Analysis of Four Hepatotoxicants Using the xCELLigence Analytical System and HepaRG Cells (030514) ¹Can Jin, ²David Steen, ²Valery Schevchenko. ²Christophe Chesne, and ¹Yama Abassi ABSTRACT ¹Biopredic International, St. Gregoire France ²ACEA Biosciences, San Diego, CA Hepatotoxicants act through different mechanisms nd kinetics, and we 1. RESULTS CONCENTRATION AND TIME-DEPENDENT EFFECTS 2. RESULTS, ACUTE DOSING MONITORING reasoned that an analytical system tcCI/ccCI after exposure intervals allowing for convenient, kinetic A. APAP (mM) B. Troglitazone (uM) C. Rosaglitazone (uM) APAP Dose (mM) monitoring of relevant effects 1.5 1.5 1.5 1.5 1 combined with metabolically 0.4 2.5 1.85 1.25 competent human hepatic cells, 5 1 5.56 2 should facilitate recognition of the risk 1 1 1 1 10 16.7 2.5 2 of hepatotoxicity. Our protocol tcCI/ccCI tcCI/ccCI tcCI/ccCI tcCI/ccCI 25 involved culturing cryopreserved, 50 4 0.5 4 0.5 0.5 0.5 50 5 differentiated HepaRG cells onto the 150 6 xCELLigence 96-well cell culture plate, 100 6 and after a four-day adaptation 450 0 8 0 200 0 0 10 period, treating them with different 24h 48h 72h 96h 168h 24h 48h 72h 96h 168h 24h 48h 72h 96h 168h 2h 5h 10h 20h concentrations of four compounds with known risk of hepatotoxicity D. Amiodarone (uM) E. CsA (uM) APAP, amiodarone, CsA, and 1.5 1.5 troglitazone-and the PPAR agonist 1 1 rosiglitazone. The Cell Index (CI), an 2.5 2.5 3. RESULTS, REPRODUCIBILITY OF tcCI/ccCI expression of the electrical impedance 1 5 1 5 (studies in July and Sept) of adherent cells as measured on tcCI/ccCI tcCI/ccCI sensors in culture plate wells by the 10 10 xCELLigence Cell Analyzer, of treated 0.5 20 0.5 APAP Dose (mM) 20 1 cells (tcCI) was compared to the CI 50 1.25-Jul from HepaRG cells exposed to a 0.5% 0 100 0 40 1.25-Sep DMSO control (ccCI) after exposure 24h 48h 72h 96h 168h 24h 48h 72h 96h 168h times of 24, 48, 72, 96, and 168 hrs, 2.5-Jul during which time the medium was tcCI/ccCI 0.5 2.5-Sep replenished every 48 hours. The 5-Jul tcCI/ccCI was also assessed at 2, 5, 10, and 20 hours after treatment with MATERIALS AND METHODS 5-Sep eight APAP concentrations to 0 10-Jul determine if damage to the cells could 20h 30h 60h 90h Cryopreserved, differentiated HepaRG cells were thawed and seeded into 96 well E-Plates containing EXPOSURE TIME (HRS) 10-Sep be detected rapidly, since MCGill et al¹ microsensors at the bottom of each well to measure electrical impedance using Real-Time Cell Analyzers from reported significant glutathione ACEA. Following a five-day adaptation period during which the media was replenished twice, HepaRG cells were depletion within three hours after exposed to test articles including 0.5% DSMO as a control for a period of seven days during which time the July Experiment Sept Experiment exposing HepaRG to APAP. medium + TA or 0.5% DMSO was changed every twoi days. During the entire period of expsure the CI values from 1.25 mM APAP (1.0; 1.02; 0.72; 0.60) 1.25 mM APAP (1.0; 1.03; 0.80; 0.64) INTRODUCTION booth DMSO control and TA-treated HepaRG cells were monitored and tcCI/ccCI was determined. 2.50 mM APAP (0.98; 1.01; 0.54; 0.36) 2.50 mM APAP (0.98; 1.00; 0.61; 0.30) 5.0 mM APAP (0.55; 0.51; 0.31; 0.11) 5.0 mM APAP (0.64; 0.55; 0.25; 0.05) 10.0 mM APAP (0.21; 0.20; 0.11; 0.02) 10.0 mM APAP (0.23; 0.17; 0.09; 0.02) - Kinetic measurement of electrical impedance of charged, adherent cells CONCLUSIONS has been shown to detect changes in Combined use of the HepaRG cells and the xCELLigence analytical system demonstrated a distinct, concentration morphology, cell contact and and exposure-time dependent effect from different hepatotoxicants when the Cell Index of treated cells was conductivity, and lipid bilayer² compared with that of control (0.5% DMSO) cells. REFERENCES -DILI is a serious concern and is often Kinetic measurement allowed for detection of hepatotoxicant effect on HepaRG cells’ impedance as it occurred 1. McGill M, Jaeschke, H, et al. Hepatology 2011: 53:974-982 difficult to predict in preclinical testing³ (results not shown). 2. Xing J, Xu X, et al. Chem.Res. Toxicol. 2005, 18, 154-161. -- APAP, amiodarone, CsA, and Different mechanisms of toxicity (i.e., metabolism-dependent, steatosis, oxidative stress, idiosyncratic necrosis, 3. Antherieu S. Guillouzo A, Robin M et al. Hepatology 2011; 53:1895- troglitazone are known to cause DILI in glutathione depletion) quantitatively affected the HepaRG cells’ impedance using the same study design. 1905 either an exposure-related or Mechanisms requiring extended exposure time for clinical toxicity to develop (steatosis) demonstrated significant 4. Kienhuis A, van der Ven L, et al. Toxicology Letters 221 (2013) 225-236. idiosyncratic fashion and through change in HepaRG cells’ impedance within 48hrs at higher concentrations. 5. Gunness P, Noor F et al. Toxic. Sci. 133 (1) 67-78. somewhat different mechanisms¹ ³ ̕⁴ ⁵ Between the glitazones with significantly different clinical risk of hepatotoxicity, a distinct concentration effect was 6. Josse’R, Guillouzo C, Guillouzo A et al. Drug Metab. Dispos. 36:1111- -- Primary human hepatocytes have been demonstrated with the “safter” rosiglitazone requiring a concentration of 450uM and exposure time of 72 hr to 1118 used for in-vitro assessment of toxicity demonstrate approximately equivalent effect of 200uM of troglitazone for 48hrs on HepaRG cells’ impedance. risk but have many limitations which the The system essentially duplicated results derived in July with APAP when the same conditions were used in a human hepatic cell lines HepaRG has September study. been shown to improve upon. ⁶