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- 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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| 2. |
- 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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| 3. |
- 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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