1.
Attoff, Kristina, et al.
(författare)
Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y
2016
Ingår i: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 35, s. 100-111
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.
2.
Hinojosa, Maria G., 1994-, et al.
(författare)
Evaluation of mRNA markers in differentiating human SH-SY5Y cells for estimation of developmental neurotoxicity
2023
Ingår i: Neurotoxicology. - : Elsevier. - 0161-813X .- 1872-9711. ; 97, s. 65-77
Tidskriftsartikel (refereegranskat) abstract
Current guidelines for developmental neurotoxicity (DNT) evaluation are based on animal models. These have limitations so more relevant, efficient and robust approaches for DNT assessment are needed. We have used the human SH-SY5Y neuroblastoma cell model to evaluate a panel of 93 mRNA markers that are frequent in Neuronal diseases and functional annotations and also differentially expressed during retinoic acid-induced differentiation in the cell model. Rotenone, valproic acid (VPA), acrylamide (ACR) and methylmercury chlo-ride (MeHg) were used as DNT positive compounds. Tolbutamide, D-mannitol and clofibrate were used as DNT negative compounds. To determine concentrations for exposure for gene expression analysis, we developed a pipeline for neurite outgrowth assessment by live-cell imaging. In addition, cell viability was measured by the resazurin assay. Gene expression was analyzed by RT-qPCR after 6 days of exposure during differentiation to concentrations of the DNT positive compounds that affected neurite outgrowth, but with no or minimal effect on cell viability. Methylmercury affected cell viability at lower concentrations than neurite outgrowth, hence the cells were exposed with the highest non-cytotoxic concentration. Rotenone (7.3 nM) induced 32 differentially expressed genes (DEGs), ACR (70 mu M) 8 DEGs, and VPA (75 mu M) 16 DEGs. No individual genes were significantly dysregulated by all 3 DNT positive compounds (p < 0.05), but 9 genes were differentially expressed by 2 of them. Methylmercury (0.8 nM) was used to validate the 9 DEGs. The expression of SEMA5A (encoding semaphorin 5A) and CHRNA7 (encoding nicotinic acetylcholine receptor subunit alpha 7) was downregulated by all 4 DNT positive compounds. None of the DNT negative compounds dysregulated any of the 9 DEGs in common for the DNT positive compounds. We suggest that SEMA5A or CHRNA7 should be further evaluated as biomarkers for DNT studies in vitro since they also are involved in neurodevelopmental adverse outcomes in humans.
3.
Axelsson, Viktoria, et al.
(författare)
Gliotoxin induces caspase-dependent neurite degeneration and calpain-mediated general cytotoxicity in differentiated human neuroblastoma SH-SY5Y cells.
2006
Ingår i: Biochem Biophys Res Commun. - 0006-291X. ; 345:3, s. 1068-74
Tidskriftsartikel (refereegranskat) abstract
In this study, a significant increase by 50% in intracellular free calcium concentration ([Ca(2+)](i)) was observed in differentiated human neuroblastoma (SH-SY5Y) cells after exposure to 0.25microM of the fungal metabolite gliotoxin for 72h. Further, the involvement of caspases and calpains was demonstrated to underlie the gliotoxin-induced cytotoxic and neurite degenerative effects. The caspase inhibitor Z-VAD-fmk almost completely reduced the neurite degeneration from 40% degeneration of neurites to 5% as compared to control. Inhibition of calpains with calpeptin significantly attenuated gliotoxin-induced cytotoxicity, determined as reduction in total cellular protein content, from 43% to 14% as compared to control cells. Western blot analyses of alphaII-spectrin breakdown fragments confirmed activity of the proteases, and that alphaII-spectrin was cleaved by caspases in gliotoxin-exposed cells. These results show that calpains and caspases have a role in the toxicity of gliotoxin in differentiated SH-SY5Y cells and that the process may be Ca(2+)-mediated.
4.
Bal-Price, Anna, et al.
(författare)
Putative adverse outcome pathways relevant to neurotoxicity
2015
Ingår i: Critical reviews in toxicology. - : Informa UK Limited. - 1040-8444 .- 1547-6898. ; 45:1, s. 83-91
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.
5.
Clemedson, Cecilia, et al.
(författare)
Development of an in vitro test battery for the estimation of acute human systemic toxicity : An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries
2002
Ingår i: ATLA (Alternatives to Laboratory Animals). - 0261-1929. ; 30:3, s. 313-321
Tidskriftsartikel (refereegranskat) abstract
The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.
6.
Delp, Johannes, et al.
(författare)
Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants
2019
Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 93:6, s. 1585-1608
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.
7.
Delp, Johannes, et al.
(författare)
Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors
2021
Ingår i: Archives of Toxicology. - : Springer. - 0340-5761 .- 1432-0738. ; 95:2, s. 591-615
Tidskriftsartikel (refereegranskat) abstract
Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.
8.
Forsby, Anna, et al.
(författare)
Using Novel In Vitro NociOcular Assay Based on TRPV1 Channel Activation for Prediction of Eye Sting Potential of Baby Shampoos
2012
Ingår i: Toxicological Sciences. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 129:2, s. 325-331
Tidskriftsartikel (refereegranskat) abstract
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.
9.
Gustafsson, Helena, et al.
(författare)
The use of the human neuroblastoma SH-SY5Y cell line for estimation of acute systemic toxicity in vitro.
2007
Konferensbidrag (refereegranskat) abstract
Acute systemic toxicity, expressed as human lethal blood peak concentration or the dose inducing 50 % lethality in an animal population (LD50), can be estimated by general cytotoxicity tests using proliferating mammalian cell lines for 70-80 % of all chemicals. The cytotoxicity for the remaining chemicals over or under estimate the LD50 values/human lethal blood peak concentrations because of their very specific molecular targets or toxicokinetic features in vivo. The objective of the EU funded integrated project “ACuteTox” is to develop a strategy in which organ-specific endpoints and toxicokinetic features are taken into consideration in the in vitro prediction of acute systemic toxicity. The human neurotblastoma SH-SY5Y cell line was used as a model for studies on neurospecific targets, which are know to be crucial for survival. All endpoints were investigated after short exposure times (minutes to an hour) at concentrations of the test chemicals that did not affect the cell viability, measured as cell membrane leakage of lactate dehydrogenase. The effects of 23-26 compounds (drugs, pesticides and industrial chemicals) were studied on the cell membrane potential (CMP), voltage dependent Ca2+ channels (VDCC), muscarinic acetylcholine receptor (mAChR) function, acetylcholinesterase (AChE) activity and noradrenalin uptake. The results showed that the CMP was altered by atropine, amphetamine, mercury chloride, methadone, nicotine, pentachlorphenol, sodium lauryl sulphate (SLS) and verapamil, where as an effect on VDCC could be detected for amphetamine, atropine, colchicine, pentachlorphenol, SLS and verapamil. The mAChR function was measured as carbachol-induced Ca2+, i.e. activation of phospholipase C. Amphetamine, pentachlorphenol, SDS and verapamil attenuated the carbachol response by 50% at concentrations about 1 mM, but the specific mAChR antagonist atropine had the same effect at 3 nM. Nicotine, caffeine, pentachlorphenol, methadone, mercury chloride, SLS and the specific inhibitors physostigmine, dichlorvos and malathion attenuated the AChE activity at significantly non-cytotoxic concentrations in SH-SY5Y cells after 60 minutes of exposure. Parathion did not inhibit the AChE activity after 60 minutes exposure, but after 48 hr, indicating that oxidation of parathion to the active inhibitor paraoxon took place in the cell culture. This phenomenon was also observed for malathion, which displayed a lower EC50 value after the prolonged exposure time. The noradrenalin uptake was affected by atropine, caffeine, carbamazepine, amphetamine, diazepam, isopropanol, methadone, SLS and verapamil. A comparison of the active concentrations with the basal cytotoxicity measured as neutral red uptake in mouse fibroblast 3T3 cells indicated that the AChE assay is useful for detection of AChE inhibitors and possibly also AChR ligands. The VDCC endpoint was useful as an alert only for verapamil and the mAChR function was only specifically affected by atropine. The noradrenalin uptake indicated a clear alert for amphetamine and methadone, which was expected, but not for the other test compounds. These results indicate that the usefulness of these endpoints in a general test battery for estimation of acute systemic toxicity is limited, except for AChE activity measurements. However, the results clearly showed that the compounds with known mechanisms (e.g. atropine, verapamil, amphetamine and methodone ) displayed expected effects on their specific endpoints.
10.
Johansson, Ylva, 1993-, et al.
(författare)
Attenuated neuronal differentiation caused by acrylamide is not related to oxidative stress in differentiated human neuroblastoma SH-SY5Y cells
2024
Ingår i: Food and Chemical Toxicology. - 0278-6915 .- 1873-6351. ; 187
Tidskriftsartikel (refereegranskat) abstract
Acrylamide (ACR) is a known neurotoxicant and developmental neurotoxicant. As a soft electrophile, ACR reacts with thiol groups in cysteine. One hypothesis of ACR induced neurotoxicity and developmental neurotoxicity (DNT) is conjugation with reduced glutathione (GSH) leading to GSH depletion, increased reactive oxygen species (ROS) production and further oxidative stress and cellular damage. In this regard, we have investigated the effect of ACR on neuronal differentiation, glutathione levels and ROS production in the human neuroblastoma SH-SY5Y cell model. After 9 days of differentiation and exposure, ACR significantly impaired area neurites per cell at non-cytotoxic concentrations (0.33 μM and 10 μM). Furthermore, 10 μM ACR dysregulated 9 mRNA markers important for neuronal development, 5 of them being associated with cytoskeleton organization and axonal guidance. At the non-cytotoxic concentrations that significantly attenuate neuronal differentiation, ACR did neither decrease the level of GSH or total glutathione levels, nor increased ROS production. In addition, the expression of 5 mRNA markers for cellular stress was assessed with no significant altered regulation after ACR exposure up to 320 μM. Thus, ACR-induced DNT is not due to GSH depletion and increased ROS production, neither at non-cytotoxic nor cytotoxic concentrations, in the SH-SH5Y model during differentiation.
