Assessment of in vitro cardiotoxicity using metabolomics and 13C metabolic flux analysis

The potential of respiration measurements, metabolomics and 13C metabolic flux analysis (13CMFA) for the determination of drug-induced cardiotoxicity was analysed. Two cardiac in vitro models, namely murine HL-1 cells and human embryonic stem cell derived cardiomyocytes (hESC-CM) were applied for this purpose. Respiration measurement in HL-1 cells upon drug treatment revealed distinct EC50 profiles. The toxicity occurred either fast or with a delay. This effect was dependant on the mechanism of toxicity of the respective drugs. Metabolite profiling of HL-1 cells in response to sub-toxic drug concentrations was carried out by using HPLC. The considered metabolites included glucose, lactate, pyruvate and amino acids. The metabolic profiles were drug class dependant, as shown by multivariate statistics, thereby allowing classification of drugs according to their mechanisms of action. 13C-MFA was carried out to determine the effect of Ca2+ channel blocker verapamil and the cytostatic drug doxorubicin on the central metabolism at concentrations which were clinically relevant and non-toxic. Verapamil-treatment resulted in a highly efficient glucose metabolism in HL-1 cells. In both HL-1 cell and hESC-CM, doxorubicin-treatment resulted in an increased oxidative metabolism, most likely to avoid ATP-depletion. The obtained results potentially have pharmacological relevancy, but also provide novel strategies for preclinical toxicity determination of new drug compounds.In dieser Arbeit wurde das Potential von Respirationsmessungen, Metabolomics-Anwendungen und 13C basierten metabolischen Flussanalysen (13C-MFA) zur Bestimmung von Medikamenteninduzierter Kardiotoxizität untersucht. Es wurden HL-1 Kardiomyozyten sowie aus humanen embryonalen Stammzellen gewonnene Herzzellen (hESC-CM) als in vitro Modelle verwendet. Die Respirationsmessungen an HL-1 Zellen ergaben je nach Medikament sehr unterschiedliche EC50-Dynamiken. Der toxische Effekt trat entweder sehr schnell oder mit einer zeitlichen Verzögerung ein. Die EC50-Dynamiken waren von den Toxizitätsmechanismen der entsprechenden Medikamente abhängig. Die Erstellung von Metabolit-Profilen in HL-1 Zellen wurde nach Gabe von subtoxischen Medikamentenkonzentrationen mittels HPLC durchgeführt. Es wurden Glukose, Laktat, Pyruvat sowie 20 Aminosäuren gemessen. Mit Hilfe multivariater statistischer Methoden konnten Medikamentenklassen-abhängige Metabolit-Profile bestimmt werden. 13C-MFA wurde angewandt, um den Einfluss des Kalziumkanal-Blockers Verapamil sowie des Zytostatikums Doxorubicin auf den Zentralstoffwechsel zu bestimmen. Die betrachteten Konzentrationen der Wirkstoffe waren sowohl klinisch relevant also auch nicht toxisch. Die Behandlung von HL-1 Zellen mit Verapamil resultierte in einer deutlich höheren Effizienz in der Glukosenutzung. Sowohl in hESC-CM als auch in HL-1 Zellen resultierte die Doxorubicin- Behandlung in einer Zunahme des oxidativen Stoffwechsels, welche wahrscheinlich der Aufrechterhaltung der intrazellulären ATP-Konzentration dient. Die erzielten Ergebnisse haben pharmakologische Relevanz und zeigen des Weiteren auch neue Strategien für präklinische Kardiotoxizitäts-Messungen von neuen Wirkstoffen

Beurteilung von in vitro Kardiotoxizität mit Hilfe von Metabolomics und 13C metabolischer Flussanalyse

The potential of respiration measurements, metabolomics and 13C metabolic flux analysis (13CMFA) for the determination of drug-induced cardiotoxicity was analysed. Two cardiac in vitro models, namely murine HL-1 cells and human embryonic stem cell derived cardiomyocytes (hESC-CM) were applied for this purpose. Respiration measurement in HL-1 cells upon drug treatment revealed distinct EC50 profiles. The toxicity occurred either fast or with a delay. This effect was dependant on the mechanism of toxicity of the respective drugs. Metabolite profiling of HL-1 cells in response to sub-toxic drug concentrations was carried out by using HPLC. The considered metabolites included glucose, lactate, pyruvate and amino acids. The metabolic profiles were drug class dependant, as shown by multivariate statistics, thereby allowing classification of drugs according to their mechanisms of action. 13C-MFA was carried out to determine the effect of Ca2+ channel blocker verapamil and the cytostatic drug doxorubicin on the central metabolism at concentrations which were clinically relevant and non-toxic. Verapamil-treatment resulted in a highly efficient glucose metabolism in HL-1 cells. In both HL-1 cell and hESC-CM, doxorubicin-treatment resulted in an increased oxidative metabolism, most likely to avoid ATP-depletion. The obtained results potentially have pharmacological relevancy, but also provide novel strategies for preclinical toxicity determination of new drug compounds.In dieser Arbeit wurde das Potential von Respirationsmessungen, Metabolomics-Anwendungen und 13C basierten metabolischen Flussanalysen (13C-MFA) zur Bestimmung von Medikamenteninduzierter Kardiotoxizität untersucht. Es wurden HL-1 Kardiomyozyten sowie aus humanen embryonalen Stammzellen gewonnene Herzzellen (hESC-CM) als in vitro Modelle verwendet. Die Respirationsmessungen an HL-1 Zellen ergaben je nach Medikament sehr unterschiedliche EC50-Dynamiken. Der toxische Effekt trat entweder sehr schnell oder mit einer zeitlichen Verzögerung ein. Die EC50-Dynamiken waren von den Toxizitätsmechanismen der entsprechenden Medikamente abhängig. Die Erstellung von Metabolit-Profilen in HL-1 Zellen wurde nach Gabe von subtoxischen Medikamentenkonzentrationen mittels HPLC durchgeführt. Es wurden Glukose, Laktat, Pyruvat sowie 20 Aminosäuren gemessen. Mit Hilfe multivariater statistischer Methoden konnten Medikamentenklassen-abhängige Metabolit-Profile bestimmt werden. 13C-MFA wurde angewandt, um den Einfluss des Kalziumkanal-Blockers Verapamil sowie des Zytostatikums Doxorubicin auf den Zentralstoffwechsel zu bestimmen. Die betrachteten Konzentrationen der Wirkstoffe waren sowohl klinisch relevant also auch nicht toxisch. Die Behandlung von HL-1 Zellen mit Verapamil resultierte in einer deutlich höheren Effizienz in der Glukosenutzung. Sowohl in hESC-CM als auch in HL-1 Zellen resultierte die Doxorubicin- Behandlung in einer Zunahme des oxidativen Stoffwechsels, welche wahrscheinlich der Aufrechterhaltung der intrazellulären ATP-Konzentration dient. Die erzielten Ergebnisse haben pharmakologische Relevanz und zeigen des Weiteren auch neue Strategien für präklinische Kardiotoxizitäts-Messungen von neuen Wirkstoffen

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 muM 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 muM) 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

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

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

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

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

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

Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells

Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.publishe