Fozia Noor

3D Organotypic Cultures of Human HepaRG Cells: A Tool for In Vitro Toxicity Studies

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.

Metabolomics in toxicology and preclinical research.

Metabolomics, the comprehensive analysis of metabolites in a biological system, provides detailed information about the biochemical/physiological status of a biological system, and about the changes caused by chemicals. Metabolomics analysis is used in many fields, ranging from the analysis of the physiological status of genetically modified organisms in safety science to the evaluation of human health conditions. In toxicology, metabolomics is the -omics discipline that is most closely related to classical knowledge of disturbed biochemical pathways. It allows rapid identification of the potential targets of a hazardous compound. It can give information on target organs and often can help to improve our understanding regarding the mode-of-action of a given compound. Such insights aid the discovery of biomarkers that either indicate pathophysiological conditions or help the monitoring of the efficacy of drug therapies. The first toxicological applications of metabolomics were for mechanistic research, but different ways to use the technology in a regulatory context are being explored. Ideally, further progress in that direction will position the metabolomics approach to address the challenges of toxicology of the 21st century. To address these issues, scientists from academia, industry, and regulatory bodies came together in a workshop to discuss the current status of applied metabolomics and its potential in the safety assessment of compounds. We report here on the conclusions of three working groups addressing questions regarding 1) metabolomics for in vitro studies 2) the appropriate use of metabolomics in systems toxicology, and 3) use of metabolomics in a regulatory context.

3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies.

Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between experimental in vitro and in vivo data. Using 3D HepaRG organotypic cultures we tested four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) for toxic effects and compared the results with 2D HepaRG and HepG2 cultures. We show that 3D HepaRG cultures are more sensitive than the other tested cultures to aflatoxin B1 which is only toxic upon metabolic activation in the liver. We observed that CYP3A4 activity is higher in the 3D HepaRG cultures compared to the 2D HepaRG cultures. Furthermore, we investigated repeated dose toxicity of chlorpromazine and assessed its effects on glucose and lactate metabolism. Sub-toxic concentrations of chlorpromazine induced significant metabolic changes in both 2D and 3D HepaRG cultures upon acute and repeated dose (3 doses) exposure. In summary, our data support the hypothesis that 3D cell culture models better mimic the in vivo tissue and improve cellular functionality. The 3D HepaRG organotypic cultures represent a high throughput system for drug toxicity screening. This system is therefore a promising tool in preclinical testing of human relevance which can allow reducing and/or replacing animal testing for drug adverse effects.

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

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. Copyright

Enhanced cellular uptake and cytotoxicity studies of organometallic bioconjugates of the NLS peptide in Hep G2 cells.

Space invaders: Organometallic fragments such as the ferrocenyl group (shown in red in the picture) help to enhance cellular entry of NLS peptides. Eventually, these nontoxic conjugates find their way to the cellular nucleus as shown by fluorescence microscopy studies in this work.

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

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.

An integrated approach to improved toxicity prediction for the safety assessment during preclinical drug development using Hep G2 cells.

Efficient and accurate safety assessment of compounds is extremely important in the preclinical development of drugs especially when hepatotoxicity is in question. Multiparameter and time resolved assays are expected to greatly improve the prediction of toxicity by assessing complex mechanisms of toxicity. An integrated approach is presented in which Hep G2 cells and primary rat hepatocytes are compared in frequently used cytotoxicity assays for parent compound toxicity. The interassay variability was determined. The cytotoxicity assays were also compared with a reliable alternative time resolved respirometric assay. The set of training compounds consisted of well known hepatotoxins; amiodarone, carbamazepine, clozapine, diclofenac, tacrine, troglitazone and verapamil. The sensitivity of both cell systems in each tested assay was determined. Results show that careful selection of assay parameters and inclusion of a kinetic time resolved assay improves prediction for non-metabolism mediated toxicity using Hep G2 cells as indicated by a sensitivity ratio of 1. The drugs with EC(50) values 100 microM or lower were considered toxic. The difference in the sensitivity of the two cell systems to carbamazepine which causes toxicity via reactive metabolites emphasizes the importance of human cell based in-vitro assays. Using the described system, primary rat hepatocytes do not offer advantage over the Hep G2 cells in parent compound toxicity evaluation. Moreover, respiration method is non invasive, highly sensitive and allows following the time course of toxicity. Respiration assay could serve as early indicator of changes that subsequently lead to toxicity.

In‐depth physiological characterization of primary human hepatocytes in a 3D hollow‐fiber bioreactor

As the major research focus is shifting to three‐dimensional (3D) cultivation techniques, hollow‐fiber bioreactors, allowing the formation of tissue‐like structures, show immense potential as they permit controlled in vitro cultivation while supporting the in vivo environment. In this study we carried out a systematic and detailed physiological characterization of human liver cells in a 3D hollow‐fiber bioreactor system continuously run for > 2 weeks. Primary human hepatocytes were maintained viable and functional over the whole period of cultivation. Both general cellular functions, e.g. oxygen uptake, amino acid metabolism and substrate consumption, and liver‐specific functions, such as drug‐metabolizing capacities and the production of liver‐specific metabolites were found to be stable for > 2 weeks. As expected, donor‐to‐donor variability was observed in liver‐specific functions, namely urea and albumin production. Moreover, we show the maintenance of primary human hepatocytes in serum‐free conditions in this set‐up. The stable basal cytochrome P450 activity 3 weeks after isolation of the cells demonstrates the potential of such a system for pharmacological applications. Liver cells in the presented 3D bioreactor system could eventually be used not only for long‐term metabolic and toxicity studies but also for chronic repeated dose toxicity assessment. Copyright

High throughput, non-invasive and dynamic toxicity screening on adherent cells using respiratory measurements.

A dynamic respiration assay based on luminescence decay time detection of oxygen for high throughput toxicological assessment is presented. The method uses 24-well plates (OxoDishes) read with the help of a sensor dish reader placed in a humidified CO(2)-incubator. Adherent primary rat hepatocytes and the human hepatic cell line Hep G2 were exposed to known toxic compounds. Dissolved oxygen concentration, a measure of respiration, was measured with an oxygen sensor optode immobilized in the centre of each well. The cells were maintained in the dishes during the assay period and can afterwards be processed for further analyses. This dynamic, non-invasive measurement allowed calculation of 50% lethal concentrations (LC(50)) for any incubation time point giving concentration-time-dependent responses without further manipulation or removal of the cells from the incubator. Toxicokinetic profiles are compared with Sulforhodamine B assay, a common cytotoxicity assay. The novel assay is robust and flexible, very easy to carry out and provides continuous online respiration data reflecting dynamic toxicity responses. It can be adapted to any cell-based system and the calculated kinetics contributes to understanding of cell death mechanisms.

Doxorubicin Increases Oxidative Metabolism in HL-1 Cardiomyocytes as Shown by 13C Metabolic Flux Analysis

Doxorubicin (DXR), an anticancer drug, is limited in its use due to severe cardiotoxic effects. These effects are partly caused by disturbed myocardial energy metabolism. We analyzed the effects of therapeutically relevant but nontoxic DXR concentrations for their effects on metabolic fluxes, cell respiration, and intracellular ATP. (13)C isotope labeling studies using [U-(13)C(6)]glucose, [1,2-(13)C(2)]glucose, and [U-(13)C(5)]glutamine were carried out on HL-1 cardiomyocytes exposed to 0.01 and 0.02 μM DXR and compared with the untreated control. Metabolic fluxes were calculated by integrating production and uptake rates of extracellular metabolites (glucose, lactate, pyruvate, and amino acids) as well as (13)C-labeling in secreted lactate derived from the respective (13)C-labeled substrates into a metabolic network model. The investigated DXR concentrations (0.01 and 0.02 μM) had no effect on cell viability and beating of the HL-1 cardiomyocytes. Glycolytic fluxes were significantly reduced in treated cells at tested DXR concentrations. Oxidative metabolism was significantly increased (higher glucose oxidation, oxidative decarboxylation, TCA cycle rates, and respiration) suggesting a more efficient use of glucose carbon. These changes were accompanied by decrease of intracellular ATP. We conclude that DXR in nanomolar range significantly changes central carbon metabolism in HL-1 cardiomyocytes, which results in a higher coupling of glycolysis and TCA cycle. The myocytes probably try to compensate for decreased intracellular ATP, which in turn may be the result of a loss of NADH electrons via either formation of reactive oxygen species or electron shunting.