Assessment Of Chemical Induced Liver Injury In Three-Dimensional HepaRG Cell Model

ObjectiveThe liver is not only the main organ of exogenous chemical metabolism,but also an important target organ for the toxic action of drugs and their metabolites.The non-nutritive substances from in vivo and in vitro,such as drugs,poisons and some metabolites in vivo,can be completely decomposed or excreted out of the body by metabolism.Over 1,100 commonly used chemical drugs,herbal medicines,natural products,vitamins,minerals,and dietary supplements have been estimated to have the potential to cause hepatotoxicity.Chemical-induced liver diseases,are the most frequent causes of acute liver failure among drug-induced liver injury,resulting in considerable mortality and great economic burden to medical care systems worldwide.Therefore,it is necessary to assess the safety of chemicals to prevent the injury for human.However,there still remains obstacles that prevent the accuracy of the hazards and risks of chemical assessment.For instance,animal models cannot accurately replicate the etiology and pathogenesis of human liver injury owing to the species differences in the expression and catalytic activities of the liver proteins and enzymes involved in drug absorption,distribution,metabolism,and excretion.The two-dimensional（Two-dimensional,2D）cell in vitro model has the advantages of high throughput and inexpensive compared to the traditional toxicity system based on whole-animal testing.But there are still obstacles,such as lack of metabolic enzyme activity and limited growth activity.Therefore,developing in vitro test platforms that can closely mimic the human hepatocytes is crucial to the assessment of chemical-induced liver injury.Three-dimensional（Three-dimentional,3D）in vitro models are emerging tools for toxicity testing and safety evaluation.Compared with traditional two-dimensional cells,3D cells have better simulate the internal environment and function of human body.As a viable alternative for human primary hepatocyte（Primary human hepatocytes,PHH）,Hepa RG cells have been increasingly used in the exploration of liver diseases and the evaluation of drug hepatotoxicity.Therefore,the Hepa RG cells were chose to establish the 3D hepatocyte model.In addition,3D models were compared the difference of metabolic function and evaluate the hepatotoxicity induced by paracetamol（Acetaminophen,APAP）and troglitazone（Troglitazone,TRO）with 2D cells.Based on the results of the hepatotoxicity evaluation,the suitable in vitro model was selected and applied to assess the safety of ricin and abrin toxins and to explore their mechanisms of toxicity.Methods1.The 3D culture of Hepa RG cells was established by ultra-low attachment method.Cells were initially seeded at density of 1,000,2,000,or 6,000 per well to promote self-aggregation of cells into spheroids.Morphological changes were recorded at1^st,3^rd,7^th,10^th,14^thday after seeding.To evaluate the metabolic competence of3D/2D-cultured Hepa RG cells,the activity and protein expression of CYP3A4 were measured by P450-Glo assay and Western blotting.2.To evaluate the hepatotoxicity induced by acetaminophen and troglitazone,2D/3D Hepa RG cells were treated with acetaminophen（0.16,0.8,4,20 m M）and troglitazone（3.125,6.25,12.5,25 and 50μM,respectively）.The cell viability was measured by Alamar blue.The mitochondrial superoxide,mitochondrial membrane potential and mitochondrial mass were detected by high connotation imaging analysis.3.To assess the metabolism of APAP in 2D and 3D Hepa RG cultures,the levels of metabolism were measured using Liquid chromatography–mass spectrometry（LC-MS）after 24 h APAP exposure.4.To evaluate the hepatotoxicity induced by ricin and abrin,2D/3D Hepa RG cells were treated with ricin（1.25,2.5,5,10,20 ng/m L）and abrin（1.25,2.5,5,10,20 ng/m L,respectively）in different time and measured the cell viability,mitochondrial superoxide and mitochondrial mass.Results1.The 3D culture of Hepa RG cells was established by ultra-low attachment method.Cells spontaneously aggregated in the 3D culture to form compact spherical units on day 3 after seeding.The initial cell densities of 1,000,2,000,and 6,000 cells produced spheroids with diameters of approximately 180,250,and 300μm,respectively,on day 7.The 3D spheroids remained stable diameter and shape throughout the rest of the 7-day experimental period.2.Comparing the difference of CYP3A4 activity and expression between 2D Hepa RG cells and 3D Hepa RG cells on the 7th day,it was found that 3D Hepa RG cells showed higher CYP3A4 activity and expression than 2D cells,suggesting that 3D Hepa RG cells had higher metabolic ability than 2D cells.3.The viability of both 3D-and 2D cultured cells was significantly decreased by APAP in a concentration-dependent manner.3D cultures showed toxicity at lower concentration（0.8 m M）.Furthermore,APAP induced mitochondrial superoxide accumulation and mitochondrial membrane potential loss in 2D/3D cells.3D cultures were more sensitive to APAP-induced mitochondrial damage than 2D cultures were,based on measurements of mitochondrial superoxide accumulation and mitochondrial membrane potential loss,suggesting that the related indexes of mitochondria can be used as sensitive biomarkers of 3D cultured Hepa RG cells.Both APAP-sulfate and APAP-GSH levels were significantly higher in 3D cultures than they were in 2D cultures,indicating that 3D Hepa RG model had better metabolic ability.It showed that3D cells had potential application value in the evaluation of drug-induced liver injury and other hepatotoxicity tests.4.Under the toxic dose of APAP in human the corresponding predicted maximum liver concentration was 1.19 m M by the PBPK model.Based on the lowest significant toxic concentrations of APAP in vitro for the mitochondrial toxicity biomarkers evaluated in 2D and 3D cultures,the 3D model was closer to the predicted value of PBPK than the 2D model,suggesting that the 3D culture was more sensitive and closer to the human liver concentration.It indicated that 3D culture was more suitable for the assessment of chemical-induced hepatotoxicity.5.The cell viability of 3D cultures was significantly decreased by troglitazone in a concentration-dependent manner.Troglitazone induced mitochondrial damage in 3D cultures as evidenced by enhanced ROS accumulation,mitochondrial membrane potential loss,and reduction of mitochondrial mass.6.The cell viability of 3D cultures was significantly decreased by ricin in a concentration-dependent manner.The IC₅₀ value was 21.98 ng/m L.mitochondrial ROS increased in 3D Hepa RG which was 148%of that of the control group at 10 ng/m L.7.The cell viability of 3D cultures was significantly decreased by abrin in a concentration-dependent manner.The IC₅₀ value was 8.73 ng/m L.Mitochondrial ROS increased in 3D Hepa RG which was 112%and 150%of the control group at 2.5 ng/m L and 10 ng/m L,respectively.Conclusion1.The 3D Hepa RG model was successfully established by ultra-low attachment method.3D Hepa RG cultures are promising models in the assessment of chemical induced liver injury.2.Ricin and abrin showed significant dose-dependent cytotoxicity and mitochondrial toxicity in 3D Hepa RG cells.Mitochondrial injury may be the key events of hepatocytotoxicity induced by ricin and abrin.