| 1. |
- Bajinskis, Ainars, et al.
(författare)
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Low-Dose/Dose-Rate gamma Radiation Depresses Neural Differentiation and Alters Protein Expression Profiles in Neuroblastoma SH-SY5Y Cells and C17.2 Neural Stem Cells
- 2011
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Ingår i: Radiation Research. - 0033-7587 .- 1938-5404. ; 175:2, s. 185-192
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Tidskriftsartikel (refereegranskat)abstract
- 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
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| 2. |
- Gustafsson, Helena, et al.
(författare)
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Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity
- 2010
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Ingår i: Toxicology and Applied Pharmacology. - : Elsevier BV. - 0041-008X .- 1096-0333. ; 245:2, s. 191-202
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Gustafsson, Helena, et al.
(författare)
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The use of the human neuroblastoma SH-SY5Y cell line for estimation of acute systemic toxicity in vitro.
- 2007
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Konferensbidrag (refereegranskat)abstract
- Acute systemic toxicity, expressed as human lethal blood peak concentration or the dose inducing 50 % lethality in an animal population (LD50), can be estimated by general cytotoxicity tests using proliferating mammalian cell lines for 70-80 % of all chemicals. The cytotoxicity for the remaining chemicals over or under estimate the LD50 values/human lethal blood peak concentrations because of their very specific molecular targets or toxicokinetic features in vivo. The objective of the EU funded integrated project “ACuteTox” is to develop a strategy in which organ-specific endpoints and toxicokinetic features are taken into consideration in the in vitro prediction of acute systemic toxicity. The human neurotblastoma SH-SY5Y cell line was used as a model for studies on neurospecific targets, which are know to be crucial for survival. All endpoints were investigated after short exposure times (minutes to an hour) at concentrations of the test chemicals that did not affect the cell viability, measured as cell membrane leakage of lactate dehydrogenase. The effects of 23-26 compounds (drugs, pesticides and industrial chemicals) were studied on the cell membrane potential (CMP), voltage dependent Ca2+ channels (VDCC), muscarinic acetylcholine receptor (mAChR) function, acetylcholinesterase (AChE) activity and noradrenalin uptake. The results showed that the CMP was altered by atropine, amphetamine, mercury chloride, methadone, nicotine, pentachlorphenol, sodium lauryl sulphate (SLS) and verapamil, where as an effect on VDCC could be detected for amphetamine, atropine, colchicine, pentachlorphenol, SLS and verapamil. The mAChR function was measured as carbachol-induced Ca2+, i.e. activation of phospholipase C. Amphetamine, pentachlorphenol, SDS and verapamil attenuated the carbachol response by 50% at concentrations about 1 mM, but the specific mAChR antagonist atropine had the same effect at 3 nM. Nicotine, caffeine, pentachlorphenol, methadone, mercury chloride, SLS and the specific inhibitors physostigmine, dichlorvos and malathion attenuated the AChE activity at significantly non-cytotoxic concentrations in SH-SY5Y cells after 60 minutes of exposure. Parathion did not inhibit the AChE activity after 60 minutes exposure, but after 48 hr, indicating that oxidation of parathion to the active inhibitor paraoxon took place in the cell culture. This phenomenon was also observed for malathion, which displayed a lower EC50 value after the prolonged exposure time. The noradrenalin uptake was affected by atropine, caffeine, carbamazepine, amphetamine, diazepam, isopropanol, methadone, SLS and verapamil. A comparison of the active concentrations with the basal cytotoxicity measured as neutral red uptake in mouse fibroblast 3T3 cells indicated that the AChE assay is useful for detection of AChE inhibitors and possibly also AChR ligands. The VDCC endpoint was useful as an alert only for verapamil and the mAChR function was only specifically affected by atropine. The noradrenalin uptake indicated a clear alert for amphetamine and methadone, which was expected, but not for the other test compounds. These results indicate that the usefulness of these endpoints in a general test battery for estimation of acute systemic toxicity is limited, except for AChE activity measurements. However, the results clearly showed that the compounds with known mechanisms (e.g. atropine, verapamil, amphetamine and methodone ) displayed expected effects on their specific endpoints.
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| 4. |
- Gyllberg, Hanna, et al.
(författare)
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Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines.
- 2006
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Ingår i: FEBS Letters. - : Elsevier. - 0014-5793 .- 1873-3468. ; 580:11, s. 2603-8
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Tidskriftsartikel (refereegranskat)abstract
- We have studied how prion infection may affect the Src kinase activity in three different neuronal cell lines, ScGT1 and ScN2a, where ScGT1 were generated in our laboratory. By immunoblotting, using clone 28 - a monoclonal antibody recognizing active Src, we have found a 32+/-6.3% and 75+/-7.7% elevation in Src activity in ScGT1 and ScN2a cells, respectively, compared to uninfected cells. Immunocomplex in vitro kinase assay confirmed the increased Src activity. The increased Src kinase activity in scrapie-infected cells was further shown to correlate to an increased level of Src protein. In addition, an important increase in the protein tyrosine phosphorylation signal was observed in ScGT1 and ScN2a cells, which was further shown to be Src-dependent, as treatment with PP2 - a Src family kinase specific inhibitor, reversed the protein tyrosine phosphorylation profile. Abnormal Src-kinase activation and subsequent protein tyrosine phosphorylation may be key elements in the neuropathology of the prion diseases.
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| 5. |
- Heléne, Lindegren, et al.
(författare)
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A NOVEL MECHANISTIC PATHWAY FOR SURFACTANT-INDUCED NOCICEPTION
- 2007
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Ingår i: 25th SSCT annual workshop.
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Konferensbidrag (populärvet., debatt m.m.)abstract
- 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. Commercial hygiene products were applied to the cells in diluted solutions and stimulation was monitored during exposure as an increase in the intracellular free Ca2+ levels by using the Fura 2 fluorescence assay and semi-HTS fluorometer for 96 well plates. Co-application with the TRPV1 antagonist capsazepine revealed if the response was specific for the receptor. The response was quantified as the product induced Ca2+ influx during 2 minutes in relation to the maximum response induced by the TRPV1 agonist capsaicin. The results show that shampoos and soaps induced a TRPV1 mediated Ca2+ influx whereas 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+ influx 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 mild eye irritating products.
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| 6. |
- Lilja, Johanna, 1980-, et al.
(författare)
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Surfactant-Induced TRPV1 Activity—A Novel Mechanismfor Eye Irritation?
- 2007
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Ingår i: Toxicological Sciences. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 99:1, s. 174-180
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Tidskriftsartikel (refereegranskat)abstract
- The pain receptor transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca2+ influx with secondary effects leading to neurogenic inflammation. Here we report specific activation of TRPV1 by detergent-containing hygiene products measured as intracellular Ca2+ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. Children products marketed as “painless” (containing lower concentration of detergents), and conditioners (without detergents) did not induce specific TRPV1 activation. Furthermore, low concentrations of the detergent sodium lauryl sulfate (SLS) dose-dependently induced Ca2+ influxes that could be addressed to TRPV1. These results reveal a novel mechanistic pathway for surfactant-induced nociception, which may be an important endpoint in in vitro test batteries as alternatives to Draize’s rabbit eye test for classification of eye irritating products.
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| 7. |
- Lindegren, Heléne, 1971-
(författare)
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Alterations in receptor expression and function in scrapie-infected neuronal cell lines
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This study shows that lipopolysaccharide (LPS) induces a robust, concentration-and time-dependent increase in nitric oxide (NO) production in the murine neuroblastoma cell line N2a by increased expression of iNOS. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was not due to inhibited enzymatic activity of iNOS but a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, major changes in the protein tyrosine phosphorylation profile of scrapie-infected hypothalamic neurons (ScGT1-1) and ScN2a, compared to their uninfected counterparts were shown, by Western blot of whole cell extracts and of anti-phosphotyrosine immunoprecipitates, with major bands corresponding to120, 150 and 180 kD. Anti-phosphotyrosine blots of lectin-purified glycoproteins, indicated that some, but not all, of the proteins in the 120 kD band are glycosylated. Two phosphoproteins of ≈ 120 kDa were identified, the receptor tyrosine kinase, fibroblast growth factor 2 (FGFR2) and the cytoplasmic non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (PYK2). In addition, scrapie-infection induces important alterations in the expression, binding and signalling of two neurotrophic receptors, the insulin-like growth factor-1 receptor (IGF-1R) and the insulin receptor (IR) in ScN2a, as compared to uninfected N2a cells. In ScN2a, the IGF-1R and IR protein levels were four- and two-fold increased, respectively, with an unexpected decrease in specific binding sites, as revealed by equilibrium binding studies. In the case of the IGF-1R, the apparent drop in binding sites was due to a seven-fold drop in IGF-1 affinity. Moreover, the IGF-1 stimulated IGF-1R tyrosine phosphorylation was increased in ScN2a when compared to the reduced affinity, possibly due to altered tyrosine kinase signalling in ScN2a. In the case of the IR, the binding affinity was unchanged, although insulin-stimulated IR tyrosine phosphorylation was increased.
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| 8. |
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| 9. |
- Lindegren, Heléne, et al.
(författare)
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Loss of lipopolysaccharide-induced nitric oxide production and inducible nitric oxide synthase expression in scrapie-infected N2a cells
- 2003
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Ingår i: Journal of neuroscience research. - : Wiley Interscience. - 0360-4012. ; 71:2, s. 291-299
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Tidskriftsartikel (refereegranskat)abstract
- In scrapie-infected cells, the conversion of the cellular prion protein to the pathogenic prion has been shown to occur in lipid rafts, which are suggested to function as signal transduction platforms. Neuronal cells may respond to bacterial lipopolysaccharide (LPS) treatment with a sustained and elevated nitric oxide (NO) release. Because prions and the major LPS receptor CD14 are colocalized in lipid rafts, the LPS-induced NO production in scrapie-infected neuroblastoma cells was studied. This study shows that LPS induces a dose- and time-dependent increase in NO release in the murine neuroblastoma cell line N2a, with a 50-fold increase in NO production at 1 g/ml LPS after 96 hr, as measured by nitrite in the medium. This massive NO release was not caused by activation of the neuronal NO synthase (nNOS), but by increased expression of the inducible NOS (iNOS) mRNA and protein. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was due not to abolished enzymatic activity of iNOS but to a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and RT-PCR. These results indicate that scrapie infection inhibits the LPS-mediated signal transduction upstream of the transcriptional step in the signaling cascade and may reflect the important molecular and cellular changes induced by scrapie infection
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| 10. |
- Östlund, Pernilla, et al.
(författare)
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Altered insulin receptor processing and function in scrapie-infected neuroblastoma cell lines
- 2001
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Ingår i: Brain Research. Molecular Brain Research. - : Elsevier. - 0169-328X .- 1872-6941. ; 97:2, s. 161-170
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Tidskriftsartikel (refereegranskat)abstract
- The underlying neurochemical changes contributing to prion-induced neurodegeneration remain largely unknown. This study showsthat scrapie infection induced a 2-fold increase of insulin receptor (IR) protein and aberrantly processed IR b-chain in scrapie-infectedN2a neuroblastoma cells (ScN2a) as measured byWestern blot of immunoprecipitated IR, in the absence of increased IR mRNA. ElevatedIR protein level was further confirmed in an independently scrapie-infected neuroblastoma cell line N1E-115 (ScN1E-115). Proliferationstudies showed that the increased IR level in ScN2a did not result in an increased insulin-mediated cell growth compared to normal N2a125 cells. Binding studies indicated that this apparent paradox was due to a 65% decrease in specific [ I]insulin binding sites in ScN2a whencompared to the amount of immunoreactive IR, although the IR binding affinity was unchanged. Analysis of insulin stimulated IR tyrosinephosphorylation showed a slight but not significant reduction in ScN2a, when related to the increased level of immunoreactive IR.However, comparing the IR tyrosine phosphorylation to the loss of binding sites in ScN2a, we demonstrated an increased IR tyrosinephosphorylation of the remaining functional IR. In addition to these differences in IR properties, the basal extracellular signal regulatedkinase-2 (ERK2) phosphorylation detected by Western blot, was significantly elevated and the insulin stimulated ERK2 phosphorylationwas subsequently decreased in ScN2a. Together, these data show that scrapie infection affects the level and processing of the IR andsignal transduction mediated by the IR in neuroblastoma cells, as well as induces an elevated basal ERK2 phosphorylation. Aberrantregulation of neuroprotective receptors may contribute to neurodegeneration in prion diseases.
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