not. A subset of the cells that showed recovery capabilities (that is, repairable damage) had not fully recovered by 72 hours. It remains unknown whether recovery would have continued beyond that or maximum, though incomplete, restoration was achieved.
Despite these limitations, the findings demonstrate the utility of High Content Imaging for detection of toxicological response and providing an insight into adaptation and resilience of in vitro cellular systems based on tipping point analysis. It is proposed that this technique has the potential for defining risk-based prioritisation of chemicals.