9 research outputs found
Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress
International audience; High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14 days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15 ”M induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5 ”M CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress
Evaluierung der Kinetik anhand von in vitro Systemen - Ein Beitrag um die Anzahl von Tierversuchen zur ToxizitĂ€tsprĂŒfung zu reduzieren
The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program âPredict-IVâ; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of âomicsâ technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling.
This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day.
Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors.
Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling.
Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions.
Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo.
Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compoundâs distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.Die Erlassung von Richtlinien und Verordnungen durch die EU fĂŒhrte zu der Entwicklung von alternativen Testmethoden als Ersatz von Tierversuchen zur Risikobewertung von Xenobiotika. Des Weiteren weisen hohe Ausfallraten von Arzneimitteln in der spĂ€ten Entwicklungsphase auf die Notwendigkeit hin, die bisher verwendeten Testmethoden der prĂ€klinischen Phase zu verbessern. Diese Punkte wurden in dem im siebten Rahmenprogramm der EU finanzierten Projekt âPredict-IVâ aufgegriffen. Ziel des Projektes war es, die Vorhersage der Arzneimittelsicherheit durch integrierte âomicsâ-Technologien, angewendet an etablierten in vitro AnsĂ€tzen, zu verbessern. Dabei standen drei Zielorgane bzgl. Arzneimittel-induzierter OrgantoxizitĂ€t im Mittelpunkt: Leber, Niere und zentrales Nervensystem, die jeweils durch Zelllinien oder primĂ€re Zellen vertreten waren. Um die in vitro generierten Dynamik-Daten mit der in vivo Situation in Korrelation zu bringen, muss die Kinetik der Testsubstanz berĂŒcksichtigt und die Ergebnisse mit Hilfe von physiologisch-basierter pharmakokinetischer Modellierung extrapoliert werden.
Ziel der vorliegenden Arbeit war es, Kinetik-Daten der gewĂ€hlten Testsubstanzen (Cyclosporin A, Adefovir dipivoxil und Cisplatin) in vitro zu erheben und bzgl. ihrer ZuverlĂ€ssigkeit sowie ihrer Relevanz verglichen mit in vivo Daten zu beurteilen. HierfĂŒr wurden kultivierte Zellen tĂ€glich bzw. jeden zweiten Tag fĂŒr zwei Wochen mit zwei verschiedenen Konzentrationen (toxisch und nicht toxisch) des Arzneimittels behandelt. Der Gehalt des applizierten Arzneimittels oder die Hauptmetaboliten wurden mittels LC MS/MS oder ICP-MS in Vehikel, Medium und Zellen sowie die vom Plastik adsorbierte Menge in Proben bestimmt, die am ersten und letzten Behandlungstag zu fĂŒnf unterschiedlichen Zeitpunkten gewonnen wurden.
Eine eindeutige Bioakkumulation von Cyclosporin A wurde in primĂ€ren Rattenhepatozyten nach Behandlung mit der hohen Konzentration festgestellt. Eine effiziente CYP3A4- und CYP3A5-vermittelte Biotransformation von Cyclosporin A wurde fĂŒr primĂ€re humane Hepatozyten sowie HepaRG Zellen beobachtet. Diese Ergebnisse stimmten mit der in vivo Situation ĂŒberein. Ratten sind aufgrund ihrer geringen CYP3A AktivitĂ€t schlechte Tiermodelle fĂŒr CYP3A-Biotransformationsstudien. Des Weiteren wurden Interassay-Schwankungen bei primĂ€ren human Hepatozyten bemerkt, die auf die biologische VariabilitĂ€t der CYP3A4- sowie CYP3A5-AktivitĂ€t zwischen den menschlichen Spendern zurĂŒckzufĂŒhren sind. Rattenhepatozyten und HepaRG Zellen hingegen wiesen Interassay-Schwankungen auf, die durch unterschiedliche Cyclosporin A Behandlungskonzentrationen zwischen den Replikaten verursacht wurden. Die Cyclosporin A Biotransformation war in HepaRG Zellen am stĂ€rksten ausgeprĂ€gt, was durch stabile und wesentlich höhere CYP3A4- und CYP3A5-AktivitĂ€t in HepaRG Zellen zu erklĂ€ren ist. ZusĂ€tzlich wurden die in vitro Clearance-Werte bestimmt und auf in vivo Clearance-Werte extrapoliert. Alle extrapolierten Werte waren zu hoch geschĂ€tzt (primĂ€re Rattenhepatozyten: 10fach, primĂ€re human Hepatpzyten: 2fach, HepaRG Zellen: 2fach). Diese Ergebnisse sollten mittels physiologisch-basierter pharmakokinetischer Modellierung sowie durch weitere Experimente ĂŒberprĂŒft werden, um zu ermitteln, ob diese hohen SchĂ€tzungen fĂŒr jedes System konstant sind und somit durch die EinfĂŒhrung von weiteren Skalierungsfaktoren verringert werden können.
Kultivierte Gehirnzellen, primĂ€re Nervenzellkulturen der Kortex von MĂ€usen und primĂ€re Hirnzellaggregate der Ratte, zeigten schnell erreichte Cyclosporin A Gleichgewichtskonzentrationen. Diese Ergebnisse deuteten auf eine Verteilung von Cyclosporin A zwischen der wĂ€ssrigen und organischen Phase hin, wobei biologische Prozesse nur eine untergeordnete Rolle spielen. Daher scheint die intrazellulĂ€re Cyclosporin A Aufnahme Transporter-vermittelt zu sein. Ergebnisse der Transporter Experimente, die an einer kĂŒnstlichen Membran und Caco-2 Zellen durchgefĂŒhrt wurden, unterstĂŒtzten diese Hypothese. Messungen der Plastikbindung von Cyclosporin A zeigten signifikante, aber fĂŒr jedes Zellsystem unterschiedliche, Adsorptionsraten, die mittels physiologisch-basierter pharmakokinetischer Modellierung berĂŒcksichtigt werden sollten.
Die Kinetik von Adefovir dipivoxil machte auf die Nachteile von in vitro Versuchen aufmerksam. FĂŒr die intrazellulĂ€re Aufnahme von Adefovir sind aktive Transportproteine nötig, die jedoch in der Nierenzelllinie RPTEC/TERT1 nicht funktionell vorhanden sind. Daher war die Aufnahme von Adefovir auf die passive Diffusion von Adefovir dipivoxil beschrĂ€nkt, das aber auch zeitabhĂ€ngig unter den experimentellen Konditionen zerfiel.
Die an RPTEC/TERT1 Zellen untersuchte Kinetik von Cisplatin deutete auf eine intrazellulĂ€re Platin-Anreicherung hin, die jedoch nicht in einer signifikanten Bioakkumulation resultierte. Möglicherweise sind innerhalb von 14 Tagen die Gleichgewichtskonzentrationen von Cisplatin noch nicht erreicht. Die Kinetikprofile von Cisplatin in Medium lieĂen einen aktiven, von der basolateralen zur apikalen Seite gerichteten Cisplatin Transport erkennen, wie schon in vivo beschrieben, wobei die Geschwindigkeit dieser Transportprozesse in vitro langsamer zu sein scheint als in der intakte Niere. Daher mĂŒssen die generierten Daten zur SchĂ€tzung von zellulĂ€ren Prozessen modelliert werden, um durch anschlieĂende Extrapolation mit in vivo Daten verglichen werden zu können.
AbschlieĂend bleibt zu sagen, dass das experimentelle Design vorteilhaft war. Wiederholte tĂ€gliche Administration von zwei unterschiedlichen Konzentrationen eines Medikaments ermöglichte die Erfassung von Bioakkumulation bei toxischen Konzentrationen sowie Biotransformation/Export bei nicht-toxischen Konzentrationen. Potenzielle Fehler, die zu einer Fehlinterpretation fĂŒhren könnten, wurden durch die exakte Bestimmung der tatsĂ€chlich applizierten Arzneimittelmenge reduziert, da nicht immer die applizierte Konzentration mit der Nominalkonzentration ĂŒbereinstimmt. DarĂŒber hinaus erwies es sich als Vorteil, die Arzneimittelkonzentrationen in den einzelnen Kompartimenten (Medium, Zellen und Plastik) zu bestimmen. Somit konnten zum einen Erkenntnisse ĂŒber die Verteilung der Substanz gewonnen werden und zum anderen FehleinschĂ€tzungen von zellulĂ€ren Prozessen, z.B. Biotransformation, verhindert werden, was letzten Endes bei die Interpretation von Kinetik-Daten behilflich ist. Jedoch, wurden auch die Grenzen von in vitro AnsĂ€tzen deutlich. FĂŒr eine korrekte Extrapolation ist es unverzichtbar, dass die untersuchten in vitro Systeme funktionierende Proteine aufweisen, die bei der untersuchten Arzneimittel-induzierten ToxizitĂ€t eine SchlĂŒsselrolle ĂŒbernehmen. AbschlieĂend kann festgehalten werden, dass es, unter BerĂŒcksichtigung der Vor- und Nachteile, von Nutzen sein kann diesen Versuchsansatz der Langzeitbehandlung zu validieren und darĂŒber hinaus auf Co Kultursysteme sowie Organ-Chips anzuwenden hinsichtlich der Entwicklung von Alternativmethoden fĂŒr ToxizitĂ€tsstudien bei wiederholter Gabe
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Tryptophan-derived, indolic metabolites possess diverse functions in Arabidopsis innate immunity to microbial pathogen infection. Here, we investigate the functional role and regulatory characteristics of indolic metabolism in Arabidopsis systemic acquired resistance (SAR) triggered by the bacterial pathogen Pseudomonas syringae. Indolic metabolism is broadly activated in both P. syringae-inoculated and distant, non-inoculated leaves. At inoculation sites, camalexin, indol-3-ylmethylamine (I3A), and indole-3-carboxylic acid (ICA) are the major accumulating compounds. Camalexin accumulation is positively affected by MYB122, and the cytochrome P450 genes CYP81F1 and CYP81F2. Local I3A production, by contrast, occurs via indole glucosinolate breakdown by PEN2- dependent and independent pathways. Moreover, exogenous application of the defense hormone salicylic acid stimulates I3A generation at the expense of its precursor indol-3-ylmethylglucosinolate (I3M), and the SAR regulator pipecolic acid primes plants for enhanced P. syringae-induced activation of distinct branches of indolic metabolism. In uninfected systemic tissue, the metabolic response is more specific and associated with enhanced levels of the indolics I3A, ICA, and indole-3-carbaldehyde (ICC). Systemic indole accumulation fully depends on functional CYP79B2/3, PEN2, and MYB34/51/122, and requires functional SAR signaling. Genetic analyses suggest that systemically elevated indoles are dispensable for SAR and associated systemic increases of salicylic acid. However, soil-grown but not hydroponically -cultivated cyp79b2/3 and pen2 plants, both defective in indolic secondary metabolism, exhibit pre-induced immunity, which abrogates their intrinsic ability to induce SAR
Development of an in vitro renal epithelial disease state model for xenobiotic toxicity testing
There is a growing impetus to develop more accurate, predictive and relevant in vitro models of renal xenobiotic exposure. As part of the EU-FP7, Predict-IV project, a major aim was to develop models that recapitulate not only normal tissue physiology but also aspects of disease conditions that exist as predisposing risk factors for xenobiotic toxicity. Hypoxia, as a common micro-environmental alteration associated with pathophysiology in renal disease, was investigated for its effect on the toxicity profile of a panel of 14 nephrotoxins, using the human proximal tubular epithelial RPTECT/TERT1 cell line. Changes in ATP, glutathione and resazurin reduction, after 14 days of daily repeat exposure, revealed a number of compounds, including adefovir dipivoxil with enhanced toxicity in hypoxia. We observed intracellular accumulation of adefovir in hypoxia and suggest decreases in the efflux transport proteins MRP4, MRP5, NHERF1 and NHERF3 as a possible explanation. MRP5 and NHERF3 were also down-regulated upon treatment with the HIF-1 activator, dimethyloxalylglycine. Interestingly, adefovir dependent gene expression shifted from alterations in cell cycle gene expression to an inflammatory response in hypoxia. The ability to investigate aspects of disease states and their influence on renal toxin handling is a key advantage of in vitro systems developed here. They also allow for detailed investigations into mechanisms of compound toxicity of potential importance for compromised tissue exposure
Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics
Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 lM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity.JRC.I.1-Chemical Assessment and Testin
Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics
Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2ÎŒM) in a daily repeat dose treating regime for up to 14days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity
Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in the complementary CNS in vitro models
The present study was performed in an attempt to develop an in vitro integrated testing strategy to evaluate neurotoxicity of drugs during development phase. A number of endpoints was analyzed using two complementary brain cell culture models, and an in vitro blood-brain barrier model after acute, sub-chronic, and repeated-dose treatments with a series of selected drugs.
The developed in vitro BBB model allowed to detect toxic effects on the BBB and to evaluate drug transport through the BBB for prediction free brain concentrations of studied drugs. The electrical activity of cortical neuronal networks recorded with a micro-electrode array was found to be a good tool to predict the neuroactivity and neurotoxicity of drugs and it is suggested as a first-step high content screening test. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found well suitable for OMICs analyzes.
The obtained data suggest that an in vitro integrated testing strategy (ITS), including toxicity to and transport through BBB, as well as metabolomics, proteomics and neuronal electrical activity, measured in stable rodent brain cell culture systems (in the future human stem cell-derived neuronal models), may considerably improve current drug-induced neurotoxicity evaluation. Robustness of this ITS has to be further evaluated with a larger number of studied drugs.JRC.I.5-Systems Toxicolog
Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress.
High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the characterized nephrotoxin Cyclosporine A (CsA) at therapeutic and supra therapeutic concentrations for 14 days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15 ”M induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein- response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5 ”M CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress.JRC.I.1-Chemical Assessment and Testin