The use of cell models in determining neuronal responses to EASs Professor Robert A. Smith School of Life Sciences University of Glasgow Scotland (UK)
Nervous System Complexity � � � � CNS and PNS � � � � Cellular heterogeneity N euronal and glial cells Synaptic contacts/neural networks � � � � Functional diversity Regional specialisation Blood Brain Barrier Motor/sensory pathways Cognitive � � � � Age Related susceptibility Developmental stages Maturation after birth Adult � � � � Acute/Chronic/Delayed Responses
Brain Vulnerability… � Potentially more susceptible to damage by � � � hazardous chemicals (including EASs) - high metabolic rate - consumes more oxygen than other tissues. � Reactive Oxygen Species (ROS) generated � � � - mitochondrial dysfunction - over activation of glutamate receptors (especially NMDA receptors) - leads to Ca 2+ influx - cell death pathways triggered
The Developing Brain: Additional challenges… � � � Blood Brain Barrier incomplete � � � � � Cell proliferation/ neurogenesis � � Cell migration � � � Neuronal cell differentiation - axon - dendrites - receptors ■ adrenergic, cholinergic, dopaminergic ■ -TH, estrogen and androgen - synaptogenesis � � � � Glial maturation
Neural targets of Endocrine Disruptors in vivo Sex steroids and hormones crucial in developing brain - Hypothalamus - Pituitary - Hippocampus - Cerebral Cortex - Cerebellum Control of Hypothalamic/neuroendocrine axis Spatial cognitive functions Memory Effects of: Insecticides - DDT (estrogen agonist) Polychorinated biphenyls (TH receptors) Phthalates (androgen antagonists)
Neurotoxicity Testing Alternatives - Initiatives Early � 31 st OHOLO Conference: Model systems in neurotoxicology: � � � alternative approaches to animal testing (1986) – Israel Shahar & Goldberg, 1987 � ECVAM Workshop: In vitro neurotoxicity testing – Italy � � � Atterwill et al., 1994 � WHO/IPCS: In vitro techniques for assessing neurotoxicity (1997) – USA � � � Harry et al., 1998 Recent � 3 rd Intl Conference on Alternatives for DNT (2011) – Italy Bal-Price et al., 2012 � Xi’an International Neurotoxicology conference (2011) – China Llorens et al., 2012 � � � � Developmental neurotoxicity testing (2011) – Japanese Teratology Society Crofton et al., 2012 � 10 th Intl Early Toxicity Screening conference (2012) – USA Neurotoxicity (opening session)
In Vitro endpoints for predicting and assessing neurotoxicity Cell proliferation Cell death Migration assays Neurite outgrowth/ network formation Protein marker expressions - Axonal - Dendrtic - Synaptic - Glial cell Receptor expression
In vitro systems available for determining neural responses � Continuous and immortalised cells lines - rodent/human - undifferentiated/differentiated � Primary neurons - mainly rodent � Brain slices - e.g. hippocampus � Stem and Progenitor cells - rodent/human - embryonic/induced pleuripotent Adherent cultures or 3D floating neurosphere masses
Cell Lines - Rodent PC12 – rat pheochromocytoma (dopaminergic) Undifferentiated - proliferation Differentiated - neurite outgrowth � Bisphenol-A (BPA) – neurites suppressed (Radio & Mundy, 2008) � � � � � BPA inhibited MAPK phosphorylation � � (Seki et al., 2011) B35 – rat neuroblastoma (cholinergic) � exposure to tetrabromo-BPA – ROS production, [ Ca2+]i � � � and caspase-3 activity increased (Hendriks et al., 2012) NB2a – mouse neuroblastoma (cholinergic) � � neurite outgrowth � � (Axelrad et al., 2003) GH3 – rat pituitary � � � � BPA induced Growth Hormone release (Dang et al., 2007; 2009) C17.2 – mouse cerebellum derivation (Lunqvist et al., 2012)
Limitation of Continuous Cell Lines However, majority established from tumours…
Primary cultured neurons Cerebral cortex Silva et al. (2006) Su ň ol et al. (2008) Briz et al. (2011) Hippocampus Matsunaga et al. (2010) Cerebellum - Granule cells Mundy et al. (2006) Su ň ol et al. (2008) Smith (2009) Briz et al. (2011) - Purkinje cells Xiong et al. (2012) Hypothalamus Iwakura et al. (2010)
Primary cultured rodent cerebellar granule cells Homogeneous neuronal cultures Prepared from post-natal pups Functional glutamate receptors by 6-8 div Functional estrogen receptors Extensive neurite production - quantitative analysis of changes Expression of neurotypic proteins - cytoskeleton - growth cones - synapses Basis of many neurotoxicity studies (Mundy et al., 2008; Bal-Price et al., 2010; Briz et al., 2011)
Still in search of the Human Dimension… Advantages of primary neurons over using cell lines established from tumours therefore - � More normal functional phenotype � � � BUT… Culture preparation with potential variability Relatively low-medium throughput screening Majority not of human origin
Human Cell Lines SH-SY5Y – neuroblastoma (dopaminergic) cells Phase 40X Phase 40X Sanfeliu et al. (1999) Cheung et al. (2009) Tuj-1 Undifferentiated Differentiated � Neurite outgrowth and network formation assays Frimat et al. (2010); van Thriel et al. (2012) � Cell membrane potential assay - AcuteTox project chemicals Gustafsson et al. (2010) NTera-2 – teratocarcinoma (cholinergic) Paquet-Durand et al. (2003) � Differentiated cells express neuronal polarity markers � � � Human origin but - lack advantage of 1˚ cultures - derived from tumours
The way forward…. Human Neural Stem Cells Immortalised stem cells (fetal brain) hNSC (De Filippis et al., 2007) ReNcell CX (Breier et al, 2008) Neuronal Precursor Cells PCBs disrupt TH-dependent (Fritsche et al., 2005; neural & glial differentiation Schreiber et al., 2010) LUHMES cells (NPCs from female fetal brain) Neurite quantification in live cells (Stiegler et al., 2011) HUB-NSC (Human umbilical cord derived) (Buzanska et al., 2005) Human Neural Crest Cells (Generated from embryonic line) (Zimmer et al., 2012) Induced Neuronal Cells (Conversion of fetal and postnatal fibroblasts) (Pang et al., 2010)
The Neurosphere Assay for Developmental Neurotoxicity Testing Human (or Rodent) Fetal NPCs Proliferation - * Quantify by several methods Migration – * NPCs leave sphere following growth factor withdrawal Differentiation – * Neuronal & glial markers - Tuj-1 (green) - O4 (red) Breier et al. (2010): Neurotox. Teratol. 32: 4-15
Polybrominated Diphenyl Ethers inhibit differentiation of hNPCs 10 µ M PBDE- 99 10 µ M PBDE- 47 Control Tuj-1 O4 Migration and differentiation of neurons and oligodendrocytes reduced following exposure to PBDEs Schreiber et al. (2010): Environ Health Perspect 118, 572-578
HUCB-NSC – Buzanska Lab ECM bioengineered printed arrays Culture environment effect - + 2% Serum Serum free PLL – undifferentiated FN – differentiated PLL Incorporate electrodes FN Potential in neurotoxicity screens proliferation differentiation Tuji-1: green Ki-67 ( proliferation ): red migration Hoechst: nuclei neurite outgrowth electrophysiology Zychowicz et al. (2011): Acta Neurobiol. Exp. 71, 12-23
hESC-derived NC cells used in MINC assay Tuj-1 Brn3a Peripheral markers Used to assay expressed in neural migration impairment cells from hESC of NC cells (MINC) following exposure to chemicals Tuj-1 Peripherin DNA Tuj-1 NeuN Untreated Treated Zimmer et al. (2010): Environ. Health Perspect. 120, 1116-1122
Other promising approaches � Genomic and metabolomic analyses � � � Gene expression changes in murine NSCs following chemical exposure Need define thresholds between adaptive v. adverse responses (Pennings et al., 2012; Theunissen et al., 2012) � Mathematical & computational initiatives ToxCast Programme (EPA) � Neuronal cells from fetal & postnatal fibroblasts � � � Transgene activation and transcription factor induction (Pang et al., 2011; Kumar et al., 2012)
Unresolved Issues… In vitro methods unable to mimic complexity of brain Yet to achieve brain-region specific human neural cells Cognitive and behavioural aspects Selection of appropriate battery of neurotoxicity tests Validation Routine High Throughput Screening
Current State of the Available Art… Evolution of neural cell models for neurotoxicity Exciting advances in human cell technologies – stem and NPCs of particular merit Reliable endpoints for testing neurotoxicity (incl. DNT) – proliferation – migration – neurite outgrowth – functional activity Data on mechanisms of action Relevance to investigation of responses to EASs
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