Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

in Journal of cellular biochemistry (2017)

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the ... [more ▼]

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 9999: 1-8, 2017. (c) 2017 Wiley Periodicals, Inc. [less ▲]

In Silico Modeling for the Prediction of Dose and Pathway-Related Adverse Effects in Humans From In Vitro Repeated-Dose Studies.

in Toxicological sciences : an official journal of the Society of Toxicology (2016), 149(1), 55-66

Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Nowadays advanced human in vitro systems, eg, metabolically competent HepaRG ... [more ▼]

Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Nowadays advanced human in vitro systems, eg, metabolically competent HepaRG cells, are used for toxicity screening. Extrapolation of in vitro toxicity to in vivo effects is possible by reverse dosimetry using pharmacokinetic modeling. We assessed long-term repeated-dose toxicity of bosentan and valproic acid (VPA) in HepaRG cells under serum-free conditions. Upon 28-day exposure, the EC50 values for bosentan and VPA decreased by 21- and 33-fold, respectively. Using EC(10) as lowest threshold of toxicity in vitro, we estimated the oral equivalent doses for both test compounds using a simplified pharmacokinetic model for the extrapolation of in vitro toxicity to in vivo effect. The model predicts that bosentan is safe at the considered dose under the assumed conditions upon 4 weeks exposure. For VPA, hepatotoxicity is predicted for 4% and 47% of the virtual population at the maximum recommended daily dose after 3 and 4 weeks of exposure, respectively. We also investigated the changes in the central carbon metabolism of HepaRG cells exposed to orally bioavailable concentrations of both drugs. These concentrations are below the 28-day EC(10) and induce significant changes especially in glucose metabolism and urea production. These metabolic changes may have a pronounced impact in susceptible patients such as those with compromised liver function and urea cycle deficiency leading to idiosyncratic toxicity. We show that the combination of modeling based on in vitro repeated-dose data and metabolic changes allows the prediction of human relevant in vivo toxicity with mechanistic insights. [less ▲]

3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies.

in Toxicological sciences : an official journal of the Society of Toxicology (2013), 133(1), 67-78

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a ... [more ▼]

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity. [less ▲]

Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review.

in Journal of Applied Toxicology (2011), 31(3), 191-205

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art ... [more ▼]

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given. [less ▲]

Metabolic flux analysis gives an insight on verapamil induced changes in central metabolism of HL-1 cells.

in Journal of biotechnology (2011), 155(3), 299-307

Verapamil has been shown to inhibit glucose transport in several cell types. However, the consequences of this inhibition on central metabolism are not well known. In this study we focused on verapamil ... [more ▼]

Verapamil has been shown to inhibit glucose transport in several cell types. However, the consequences of this inhibition on central metabolism are not well known. In this study we focused on verapamil induced changes in metabolic fluxes in a murine atrial cell line (HL-1 cells). These cells were adapted to serum free conditions and incubated with 4 muM verapamil and [U-(1)(3)C(5)] glutamine. Specific extracellular metabolite uptake/production rates together with mass isotopomer fractions in alanine and glutamate were implemented into a metabolic network model to calculate metabolic flux distributions in the central metabolism. Verapamil decreased specific glucose consumption rate and glycolytic activity by 60%. Although the HL-1 cells show Warburg effect with high lactate production, verapamil treated cells completely stopped lactate production after 24 h while maintaining growth comparable to the untreated cells. Calculated fluxes in TCA cycle reactions as well as NADH/FADH(2) production rates were similar in both treated and untreated cells. This was confirmed by measurement of cell respiration. Reduction of lactate production seems to be the consequence of decreased glucose uptake due to verapamil. In case of tumors, this may have two fold effects; firstly depriving cancer cells of substrate for anaerobic glycolysis on which their growth is dependent; secondly changing pH of the tumor environment, as lactate secretion keeps the pH acidic and facilitates tumor growth. The results shown in this study may partly explain recent observations in which verapamil has been proposed to be a potential anticancer agent. Moreover, in biotechnological production using cell lines, verapamil may be used to reduce glucose uptake and lactate secretion thereby increasing protein production without introduction of genetic modifications and application of more complicated fed-batch processes. [less ▲]

Metabolic profiling using HPLC allows classification of drugs according to their mechanisms of action in HL-1 cardiomyocytes.

in Toxicology and applied pharmacology (2011), 252(2), 183-91

Along with hepatotoxicity, cardiotoxic side effects remain one of the major reasons for drug withdrawals and boxed warnings. Prediction methods for cardiotoxicity are insufficient. High content screening ... [more ▼]

Along with hepatotoxicity, cardiotoxic side effects remain one of the major reasons for drug withdrawals and boxed warnings. Prediction methods for cardiotoxicity are insufficient. High content screening comprising of not only electrophysiological characterization but also cellular molecular alterations are expected to improve the cardiotoxicity prediction potential. Metabolomic approaches recently have become an important focus of research in pharmacological testing and prediction. In this study, the culture medium supernatants from HL-1 cardiomyocytes after exposure to drugs from different classes (analgesics, antimetabolites, anthracyclines, antihistamines, channel blockers) were analyzed to determine specific metabolic footprints in response to the tested drugs. Since most drugs influence energy metabolism in cardiac cells, the metabolite "sub-profile" consisting of glucose, lactate, pyruvate and amino acids was considered. These metabolites were quantified using HPLC in samples after exposure of cells to test compounds of the respective drug groups. The studied drug concentrations were selected from concentration response curves for each drug. The metabolite profiles were randomly split into training/validation and test set; and then analysed using multivariate statistics (principal component analysis and discriminant analysis). Discriminant analysis resulted in clustering of drugs according to their modes of action. After cross validation and cross model validation, the underlying training data were able to predict 50%-80% of conditions to the correct classification group. We show that HPLC based characterisation of known cell culture medium components is sufficient to predict a drug's potential classification according to its mode of action. [less ▲]

Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2.

in Toxicology and applied pharmacology (2009), 240(3), 327-36

Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given ... [more ▼]

Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC(50) values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of these drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs. [less ▲]