multiparametric hepatotoxicity screening in hepg2 cells ...€¦ · multiparametric hepatotoxicity...
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MultiparaMetric Hepatotoxicity Screening in Hepg2 cellS witH coMpariSon in priMary HepatocyteSK.F. Marcoe, Y. Ovechkina, R. Keyser, P. TB. Nguyen, C. O’Day
MDS Pharma Services – Bothell, WA, USA
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Amiodarone Valproic acid Amitriplyline
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introDuctionRecognition of drug-induced hepatotoxic potential early in the drug development cascade creates opportunities for ranking and prioritizing, or developing alternatives with lower toxicity. Detection of hepatic apoptosis induction, phospholipidosis and neutral lipid accumulation late in the drug development pipeline has contributed to late-stage drug failures. Development of cellular systems capable of assessing mechanisms of cytotoxicity with a high level of specificity for identification of hepatotoxic drugs can provide predictive early toxicity assessment and ultimately improve the safety profile of new therapies. In this study, multiplexed high content screening (HCS) with automated fluorescence microscopy and image analysis in a 384-well microtiter plate format coupled with a proprietary data analysis tool was use to enable more comprehensive predictive early toxicity assessment previously only attainable in more complex in vivo models. We developed a robust and rapid throughput screening system using HepG2 cells that allows early assessment of acute and chronic mechanisms of hepatotoxicity and have extended the application to include testing in human primary hepatocytes. Compounds with known hepatotoxicities were tested to validate the capabilities of this multiparametric HCS system in identifying and quantifying toxicities relevant to cell proliferation, apoptosis, cell cycle, steatosis, and phospholipidosis. In addition, we developed a 384-well format MTS assay to provide comparison of cytotoxicity results in a single parameter data output assay and our multiplexed high content screening platform. This cost-effective extensive multiplexed platform for predictive assessment in either HepG2 or human primary hepatocytes delivers a more sensitive approach to detection of end-point-specific drug hepatotoxicities.
MetHoDSCell Culture Human hepatocellular carcinoma cell line (HepG2) was grown in MEM, 10% FBS, 1% NEAA, 1% Alanyl-L-Glutamine and 1% sodium pyruvate in a humidified atmosphere of 5% CO2 at 37oC. Cells were thawed from working stocks and passed once prior to seeding into 384-well plates. Cryo-preserved human primary hepatocytes (CELLZDIRECT) from two donors were thawed in hepatocyte culture medium with supplements and growth factors (LONZA), treated with a percoll separation step and immediately seeded into 384-well plates in the same media. Within 24 hours of plating these cells, the media was exchanged with fresh media. Prior to compound addition, the media was exchanged again.
HCS Multiplexed Hepatotoxicity Assay Caspase-3 activation, a marker of apoptosis, phospho-histone-3, a marker of mitosis, and nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 2x103 cells per well with complete growth media into 384-well tissue culture plates and incubated in a humidified atmosphere of 5% CO2 at 37oC. Test compounds were serially diluted 3-fold over 10 concentrations and added 24 hours post cell seeding with a final assay concentration of 0.5% DMSO. Following an additional 72 hour incubation in the humidified atmosphere of 5% CO2 at 37oC, cells were fixed and immunolabeled with anti-active caspase-3 for detection of apoptosis and anti-phospho-histone-3 for detection of cell cycle and stained with a nuclei dye for cell proliferation quantification. Automated fluorescence microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective.
HCS Multiplexed Hepato-Lipid Accumulation Assay Intracellular phospholipids, a marker of phospholipidosis, intracellular neutral lipid, a marker of steatosis, and nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 4x103 cells per well with complete growth media and human hepatocytes were seeded at 15X103 to 20X103 cells per well with hepatocyte culture medium with supplements and growth factors into 384-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% CO2 at 37oC for 24 and 36 hours, respectively. For detection of phospholipid accumulation a fluorescently-labeled phospholipid (Invitrogen, H34350) was added to the cells with test compounds serially diluted 3-fold over 10 concentrations in a final assay concentration of 0.5% DMSO. Following an additional 24 hour incubation for human hepatocytes and 24 and 48 hour incubation for HepG2 in a humidified atmosphere of 5% CO2 at 37oC, cells were fixed and stained with a neutral lipid dye (Invitrogen, H34476) for neutral lipid detection and a nuclei dye for cell proliferation quantification in HepG2 cells and nuclei location in human hepatocytes. Before fixation the plated human primary hepatocytes were washed to remove unattached dead cells so that only the viable attached cells were fixed and stained. Automated fluorescence microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective.
MTS Assay Cell viability was measured using the CellTiter MTS/PMS cell proliferation assay (Promega). The number of live cells was determined by the uptake of MTS/PMS dyes. HepG2 cells were seeded at 2x103 cells per well into 384-well tissue culture plates in MEM without phenol red, 10% FBS, 1% NEAA, 1% Alanyl-L-Glutamine and 1% sodium pyruvate and incubated in a humidified atmosphere of 5% CO2 at 37oC. Human hepatocytes were seeded at 15x103 and 20x103 cells per well with hepatocyte culture medium with supplements and growth factors into 384-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% CO2 at 37oC. Test compounds were serially diluted 3-fold over 10 concentrations and added 24 and 36 hours, respectively, post cell seeding with a final assay concentration of 0.5% DMSO. Following an additional 72 hour incubation for HepG2 cells and 24 hour incubation for human hepatocytes in the humidified atmosphere of 5% CO2 at 37oC, MTS/PMS solution was added to all wells on the plate and the plates were returned to the incubator. The absorbance of the formazan product was measured at 1 hour for HepG2 cells and 4 hours for human hepatocytes on an Analyst microplate reader (MDS Analytical) using the A490 absorbance.
Data Analysis For HCS assay twelve bit tiff images were acquired with InCell Analyzer 1000 3.2 and analyzed with Developer Toolbox 1.6 software. IC50 and EC50 values were calculated using nonlinear regression to fit data to the dose-response, one-site model where: y = A + [(B – A)/(1 + ((C/x) ^ D))]. Curve-fitting, EC50 / IC50 calculations and report generation were performed using a custom data reduction engine MathIQ based software (AIM). For the MTS assay the corrected absorbance values were obtained by subtracting the averaged 490 nm absorbance value of control wells without cells containing the same volume of culture medium and MTS/PMS solution from the experimental wells. EC50 values were calculated using nonlinear regression to fit data to a Dose-Response, One-Site Model, where: y = A + [(B – A)/1 + ((C/x) ^ D))]. Curve-fitting and EC50 / IC50 calculations were performed using XLFit™ software (IDBS).
Measured Parameters For HepG2 cells cell proliferation was measured by the signal intensity of the incorporated nuclear dye. The cell proliferation output was referred to as the relative cell count. To determine the cell proliferation end point, the cell proliferation data output was transformed to percent of control (POC) using the following formula:
Percent of Control = relative cell count (compound wells) x 100
relative cell count (vehicle wells)
Relative cell count IC50 is the test compound concentration that produces 50% of the cell proliferation inhibitory response or 50% cytotoxicity level. A relative cell count EC50 is the test compound concentration that produces 50% of the maximum effective response that occurs at the curve inflection point. For human primary hepatocytes attached live cells were measured by the signal intensity of the incorporated nuclear dye. The live cell output was referred to as the percent of attached live cells. To determine the live cell end point, the live cell data output was transformed to percent of control (POC) using the following formula:
Percent of Control = relative attached live cell count (compound wells) x 100
relative attached live cell count (vehicle wells)
Relative attached live cell count IC50 is the test compound concentration that produces 50% of the cell detachment response or 50% cytotoxicity level. The output of each biomarker is fold increase over vehicle background normalized to the relative cell count in each well. Concentrations of test compound that cause a 5 or more fold induction in the caspase-3 signal were determined to induce significant apoptotic induction. When the fold induction of the phospho-histone-3 signal over background is ~1, there was “no effect” on the cell cycle. Two or more fold increase in phospho-histone-3 signal over vehicle background indicated significant test compound induction of mitotic block. Two or more fold decrease in the phospho-histone-3 signal may indicate G1/S block. Wells with concentrations higher than the relative cell count IC90 are eliminated from the phospho-histone-3 and caspase-3 analyses. Concentrations of test compound that cause a 5 or more fold induction of the labeled-phospholipid signal represent significant phospholipidosis induction. Concentrations of test compound which cause a 5 or more fold induction of the neutral lipid signal represent significant neutral lipid accumulation. In HepG2 cells the wells with concentrations higher than the relative cell count IC50 were eliminated from the phospholipidosis and neutral lipid analyses. In human primary hepatocytes the wells with concentrations higher than the IC90 were eliminated from the phospholipidosis and neutral lipid analyses. For the MTS assay background 490 nm absorbance was read from control wells without cells containing the same volume of culture medium and MTS/PMS solution as in the experimental wells. The averaged 490 nm absorbance from the background wells was subtracted from all absorbance values to yield corrected absorbances. All tests were performed in a 384-well microtiter plate format in triplicate.
concluSionDevelopment of in vitro multiplexed HCS platforms has enabled more comprehensive predictive early toxicity assessments previously only attainable in more complex in vivo models. The multiplexed HCS platform with automated fluorescence microscopy and image analysis in a 384-well microtiter plate format described here is sensitive and reproducible in identifying multiple mechanisms of drug-induced toxicity in HepG2 cells as well as human primary hepatocytes. High concordance was found with results obtained with the 384-well formatted MTS assay and the reported toxicity profiles for the tested compounds. Further, the MTS assay results confirmed our novel approach of reporting cell viability in human primary hepatocytes as % of attached live cells. Our high-throughput in vitro multiplexed hepatotoxicity screen coupled with a proprietary data analysis tool offers comprehensive predictive information allowing pre-selection of drug scaffold designs with long-term toxicity considerations and provides evaluation in normal primary hepatocytes. This HCS platform can be readily adapted and expanded to include other biomarkers for end-point-specific drug hepatotoxicities.
reFerenceS1. Abraham VC, Towne DL, Waring JF, Warrior U, Burns DJ. Applications of a high-content multiparameter cytotoxicity assay to prioritize compounds based on
toxicity potential in humans. J Biomol Screen 2008;Jul;13(6):527-537.
2. O’Brien PJ, Irwin W, Daiz D, et al. High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Arch Toxicol. 2006;80:580-604.
3. Wang K, Shindoh H, Inoue T, Horii I. Advantages of in vitro cytotoxicity testing by using primary rat hepatocytes in comparison with established cell lines. Journal of Toxicological Sciences 2002;27(3):229-237.
4. Yang A, Cardona DL, Barile FA. Subacute cytotoxicity testing with cultured human lung cells. Toxicology in Vitro 2002;16:33-39.
5. Atienzar F, Gerets H, Dufrane S, et al. Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells. Toxicological Sciences 2007;96(1):101-114.
6. Sawada H, Takami K, Asahi S. A toxicogenomic approach to drug-induced phopholipidosis. Analysis of its induction mechanism and establishment of a novel in vitro screening system. Toxicol. Sci. 2005;83:282-292.
7. Morelli JK, Buehrle M, Pognan F, et al. Validation of an in vitro screen for phospholipidosis using a high-content biology platform. Cell Biology and Toxicology 2006;22:15-27.
8. McMillian MK, Grant ER, Zhong Z, et al. Nile red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis. In vitro & Molecular Toxicology 2001;14(3):177.
9. Xu JJ, Diaz D, O’Brien PJ. Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential. Chemico-Biological Interactions 2004;150:115-128.
10. LeCluyse, E, et al. Isolation and culture of primary hepatocytes. Methods Mol Biol 2005;290:207-229.
HcS MultiparaMetric aSSay in Hepg2 cellS
HCS Multiparameter Assay, representative curves for cell proliferation in HepG2 cells after 24, 48 and 72 hours of compound exposure are shown.
The effect of compound exposure time on the cell proliferation parameter of the HCS multiplexed screening assay for each compound tested in HepG2 cells, (n = 3). IC50 curves generated at 48 and 72 hours more predictive of toxicity end points.
The relative cell count IC50 (half maximal inhibitory constant) and EC50 (half maximal effective constant) values measure cell proliferation. All values are given as the mean ± s.e.m.
HCS Multiparameter Assay, representative curves for cell proliferation, apoptosis induction, and cell cycle block in HepG2 cells after 72 hours of compound exposure are shown.
MTS Assay, representative curves for cell viability in HepG2 cells after 72 hours of compound exposure are shown.
HCS multiparameter assay data (cell proliferation, apoptosis, and cell cycle) in comparison to MTS assay data (cell viability) for each compound tested after 72 hour exposure in HepG2 cells, (n = 3) with comparison of published cytotoxicity studies. HCS data provides mechanistic cytotoxicity information.
The relative cell count IC50 (half maximal inhibitory constant) values measure cell proliferation. Compound concentrations are indicative of a 5-fold apoptosis induction in activated caspase-3 signal and a 2-fold change in phospho-histone-3 signal (mitosis marker) over vehicle background. MTS IC50 values measure cell viability. All values are given as the mean ± s.e.m.
The HCS multiplexed assay cell proliferation and phospholipidosis parameters for each compound after 24 and 48 hour exposures to HepG2 cells, (n = 3). Results for phospholipid induction at 48 hours more reliable.
The relative cell count IC50 (half maximal inhibitory constant) values measure cell proliferation. Compound concentrations are indicative of 5-fold phospholipidosis induction over vehicle background. All values are given as the mean ± s.e.m.
The HCS multiplexed cell proliferation and neutral lipid parameters for each compound after 24 and 48 hour exposures in HepG2 cells, (n = 3). Testing at 24 hours insufficient time period for neutral lipid detection can generate false negative data.
The relative cell count IC50 (half maximal inhibitory constant) values measure cell proliferation. Compound concentrations are indicative of 5-fold neutral lipid accumulation over vehicle background. All values are given as the mean ± s.e.m.
The relative attached live cell count IC50 (half maximal inhibitory constant) value measures cell detachment. Compound concentrations are indicative of 5-fold phospholipid and neutral lipids over vehicle background. MTS IC50 values measure cell viability. All values are given as the mean ± s.e.m.
MTS Assay, representative curves for cell viability in human primary hepatocytes after 24 hours of compound exposure are shown.
HCS multiparameter assay data (percent of attached live cells, phospholipidosis, and neutral lipids) versus MTS assay data (cell viability) for each compound after 24 hour exposure to human primary hepatocytes (HPH) from different donors, (n = 2). The HCS multiparameter assay performed in normal human primary hepatocytes offers relevant comprehensive predictive hepatotoxicity information.
HCS Multiparameter Assay, representative curves for attached live cells, phospholipid induction, and neutral lipid accumulation in human primary hepatocytes after 24 hours of compound exposure are shown.
HcS MultiparaMetric aSSay in HuMan priMary HepatocyteS
HCS Multiparameter Assay, representative curves for cell proliferation, phospholipid induction, and neutral lipid accumulation in HepG2 cells after 48 hours of compound exposure are shown.
HCS multiparameter assay data (cell proliferation, phospholipidosis induction, and neutral lipid accumulation) for each compound tested after 48 hour exposure to HepG2 cells, (n = 3) with comparison of published lipidosis studies.
The relative cell count IC50 (half maximal inhibitory constant) values measure cell proliferation. Compound concentrations are indicative of 5-fold phospholipid induction and neutral lipid accumulation over vehicle background. All values are given as the mean ± s.e.m.
HCS Multiparameter Assay, representative images for attached live cells, phospholipid induction and neutral lipid accumulation in human primary hepatocytes.
Amiodarone Valproic acid Amitriplyline
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Concentration microM0.01 1 100
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Concentration microM0.01 1 100
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