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1.
Front Bioeng Biotechnol ; 9: 797076, 2021.
Article in English | MEDLINE | ID: mdl-34957083

ABSTRACT

Research on pathogenic organisms is crucial for medical, biological and agricultural developments. However, biological agents as well as associated knowledge and techniques, can also be misused, for example for the development of biological weapons. Potential malicious use of well-intended research, referred to as "dual-use research", poses a threat to public health and the environment. There are various international resources providing frameworks to assess dual-use potential of the research concerned. However, concrete instructions for researchers on how to perform a dual-use risk assessment is largely lacking. The international need for practical dual-use monitoring and risk assessment instructions, in addition to the need to raise awareness among scientists about potential dual-use aspects of their research has been identified over the last years by the Netherlands Biosecurity Office, through consulting national and international biorisk stakeholders. We identified that Biorisk Management Advisors and researchers need a practical tool to facilitate a dual-use assessment on their specific research. Therefore, the Netherlands Biosecurity Office developed a web-based Dual-Use Quickscan (www.dualusequickscan.com), that can be used periodically by researchers working with microorganisms to assess potential dual-use risks of their research by answering a set of fifteen yes/no questions. The questions for the tool were extracted from existing international open resources, and categorized into three themes: characteristics of the biological agent, knowledge and technology about the biological agent, and consequences of misuse. The results of the Quickscan provide the researcher with an indication of the dual-use potential of the research and can be used as a basis for further discussions with a Biorisk Management Advisor. The Dual-Use Quickscan can be embedded in a broader system of biosafety and biosecurity that includes dual-use monitoring and awareness within organizations. Increased international attention to examine pathogens with pandemic potential has been enhanced by the current COVID-19 pandemic, hence monitoring of dual-use potential urgently needs to be encouraged.

2.
Reprod Toxicol ; 56: 77-86, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-26072468

ABSTRACT

Embryonic stem cell tests (EST) are considered promising alternative assays for developmental toxicity testing. Classical mouse derived assays (mEST) are being replaced by human derived assays (hEST), in view of their relevance for human hazard assessment. We have compared mouse and human neural ESTn assays for neurodevelopmental toxicity as to regulation of gene expression during cell differentiation in both assays. Commonalities were observed in a range of neurodevelopmental genes and gene ontology (GO) terms. The mESTn showed a higher specificity in neurodevelopment than the hESTn, which may in part be caused by necessary differences in test protocols. Moreover, gene expression responses to the anticonvulsant and human teratogen valproic acid were compared. Both assays detected pharmacological and neurodevelopmental gene sets regulated by valproic acid. Common significant expression changes were observed in a subset of homologous neurodevelopmental genes. We suggest that these genes and related GO terms may provide good candidates for robust biomarkers of neurodevelopmental toxicity in hESTn.


Subject(s)
Anticonvulsants/toxicity , Embryonic Stem Cells/drug effects , Gene Expression Regulation, Developmental/drug effects , Neural Stem Cells/drug effects , Neurogenesis/drug effects , Valproic Acid/toxicity , Animals , Cells, Cultured , Cluster Analysis , Dose-Response Relationship, Drug , Embryonic Stem Cells/metabolism , Gene Expression Profiling/methods , Genetic Markers , Humans , Mice , Neural Stem Cells/metabolism , Neurogenesis/genetics , Oligonucleotide Array Sequence Analysis , Risk Assessment , Species Specificity , Time Factors , Toxicity Tests/methods
3.
Toxicol Sci ; 146(2): 311-20, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25979313

ABSTRACT

Differentiating pluripotent stem cells in vitro have proven useful for the study of developmental toxicity. Here, we studied the effects of anticonvulsant drug exposure in a human embryonic stem cell (hESC)-based neurodevelopmental toxicity test (hESTn). During neural differentiation the cells were exposed, for either 1 or 7 days, to noncytotoxic concentration ranges of valproic acid (VPA) or carbamazepine (CBZ), antiepileptic drugs known to cause neurodevelopmental toxicity. The effects observed on gene expression and correlated processes and pathways were in line with processes associated with neural development and pharmaceutical mode of action. In general, VPA showed a higher number of genes and molecular pathways affected than CBZ. The response kinetics differed between both compounds, with CBZ showing higher response magnitudes at day 1, versus VPA at day 7. With this study, we demonstrated the potential and biological relevance of the application of this hESC-based differentiation assay in combination with transcriptomics, as a tool to study neurodevelopmental toxicity.


Subject(s)
Anticonvulsants/toxicity , Carbamazepine/toxicity , Embryonic Stem Cells/drug effects , Gene Expression Regulation/drug effects , Nervous System/drug effects , Valproic Acid/toxicity , Humans , Nervous System/embryology , Principal Component Analysis
4.
Reprod Toxicol ; 55: 11-9, 2015 Aug 01.
Article in English | MEDLINE | ID: mdl-25461900

ABSTRACT

Previously we showed a battery consisting of CALUX transcriptional activation assays, the ReProGlo assay, and the embryonic stem cell test, and zebrafish embryotoxicity assay as 'apical' tests to correctly predict developmental toxicity for 11 out of 12 compounds, and to explain the one false negative [7]. Here we report on applying this battery within the context of grouping and read across, put forward as a potential tool to fill data gaps and avoid animal testing, to distinguish in vivo non- or weak developmental toxicants from potent developmental toxicants within groups of structural analogs. The battery correctly distinguished 2-methylhexanoic acid, monomethyl phthalate, and monobutyltin trichloride as non- or weak developmental toxicants from structurally related developmental toxicants valproic acid, mono-ethylhexyl phthalate, and tributyltin chloride, respectively, and, therefore, holds promise as a biological verification model in grouping and read across approaches. The relevance of toxicokinetic information is indicated.


Subject(s)
Animal Testing Alternatives , Teratogens/toxicity , Toxicity Tests/methods , Animals , Cell Line , Cells, Cultured , Embryo, Nonmammalian/drug effects , Embryonic Stem Cells/drug effects , Genes, Reporter , Humans , Mice , Receptors, Estrogen/metabolism , Reproduction , Teratogens/classification , Teratogens/pharmacokinetics , Toxicokinetics , Zebrafish/embryology
5.
Toxicol In Vitro ; 29(3): 449-57, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25524013

ABSTRACT

Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as ßIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of ßIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.


Subject(s)
Anticonvulsants/pharmacology , Cell Differentiation/drug effects , Embryonic Stem Cells/drug effects , Gene Expression/drug effects , Neurons/drug effects , Animals , Carbamazepine/toxicity , Embryonic Stem Cells/metabolism , Fibroblasts , Humans , Mice , RNA/biosynthesis , Reelin Protein , Valproic Acid/toxicity
6.
Toxicology ; 324: 76-87, 2014 Oct 03.
Article in English | MEDLINE | ID: mdl-25089837

ABSTRACT

Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST.


Subject(s)
Cell Differentiation/drug effects , Embryonic Stem Cells/drug effects , Energy Metabolism/drug effects , Toxicology , Animals , Cell Differentiation/genetics , Cell Line , Cluster Analysis , Databases, Genetic , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/pathology , Energy Metabolism/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Genomics , Humans , Mice , Risk Assessment , Time Factors , Toxicology/methods
7.
Toxicol Lett ; 225(3): 342-9, 2014 Mar 21.
Article in English | MEDLINE | ID: mdl-24480513

ABSTRACT

The embryonic stem cell test (EST) is applied as a model system for detection of embryotoxicants. The application of transcriptomics allows a more detailed effect assessment compared to the morphological endpoint. Genes involved in cell differentiation, modulated by chemical exposures, may be useful as biomarkers of developmental toxicity. We describe a statistical approach to obtain a predictive gene set for toxicity potency ranking of compounds within one class. This resulted in a gene set based on differential gene expression across concentration-response series of phthalatic monoesters. We determined the concentration at which gene expression was changed at least 1.5-fold. Genes responding with the same potency ranking in vitro and in vivo embryotoxicity were selected. A leave-one-out cross-validation showed that the relative potency of each phthalate was always predicted correctly. The classical morphological 50% effect level (ID50) in EST was similar to the predicted concentration using gene set expression responses. A general down-regulation of development-related genes and up-regulation of cell-cycle related genes was observed, reminiscent of the differentiation inhibition in EST. This study illustrates the feasibility of applying dedicated gene set selections as biomarkers for developmental toxicity potency ranking on the basis of in vitro testing in the EST.


Subject(s)
Embryonic Stem Cells/drug effects , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/drug effects , Phthalic Acids/toxicity , Toxicity Tests/methods , Abnormalities, Drug-Induced/etiology , Abnormalities, Drug-Induced/genetics , Algorithms , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Dose-Response Relationship, Drug , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/physiology , Mice , Predictive Value of Tests , Random Allocation , Teratogens/toxicity
8.
Methods Mol Biol ; 947: 375-82, 2013.
Article in English | MEDLINE | ID: mdl-23138917

ABSTRACT

The embryonic stem cell test is an animal-free alternative test method for developmental toxicity. Mouse embryonic stem cells are cultured in a hanging drop method to form embryoid bodies. These embryoid bodies, when plated on tissue culture dishes, differentiate to form contracting myocardial cell foci within 10 days. Inhibition of cardiomyocyte differentiation by test compounds serves as the end point of the assay, as monitored by counting contracting muscle foci under the microscope.


Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Toxicity Tests/methods , Animals , Cell Culture Techniques , Cell Differentiation/drug effects , Cell Line , Cytotoxins/toxicity , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Time Factors
9.
Reprod Toxicol ; 35: 81-8, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22813628

ABSTRACT

The embryonic stem cell test (EST) is based on compound-induced inhibition of cardiomyocyte differentiation of pluripotent stem cells. We examined the use of transcriptomics to assess concentration-effect relationships and performed potency ranking within a chemical class. Three embryotoxic phthalate monoesters, monobutyl phthalate (MBuP), monobenzyl phthalate (MBzP) and mono-(2-ethylhexyl) phthalate (MEHP) and the non-embryotoxic monomethyl phthalate (MMP) were studied for their effects on gene expression. Effects on gene expression were observed at concentrations that did not inhibit cardiomyocyte differentiation or induce cytotoxicity. The embryotoxic phthalate monoesters altered the expression of 668 commonly expressed genes in a concentration-dependent fashion. The same potency ranking was observed for morphology and gene expression (MEHP>MBzP>MBuP>MMP). These results indicate that integrating transcriptomics provides a sensitive method to measure the dose-dependent effects of phthalate monoester exposure and enables potency ranking based on a common mode of action within a class of compounds. Transcriptomic approaches may improve the applicability of the EST, in terms of sensitivity and specificity.


Subject(s)
Embryonic Stem Cells/drug effects , Gene Expression Regulation/drug effects , Myocytes, Cardiac/drug effects , Phthalic Acids/toxicity , Plasticizers/toxicity , Animals , Cell Differentiation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Mice , Myocytes, Cardiac/cytology , Oligonucleotide Array Sequence Analysis
10.
Free Radic Biol Med ; 51(3): 719-25, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21658444

ABSTRACT

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~250 µl volumes, at -80°C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at -80°C, unless a cryopreservative is present. Our "small volume" approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.


Subject(s)
Biomarkers/analysis , Cryopreservation , DNA Damage/genetics , DNA/metabolism , Leukocytes, Mononuclear/metabolism , Blood Specimen Collection , Cell Line , Comet Assay/methods , Comet Assay/trends , DNA Damage/immunology , Humans , Leukocytes, Mononuclear/pathology , Oxidative Stress
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