Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity

Abstract

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.

Toxicological Comparison of Mancozeb and Zoxamide Fungicides at Environmentally Relevant Concentrations by an In Vitro Approach

“…Overall, this evidence was suggestive of the activation of the aryl hydrocarbon receptor (AhR), which regulates the transcription of NRF2 and CYP1A1 [36,37], both involved in the control of the expression of anti-oxidant enzymes that, as last, act as pro-oxidants and lead to the oxygenation of exogenous compounds, thus contributing to the production of ROS. Depending on their balance, cells may undergo to oxidative stress, cell toxicity and finally to death or not [38,39]. Although limited effects on ROS production were observed, cytotoxicity and apoptosis were significantly induced by MZ in both cell lines, thus an imbalance toward CYP1A1 activation may be hypothesized.…”

Synthesis of Poly(Dimethylmalic Acid) Homo- and Copolymers to Produce Biodegradable Nanoparticles for Drug Delivery: Cell Uptake and Biocompatibility Evaluation in Human Heparg Hepatoma Cells

“…In order to assess the biocompatibility of the NPs produced in this work, we used the human HepaRG hepatoma cell line. The HepaRG cells are undifferentiated bipotent progenitor cells actively proliferating that have the ability to differentiate into quiescent hepatocyte-like cells [ 43 , 44 ], which express most of the liver specific functions including drug metabolism enzymes [ 62 , 63 , 64 , 65 ]. The HepaRG cells are considered to be one of the most relevant hepatic cell models to examine the cytotoxic effect of xenobiotics both in proliferating progenitor and differentiated quiescent cells as well as the cellular uptake of polymeric NPs [ 53 ].…”

“…Such difference and behavior can be explained by the strong phenotypical changes between progenitor and differentiated HepaRG cells. In contrast to progenitor HepaRG cells that actively proliferate but show low expression levels of liver specific functions, differentiated hepatocyte-like HepaRG cells are quiescent and metabolically competent cells expressing most phase I, II, and III detoxifying enzymes [62][63][64]. It can be postulated that the detoxifying machinery of differentiated HepaRG cells catabolize and eliminate the polymers and organic compounds used during the synthesis and/or that the (co)polymers are more toxic towards proliferating cells.…”

Noninvasive Evaluation of HepaRG Aggregates during Drug‐Induced Intrahepatic Cholestasis Using Optical Coherence Tomography

“…Bioimaging techniques are required to understand the liver‐specific structural and functional characteristics of the in vitro 3D spheroid model, as well as fluorescent probes for the in vivo and in vitro bioimaging. [ 5,6 ] Confocal laser scanning microscopy (CLSM) allows the high‐resolution imaging of specific proteins through fluorescence staining and is used to visualize the location of specific protein markers. However, CLSM requires invasive processes, such as permeabilization and optical clearing, for the observation of deep tissues.…”