| 1. |
- Bal-Price, Anna, et al.
(författare)
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Putative adverse outcome pathways relevant to neurotoxicity
- 2015
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Ingår i: Critical reviews in toxicology. - : Informa UK Limited. - 1040-8444 .- 1547-6898. ; 45:1, s. 83-91
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Forskningsöversikt (refereegranskat)abstract
- The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.
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| 2. |
- Hamm, Jon, et al.
(författare)
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Alternative approaches for identifying acute systemic toxicity : Moving from research to regulatory testing
- 2017
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Ingår i: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 41, s. 245-259
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Forskningsöversikt (refereegranskat)abstract
- Acute systemic toxicity testing provides the basis for hazard labeling and risk management of chemicals. A number of international efforts have been directed at identifying non-animal alternatives for in vivo acute systemic toxicity tests. A September 2015 workshop, Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing, reviewed the state-of-the-science of non-animal alternatives for this testing and explored ways to facilitate implementation of alternatives. Workshop attendees included representatives from international regulatory agencies, academia, nongovernmental organizations, and industry. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements. Attendees particularly noted the need to characterize variability in reference data to evaluate new approaches. They also noted the importance of understanding the mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Workshop breakout groups explored different approaches to reducing or replacing animal use for acute toxicity testing, with each group crafting a roadmap and strategy to accomplish near-term progress. The workshop steering committee has organized efforts to implement the recommendations of the workshop participants.
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| 3. |
- Attoff, Kristina, et al.
(författare)
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Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y
- 2016
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Ingår i: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 35, s. 100-111
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Tidskriftsartikel (refereegranskat)abstract
- Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10 fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10 pM. Acrylamide significantly reduced the number of neurons starting at 1 mu M and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.
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| 4. |
- Attoff, Kristina, 1985-
(författare)
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Cell models for evaluation of adult and developmental neurotoxicity : Focus on acrylamide
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is aimed at summarizing some of the alternative in vitro methods and models that have been used to study both adult and developmental neurotoxicity (DNT), and also to pinpoint some of the important aspects of using alternative in vitro methods. The aim of the papers included in this thesis was to challenge the hypothesis that neurotoxicity and DNT of chemicals can be studied using robust endpoints for proliferation and neural differentiation, such as neurite outgrowth, mRNA expression and protein expression, in two different cell lines. The aim was also to characterize the two cell lines and identify marker genes important for differentiation and to evaluate if these markers could be used as indicators for DNT. The hypothesis being that any chemical that change the expression of important genes for the developmental process could possibly result in DNT for the cells. The current developmental neurotoxicity testing guidelines, using animal models, are time consuming, expensive, ethically questionable and have relatively low sensitivity. Because of this, there has been a paradigm shift towards developing and using alternative methods capable of testing and screening large number of substances. The next generation of developmental neurotoxicity testing is predicted to consist of both in silico and in vitro testing that have to be used in a combined fashion so that it will generate a more rapid and efficient toxicity testing. The idea is to use a battery of refined endpoint studies that identify the specific toxicity of a compound, discriminate between different neural subpopulations and the different stages of neural differentiation. The use of transcriptomic approaches has been suggested as an example of such an endpoint. In this thesis we have evaluated the human neuroblastoma cell line SH-SY5Y and the murine neural progenitor cell line C17.2 in their ability to detect neurotoxic and developmental neurotoxic compounds. We have evaluated this by using functional endpoints, such as neurite outgrowth, cell membrane potential and phenotype ratios. We have also studied the effect of selected chemicals on the levels of mRNA markers specific for different neural cell populations or for neural differentiation in general. We have performed whole genome gene expression on the two cell lines during differentiation and identified and selected a limited number of genes that have been evaluated for their ability to detect developmental neurotoxicity. Both cell lines showed that they have the capability to identify neurotoxic and developmental neurotoxic compounds and could possibly serve as an addition to the testing battery of neurotoxicity in the future. Some of the focus of this thesis has been directed towards the neurodevelopmental effects of the neurotoxic compound acrylamide. Most people get exposed to acrylamide through food consumption and from environmental pollution. Since acrylamide crosses the placental barrier, it creates a risk for developmental consequences. We found that acrylamide affected both cell proliferation and differentiation in both cell lines. Acrylamide affected both neuronal and the glial phenotypes in the C17.2 cell line. We also revealed that acrylamide attenuated neural differentiation at concentrations that were seven orders of magnitude lower than the estimated plasma concentration of free acrylamide in the fetus. Low concentrations of acrylamide altered the gene expression of several genes involved in the retinoic acid signaling as well as the CREB signaling pathways during retinoic acid driven differentiation in the SH-SY5Y cells. Since sub-micromolar concentrations seem to inhibit the differentiation process in both cell lines, developmental neurotoxicity induced by daily intake of acrylamide is a matter of concern. We found that the C17.2 cell line could function as a good model for detecting acute neurotoxicity by evaluating the cell membrane potential of the cells in combination with gene expression of neural and stress marker genes.
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| 5. |
- Attoff, Kristina, 1985-
(författare)
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In vitro developmental neurotoxicity of acrylamide
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The number of children with neurodevelopmental disorders is increasing worldwide which makes it a public concern. Exposure to environmental chemicals has been reported as a source of developmental neurotoxicity. There is also an increase in the number of chemicals reaching the global market each year and currently there are thousands of substances that have not yet been tested for developmental neurotoxicity. The current developmental neurotoxicity testing guidelines are time consuming, expensive, require a lot of animals and have relatively low sensitivity understanding for the mechanisms of toxicology. The field of developmental neurotoxicity testing is in need of a paradigm shift to the use of alternative in vitro methods capable of testing and screening large number of substances. The next generation developmental neurotoxicity testing will consist of both in silico and in vitro testing that has to be used in a combined fashion so that it will generate a more rapid and efficient toxicity testing. The methods need to be standardized between laboratories so that reproducible data can be obtained. Simple endpoints will simply not be enough for in vitro developmental neurotoxicity testing models. Rather, a battery of more refined endpoints that pinpoints the specific toxicity of a compound, discriminate between different neural subpopulations and different stages of neural differentiation is crucial for success. The use of mRNA biomarkers could be a good example of such an endpoint, and have been suggested to be valuable in detecting developmental neurotoxicity. This thesis will give a broad overview of different alternative in vitro models for developmental neurotoxicity. Developmental neurotoxicity of acrylamide was investigated by using selected cell models and endpoints. Acrylamide is a well-known neurotoxic compound and most people get exposed to the compound by food consumption and from environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed and the risk for adverse effects in the developing nervous system is overwhelming. The neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as indicators for developmental neurotoxicity. The reduced neurite outgrowth in the SH-SY5Y cell model occurred at up to seven orders of magnitude lower than what have been previously shown for different neural cell systems. Acrylamide also affected the differentiation process in both neurons and glia cells in the C17.2 cell line. We show that acrylamide attenuated neural differentiation at seven orders of magnitude lower concentrations than the estimated plasma concentration of free acrylamide in the fetus. The fact that low concentrations seem to delay the differentiation process in both cell lines, raises cause for an alarm for developmental neurotoxicity induced by acrylamide.
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| 6. |
- Attoff, Kristina, et al.
(författare)
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Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity
- 2017
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Ingår i: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 12:12
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Tidskriftsartikel (refereegranskat)abstract
- Despite its high relevance, developmental neurotoxicity (DNT) is one of the least studied forms of toxicity. Current guidelines for DNT testing are based on in vivo testing and they require extensive resources. Transcriptomic approaches using relevant in vitro models have been suggested as a useful tool for identifying possible DNT-generating compounds. In this study, we performed whole genome microarray analysis on the murine progenitor cell line C17.2 following 5 and 10 days of differentiation. We identified 30 genes that are strongly associated with neural differentiation. The C17.2 cell line can be differentiated into a co-culture of both neurons and neuroglial cells, giving a more relevant picture of the brain than using neuronal cells alone. Among the most highly upregulated genes were genes involved in neurogenesis (CHRDL1), axonal guidance (BMP4), neuronal connectivity (PLXDC2), axonogenesis (RTN4R) and astrocyte differentiation (S100B). The 30 biomarkers were further validated by exposure to non-cytotoxic concentrations of two DNT-inducing compounds (valproic acid and methylmercury) and one neurotoxic chemical possessing a possible DNT activity (acrylamide). Twenty-eight of the 30 biomarkers were altered by at least one of the neurotoxic substances, proving the importance of these biomarkers during differentiation. These results suggest that gene expression profiling using a predefined set of biomarkers could be used as a sensitive tool for initial DNT screening of chemicals. Using a predefined set of mRNA biomarkers, instead of the whole genome, makes this model affordable and high-throughput. The use of such models could help speed up the initial screening of substances, possibly indicating alerts that need to be further studied in more sophisticated models.
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| 7. |
- Cotgreave, Ian, et al.
(författare)
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Pyriproxifen and microcephaly: an investigation of potential ties to the ongoing "Zika epidemic"
- 2016
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- As part of the Swetox mission to react to emerging concerns in chemical health and environmental safety, a preliminary litterature investigation was undertaken to gather all readily available scientific information on PPF with respect to safety assessment, in order to better understand potential links between chemical exposure and the devopment of microcephaly in affected areas. Therefore the contents of the report do not constitute an attempt at either questioning the use of existing regulatory data in the manner prescribed by international regulatory proceedures, or as a new risk assessment, based on the scientific information and concepts discussed. Here we report our findings, with particular emphasis on exisiting regulatory information, potential for lack of translation of results from regulatory animal testing to humans, lack of human exposure data and suggestions on plausible mode(s) of action of PPF in human neurodevelopmental adversities such as microcephaly.
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| 8. |
- Delp, Johannes, et al.
(författare)
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Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants
- 2019
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 93:6, s. 1585-1608
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Tidskriftsartikel (refereegranskat)abstract
- Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose <-> galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.
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| 9. |
- Knudsen, Lisbeth E., et al.
(författare)
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Nordic symposium on toxicology and pharmacology without animal experiments-Will it be possible in the next 10 years?
- 2019
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Ingår i: Basic & Clinical Pharmacology & Toxicology. - : Wiley. - 1742-7835 .- 1742-7843. ; 124:5, s. 560-567
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Forskningsöversikt (refereegranskat)abstract
- Toxicological and pharmacological information from human cells and tissues provides knowledge readily applicable to human safety assessment and to the efficacy assessment of pharmaceuticals. The 3R principle in animal studies includes the use of human material in the R of Replacement. The Reduction and Refinement Rs are related to animal use. Knowledge of the 3Rs and successful 3R methods are a prerequisite for the Reduction of animal experiments in the future. More collaboration among researchers using experimental animals and those working in vitro is necessary with mutual respect. The OECD Guidelines for the Testing of Chemicals have included the animal-free part of the 3Rs in guidances for the development and reporting of Adverse Outcome Pathways (AOPs), which is to be part of the Integrated Approaches to Testing and Assessment (IATA). The 3R centres established to help fulfil the Directive 2010/63/EU play an important role to promote the 3Rs and in the development of animal-free toxicology. Research centres in each Nordic country are founded upon solid research activities in cell and organ toxicity, including major EU programmes to promote 3Rs and implementation of good practices and methods broadly in all stakeholders of industry, regulators and academia. In the light of this, the Nordic Symposium on Toxicology and Pharmacology without Animal Experiments addressed more adopted/modified test guidelines or new test guidelines for new end-points, or hazard challenges, new in vitro 3D models, speeding up transfer of knowledge from research to regulation to understand AOP and towards IATA.
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| 10. |
- Leist, Marcel, et al.
(författare)
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Adverse outcome pathways : opportunities, limitations and open questions
- 2017
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 91:11, s. 3477-3505
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Tidskriftsartikel (refereegranskat)abstract
- Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event erelationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.
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