High Content Toxicology: CellCiphr^® Premier

Figure 1a
Reactive Oxygen Species (ROS) Assay. Rat hepatocytes were stained with Hoechst and CM-H₂DCFDA after 4 hour incubation with vehicle (DMSO) or Compound 1, which induces ROS. CM-H₂DCFDA enters the cell and reacts with ROS to create a fluorescent signal. ROS levels were determined using the ArrayScan^® VTI.

Figure 1b
Glutathione (GSH) Assay. Rat hepatocytes were stained with Hoechst and monochlorobimane after 18 hour incubation with vehicle (DMSO) or Compound 2, which depletes GSH. Monochlorobimane reacts with cellular GSH. GSH levels were determined in the cytoplasm of the cells using the ArrayScan^® VTI.

Figure 1c
Representative heat maps for CellCiphr^® Premier. The heatmaps demonstrate the pattern of endpoint response for the six compounds in a set. The AC₅₀ of the endpoint response is shown using a color scale from red (highly toxic) to green (safe) with the bar at the right showing the full range of the AC₅₀ scale. The heatmaps demonstrate various biomarkers at different time points giving rise to a ‘toxicity risk fingerprint’ for each compound. These data can be used to determine the mechanistic pathways causing the toxic response and the time course over which it occurs. The AC₅₀ data from the different endpoints, for example GSH and ROS activity, are represented by specific boxes on the heatmaps, as demonstrated by the grey connectors that link to the detailed images shown in Figures 1a and 1b.

Table 1
Sensitivity and Specificity of CellCiphr^® Premier to Predict Human Hepatotoxicity.

A blind set of 47 compounds were screened in CellCiphr^® Premier to establish the sensitivity and specificity of the model. Data generated were compared with existing data from the HIAT method. Mechanistic endpoints were normalized for C_max to predict toxicity relative to exposure. The compounds chosen were known to be poorly sensitive (i.e.: they were mainly false negatives) in the HIAT method. In summary, the CellCiphr^® Premier method shows greatly increased sensitivity over the HIAT method for this set of compounds.