| 1. |
- Attoff, Kristina, et al.
(author)
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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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In: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 35, s. 100-111
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Journal article (peer-reviewed)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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| 2. |
- Attoff, Kristina, 1985-, et al.
(author)
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Acrylamide alters CREB and retinoic acid signaling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
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Other publication (other academic/artistic)abstract
- Acrylamide is a known neurotoxic compound that we get exposed to through food and through the environment. It can cross the placental barrier as well as the blood-brain barrier resulting in exposure of the fetus and the infant child. We used the human neuroblastoma cell line SH-SY5Y to study the effects of non-cytotoxic acrylamide exposure during 9 days of differentiation on two differentially important signaling pathways, i.e. the retinoic acid receptor (RAR) and cAMP response element-binding protein (CREB) signaling in neurons. Our results showed that exposure of non-cytotoxic concentrations of acrylamide during 9 days of differentiation induced altered expression of multiple genes that are part of the CREB and RAR activation pathways, e.g. cellular retinoic acid binding protein 1, retinol binding protein 7, CREB5 and fibroblast growth factor receptor 2. Other well-established neuronal markers such as brain-derived neurotrophic factor, syntaxin binding protein 2, transforming growth factor beta 1, the dopaminergic markers monoamine oxidase A and dopamine receptor D2 as wells as the cholinergic marker choline O-acetyltransferase were also significantly altered by acrylamide. Our results reveal that acrylamide interferes with crucial pathways involved in neuronal differentiation in vitro and raise concerns over the potential toxic outcomes in humans.
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| 3. |
- Attoff, Kristina, et al.
(author)
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Acrylamide alters CREB and retinoic acid signalling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
- 2020
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- Acrylamide (ACR) is a known neurotoxicant which crosses the blood-brain barrier, passes the placenta and has been detected in breast milk. Hence, early-life exposure to ACR could lead to developmental neurotoxicity. The aim of this study was to elucidate if non-cytotoxic concentrations of ACR alter neuronal differentiation by studying gene expression of markers significant for neurodevelopment in the human neuroblastoma SH-SY5Y cell model. Firstly, by using RNASeq we identified two relevant pathways that are activated during 9 days of retinoic acid (RA) induced differentiation i.e. RA receptor (RAR) activation and the cAMP response element-binding protein (CREB) signalling pathways. Next, by qPCR we showed that 1 and 70 mu M ACR after 9 days exposure alter the expression of 13 out of 36 genes in the RAR activation pathway and 18 out of 47 in the CREB signalling pathway. Furthermore, the expression of established neuronal markers i.e. BDNF, STXBP2, STX3, TGFB1 and CHAT were down-regulated. Decreased protein expression of BDNF and altered ratio of phosphorylated CREB to total CREB were confirmed by western blot. Our results reveal that micromolar concentrations of ACR sustain proliferation, decrease neurite outgrowth and interfere with signalling pathways involved in neuronal differentiation in the SH-SY5Y cell model.
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| 4. |
- Attoff, Kristina, 1985-
(author)
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Cell models for evaluation of adult and developmental neurotoxicity : Focus on acrylamide
- 2019
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Doctoral thesis (other academic/artistic)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-
(author)
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In vitro developmental neurotoxicity of acrylamide
- 2016
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Licentiate thesis (other academic/artistic)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.
(author)
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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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In: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 12:12
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Journal article (peer-reviewed)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. |
- Cediel-Ulloa, Andrea, et al.
(author)
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The C17.2 cell line as testing system for endocrine disruption-induced developmental neurotoxicity
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Other publication (other academic/artistic)abstract
- Hormone signaling plays an essential role during fetal life and is vital for brain development. Endocrine-disrupting chemicals (EDCs) can interfere with the hormonal milieu in this critical time period, disrupting key neurodevelopmental processes. Hence, there is a need for the development of assays that evaluate developmental neurotoxicity (DNT) induced by an endocrine mode of action. Herein, we evaluated the applicability of the neural progenitor C17. 2 cell-line, as an in vitro test method to aid in the detection of endocrine disruption induced DNT. For this, C17.2 cells were exposed during 10 days of differentiation to (ant)agonists of the thyroid hormone (Thr), glucocorticoid (Gr), retinoic acid (Rar), retinoic x (Rxr), oxysterols (Lxr), estrogen (Er), androgen (Ar), and peroxisome proliferator activated delta (Ppard) receptors, as well as to the agonist of the vitamin D (Vdr) receptor. Upon exposure and differentiation, the cells were incubated with Hoechst (nuclear staining) and subsequently stained for βIII-tubulin (neuronal marker) by immunofluorescence. Automated imaging was carried out with a 10X objective lens using an ImageXpress micro xls system (Molecular Devices) and image analysis was performed with MetaXpress® software (Molecular Devices). The C17.2 cells were responsive to the Rar and Rxr agonists which decreased neurite outgrowth, branching and neuronal differentiation as well as to the Rar antagonist which increased neurite outgrowth and branching. With this approach, we have identified that the C17.2 cells are responsive to Gr, Rar, Rxr, and Pparβ/δ, hence contributing to the development of reliable and transferable test methods for hazard assessment of EDCs.
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| 8. |
- Lundqvist, Jessica, et al.
(author)
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Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity
- 2017
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In: Applied In Vitro Toxicology. - : Mary Ann Liebert Inc. - 2332-1539 .- 2332-1512. ; 3:2, s. 154-162
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Journal article (peer-reviewed)abstract
- Using general cytotoxicity assays in combination with in vitro tests for organ-specific toxicity has been proposed as an alternative approach to animal tests for estimation of acute systemic toxicity. Here, we present the C17.2 neural progenitor cell line as an option for estimation of acute neurotoxicity. The C17.2 cells were differentiated for 6 days in serum-free N2 medium with brain-derived neurotrophic factor and nerve growth factor to a mixed culture of neurons and astrocytes. The cells were then exposed to noncytotoxic concentrations of acetylsalicylic acid, atropine, digoxin, ethanol, nicotine, or strychnine for 48 hours and the mRNA levels of glial fibrillary acidic protein, βIII-tubulin, and heat shock protein 32 were analyzed as biomarkers for astrocytes, neurons, and cellular stress respectively. As a functional endpoint, the cell membrane potential (CMP) was monitored after acute addition of each compound to the differentiated C17.2 cells, by using the fluorescent FLIPR® membrane potential assay. Nicotine [3.2E-04 M], atropine [1.2E-05 M], or strychnine [6.4E-05 M] resulted in altered gene expression of at least one biomarker for each compound, indicating alerts for neurotoxicity. The three compounds also induced depolarization of the CMP at the lowest observed effect concentrations 9.5E-05 M of nicotine, 1.5E-05 M of atropine, and 6.9E-07 M of strychnine. The non-neurotoxic compounds acetylsalicylic acid, ethanol, and digoxin did neither affect the mRNA levels, nor the CMP. This study showed that the differentiated C17.2 cells might be useful for estimation of acute neurotoxicity by analyzing expression of mRNA biomarkers and CMP alterations.
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| 9. |
- van der Stel, Wanda, et al.
(author)
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New Approach Methods (NAMs) Supporting Read-Across : Two Neurotoxicity AOP-based IATA Case Studies
- 2021
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In: Altex. - : ALTEX Edition. - 1868-596X .- 1868-8551. ; 38:4, s. 615-635
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Journal article (peer-reviewed)abstract
- Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides - rotenoids and strobilurins - each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts - including transcriptomics - in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.
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