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Chauvigne F.,French Institute of Health and Medical Research | Plummer S.,CXR Biosciences | Lesne L.,French Institute of Health and Medical Research | Cravedi J.-P.,French National Institute for Agricultural Research | And 3 more authors.
PLoS ONE | Year: 2011

Exposure to phthalates in utero alters fetal rat testis gene expression and testosterone production, but much remains to be done to understand the mechanisms underlying the direct action of phthalate within the fetal testis. We aimed to investigate the direct mechanisms of action of mono-(2-ethylhexyl) phthalate (MEHP) on the rat fetal testis, focusing on Leydig cell steroidogenesis in particular. We used an in vitro system based on the culture for three days, with or without MEHP, of rat fetal testes obtained at 14.5 days post-coitum. Exposure to MEHP led to a dose-dependent decrease in testosterone production. Moreover, the production of 5 alpha-dihydrotestosterone (5α-DHT) (-68%) and androstenedione (-54%) was also inhibited by 10 μM MEHP, whereas 17 alpha-hydroxyprogesterone (17α-OHP) production was found to increase (+41%). Testosterone synthesis was rescued by the addition of androstenedione but not by any of the other precursors used. Thus, the hormone data suggested that steroidogenesis was blocked at the level of the 17,20 lyase activity of the P450c17 enzyme (CYP17), converting 17α-OHP to androstenedione. The subsequent gene expression and protein levels supported this hypothesis. In addition to Cyp17a1, microarray analysis showed that several other genes important for testes development were affected by MEHP. These genes included those encoding insulin-like factor 3 (INSL3), which is involved in controlling testicular descent, and Inha, which encodes the alpha subunit of inhibin B. These findings indicate that under in vitro conditions known to support normal differentiation of the fetal rat testis, the exposure to MEHP directly inhibits several important Leydig cell factors involved in testis function and that the Cyp17a1 gene is a specific target to MEHP explaining the MEHP-induced suppression of steroidogenesis observed. © 2011 Chauvigné et al.

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.

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.

Lucas C.D.,Queens Medical Research Institute | Allen K.C.,Queens Medical Research Institute | Dorward D.A.,Queens Medical Research Institute | Hoodless L.J.,Queens Medical Research Institute | And 7 more authors.
FASEB Journal | Year: 2013

Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti-inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced timeand concentration-dependent neutrophil apoptosis (apigenin, EC50=12.2 μM; luteolin, EC50=14.6 μM; and wogonin, EC50=28.9 μM). Induction of apoptosis was caspase dependent, as it was blocked by the broadspectrum caspase inhibitor Q-VD-OPh and was associated with both caspase-3 and caspase-9 activation. Flavone-induced apoptosis was preceded by down-regulation of the prosurvival protein Mcl-1, with proteasomal inhibition preventing flavone-induced Mcl-1 down-regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte-macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin-induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis-inducing anti-inflammatory, proresolution agents. © The Author(s).

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.

Warnasuriya G.D.,University of Dundee | Elcombe B.M.,CXR Biosciences | Foster J.R.,Astrazeneca | Elcombe C.R.,CXR Biosciences
Archives of Toxicology | Year: 2010

Short-chain chlorinated paraffins (SCCPs) cause kidney tumours in male rats, but not in female rats or mice of either sex. Male rat-specific tumours also occur in rats dosed with a range of compounds including 1,4-dichlorobenzene (DCB) and d-limonene (DL). These compounds bind to a male rat-specific hepatic protein, alpha-2-urinary globulin (α2u), and form degradationresistant complexes in the kidney. The resulting accumulation of α2u causes cell death and sustained regenerative cell proliferation, which in turn leads to the formation of renal tumours. To investigate whether the SCCP, Chlorowax 500C (C500C), causes tumours via the accumulation of α2u male rats were orally dosed with either C500C (625 mg/kg of body weight), DCB (300 mg/kg of body weight), or DL (150 mg/kg of body weight) for 28 consecutive days. An increase in renal α2u and cell proliferation was observed in DCB- and DL-treated rats but not in C500C-treated rats. C500C caused peroxisome proliferation and a down-regulation of α2u synthesis in male rat liver. This down-regulation occurred at the transcriptional level. Since less α2u was produced in C500C-treated rats, there was less available for accumulation in the kidney hence a typical α2u nephropathy did not appear. However, the administration of a radiolabelled SCCP, [14C]polychlorotridecane (PCTD), to male rats demonstrated its binding to renal α2u. Thus, it is possible that SCCPs bind to a2u and cause a slow accumulation of the protein in the kidney followed by delayed onset of α2u nephropathy. As a consequence of these findings in the current experiments, while evidence exists implicating α2u-globulin in the molecular mechanism of action of the C500C, the classic profile of a α2u-globulin nephropathy seen with other chemicals such as DCB and DL was not reproduced during this experimental protocol. © Springer-Verlag 2009.

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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