[Show abstract][Hide abstract] ABSTRACT: The mouse embryonic stem D3 (ES-D3) cell differentiation assay is based on the morphometric measurement of cardiomyocyte differentiation and is a promising tool to detect developmental toxicity of compounds. The BeWo transport model, consisting of BeWo b30 cells grown on transwell inserts and mimicking the placental barrier, is useful to determine relative placental transport velocities of compounds. We have previously demonstrated the usefulness of the ES-D3 cell differentiation assay in combination with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies of a set of reference azole compounds. To further evaluate this combined in vitro toxicokinetic and toxicodynamic approach, we combined ES-D3 cell differentiation data of six novel triazoles with relative transport rates obtained from the BeWo model and compared the obtained ranking to the developmental toxicity ranking as derived from in vivo data. The data show that the combined in vitro approach provided a correct prediction for in vivo developmental toxicity, whereas the ES-D3 cell differentiation assay as stand-alone did not. In conclusion, we have validated the combined in vitro approach for developmental toxicity, which we have previously developed with a set of reference azoles, for a set of six novel triazoles. We suggest that this combined model, which takes both toxicodynamic and toxicokinetic aspects into account, should be further validated for other chemical classes of developmental toxicants.
Full-text · Article · Jun 2015 · Archives of Toxicology
[Show abstract][Hide abstract] ABSTRACT: Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfil regulatory requirements. These assessments often involve animal testing but ethical, cost and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. Here, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell derived (SCD)-systems in toxicology, with a focus on industrial applications. SCD-systems, in particular iPSC-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing non-clinical data for the understanding of in vitro to in vivo translation. SCD-systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD-systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD-cells are still lacking. Organotypical, 3D-culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support co-culture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD-toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD-in vitro methods undergo appropriate validation and become accepted in the regulatory arena.
No preview · Article · Feb 2015 · Stem Cells and Development
[Show abstract][Hide abstract] ABSTRACT: The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up4U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up4U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up4U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up4U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up4U after stimulation with shear stress. Mean total plasma Up4U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L(-1)). In conclusion, Up4U is a novel strong human endothelium-derived angiogenic factor.
[Show abstract][Hide abstract] ABSTRACT: This chapter contains sections titled: Introduction Endocrine Disruption: Definitions Current Regulatory Situation in the EU US EPA Endocrine Disruptor Screening Program and OECD Conceptual Framework for the Testing and Assessment of Endocrine-Disrupting Chemicals ECETOC Approach Methods to Assess Endocrine Modes of Action and Endocrine-Related Adverse Effects in Screening and Regulatory Contexts Proposal for Decision Criteria for EDCs: Regulatory Agencies References
[Show abstract][Hide abstract] ABSTRACT: The Threshold Toxicological Concern (TTC) is based on the concept that reasonable assurance of safety can be given if exposure is sufficiently low. We report on the evaluation of BASF's data for oral developmental toxicity studies in rabbits with 48 NOAEL values for maternal and developmental toxicity. The 5th percentile of the NOAEL distributions was calculated to be 5mg/kgbw/d for both maternal and developmental toxicity. From literature 56 compounds tested in rabbits were taken and combined with values from BASF's studies. The 5th percentile value for developmental toxicity of these 104 studies (mostly active ingredients) was 2mg/kgbw/d. Thus, a TTC value of 4μg/kgbw/d was calculated using a safety factor of 500 to account for relatively small database. This value is in the same range as the TTC value for developmental toxicity in rats of 8μg/kgbw/d. The lower value may serve as guidance to determine whether further evaluation is needed or whether to rely on a TTC value for industrial chemicals or low concentration (environmental) contaminants if exposure is sufficiently low. A comparison of 30 compounds tested at BASF in both species, suggests that rabbits are not more sensitive than rats. We encourage others to publish data on rabbit developmental toxicity.
No preview · Article · Jun 2012 · Regulatory Toxicology and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. Here, we present an alternative testing paradigm for detection of endocrine modes of action that replace and reduce animal testing through refinement. Receptor mediated endocrine effects were assessed using the yeast-based receptor-mediated transcriptional activation YES/YAS assays and effects on steroid hormone biosynthesis were assessed using the human cell line H295R in the steroidogenesis assay. In our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile in addition to classical parameters such as histopathology. The combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. Thus, this proposal constitutes a potentially attractive alternative to EPA's Endocrine Disruptor Screening Program to identify mammalian, systemic endocrine modes of action. Data on 14 reference substances for which the in vitro YES/YAS and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine modes of action are presented here.
No preview · Article · Apr 2012 · Regulatory Toxicology and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: MDMA consumers are young people of childbearing age. Consequently, developmental exposure to this drug is a potential public health concern. Several studies have addressed MDMA neurotoxicity in adults; however, knowledge of the effects of MDMA on developing embryos is limited. After administration, MDMA is metabolized species specifically via two main pathways. One leads to the formation of MDA and the other to the formation of HHMA. Here we evaluated the embryotoxic effects of MDMA, and also those of MDA, a main metabolite of MDMA in rats, and HHMA, a main metabolite in humans. For this purpose, we used the whole embryo culture (WEC). Our results show a concentration-dependent embryotoxic effect of MDMA, MDA and HHMA at a concentration range of 25-50μg/ml. The embryotoxic potential of the parent compound and the two metabolites was comparable in vitro.
No preview · Article · Feb 2012 · Reproductive Toxicology
[Show abstract][Hide abstract] ABSTRACT: Myocardium is very sensitive to gravitational changes. During a spaceflight cardiovascular atrophy paired with rhythm problems and orthostatic intolerance can occur. The aim of this study was to investigate the impact of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on cardiac fibroblasts (CF) grown under altered gravity conditions.
We examined the influence of exposure to a Random Positioning Machine (RPM) on CF, derived from porcine hearts. We focused on growth, extracellular matrix protein (ECMP) synthesis and apoptosis.
When cultured on a RPM, CF began to form 3D spheroids within 24h, irrespective of growth factor treatment. Exposure to RPM induced an increased synthesis of ECMP and also resulted in elevated apoptosis in adherent CF as measured by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling (TUNEL) analysis, 4',6-diamidino-2-phenylindole (DAPI) staining, and caspase-3 detection. bFGF and VEGF significantly decreased the amount of ECMP (collagen type I, III, chondroitin sulfate) in 1g and RPM cultures, and also significantly reduced the amount of apoptotic CF as well as caspase-3.
Altered gravity conditions on a RPM induced 3D growth, elevated ECMP synthesis and apoptosis in cardiac fibroblasts. Growth factor treatment attenuated programmed cell death and ECMP secretion.
No preview · Article · Jan 2010 · Cellular Physiology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: An inflammatory reaction at the site of infusion is a common clinical problem that is observed after the intravenous application of antibiotics and other drugs. The pathomechanism of this infusion-related phlebitis is not fully understood. We analyzed the effects of the three macrolide antibiotics erythromycin, clarithromycin and azithromycin on human endothelial cells in vitro. As a positive control quinupristin/dalfopristin was studied. The cytotoxicity of all substances was analyzed by a modified MTT cytotoxicity assay with 3T3-fibroblasts and EA.hy 926 endothelial cells. Cells were incubated for 10 days with the antibiotics. After adding MTT the optical density was measured which correlates with cell death. Clarithromycin exhibited the strongest cytotoxic effect on EA.hy 926 cells (EC(50) 30 mg/L), followed by azithromycin (EC(50) 40 mg/L), a cytotoxic effect of erythromycin could only be observed at much higher concentrations (EC(50) 310 mg/L). The reaction of the endothelial cells was further analyzed in detail by means of flow cytometry. For these experiments the endothelial cell line EA.hy 926 as well as primary cells (HUVEC) were used. The antigens were stained with fluoresceinisothiocyanat- or phycoerythrin-conjugated monoclonal antibodies for the following surface antigens: CD34, E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106). Cells were incubated with the antibiotics at concentrations ranging from 100 to 800 mg/L (clarithromycin and azithromycin) and from 200 to 1,200 mg/L (erythromycin). These concentrations occur under therapeutic conditions at the site of infusion. Cells were incubated for 2 h and analysis was carried out after an additional culture period of 22 h without test compounds. A significantly enhanced expression of all four antigens was observed which was most pronounced at 800 mg/L (erythromycin), 600 mg/L (azithromycin) and 400 mg/L (clarithromycin). A concentration of 800 mg/L erythromycin medium caused an increase of the expression of CD34 (+6%), E-selectin (+5%), ICAM-1 (+14%) and VCAM-1 (+5%). At lower concentrations (600 mg/L) azithromycin provokes a stronger upregulation of the proinflammatory antigens: CD34 (+17%), E-selectin (+18%), ICAM-1 (+27%) and VCAM-1 (+17%). At a concentration of 400 mg/L medium clarithromycin induced a similar effect as erythromycin at twice this concentration: CD34 (+5%), E-selectin (+7%), ICAM-1 (+23%) and VCAM-1 (+4%). Reactions of the HUVECs were less pronounced than those of the EA.hy 926 cells. Cell surface markers involved in interactions between endothelial cells and leukocytes proved to be useful markers to study differences in the proinflammatory potential of the three macrolides. By analysing the upregulation of these antigens on EA.hy 926 cells in vitro the risk of phlebitis could be predictable for other drugs as well.
No preview · Article · May 2009 · Archives of Toxicology
[Show abstract][Hide abstract] ABSTRACT: N-methyl-2-pyrrolidone (NMP), which undergoes extensive biotransformation, has been shown in vivo to cause developmental toxicity and, especially after oral treatment, malformations in rats and rabbits. Data are lacking as to whether the original compound or one of its main metabolites is responsible for the toxic effects observed. Therefore, the relative embryotoxicity of the parent compound and its metabolites was evaluated using rat whole embryo culture (WEC) and the balb/c 3T3 cytotoxicity test. The resulting data were evaluated using two strategies; namely, one based on using all endpoints determined in the WEC and the other including endpoints from both the WEC and the cytotoxicity test. On basis of the first analysis, the substance with the highest embryotoxic potential is NMP, followed by 5-hydroxy-N-methyl-pyrrolidone (5-HNMP), 2-hydroxy-N-methylsuccinimide (2-HMSI) and N-methylsuccinimide (MSI). Specific dysmorphogeneses induced by NMP and 5-HNMP were aberrations in the head region of the embryos, abnormal development of the second visceral arches and open neural pores. The second evaluation strategy used only two endpoints of the WEC, i.e. the no observed adverse effect concentration (NOAEC(WEC)) and the lowest concentration leading to dysmorphogenesis in 100% of the cultured embryos (IC(Max WEC)). In addition to these WEC endpoints the IC(50 3T3) from the cytotoxicity test (balb/c 3T3 fibroblasts) was included in the evaluation scheme. These three endpoints were applied to a prediction model developed during a validation study of the European Centre for the Validation of Alternative Methods (ECVAM) allowing the classification of the embryotoxic potential of each compound into three classes (non-, weakly- and strongly embryotoxic). Consistent results from both evaluation strategies were observed, whereby NMP and its metabolites revealed a direct embryotoxic potential. Hereby, only NMP and 5-HNMP induced specific embryotoxic effects and were classified as weakly embryotoxic, whereas the other two metabolites, 2-HMSI and MSI, were determined to be non-embryotoxic.
No preview · Article · Apr 2009 · Toxicology and Applied Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells (ECs) form three-dimensional (3D) aggregates without any scaffold when they are exposed to microgravity simulated by a random positioning machine (RPM) but not under static conditions at gravity. Here we describe a delayed type of formation of 3D structures of ECs that was initiated when ECs cultured on a desktop RPM remained adherent for the first 5 days but spread over neighboring adherent cells, forming little colonies. After 2 weeks, tube-like structures (TSs) became visible in these cultures. They included a lumen, and they elongated during another 2 weeks of culturing. The walls of these TSs consisted mainly of single-layered ECs, which had produced significantly more beta(1)-integrin, laminin, fibronectin, and alpha-tubulin than ECs simultaneously grown adhering to the culture dishes under microgravity or normal gravity. The amount of actin protein was similar in ECs incorporated in TSs and in ECs growing at gravity. The ratio of tissue inhibitor of metalloproteinases-1 to matrix metalloproteinase-2 found in the supernatants was lower at the seventh than at the 28th day of culturing. These results suggest that culturing ECs under conditions of modeled gravitational unloading represents a new technique for studying the formation of tubes that resemble vascular intimas.
No preview · Article · Mar 2009 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Fibroblast growth factors interact with appropriate endothelial cell (EC) surface receptors and initiate intracellular signal cascades, which participate in modulating blood vessel growth. EC, upon exposure to basic fibroblast growth factors (bFGFs) undergo profound functional alterations, which depend on their actual sensitivity and involve gene expression and de novo protein synthesis. We investigated the effects of bFGF on signaling pathways of EA.hy926 cells in different environments. EC were cultured under normal gravity (1 g) and simulated microgravity (micro g) using a three-dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin-1 (ET-1) and TGF-beta(1) expression. Microgravity reduced eNOS mRNA within 24 h. Moreover, a six- to eightfold higher amount of IL-6 and IL-8 was secreted within 24 h micro g. In addition, microgravity induced a duplication of NF-kappaB p50, while p65 was quadrupled. At 1 g, bFGF application (4 h) reduced ET-1, TGF-beta(1) and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk-1, Flt-1, the release of IL-6, IL-8, and TGF-beta(1). Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h micro g, bFGF decreased TGF-beta(1), eNOS, and ET-1 gene expression. After 24 h micro g, bFGF elevated fibronectin, Flk-1 and Flt-1 protein, and reduced IL-6 and IL-8 compared with vehicle treated micro g cultures. In micro g, bFGF enhanced NF-KappaB p50 by 50%, Bax by 25% and attenuated p65, activation of caspase-3 and annexin V-positive cells. bFGF differently changes intracellular signals in ECs depending whether it is applied under microgravity or normal gravity conditions. In microgravity, bFGF contributes to protect the EC from apoptosis.
No preview · Article · Jul 2008 · Journal of Cellular Biochemistry
[Show abstract][Hide abstract] ABSTRACT: To assess the effect of nebivolol, a highly selective third generation beta1-adrenoceptor antagonist with an endothelium-dependent vasodilatory action, on smoking-induced endothelial dysfunction.
This open-label study examined the effect of 14 daily doses of 5 mg nebivolol on forearm blood flow in 21 healthy, young, male, light smokers (< or =5 cigarettes/day), measured by plethysmography on Days 1, 7, and 14. The primary endpoint was the difference in forearm blood flow after smoking one standard cigarette from baseline (Day 1) until treatment end on Day 14. Secondary outcomes included the difference in forearm blood flow between Day 1 and Day 7 compared with Day 14 before and after smoking, the effect of nebivolol on blood coagulation parameters, high-sensitive-C-reactive protein (hs-CRP), and the safety and tolerability of nebivolol.
Nebivolol for 14 days did not significantly affect forearm blood flow after smoking. On Day 7 of nebivolol treatment, forearm blood flow after smoking was significantly greater than blood flow before smoking (increase of 0.44 mL/min; p = 0.00656). Serum level ofhs-CRP showed a marked decrease from Day 1 to Day 14. No changes in coagulation parameters were observed over the course of nebivolol treatment. Nebivolol was well tolerated throughout the study.
The increase in forearm blood flow and the marked decrease in hs-CRP over 14 days of treatment suggest that nebivolol has a positive effect on endothelial function in light smokers, but larger studies are required to confirm these observations.
Full-text · Article · Feb 2008 · Vascular Health and Risk Management
[Show abstract][Hide abstract] ABSTRACT: Infusion phlebitis is a common clinical problem that is observed with some antimicrobial agents, when being administered intravenously. In this study, cultured murine fibroblasts and immortalised human endothelial cells were exposed to three antibiotics at clinically relevant concentrations to assess their toxic potential in two established cytotoxicity assays. BALB/c 3T3 fibroblasts and Eahy926 endothelial cells were exposed to quinupristin/dalfopristin (QD), erythromycin and levofloxacin at increasing concentrations. For assessment of cytotoxicity the cells were incubated with neutral red (NR) or stained with crystal violet (CV). Measurements were done by photometry. At the concentration range tested QD and erythromycin showed a concentration-dependent cytotoxic effect in both cell cultures. In 3T3 cells the half-maximal effect concentration (EC50) was 20 mg/l for QD and 340 mg/l for erythromycin in the NR uptake test and 12 and 200 mg/l, respectively, in the CV assay. In Eahy926 cells the EC50 was 50 mg/l for QD and 880 mg/l for erythromycin in the NR uptake test and 40 and 750 mg/l, respectively, in the CV assay. No EC50 could be established in both cell types for levofloxacin. Eahy926 cells were less sensitive to cytotoxic stimuli than 3T3 fibroblasts. Cytotoxic effects in both cell cultures occurred in the following order: QD > erythromycin > levofloxacin. This ranking correlates well with the frequency of local adverse effects observed with the infusion of these antibiotics in patients. Thus, these in vitro assays may serve as an estimate for the prediction of local tolerability of antibiotics when administered parenterally.
No preview · Article · Jun 2007 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: The number of candidate chemicals or drugs for registration and authorization is increasing at a fast rate and only few of the existing substances have been tested for teratogenicity to date. Therefore, there is high pressure on authorities to accept models like the whole embryo culture as a screening system for safety evaluation procedures. In view of this background the gradual development of the whole embryo culture into a standardized, scientifically validated tool for developmental toxicology during the last 70 years is summarized. The methodological development of the culture technique is described with the completion, improvement and refinement of the basic culture method as main intention. Special attention was paid to different culture techniques, culture media, gassing schedules, and evaluation strategies. Furthermore the importance of taking "in vitro pharmacokinetics" into consideration when a comparison of in vitro/in vivo results from embryotoxicity testing is intended, is stressed. Additionally, the demonstration of the broad spectrum of useful scientific applications when using this culture system in combination with sophisticated analytical techniques is demonstrated. Finally, an overview on different strategies for the validation of this culture system as an in vitro embryo toxicity test is provided and the officially accepted formal validation process for this application is summarized. The successful validation makes the whole embryo culture a complex in vitro embryotoxicity test with high accuracy and predictability. This robust in vitro system modelling the main phase of rodent organogenesis with a high reproducibility is valuable enough to attract special attention in related scientific fields.
No preview · Article · Feb 2006 · Current Pharmaceutical Design