Anna Forsby

Integration of in vitro neurotoxicity data with biokinetic modelling for the estimation of in vivo neurotoxicity.

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 (EC 20) 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 diazepams 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. Human & Experimental Toxicology (2007) 26, 333—338

Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery

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.

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

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.

Insulin-like growth factor type 1 prevents hyperglycemia-induced uncoupling protein 3 down-regulation and oxidative stress

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.

Putative adverse outcome pathways relevant to neurotoxicity

Abstract The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

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.

Insulin and insulin-like growth factor type-I up-regulate the vanilloid receptor-1 (TRPV1) in stably TRPV1-expressing SH-SY5Y neuroblastoma cells

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 hr 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 a 50% Ca2+ increase (EC50) after insulin and IGF‐I treatments were significantly lowered compared with 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.

Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests

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 7 days, 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.

TRPV1 expression and activity during retinoic acid-induced neuronal differentiation

The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca(2+)-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 re-organisation 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 Ca(2+) concentration by 30% as well as the relative capsaicin-induced Ca(2+) influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca(2+) homeostasis.

GABAA receptor and cell membrane potential as functional endpoints in cultured neurons to evaluate chemicals for human acute toxicity

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.