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1.
Altern Lab Anim ; 39(2): 147-71, 2011 May.
Article in English | MEDLINE | ID: mdl-21639679

ABSTRACT

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.


Subject(s)
Animal Testing Alternatives , Drug Evaluation, Preclinical/methods , Embryonic Stem Cells , Hepatocytes/drug effects , Toxicity Tests/methods , Animals , Bioreactors , Biotransformation , Cell Differentiation , Cell Line , Cell Respiration , Enzyme Induction , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Liver/drug effects , Liver/metabolism , Metabolic Networks and Pathways , Rats
2.
Cardiovasc Toxicol ; 9(3): 107-25, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19572114

ABSTRACT

Cardiotoxicity is among the leading reasons for drug attrition and is therefore a core subject in non-clinical and clinical safety testing of new drugs. European Centre for the Validation of Alternative Methods held in March 2008 a workshop on "Alternative Methods for Drug-Induced Cardiotoxicity" in order to promote acceptance of alternative methods reducing, refining or replacing the use of laboratory animals in this field. This review reports the outcome of the workshop. The participants identified the major clinical manifestations, which are sensitive to conventional drugs, to be arrhythmias, contractility toxicity, ischaemia toxicity, secondary cardiotoxicity and valve toxicity. They gave an overview of the current use of alternative tests in cardiac safety assessments. Moreover, they elaborated on new cardiotoxicological endpoints for which alternative tests can have an impact and provided recommendations on how to cover them.


Subject(s)
Animal Testing Alternatives/methods , Cardiotoxins , Drug-Related Side Effects and Adverse Reactions , Health Planning Guidelines , Animal Testing Alternatives/trends , Animals , Animals, Laboratory , Arrhythmias, Cardiac/chemically induced , Arrhythmias, Cardiac/prevention & control , Cardiotoxins/adverse effects , Cardiotoxins/toxicity , Disease Models, Animal , Drug-Related Side Effects and Adverse Reactions/chemically induced , Drug-Related Side Effects and Adverse Reactions/prevention & control , Education/trends , Europe , Humans
3.
Anal Biochem ; 387(2): 271-5, 2009 Apr 15.
Article in English | MEDLINE | ID: mdl-19454238

ABSTRACT

A novel cell-based fluorometric sensor system for toxicity monitoring is described, which uses functional spontaneously contracting cardiomyocytes (HL-1 cell line) as the biological recognition element. Based on these highly specialized cells, it has the potential of providing a sensitive and relevant analytical in vitro toxicity testing method. The system was configured by propagating the surface-attaching HL-1 cardiomyocytes in the wells of a 96-well microtiter plate and connecting the plate via an optical fiber to a fluorescence spectrometer capable of excitation-emission matrix scanning. The fluorescence data were analyzed using a conventional spectral analysis software program. The performance of the system for detection of general cytotoxicity to the cells was evaluated using three well-known drugs: verapamil, quinidine, and acetaminophen. The dose-response curves were assessed and the EC(50) values were determined (0.10+/-0.007, 0.23+/-0.025, and 12.32+/-2.40 mM, respectively). Comparison with in vitro and in vivo reference data for the drugs showed good correlations, suggesting that this cell-based sensor system could be a useful tool in pharmacological in vitro drug testing.


Subject(s)
Myocytes, Cardiac/drug effects , Spectrometry, Fluorescence/methods , Toxicity Tests/methods , Animals , Cells, Cultured , Mice , Reproducibility of Results
4.
J Pharmacol Toxicol Methods ; 55(1): 35-48, 2007.
Article in English | MEDLINE | ID: mdl-16581270

ABSTRACT

INTRODUCTION: Drug-induced QT interval prolongation may lead to ventricular arrhythmias. The aim of the study was to optimize QT interval data processing to quantify drug-induced QT interval prolongation in the telemetry instrumented conscious dog model. METHODS: The test substances cisapride, dofetilide, haloperidol, and terfenadine and corresponding vehicles were given to male and female beagle dogs during two consecutive 90-min intravenous infusions. Cardiovascular parameters were recorded for 24 h and exposure to the drugs was measured. The delayed response in the QT interval after an abrupt change in heart rate was investigated. Eight mathematical models to describe the QT interval-heart rate relationship were compared and different sets of covariates were used to quantify the drug-induced effect on the QT interval. RESULTS: After an abrupt decrease in heart rate, a 75% adaptation of the QT interval was reached after 54+/-9 s. A linear model was preferred to correct the drug-induced effect on the QT interval for heart rate, vehicle effect, serial correlation, plasma concentration and time of day. All test substances significantly prolonged the QT interval. DISCUSSION: To optimize the processing of QT interval data, the delay in QT interval response after an abrupt change in heart rate should be considered. The QT interval-heart rate relationship and vehicle response were individual-specific and corrections were therefore made individually. When estimating the drug-induced effect on the QT interval it is considered advantageous to use plasma concentration as a covariate, as well as adjusting for vehicle effect and serial correlation in measurements. The conscious dog model detected significant increases in the QT interval for all test substances investigated.


Subject(s)
Cisapride/pharmacology , Electrocardiography/drug effects , Long QT Syndrome/chemically induced , Long QT Syndrome/diagnosis , Algorithms , Animals , Blood Pressure/drug effects , Cisapride/pharmacokinetics , Dogs , Electrocardiography/methods , Electronic Data Processing , Female , Haloperidol/pharmacokinetics , Haloperidol/pharmacology , Heart Rate/drug effects , Male , Phenethylamines/pharmacokinetics , Phenethylamines/pharmacology , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology , Telemetry/methods , Terfenadine/pharmacokinetics , Terfenadine/pharmacology
5.
FEBS Lett ; 579(25): 5680-4, 2005 Oct 24.
Article in English | MEDLINE | ID: mdl-16219308

ABSTRACT

To examine whether cold-induced vascular endothelial growth factor (VEGF) gene expression in brown adipose tissue involved generation of hypoxic oxygen levels by thermogenic processes, we cold-exposed wild-type mice, as well as uncoupling protein-1 (UCP1)-ablated mice in which no thermogenesis in brown adipocytes can be induced. Cold exposure stimulated VEGF expression in both wild-type and UCP1-ablated mice. Unexpectedly, the effect was 3-fold higher in UCP1-ablated animals, whereas cultured brown adipocytes from both genotypes responded identically to norepinephrine stimulation. These results demonstrate that generation of low oxygen levels does not contribute to cold-induced VEGF expression in brown adipose tissue, but the results are consistent with an adrenergic regulation of expression.


Subject(s)
Adipose Tissue, Brown/metabolism , Cold Temperature , Gene Expression Regulation , Thermogenesis , Vascular Endothelial Growth Factor A/genetics , Anaerobiosis , Animals , Carrier Proteins/genetics , Ion Channels , Membrane Proteins/genetics , Mice , Mice, Mutant Strains , Mitochondrial Proteins , Mutation , Oxygen Consumption , RNA, Messenger/analysis , RNA, Messenger/metabolism , Thermogenesis/genetics , Uncoupling Protein 1
6.
Biochem Biophys Res Commun ; 337(1): 121-6, 2005 Nov 11.
Article in English | MEDLINE | ID: mdl-16171784

ABSTRACT

The cellular response to hypoxic stress is mainly mediated via activation of the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). In the present study, the sympathetically controlled brown adipose tissue was used to investigate the effect of norepinephrine on HIF-1alpha gene expression. Norepinephrine increased HIF-1alpha mRNA levels in cultured brown adipocytes, whereas the hypoxia-mimic cobalt was without effect. Cold exposure of mice increased HIF-1alpha gene expression in brown adipose tissue. In UCP1-ablated mice, which are incapable of inducing thermogenic oxygen consumption in brown adipose tissue, cold exposure generated a significantly higher elevation of HIF-1alpha mRNA levels than in wild-type. These results demonstrate that cold-induced HIF-1alpha gene expression is independent of thermogenic oxygen consumption leading to hypoxia, but is consistent with a norepinephrine regulation of HIF-1alpha gene expression. Thus, by elevating HIF-1alpha gene expression, norepinephrine may mediate an increased potential to respond to hypoxia in brown adipose tissue and possibly in other tissues.


Subject(s)
Adipocytes/metabolism , Adipose Tissue, Brown/metabolism , Norepinephrine/pharmacology , Transcription Factors/metabolism , Adipocytes/drug effects , Adipose Tissue, Brown/cytology , Animals , Cell Hypoxia , Cells, Cultured , Cold Temperature , Female , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Mice, Inbred C57BL , RNA, Messenger/metabolism , Transcription Factors/genetics , Up-Regulation
7.
Biochem J ; 364(Pt 1): 73-9, 2002 May 15.
Article in English | MEDLINE | ID: mdl-11988078

ABSTRACT

Because of the central role of adrenergic mechanisms in the expression of crucial genes during brown adipocyte differentiation, we examined the activation (phosphorylation) of CREB (cAMP-response-element-binding protein) in mouse brown adipocytes in primary culture. We found that noradrenaline ('norepinephrine') stimulated CREB phosphorylation rapidly (maximum effect in < or =5 min with slow decay) and efficiently (EC(50), 6 nM). The increase in CREB phosphorylation coincided with increased expression of an artificial cAMP-response-element-containing reporter construct. CREB phosphorylation was partly inhibitable, both by the beta-adrenergic antagonist propranolol and by the alpha(1)-adrenergic antagonist prazosin. Adenylate cyclase hyperactivation (by forskolin) could stimulate CREB phosphorylation to the same extent as noradrenaline. The alpha(1)-adrenergic agonist cirazoline also increased CREB phosphorylation. An increase in intracellular [Ca(2+)] had, however, no effect, but protein kinase C activation by PMA was a potent stimulator. The cirazoline-stimulated (alpha(1)-adrenergic) CREB phosphorylation was inhibited by a desensitizing pretreatment with PMA, demonstrating that the alpha(1)-stimulation was mediated via protein kinase C activation; neither Src nor extracellular-signal-regulated kinases 1 and 2 activation was involved in the signalling process. We conclude that CREB phosphorylation in brown adipocytes is mediated not only through the classical beta-adrenergic/cAMP pathway but also through a novel alpha(1)-adrenergic/protein kinase C/CREB pathway, which has not been described previously in any tissue.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Protein Kinase C/metabolism , Receptors, Adrenergic, alpha-1/metabolism , Adipocytes/metabolism , Adrenergic alpha-Agonists/pharmacology , Animals , Carcinogens , Cyclic AMP/metabolism , Enzyme Activation , Imidazoles/pharmacology , Immunoblotting , Luciferases/metabolism , MAP Kinase Signaling System , Male , Mice , Models, Biological , Norepinephrine/pharmacology , Phosphorylation , Protein Binding , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology , Time Factors , Transfection
8.
Exp Cell Res ; 274(2): 207-15, 2002 Apr 01.
Article in English | MEDLINE | ID: mdl-11900481

ABSTRACT

We have examined whether a qualitative switch occurs in the response of the ribonucleotide reductase (RNR) genes to the effect of the physiological cAMP-elevating agent norepinephrine (NE) during the development of brown adipocytes. Basal expression of the genes for both RNR subunits, R1 and R2, was high in proliferating cells, but was markedly down-regulated in parallel with adipocyte differentiation. NE stimulation, which promotes DNA synthesis and proliferation of brown preadipocytes, resulted in an increased expression of the R2 gene in proliferating cells (1.6-fold), but was without effect on R1 expression. In contrast, NE stimulation of confluent differentiating brown adipocytes reduced both R1 and R2 expression. The NE stimulation of R2 expression in preadipocytes was mimicked by forskolin and abolished by H89, demonstrating mediation via cAMP and protein kinase A (PKA). Also, inhibitors of Src and of Erk1/2 kinases markedly reduced NE-stimulated R2 expression. We conclude that adrenergic stimulation of brown adipocytes by NE specifically elevates expression of the RNR subunit R2 gene in the proliferative stage of brown adipocyte development, the mediating pathway being a cAMP/PKA cascade further involving Src and the MAP kinase Erk1/2. These results suggest that adrenergic stimulation of brown adipocyte proliferation may act at the level of gene expression of the limiting subunit for RNR activity, R2, and demonstrate a qualitative switch in the response of the R2 gene to cAMP-elevating agents as a consequence of the switch from proliferating to differentiating cell status.


Subject(s)
Adipocytes/enzymology , Adipose Tissue, Brown/enzymology , Adipose Tissue, Brown/growth & development , Cell Differentiation/physiology , Cell Division/physiology , Gene Expression Regulation, Developmental/physiology , Norepinephrine/metabolism , Ribonucleotide Reductases/genetics , Adipocytes/cytology , Adipocytes/drug effects , Adipose Tissue, Brown/cytology , Animals , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Developmental/drug effects , Mice , Mice, Inbred Strains , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Norepinephrine/pharmacology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Up-Regulation/drug effects , Up-Regulation/physiology , src-Family Kinases/drug effects , src-Family Kinases/metabolism
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