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WP7.1: Improving in vitro/in vivo correlations – Neurotoxicology To examine responses for the nervous system in WP7.1, the first 26 ACuteTox reference chemicals (neurotoxic and non-neurotoxic) were studied in native or differentiated human neuroblastoma SH-SY5Y cells, primary cultures of mouse or rat neurons, and mature reaggregated rat brain cells by using around 40 different endpoints (see below). The results showed that the broad collection of assays could in a very good way predict the neurotoxic compounds. However, none of the individual tests could be identified as a “stand alone” assay. Therefore, we carried out the extended evaluation by studying the effect of 33 additional reference chemicals on the following endpoints, which were identified as good candidates from the introductory phase of the project: · · · · · · Caspase-3 gene expression in primary cultures of cerebellar granule cells (P37) Cell markers for neurons (NF-H), astrocytes (GFAP), oligodendrocytes (MBP) and cellular stress (HSP32) by measuring gene expression in reaggregate cultures (P20) Total RNA synthesis and glucose uptake in reaggregate cultures (P20) Acetylcholine esterase (AChE) activity in SH-SY5Y cells (P5) GABAA receptor in primary cultures of cortical neurons (P13) Cell membrane potential (CMP) in SH-SY5Y cells (P16) A brief description of the neurotoxicity endpoints and assays used in WP7.1: Rat cerebellar granule cells (P4) Endpoints evaluated using rat cerebellar granule cells: 1. Cytotoxic effects of test chemicals after 72 hr of exposure using the Alamar Blue assay 2. The results did not differ from the 3T3 cytotoxicity test (see WP2), i.e. the endpoint did not contribute to a better correlation to acute systemic toxicity 3. Changes in mitochondrial membrane potential using the JC-1 assay 4. Study of biomarkers for GFAP and neurofilaments (NF) using ELISA assay. The assay had a poor reproducibility. Also, the results obtained indicated that this endpoint seemed more related to subchronic neurotoxicity than to acute systemic lethality. 1. 2. 3. 4. Human neuroblastoma SH-SY5Y cells, mouse cortical neurons cells (P5) Endpoints evaluated using SH-SY5Y cells and mouse cortical neuron cells: Cytotoxic activity of reference chemicals by measuring the total intracellular lactate dehydrogenase (LDH) activity in SH-SY5Y cells, primary cortical neuron (PCN) cultures from mouse, and mouse brain slices after 24 hr of exposure. Inhibitory effects of the compounds on voltage-operated calcium channels (VOCC) in PCN cultures after 24 hr of exposure by investigating the ability of a test compound to protect the cytotoxic effects induced by of 120 mM KCl. Isopropyl alcohol, parathion, pentachlorophenol and SLS were shown to block VOCC but only at high concentrations Analysis of effects on gene expression in PCN cultures by using primers for bax, bcl2 (isof 1), bcl2 (isof2), bcl-xL, myc, p53 and ngf. Investigations of the effects on acetylcholine esterase (AChE) activity with purified bovine erythrocyte enzyme after 10 minutes of exposure of the reference chemical. ACuteTox 1 Primary mouse cortical neurons (P13) Endpoints evaluated using primary mouse cortical neurons (PCN): 1. Effects on cell membrane potential (CMP) using the fluorophore probe FMP (Molecular Devices®). Changes in CMP resulting in a more positive potential (depolarization) results in an increased fluorescence signal of the probe. Results have been reported as the concentrations that produced 20%, 50% and 80% (EC20, 50 and 80) of the maximum depolarizing effects induced by 30 mM KCl. Effects on Na+ channel function were investigated by studying veratridine-induced depolarization of CMP after exposure with the test chemicals. Only carbamazepine and D-amphetamine displayed an inhibitory effect on Na+ channel function. 2. GABAA receptor function was analysed by GABA activated 36Cl- influx. Both facilitated and attenuated receptor function were studied. This was a promising assay which gave most alerts as compared to other assays performed by P13 and was selected for the second phase of the project. 3. Effects on GABA uptake in PCN and on glutamate uptake in primary cerebellar granule cells. Human neuroblastoma SH-SY5Y cells (P16) Endpoints evaluated using human neuroblastoma SH-SY5Y cells: 1. The effects on CMP in differentiated SH-SY5Y cells by using the FMP fluorescence probe (Molecular Devices®). The CMP response was presented as the percent increase/decrease in FMP fluorescence in relation to the response of the addition of 30 mM KCl. The CMP assay indicated several alerts, which were also under-predicted by the general cytotoxicity assay 3T3-NRU (WP2), and was selected for the second phase of the project. 2. The function of muscarinic acetylcholine receptor (mAChR) in differentiated SH-SY5Y cells by measuring the change in [Ca2+]i with the Ca2+ indicator FURA-2. To stimulate the cells, carbachol was applied and the change in [Ca2+]i was measured fluorometrically. Only the mAChR antagonist atropine was identified as an alert. 3. The function of VOCC in differentiated SH-SY5Y cells by measuring the change in [Ca2+]i with FURA-2. To stimulate the cells, 10 mM KCl was applied and the change in [Ca2+]i was measured fluorometrically Pentachlorophenol, sodium lauryl suphate and verapamil affected VOCC function, but at the same or higher concentration indicated in the 3T3-NRU cytotoxicity assay (see WP2). As expected verapamil blocked the calcium signal. 4. Effects on AChE activity. The cells were exposed with all test chemicals for 1 hr, but the esterase inhibitors were also applied for 24 and 72 hrs in order to investigate the role of various exposure times. Effects were observed for parathion, malathion, dichlorvos and physostigmine after 24 and 72 hr of exposure. 24 hr exposure with malathion or dichlorvos gave the same IC20 and IC50 values as for 1 h. The inhibitory effect was enhanced with time for parathion, but prolonged exposure with physostigmine as well as 72 hr with dichlorvos attenuated the inhibitory activity. 5. Uptake of [3H]-Noradrenalin (NA). Imipramine, a NA-transporter inhibitor, was used as a positive control for inhibition of specific uptake and as a criterion for results acceptance. Nine compounds were identified, of them five alerts, including amphetamine and methadone. ACuteTox 2 Re-aggregated rat brain cells (P20) Aggregating brain cell cultures were prepared at a rate of about one batch per month using mechanically dissociated embryonic rat brain (telenchephalon plus mesencephalon), and grown in serum-free, chemically defined medium. Cultures were exposed to the reference chemicals for 40-44 hr at day 17 to 21. 1. Cell-specific enzyme activities were analysed: Choline acetyl transferase, glutamine synthetase, glutamic acid decarboxylase and CNP. All chemicals except acetaminophene, acetyl salicylic acid, atropine and nicotine displayed activity in one or more assay at 1 mM or lower. 2. Metabolic activity: glucose uptake, protein synthesis and RNA synthesis 3. The mRNA expression of the following genes was studied as endpoints using quantitative RT-PCR: β-actin (reference), GFAP, NF-H, NF-M, MBP, PPARγ, HSP32, and iNOS. HSP32, NF-H and GFAP showed to be sensitive indicators of toxicity. The combination of the 3 endpoints together with MBP expression and metabolic activity measurement was promising as biomarkers of acute systemic toxicity and these endpoints were selected for the second phase of the project, testing 33 additional reference chemicals. Rat cerebellar granule cells (P37) Endpoints evaluated using rat cerebellar granule cells: 1. Cytotoxicity was analysed by the MTT assay after 24 hrs of exposure. Only 6 of 23 chemicals displayed toxicity to the neurons 2. Oxidative stress was measured by ROS production after 30 min of exposure. No chemicals gave any ROS production. 3. Effects on glutamate receptor activity was analysed by measuring the effects of increasing concentrations of the test chemicals on glutamate-induced Ca2+ influx. Digoxin, mercury chloride, pentachlorophenol and SLS inhibited glutamate receptor function. 4. The microassay technology (TaqMan) was used for quantitative mRNA expression analyses of 31 genes in rat cerebellar granule cells after 16 hr of exposure. The expression of each gene was related to glyceraldehyd-3-phosphate dehydrogenase (GAPDH). LOEC was defined as the lowest concentration that induced an up- or a down regulation in each gene. 5. Caspase 3 showed to be a promising biomarker of acute systemic toxicity, displaying nicotine, atropine, digoxin and malathion as alerts. This endpoint was selected for the second phase of the project. The predictive capacity of the selected neurotoxicity tests for human toxicity The data obtained from the selected neurotoxicity were analysed together with the general cytotoxicity data determined in the 3T3-NRU assay (WP2) and compared with data for the human lethal blood concentration (WP1) (Figure 1). ACuteTox 3 Figure 1. Correlation between the general cytotoxicity in the 3T3-NRU assay in combination with neurospecific toxicity and the human lethal blood concentration for the ACuteTox reference chemicals. a) Lowest LOEC of the multi-parameter endpoint (NFH, GFAP, MBP, HSP32 mRNA expression, glucose uptake, mRNA synthesis) in AGGR, b) LOEC of altered Caspase-3 expression in CGC, c) LOEC of altered CMP in differentiated SH-SY5Y cells, d) IC20 of GABAA receptor function in PCN, and e) IC50 of AChE activity in SH-SY5Y cells. The numbering is referring to the chemicals’ identity in the project. The toxicity is indicated as the negative logarithm of the neurotoxic concentrations, IC50 (3T3NRU) and LC50 (mol/L). Filled circles: 3T3-NRU, opened triangles: neurospecific endpoints ACuteTox 4 or 3T3-NRU when pIC50>pNTC or pNTC/pIC50 is missing. The line illustrates the 1:1 correlation between the pIC50/pNTC and pLC50. (From Forsby et al., 2009. Toxicology In Vitro, 23, 1564-1569). Publications Aschner M., Levin E.D., Suñol C., Olopade J.O., Helmcke K.J., Avila D.S., Sledge D., Ali R.H., Upchurch L., Donerly S., Linney E., Forsby A., Ponnoru P., Connor J.R. GeneEnvironment Interactions: Neurodegeneration in Non-Mammals and Mammals. Neurotoxicology, In press, doi:10.1016/j.neuro.2010.03.008. (2010). Folch J., Alvira D., López-Querol M., Tajes M., Sureda F.X ., Forsby A., Rimbau V., Camins A., Pallàs M. Evaluation of transcriptional activity of caspase 3 gene as a marker of acute neurotoxicity in rat cerebellar granular cells. Toxicology in Vitro. 24, 465-71. (2009). Folch J., Yeste-Velasco M., Alvira D., de la Torre A.V., Bordas M., López M., Sureda F.X., Rimbau V., Camins A., Pallàs M. 2009. Evaluation of pathways involved in pentachlorophenol-induced apoptosis in rat neurons. Neurotoxicology 30(3):451-8 (2009). Forsby A., Bal-Price A., Camins A., Coecke S., Fabre N., Gustafsson H., Honegger P., Kinsner-Ovaskainen A., Pallas M., Rimbau V., Rodríguez-Farré E., Suñol C., Vericat J.A. and Zurich M.G. Neuronal in vitro models for the estimation of acute systemic toxicity. Toxicology In Vitro, 23, 1564-9. (2009). Galofré M., Babot Z., García D.A., Iraola S., Rodríguez-Farré E., Forsby A., Suñol C. 2009. GABAA receptor and cell membrane potential as functional endpoints in cultured neurons to evaluate chemicals for human acute toxicity. Neurotoxicology and Teratology 32, 52–61 (2010) Gustafsson H., Runesson J., Lundqvist J., Lindegren H., and Forsby A. Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity. Toxicology and Applied Pharmacology, In press, doi:10.1016/j.taap.2010.02.018. (2010). ACuteTox 5