Cellular Neurotoxicity and Nociception

Research Projects

1. Neurotoxicity determined in vitro

2. The NociOcular test: TRPV1-expressing cells as a model for eye-irritation

PhD students

Selected publications:

A selection from Stockholm University publication database

• Does the food processing contaminant acrylamide cause developmental neurotoxicity? A review and identification of knowledge gaps

  2021. Birgitte Lindeman (et al.). Reproductive Toxicology 101, 93-114

  Article

  There is a worldwide concern on adverse health effects of dietary exposure to acrylamide (AA) due to its presence in commonly consumed foods. AA is formed when carbohydrate rich foods containing asparagine and reducing sugars are prepared at high temperatures and low moisture conditions. Upon oral intake, AA is rapidly absorbed and distributed to all organs. AA is a known human neurotoxicant that can reach the developing foetus via placental transfer and breast milk. Although adverse neurodevelopmental effects have been observed after prenatal AA exposure in rodents, adverse effects of AA on the developing brain has so far not been studied in humans. However, epidemiological studies indicate that gestational exposure to AA impair foetal growth and AA exposure has been associated with reduced head circumference of the neonate. Thus, there is an urgent need for further research to elucidate whether pre- and perinatal AA exposure in humans might impair neurodevelopment and adversely affect neuronal function postnatally. Here, we review the literature with emphasis on the identification of critical knowledge gaps in relation to neurodevelopmental toxicity of AA and its mode of action and we suggest research strategies to close these gaps to better protect the unborn child.

  Read more about Does the food processing contaminant acrylamide cause developmental neurotoxicity? A review and identification of knowledge gaps

• Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons

  2021. Dominik Loser (et al.). Archives of Toxicology

  Article

  Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+](i)) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of alpha 7 and non-alpha 7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+](i) signaling at 10-100 mu M. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the alpha 7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1-10 mu M) blunted the signaling response of nicotine. The pesticides (at 3-30 mu M) also blunted the response to the non-alpha 7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.

  Read more about Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons

• Acrylamide alters CREB and retinoic acid signalling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line

  2020. Kristina Attoff (et al.). Scientific Reports 10 (1)

  Article

  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.

  Read more about Acrylamide alters CREB and retinoic acid signalling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line

• The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

  2020. Alice Krebs (et al.). Archives of Toxicology 94 (7), 2435-2461

  Article

  Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

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• Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

  2019. Johannes Delp (et al.). Archives of Toxicology 93 (6), 1585-1608

  Article

  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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• Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity

  2017. Jessica Lundqvist (et al.). Applied In Vitro Toxicology 3 (2), 154-162

  Article

  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.

  Read more about Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity

• 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. Kristina Attoff (et al.). PLOS ONE 12 (12)

  Article

  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.

  Read more about Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity

• Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y

  2016. Kristina Attoff (et al.). Toxicology in Vitro 35, 100-111

  Article

  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.

  Read more about Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y

• Alternative approaches for identifying acute systemic toxicity

  2017. Jon Hamm (et al.). Toxicology in Vitro 41, 245-259

  Article

  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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• In vitro acute and developmental neurotoxicity screening

  2017. Bela Z. Schmidt (et al.). Archives of Toxicology 91 (1), 1-33

  Article

  Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

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• Putative adverse outcome pathways relevant to neurotoxicity

  2015. Anna Bal-Price (et al.). Critical reviews in toxicology 45 (1), 83-91

  Article

  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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• Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests

  2013. Jessica Lundqvist (et al.). Toxicology in Vitro 27 (5), 1565-1569

  Article

  Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.

  Read more about Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests

• Predicting eye stinging potential of baby shampoos by assessing TRPV1 channel activity

  2012. Anna Forsby (et al.). Toxicology Letters 211, S113-S113

  Article

  The Transient Receptor Potential Vanilloid type 1 (TRPV1) receptor is one of the most well characterized pain-inducing receptors. The purpose of this study was to predict human eye stinging of 19 baby bath and shampoo formulations by studying TRPV1 activity. The NociOcular test, a novel recombinant neuronal in vitro model with high expression of functional TRPV1 channels was used to test shampoo formulations containing surfactants, preservatives, and fragrances (sodium laureth sulfate, cocoamidopropylbetaine, cocoglucoside, sodium benzoate, quaternium-15, etc.). The increase in intracellular free Ca²⁺ was analysed by fluorescence during exposure. TRPV1-specific Ca²⁺ influx was abolished when the TRPV1 channel antagonist capsazepine was applied to the cells prior to shampoo samples. The positive control, i.e. adult shampoo, was the most active sample tested in the NociOcular test and also induced the worst stinging sensation. The negative control, i.e. marketed baby shampoo, was negative in both tests. Seven of the formulations induced stinging in the human test, and of those six were positive in the NociOcular test. Twelve of the formulations were classified as non-stinging in the human test, and of those 10 were negative in the NociOcular test. None of the established in vitro tests for eye irritation were able to correctly predict the human stinging sensation of the baby products. Our data support that the TRPV1 channel is a principle mediator of eye stinging sensation induced by baby bath and shampoo formulations and that the NociOcular test may be a valuable in vitro tool to predict human eye stinging sensation.

  Read more about Predicting eye stinging potential of baby shampoos by assessing TRPV1 channel activity

• Using Novel In Vitro NociOcular Assay Based on TRPV1 Channel Activation for Prediction of Eye Sting Potential of Baby Shampoos

  2012. Anna Forsby (et al.). Toxicological Sciences 129 (2), 325-331

  Article

  The transient receptor potential vanilloid type 1 (TRPV1) channel is one of the most well-characterized pain-inducing receptors. The purpose of this study was to predict human eye stinging of 19 baby bath and shampoo formulations by studying TRPV1 activity, as measured by increase in intracellular free Ca2+. The NociOcular test, a novel recombinant neuronal in vitro model with high expression of functional TRPV1 channels, was used to test formulations containing a variety of surfactants, preservatives, and fragrances. TRPV1-specific Ca2+ influx was abolished when the TRPV1 channel antagonist capsazepine was applied to the cells prior to shampoo samples. The positive control, an adult shampoo that contains cocamide monoethanolamine (CMEA), a known stinging ingredient, was the most active sample tested in the NociOcular test. The negative control, a marketed baby shampoo, was negative in the NociOcular and human tests. Seven of the formulations induced stinging in the human test, and of those six were positive in the NociOcular test. Twelve formulations were classified as nonstinging in the human test, and of those ten were negative in the NociOcular test. There was no correlation between the clinical stinging results for the baby formulations and the data generated from other in vitro eye irritation assays (cytosensor microphysiometer, neutral red uptake, EpiOcular, transepithelial permeability). Our data support that the TRPV1 channel is a principal mediator of eye-stinging sensation induced by baby bath and shampoo formulations and that the NociOcular test may be a valuable in vitro tool to predict human eye stinging sensation.

  Read more about Using Novel In Vitro NociOcular Assay Based on TRPV1 Channel Activation for Prediction of Eye Sting Potential of Baby Shampoos

• Low-Dose/Dose-Rate gamma Radiation Depresses Neural Differentiation and Alters Protein Expression Profiles in Neuroblastoma SH-SY5Y Cells and C17.2 Neural Stem Cells

  2011. Ainars Bajinskis (et al.). Radiation Research 175 (2), 185-192

  Article

  The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose gamma-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs gamma rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate gamma rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism. (C) 2011 by Radiation Research Society

  Read more about Low-Dose/Dose-Rate gamma Radiation Depresses Neural Differentiation and Alters Protein Expression Profiles in Neuroblastoma SH-SY5Y Cells and C17.2 Neural Stem Cells

• Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity

  2010. Helena Gustafsson (et al.). Toxicology and Applied Pharmacology 245 (2), 191-202

  Article

  The objective of the EU-funded integrated project ACuteTox is to develop a strategy in which general cytotoxicity, together with organ-specific toxicity and biokinetic features, are used for the estimation of human acute systemic toxicity. Our role in the project is to characterise the effect of reference chemicals with regard to neurotoxicity. We studied cell membrane potential (CMP), noradrenalin (NA) uptake, acetylcholine esterase (AChE) activity, acetylcholine receptor (AChR) signalling and voltage-operated calcium channel (VOCC) function in human neuroblastoma SH-SY5Y cells after exposure to 23 pharmaceuticals, pesticides or industrial chemicals. Neurotoxic alert chemicals were identified by comparing the obtained data with cytotoxicity data from the neutral red uptake assay in 3T3 mouse fibroblasts. Furthermore, neurotoxic concentrations were correlated with estimated human lethal blood concentrations (LC50). The CMP assay was the most sensitive assay, identifying eight chemicals as neurotoxic alerts and improving the LC50 correlation for nicotine, lindane, atropine and methadone. The NA uptake assay identified five neurotoxic alert chemicals and improved the LC50 correlation for atropine, diazepam, verapamil and methadone. The AChE, AChR and VOCC assays showed limited potential for detection of acute toxicity. The CMP assay was further evaluated by testing 36 additional reference chemicals. Five neurotoxic alert chemicals were generated and orphendrine and amitriptyline showed improved LC50 correlation. Due to the high sensitivity and the simplicity of the test protocol, the CMP assay constitutes a good candidate assay to be included in an in vitro test strategy for prediction of acute systemic toxicity.

  Read more about Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity

• Anionic linear aliphatic surfactants activate TRPV1

  2009. H. Lindegren (et al.). Toxicology in Vitro 23 (8), 1472-1476

  Article

  The transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca²⁺ influx in sensory C-fibres with secondary effects leading to neurogenic inflammation in the surrounding tissue. We have earlier reported specific activation of TRPV1 by surfactant-containing hygiene products. We have continued this project by investigating activation of the TRPV1 by shampoo and soap ingredients in low concentrations measured as intracellular Ca²⁺ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. As a TRPV1 specific control, the TRPV1 antagonist capsazepine was used. The response was quantified as the product induced Ca²⁺ influx during 2 min in relation to the maximum response induced by the TRPV1 agonist capsaicin. The results show that anionic alkyl linear surfactant ingredients such as sodium lauryl sulphate, sodium laureth sulphate, ammonium lauryl sulphate, sodium C12-15 pareth sulphate and N-lauroylsarcosine concentration-dependently induced Ca²⁺ influx that could be addressed to TRPV1. The cationic surfactants benzalkonium chloride and cetylpyridinium chloride induced a Ca²⁺ influx that was not TRPV1 mediated as well as the zwitterionic surfactant cocamidopropyl betaine, the non-linear anionic surfactant sodium deoxycholate and the non-ionic surfactant Triton-X. These results reveal a new mechanistic pathway for surfactant-induced nociception.

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• Neuronal in vitro models for the estimation of acute systemic toxicity.

  2009. A Forsby (et al.). Toxicology in vitro : an international journal published in association with BIBRA 23 (8), 1564-1569

  Article

  The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

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• TRPV1 expression and activity during retinoic acid-induced neuronal differentiation

  2009. Johanna EL Andaloussi-Lilja, Jessica Lundqvist, Anna Forsby. Neurochemistry International 55 (8), 768-774

  Article

  The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca²⁺-permeable channel primarily expressed in dorsal root ganglion neurons. Besides its function in thermogenic nociception and neurogenic inflammation, TRPV1 is involved in cell migration, cytoskeleton reorganisation and in neuronal guidance.  To explore the TRPV1 level and activity during conditions for neuronal maturation, TRPV1-expressing SHSY5Y neuroblastoma cells were differentiated into a neuronal phenotype using all-trans-retinoic acid (RA). We show that RA highly up-regulated the total and cell surface TRPV1 protein expression but the TRPV1 mRNA level was unaffected. The up-regulated receptors were localised to the cell bodies and the developed neurites. Furthermore, RA increased both the basal intracellular free Ca²⁺ concentration by 30 % as well as the relative capsaicin-induced Ca²⁺ influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca²⁺ homeostasis.

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• Surfactant-Induced TRPV1 Activity—A Novel Mechanismfor Eye Irritation?

  2007. Johanna Lilja, Helene Lindegren, Anna Forsby. Toxicological Sciences 99 (1), 174-180

  Article

  The pain receptor transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway.  Activation of the receptor causes a Ca²⁺ influx with secondary effects leading to neurogenic inflammation. Here we report specific activation of TRPV1 by detergent-containing hygiene products measured as intracellular Ca²⁺ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. Children products marketed as “painless” (containing lower concentration of detergents), and conditioners (without detergents) did not induce specific TRPV1 activation. Furthermore, low concentrations of the detergent sodium lauryl sulfate (SLS) dose-dependently induced Ca²⁺ influxes that could be addressed to TRPV1. These results reveal a novel mechanistic pathway for surfactant-induced nociception, which may be an important endpoint in in vitro test batteries as alternatives to Draize’s rabbit eye test for classification of eye irritating products.

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Show all publications by Anna Forsby at Stockholm University