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  • 1.
    Andres, M I
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Walum, E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Polygodial-induced noradrenaline release in human neuroblastoma SH-SY5Y cells.1997Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 11, nr 5, s. 509-11Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Polygodial is a natural sesquiterpene which exhibits pronounced pungency and a powerful antifeedant activity. At low concentrations, which do not alter general cell membrane permeability, polygodial increases the intracellular concentration of free calcium ([Ca(2+)](i)). Sensory neurotransmission depends on noradrenaline (NA) release, and vesicular exocytosis, in turn, is dependent on an increase in [Ca(2+)](i). The nociceptive response induced by polygodial could therefore be directly linked to intracellular calcium levels. Consequently, the objective of this work was to investigate the effect of polygodial on NA release. The human neuroblastoma cell line SH-SY5Y was selected as an in vitro model for sensory neurones. Semiconfluent cells were preloaded with tritiated NA ([(3)H]NA). After 3 min exposure of polygodial to the cells, released and unreleased radioactivity were measured. Polygodial induced a significant [(3)H]NA release at concentrations between 0.1 and 0.5 mug/ml with a maximum effect at 0.2 mug/ml (40% increased release of [(3)H]NA as compared with unstimulated control cells). No polygodial-induced transmitter release was seen at 3.5 and 5 mug/ml. For comparison, carbachol (1 rim) increased [(3)H]NA release by 10% and the KCl-induced (100 mm) [(3)H]NA release increased by 8% as compared with unstimulated SH-SY5Y cells. In conclusion polygodial, at the concentrations 0.1-0.5 mug/ml (equal to 0.4-2 mum), induces NA release which is dependent on polygodial-induced increase in [Ca(2+)](i).

  • 2. Aschner, Michael
    et al.
    Levin, Edward D.
    Suñol, Cristina
    Olopade, James O.
    Helmcke, Kirsten J.
    Avila, Daiana S.
    Sledge, Damiyon
    Ali, Rahim H.
    Upchurch, Lucia
    Donerly, Susan
    Linney, Elwood
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ponnuru, Padmavathi
    Connor, James R.
    Gene-environment interactions: Neurodegeneration in non-mammals and mammals2010Inngår i: Neurotoxicology, ISSN 0161-813X, E-ISSN 1872-9711, Vol. 31, nr 5, s. 582-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The understanding of how environmental exposures interact with genetics in central nervous system dysfunction has gained great momentum in the last decade. Seminal findings have been uncovered in both mammalian and non-mammalian model in large result of the extraordinary conservation of both genetic elements and differentiation processes between mammals and non-mammalians. Emerging model organisms, such as the nematode and zebrafish have made it possible to assess the effects of small molecules rapidly, inexpensively, and on a miniaturized scale. By combining the scale and throughput of in vitro screens with the physiological complexity and traditional animal studies, these models are providing relevant information on molecular events in the etiology of neurodegenerative disorders. The utility of these models is largely driven by the functional conservation seen between them and higher organisms, including humans so that knowledge obtained using non-mammalian model systems can often provide a better understanding of equivalent processes, pathways, and mechanisms in man. Understanding the molecular events that trigger neurodegeneration has also greatly relied upon the use of tissue culture models. The purpose of this summary is to provide-state-of-the-art review of recent developments of non-mammalian experimental models and their utility in addressing issues pertinent to neurotoxicity (Caenorhabditis elegans and Danio rerio). The synopses by Aschner and Levin summarize how genetic mutants of these species can be used to complement the understanding of molecular and cellular mechanisms associated with neurobehavioral toxicity and neurodegeneration. Next, studies by Suñol and Olopade detail the predictive value of cultures in assessing neurotoxicity. Suñol and colleagues summarize present novel information strategies based on in vitro toxicity assays that are predictive of cellular effects that can be extrapolated to effects on individuals. Olopade and colleagues describe cellular changes caused by sodium metavanadate (SMV) and demonstrate how rat primary astrocyte cultures can be used as predicitive tools to assess the neuroprotective effects of antidotes on vanadium-induced astrogliosis and demyelination.

  • 3.
    Attoff, Kristina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gliga, Anda
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swetox, Karolinska Institutet, Sweden.
    Norinder, Ulf
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swetox, Karolinska Institutet, Sweden.
    Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity2017Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, nr 12, artikkel-id e0190066Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 4.
    Attoff, Kristina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kertika, Dimitra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oredsson, S.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Stockholm Univ, Dept Neurochem, S-10691 Stockholm, Sweden.
    Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y2016Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 35, s. 100-111Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 5.
    Attoff, Kristina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Stockholm University.
    Kertika, Dimitra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oredsson, Stina
    Lund University.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5YManuskript (preprint) (Annet vitenskapelig)
  • 6.
    Axelsson, V
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pikkarainen, K
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells.2006Inngår i: Cell Biology and Toxicology, ISSN 0742-2091, E-ISSN 1573-6822, Vol. 22, nr 2, s. 127-36Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Gliotoxin is a fungal second metabolite produced by diverse species that can be found in compost, stored crops, moist animal feed and sawdust. The role of glutathione in gliotoxin-induced toxicity was studied in order to elucidate the toxic mechanisms leading to neurite degeneration and cell death in differentiated human neuroblastoma (SH-SY5Y) cells. After 72 h of exposure to gliotoxin, moderate cytotoxicity was induced at 0.1 micromol/L, which was more severe at higher concentrations. A reduction in the number of neurites per cell was also observed. By decreasing the level of intracellular glutathione with L: -buthionine-sulfoxamine (BSO) a specific inhibitor of glutathione synthesis, the cytotoxic effect of gliotoxin was significantly attenuated. The gliotoxin-induced cytotoxicity was also slightly reduced by the antioxidant vitamin C. However, the neurite degenerative effect was not altered by BSO, or by vitamin C. A concentration-dependent increase in the ratio between oxidized and reduced forms of glutathione, as well as the total intracellular glutathione levels, was noted after exposure to gliotoxin. The increase of glutathione was also reflected in western blot analyses showing a tendency for the regulatory subunit of gamma-glutamylcysteine synthetase to be upregulated. In addition, the activity of glutathione reductase was slightly increased in gliotoxin-exposed cells. These results indicate that glutathione promotes gliotoxin-induced cytotoxicity, probably by reducing the ETP (epipolythiodioxopiperazine) disulfide bridge to the dithiol form.

  • 7.
    Axelsson, Viktoria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holback, Sofia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sjögren, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gustafsson, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gliotoxin induces caspase-dependent neurite degeneration and calpain-mediated general cytotoxicity in differentiated human neuroblastoma SH-SY5Y cells.2006Inngår i: Biochem Biophys Res Commun, ISSN 0006-291X, Vol. 345, nr 3, s. 1068-74Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 8.
    Bajinskis, Ainars
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Lindegren, Helene
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Lotta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Harms-Ringdahl, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Low-Dose/Dose-Rate gamma Radiation Depresses Neural Differentiation and Alters Protein Expression Profiles in Neuroblastoma SH-SY5Y Cells and C17.2 Neural Stem Cells2011Inngår i: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404, Vol. 175, nr 2, s. 185-192Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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

  • 9. Bal-Price, Anna
    et al.
    Crofton, Kevin M.
    Sachana, Magdalini
    Shafer, Timothy J.
    Behl, Mamta
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hargreaves, Alan
    Landesmann, Brigitte
    Lein, Pamela J.
    Louisse, Jochem
    Monnet-Tschudi, Florianne
    Paini, Alicia
    Rolaki, Alexandra
    Schrattenholz, Andre
    Sunol, Cristina
    van Thriel, Christoph
    Whelan, Maurice
    Fritsche, Ellen
    Putative adverse outcome pathways relevant to neurotoxicity2015Inngår i: Critical reviews in toxicology, ISSN 1040-8444, E-ISSN 1547-6898, Vol. 45, nr 1, s. 83-91Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    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.

  • 10. Clemedson, Cecilia
    et al.
    Kolman, Ada
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The integrated acute systemic toxicity project (ACuteTox) for the optimisation and validation of alternative in vitro tests.2007Inngår i: ATLA (Alternatives to Laboratory Animals), ISSN 0261-1929, Vol. 35, nr 1, s. 33-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The ACuteTox project is designed to replace animal testing for acute systemic toxicity, as is widely used today for regulatory purposes, by using in vitro and in silico alternatives. In spite of the fact that earlier studies on acute systemic toxicity demonstrated a good correlation between in vitro basal cytotoxicity data (the 50% inhibitory concentration [IC50]) in human cell lines and rodent LD50 values, and an even better correlation between IC50 values and human lethal blood concentrations, very few non-animal tests have been accepted for general use. Therefore, the aim of the ACuteTox project is to adapt new testing strategies, for example, the implementation of new endpoints and new cell systems for toxicity screening, organ-specific models, metabolism-dependent toxicity, tissue absorption, distribution and excretion, and computer-based prediction models. A new database, AcuBase, containing descriptions and results of in vitro tests of the 97 reference chemicals, as well as the results of animal experimentation, and human acute toxicity data, will be generated within the framework of ACuteTox. Scientists from 13 European countries are working together and making efforts to find the most appropriate testing strategies for the prediction of human acute systemic toxicity, and also to select a robust in vitro test battery for cytotoxicity testing of chemicals.

  • 11. Clemedson, Cecilia
    et al.
    Nordin-Andersson, Marika
    Bjerregaard, Henning F.
    Clausen, Jørgen
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gustafsson, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hansson, Ulrika
    Isomaa, Boris
    Jørgensen, Carsten
    Kolman, Ada
    Kotova, Natalia
    Krause, Gunter
    Kristen, Udo
    Kurppa, Kalle
    Romert, Lennart
    Scheers, Ellen
    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 Batteries2002Inngår i: ATLA (Alternatives to Laboratory Animals), ISSN 0261-1929, Vol. 30, nr 3, s. 313-321Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 12. Dejongh, J
    et al.
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Houston, J B
    Beckman, M
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Combes, R
    Blaauboer, B J
    An Integrated Approach to the Prediction of Systemic Toxicity using Computer-based Biokinetic Models and Biological In vitro Test Methods: Overview of a Prevalidation Study Based on the ECITTS Project.1999Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 13, nr 4-5, s. 549-54Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Chemical toxicity was estimated by integrating in vitro study results with physiologically-based biokinetic models for eight neurotoxic compounds (benzene, toluene, lindane, acrylamide, parathion/oxon, caffeine, diazepam and phenytoin). In vitro studies on general and specific neurotoxicity were performed and biotransformation and tissue-blood distribution studies were used in modelling the biokinetic behaviour of the compounds. Subsequently, neurotoxicity was estimated from the integrated in vitro and kinetic studies. These results were compared with in vivo data from the literature on minimal neurotoxicity for these compounds, such as lowest-observed-effect levels (LOELs). The discrepancy between estimated and experimental LOELs ranged from 2- to 10-fold. LOEL estimates for compounds with a relatively low toxicity were more accurate than for compounds with a relatively high toxicity. LOELs for the most active compounds could only be established after consideration of additional in vitro results from the literature. The present study has generated encouraging results on the risk assessment of chemicals from in vitro studies and computer simulations and has identified some key directions for future research.

  • 13. DeJongh, J
    et al.
    Nordin-Andersson, M
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ploeger, B A
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Estimation of systemic toxicity of acrylamide by integration of in vitro toxicity data with kinetic simulations.1999Inngår i: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 158, nr 3, s. 261-268Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Neurodegenerative properties of acrylamide were studied in vitro by exposure of differentiated SH-SY5Y human neuroblastoma cells for 72 h. The number of neurites per cell and the total cellular protein content were determined every 24 h throughout the exposure and the subsequent 96-h recovery period. Using kinetic data on the metabolism of acrylamide in rat, a biokinetic model was constructed in which the in vitro toxicity data were integrated. Using this model, we estimated the acute and subchronic toxicity of acrylamide for the rat in vivo. These estimations were compared to experimentally derived lowest observed effect doses (LOEDs) for daily intraperitoneal exposure (1, 10, 30, and 90 days) to acrylamide. The estimated LOEDs differed maximally twofold from the experimental LOEDs, and the nonlinear response to acrylamide exposure over time was simulated correctly. It is concluded that the integration of the present in vitro toxicity data with kinetic data gives adequate estimates of acute and subchronic neurotoxicity resulting from acrylamide exposure.

  • 14.
    EL Andaloussi-Lilja, Johanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    TRPV1 expression and activity during retinoic acid-induced neuronal differentiation2009Inngår i: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 55, nr 8, s. 768-774Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 15.
    Folch, Jaume
    et al.
    Unitat de Bioquimica, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Tarragona, Spain.
    Alvira, Daniel
    Unitat de Farmacologia i Farmacognòsia and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona, Spain.
    López-Querol, Marta
    Unitat de Farmacologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Tarragona, Spain.
    Tajes, Marta
    Unitat de Farmacologia i Farmacognòsia and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona, Spain.
    Sureda, Francesc X
    Unitat de Farmacologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Tarragona, Spain.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rimbau, Víctor
    Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona, Spain.
    Camins, Antoni
    Unitat de Farmacologia i Farmacognòsia and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona, Spain.
    Pallàs, Mercè
    Unitat de Farmacologia i Farmacognòsia and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona, Spain.
    Evaluation of transcriptional activity of caspase-3 gene as a marker of acute neurotoxicity in rat cerebellar granular cells2010Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 24, nr 2, s. 465-471Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Caspase-3 is a key protein involved in the classical apoptosis mechanism in neurons, as in many other cells types. In the present research, we describe the transcriptional activity of caspase-3 gene as a marker of acute toxicity in a primary culture model of rat cerebellar granule neurons (CGNs). CGNs were incubated for 16h in complete medium containing the chemicals at three concentrations (10, 100 μM and 1 mM). A total of 48 different compounds were tested. Gene transcriptional activity was determined by low-density array assays, and by single Taqman caspase-3 assays. Results from the PCR arrays showed that the caspase-3 gene was up-regulated when CGNs were exposed to neurotoxic chemicals. Significative correlations were found between the transcriptional activity of caspase-3 and the activity of some other genes related to apoptosis, cell-cycle and ROS detoxification. In our experiments, acute exposure of CGNs to well-documented pro-apoptotic xenobiotics modulated significantly caspase-3 gene expression, whereas chemicals not related to apoptosis did not modify caspase-3 gene expression. In conclusion, acute exposure of CGNs to neurotoxic compounds modulates the transcriptional activity of genes involved in the classical apoptotic pathway, oxidative stress and cell-cycle control. Transcriptional activity of caspase-3 correlates significantly with these changes and it could be a good indicator of acute neurotoxicity.

  • 16.
    Forsby, A
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bal-Price, A K
    Camins, A
    Coecke, S
    Fabre, N
    Gustafsson, H
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Honegger, P
    Kinsner-Ovaskainen, A
    Pallas, M
    Rimbau, V
    Rodríguez-Farré, E
    Suñol, C
    Vericat, J A
    Zurich, M G
    Neuronal in vitro models for the estimation of acute systemic toxicity.2009Inngår i: Toxicology in vitro : an international journal published in association with BIBRA, ISSN 1879-3177, Vol. 23, nr 8, s. 1564-1569Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 17.
    Forsby, A
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Walum, E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Polygodial induces inositol phosphate turnover in human neuroblastoma SH-SY5Y cells.1996Inngår i: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 217, nr 1, s. 50-4Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The pungent sesquiterpenoid unsaturated dialdehydes polygodial and isovelleral, have previously been shown to increase the intracellular free calcium concentration [Ca2+]i in human neuroblastoma SH-SY5Y cells, partly by a release from intracellular Ca2+ stores, whereas the non-pungent compound epipolygodial, had no effect on the [Ca2+]i. In this study, we investigated the effect of isovelleral, polygodial and epipolygodial on inositol phosphate (IP) formation on the assumption that there might be a correlation between the release of intracellular Ca2+ and pungency of the compounds. It was found that polygodial induced IP mobilization in a concentration dependent way, whereas isovelleral had no effect on the IP formation in the SH-SY5Y cells. Phosphoinositide (PPI) turnover was activated by epipolygodial, but only at concentrations 40-fold higher than for polygodial, which emphasizes the importance of the correct stereometry in the dialdehyde configuration for the biological activity of polygodial. The polygodial-induced formation of IP1 was reduced by 71% under extracellular calcium-free conditions, which suggests feedback interactions between the IP formation and the increase in [Ca2+]i to account for a periodic activation of phospholipase C(PLC).

  • 18.
    Forsby, A
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Walum, E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sterner, O
    The effect of six sesquiterpenoid unsaturated dialdehydes on cell membrane permeability in human neuroblastoma SH-SY5Y cells.1992Inngår i: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 84, nr 1, s. 85-95Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The effect of six sesquiterpenes containing an unsaturated dialdehyde functionality, on cell membrane permeability in the human neuroblastoma cell line SH-SY5Y has been studied. The kinetics of the membrane leakage after addition of the sesquiterpenes were determined by measuring the efflux of radioactivity from cells preloaded with tritiated 2-deoxyglucose. The concentrations that gave 5% and 20% efflux of radioactivity as compared with control cells (EC5 and EC20) were determined for each compound. In spite of the structural similarities between the compounds, the effects on cell membrane permeability varied considerably. EC20 for polygodial, which is the most active compound, is 2.5 microM after 20-min incubation, but no leakage could be determined for merulidial even at concentrations as high as 4 mM. Rather, this compound seems to stabilize or fix the cell membrane and a lower efflux of radioactivity was observed as compared to the control cells. A quantitative structure-activity relationship analysis for the five active compounds showed a good correlation between the membrane leakage activity and certain chemical characteristics. Structural features strongly correlated with high activity were found to be: The geometry and the atomic charges of the unsaturated dialdehyde functionality, the dipole moment, the energy difference between the lowest unoccupied molecular orbital and the highest occupied molecular orbital and the lipophilicity.

  • 19.
    Forsby, A
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Witt, R
    Walum, E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sesquiterpenoid unsaturated dialdehydes increase the concentration of intracellular free Ca2+ in human neuroblastoma SH-SY5Y cells.1994Inngår i: Natural toxins (Print), ISSN 1056-9014, E-ISSN 1522-7189, Vol. 2, nr 2, s. 89-95Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The effect of the three sesquiterpenoid unsaturated dialdehydes--polygodial, isovelleral, and epipolygodial--on intracellular free Ca2+ concentration, [Ca2+]i, was investigated in the human neuroblastoma cell line SH-SY5Y. [Ca2+]i was measured by the Fura-2 spectrophotofluorometric technique in multi-cell and single-cell experiments. Polygodial and isovelleral induced an increase in [Ca2+]i at low concentrations (0.1 and 0.5 micrograms/ml, respectively) but epipolygodial affected [Ca2+]i only at a high concentration (10 micrograms/ml). The results indicated that there was a relationship between the effect on [Ca2+]i and the pungency of the sesquiterpenes tested. Experiments in a Ca(2+)-free extracellular solution showed that Ca2+ was also released from intracellular calcium stores. Images from single-cell experiments indicated that polygodial induced fluctuations in the [Ca2+]i in some cells. The mechanism behind the sesquiterpene induced increase of the [Ca2+]i was not identified but a possible mobilization of inositol phosphates is considered.

  • 20.
    Forsby, Anna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Blaauboer, Bas
    Integration of in vitro neurotoxicity data with biokinetic modelling for the estimation of in vivo neurotoxicity.2007Inngår i: Human and Experimental Toxicology, ISSN 0960-3271, E-ISSN 1477-0903, Vol. 26, nr 4, s. 333-338Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Risk assessment of neurotoxicity is mainly based on in vivo exposure, followed by tests on behaviour, physiology and pathology. In this study, an attempt to estimate lowest observed neurotoxic doses after single or repeated dose exposure was performed. Differentiated human neuroblastoma SH-SY5Y cells were exposed to acrylamide, lindane, parathion, paraoxon, phenytoin, diazepam or caffeine for 72 hours. The effects on protein synthesis and intracellular free Ca2+ concentration were studied as physiological endpoints. Voltage operated Ca2+ channel function, acetylcholine receptor function and neurite degenerative effects were investigated as neurospecific endpoints for excitability, cholinergic signal transduction and axonopathy, respectively. The general cytotoxicity, determined as the total cellular protein levels after the 72 hours exposure period, was used for comparison to the specific endpoints and for estimation of acute lethality. The lowest concentration that induced 20% effect (EC20) obtained for each compound, was used as a surrogate for the lowest neurotoxic level (LOEL) at the target site in vivo. The LOELs were integrated with data on adsorption, distribution, metabolism and excretion of the compounds in physiologically-based biokinetic (PBBK) models of the rat and the lowest observed effective doses (LOEDs) were estimated for the test compounds. A good correlation was observed between the estimated LOEDs and experimental LOEDs found in literature for rat for all test compounds, except for diazepam. However, when using in vitro data from the literature on diazepam's effect on gamma-amino butyric acid (GABA)A receptor function for the estimation of LOED, the correlation between the estimated and experimental LOEDs was improved from a 10,000-fold to a 10-fold difference. Our results indicate that it is possible to estimate LOEDs by integrating in vitro toxicity data as surrogates for lowest observed target tissue levels with PBBK models, provided that some knowledge about toxic mechanisms is known.

  • 21.
    Forsby, Anna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Norman, Kimberly
    EL Andaloussi-Lilja, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Wojcik, Beata
    Walczak, Vincent
    Curren, Rodger
    Martin, Katharine
    Tierney, Neena
    Predicting eye stinging potential of baby shampoos by assessing TRPV1 channel activity2012Inngår i: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 211, s. S113-S113Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 22.
    Forsby, Anna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Norman, Kimberly G.
    EL Andaloussi-Lilja, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Walczak, Vincent
    Curren, Rodger
    Martin, Katharine
    Tierney, Neena K.
    Using Novel In Vitro NociOcular Assay Based on TRPV1 Channel Activation for Prediction of Eye Sting Potential of Baby Shampoos2012Inngår i: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 129, nr 2, s. 325-331Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 23.
    Galofré, Mireia
    et al.
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    Babot, Zoila
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    García, Daniel A
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona,.
    Iraola, Susana
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona,.
    Rodríguez-Farré, Eduard
    Department of Brain Ischemia and Neurodegeneration, IIBB, CSIC-IDIBAPS, Spain and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Suñol, Cristina
    Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, and CIBER Epidemiología y Salud Pública (CIBERESP), Spain.
    GABA(A) receptor and cell membrane potential as functional endpoints in cultured neurons to evaluate chemicals for human acute toxicity2010Inngår i: Neurotoxicology and Teratology, ISSN 0892-0362, E-ISSN 1872-9738, Vol. 32, s. 52-61Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Toxicity risk assessment for chemical-induced human health hazards relies mainly on data obtained from animal experimentation, human studies and epidemiology. In vitro testing for acute toxicity based on cytotoxicity assays predicts 70 - 80% of rodent and human toxicity. The nervous system is particularly vulnerable to chemical exposure which may result in different toxicity features. Acute human toxicity related to adverse neuronal function is usually a result of over-excitation or depression of the nervous system. The major molecular and cellular mechanisms involved in such reactions include GABAergic, glutamatergic and cholinergic neurotransmission, regulation of cell and mitochondrial membrane potential, and those critical for maintaining central nervous system functionality, such as controlling cell energy. In this work, a set of chemicals that are used in pharmacy, industry, biocide treatments or are often abused by drug users are tested for their effects on GABA(A) receptor activity, GABA and glutamate transport, cell membrane potential and cell viability in primary neuronal cultures. GABA(A) receptor function was inhibited by compounds for which seizures have been observed after severe human poisoning. Commonly abused drugs inhibit GABA uptake but not glutamate uptake. Most neurotoxins altered membrane potential. The GABA(A) receptor, GABA uptake and cell membrane potential assays were those that identified the highest number of chemicals as toxic at low concentrations. These results show that in vitro cell assays may identify compounds that produce acute neurotoxicity in humans, provided that in vitro models expressing neuronal targets relevant for acute neural dysfunctions are used.

  • 24.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adamson, Lars
    Hedander, Jan
    Walum, Erik
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin-like growth factor type 1 upregulates uncoupling protein 3.2001Inngår i: Biochem Biophys Res Commun, ISSN 0006-291X, Vol. 287, nr 5, s. 1105-11Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor. Copyright 2001 Academic Press.

  • 25.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Adamson, Lars
    Hedander, Jan
    Walum, Erik
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin-like growth factor type 1 up-regulates uncoupling protein 32001Inngår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 287, nr 5, s. 1105-1111Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.

  • 26.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Axelsson, Viktoria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The use of the human neuroblastoma SH-SY5Y cell line for estimation of acute systemic toxicity in vitro.2007Konferansepaper (Fagfellevurdert)
    Abstract [en]

    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.

  • 27.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Axelsson, Viktoria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity2010Inngår i: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 245, nr 2, s. 191-202Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 28.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Söderdahl, Therése
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jönsson, Gunn
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bratteng, Jan-Ove
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin-like growth factor type 1 prevents hyperglycemia-induced uncoupling protein 3 down-regulation and oxidative stress2004Inngår i: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 77, nr 2, s. 285-291Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uncoupling proteins (UCPs) have been reported to decrease the mitochondrial production of reactive oxygen species (ROS) by lowering the mitochondrial inner membrane potential (MMP). We have previously shown that UCP3 expression is positively regulated by insulin-like growth factor-1 (IGF-1). The aim of this study was to investigate the role of UCPs in IGF-1-mediated protection from hyperglycemia-induced oxidative stress and neurodegeneration. Human neuroblastoma SH-SY5Y cells were differentiated with retinoic acid for 6 days, after which exposure to 8, 30, or 60 mM glucose with or without 10 nM IGF-1 was started. After 48-72 hr, the number of neurites per cell, UCP3 protein expression, MMP, and intracellular levels of ROS and total glutathione were examined. These studies showed that glucose concentration-dependently reduced the number of neurites per cell, with a 50% reduction at 60 mM. In parallel, the UCP3 protein expression was down-regulated, and the MMP was raised 3.5-fold, compared with those in cells incubated with 8 mM glucose. Also, the ROS levels were increased, showing a twofold maximum at 60 mM glucose. This was accompanied by a twofold elevation of total glutathione levels, confirming an altered cellular redox state. IGF-1 treatment prevented the glucose-induced neurite degeneration and UCP3 down-regulation. Furthermore, the MMP and the intracellular levels of ROS and glutathione were normalized to those of control cells. These data indicate that IGF-1 may protect from hyperglycemia-induced oxidative stress and neuronal injuries by regulating MMP, possibly by the involvement of UCP3.

  • 29.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tamm, Christoffer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 32004Inngår i: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 88, nr 2, s. 462-468Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell-survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1) in human neuroblastoma SH-SY5Y cells. Northern blot analysis and RT-PCR showed that treatment with 10 nm IGF-1 increased the UCP3 mRNA levels 2.5-fold after 5 h. Co-incubation with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002 prohibited IGF-1-mediated induction of both UCP3 mRNA and protein in a concentration-dependent manner, with a complete blockage at 1 μm, as shown by RT-PCR and western blot analyses. The mitogen-activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 μm, however, this concentration only partly inhibited the protein expression. We conclude that IGF-1 enhanced UCP3 expression at transcriptional level, primarily through the PI3-kinase-dependent pathway and partly through the MAP kinase pathway.

  • 30.
    Gustafsson, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tamm, Christoffer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 3.2004Inngår i: J Neurochem, ISSN 0022-3042, Vol. 88, nr 2, s. 462-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell-survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1) in human neuroblastoma SH-SY5Y cells. Northern blot analysis and RT-PCR showed that treatment with 10 nm IGF-1 increased the UCP3 mRNA levels 2.5-fold after 5 h. Co-incubation with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002 prohibited IGF-1-mediated induction of both UCP3 mRNA and protein in a concentration-dependent manner, with a complete blockage at 1 microm, as shown by RT-PCR and western blot analyses. The mitogen-activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 microm, however, this concentration only partly inhibited the protein expression. We conclude that IGF-1 enhanced UCP3 expression at transcriptional level, primarily through the PI3-kinase-dependent pathway and partly through the MAP kinase pathway.

  • 31. Hamm, Jon
    et al.
    Sullivan, Kristie
    Clippinger, Amy J.
    Strickland, Judy
    Bell, Shannon
    Bhhatarai, Barun
    Blaauboer, Bas
    Casey, Warren
    Dorman, David
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swedish Toxicology Sciences Research Center (Swetox), Sweden.
    Garcia-Reyero, Natàlia
    Gehen, Sean
    Graepel, Rabea
    Hotchkiss, Jon
    Lowit, Anna
    Matheson, Joanna
    Reaves, Elissa
    Scarano, Louis
    Sprankle, Catherine
    Tunkel, Jay
    Wilson, Dan
    Xia, Menghang
    Zhu, Hao
    Allen, David
    Alternative approaches for identifying acute systemic toxicity: Moving from research to regulatory testing2017Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 41, s. 245-259Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    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.

  • 32. Kinsner-Ovaskainen, Agnieszka
    et al.
    Clemedson, Cecilia
    Cole, Thomas
    Clothier, Richard
    Coecke, Sandra
    O’Connor, José-Enrique
    Blaauboer, Bas
    Gómez-Lechón, Maria-José
    Vericat, Joan-Albert
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ryan, Michael
    Castell, José
    Risteli, Leila
    Wendel, Albrecht
    Optimisation and pre-validation of an in vitro test strategy for predicting human acute toxicity: Progress of the “A-Cute-Tox” project2007Konferansepaper (Annet vitenskapelig)
  • 33. Leist, Marcel
    et al.
    Ghallab, Ahmed
    Graepel, Rabea
    Marchan, Rosemarie
    Hassan, Reham
    Hougaard Bennekou, Susanne
    Limonciel, Alice
    Vinken, Mathieu
    Schildknecht, Stefan
    Waldmann, Tanja
    Danen, Erik
    van Ravenzwaay, Ben
    Kamp, Hennicke
    Gardner, Iain
    Godoy, Patricio
    Bois, Frederic Y.
    Braeuning, Albert
    Reif, Raymond
    Oesch, Franz
    Drasdo, Dirk
    Höhme, Stefan
    Schwarz, Michael
    Hartung, Thomas
    Braunbeck, Thomas
    Beltman, Joost
    Vrieling, Harry
    Sanz, Ferran
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swetox, Karolinska Institutet, Sweden.
    Gadaleta, Domenico
    Fisher, Ciaran
    Kelm, Jens
    Fluri, David
    Ecker, Gerhard
    Zdrazil, Barbara
    Terron, Andrea
    Jennings, Paul
    van der Burg, Bart
    Dooley, Steven
    Meijer, Annemarie H.
    Willighagen, Egon
    Martens, Marvin
    Evelo, Chris
    Mombelli, Enrico
    Taboureau, Olivier
    Mantovani, Alberto
    Hardy, Barry
    Koch, Bjorn
    Escher, Sylvia
    van Thriel, Christoph
    Cadenas, Cristina
    Kroese, D.
    van de Water, Bob
    Hengstler, Jan G.
    Adverse outcome pathways: opportunities, limitations and open questions2017Inngår i: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 91, nr 11, s. 3477-3505Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 34.
    Lilja, Johanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Development of a sensory neuronal cell model for the estimation of mild eye irritation.2004Inngår i: Altern Lab Anim, ISSN 0261-1929, Vol. 32, nr 4, s. 339-43Artikkel i tidsskrift (Annet vitenskapelig)
  • 35.
    Lilja, Johanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Laulund, Frida
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Insulin and Insulin-Like Growth FactorType-I Up-Regulate the Vanilloid Receptor-1(TRPV1) in Stably TRPV1-ExpressingSH-SY5Y Neuroblastoma Cells2007Inngår i: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 85, nr 7, s. 1413-1419Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The capsaicin receptor, transient receptor potential, vanilloid type 1 (TRPV1), is a Ca2+ permeable ion channel activated by noxious stimuli eliciting pain. Several reports have shown modulation of TRPV1 activity and expression by neuronal growth factors. Here, we study the long-term effects on TRPV1 expression mediated by insulin like growth factor type-I (IGF-I) and insulin in a stably TRPV1-expressing SH-SY5Y neuroblastoma cell line. We show that after 72 h of 10 nM IGF-I or insulin exposure, the TRPV1 protein level was up-regulated 2.5 and 2-fold, respectively. By blocking phosphatidylinositol-3-kinase (PI(3)K) or mitogen-activated protein kinase (MAPK) signaling we concluded that the increase in total TRPV1 protein content induced by IGF-I was controlled by PI(3)K signaling whereas insulin seemed to regulate TRPV1 protein expression via both PI(3)K and MAPK pathways. Inhibiting protein kinase C (PKC) blocked the effects of both IGF-I and insulin. Furthermore, the concentrations causing 50 % Ca2+ increase (EC50) after insulin and IGF-I-treatments were significantly lowered compared to untreated cells. We conclude that IGF-I and insulin enhance TRPV1 protein expression and activity, and impaired pain sensation might result from distorted TRPV1 regulation in the peripheral nervous system.

  • 36.
    Lilja, Johanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindegren, Helene
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Surfactant-Induced TRPV1 Activity—A Novel Mechanismfor Eye Irritation?2007Inngår i: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 99, nr 1, s. 174-180Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 37.
    Lindegren, H.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mogren, H.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi-Lilja, J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Anionic linear aliphatic surfactants activate TRPV1: a possible endpoint for estimation of detergent induced eye nociception?2009Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 23, nr 8, s. 1472-1476Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 38.
    Lundqvist, Jessica
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Christina, Svensson
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kristina, Attoff
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swetox, Karolinska Institutet, Sweden.
    Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity2017Inngår i: Applied In Vitro Toxicology, ISSN 2332-1539, Vol. 3, nr 2, s. 154-162Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 39.
    Lundqvist, Jessica
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi-Lilja, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Svensson, Christina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gustafsson Dorfh, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests2013Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 27, nr 5, s. 1565-1569Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 40.
    Nordin-Andersson, M
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Heldring, N
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dejongh, J
    Kjellstrand, P
    Walum, E
    Neurite degeneration in differentiated human neuroblastoma cells.1998Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 12, nr 5, s. 557-60Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have studied neurite degeneration in differentiated human neuroblastoma (SH-SY5Y) cells. The axonopathy-inducing potency in vitro of caffeine, diazepam, methylmercury chloride (MeHg), triethyltin chloride (TET) and acrylamide (ACR) was elucidated. After 72 hours of exposure the neurite degeneration was determined (by morphological quantification) as well as the total protein content (general cytotoxicity). The concentrations that caused 20% reduction of number of neurites (ND(20)) for ACR (250+/-36 mum) and TET (0.097+/-0.03 mum) was significantly lower, 63% and 35%, respectively (P</=0.005), as compared to corresponding inhibition of general cytotoxicity (IC(20)). The effects of TET on the neurites may be related to the disturbance in Ca(2+)-signalling, and thus a secondary event. The ND(20)s for caffeine and diazepam, which are compounds without a known neurite degenerative potency in vivo, were higher as compared with the IC(20). For MeHg which is an extremely cyto- and neurotoxic compound the ND(20) was not statistically different from the IC(20), indicating that degeneration of the neurites is not a primary effect. This study indicates that the SH-SY5Y-neurite degeneration model is useful for the identification of axonopathy-inducing substances.

  • 41.
    Nordin-Andersson, M
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Walum, E
    Kjellstrand, P
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery.2003Inngår i: Cell Biology and Toxicology, ISSN 0742-2091, E-ISSN 1573-6822, Vol. 19, nr 1, s. 43-51Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca2+]i) and receptor-activated (carbachol, 0.1 mmol/L) Ca2+ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca2+]i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca2+]i plays a significant role for acrylamide-induced axonopathy.

  • 42. Prieto, Pilar
    et al.
    Blaauboer, Bas J
    de Boer, Albertus Gerrit
    Boveri, Monica
    Cecchelli, Romeo
    Clemedson, Cecilia
    Coecke, Sandra
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Galla, Hans-Joachim
    Garberg, Per
    Greenwood, John
    Price, Anna
    Tähti, Hanna
    Blood-brain barrier in vitro models and their application in toxicology. The report and recommendations of ECVAM Workshop 49.2004Inngår i: ATLA (Alternatives to Laboratory Animals), ISSN 0261-1929, Vol. 32, nr 1, s. 37-50Artikkel i tidsskrift (Fagfellevurdert)
  • 43. Scheers, Ellen M
    et al.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dierickx, Paul J
    Cytotoxicity of amino alcohols to rat hepatoma-derived Fa32 cells.2002Inngår i: ATLA (Alternatives to Laboratory Animals), ISSN 0261-1929, Vol. 30, nr 3, s. 309-12Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Amino alcohols are used as emulsifying agents in dry-cleaning soaps, wax removers, cosmetics, paints and insecticides. The cytotoxicities of 12 amino alcohols, which differed in chain length, position of the amino and alcohol groups, and the presence of an additional phenyl group, were determined by the neutral red uptake inhibition assay with normally cultured, glutathione-depleted or antioxidant-enriched Fa32 rat hepatoma-derived cells. Glutathione depletion and antioxidant enrichment were achieved by including 50(M L-buthionine-S,R-sulphoximine (BSO) or 100(M (-tocopherol acetate (vitamin E) in the culture medium for 24 hours before and during the assay. The cytotoxicity of the amino alcohols observed after treatment for 24 hours was expressed as the concentration of compound needed to induce a 50% reduction in neutral red uptake (NI50). The observed NI50 values ranged from 3mM to 30mM. The individual stereoisomers and a racemic mixture of 1-amino-2-propanol exhibited similar cytotoxicities (with normally cultured Fa32 cells, and vitamin E- and BSO-treated cultures). Similar NI50 values for D-(+)-2-amino-1-propanol, 3-amino-1-propanol and the L-, D- or DL- forms of 1-amino-2-propanol, indicated that the position of the amino group had little influence on the cytotoxicities of the amino alcohols. In contrast, the position of the hydroxyl group appeared to play an important role for the toxicity of the compound, as indicated by the significantly different NI50 values for 4-amino-1-butanol and 4-amino-2-butanol. An additional phenyl group greatly increased the cytotoxicity of 2-amino-1,3-propanediol. For most of the compounds, cytotoxicity increased when GSH was depleted, and decreased when the cells were enriched with vitamin E. This indicated that most of the tested chemicals interact with GSH, either directly or indirectly, by processes which generate oxygen free-radicals. Decreased toxicity was found for most of the chemicals administered to vitamin E-enriched cells, indicating that reactive oxygen species could be involved in the toxicity of the amino alcohols.

  • 44. Schmidt, Bela Z.
    et al.
    Lehmann, Martin
    Gutbier, Simon
    Nembo, Erastus
    Noel, Sabrina
    Smirnova, Lena
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swedish Toxicology Research Center (Swetox), Sweden.
    Hescheler, Juergen
    Avci, Hasan X.
    Hartung, Thomas
    Leist, Marcel
    Kobolak, Julianna
    Dinnyes, Andras
    In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities2017Inngår i: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 91, nr 1, s. 1-33Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    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.

  • 45.
    Yeste-Velasco, Marc
    et al.
    Facultat de Farmàcia, Universitat de Barcelona,.
    Alvira, Daniel
    Sureda, Francesc X
    Rimbau, Victor
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pallàs, Mercè
    Camins, Antoni
    Folch, Jaume
    DNA low-density array analysis of colchicine neurotoxicity in rat cerebellar granular neurons.2008Inngår i: Neurotoxicology, ISSN 0161-813X, E-ISSN 1872-9711, Vol. 29, nr 2, s. 309-17Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytoskeletal alteration is a key factor in neurodegenerative processes like Alzheimer's or Parkinson's disease. Colchicine is a microtubule-disrupting agent that binds to tubuline, inhibiting microtubule assembly, and which triggers apoptosis. The present research describes the transcriptional activation of molecules related to alternative forms of apoptosis, in an acute colchicine model of apoptosis in rat cerebellar granule neurons (CGNs). Treatment with colchicine up-regulated significantly the activity of genes related to oxidative stress: glutathione peroxidase 1 and catalase; altered significantly genes related to cell cycle control (cyclin D1 and cyclin-dependent kinase 2), genes related to classical apoptosis pathway (caspase 3) and a neuronal cell-related gene (pentraxin 1). Colchicine treatment also down-regulated the gene expression of calpain 1. In conclusion, our experiments demonstrate that the cell damage caused by exposure to colchicine activates the classical apoptosis pathway, but also promotes the up-regulation of several genes related to oxidative stress and cell cycle control. Present data may help to a better understanding of the molecular mechanisms involved in cytoskeletal degradation-induced apoptosis in neurons.

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