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Schenk B.,Institute of Experimental and Clinical Pharmacology and Toxicology | Weimer M.,German Cancer Research Center | Bremer S.,European Commission - Joint Research Center Ispra | van der Burg B.,BioDetection Systems B.V. | And 9 more authors.
Reproductive Toxicology | Year: 2010

ReProTect is a project within the 6th European Framework Program which has developed alternative methods aimed to reduce or replace animal experimentation in the field of reproductive toxicology. In its final year, a ring trial, named the " Feasibility Study" , was conducted, in which 10 blinded chemicals with toxicologically well-documented profiles were analyzed by employing a test battery of 14 in vitro assays. EC50 (half maximal effective concentration) or equivalent endpoints were determined and the test compounds were ranked relative to chemicals previously assayed in the tests of the battery. This comparative analysis together with a weight of evidence approach allowed a robust prediction of adverse effects on fertility and embryonic development of the 10 test chemicals in vivo. In summary, the vast majority of the predictions made based on the in vitro results turned out to be correct when compared to the whole animal data. The procedure used here, a nearest neighbor analysis coupled with a weight of evidence approach, may guide future activities in the field of alternative toxicity testing. © 2010 Elsevier Inc.


Gougelet A.,University of Paris Descartes | Gougelet A.,French Institute of Health and Medical Research | Torre C.,University of Paris Descartes | Torre C.,French Institute of Health and Medical Research | And 25 more authors.
Hepatology | Year: 2014

β-catenin signaling can be both a physiological and oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20%-40% of hepatocellular carcinomas (HCCs) with specific metabolic features. We decipher the molecular determinants of β-catenin-dependent zonal transcription using mice with β-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T-cell factor (Tcf)-4 and β-catenin, transcriptome, and metabolome. We find that Tcf-4 DNA bindings depend on β-catenin. Tcf-4/β-catenin binds Wnt-responsive elements preferentially around β-catenin-induced genes. In contrast, genes repressed by β-catenin bind Tcf-4 on hepatocyte nuclear factor 4 (Hnf-4)-responsive elements. β-Catenin, Tcf-4, and Hnf-4α interact, dictating β-catenin transcription, which is antagonistic to that elicited by Hnf-4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by β-catenin, partly through xenobiotic nuclear receptors. Conclusions: β-catenin patterns the zonal liver together with Tcf-4, Hnf-4α, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with β-catenin mutational activation. © 2014 by the American Association for the Study of Liver Diseases.


Gerding D.N.,Loyola University Chicago | Johnson S.,Loyola University Chicago | Rupnik M.,Institute of Public Health Maribor | Aktories K.,Institute of Experimental and Clinical Pharmacology and Toxicology
Gut microbes | Year: 2014

Binary toxin (CDT) is frequently observed in Clostridium difficile strains associated with increased severity of C. difficile infection (CDI). CDT belongs to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb which binds to host cells and translocates CDTa into the cytosol. CDTb is activated by serine proteases and binds to lipolysis stimulated lipoprotein receptor. ADP-ribosylation induces depolymerization of the actin cytoskeleton. Toxin-induced actin depolymerization also produces microtubule-based membrane protrusions which form a network on epithelial cells and increase bacterial adherence. Multiple clinical studies indicate an association between binary toxin genes in C. difficile and increased 30-d CDI mortality independent of PCR ribotype. Further studies including measures of binary toxin in stool, analyses of CDI mortality caused by CDT-producing strains, and examination of the relationship of CDT expression to TcdA and TcdB toxin variants and PCR ribotypes are needed.


Bock K.W.,Institute of Experimental and Clinical Pharmacology and Toxicology
Biochemical Pharmacology | Year: 2016

UDP-glycosyltransferases (UGTs) are major phase II enzymes of a detoxification system evolved in all kingdoms of life. Lipophilic endobiotics such as hormones and xenobiotics including phytoalexins and drugs are conjugated by vertebrates mainly with glucuronic acid, by invertebrates and plants mainly with glucose. Plant-herbivore arms-race has been the major driving force for evolution of large UGT and other enzyme superfamilies. The UGT superfamily is defined by a common protein structure and signature sequence of 44 amino acids responsible for binding the UDP moiety of the sugar donor. Plants developed toxic phytoalexins stored as glucosides. Upon herbivore attack these conjugates are converted to highly reactive compounds. In turn, animals developed large families of UGTs in their intestine and liver to detoxify these phytoalexins. Interestingly, phytoalexins, exemplified by quercetin glucuronides and glucosinolate-derived isocyanates, are known insect attractant pigments in plants, and antioxidants, anti-inflammatory and chemopreventive compounds of humans. It is to be anticipated that phytochemicals may provide a rich source in beneficial drugs. © 2015 Elsevier Inc. All rights reserved.


Uibel F.,Institute of Experimental and Clinical Pharmacology and Toxicology | Schwarz M.,Institute of Experimental and Clinical Pharmacology and Toxicology
Reproductive Toxicology | Year: 2015

The ReProGlo assay was developed in 2009 to predict embryotoxic potential of drugs and chemicals by use of a stem cell-based in vitro system. It utilizes a luciferase reporter to detect drug-induced alterations in the canonical Wnt/β-catenin signaling pathway, which is involved in regulation of early embryonic development. It allows the simultaneous determination of cell viability and luciferase reporter activity in a high throughput format. The present study was conducted within the framework of the EU ChemScreen-project. It (1) enlarges the original number of test-compounds from 17 to now 80, (2) introduces a new classification scheme and (3) anchors the results against a prediction scheme based on structural features of chemicals. The assay is applicable as stand-alone for priority setting or in a test battery. © 2014 Elsevier Inc.


Bock K.W.,Institute of Experimental and Clinical Pharmacology and Toxicology
Biochemical Pharmacology | Year: 2016

Target cells and molecular targets responsible for dioxin-mediated chloracne, the hallmark of dioxin toxicity, are reviewed. The dioxin TCDD accumulates in sebum, and thereby persistently activates the Ah receptor (AhR), expressed in bipotential stem/progenitor cells of the sebaceous gland. AhR operates in cooperation with other transcription factors including c-Myc, Blimp1 and ß-Catenin/TCF: c-Myc stimulates exit of stem cells from quiescence to proliferating sebocyte progenitors; Blimp1 is a major c-Myc repressor, and ß-Catenin/TCF represses sebaceous gland differentiation and stimulates differentiation to interfollicular epidermis. TCDD has been demonstrated to induce Blimp1 expression in the sebocyte stem/progenitor cell line SZ95, leading to sebocyte apoptosis and proliferation of interfollicular epidermis cells. These findings explain observations in TCDD-poisoned individuals, and identify target cells and molecular targets of dioxin-mediated chloracne. They clearly demonstrate that the AhR operates in a cell context-dependent manner, and provide hints to homeostatic functions of AhR in stem/progenitor cells. © 2016.


PubMed | Institute of Experimental and Clinical Pharmacology and Toxicology
Type: | Journal: Reproductive toxicology (Elmsford, N.Y.) | Year: 2015

The ReProGlo assay was developed in 2009 to predict embryotoxic potential of drugs and chemicals by use of a stem cell-based in vitro system. It utilizes a luciferase reporter to detect drug-induced alterations in the canonical Wnt/-catenin signaling pathway, which is involved in regulation of early embryonic development. It allows the simultaneous determination of cell viability and luciferase reporter activity in a high throughput format. The present study was conducted within the framework of the EU ChemScreen-project. It (1) enlarges the original number of test-compounds from 17 to now 80, (2) introduces a new classification scheme and (3) anchors the results against a prediction scheme based on structural features of chemicals. The assay is applicable as stand-alone for priority setting or in a test battery.


PubMed | Institute of Experimental and Clinical Pharmacology and Toxicology
Type: | Journal: Biochemical pharmacology | Year: 2016

UDP-glycosyltransferases (UGTs) are major phase II enzymes of a detoxification system evolved in all kingdoms of life. Lipophilic endobiotics such as hormones and xenobiotics including phytoalexins and drugs are conjugated by vertebrates mainly with glucuronic acid, by invertebrates and plants mainly with glucose. Plant-herbivore arms-race has been the major driving force for evolution of large UGT and other enzyme superfamilies. The UGT superfamily is defined by a common protein structure and signature sequence of 44 amino acids responsible for binding the UDP moiety of the sugar donor. Plants developed toxic phytoalexins stored as glucosides. Upon herbivore attack these conjugates are converted to highly reactive compounds. In turn, animals developed large families of UGTs in their intestine and liver to detoxify these phytoalexins. Interestingly, phytoalexins, exemplified by quercetin glucuronides and glucosinolate-derived isocyanates, are known insect attractant pigments in plants, and antioxidants, anti-inflammatory and chemopreventive compounds of humans. It is to be anticipated that phytochemicals may provide a rich source in beneficial drugs.


PubMed | Institute of Experimental and Clinical Pharmacology and Toxicology
Type: | Journal: Biochemical pharmacology | Year: 2016

Despite decades of intensive research physiologic Ah receptor (AHR) functions are not yet elucidated. Challenges include marked species differences and dependence of AHR function on the cell type and cellular context. Hints to physiologic functions may be derived (i) from feedback loops between endogenous ligands and substrates of major target enzymes such as CYP1A1 and UGT1A1, and (ii) from dioxin toxicity in human individuals. For example, dioxin-mediated chloracne is probably due to dysregulated homeostasis of sebocyte stem/progenitor cells. Dioxin-mediated inflammatory responses may be due to complex dysregulation of hematopoiesis. Comparison of AHR functions with those of PXR and its target enzyme CYP3A4 may be helpful to emphasize AHR functions in specialized cells: PXR is known to be mainly involved in regulation of systemic metabolism of endo- and xenobiotics. However, AHR may be mostly controlling local homeostasis of signals in specialized cells such as stem/progenitor cells. Accumulating evidence suggests that knowledge about physiologic AHR functions may stimulate drug development.


PubMed | Institute of Experimental and Clinical Pharmacology and Toxicology
Type: | Journal: Biochemical pharmacology | Year: 2016

Target cells and molecular targets responsible for dioxin-mediated chloracne, the hallmark of dioxin toxicity, are reviewed. The dioxin TCDD accumulates in sebum, and thereby persistently activates the Ah receptor (AhR), expressed in bipotential stem/progenitor cells of the sebaceous gland. AhR operates in cooperation with other transcription factors including c-Myc, Blimp1 and -Catenin/TCF: c-Myc stimulates exit of stem cells from quiescence to proliferating sebocyte progenitors; Blimp1 is a major c-Myc repressor, and -Catenin/TCF represses sebaceous gland differentiation and stimulates differentiation to interfollicular epidermis. TCDD has been demonstrated to induce Blimp1 expression in the sebocyte stem/progenitor cell line SZ95, leading to sebocyte apoptosis and proliferation of interfollicular epidermis cells. These findings explain observations in TCDD-poisoned individuals, and identify target cells and molecular targets of dioxin-mediated chloracne. They clearly demonstrate that the AhR operates in a cell context-dependent manner, and provide hints to homeostatic functions of AhR in stem/progenitor cells.

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