The utility of HepG2 cells to identify direct mitochondrial dysfunction in the absence of cell death.

Drug-induced mitochondrial dysfunction has been hypothesized to be an important determining factor in the onset of drug-induced liver injury. It is essential to develop robust screens with which to identify drug-induced mitochondrial toxicity and to dissect its role in hepatotoxicity. In this study we have characterised a mechanistically refined HepG2 model, using a panel of selected hepatotoxicants and non-hepatotoxicants. We have demonstrated that acute metabolic modification, via glucose-deprivation over a 4 h period immediately prior to compound addition, is sufficient to allow the identification of drugs which induce mitochondrial dysfunction, in the absence of cell death over a short exposure (2 – 8 h) using a plate-based screen to measure cellular ATP content and cytotoxicity. These effects were verified by measuring changes in cellular respiration, via oxygen consumption and extracellular acidification rates. Overall, these studies demonstrate the utility of HepG2 cells for the identification of mitochondrial toxins which act directly on the electron transport chain and that the dual assessment of ATP content alongside cytotoxicity provides an enhanced mechanistic understanding of the causes of toxicity

Acute Metabolic Switch Assay Using Glucose/Galactose Medium in HepaRG Cells to Detect Mitochondrial Toxicity.

Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantages of working with a cell line, therefore, are considered an alternative for HepG2 cells in drug toxicity screening. A method is described here using HepaRG cells in an acute metabolic switch assay utilizing specific glucose/galactose media, a combined ATP-protein-LDH assay measuring three endpoints from one 96-well plate, and a criteria to label a compound as a mitochondrial toxin. © 2019 by John Wiley & Sons, Inc

Inhibition of tumourigenicity of small cell lung cancer cells by suppressing Id3 expression.

Id3 is over-expressed in small cell lung cancer (SCLC). To test whether the tumourigenicity of SCLC cells can be inhibited by suppressing Id3 expression, we transfected siRNA into SCLC cell line GLC-19 and established two sublines (G-Id3-1 and G-Id3-7) which expressed only 30% of the level of Id3 measured in control transfectants. Suppression of Id3 expression in both G-Id3-1 and G-Id3-7 cells produced significant reductions in proliferation rates and in numbers of colonies formed in soft agar assay. When G-Id3-1, G-Id3-7 and the control transfectants were inoculated subcutaneously into 3 groups (8 each) of nude mice, respectively, all (100%) inoculated animals produced tumours. Although there was no difference in tumour incidents amongst the 3 groups, significant reductions were observed in both size and weight of tumours produced by either G-Id3-1 or G-Id3-7 cells. While the final average volume of tumours produced in control group was 1012.1+/-394 mm(3), it was significantly reduced (p2.4-fold higher than that in control. The results in this study suggest that highly expressed Id3 in SCLC cells may be an important therapeutic target for tumour suppression

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile

© 2016 The Author(s)The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems

Identification of Id family proteins as promoting factors in tumourigenesis of small cell lung cancer

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Data from: Identification of the additional mitochondrial liabilities of 2-hydroxyflutamide when compared to its parent compound, flutamide in HepG2 cells

The androgen receptor antagonist, flutamide, is strongly associated with idiosyncratic drug-induced liver injury (DILI). Following administration, flutamide undergoes extensive first-pass metabolism to its primary metabolite, 2-hydroxyflutamide. Flutamide is a known mitochondrial toxicant; however there has been limited investigation into the potential mitochondrial toxicity of 2-hydroxyflutamide and its contribution to flutamide-induced liver injury. In this study we have used the acute glucose or galactose-conditioning of HepG2 cells to compare the mitochondrial toxicity of flutamide, 2-hydroxyflutamide and the structurally-related, non-hepatotoxic androgen receptor antagonist, bicalutamide. Compound-induced changes in mitochondrial oxygen consumption rate were assessed using Seahorse technology. Permeabilization of cells and delivery of specific substrates and inhibitors of the various respiratory complexes provided more detailed information on the origin of mitochondrial perturbations. These analyses were supported by assessment of downstream impacts including changes in cellular NAD+/NADH ratio. Bicalutamide was not found to be a mitochondrial toxicant, yet flutamide and 2-hydroxyflutamide significantly reduced basal and maximal respiration. Both flutamide and 2-hydroxyflutamide significantly reduced respiratory complex I-linked respiration, though 2-hydroxyflutamide also significantly decreased complex II and V-linked respiration; liabilities not demonstrated by the parent compound. This study has identified for the first time, the additional mitochondrial liabilities of the major metabolite, 2-hydroxyflutamide compared with its parent drug, flutamide. Given the rapid production of this metabolite upon administration of flutamide, but not bicalutamide, we propose that the additional mitochondrial toxicity of 2-hydroxyflutamide may fundamentally contribute to the idiosyncratic DILI seen in flutamide-treated, but not bicalutamide-treated patients

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Data used to created Figures 5-9

The utility of HepaRG cells for bioenergetic investigation and detection of drug-induced mitochondrial toxicity

The importance of mitochondrial toxicity in drug-induced liver injury is well established. The bioenergetic phenotype of the HepaRG cell line was defined in order to assess their suitability as a model of mitochondrial hepatotoxicity. Bioenergetic phenotyping categorised the HepaRG cells as less metabolically active when measured beside the more energetic HepG2 cells. However, inhibition of mitochondrial ATP synthase induced an increase in glycolytic activity of both HepaRG and HepG2 cells suggesting an active Crabtree Effect in both cell lines. The suitability of HepaRG cells for the acute metabolic modification assay as a screen for mitotoxicity was confirmed using a panel of compounds, including both positive and negative mitotoxic compounds. Seahorse respirometry studies demonstrated that a statistically significant decrease in spare respiratory capacity is the first indication of mitochondrial dysfunction. Furthermore, based upon comparing changes in respiratory parameters to those of the positive controls, rotenone and carbonyl cyanide m-chlorophenyl hydrazone, compounds were categorised into two mechanistic groups; inhibitors or uncouplers of the electron transport chain. Overall, the findings from this study have demonstrated that HepaRG cells, despite having different resting bioenergetic phenotype to HepG2 cells are a suitable model to detect drug-induced mitochondrial toxicity with similar detection rates to HepG2 cells

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 μM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants