High-content analysis of in vitro hepatocyte injury induced by various hepatotoxicants

Abstract

In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.

Keywords: HepG2 cell; Hepatotoxicity; High-content analysis; Human primary hepatocyte; Multi-parameter cell-based assay.

Fig. 1. High-content analysis of HepG2 cells exposed to various hepatotoxicants. Cells were treated for 24 h with the indicated doses of the following hepatotoxicants: acetaminophen (AAP; 5, 10, and 20 mM), aflatoxin B1 (AFB1; 10, 20, and 50 µM), amiodarone HCl (ADR; 3, 10, and 30 µM), cycloheximide (CHM; 10, 100, and 200 µM), cyclophosphamide monohydrate (CPP; 3, 10, and 30 mM), etoposide (ETP; 10, 20, and 50 µM), lovastatin (LVT; 12.5, 25, and 50 µM), orphenadrine hydrochloride (OPN; 10, 100, and 200 µM), t-butylhydroperoxide (TBHP; 10, 100, and 200 µM), and tetracycline (TC; 10, 100, and 300 µM). The data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Data are presented as a percentage of the vehicle control (mean ± SE of triplicate wells). ^*p < 0.05 compared to the vehicle control.

Fig. 2. Representative images from high-content analysis of HepG2 cells exposed to different hepatotoxicants. Cells were treated for 24 h with vehicle control (0.5% dimethyl sulfoxide [DMSO]) or different hepatotoxicants: acetaminophen (APP) 20 mM, aflatoxin B1 (AFB1) 50 µM, amiodarone HCl (ADR) 30 µM, cycloheximide (CHM) 200 µM, cyclophosphamide monohydrate (CPP) 30 mM, etoposide (ETP) 50 µM, lovastatin (LVT) 50 µM, orphenadrine hydrochloride (OPN) 200 µM, t-butylhydroperoxide (TBHP) 200 µM, and tetracycline (TC) 300 µM. Different fluorescence images (20× objective) of each compound tested were obtained from the same field. The number of cells was reduced in all treatments, and TMRM intensity was decreased by most hepatotoxicants.

Fig. 3. High-content analysis of human primary hepatocytes exposed to different hepatotoxicants. Cells were treated for 24 h with various doses of different hepatotoxicants: acetaminophen (AAP; 5, 10, and 20 mM), aflatoxin B1 (AFB1; 10, 20, and 50 µM), amiodarone HCl (ADR; 3, 10, and 30 µM), cycloheximide (CHM; 10, 100, and 200 µM), cyclophosphamide monohydrate (CPP; 3, 10, and 30 mM), etoposide (ETP; 10, 20, and 50 µM), lovastatin (LVT; 12.5, 25, and 50 µM), orphenadrine hydrochloride (OPN; 10, 100, and 200 µM), t-butylhydroperoxide (TBHP; 10, 100, and 200 µM), and tetracycline (TC; 10, 100, and 300 µM). The data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Data are presented as a percentage of the corresponding vehicle control (mean ± SE of triplicate wells). ^*p < 0.05 compared to the vehicle control.

Fig. 4. Representative images from high-content analysis of human primary hepatocytes exposed to various hepatotoxicants. Cells were treated for 24 h with vehicle control (0.5% dimethyl sulfoxide [DMSO]) or the indicated hepatotoxicants: acetaminophen (APP) 20 mM, aflatoxin B1 (AFB1) 50 µM, amiodarone HCl (ADR) 30 µM, cycloheximide (CHM) 200 µM, cyclophosphamide monohydrate (CPP) 30 mM, etoposide (ETP) 50 µM, lovastatin (LVT) 50 µM, orphenadrine hydrochloride (OPN) 200 µM, t-butylhydroperoxide (TBHP) 200 µM, or tetracycline (TC) 300 µM. Different fluorescence images (20× objective) of each compound tested were obtained from the same field. Number of cells and TMRM intensity were decreased by most hepatotoxicants.