Snodland, United Kingdom
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Braeuning A.,University of Tübingen | Gavrilov A.,University of Tübingen | Brown S.,CXR Biosciences | Roland Wolf C.,University of Dundee | And 2 more authors.
Toxicological Sciences | Year: 2014

The nuclear receptors CAR (constitutive androstane receptor) and possibly PXR (pregnane X receptor) mediate the hepatic effects of phenobarbital (PB) and similar-acting compounds. Although PB is a potent nongenotoxic tumor promoter in rodent liver, epidemiological data fromepilepsy patients treated with phenobarbital do not show a specific role of PB in human liver cancer risk. That points to species differences in the susceptibility to tumor promotion by PB, which might be attributed to divergent functions of the PB receptorsCARand PXRin mice and humans. In the present study,male transgenicmice expressing humanCARand PXRwere used to detect possible differences between wild-type (WT) and humanized mice in their response to CAR activation in a tumor initiation/promotion experiment with a single injection of the tumor initiator N-nitrosodiethylamine preceding chronic PB treatment for 10 months. Analysis of liver tumor burden revealed that PB strongly promoted the outgrowth of hepatocellular adenoma driven by activated β-catenin in WT mice, whereas the tumorpromoting effect of PB was much less pronounced in the humanized group. In conclusion, the present findings demonstrate that human CAR and PXR support tumor promotion by PB in mouse liver, but to a significantly lesser extent than theWT murine receptors. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology.


Elcombe C.R.,CXR Biosciences | Elcombe B.M.,CXR Biosciences | Foster J.R.,Astrazeneca | Chang S.-C.,3M | And 2 more authors.
Toxicology | Year: 2012

The present study investigated the potential role for activation of PPARα and CAR/PXR by potassium PFOS (K+PFOS) with respect to the etiology of hepatic hypertrophy and hepatocellular adenoma in rats. Male Sprague-Dawley rats were fed K+PFOS (20 or 100ppm) for either 1, 7, or 28 days. Wyeth 14,643 (Wy 14,643, 50ppm) and phenobarbital (PB, 500ppm) were the controls for PPARα and CAR/PXR activation, respectively. Measurements included: plasma ALT, AST, cholesterol, triglycerides, and glucose; liver protein and DNA content; liver activities of palmitoyl CoA oxidase (ACOX), Cyp4A, CYP2B, and CYP3A; induction of liver CYP4A1, CYP2E1, CYP2B1/2, and CYP3A1 proteins (SDS-PAGE and Western blots); liver and thyroid microscopic histopathology, apoptotic index, and cell proliferation index. Terminal body weight was decreased by K+PFOS (100ppm) and Wy 14,643. All test-compound treatments increased liver weight. Plasma lipids were decreased by both PFOS and Wy 14,643. After treatment for 1 day, K+PFOS (100ppm), PB, and Wy 14,643 increased mean hepatic DNA concentration and total hepatic DNA, and total DNA remained elevated after treatment for 7 days and 28 days (PB and Wy 14,643 only). Hepatic P450 concentration was elevated after 7 and 28 days by K+PFOS and by PB. K+PFOS and Wy 14,643 increased liver activities of ACOX and CYP4A as well as increased liver CYP4A1 protein. By 28 days of treatment, K+PFOS and PB increased liver activities of CYP2B and CYP3A as well as increased liver CYP2B1/2 and CYP3A1 proteins, and Wy 14,643 increased CYP2B enzyme activity to a slight extent. All test compounds increased the liver cell proliferative index and decreased the liver apoptotic index. No histological changes of the thyroid were noted; however, PB and WY increased thyroid follicular cell proliferation index (seven-day treatment only), while K+PFOS did not. The thyroid follicular cell apoptotic index did not differ between groups. The hepatomegaly and hepatocellular adenoma observed after dietary exposure of Sprague-Dawley rats to K+PFOS likely are due to the increased expression of xenosensor nuclear receptors PPARα and CAR/PXR. Given the markedly lower or absent response of human hepatocytes to the proliferative stimulus from activation of PPARα and CAR/PXR, the hepatocellular proliferative response from activation of these receptors by PFOS observed in rats is not expected to be of human relevance. © 2012 Elsevier Ireland Ltd.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: HEALTH.2010.4.2-9-1 | Award Amount: 9.40M | Year: 2011

In the development of products for use by humans it is vital to identify compounds with toxic properties at an early stage of their development, to avoid spending time and resource on unsuitable and potentially unsafe candidate products. Human pluripotent stem cell lines offer a unique opportunity to develop a wide variety of human cell-based test systems because they may be expanded indefinitely and triggered to differentiate into any cell type. SCR&Tox aims at making use of these two attributes to provide in vitro assays for predicting toxicity of pharmaceutical compounds and cosmetic ingredients. The consortium has been designed to address all issues related with biological and technological resources to meet that goal. In order to demonstrate the value of pluripotent stem cells for toxicology, the consortium will focus on four complementary aspects: Relevance i.e. establishing and maintaining discrete cell phenotypes over long-term cultures; providing large versatility to adapt to assays of specific pathways. Efficiency for i) automated cell production and differentiation, ii) cell engineering for differentiation and selection iii) multi-parametric toxicology using functional genomic, proteomic and bioelectronics. Extension i.e. i) scalability through production of cells and technologies for industrial-scale assays, and ii) diversity of phenotypes (5 different tissues), and of genotypes (over 30 different donors). Normalization validation and demonstration of reproducibility and robustness of cell-based assays on industrial-scale platforms, to allow for secondary development in the pharmaceutical and cosmetic industry. SCR&Tox will be intricately associated to other consortia of the Alternative Testing call, sharing biological, technological and methodological resources. Proof of concept of the proposed pluripotent stem cell-based assays for toxicology will be provided on the basis of toxicity pathways and test compounds identified by other consortia.


Bell C.C.,Karolinska Institutet | Lauschke V.M.,Karolinska Institutet | Vorrink S.U.,Karolinska Institutet | Palmgren H.,Astrazeneca | And 4 more authors.
Drug Metabolism and Disposition | Year: 2017

Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems-hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and threedimensional primary human hepatocyte (PHH) spheroids-at transcriptional and functional levels in a multicenter study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8148 of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG cells and hepatocytes derived from induced pluripotent stem cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic, or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in humans. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.


Donaldson K.,University of Edinburgh | Schinwald A.,University of Edinburgh | Murphy F.,Dong - A University | Cho W.-S.,MRC Toxicology Unit | And 3 more authors.
Accounts of Chemical Research | Year: 2013

In all branches of toxicology, the biologically effective dose (BED) is thefraction of the total dose of a toxin that actually drives any toxic effect. Knowledge of the BED has a number of applications including in building structure-activity relationships, the selection of metrics, the design of safe particles, and the determination of when a nanoparticle (NP) can be considered to be "new" for regulatory purposes. In particle toxicology, we define the BED as "the entity within any dose of particles in tissue that drives a critical pathophysiogically relevant form of toxicity (e.g., oxidative stress, inflammation, genotoxicity, or proliferation) or a process that leads to it."In conventional chemical toxicology, researchers generally use the mass as the metric to describe dose (such as mass per unit tissue or cells in culture) because of its convenience. Concentration, calculated from mass, may also figure in any description of dose. In the case of a nanoparticle dose, researchers use either the mass or the surface area. The mass of nanoparticles is not the only driver of their activity: the surfaces of insoluble particles interact with biological systems, and soluble nanoparticles can release factors that interact with these systems. Nanoparticle shape can modify activity.In this Account, we describe the current knowledge of the BED as it pertains to different NP types. Soluble toxins released by NPs represent one potential indicator of BED for wholly or partially soluble NPs composed of copper or zinc. Rapid dissolution of these NPs into their toxic ions in the acidic environment of the macrophage phagolysosome causes those ions to accumulate, which leads to lysosome destabilization and inflammation. In contrast, soluble NPs that release low toxicity ions, such as magnesium oxide NPs, are not inflammogenic. For insoluble NPs, ζ potential can serve as a BED measurement because the exposure of the particle surface to the acidic milieu of the phagolysosome and interactions with the lysosomal membrane can compromise the integrity of the NPs. Researchers have explored oxidative potential of NPs most extensively as an indicator of the BED: the ability of an NP to cause oxidative stress in cells is a key factor in determining cell toxicity, inflammogenicity, and oxidative DNA adduct formation. Finally we discuss BEDs for high aspect ratio nanoparticles because long fibers or nanoplatelets can cause inflammation and further effects. These consequences arise from the paradoxically small aerodynamic diameter of fibers or thin platelets. As a result, these NPs can deposit beyond the ciliated airways where their extended dimensions prevent them from being fully phagocytosed by macrophages, leading to frustrated phagocytosis. Although knowledge is accumulating on the BED for NPs, many questions and challenges remain in understanding and utilizing this important nanotoxicological parameter. © 2012 American Chemical Society.


Scheer N.,TaconicArtemis | Kapelyukh Y.,CXR Biosciences | Chatham L.,University of Dundee | Rode A.,TaconicArtemis | And 3 more authors.
Molecular Pharmacology | Year: 2012

Compared with rodents and many other animal species, the human cytochrome P450 (P450) Cyp2c gene cluster varies significantly in the multiplicity of functional genes and in the substrate specificity of its enzymes. As a consequence, the use of wild-type animal models to predict the role of human CYP2C enzymes in drug metabolism and drug-drug interactions is limited. Within the human CYP2C cluster CYP2C9 is of particular importance, because it is one of the most abundant P450 enzymes in human liver, and it is involved in the metabolism of a wide variety of important drugs and environmental chemicals. To investigate the in vivo functions of cytochrome P450 Cyp2c genes and to establish a model for studying the functions of CYP2C9 in vivo, we have generated a mouse model with a deletion of the murine Cyp2c gene cluster and a corresponding humanized model expressing CYP2C9 specifically in the liver. Despite the high number of functional genes in the mouse Cyp2c cluster and the reported roles of some of these proteins in different biological processes, mice deleted for Cyp2c genes were viable and fertile but showed certain phenotypic alterations in the liver. The expression of CYP2C9 in the liver also resulted in viable animals active in the metabolism and disposition of a number of CYP2C9 substrates. These mouse lines provide a powerful tool for studying the role of Cyp2c genes and of CYP2C9 in particular in drug disposition and as a factor in drug-drug interaction. Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics.


Scheer N.,TaconicArtemis | Kapelyukh Y.,CXR Biosciences | McEwan J.,CXR Biosciences | Beuger V.,TaconicArtemis | And 4 more authors.
Molecular Pharmacology | Year: 2012

The highly polymorphic human cytochrome P450 2D6 enzyme is involved in the metabolism of up to 25% of all marketed drugs and accounts for significant individual differences in response to CYP2D6 substrates. Because of the differences in the multiplicity and substrate specificity of CYP2D family members among species, it is difficult to predict pathways of human CYP2D6-dependent drug metabolism on the basis of animal studies. To create animal models that reflect the human situation more closely and that allow an in vivo assessment of the consequences of differential CYP2D6 drug metabolism, we have developed a novel straightforward approach to delete the entire murine Cyp2d gene cluster and replace it with allelic variants of human CYP2D6. By using this approach, we have generated mouse lines expressing the two frequent human protein isoforms CYP2D6.1 and CYP2D6.2 and an as yet undescribed variant of this enzyme, as well as a Cyp2d cluster knockout mouse. We demonstrate that the various transgenic mouse lines cover a wide spectrum of different human CYP2D6 metabolizer phenotypes. The novel humanization strategy described here provides a robust approach for the expression of different CYP2D6 allelic variants in transgenic mice and thus can help to evaluate potential CYP2D6-dependent interindividual differences in drug response in the context of personalized medicine. Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics.


The molecules of formula (1) are useful in treating diabetes, obesity, hypercholesterolaemia, hyperlipidaemia, cancer, inflammation of other conditions in which modulation of lipid or eicosanoid status or functions may be desirable. Formula (1): Z^(1)-X-Z^(2) wherein (a) Z^(1) represents CO^(2)H or a derivative thereof; (b) Z^(2) represents F, H, -CO_(2)H or a derivative thereof; and (c) X represents fluorinated alkylene; or a solvate thereof, for example a perfluorinated fatty acid or derivative thereof.


The molecules of formula (I) are useful in treating diabetes, obesity, hypercholesterolaemia, hyperlipidaemia, cancer, inflammation or other conditions in which modulation of lipis of eicosanoid status or functions may be desirable. Formula (I): Z^(1)Z^(1)Z^(2 )wherein a) Z^(1 )represents CO_(2)H or a derivative thereof; b) Z^(2 )represents F, H, CO_(2)H or a derivative thereof; and c) X represents fluorinated alkylene; or a solvate thereof, for example a perfluorinated fatty acid or derivative thereof.

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