Mechanistic Classification of Toxicants Using Differential Impedance‐Based Real Time Cytotoxicity Profiles in Primary Human Hepatocytes

Impedance‐based real time cytolethality profiling has become an important lead optimization toxicology tool in pharmaceutical discovery. This technique offers the advantage of continuous, non‐destructive monitoring of changes in electrical impedance (reported as cellular index or CI) that correlates to alterations in cell health, morphology, and proliferation of adherent primary cells as well as cell lines. In this study, we used a panel of reference toxicants representing several distinct mechanisms of hepatotoxicity and evaluated their impact on CI profiles of cultured primary human hepatocytes (PHH). The mechanisms of action included: apoptosis (staurosporine; ceramide c2), cytoskeletal disruption (phalloidin; colchicine), mitochondrial inhibition (antimycin A; carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP)), plasma membrane perforation (Triton X‐100), and general cellular detachment (trypsin digestion). PHH donor to donor variability (n= 6 donors) was assessed using chlorpromazine, which acts through two cytotoxicity mechanisms: oxidative stress via quinoneimine formation and changes in mitochondrial membrane potential. Cryopreserved PHH were thawed and cultivated using conventional conditions in Williams E‐based media. Cells were treated with eight concentrations of each toxicant or vehicle (0.1 to 0.5% DMSO) in electrode coated 96‐well plates and monitored with an xCELLigence real‐time cell analyzer (Acea Biosciences) for up to 3 days. Treatment of PHH with surfactant (Triton X‐100) or trypsin resulted in an immediate decline of normalized cellular index (NCI). Mitochondrial function inhibitors caused a rapid decrease of NCI at doses as low as 1.6 and 3.1 μM. Staurosporine and ceramide treatment (apoptosis inducers) decreased NCI (IC 50 =1 to 6 μM), but over an extended time (>10 h) compared to the mitochondrial toxicants. Interestingly, exposure to cytoskeletal disruptors led to a gradual increase in NCI over 3 days, plausibly due to subtle alterations in cell shape. There was a high degree of inter‐individual variability in response to chlorpromazine treatment suggesting that cryopreserved PHH should be characterized using a positive control before an impedance‐based compound evaluation. Altogether, these results are useful to understand differences in impedance profiles based on compound toxicity mechanisms and can be compared to profiles from related liver‐derived primary cells and cell lines. Support or Funding Information All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.