Abstract
Figures and Tables
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.
![jvs-20-34-g001](/upload/SynapseData/ArticleImage/0118jvs/jvs-20-34-g001.jpg)
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.
![jvs-20-34-g002](/upload/SynapseData/ArticleImage/0118jvs/jvs-20-34-g002.jpg)
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.
![jvs-20-34-g003](/upload/SynapseData/ArticleImage/0118jvs/jvs-20-34-g003.jpg)
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.
![jvs-20-34-g004](/upload/SynapseData/ArticleImage/0118jvs/jvs-20-34-g004.jpg)
Table 1
Hepatotoxicants selected by mechanism of action
![jvs-20-34-i001](/upload/SynapseData/ArticleImage/0118jvs/jvs-20-34-i001.jpg)
AAP, acetaminophen; AFB1, aflatoxin B1; ADR, amiodarone HCl; CHM, cycloheximide; CPP, cyclophosphamide monohydrate; ETP, etoposide; LVT, lovastatin; OPN, orphenadrine hydrochloride; TBHP, t-butylhydroperoxide; TC, tetracycline; CAS, Chemical Abstracts Service; AP, apoptosis; BA, bioactivation; OS, oxidative stress damage; MI, mitochondria impairment; CA, calcium homeostasis; IC50, 50% inhibitory concentration. *These endpoints represent the mechanisms of hepatotoxicity reviewed by Gómez-Lechón et al. [6], of which some mechanisms (bold) are in agreement with the articles published by Tolosa et al. [28]. †These values were determined under our experimental conditions (unpublished data).
Table 2
Comparison of cytotoxic effects in HepG2 cells and human primary hepatocytes (hPHs)
![jvs-20-34-i002](/upload/SynapseData/ArticleImage/0118jvs/jvs-20-34-i002.jpg)
Hepatocyte injury was measured by high-content analysis using multi-parameter cell-based assays: mitochondrial membrane potential (TMRM), cytosolic free calcium ion (Fluo-4AM), and lipid peroxidation (BODIPY). Criteria for phenotypic changes in nuclear size were as follows: statistical significance and ≥ 10% variation in comparison with the vehicle control. Other cytotoxic effects were based on statistical significance and ≥ 20% variation. AAP, acetaminophen; AFB1, aflatoxin B1; ADR, amiodarone HCl; CHM, cycloheximide; CPP, cyclophosphamide monohydrate; ETP, etoposide; LVT, lovastatin; OPN, orphenadrine hydrochloride; TBHP, t-butylhydroperoxide; TC, tetracycline; ↑, statistically significant increase with variation; ↓, statistically significant decrease with variation; −, no change; +, good agreement in more than two parameters between HepG2 cells and hPHs; ~, good agreement in zero or one parameters.
Acknowledgments
References
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