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Doctoral thesis from the Department of Physiology, The Wenner-Gren Institute,. Stockholm University, Stockholm, Sweden. Developmental Neurotoxicity Testing.
Developmental Neurotoxicity Testing Using In vitro Approaches Helena Högberg

Doctoral thesis from the Department of Physiology, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

Developmental Neurotoxicity Testing Using In vitro Approaches

Helena Högberg

Stockholm 2009

©Helena Högberg, Stockholm 2009 Picture on cover: Culture of rat primary cortical neurons seeded on MEAs ISBN 978-91-7155-941-8 Printed in Sweden by Universitetsservice AB, Stockholm 2009 Distributor: Stockholm University Library

“If the human brain were so simple that we could understand it, we would be so simple that we couldn't.” Emerson M. Pugh

“I not only use all the brains that I have, but all that I can borrow.” Woodrow Wilson

To my family and friends

ABSTRACT

There is a great concern about children’s health as the developing brain in foetuses and children is much more vulnerable to injury caused by different classes of chemicals than the adult brain. This vulnerability is partly due to the fact that the adult brain is well protected against chemicals by the blood brain barrier (BBB) and children have increased absorption rates and diminished ability to detoxify many exogenous compounds, in comparison to that of adults. Moreover, the development of the central nervous system (CNS) is a very complex process involving several different important events, e.g. proliferation, migration and differentiation of cells. These events are occurring within a strictly controlled time frame and therefore create different windows of vulnerability. Furthermore, the brain consists of numerous different cell types (neuronal, glial and endothelial cells) that have specific functions. The development of each cell type occurs within a specific time window and is therefore susceptible to environmental disturbances at different time periods. Evidence indicates that exposure to industrial chemicals, pesticides or drugs, contributes to the increasing incidence of neurodevelopment disorders. However, due to lack of studies only a few industrial chemicals have been identified as developmental neurotoxicants so far. The current developmental neurotoxicity (DNT) guidelines (OECD TG 426 and US EPA 712-C-98-239) are based entirely on in vivo studies that are time consuming, complex, costly and not suitable for the testing of a high number of chemicals. Applying alternative approaches such as in silico, in vitro and non-mammalian models as a part of an integrated test strategy, could speed up the process of DNT evaluation and reduce and refine animal usage. Both in vitro and non-mammalian test systems offer the possibility of providing an early screening for a large number of chemicals, and could be particularly useful in characterising the compound-induced mechanism of toxicity of various developmental processes. This thesis has characterised two primary neuronal cultures (cerebellar granule cells (CGCs) and cortical neuronal cultures) and identified them as relevant models for DNT testing, since the key processes of brain development are present, such as cell proliferation, migration and neuronal/glial differentiation. Furthermore, two emerging technologies (gene expression and electrical activity) have been evaluated and were identified as promising tools for in vitro DNT assessment. In combination with other assays they could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by prioritising chemicals with DNT potential for further testing.

This thesis is based on the following papers, which are referred to in the text by their respective Roman numerals. I.

Hogberg, H. T., Kinsner-Ovaskainen, A., Hartung, T., Coecke, S., and Bal-Price, A. K. (2009) Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides. Toxicology and Applied Pharmacology 235, 268-286.

II.

Hogberg, H. T., Kinsner-Ovaskainen, A., Coecke, S., Hartung, T., and Bal-Price, A. K. (2009) mRNA expression is a relevant tool to identify developmental neurotoxicants using an in vitro approach. Toxicological Sciences (in press)

III.

Hogberg, H.T., and Bal-Price, A.K. (2009) Comparative toxicity induced by domoic acid in immature and mature mixed primary cultures of cerebellar granule cells: involvement of AMPA and NMDA receptors. Manuscript

IV.

Hogberg, H.T., Sobanski, T., Novellino, A., Whelan, M., and Bal-Price, A. K. (2009) Application of micro electrode arrays (MEAs) as an emerging technology for domoic acid induced developmental neurotoxicity evaluation in primary cultures of rat cortical neurons. Manuscript

It also refers to the following papers. Buzanska, L., Sypecka, J., Molteni, S.N., Compagnoni, A., Hogberg, H.T., Del Torchio, R., Domanska-Janik, K., Zimmer, J., and Coecke, S. (2009) A Human Stem Cell Based Model For Identifying Adverse Effects Of Organic And Inorganic Chemicals On The Developing Nervous System. Stem Cells (in press) Bal-Price, A.K., Hogberg, H.T., Buzanska, L., and Coecke, S. (2009) Relevance of in vitro neurotoxicity testing for regulatory requirements: Challenges to be considered. Neurotoxicology and Teratology (in press)

CONTENTS

1. Introduction ............................................................................................................................................. 13 1.1 Bases for a concern about children’s health ..................................................................................... 13 1.2 Vulnerability of the developing brain .............................................................................................. 13 1.3 Major neurodevelopmental disorders ............................................................................................... 14 1.3.1 Mental retardation..................................................................................................................... 14 1.3.2 Schizophrenia ........................................................................................................................... 15 1.3.3 Fetal alcohol syndrome ............................................................................................................. 15 1.3.4 Attention deficit hyperactivity disorders .................................................................................. 15 1.3.5 Minamata disease...................................................................................................................... 16 1.3.6 Autism ...................................................................................................................................... 16 1.4 European policy to protect children’s health .................................................................................... 16 2. Characterisation of regulatory requirements for DNT testing ................................................................ 18 2.1 Existing DNT guidelines .................................................................................................................. 18 2.2 Testing requirements ........................................................................................................................ 19 2.3 The main endpoints .......................................................................................................................... 19 2.3.1 Neurobehavioral testing ............................................................................................................ 19 2.3.2 Neuropathology evaluation ....................................................................................................... 20 2.3.3 Pharmacokinetics testing .......................................................................................................... 20 2.4 Implementation of the guidelines ..................................................................................................... 20 2.4.1 Animal model ........................................................................................................................... 20 2.4.2 Exposure route and dose ........................................................................................................... 21 2.4.3 Test performance ...................................................................................................................... 21 2.5 Interpretation of data and application to risk assessment ................................................................. 22 3. Environmental exposure to potential DNT toxicants .............................................................................. 23 3.1 Exposure to recognised DNT industrial chemicals .......................................................................... 24 3.1.1 Methyl mercury ........................................................................................................................ 24 3.1.2 Lead .......................................................................................................................................... 24 3.1.3 Arsenic ...................................................................................................................................... 24 3.1.4 Polychlorinated biphenyls ........................................................................................................ 25 3.1.5 Toluene and ethanol (solvents) ................................................................................................. 25 3.2 Exposure to pesticides ...................................................................................................................... 25 4. Alternative in vitro and non-mammalian models relevant to DNT evaluation ....................................... 27 4.1 Cell lines........................................................................................................................................... 27 4.1.1 Phaeochromocytomas (PC12 cells) .......................................................................................... 28 4.1.2 Neuroblastomas ........................................................................................................................ 28 4.2 Primary cultures ............................................................................................................................... 29 4.2.1 Cerebellar granule cells ............................................................................................................ 29 4.2.2 Cultures of cortical neurons ...................................................................................................... 30 4.3 Neural stem cells .............................................................................................................................. 31 4.3.1 Embryonic stem cells ................................................................................................................ 32 4.3.2 Bone marrow derived somatic stem cells ................................................................................. 33 4.3.3 Human umbilical cord blood derived neuronal stem cells........................................................ 33 4.4 Non-mammalian animals ................................................................................................................. 34 4.4.1 Sea urchin ................................................................................................................................. 34 4.4.2 Caenorhabditis elegans ............................................................................................................ 34 4.4.3. Danio rerio .............................................................................................................................. 35

5. Key cellular processes of brain development and methods for their in vitro evaluation ........................ 36 5.1 Development of the brain in utero ................................................................................................... 36 5.2 Proliferation ...................................................................................................................................... 37 5.3 Cell migration ................................................................................................................................... 37 5.4 Neuronal differentiation ................................................................................................................... 38 5.4.1 Elaboration of neuronal networks ............................................................................................. 38 5.4.2 Synaptogenesis ......................................................................................................................... 39 5.5 Glial function.................................................................................................................................... 39 5.6 Myelination ...................................................................................................................................... 40 5.7 Apoptosis .......................................................................................................................................... 41 6. Application of Emerging Technologies with the potential to advance DNT testing .............................. 42 7. Incorporation of alternative approaches into a DNT testing strategy for regulatory purposes ............... 44 8. Summary and Conclusions ..................................................................................................................... 46 9. Acknowledgements ................................................................................................................................. 48 10. References ............................................................................................................................................. 50

ABBREVIATIONS

ADHD ADP ATP BBB BM BrdU cAMP CDC C. elegans CGCs CNS 3D DNA DNT EC50 EChA EDF EFSA ELISA EMEA EPA ESCs EU-TGD FAS FDA FQPA GABA GD GFAP GFP HUCB-NSC IPCS IQ LIF MAG MAM MEA mRNA MTT NAS NCAM NGF NMDA NMR NOAEL NRC NSCs

Attention Deficit Hyperactivity Disorders Adenosine di-phosphate Adenosine tri-phosphate Blood Brain Barrier Bone Marrow Bromodeoxyuridine Cyclic Adenosine mono-phosphate Centers for Disease Control Caenorhabditis elegans Cerebellar Granule Cells Central Nervous System Three Dimensional Deoxyribonucleic acid Developmental Neurotoxicity Half maximal effective concentration European Chemicals Agency Environmental Defense Fund European Food Safety Authority Enzyme-linked immunosorbent assay European Medicines Agency Environmental Protection Agency Embryonic derived Stem Cells EU Technical Guidance Document Fetal Alcohol Syndrome Food and Drug Administration Food Quality Protection Act Gamma-aminobutyric acid Gestational Day Glial Fibrillary Acidic Protein Green Fluorescent Protein Human Umbilical Cord Blood derived Neuronal Stem Cells International Programme on Chemical Safety Intelligence Quotient Leukemia Inhibitory Factor Myelin Associated Glycoprotein Methylazooxylmethanol Micro Electrode Array Messenger Ribonucleic acid 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide National Academia of Science Neural Cell Adhesion Molecule Nerve Growth Factors ionotropic N-methyl D-aspartate glutamate Nuclear Magnetic Resonance No Observable Adverse Effect Level National Research Council Neural Stem Cells

OECD PCBs PCR PKC PNS PSD-95 REACH RNA RNAi S100β SCF SSCs TG TUNEL

Organisation for Economic Co-operation and Development Polychlorinated Biphenyls Polymerase Chain Reaction Protein Kinase C Peripheral Nervous System Post Synaptic Density protein-95 Registration, Evaluation, Authorisation of Chemicals Ribonucleic acid Ribonucleic acid interference S100 protein, Beta polypeptide Scientific Committee for Food Somatic Stem Cells Test Guideline Terminal deoxynucleotidyl Transferase dUTP1

1. INTRODUCTION

The European Centre for the Validation of Alternative Methods (ECVAM), where the work for this thesis was performed, develops, optimises and validates in vitro test methods that can replace, reduce or refine (3 R’s principles) animal experiments for regulatory purposes (Russell and Burch, 1959). These tests that mimic specific cell, tissue or organ toxicity are used to evaluate topical or systemic exposure following up mainly the Organisation for Economic Co-operation and Development (OECD) test guidelines for human health and ecotoxicological endpoints. In line with ECVAMs recommendations (Worth and Balls, 2002), this thesis has subsequently investigated the use of primary neuronal cultures and emerging technologies for in vitro developmental neurotoxicity (DNT) testing. This Ph.D. thesis gives an overview of the current status of DNT testing and also discusses my contributions to the field.

1.1 Bases for a concern about children’s health There is a great concern about children’s health as the developing central nervous system (CNS), considered an important target of toxicity (in foetuses and children), is much more vulnerable to injury from different classes of chemicals than the adult CNS. Indeed, evidence indicates that exposure to industrial chemicals, pesticides or drugs, is contributing to the increasing incidences of neurodevelopmental disorders (Boyle et al., 1994; Grandjean and Landrigan, 2006; Lein et al., 2007; Schettler, 2001). Today one out of every six children has developmental disabilities and many of them are neurodevelopmental disorders such as learning disabilities, dyslexia, attention deficits, hyperactivity disorders and autism (Boyle et al., 1994; Grandjean and Landrigan, 2006; Lein et al., 2007; Schettler, 2001). According to the National Academy of Sciences (NAS) 12 % of children under the age of 18 years are suffering from one or more mental disorders (NAS, 1990). Furthermore, a report from NAS suggests that 28 % of all major developmental disorders in children are linked entirely or partly to environmental exposures (NRC, 2000a). A vast amount of papers in the peer-reviewed scientific literature suggests that the exposure to toxic substances during development should be considered as a non-negligible risk factor for triggering neurodevelopmental disorders in children. However, not all regulatory or industrial communities share this opinion. This is mainly due to the fact that this could trigger increased testing requirements. Furthermore, not all experts agree that conventional animal tests represent the best test system to model DNT effects on the developing human brain. This puts pressure on all stakeholders involved to look for alternative methods (in vitro, in silico and non-mammalian models) that could accelerate the process of DNT testing for regulatory requirements.

1.2 Vulnerability of the developing brain It is well known that the developing brain in foetuses and children is more susceptible to chemical exposure than the adult brain. Indeed, low doses of chemicals that are not harmful to mature CNS can cause developmental neurotoxicity (Claudio et al., 2000; Eriksson, 1997; Tilson, 2000). This is partly due to the fact that the adult brain is well protected against chemicals by the blood brain barrier (BBB). However, as long as the critical processes of angiogenesis in the growing brain are progressing (until about six month after birth) the BBB is still immature and does not fully protect the developing brain (Adinolfi, 1985; Tilson, 2000). Even though the developing brain is not completely unprotected - the placental barrier protects the foetus, while the infant is protected by the blood-milk barrier - many chemicals such as metals, low-molecular weight and lipophilic compounds are fully capable of crossing these barriers (David et al., 1972; DeKoning and Karmaus, 2000; Sakamoto et al., 2004). Lipophilic chemicals can also accumulate in maternal adipose tissue and be transferred to the infant via breast milk, resulting in an infant expo13

sure exceeding the mother’s exposure value by 100-fold on the basis of body weight (Jensen, 1983; Landrigan et al., 2002). Furthermore, there are several substances that have been reported to have higher specificity for the foetal blood than for the blood of the mother due to different protein compartments giving much higher exposure concentrations to the foetus (Amin-Zaki et al., 1976; Dickinson et al., 1979; Nau et al., 1984; Sakamoto et al., 2004). The development of the CNS is a very complex process involving several different important events, e.g. proliferation, migration and differentiation of cells. These events are occurring within strictly controlled time frames and therefore each event creates different windows of vulnerability to xenobiotic exposure (Rice and Barone, 2000; Rodier, 1994; Rodier, 1995). Furthermore, the brain consists of many different cell types (e.g. neuronal, glial and endothelial cells) that have specific functions and different roles. Also each cell type is produced at a specific moment during the development and is therefore susceptible to environmental disturbances at different time periods. Some events take place during a very short time period and interference by chemicals during these stages could have serious consequences for the individual. The vulnerability of the developing brain depends on whether a toxicant reaches the target and the period of exposure. Before or after an organ is developed it is in general less sensitive to environmental perturbation than during development. The susceptibility of infants and children to chemicals is further enhanced by their increased absorption rates and diminished ability to detoxify many exogenous compounds, in comparison to that of adults (Ginsberg et al., 2004; NRC, 2000a).

1.3 Major neurodevelopmental disorders Substance perturbation during development of the CNS could lead to brain injury and serious health effects. The definition of developmental neurotoxicity (DNT) is the adverse effects of xenobiotics on the nervous system associated with exposure during development. These adverse effects may be expressed at any time during the life span of the exposed individual (Coecke et al., 2007). Neurodevelopmental disorders are defined as pathological alterations of the developing nervous system induced by genetic defects, infections or specific environmental factors. The diagnostic criteria are defined in three different domains, morphologic, neurologic and psychiatric. The morphological group is defined as gross and microscopic alterations in the post mortem brain such as malformations, acquired lesions and metabolic disorders (Friede, 1989). The neurological group is diagnosed based on neuronal exams that determine the anatomic site of a lesion, gaining information on its pathological condition (Willis and Grossman, 1973), while the physiological domain is defined by the presence of several related behavioral abnormalities (American Psychiatric Association, 1994). However, there are numerous difficulties to draw clear parallels between the different diagnostic criteria and to associate them with a particular syndrome, e.g. due to high frequency of co-existence of different disorders within an individual.

1.3.1 Mental retardation Approximately 2.5 % of infants are suffering from mental retardation (Slikker JRr. and Chang, 1998). The criterion for this disorder is defined as an IQ of 70-75 or less and impaired adaptive behavior. The most common cause of mental retardation (about 30 % of the patients) is chromosomal aberrations such as Down’s syndrome (20 %) and fragile X syndrome (10 %). Other cognitive abnormalities are nervous system malformations and hormone and metabolic disorders. Of these about 15-20 % depends on infections and teratogens. However, interactions between genetic and environmental factors significantly contribute to these disorders (Gibbons et al., 1995; Liu and Elsner, 1995; Matilainen et al., 1995; Simonoff et al., 1996). Exposure to chemicals that have been associated with mental retardation are ethanol, lead (Beattie et al., 1975; Moore et al., 1977), methyl mercury (Mendola et al., 2002; Trasande et al., 2006), barbiturates (Slikker JRr. and Chang, 1998) and cadmium (Marlowe et al., 1983). Based on imaging studies morphological alterations associated with mental retardation are commonly reported in the hippocampus and cerebellum, e.g. alteration in dendritic spines (Marin-Padilla, 1972; Purpura, 1974). Other reported changes are increased reactive oxygen species, alterations in RNA processing leading to increased transcript mutations and activation of several intracellular signaling cascades. The ranges of these morphological changes are mostly occurring during early morphogenesis. One classic 14

example leading to mental retardation is hypothyroidism (decreased thyroid hormone) which causes cells to fail to make the appropriate connections (Eayrs, 1955).

1.3.2 Schizophrenia Schizophrenia is a neurobehavioral disorder characterised by a delay in neurodevelopment and generalised cognitive deficit. Approximately 0.4-0.6 % of the population is affected and the diagnosis is generally based on behavioral symptoms that can include all the five senses (sight, hearing, taste, smell and touch). However, most commonly apparent are auditory hallucinations, paranoid or bizarre delusions, disorganised speech and thinking with significant social or occupational dysfunction (Goldner et al., 2002). Structural alterations have been reported in the brain of schizophrenia patients and are including connectivity impairments in the area of the social brain and cerebellum, ventricular enlargement, decrease in cortical and sub-cortical volume, increased cerebral symmetry and alterations in cerebral architectonics. In addition, processes of myelinsation, absence of gliosis (Dwork, 1997; Heckers, 1997) and disruption in the normal cell death have been proposed to play a major role in the pathogenesis of a schizophrenic breakdown (Mala, 2008). The abnormalities in the development and maturation of the brain seem to begin prenatal but may continue throughout childhood (Woods, 1998). The cause is still un-known but both genetic and environmental factors seem to play a significant role. For example infections (Barr et al., 1990; Brown et al., 2009), malnutrition (Hoek et al., 1996; Susser et al., 1996) and exposure to lead (Opler and Susser, 2005) during pregnancy may increase the risk for later developing schizophrenia.

1.3.3 Fetal alcohol syndrome Fetal Alcohol Syndrome (FAS) was identified in the middle of the 1970’s and is a syndrome occurring in 1 of 800 births in the general population. However, up to 1 of 90 births with FAS symptoms is reported from groups with lower socioeconomic status (Abel, 1995). These observed differences between groups could be due to different alcohol patterns, genetic susceptibility to alcohol effects, life style factors or an inherent process of identification of children with FAS. The main cause of FAS is exposure to ethanol during the fetal development that consequently leads to birth defects, including facial dysmorphia, growth retardation and neuro-cognitive defects. These children often suffer from difficulties with learning, memory attention and behavioral and intellectual dysfunctions such as hyperactivity, delayed motor development and mental retardation (May and Gossage, 2001). The cellular events behind FAS are proposed to be mediated through multiple mechanisms e.g. alteration in proliferation, neuronal migration, dendritic growth, conductivity among neurons and even neuronal cell death (Miller, 1993). Moreover, ethanol is proposed to interfere with the brain cell metabolism and alters gene expression. Especially the hippocampus is known to be highly vulnerable to ethanol exposure and above all during the brain growth spurt period (Pauli et al., 1995; Tomlinson et al., 1998).

1.3.4 Attention deficit hyperactivity disorders The number of children diagnosed for Attention Deficit Hyperactivity Disorders (ADHD) is continuously increasing and chemical exposure might be one of the causes. For example children born to smoking mothers (Kukla et al., 2008; Laucht and Schmidt, 2004), children exposed to manganese (Bouchard et al., 2007), lead (Braun et al., 2006) or mercury (Cheuk and Wong, 2006) are more likely to develop ADHD than non-exposed groups. It is reported that as many as 5-10 % of all children are affected (Lederer et al., 1991) and it is about five times more common in boys than in girls. The reason behind this disproportional distribution is not known, though studies suggest it may be due to subjective prejudice (Sciutto and Nolfi, 2004). Some of the primary symptoms in ADHD are hyperactivity, decreased attention span, learning disabilities, perceptual problems, impulsivity and difficulties to focus, to sustain, encode and shift attention (Entwistle, 2000). To diagnose ADHD several models are used, e.g. the three compartment model (Ruff and Rothbart, 1996), which includes, selectivity, state of engagement and higher level control. The diagnose is based on observed behavioral responses such as facial expression, duration of looking, head turning, reaction time and speech performance. The symptoms are normally disappearing 15

at the time of puberty but some effects, like decreased attention span and emotional problems, might persist also into adulthood (Lederer et al., 1991). ADHD has been suggested to occur due to a disturbance in cathecholaminergic neurotransmission, with particular emphasis on dopamine (Takeuchi, 2008; Volkow et al., 2009). However, the mechanistic pathways behind it need to be further explored.

1.3.5 Minamata disease Minamata disease was identified in Minamata Bay, Japan, in the middle of the 1950’s. The disease was later related to foetal exposure to methyl mercury occurring via trans-placenta transfer of mercury to the foetus (Harada, 1995). In the 1970’s there was another massive outbreak of Minamata disease in both adults and infants, this time in Iraq. Characteristic neurological symptoms associated with the disease are mental retardation and cerebral palsy, dysorthria ataxia (cerebellar symptoms), deformity of limbs, primitive reflexes, hyper salivation and stunted body growth. Although there have been many cases with this syndrome only a few autopsy investigations have been performed. However, the characteristic neuropathological changes found are decrease or malformation of cortical nerve cells, reduction of cerebellar granule cell layer and abnormality in neural cyto-architecture. These changes are believed to be due to the interference of methyl mercury with the neuronal migration during brain development, which could lead to abnormal synaptogenesis and neuronal dysfunction. The cellular mechanisms are likely due to the disruption of cell membranes and their associated surface molecules (such as neural cell adhesion molecule, NCAM) and the disruption of intracellular neuroskeleton proteins (e.g. tubulin and microtubule-associated proteins) (Choi et al., 1978; Graff et al., 1993). In addition, methyl mercury exposure has been reported to interact with glial cells, which as well could affect neuronal migration (Choi, 1986).

1.3.6 Autism Autism is a lifelong neurodevelopment disorder that occurs in 1 of 150 children (CDC 2007). Boys are affected four times more often than girls. It is characterised by early onset of impairments in social interaction and communication, and unusual stereotyped behaviors (Geier et al., 2008). Children with autism have difficulties to learn and therefore generally show little interest in the world or people around them. The diagnosis of autism is based exclusively on developmental patterns and behavioral observations (CDC, 2007). Animal studies of autism are difficult since the observed behavioral patterns are exclusive to humans, such as language (Tager-Flusberg, 1992), associative pointing (Osterling and Dawson, 1994) and imitation (Smith and Bryson, 1994). This is one reason why the cause behind autism is still not understood, even though it is believed to be initiated by a disturbance of the brain development around the time of neural tube closure. Moreover, the cerebellum seems to be the structure most widely recognised as being abnormal in cases with autism. Some studies have reported different histology in the autistic brain such as a reduction in granule cells and changes in the configuration of the superior olive (Bauman and Kemper, 1985). However, one cellular finding that has been confirmed in several labs is the reduced numbers of Purkinje cells in the cerebellum (Bauman and Kemper, 1985; Ritvo et al., 1986). There is clear evidence that autism can be induced by some teratogenes, e.g. intrauterine rubella infection (Chess, 1971) and postnatal herpes simplex infection (DeLong et al., 1981). Moreover, exposure to mercury (Burbacher et al., 1990), lead (Laughlin et al., 1991), thalidomide (Stromland et al., 1994) or valproate (Christianson et al., 1994) during the fetal period and/or the early childhood has been proposed to contribute to the development of autism (Geier et al., 2008). One particular case where there is a lot of controversy is the causal relationship of thimerosal (contains mercury) and autism (Ip et al., 2004). However, the majority of autism cases are thought to be of genetic origin.

1.4 European policy to protect children’s health Systematic guideline-based testing of new chemicals for toxicity before marketing has only been required in recent years and many widely used chemicals were never sufficiently assessed for their human 16

and environmental safety (Yan et al., 2002). This has lead to upcoming changes in the chemical regulation in the western societies attempting to close the gap in knowledge of the toxic effects of chemicals. In Europe, a new regulation known as REACH (Registration, Evaluation, Authorisation of Chemicals), has been introduced and it entered into force in June 2007. The aim of REACH is to improve the protection of human health and the environment through the better and earlier identification of toxic properties of chemicals. The REACH legislation includes a systematic evaluation of chemicals that are produced in significant quantities within the European Union. It requires that producers and importers register all chemicals, produced in volumes greater than 1 ton per year, at the new European Chemicals Agency (EChA) based in Helsinki, Finland. This includes information on their properties, users risks and safe ways of handling. The chemicals of very high concern (e.g. bio-accumulative, carcinogenic, mutagenic and reproductive toxic compounds) require specific authorisations before usage. Chemicals causing unmanageable risks will be phased out in the European Union by partial or total bans (European Commission 2006). In the REACH testing scheme there is no direct requirement for DNT testing, although it is mentioned in the law text. Currently there is a desire amongst certain organisations to increase the DNT testing requirements. They also want to state that when any noticeable neurobehavioral changes are observed during systemic toxicity evaluation, then tests for neurotoxicity and/or DNT should be performed according to the existing OECD test guidelines (OECD, 1997; OECD, 2007). Further testing should identify the chemicals with possible DNT effects and finally lead to the restricted use and control of the risk of exposure (Grandjean and Landrigan, 2006).

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2. CHARACTERISATION OF REGULATORY REQUIREMENTS FOR DNT TESTING

Currently more than 100.000 industrial chemicals are manufactured, with another 2.000-3.000 new chemicals registered every year. According to the Environmental Defense Fund (EDF) about 70 % of the high production volume (HPV) chemicals (more than 450 ton per year, about 3.000 chemicals) are lacking minimal safety data for health hazards, and among them neurotoxic effects are not evaluated (US EPA, 1998b). Similarly, chemicals that have been recognised as developmental neurotoxicants are notably few because of lack of testing. Moreover, toxicokinetics in the developing animal differ from the adult for instance in absorption, distribution, metabolism and excretion, so several assumptions have to be made. When estimating the health risks for children exposed to chemicals, specific safety factors have to be applied to the adult toxicity data. Currently DNT testing is only recommended on a case-by-case basis and at regulatory level discussions are ongoing to establish a number of criteria in the different regulatory frameworks (for chemicals, pesticides, food additives etc.) to be used as triggers for deciding when testing is needed (Coecke et al., 2007). The existing guidelines for DNT regulatory requirements are based on in vivo animal studies which are complex, costly, time consuming and often provide limited mechanistic information. In addition, the interpretation of the data from these studies can be difficult to evaluate and often does not provide sufficient amount of information to facilitate regulatory decision-making.

2.1 Existing DNT guidelines The guidelines are developed to serve as a general framework to assess DNT and address a number of study design issues (Francis, 1992). They should be suitable for testing of any chemical and provide consistency, but also flexibility in the specific methodology used. This means that chemicals used in different regulatory frameworks (pesticides, insecticides, food additives, cosmetics, industrial chemicals, nanoparticles) do not always have to undergo the same testing. In 1991 the US Environmental Protection Agency (EPA) issued the first guideline for DNT (US EPA OPPTS Developmental Neurotoxicity Testing Guideline 870.6300 § 83-6) that was revised and published in 1998 (US EPA, 1998a). The guideline was founded upon an extensive scientific database including between-laboratory validation studies (Makris et al., 2009). However, since children are considered a susceptible population they require much more extensive evaluations of potential risks, and US EPA recommended the inclusion of a DNT study for all chemical food-used pesticides (FQPA 1998). The recommendation was expanded to include all organophosphates insecticides (US EPA 1999) and 2002 US EPA required registrants to perform DNT studies for a wide range of pesticides that showed evidence of neurotoxicity. In 1995 the Organisation for Economic Co-operation and Development (OECD) initiated the development of the OECD test guideline 426, using the US EPA guideline as a template. It was recently finalised and adopted by the OECD council (OECD, 2007). However, only a limited number of chemicals have been tested according to these guidelines so far. In the European Union (EU), the Scientific Committee for Food (SCF) has recommended that appropriate experts should set the criteria for when DNT testing is necessary. Preclinical DNT studies for human pharmaceuticals are based on the guideline ICH S7A (ICH, 2000) from the European Medicines Agency (EMEA) and the US Food and Drug Administration (FDA).

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2.2 Testing requirements At the present time there are no general requirements for DNT testing of chemicals or pesticides prior to their registration and use (Claudio et al., 2000). Instead the evaluation is based on a weight-of-evidence approach to determine when testing should be recommended. This is done by gathering available data from all toxicity studies and also information of potential human exposure. The decision, whether a chemical should be recognised as a possible DNT trigger that would require DNT studies, can for example be based on observations of neurological effects or induced structural abnormalities of the CNS. Chemical triggers can be adult neurotoxicants, hormonally active peptides and amino acids, or chemicals that are structurally similar to other chemicals with these effects. However, due to commonly used experimental designs, the relevant data that is needed to trigger a DNT evaluation is currently not always available. In the EU Technical Guidance Document (EU-TGD) for Risk Assessment reproductive toxicity testing for new substances and biocides are required by performing a two-generation study (OECD, 2001b) and a prenatal developmental toxicity study (teratogenicity) in two species (rat and rabbit) (OECD, 2001a). However, the testing can be reduced or enhanced depending on different factors, such as structural similarity with known reproductive toxicants, concerns for endocrine disruption, results from other toxicity studies and the anticipated use and human exposure patterns. Chemicals with widespread human exposure would primarily be tested for reproductive toxicity and adult neurotoxicity before making a decision for further DNT studies. However, regulators only require reproductive and developmental studies for food-used pesticides. In addition, adult neurotoxicological studies are only demanded if certain triggers have been found, such as organophosphates or pesticides with structural similarities to a substance that causes delayed neurotoxicity. Many chemicals will probably meet the criteria for DNT testing, but since we already know the limitations in the data set for several chemicals, these requirements might not be sufficient to protect our children from exposure to potential DNT toxicants. The numbers of chemicals tested so far for DNT following either the EPA or the OECD guidelines are about 110 (where 73 are pesticides) (Makris et al., 2009). However, many more chemicals have been recommended to be tested by EPA review committees and the chemicals already tested did not include all suggested endpoints. However, as more data is collected, the criteria for DNT testing requirements may be changed.

2.3 The main endpoints Risk assessment of a potential developmental neurotoxicant requires careful consideration of the selection of endpoints, as well as dosing route, time of exposure and choice of animal model. The existing guidelines are all based on in vivo animal experiments where observations are made to detect gross neurological and behavioral abnormalities, including the assessment of physical development. Subsequently, a core battery of tests was established to detect postnatal developmental disorders in rats. The recommended endpoints can be divided into three groups; neurobehavioral testing, neuropathology and pharmacokinetics.

2.3.1 Neurobehavioral testing The neurobehavioral testing includes motor function, sensory function, and associative learning and memory. The selected endpoints should be chosen according to study goals and objectives. The general motor activity test should be able to characterise the typical exploratory and asymptotic portions of a rodent’s general motor activity and to assess the rate of habituation (Tyl et al., 2008). The methods studied should be able to measure both decreases and increases in activity with reference chemicals, as well as habituation within the test session (US EPA 1998a). Additionally, the test should consider any kind of movement, e.g. horizontal movements and tremor, and should include measurements of overall activity, habituation and ontogeny. Motor activity is vulnerable to perturbation by a variety of experimental factors including developmental delays. The current EPA guideline also request a test of auditory startle response and habituation, an apical test of sensory motor function which measures sensory-evoked motor reflex that involves four to five neurons from the cochlea via the brainstem to ventral horn motor neurons (Davis et al., 1982; Koch, 1999). It 19

should be kept in mind that other underlying behavioral systems may also affect appearances and measurements of auditory startle, in particular motor functions. Cognitive function is one of the basic processes that can potentially be disrupted by chemical exposure during development. Memory and learning testing need to assess either changes across several repeated learning trials, or one single trial with reference to a condition that controls for non-associative effects of the training experience. In addition to the original learning, these tests should include some measurement of short- or long-term memory. The choices of methods are flexible even though relatively few types of tests are normally used (Tyl et al., 2008). The most commonly used tests for learning and memory determination are the passive avoidance and water maze tests. The passive avoidance test involves training the animal to avoid punishment (normally an electric shock) by limiting the normal behavior (such as exploratory behavior) (Johns Hopkins University, 2009), while the water maze test measures parameters and time before the animal finds a hidden exit from a small pool of water (Morris, 1984). The tests are repeated to evaluate the memory and learning abilities of the animal. However, especially the water maze test is not always sensitive enough to detect any effects due to a high variability between individuals (Tanimura, 1992).

2.3.2 Neuropathology evaluation The neuropathology evaluation is addressing DNT effects based on morphological and histopathology endpoints stretching from simple brain weight observations to more complex histopathological measurements (Kaufmann and Groters, 2006). The test guidelines describe which endpoint to choose and how the neuropathological evaluation should be performed, e.g. methods for fixation, processes of tissue sampling, regions of the nervous system to be examined and procedures for semi-quantitative analysis (OECD, 2007; US EPA 1998a). One proposed endpoint is staining for detection of cellular alterations, such as vacuolation, degeneration, necrosis and astrocytic proliferation (which is a stereotypic indication of brain injury). Another important endpoint is the measurement of cholinesterase activity. Cholinesterase is suggested to play an important role in the neural development mainly through pathways critical for neuronal outgrowth. Chemicals that interfere with acetylcholinesterase, e.g. organophosphate pesticides, might give permanent damage to the developing brain.

2.3.3 Pharmacokinetics testing The pharmacokinetic testing contains mainly analytical chemical techniques and modeling approaches to reduce uncertainty in the extrapolation of results from animals to humans. However, at present these tests have only been proposed to be included in the DNT testing. To identify potential DNT chemicals the design of the study, as well as, the selection of endpoints are of great importance. However, most of the endpoints in behavioral studies are functionally based and these often have a low sensitivity detecting subtle damages in the brain. Still, the behavioral endpoints are recognised to be much more sensitive than for example reproductive or adult neurotoxic endpoints (Elsner, 1986; Elsner et al., 1986). A study that tested some selected chemicals with DNT potential, using the DNT guidelines, could identify all these chemicals, although the toxic doses could vary across species (Francis et al., 1990).

2.4 Implementation of the guidelines 2.4.1 Animal model The development of the mammalian nervous system in different species is following more or less the same sequence of events and is comparable to humans. The choice of animal species and strain is flexible. However, the rat is normally the first choice of experimental animal when performing DNT studies as the background information on normal behavior is well characterised. Generally a new-born rat is comparable in brain development to a human foetus at the end of the second trimester of gestation (3-6 month), while a 16-19 day old rat is comparable to a human infant at term (Rice and Barone, 2000). 20

The rat is, in general, considered a suitable model to predict human risks. However, there are some differences that should be taken in consideration. For example the “brain growth spurt” (period of enhanced brain growth) takes place postnatally in rats while in humans it starts before birth and continues until 18 month postnatal. In this case it might be more relevant to compare in utero exposure of humans via the placental barrier with lactational exposure of rats via the blood-milk barrier. A common way to overcome inter-species uncertainties is to add an arbitrary safety factor of 10 to the obtained animal results assuming a better prediction of the risks for humans.

2.4.2 Exposure route and dose The exposure route is most frequently oral, but can occasionally be by gavage, dermal or inhalation. Attention has to be paid to the route of exposure since a treatment that induces stress in the animal might also have negative effects on the developing nervous system. When postnatal exposure is evaluated, possible effects of the chemical on maternal behavior, such as milk quality and quantity and pup suckling behavior, should be determined. Some general issues for DNT testing have been discussed. For example, specific effects are often dependent upon the dose and the time of exposure, and different exposure scenarios may give different adverse effects. It should also be mentioned that the offspring is typically not directly exposed to the chemical but only via the mother, transplacentally or through the breast milk. This makes it difficult to conclude the factual exposure of the pups and does not incorporate other exposure possibilities such as surface contaminations (Kaufmann, 2003). At least three levels of exposure doses of the chemical should be tested. The highest dose should produce maternal toxicity but not result in more than a 10 % decrease of maternal weight, while the lowest dose should not produce any observable toxic effects (OECD, 2007; US EPA 1998a). The animals should be exposed from day 6 of gestation until day 10 (US EPA 1998a) or 21 postnatal (OECD, 2007). However, an exposure only until day 10 postnatal may be insufficient to detect all DNT effects and ensure a proper risk assessment.

2.4.3 Test performance The guidelines recommend the use of at least 20 litters per dose group (10 animals per sex) with minimum three treatments and one control group (Tyl et al., 2008). Moreover, the test study needs to incorporate positive control data (performed within the same laboratory and during the last few years) to determine the sensitivity of the methods used (Crofton et al., 2008), but these data do not need to include prenatal exposure. In the classical DNT approach, pups are observed in an open field and many different parameters are documented, including autonomic, motor and sensory functions. Separate groups of pups are used for the testing of complex functions, such as learning and memory and for neuropathology examinations, including brain weight measurements and morphology studies. These observations are normally made at different time points up to 60 days postnatal. However, the animal is still considered as young at this age and this time frame might not be long enough to detect changes that occur later in life. The maternal animals are observed daily for signs of toxicity and are weighed at least weekly during pregnancy and on the day of delivery plus at 11 and 21 days postnatal. The offspring is observed daily to detect any behavior or neurological abnormalities. The weight is measured at birth, at day 4, 11, 17 and 21 postnatal, and later at least every second week. For behavioral tests, one female and one male pup are selected from each litter. According to the OECD guideline the motor activity tests should be performed once before day 21 postnatal, once between days 21-35 and once between days 60-75. Motor and sensory functions and learning and memory tests should be carried out in both young (between days 21-35) and adult animals (between days 60-75). Furthermore, neuropathological and brain weight studies (10 animals/sex/dose) are required on day 11 or 22 postnatal and at the end of the study (Kaufmann, 2006). The estimation is that about 1.000 rat pups are used for one DNT study and as a minimum 140 mated females are needed to produce enough pups. Of these 1.000 pups approximately 640 are kept for at least 3 weeks and 240 pups are kept up to the young adult step of 60 days postnatal. Together with the prenatal period, a DNT study lasts for 3 month.

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Taking all these factors into consideration results in DNT studies being complex, time consuming, costly and laboratory demanding. A proposed solution could be to simplify the DNT testing and decrease the amount of animals used. This could be done by developing a first-tier screening paradigm where the incorporation of a high throughput screening test batteries, based on in vitro testing, would allow the rapid screening of a large number of chemicals and flag those that have the potential to cause DNT effects (Coecke et al., 2007; Lein et al., 2007).

2.5 Interpretation of data and application to risk assessment Once the DNT studies have been performed, the data has to be interpreted. Various reviews are providing guidance for evaluating DNT data, including data interpretation, data variability, positive control data and statistical analysis (Crofton et al., 2008; Fitzpatrick et al., 2008; Holson et al., 2008; Raffaele et al., 2008; Tyl et al., 2008). The reliability of the result has first to be assessed before the relevance can be determined. Primarily, the variability of the parameters measured should be understood and taken into consideration in order to evaluate the relevance of the observed changes (Fitzpatrick et al., 2008). The variability can be intrinsic (biological), meaning natural variability among or within individuals, or extrinsic (factors outside the individual). High variability can obscure treatment related effects, whereas a lack of variability can result in statistically significant findings even when the effects might be within the normal range. When performing the statistical analyses there are a number of factors of concern which could lead to errors, such as litter allocation using sex as a factor, using repeated measurements or wrong statistical assumptions (Fitzpatrick et al., 2008). The obtained data can be either behavioral changes or morphological alterations of the brain. Generally, a pattern of effects is more convincing evidence of DNT than one or a few unrelated changes. Moreover, irreversible effects are clearly serious while reversible ones might be of less concern. However, reversible effects might not always be easily determined since the brain possesses reserve capacity, which may compensate for neuronal damage. In this case, the reserve capacity of the brain would be decreased and should therefore be considered as an irreversible effect. The results can sometimes be difficult to interpret and regulators are generally advised to do their own evaluation or have it evaluated by other experts than the study directors. In some cases, a second study in another species or a different endpoint can be demanded, especially if there are extensive exposure risks of children or women at child bearing age. In addition to using DNT data for regulatory decisions, some regulatory agencies have also incorporated into their decision making an additional set of uncertainty factors because of the absence of relevant DNT data (Makris et al., 2009). Regulatory appraisal, such as no observable adverse effect level (NOAEL), and any subsequent risk assessment should be based on the most sensitive endpoint, whether it is in the mother or the offspring. The relationship between the effects and the dose may be complex (e.g. U-shaped, inverted U-shaped), but it must be treatment related. In most cases when DNT testing has been properly performed, a lower NOAEL value has been found as compared to other tests such as reproductive and adult neurotoxicity ones. This indicates more sensitive endpoints in the DNT testing protocols than in the previous tests (Makris et al., 2009). Official statistics regarding the use of these studies have not yet been published but a preliminary survey showed that out of 19 pesticides recommended for DNT tests only 5 of the studies were used for risk estimations (Hass, 2006). If the obtained results are clearly positive, the classification of the chemical should be toxic to reproduction. However, for many regulators these behavioral endpoints are quite new and might not always be as recognised as the well established ones, e.g. malformations.

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3. ENVIRONMENTAL EXPOSURE TO POTENTIAL DNT TOXICANTS

Currently a large amount of evidence is available illustrating that foetuses’ and children’s brains are more vulnerable to chemical exposure than the adult brain (Rice and Barone, 2000; Rodier, 1994; Rodier, 1995). Moreover, the unique behavior and activities of children place them in greater risk for environmental exposure to xenobiotics (Weiss et al., 2004). An efficient way to prevent children’s environmental exposure would be obligatory testing of chemicals before allowing them on the market. Unfortunately, for 80 % of the high production volume chemicals (HPV) (450 ton/year) there is currently no information about developmental or neurodevelopmental toxicity (US EPA, 1998b). For this reason there are only five substances recognised as developmental neurotoxicants: methyl mercury, lead, arsenic, polychlorinated biphenyls (PCBs) and toluene (Grandjean and Landrigan, 2006) (Fig. 1). The lack of information about potential neurotoxic/DNT effects is the main obstacle in preventing the neurodevelopmental disorders induced by neurotoxicants. n= 5 Developmental neurotoxic in humans

n = 201 Neurotoxic in humans

n > 1.000 Neurotoxic in animal experiments

n > 100.000 Chemical Universe Fig. 1. A representation of the extent of knowledge on neurotoxic and DNT chemicals (adapted and modified from Grandjean and Landrigan, 2006). Only five industrial chemicals have been proven to cause developmental neurotoxicity in humans.

Many substances are known to be toxic for the adult nervous system and these are of particular concern regarding their potential to cause DNT effects after early-life exposure (NRC, 2000a). Several animal studies have shown that various chemicals can cause DNT at low doses that are normally not harmful to mature organisms (Eriksson, 1997; Tilson, 2000)), which is due to the higher vulnerability of children in comparison to adults (see chapter 1.2). This chapter will briefly characterise exposure to industrial chemicals and pesticides with a special emphasis on heavy metals, as they have the potential to trigger DNT effects, which in turn could lead to possible neurodevelopmental disorders. Metals can easily cross the placenta as it is not an effective barrier against environmental chemicals (Foltinova et al., 2007; Lauwerys et al., 1978). In addition, the biological life times of heavy metals are very long, which lead to accumulation and increased exposure risk as the elimination from the environment is slow.

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3.1 Exposure to recognised DNT industrial chemicals 3.1.1 Methyl mercury DNT effects of methyl mercury became evident in the 1960’s in Minamata Bay, Japan, where many infants were born with spasticity, blindness and mental retardation. The cause of the epidemic was identified as methyl mercury exposure of the foetuses via transplacental transfer of mercury from the mothers that consumed fish caught in contaminated waters. The water contamination was later found to arise from plastic industrial plants (Harada, 1995). The mothers of the poisoned children had less serious symptoms, if any at all. This could be due to the higher affinity of methyl mercury to foetal hemoglobin compared to that of pregnant women, which can give rise to about 25 % higher foetal methyl mercury exposure levels (Amin-Zaki et al., 1976). The toxicity of methyl mercury is mainly thought to affect the neuronal differentiation and migration due to disruption of cell skeleton proteins and cell membranes (Choi et al., 1978; Graff et al., 1993). Glial cells are in general spared from direct damage, although reactive gliosis might occur (Moller-Madsen, 1990; Schionning and Moller-Madsen, 1992; Tiffany-Castiglion and Qian, 2001) (Paper II). Today, human exposure to methyl mercury is primarily via consumption of contaminated fish and seed grains treated with mercury-containing fungicides (Ip et al., 2004; WHO 1990). Children are also exposed to mercury through thimerosal, a preservative added to many vaccines (Ip et al., 2004). However, the use of thimerosal has been restricted even though it still remains in some vaccines and in some countries (Ball et al., 2001). The observed toxicity of methyl mercury has led food safety authorities to issue dietary advices and agencies now seek to control and restrict the release of mercury into the environment. Still, about 500.000 children in the US are born every year with blood mercury levels above 5.8 µg/L, a level that has been associated with cognitive deficits (Trasande et al., 2005). In fact, several studies have reported that even lower concentrations of methyl mercury can result in neurotoxic effects, such as cognitive and motor dysfunctions (Amin-Zaki et al., 1976; Dolbec et al., 2000; Grandjean et al., 1997; Lebel et al., 1998; Levin et al., 2003; NRC, 2000b).

3.1.2 Lead Lead toxicity was reported in young children that were playing on verandas with peeling paint already 100 years ago (Gibson 2005). Back then the poisoning was thought to be an acute illness, from which a child either recovered or died. First in the 1940’s was lead poisoning identified as giving long-term effects, such as learning and behavioral disabilities (Byers and Lord, 1943). Despite the observed toxicity in children, lead continued to be used in many products, e.g. petrol, paints and ceramic glazes. In the 1970’s, increased blood-lead concentrations in children were associated with numerous adverse effects in the CNS, including problems with memory, concentration, cognition and behavior (Finkelstein et al., 1998; Needleman et al., 1979). Lead is predominantly affecting glial maturation (Lindahl et al., 1999; TiffanyCastiglioni et al., 1989; Zurich et al., 2002) that could in turn have an affect on the neuronal development processes, such as neurite outgrowth, migration, synaptic elaboration and cell-cell interactions (Goritz et al., 2002; Hatten and Liem, 1981; Pfrieger and Barres, 1997; Wang et al., 1994) (Paper II). Due to the increased amount of evidence of lead toxicity, many sources of lead exposure have become controlled, but not all sources and countries. Although the efforts to reduce lead levels in the ecosystem (e.g. by the use of lead-free gasoline) have significantly reduced median lead-blood concentrations (Silbergeld, 1997), the risks associated with environmental exposure still remains because of the long biological half-life of lead (Cory-Slechta, 1990). EPA and Centers for Disease Control (CDC 1991), have defined the highest acceptable lead-blood level in children as 10 µg/dl. However, several studies have reported impaired cognitive functions at significantly lower concentrations (Bellinger, 2008; Chiodo et al., 2007; Jusko et al., 2008; Surkan et al., 2007).

3.1.3 Arsenic DNT due to arsenic exposure was discovered 1955 in Japan, as consumption of arsenic contaminated milk powder resulted in over 12.000 cases of poisoning and 131 deaths (Dakeishi et al., 2006). The group 24

of children exposed to the contaminated milk was later shown to have a ten-fold increase in mentallyretarded individuals compared to non-exposed children. Furthermore, these arsenic-exposed children had poor school records, emotional disturbances and abnormal or borderline encephalogram findings. Arsenic specifically targets sulfhydrile groups, which have the potential to disrupt ATP production and other cellular processes (Petres et al., 1977). Human arsenic exposure occurrs mainly via drinking water and soil pollution, either from natural sources or from industrial pollution. Furthermore, arsenic is used as a treatment of patients with acute promyelocytic leukemia (Antman, 2001; Jing et al., 2001). In many parts of the world, such as South East Asia (Rahman et al., 2001) and South America (Blanco et al., 2006; Ferreccio and Sancha, 2006) arsenic water contamination is a major health concern and children exposed to arsenic polluted water have shown increased numbers of cognitive deficits. Despite the evidence of DNT caused by arsenic, regulatory actions do not stress the need to protect children from this (DNT) substance (IPCS 2001; NRC, 2001).

3.1.4 Polychlorinated biphenyls Human toxicity due to polychlorinated biphenyls (PCBs) was first well described in industrial exposures studies (Smith et al., 1982), but then the neurologic effects were considered minor. The first cases of developmental toxicity were observed in Asia in the 1970’s, where cooking oil had been contaminated by PCBs and related substances during manufacturing (Yamashita and Hayashi, 1985). PCBs have been widely used in many applications, especially as insulators in electrical equipment. However, due to the toxicological effects, the use of PCBs has been banned in most nations sine the 1980’s (EFSA, 2005). Still they persist in the environment, contaminating the atmosphere and ground water, making PCBs remain a focus of attention. Exposure to PCBs can cause hyporeflexia, psychomotor delays, delayed cognitive development and IQ deficits (Jacobson and Jacobson, 1996; Patandin et al., 1999; Stewart et al., 2000). The toxic mechanisms of PCBs are diverse, e.g. interaction with the transcription factor aryl hydrocarbon receptor, altered intracellular signaling processes, alteration of thyroid hormone metabolism and interference with thyroid hormone induced gene transcription (Bandiera et al., 1982; Kodavanti, 2005; Morse et al., 1996; Royland and Kodavanti, 2008). Some PCBs are also reported to decrease thyroid hormone levels and alter neurotransmitter levels, for example a decreased dopamine synthesis (Roth-Harer et al., 2001; Simon et al., 2007). Since thyroid hormone is essential for normal brain development, interference with the hormone homeostasis is of particular concern.

3.1.5 Toluene and ethanol (solvents) Solvents (e.g. ethanol and toluene) are well-known to cause neurotoxicity in adults as observed from acute poisoning cases and occupational studies (White and Proctor, 1997). Infants of women abusing alcohol or toluene have been diagnosed with FAS or fetal solvents syndrome (Schardein, 2000). Both syndromes give similar symptoms such as mental disability (Toutant and Lippmann, 1979), cerebellar dysfunctions (Streicher et al., 1981), delayed motor and cognitive development (Goodwin, 1988; Jones and Balster, 1998), hypotonia, developmental delays and CNS dysfunctions (Hersh, 1989). Conversely, ethanol has been shown to cause neuronal death, while no such evidence is available for toluene or its metabolites. However, alterations in astrocyte proliferation and maturation have been reported (mainly from in vitro studies) which could give rise to the CNS effects observed in these syndromes (Burry et al., 2003; Gotohda et al., 2000; Yamaguchi et al., 2002). Moreover, toluene has been proposed to interfere with the neurogenesis process through disruption of synapse formation and maintenance (Lin et al., 2009).

3.2 Exposure to pesticides Since many pesticides are developed to target the nervous system of different organisms their effects on the human brain are of great concern, in particular the immature brain. The acute neurotoxicity of pesticides is well-known from occupational exposure studies, poisoning events and suicide data (Kimbrough 25

et al., 1989). Furthermore, DNT effects, such as reduced short-term memory, hand-eye coordination, drawing ability and visuospatial deficits have been observed in several studies (Grandjean et al., 2006; Guillette et al., 1998; Ruckart et al., 2004). Insecticides are especially known to induce neurotoxic effects. They are divided into several different classes and the most widely used are the cholinesterase inhibitors, organophosphates and carbamates. They were developed in the 1940’s and 50’s (Costa, 1988) and their main target is the acetylcholinesterse enzyme. Inhibition of this enzyme causes accumulation of the neurotransmitter acetylcholine at the cholinergic synapses leading to overstimulation of cholinergic receptors that can give various effects in the CNS. Young animals and presumably children are more sensitive to the acute toxicity of these inhibitors than adults, possibly due to lower detoxication abilities (Costa, 2006; Mileson et al., 1998; Pope, 1999; Thullbery et al., 2005). In addition, the adverse effects on the developing brain could also be mediated by additional mechanisms, such as damage to DNA and RNA synthesis (Crumpton et al., 2000a; Crumpton et al., 2000b; Song et al., 1997), deregulation of signal transduction pathways (Ehrich, 1995; Song et al., 1997), oxidative stress (Crumpton et al., 2000b) and astroglial cell proliferation (Garcia et al., 2001; Guizzetti et al., 2005) (Paper I). Another relatively common class of insecticides is pyrethroids that are used both in agriculture and in the household. Their main mechanism is binding to sodium channels that slow down the inactivation of the channels, causing a stable hyper-excitable state. However, there are only a few reports of human poisoning (Bradberry et al., 2005). There are several other insecticides with a potential to induce DNT through different mechanisms such as interference with neurotransmitters (Beeman and Matsumura, 1973; Cole and Casida, 1986) and inhibition of ATPase or the mitochondrial respiratory chain (Costa et al., 2008; Desaiah, 1982). Herbicides and fungicides are in general less toxic for the brain than insecticides, with the exceptions of paraquat (herbicide) and dithiocarbamastes (fungicides). Paraquat induces neurodegeneration of dopaminergic neurons (Castello et al., 2007; Thiruchelvam et al., 2002; Wu et al., 2005) and can cause oxidative stress, while dithiocarbamates affect the thyroid function, which can lead to serious damage as thyroid hormones play an essential role in the brain development (Marinovich et al., 1997). Since the development of the brain is based on precise events in space and time (such as proliferation, migration and differentiation) that are strictly controlled, e.g. by neurotransmitters, electrical activity and hormones, the mechanisms of pesticides identified above are likely to have a potential to induce DNT (Paper I). There are more than 600 pesticides registered on the market, including insecticides, fungicides and rodenticides, and several of these are produced in high volumes (Grandjean and Landrigan, 2006). Even though the uses of many pesticides are restricted and an increase in DNT testing is demanded, the general lack of DNT data for agricultural chemicals is of particular concern because of their widespread use and ubiquitous exposure (Whyatt et al., 2004). A population based study has reported that over 90 % of the children in the US have detectable urinary remains of neurotoxic organophosphate pesticides (Schettler, 2001). This exposure might be a risk to children’s health, as these substances are suggested to contribute to developmental disorders such as attention deficits, hyperactivity disorders, autism and learning disabilities (Costa et al., 2008; Jurewicz and Hanke, 2008; Shafer et al., 2005).

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4. ALTERNATIVE IN VITRO AND NON-MAMMALIAN MODELS RELEVANT TO DNT EVALUATION

Increasing complexity

In vitro models of the nervous system have been in use for many years in basic research (but not for regulatory purposes) and provide an important tool for functional studies at both cellular and molecular levels (Silva et al., 2006). The major advantage of in vitro cell-based models is the ability to reproduce, to a great extent, discrete stages of CNS development and maturation in a simplified way. The reduced complexity as compared to in vivo makes it easier to detect changes in key cellular developmental processes, such as proliferation, migration and differentiation. These processes are well understood at the cellular and molecular levels and are surprisingly similar to those in vivo. At the same time in vitro models do have some limitations, e.g. diminished metabolic capability, loss of complex interactions between cells, loss of coordinated expression of molecular and cellular events in a time and region dependent manner, and changed three dimensional cytoarchitecture (Rice and Barone, 2000; Rodier, 1994; Rodier, 1995). It is important to be aware of these disadvantages and take them into consideration when interpreting the results (Bal-Price et al., 2008). In general, the level of similarity to the in vivo situation is directly related to the complexity of the in vitro model, starting from simple neuronal or glial cell lines where usually only one cell type is present, to more complex models such as monolayer of primary mixed neuronal cultures or three-dimensional models, e.g. brain slices or re-aggregating primary cultures (Fig. 2). Additionally, non-mammalian animal species can be used to address some of the behavioral endpoints that are considered by regulators as crucial for the neurotoxicity risk assessment. These alternative systems (in vitro and non-mammalian models) all have their advantages and disadvantages that will be discussed in this chapter.

Tissue slices and explants 3D models: aggregates Primary cells: monolayer cultures

Human cell lines Increasing information Fig. 2. Diagram of possible in vitro models for DNT testing.

4.1 Cell lines The simplest alternative model, the cell line, is a population of cells that can be maintained in culture for an extended period of time. Neuronal cell lines normally originate from a single common ancestor cell (clonal) and are often derived from tumours, e.g. phaeochromocytomas (adrenal medullary tumour) (Greene and Tischler, 1976) and neuroblastomas (Augusti-Tocco and Sato, 1969). However, a recently

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developed technique to introduce oncogenes into primary cultures through retro viruses has opened up new possibilities (Bartlett et al., 1988). There are several neuronal cell lines commercially available (James and Wood, 1992) and many of them have been used for DNT studies (Jameson et al., 2006; Jenkins et al., 2004; Lau et al., 2006; Qiao et al., 2001; Radio and Mundy, 2008; Slotkin and Seidler, 2009). The cell lines are relatively easy to maintain and some of them can be indefinitely propagated. Moreover, they provide a homogenous population of cells that can divide rapidly and be continuously subcultured to provide large numbers of cells in a short period of time. The cells can normally be differentiated into neuronal-like cells by the addition of various inducers and growth factors into the medium. This makes it possible to control the onset of development and adjust the experimental design to the needs of the DNT study. These advantages make cell lines suitable for high throughput screening of many substances. Moreover, many cell lines are derived from human sources which might be an advantage as the goal is to predict human toxicity. However, there is not yet sufficient data showing that human cell lines are better to predict human DNT than cell lines of other species. It should be kept in mind that these cells are usually immortalised and do not replicate some of the more complex properties that can be seen in the original primary cell. Many properties might be lost or differ from the in vivo situation. Two of the most commonly used neuronal cell lines are further described below.

4.1.1 Phaeochromocytomas (PC12 cells) One commonly used neuronal cell line is the PC12, derived from rat phaeochromocytoma cells of the adrenal gland (Greene and Tischler, 1976). Undifferentiated PC12 cells (grown in serum-containing medium) resemble immature adrenal chromaffin cells that are polygonal in shape and lack neurite-like processes (Tischler and Greene, 1978). In the presence of nerve growth factors (NGF), the cells differentiate into neuronal-like cells with the properties of mature sympathetic neurons, such as electrical excitability, secretion of neurotransmitters (dopamine, noradrenalin and acetylcholine), expression of cholinergic and ionotropic N-methyl D-aspartate glutamate (NMDA) receptors (Casado et al., 1996), and expression of acetylcholine-esterase (Dichter et al., 1977; Fujita et al., 1989). During differentiation the cells change morphology and start to extend neurites within 24-48 hours after exposure to the differentiation factors and they attain maximal neurite length within a week (Das et al., 2004). However, the neurites do not have the specific character of axons and dendrites, and the cells do not form functional synapses. The PC12 cells can relatively easy be genotypically and phenotypically manipulated which enhance the possibilities to study specific toxic mechanisms. The PC12 cell line has been widely used in different neurobiological (Greene and Tischler, 1976), as well as, toxicological studies, e.g. morphological evaluation of cell differentiation by measurement of the neurite outgrowth (Radio et al., 2008; Radio et al., 2009), mechanisms of toxicity involved in apoptosis, mitochondrial dysfunction, disturbance in neurotransmitter synthesis and vesicle release (Gartlon et al., 2006; van Vliet et al., 2007a). The PC12 cell line, in particular, has been used to assess involvement of NGF-activated signaling pathways (Parran et al., 2003; Parran et al., 2004; Williams et al., 2000) and cholinesterase inhibition (Das and Barone S Jr, 1999) as possible mechanisms by which toxicants can affect the neuronal differentiation.

4.1.2 Neuroblastomas Many neuronal cell lines have been derived from tumors arising from immature nerve cells (neuroblastomas) in both rodents and humans. There are several different neuroblastoma cell lines, such as rat B50, mouse NB2a and N2a, and human IMR-32, SH-SY5Y and SK-N-SH, that can differentiate into neuronallike cells with the aid of diverse inducing factors, such as retinoic acid, dibutyryl cAMP and nerve growth factors, or by reduced serum content (Augusti-Tocco and Sato, 1969; Greene and Tischler, 1976; Ross and Biedler, 1985). Depending on the cell line, the differentiated cells can express cholinergic, adrenergic and/or dopaminergic markers. Various inducing factors can also stimulate the cells into different morphologies, e.g. NB2a cells in the presence of dibutyryl cAMP form neurites with axon properties, while the presence of retinoic acid results in a more extensive network of branching neurites with dendrite properties (Shea et al., 1985; Shea et al., 1988). The human neuroblastoma cell lines SK-N-SH and the subclone SH-SY5Y are derived from a metastatic neuroblastoma localised within the bone marrow (Biedler et al., 1973). The SH-SY5Y cells express 28

acetylcholine esterase and have therefore been widely used to investigate neurotoxicity of acetylcholine esterase inhibitors such as organophosphates (Ehrich, 1995). Moreover, in contrast to the PC12, they can form functional synapses (Pahlman et al., 1990). The neuroblastoma cell lines have been used in several DNT studies evaluating different key processes, such as neuronal migration (Miller et al., 2006) and differentiation including network elaboration (Hong et al., 2003; Radio and Mundy, 2008) and synaptogenesis (Chamniansawat and Chongthammakun, 2009). However, one of the limitations with these models is that the toxic response to the same chemical differs between different neuroblastoma cell lines, which make it difficult to interpret the obtained data (Radio and Mundy, 2008). For example exposure to lead has shown contradictory responses, inducing decreased or increased neurite outgrowth depending on the cell line. Moreover, they do not replicate some of the more complex properties observed in primary cultures of neurons.

4.2 Primary cultures Neuronal primary cultures are harvested from the peripheral nervous system (PNS) or CNS of a living organism and are maintained in culture for at least 24 hours (Aschner and Syversen, 2004). The advantages of primary cultures are that many developmental key processes can be largely followed and that the functional capacity of both cell types (neuronal and glial) can be maintained. Moreover, processes that play important roles for normal and abnormal cell development and maturation can be addressed, such as regional specificity, expression of receptors and neurotransmitters, and neuronal-glial interactions. The cultures can either consist of one predominant neuronal cell type or a mixture of different neuronal populations. Furthermore, the cultures can be made pure neuronal by blocking the proliferation of glia, or mixed neuronal-glial cultures. These characteristics make it possible to determine the specific sensitivity to chemical perturbation in different neuronal and glia cell types (Paper I, II and III). The presence of astrocytes is known to modify the neuronal response to toxicants and it has been identified as an important contributing factor to neurotoxicity. Primary neuronal cultures are often used to investigate cellular mechanisms of toxicity observed after in vivo chemical exposure (Paper III). However, the isolated cells may develop altered appearances, functions, and have different levels of cell-cell interaction and usually also a dramatically decreased metabolism, that could result in an altered response to test chemicals when compared to the in vivo situation (Costa, 1998). Furthermore, primary neuronal cultures consist predominantly of post-mitotic neurons which do not proliferate, giving the model a limited life span. Consequently, the cultures always need to be freshly isolated and may not be fully suitable for highthroughput screening. Lately it has become evident that more complex models, such as three-dimensional test systems and brain slices, are necessary as they offer the possibility to extend the set of relevant endpoints and can replicate the in vivo situation more convincingly. However, this approach is very time consuming, especially if large numbers of chemicals need to be tested (Costa, 1998; Harry et al., 1998). Neuronal primary cultures from the PNS and CNS can be derived from many different regions, such as superior cervical ganglion, dorsal ganglion, hippocampus, cortex, cerebellum, midbrain or sub-ventricular zone. Cultures from the two most frequently used rat brain structures (cerebellum and cortex) will be further presented.

4.2.1 Cerebellar granule cells Dissociated primary cultures of rat cerebellar granule cells (CGCs) are the most widely used in vitro system in neurobiology (Burgoyne et al., 1988; Costa et al., 2007; Harry et al., 1998; Kane et al., 2008; Pearce et al., 1987; Radio et al., 2009) (Paper I, II and III). The model is well characterised through its extensive use in studies of neuronal development, function and pathology (Contestabile, 2002). CGCs are also known to have many signaling pathways that can execute responses to toxicants. This is the reason for that primary cultures generally are considered more sensitive than cell lines (Contestabile, 2002; Costa, 1998; Harry et al., 1998), however, not in all cases (Gartlon et al., 2006). The primary cultures of CGCs are isolated from 2-10 days old pups since neurogenesis for the granule cells in the rat cerebellum takes place postnatally (P) between P0 and P10 (Altman, 1987; Altman and Bayer, 1978). The culture can either be mixed neuronal-glial, consisting of around 85 % neurons, 15 % 29

astrocytes and 5 % microglia (Paper I and II), or almost pure neuronal by adding agents that are blocking glia proliferation. The CGCs predominantly consist of granule neurons that are mainly glutaminergic and have a high expression of glutamate excitatory NMDA receptors (Contestabile, 2002). Moreover, a few Purkinje cells expressing gamma-aminobutyric acid (GABA) neurotransmitters are also present in CGC cultures. The cells can be kept in culture for several weeks and are considered mature after approximately 8-10 days in vitro (Privat et al., 1974). The cell morphology is dramatically changing over time (Fig. 3) and several key developmental processes can be measured, such as precursor commitment, glial proliferation and neuronal differentiation and maturation (Paper I, II and III).

A

B

1 DIV C

12 DIV D

4 DIV

12 DIV

Fig. 3. Maturation of mixed neuronal-glial cultures of CGCs. Cell morphology (A and B) in phase-contrast microscopy and (C and D) by immunocytochemistry of GFAP (red) and NF-200 (green) co-stained with Hoescht 33342.

4.2.2 Cultures of cortical neurons Cultures of rat cortical neurons are normally isolated from 17-19 days old foetuses. The foetal brain increases the capacity of neuronal cultures to establish more connections under in vitro conditions compared to the tissue isolated from older animals. The cultures consist of all the various cell types in the CNS, such as neurons, astrocytes, oligodendrocytes and microglia (Honegger and Werffeli, 1988). During the first weeks in vitro, proliferation of neural precursor and glial cells, as well as neuronal differentiation can be observed (Fig. 4) (Paper IV). The system is considered mature after approximately three to four weeks in culture and can be maintained for an exceptional long time (several months). The main neuronal types present in cortical neuronal cultures are pyramidal and stellate neurons and several neurotransmitters have been identified, such as acetylcholine, glutamate, GABA, dopamine and serotonin (Honegger et al., 1979; Kriegstein and Dichter, 1983). The in vitro cortical neurons have both morphological and physiological properties which are remarkably similar to cortical neurons in situ (Dichter et al., 1977). The model has been widely used for neurotoxicity studies (Eskes et al., 2002; van Vliet et al., 2008; Zurich et al., 2002), including electrophysiological recordings (Gramowski et al., 2000; Gramowski et al., 2006; van Vliet et al., 2007b) and DNT studies (Bingham et al., 2004; Qiu et al., 2006) (Paper IV).

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4 DIV C

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21 DIV E

28 DIV F

7 DIV

28 DIV

Fig. 4. Maturation of mixed neuronal-glial cortical cultures seeded on MEA chips. Cell morphology (A and B) in phase-contrast microscopy, (C and D) by immunocytochemistry of GFAP (red) and NF-200 (green) co-stained with Hoescht 33342 and (E and F) by immunocytochemistry of Map-2 (red) and nestin (green) co-stained with Hoescht 33342.

Cortical neurons can be cultured as dissociated monolayer or re-aggregating three-dimensional model. Dissociated monolayer cultures are less complex than three-dimensional models but easier to maintain and more suitable for morphological and large scale studies. The re-aggregating cultures are generated spontaneously under continuous gyratory agitation resulting in the development of an in vivo-like threedimensional cell architecture with well-differentiated neuronal processes and enhanced cell-cell interactions, myelination and synaptogenesis (Eskes et al., 2003). However, the model is not suitable for studies at single cell level and the variation between aggregates can be large due to different size of the aggregates and varying proportions of the cell types. Cortical cells isolated from 2-10 days old rat pups can generate pure glial cell cultures (astrocytes, oligodendrocytes and microglia). Depending on the culture medium condition, pure oligodendrocyte cultures (Almazan et al., 2000) or mixed astrocyte-microglia can be obtained (Kinsner et al., 2005). The astrocytes and microglia can later be separated by gentle shaking allowing microglia to detach from the astrocyte layer. This procedure allows performance of toxicity tests on pure populations of astrocytes or microglia.

4.3 Neural stem cells Neural stem cells (NSCs) or progenitor cells have the capacity of self-renewal and can generate all three major cell types of the CNS; neurons, astrocytes and oligodendrocytes (Doetsch, 2003; Temple, 2001; Zhang et al., 2001). They can be derived from several species including rodents and humans. The three main sources of stem cells are pluripotent embryonic stem cells isolated from blastocyst, human umbilical cord blood derived stem cells or multipotent somatic progenitors derived from bone marrow or other tissues including CNS. However, NSCs isolated from the CNS only have a limited life span and either differentiate spontaneously or enter an irreversible growth arrest after a finite number of cell divisions (Caldwell et al., 2001). Recently, NSCs of human origin have been immortalised to generate commercially available clonal NSC lines that can divide infinity (Seaberg and van der, 2003). These cell lines are promising in vitro models for neurotoxicity testing (Klemm and Schrattenholz, 2004) and there is hope for that these cells can acquire the complexity of primary neurons and be used for DNT testing (Bal31

Price et al., 2008; Coecke et al., 2007). The major advantage of human cell lines is that the results do not require extrapolation from animal data to the human situation. However, before the stem cell models can be used for routine screening, they should be standardised and carefully validated to evaluate their capacity for predicting human DNT effects (Hartung et al., 2004). Indeed, some laboratories have shown promising results with stem cells differentiated into functional neurons and glial cells that can have possible applications for DNT testing (Breier et al., 2009; Leist et al., 2008; Moors et al., 2009; Stummann et al., 2007; Stummann et al., 2009; Tamm et al., 2006), including our own study (Buzanska et al., 2009).

4.3.1 Embryonic stem cells Embryonic derived stem cells (ESCs) are undifferentiated cells with the capacity to develop into cells from all the three primary germ layers and to further differentiate into the numerous specialised cell types of the body, including neuronal and glial cells. Currently, the mouse embryonic stem cell test is the only system based on a mammalian cell line, which has been validated as an alternative for in vivo embryotoxicity testing (Genschow et al., 2004). The commitment of these cells into neuronal-like ones might extend the use of the test and make them a promising model for DNT studies (Hareng et al., 2005). ESCs of human origin would be especially advantageous as this would avoid extrapolation between species. Although ESCs from both mouse and human have similar characteristics, they need different conditions to be successfully differentiated. Two basic approaches have been identified, the embryoid body formation and the default pathway hypothesis. The idea of the first approach is to mimic the environment of the embryo by first forming embryoid body intermediates. The mouse cells are placed in a suspension culture in the absence of the growth factor leukemia inhibitory factor (LIF), which promotes maintenance of the pluripotental state (Smith et al., 1988). Within 2-4 days the cells form aggregates that resemble the anterior prestreak stage embryo with the ability to generate derivatives of all three primary germ layers (Keller, 2005). Subsequently, under the influence of neuronal differentiation factors, e.g. retinoic acid, growth factors, and fibronectin, the cells are directed into neurons and glia. The default pathway hypothesis proposes that the absence of signals mediates the decision of the cells to become neural or non-neural. The differentiation occurs directly, without including the embryoid body formation, but includes an intermediate cell type, the primitive NCS. This cell type can be achieved through different protocols, using defined medium or by co-culturing with stromal cells. ESCs are directed into NSCs, as selective survival of these cells is promoted by modified medium conditions. However, the differentiation into pure NSCs is rare and the cultures generally include the presence of other cell types. The co-culture with stromal cells-concept is based on the findings that the survival, proliferation and differentiation of hematopoietic stem cells rely upon interactions with bone marrow stromal cells (Kaushansky, 2006). A variety of different stromal cells have been shown to also have the ability to direct the differentiation of mouse and human ESCs into NSCs. The cells need to be plated at low density to avoid cell-cell interactions and in the absence of serum, as serum is suggested to contain an inhibitor of the NSC transition. The factors provided by the stromal cells to induce neural differentiation are still not identified, but it is suggested that both a soluble factor and the direct contact between ESCs and stromal cells may be required (Kawasaki et al., 2000). ESC cultures based on various protocols have been successfully differentiated into a variety of specific neuronal and glial cell derivatives, including oligodendrocytes (Brustle et al., 1999), dopaminergic, glutaminergic and GABAergic neurons (Barberi et al., 2003; Bibel et al., 2004; Kawasaki et al., 2000; Lee et al., 2000). However, most of these protocols require further optimisation, as the maintenance of neural stem cells is a complex process which may not always result in a high percentage of fully differentiated cells. During the last years, ESCs derived phenotypes have been used in several applications including pharmacological, cytotoxicological, embryotoxicological and DNT approaches (Bremer and Hartung, 2004; Leist et al., 2008; Rohwedel et al., 2001; Stummann et al., 2007; Stummann et al., 2009; Tamm et al., 2006). The use of ESCs is critical since they could detect DNT effects during the early embryo development stage, which is not covered by many other primary or cell line models.

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4.3.2 Bone marrow derived somatic stem cells Somatic stem cells (SSCs) derived from bone marrow (BM) can be cultured in vitro and provide progenitors that are capable of self-renewal and differentiate into neurons and glia. Different culture conditions have been used to induce neural differentiation of BM-SSCs, e.g. presence of retinoic acid, growth factors, and β-mercaptoethanol (Hung et al., 2002; Ning et al., 2009; Sanchez-Ramos et al., 2000). In general, the various protocols generate both neuronal and glial-like cells, but exclusively glial or neuronal populations have also been reported in some studies (Nakano et al., 2001; Woodbury et al., 2000). In the presence of a differentiation medium, the morphology of the cells change into neuronal-like with the expression of several neuronal proteins, including neural precursor marker (nestin), immature neuronal markers (neuronal nuclei and β-tubulin III) and more mature neuronal marker (neurofilament) (Hung et al., 2002; Sanchez-Ramos et al., 2000). Moreover, the BM-SSCs have been differentiated into electrically active neural cells with functioning membrane currents and calcium channels (Hung et al., 2002). The model could be suitable for DNT studies of early, as well as, advanced human neuronal stages of developments, such as cell proliferation, migration and differentiation. However, the use of BM-SSCs is more restricted than of ESCs when it comes to growth and differentiation, as they are present in limited amounts and are difficult to isolate and expand. They also differentiate into neural cell types with a lower efficiency and produce less cell types, demonstrating a limited potential (Colombo et al., 2006). Moreover, BM-SCSs seem to have a shorter life span, even in the presence of mitogenic growth factors. These factors could be a limitation, as it is difficult to generate the large amount of cells that are needed for performing highthroughput toxicity screening.

4.3.3 Human umbilical cord blood derived neuronal stem cells SSCs can also be derived from human cord blood, where they exist in a much higher frequency than in adult bone marrow (Lu et al., 1996). These cells have similar characteristics as ESCs, but would not raise the ethical concerns associated with the use of foetal or embryonic tissue (Habich et al., 2006; McGuckin et al., 2005). Recently a human umbilical cord blood derived NSC (HUCB-NSC) line has been established, which have the potential for a stable growth rate, the ability for self-renewal and also the possibility to maintain a differentiating potential into neurons, astrocytes and oligodendrocytes (Buzanska et al., 2002). The advantages of this model are that it is derived from human cord blood and can be maintained in culture at different developmental stages depending on the culturing conditions (Buzanska et al., 2005). In serum-free medium, the cultures consist of proliferating, non-differentiated cells forming aggregates or neurospheres, while low-serum medium promotes a heterogeneous population of proliferating cells already committed to neural phenotypes. After receiving adequate stimulation by different neuromorphogens, such as retinoic acid, brain-derived neurotrophic factor or di-butyryl cAMP, the cells can give rise to differentiated neurons, astrocytes and oligodendrocytes. Differentiated cells show advanced morphological maturation into neuronal linage with a high expression of neural-specific genes and proteins, such as neuron-specific cytoskeletal markers and numerous neurotransmitter receptors (Buzanska et al., 2006; Buzanska et al., 2009). Furthermore, electrophysiological studies have shown the presence of functional voltage and ligand-gated channels, similar to those identified in other immature neuronal systems (Sun et al., 2005). In two-dimensional culture conditions the cells do not show any action potential related to the activity of voltage-gated sodium channels, which clearly indicates that this process is incomplete and that other factors or conditions are required for these cells to fully differentiate into neurons. However, when HUCB-NSC have been cultured and differentiated in three-dimensional conditions on protein scaffolds, they have shown spontaneous field activity related to action potential, indicating the presence of functional neuronal networks (Jurga et al., 2009). The model could serve as an important tool for DNT as the whole range of neurodevelopment processes, such as proliferation, migration, differentiation, synaptogenesis and apoptosis, can be studied (Buzanska et al., 2005). Indeed, testing of DNT chemicals in this model has already been performed with promising results (Buzanska et al., 2009).

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4.4 Non-mammalian animals Since the development of the nervous system is very complex, there are concerns that simple in vitro systems will have difficulties in predicting all in vivo effects. Moreover, behavioral tests are considered crucial by regulators for DNT risk assessment and these endpoints cannot be measured in vitro. For this reason simple non-mammalian organisms have been explored as possible models for DNT testing. As the fundamental principles of key cellular events during brain development are remarkably conserved across species, this seems to be a promising approach. The use of alternative species will allow direct testing of cause-effect relationships between toxicant effects on biochemical, cellular and molecular endpoints, and moreover, behavioral endpoints can be included (Lein et al., 2007). Some of the proposed candidates for DNT alternatives are presented below, listed in order of increasing evolutionary complexity.

4.4.1 Sea urchin The sea urchin is one of the few organism models that is considered as promising for DNT studies. Its embryonic development has been studied for over a century, and the key cellular processes are wellknown. Numerous signaling systems, such as cholinergic, serotoninergic, dopaminergic and monaminergic, are playing important roles for these processes (Buznikov et al., 1996; Falugi et al., 2008). The sea urchin can be studied during the embryonic, larvae and adult stage. The model is favourable for observations due to the high amounts of eggs produced, which are transparent with the size of 80-100 µm and suitable for microscopic studies (Giusti et al., 1997). Morphological-, biochemical-, and immunohistochemical-analyses of the embryonic and larvae development can easily be conducted during the exposure to toxicants. The sea urchin has been widely used for testing of environmental toxicity, fertility toxicity, embryotoxicity and lately also neurotoxicity (Falugi et al., 2008). In particular the sea urchin is suitable for toxicological studies following pesticide exposure, e.g. organophosphates, as their main target, acetylcholine esterase, is expressed (Buznikov et al., 2001; Buznikov et al., 2007; Qiao et al., 2001; Qiao et al., 2003). In addition, the genome of the sea urchin reveals a high degree of homologies to mammals. The model is promising for use in the screening of neurotoxicants that might target the developing mammalian brain, as neurotransmitters are used as embryonic growth regulatory signals (Buznikov et al., 2007).

4.4.2 Caenorhabditis elegans The nematode Caenorhabditis elegans (C. elegans) is very well characterised, particularly its nervous system, and it has been used in numerous biological and toxicological studies. The homologues with human genes are 60-80% and as a toxicological model the C. elegans has been shown to be predictive of mammalian toxicity (Braungart et al., 2004; Ishiguro et al., 2001; Ved et al., 2005). Many studies have reported similar toxicological responses as in mammalian models for different chemicals, such as pesticides (Cole et al., 2004), metals (Roh et al., 2006) and numerous other agents (Leung et al., 2008; Peterson et al., 2008), also when used in DNT studies (Xing et al., 2009). With its small size (approximately 1 mm) and its ability to grow in a small space, most often an agar plate, the C. elegans is a relatively easy and inexpensive model to maintain. Its life cycle is short, around three days, and its life span about three weeks. The adult lays eggs that hatch and undergo four larval stages before becoming mature individuals able to lay their own eggs (Hope, 1999). The worms are mainly hermaphrodites and are capable to generate about 300 progeny. However, males are rarely present which makes it possible to do genetic experiments as various strains can be crossed. Furthermore, the possibility to generate knock-out worms and use them in combination with various techniques, such as RNAi, microarrays and GFP-tagging, have greatly enhanced the potential to investigate molecular pathways in toxicity (Boyd et al., 2007). Additionally, as the nematode is transparent, it is suitable for microscopic analysis, and the model can be frozen indefinitely making it easy to preserve several stocks. Taking these advantages into consideration makes the model ideal for DNT testing using highthroughput approaches. Combining robotic and automatic workstations will allow several parameters, such as behavioral (locomotion, chemotaxis and feeding), molecular and morphological endpoints, to be measured (Boyd et al., 2007). 34

4.4.3. Danio rerio Danio rerio, the zebra fish, is a vertebrate model that has many advantages for DNT screening studies. It is small and transparent, has a rapid development and high reproducibility, and it is relatively economic (Grunwald and Eisen, 2002; Hill et al., 2005; Zon, 1999; Zon and Peterson, 2005). The life cycle of the zebra fish is fast with gastrulation beginning already about 6 hours after fertilisation, and embryogenesis being essentially completed after 96 hours and by this time most of the organs are functioning. The neurogenesis begins around the onset of gastrulation (Ton et al., 2006) and is maximal during the first 48 hours, but further differentiation of the CNS continues for several hours. By 96 hours all neurons in the adult can be identified. As the embryo is transparent and develops rapidly, development of specific neurons and axons can be visualised live using light microscopy (Gilmour et al., 2004; Graeden and Sive, 2009). The model is hence useful for immunohistochemistry, in situ hybridisation, calcium imagining and patch clamp recordings. The zebra fish can also be used for behavioral studies since it can be trained to perform tasks involving learning and memory (Eddins et al., 2009b; Eddins et al., 2009a; Levin et al., 2003). Many aspects of the brain development are conserved between zebra fish and mammals, and the model has been used in several DNT studies (Fan et al., 2009; Ton et al., 2006). Some correlations have been made between motor activity in mammals and the direct effects seen on motor neurons or catecholaminergic neurons in zebra fish. Moreover, developmental phenotypes seen in zebra fish are quite similar to mammalian phenotypes (Ton et al., 2006). Some endpoints that are easy to measure in zebra fish, as for example total cell death in the brain or visual assessment of axons, are not feasible to measure in mammals which makes the model even more attractive.

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5. KEY CELLULAR PROCESSES OF BRAIN DEVELOPMENT AND METHODS FOR THEIR IN VITRO EVALUATION

The development of the CNS is more commonly disrupted by teratogenic agents as compared to other developing organs (Rodier, 1994; Rodier, 1995). To understand why the CNS is so vulnerable to xenobiotic disturbance, it is necessary to have some basic understanding of how the CNS develops. The formation of the CNS involves several critical key cellular processes, such as precursor cell differentiation, neuronal and glial migration, neuronal and glial differentiation, neurite elaboration, synaptogenesis, apoptosis, formation of neuronal network connections, blood-brain barrier formation and maturation. The generation of neurons and cell migration start in humans at six weeks of gestation and the development of the brain continues into postnatal years. The weight of the brain is increasing even after puberty until approximately 20 years of age (Rodier, 1994). In general, a group of neurons formed in embryonic life, migrates to a new location, begins to grow and becomes specialised. These processes are followed by glial events, such as myelination (Rodier, 1994). Disruption of any of these cellular processes could lead to developmental neurotoxicity. Interestingly, the fundamental principles underlying these key cellular events of brain development are remarkably conserved across species ranging from nematodes to humans and are supporting the use of alternative models (in vitro and non-mammalian animals).

5.1 Development of the brain in utero The development of the CNS starts in the early embryogenesis through a process called neurulation (Rice and Barone, 2000). At approximately two weeks of gestation (in humans), the epidermal tissue forms the neural plate that folds outwards along its central axis to form the neural grooves that soon begin to move together and close to form the neural tube (completed around 26-28 gestational days (GD) in humans and GD 10.5-11 in rats). The neural tube separates from the overlaying ectoderm that will later become a continuous surface over the embryo and differentiate into the epidermis of the skin. During the formation of the neural tube a population of cells separate from the surface ectoderm to form the neural crest cells. These cells give rise to numerous cell types, e.g. the sensory ganglia of spinal and cranial nerves, Schwann cells, the meningeal covering of the brain and spinal cord, and some skeletal and muscles components of the head. An interruption of the neurodevelopment during this early period can result in severe abnormalities of the brain and spinal cord, e.g. spina bitida (divided spine), and abnormal changes of the lower extremities. Folic acid taken during early gestation, or prenatally, decreases the prevalence of neural tube defects. However, despite large-scale prevention programs stimulating the use of folic acid during pregnancy, approximately 1 in every 1.000 births in the US registers neural tube defects (Reznik and Minkov, 1991). Specific areas of the CNS begin to form with the neurogenesis and migration of cells in the forebrain, midbrain and hindbrain during the first month of gestation in humans and around 9.5 GD in rats (Rice and Barone, 2000). In the forebrain (prosencephalon), the diencephalon includes the hypothalamic and thalamic nuclei and develops during the late embryonic and early foetal periods. The cortical structures of the neocortex and hippocampal formation develop mainly during the foetal period in both humans and rats. The midbrain (mesencephalon) develops during both the embryonic and foetal period. The hindbrain consists of the pons, medulla and cerebellum, where the pons and medulla include the brainstem motor and sensory nuclei. The development of the cerebellum differs from the other hindbrain structures as it is developed much later and continues to develop postnatally, both in humans and rats. Subsequently other 36

developing processes follow, such as proliferation, migration, differentiation, synaptogenesis, apoptosis and myelination (Herschkowitz et al., 1997; Herschkowitz et al., 1999). Chemical disturbance of the brain development in utero can be studied by the use of live light microscopy techniques and transparent non-mammalian species, such as nematode, sea urchin and zebra fish (Boyd et al., 2007; Gilmour et al., 2004; Giusti et al., 1997; Graeden and Sive, 2009; Gualda et al., 2008). By using simple microscopic techniques it is possible to determine individual cells in vivo across a broad range of developmental stages. Furthermore, transgenic models that express fluorescent reporter genes make it possible to visualise dynamic changes in gene expression and detailed morphogenetic movements as they occur live in the developing embryo (Guo et al., 2008; Megason, 2009; Wilt et al., 2008).

5.2 Proliferation Since the CNS consists of a large amount of cells that proliferate over an extended period of time, it is vulnerable at many more stages than any other developing organs. Not only the long development time but also the many different cell types of the brain make it much more sensitive since a loss of one cell type might lead to serious damage. Proliferation occurs at different time periods for different structures and cell types, and some neurons are generated in a very short time, in weeks or even days. Large motor neurons are produced first, such as those stimulating muscles, while the sensory neurons are produced later on (Rodier, 1980). Nuclear groups in the brain stem and diencephalon tend to form early, while in complex layered structures like the cerebral cortex, hippocampus and cerebellum cells are added over a long period. Most progenitor cells start by undergoing symmetric division giving rise to two daughter cells that adopt the fate of the progenitor. As the development proceeds the cells begin to divide asymmetrically and the amount of cells that begin to differentiate into neurons increases, while the proportion of cells remaining as progenitors decreases (Caviness, Jr. and Takahashi, 1995). The number of neurons are mainly increasing during the development, while the proliferation in the mature brain is limited and the possibilities of repairing injuries, even during development, are reduced (Rodier, 1994). Many chemicals known to induce brain injury, such as methylazooxylmethanol (MAM) (Yamanaka and Obata, 2004), ethanol (Miller, 1993), the organophosphate chlorpyrifos (Guizzetti et al., 2005) and methyl mercury (Rodier et al., 1984), are interfering with cell proliferation and can decrease or completely rule out a certain cell type, while other types formed after or before exposure might appear normal. Cell proliferation can be measured by different techniques in various in vitro models and is used as one of the most specific endpoints for DNT evaluation. A commonly used assay is the bromodeoxyuridine (BrdU) incorporation where analogues nucleotides are integrated into newly synthesised DNA permitting indirect detection of rapidly proliferating cells by fluorescent labeled anti-BrdU antibodies (Go et al., 2009; Yang et al., 2009). Cytotoxicity assays, such as MTT and alamar blue, are also sometimes used to measure proliferation (Fang et al., 2009; Go et al., 2009). However, these assays do not distinguish between cell death and inhibited cell proliferation which is of particular importance in DNT studies.

5.3 Cell migration Recently proliferated cells move away from their place of origin to their final position in the brain. The migration process takes place through a rearrangement of cytoskeletal components in response to extracellular signals, e.g. neurotransmitter release (Spitzer, 2006), and the process is mediated by numerous intracellular signaling pathways. The cell extends a leading neurite that expands and contracts as it explores the microenvironment, followed by translocation of the nucleus and the rest of the cell body (Ayala et al., 2007). The neurons are normally migrating from the inner layer to the outer surface of the brain, meaning that the deeper layers are formed first, and the migrating distance can be substantial. In most brain regions, the branched radial fibers arising from astroglial cells (Bergmann glia in the immature cerebellum) are guiding the immature migrating neurons to their proper position and directing the axons and dendrites into a precise network architecture (Hatten and Liem, 1981; Wang et al., 1994). 37

The cell migration process occurs at different time points for different cell types. Since the migration process can continue for several months after birth, the brain is especially sensitive to injuries during a long period. Moreover, the conditions necessary for cell migration in the brain are only present during this developmental period, so if migration is not accomplished on time, it remains incompleted. Groups of misplaced neurons often cause serious injuries to the brain. Once in the wrong position, the neuron has difficulties making connections to other cells leading to the impaired functional development. Damage to glial cells by toxicants (e.g. lead exposure) (Tiffany-Castiglioni et al., 1989) (Paper II) or disruption of the cell proliferation, like in the case of exposure to MAM (Yamanaka and Obata, 2004), ethanol (Miller, 1993) or methyl mercury (Choi et al., 1978; Choi, 1986; Rodier et al., 1984), are events liable to affect the migration process and might cause misplacement of the neuronal cells. The Boyden chamber is a commonly used assay for in vitro migration studies in cell lines, primary cultures and neuronal stem cells (de la Monte et al., 2009; Magge et al., 2009). The chamber consists of two medium filled compartments separated by a micro-porous membrane. The cells are placed in the upper compartment and they migrate in controlled conditions through the pores of the membrane into the lower compartment in which chemotactic agents are present. After an appropriate incubation time the cells are fixed and the number of cells that have migrated is determined. The chemical interruption of migration has also been studied using automatic time laps recordings of cells plated on distance marked coverslips (Sass et al., 2001), or by measuring radial migration away from adherent neurospheres (Moors et al., 2009). New technologies, such as specially designed nanopatterns, are currently also proposed as tools to study migration (Ruiz et al., 2008b; Ruiz et al., 2008a).

5.4 Neuronal differentiation The differentiation likely begins as soon as neuronal precursors complete their last division and are primed for movement to their terminal location (Eagleson et al., 1997; Geschwind and Galaburda, 1985; McConnell, 1990). During the migration the expression of particular genes is initiated, these will influence the neuronal differentiation, including outgrowths of axons and dendrites, expression of neurotransmitters and receptors, and maturation of electrical excitability. Many environmental substances have been reported to affect these processes and chemicals that alter proliferation or/and migration, such as ethanol (Miller, 1993), nicotine (Slotkin et al., 1993), methyl mercury (Rodier et al., 1984) and lead (TiffanyCastiglioni et al., 1989), also often result in disrupted cell differentiation.

5.4.1 Elaboration of neuronal networks One aspect of neuronal differentiation is the targeting of axons and dendrites to form a neuronal network that initiates synapses with other neurons, muscles and glands. The elongation and branching of the network is directed by different molecules, such as neurotransmitters, hormones and Ca2+, but also by substrate attachment molecules and electrical activity. Guidance of the neuronal arrangement by both substrates and diffusible molecules require expression of appropriate receptors (Spitzer, 2006). The elaboration of neurite outgrowth seems to depend on transcriptional programming regulating the expression of many intracellular signaling pathways (Goldberg, 2004). As the neurons mature, the neurite (axons and dendrites) processes will increase in length and complexity. Normally one of the neurites grows more rapidly in length and acquires axonal characteristics, while the remaining extensions elongate slower and becomes dendrites (Dotti et al., 1987; Dotti et al., 1988). The connections between cells are formed in far greater numbers than necessary during development and these are later slowly reduced to the required level. However, little is known about the molecular processes behind this phenomenon. The complexity of the network formation makes it a vulnerable process for chemical perturbation and any interference with e.g. gene expression, membrane receptors and ion channels or intracellular signaling, can affect the neurite elongation and growth (Audesirk and Audesirk, 1998). Morphological and biochemical endpoints to measure neuronal network elaboration are suited to automated high throughput screening techniques. Neurite outgrowth has been evaluated using microscopic assessment of the morphological changes ranging from simple measurements, such as the number of cells exhibiting neurites, to characterising the extensions by number of neurites, or the length and 38

branching of axons and dendrites. These techniques have been widely used in several different in vitro models, such as cell lines, primary cultures and stem cells (Radio and Mundy, 2008). Network elaboration can also be studied by evaluating biochemical markers, e.g. the neuronal cytoskeleton proteins, neurofilaments, microtubule-associated protein 2 (Map-2) and microtubules, at either protein or mRNA level (Kitaoka et al., 2009; Stummann et al., 2007; Vollner et al., 2009) (Paper I, II and III).

5.4.2 Synaptogenesis To achieve neuronal mature function, the neurons must form cell-cell connections through synapses. In rats this process occurs over the first three weeks postnatal (van Eden et al., 1990) and in humans through adolescence (Mrzljak et al., 1990; Uylings and van Eden, 1990). Neurons generally have thousands of synapses and retain their ability to form new synapses throughout life. However, the developmental period is critical for the formation of the basic network of the nervous system. The formation of synapses (synaptogenesis) includes biochemical and morphological changes in both pre- and postsynaptic elements, where the level of activity of a neuron seems to influence the development of receptors in the cells to which it projects (Spitzer, 2006). Moreover, the productions of neurotransmitters appear to be controlled by the stimulation the cell receives at particular stages of development. In the adult nervous system, neurotransmitters are mainly working as cell-cell signaling molecules in the synapses. However, during development they also affect further differentiation, e.g. expression of different neurotransmitters and receptors. Exposure to chemicals that modify neurotransmitter systems, like psycho-pharmaceuticals and pesticides, may therefore have serious effects on the developing nervous system. Other chemicals that are reported to modify synaptogenesis are nicotine (Shingo and Kito, 2005), ethanol (Sanderson et al., 2009), toluene (Lin et al., 2009), methyl mercury (Soares et al., 2003), parathion (Veronesi and Pope, 1990) and PCBs (Kodavanti, 2005). Synapse formation and maturation in different in vitro models can be evaluated by immunostaining of presynaptic markers (e.g. synaptophysin and synapsin) (Thiel, 1993) and postsynaptic markers (e.g. post synaptic density protein-95 (PSD-95) and 411B) (Buckby et al., 2004; Loessner et al., 1988). Moreover, neurotransmitter release can be studied by measuring the uptake and release of fluorescent dyes during presynaptic activity (e.g. FM1-43) (VijayRaghavan, 1995). In spontaneously electrically active in vitro models, electrophysiological techniques can be another option to evaluate the neuronal network development with functional synapses. Any disturbance of the synaptogenesis process induced by chemical exposure would most likely alter the electrical activity (Bal-Price et al., 2008) (Paper IV).

5.5 Glial function One of the important events during development of the CNS is the proliferation and maturation of glial cells (microglia, radial glia, astrocytes and oligodendrocytes). Radial glia and microglia development occurs in parallel with neurogenesis in most structures of the brain. Radial glia cells are considered stem cell-like and will later give rise to both neurons and astrocytes depending on signaling input (Choi and Lapham, 1978; Tamamaki et al., 2001). During brain development they provide a scaffold for radial migration of neuroblasts. Microglia are derived from brain sources (the mesoderm) outside the brain and act as a brain macrophages to clean up dead cells and cellular debris after apoptosis (Cuadros and Navascues, 1998). Astrocytes and oligodendrocytes mainly develop after the initial wave of neurogenesis and are often appearing later than neurons in a given structure (Rice and Barone, 2000). The development of the astrocytes are mainly determined by growth factors, neurotransmitters and cytokines (Bhat, 1995; Post and Brown, 1996) and numerous intracellular pathways are involved in the mitogenic effect on astrocytes. The development of oligodendrocytes is mainly influenced by molecules, such as platelet derived growth factors or insulin-like growth factors, where protein kinase C (PKC) is playing a relevant role (Bhat, 1995). The function of the astrocytes in the adult brain is to maintain ionic and trophic balance of the extracellular milieu. However, during CNS development astrocytes also provide guidance for axons and the assembly of synapses, and they give further cues for neuronal proliferation, migration and differentiation (Aschner et al., 1999). 39

Chemical interference with any astrocytic function might affect the neuronal development and function. Some chemicals, such as lead, tri-methyl tin and methyl mercury, have been shown to induce neurotoxicity through astrocytes. Furthermore, both astrocytes and microglia can become reactive to neuronal damage. It is well-known that once activated, these glial cells secrete a variety of proinflammatory and neurotoxic factors, such as cytokines and free radicals (Bal-Price and Brown, 2001), which can enhance the neuronal damage. In the adult system this is as a response clearly identified as a sensitive pathological indicator, while in the developing brain it might not always be initiated to the same degree. Proliferation and differentiation of glial cells can be measured by cell specific markers for astrocytes, oligodendrocytes and microglia on either protein (e.g. immunocytochemistry and western blot analysis) or mRNA level (e.g. PCR or microarray analysis) (Paper I, II and III). One of the functions of astrocytes, glutamate uptake and transformation into glutamine, can be measured by biochemical assays (Bal-Price et al., 2002; Ross et al., 2000), while microglia functions can be evaluated by time laps recordings of phagocytosis using phase contrast or fluorescent images (unpublished data). Furthermore, glial inflammatory responses can be measured by analysing cytokine secretion and the production of free radicals (BalPrice and Brown, 2001; Kinsner et al., 2006).

5.6 Myelination Coating of axons by glial tissue sheets (myelination) of oligodendrocytes (in the CNS) and Schwann cells (in the PNS) provide electrical insulation and make transmissions along the axon more rapid (Baumann and Pham-Dinh, 2001). The peak level of myelin synthesis takes place in the second week postnatal in rats and during the last trimester in humans (Rice and Barone, 2000). However, the myelin formation continues during development into adulthood making the nervous system sensitive to myelination disrupters for an exceptionally long period (Hunter et al., 1997; Wiggins, 1982). Oligodendrocyte precursors arise from multiple foci distributed along the neural tube. The progenitor cells migrate from the neural tube into what will become the white matter of the brain (Miller, 1996). The differentiation of oligodendrocytes follows the axogenesis and is believed to be mediated by complex trophic signals between the oligodendrocytes and neurons. During differentiation into mature oligodendrocytes the morphology changes to a large network of branching processes (Deng and Poretz, 2003). Since one oligodendrocyte is myelinating several axons, the location for the branching processes is important so that it can reach the target axons. It has been suggested that oligodendroglial processes send repellent signals between each other, and attractive signals between the process and the axon to fill unoccupied space and facilitate complete myelination (Simons and Trotter, 2007). The myelination process represents a complex series of metabolic and cell biological events involving intracellular recognition and interaction, adhesion, synthesis, sorting and assembly of specialised myelin membranes and axonal ion channel reorganisation. Failure of these events to complete within their developmental windows may have permanent deleterious effects. Since oligodendrocytes are operating near their metabolic capacity, they are especially sensitive to chemical disturbances and alternations in oligodendrocyte proliferation. A reduction of axonal development or myelin formation may lead to neuronal functional damage (Wiggins, 1986). The myelination process is especially vulnerable during the late gestational development in humans and postnatal in rats. Malnutrition and alterations of thyroid hormone homeostasis during this time span might affect the myelination process and cause persistent adverse outcomes (Rogister et al., 1999). Ethanol (Okamoto et al., 2006), tellurium (Bouldin et al., 1988) and lead (David and Subramaniam, 2005; Harry et al., 1985; Rothenberg et al., 1994; Tiffany-Castiglioni et al., 1986; Tiffany-Castiglioni, 1993) are chemicals proposed to have an effect on the myelination process. The myelin formation is most often studied using microscopic analysis (including electron microscopy) of the immunocytochemistry for specific proteins, such as myelin basic protein (MBP), protein zero, myelin associated glycoprotein (MAG) and peripheral myelin protein 22 (Felitsyn, 2008; Pfeiffer, 1993). The same markers can also be evaluated by other protein assays (e.g. enzyme-linked immunosorbent assay (ELISA) and western blot) or at mRNA level using e.g. PCR or microarray assays.

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5.7 Apoptosis In many structures of the CNS neurons proliferate in higher numbers than needed, why the increase of cells are followed by a wave of programmed cell death (apoptosis) to establish the correct final number of neurons (Raff, 1993; Oppenheim, 1991). This process is mediated by gene induction, with the synthesis of signal molecules, and is completed with the activation of caspases (Gorman et al., 1998). Several mammalian caspases have been identified and a number of them and their regulators have been shown to be vital for mammalian development (Cecconi et al., 1998; Madden and Cotter, 2008). Caspases are activated by cleavage, and after being activated they continue to cleave and activate other caspases. Finally they proceed to process key structural and nuclear proteins causing the disassembly and death of the cell. Two major caspase pathways have been described, the intrinsic pathway initiated by the release of cytochrome c from the mitochondria into the cytosol, and the extrinsic pathway initiated by binding to the plasma-membrane death receptors (Madden and Cotter, 2008; Shield and Mirkes, 1998; Wajant, 2002). Apoptosis occurs both prenatally in proliferative zones, and postnatally in neuronal and glial postmitotic cells. One factor believed to determine if a neuronal cell is going to apoptosis or not, is the number and kind of connections it has (Shield and Mirkes, 1998). The failure of the apoptosis process has been reported as a possible cause of autism in some individuals (Corbett et al., 2007; Sacco et al., 2007). Chemical exposure may shift the tightly regulated balance of neurotrophic signals regulating apoptosis and cause an increase or decrease in the cell numbers of a region of the nervous system. However, the effect of environmental substances on apoptosis in the developing brain has not been studied to a great extent. There are several existing techniques to measure apoptosis in vitro, such as caspase activity assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), DNA fragmentation, and annexin V- and mitochondrial-assays. These assays are suitable both for cell lines, primary cultures and neuronal stem cell. Caspase activity assays measure the proteolytic activity of pro-apoptotic caspase enzymes by the addition of a substrate to the cell lysate, that is then cleaved by activated caspases and gives a fluorescent, luminescent or calorimetric signal (Bal-Price and Brown, 2000). TUNEL staining and DNA fragmentation assays measure DNA fragmentation by the incorporation of analogue nucleotides which are either labeled or detected by antibodies (Gavrieli et al., 1992). Moreover, apoptotic cells can be identified by specific immunocytochemistry for the calcium binding protein annexin V (Koopman et al., 1994). During apoptosis annexin V is translocated from the inner surface of the cytoplasmic membrane to the outer surface, enabling to be detected by immunostaining. A promising tool for toxicity evaluation by rapid high throughput screening is the mitochondrial ADP/ATP ratio assay, which is sensitive and can detect several highly energy dependent processes, such as apoptosis, necrosis, growth arrest and cell proliferation (Bradbury et al., 2000).

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6. APPLICATION OF EMERGING TECHNOLOGIES WITH THE POTENTIAL TO ADVANCE DNT TESTING

The main problem in the development of a test strategy with predictive capacity for DNT is the complexity of the human nervous system. Since chemicals induce toxicity through various mechanisms, different models and endpoints have to to be applied. However, a global effort is needed to identify how the information currently gained from these different models and their respective endpoints can be used to build up an intelligent and reliable DNT testing strategy for regulatory purposes. During the last decade new emerging technologies have entered into the toxicology field (MacGregor, 2003; Pognan, 2004) that could speed up the testing and identification of critical DNT information, either by allowing evaluation of many chemicals (highthroughput screening), or by compiling large amounts of complex information from one test approach for each substance (highcontent screening). Robotised testing platforms could allow screening of a high number of chemicals under well standardised conditions using a simple model and endpoint, which would be useful especially for initial screening. However, DNT often is a result of the perturbance of various critical pathways and therefore one simple test method probably would not capture all of those critical aspects. In this case, a pragmatic and realistic approach would be to select an endpoint that could cover many different DNT processes. A useful tool here could be “omics” technologies, which includes genomics, transcriptomics, proteomics and metabolomics. The complete analysis of an organism’s response to a perturbation on the genome, transcriptome, proteome and metabolome level could lead to a better understanding of the mechanisms in complex systems (Dettmer et al., 2007). The various “omics” approaches differ in what and how things are measured, e.g. microarray chips, PCR, gel electrophoresis, mass spectroscopy and nuclear magnetic resonance (NMR). While genomics, transcriptomics and proteomics address the phenotypic adaption of a cell, metabolic profiling can give information on alterations in the cell physiology. The easiest and most commonly applied technique is an analysis on the transcriptome level (mRNA) using microarrays or PCR techniques. This approache provides a simple and sensitive tool to measure specific toxic responses to chemical perturbation, normally before any other changes will be observed (Paper I, II and III). Several DNT studies, both in vivo and in vitro, using gene expression as an endpoint have reported similar results as compared to effects observed in humans (Bachmann et al., 2009; Fan et al., 2009; Hong et al., 2009; Jergil et al., 2009; Royland and Kodavanti, 2008; Stummann et al., 2007; Stummann et al., 2009; Vahidnia et al., 2007) (Paper I, II and III). Moreover, evaluation of the expression of different specific genes can cover many of the key cellular events that could be affected in DNT, such as proliferation, neuronal differentiation, glial function, myelination and apoptosis. In this thesis several potential DNT chemicals, as well as chemicals with no observed neurotoxic effects, have been detected using gene expression (Table 1), qualifying it as a promising tool for DNT testing (Paper I, II and III). However, mRNA levels do not necessarily need to correlate with proteins levels and additional proteomics studies might be required to confirm results.

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Table. 1. The lowest concentration resulting in observed changes in mRNA levels for neuronal, astrocytic or precursor markers. Non-DNT chemicals were tested at concentrations up to 250 µM (paracetamol) and 500 µM (aspirin) (Paper I, II and III).

DNT chemicals

Neuronal markers

Astrocytic markers

Precursor markers

Methyl mercury Lead Tri-methyl tin Valproic acid Domoic acid Paraquat Parathion Dichlorvos Pentachlorophenol Cycloheximide

0.05 µM --2 µM 130 µM 5 µM 0.63 µM 25 µM 50 µM 50 µM 0.01 µM

--5 µM ----------75 µM 50 µM 0.05 µM

0.05 µM --2 µM 130 µM 40 µM 2.5 µM 50 µM --50 µM ---

Non-DNT chemicals Paracetamol Aspirin

Neuronal markers -----

Astrocytic markers -----

Precursor markers -----

Metabolomics is the systematic study of the unique biochemical fingerprints that cellular processes leave behind, and it provides data on changes in the cell physiology, e.g. after chemical treatment. This approach is a promising technique for getting data on toxicity (Craig et al., 2006; Griffin and Bollard, 2004; Lindon et al., 2003; van Vliet et al., 2008), disease processes (Griffin, 2003; Lindon et al., 2004), aging (Wang et al., 2007) and drug development (Lindon et al., 2007). So far the method is mainly based on samples from in vivo studies, including blood plasma, urine, saliva or cells. However, a recent study has applied metabolomics using an in vitro approach for neurotoxicity evaluation with promising results (van Vliet et al., 2008), which suggests that metabolomics could also be a useful tool for DNT testing. Nevertheless, direct extrapolation from in vitro metabolomics data to the in vivo situation should be carefully performed as cell cultures can differ in their metabolism and kinetics. The “omics” techniques might provide new biomarkers of specific pathways of toxicity and facilitate identification of the mechanisms of different chemicals. Another promising approach for neurotoxicity assessment is to measure the electrical activity using micro electrode arrays (MEAs). The first recordings using MEAs were performed in the 1980’s on neurons taken from the spinal cords of mice (Gross et al., 1982). Since then the field of electrophysiology using MEAs has advanced rapidly resulting in better and easier recording systems. The most commonly used in vitro systems for electrical recordings are hippocampus slices and primary dissociated cultures, normally from spinal cord or cortex. These cultures have shown responses similar to those in vivo when exposed to neurotransmitters and their specific agonists and antagonists (Gramowski et al., 2000; Keefer et al., 2001b; Keefer et al., 2001a; Martinoia et al., 2005; Streit, 1993). Moreover, primary cultures grown on MEAs have been used in several studies of pharmacological and toxicological responses (Gramowski et al., 2000; Gramowski et al., 2006; Gross et al., 1997; Keefer et al., 2001b; Morefield et al., 2000; Pancrazio et al., 2003; Parviz and Gross, 2007; Sundstrom et al., 2005; van Vliet et al., 2008). For these in vitro measurements, EC50 values have been established which are in the same range as those published in other toxicology studies and are generally in agreement with those obtained from animal experiments (Gross et al., 1997; Xia et al., 2003; Xia and Gross, 2003). The MEA technique provides a functional and neuronal specific endpoint that until now mainly has been used in basic research. However, this approach has also shown promising results for DNT studies (Paper IV). Indeed, many mechanisms causing DNT, such as disruption of cell proliferation, migration and differentiation, will eventually affect the electrical activity. Consequently, MEA measurements could cover many developmental endpoints and hence be a suitable tool for DNT testing. Furthermore, the suppliers of MEA chips have lately increased the amount of recording chambers per chip which could speed up the testing of large numbers of chemicals.

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7. INCORPORATION OF ALTERNATIVE APPROACHES INTO A DNT TESTING STRATEGY FOR REGULATORY PURPOSES

Today it would be too ambitious to pretend that an alternative DNT strategy could completely replace the in vivo test methods. However, by applying alternative approaches, such as in silico, in vitro and nonmammalian models, as a part of an integrated testing strategy, the process of DNT evaluation could be speeded up and the animal usage reduced and refined (Coecke et al., 2007; Lein et al., 2007). Both in vitro and non-mammalian test systems offer the possibility of providing early screening of a large number of chemicals, and could be useful in characterising the mechanisms of action or the developmental processes that are particularly affected by the test chemicals.

Tier 1

Are there sufficient data?

no

Generate data

yes

Tier 2

Is there a concern for DNT?

lower (possible no)

Data generated as resources/ new information available

high

Tier 3

Are data sufficient to make a regulatory decision?

yes

Regulatory Action STOP

no Further testing

Fig. 4. Scheme of the different steps in identifying DNT hazards (adapted and modified from Coecke et. al., 2007).

A test battery including robust in silico, in vitro and non-mammalian models, as well as, in vivo systems could be the most appropriate way of providing added value from the alternative approaches. Alternative methods could initially be used as a part of an intelligent testing strategy to flag chemicals with DNT potential, which subsequently could be prioritised for further testing using in vivo methods. A tiered test scheme could be used based on sequential assessments, where a result on one tier is used to determine the next step. It is usually a decision-tree type of testing; after each step, the information is assessed to determine whether a prediction of DNT risks can be made or whether further testing/analysis needs to be done (Fig. 4). The tiered approach uses existing compound knowledge to predict toxic effects and select further appropriate test methods. Tier 1 incorporates all existing data of a chemical, such as animal studies, in vitro studies, exposure information, epidemiology information, intended use, chemical structure and physiochemical data (Coecke et al., 2007). The information is evaluated to decide if the existing data is sufficient for making a decision about the DNT concern. If important information is missing, new in vivo data should be generated via the existing OECD test guidelines and complemented with in vitro testing. 44

When enough data is obtained during the tier 2 assessment, a decision is taken whether there is a DNT concern with high or low priority for further testing. High priority substances could, for example, be tested for pre- or/and post-natal exposure (e.g. detection of a chemical in breast milk) to obtain DNT relevant information. If available data supports a high concern for DNT, tier 3 testing should be carried out. If there is no evidence for a DNT potential, the chemical can be recognised as a low priority for further testing. Based on the available data and regulatory requirements, tier 3 could include specific tests including in silico, in vitro, non-mammalian or in vivo mammalian testing before a final decision of the DNT potential can be determined. Acrylamide Aldicarb Allethrin Aluminum Amino-nicotinamide(6-) Amphetamine(d-) Aspartame Azocytidine Benomyl Benzene Bioallethrin Bis(tri-n-butyltin)oxide Butylated hydroxy anisol Butylated hydroxytoluene Carbamazepine Carbon monoxide Chlordecone Chlordiazepoxide Chlorine dioxide Chlorpromazine Colcemid Colchicine Cytocine arabinoside DEET Diamorphine hydrochloride

Diazepam Diazinon Dieldrin Diethylene glycol diethyl ether Diethylstilbestrol Epidermal Growth Factor Ethylene thiourea Flourouracil(5-) Fluoride Haloperiodol Halothane Heptachlor Hexachlorobenzene Hydroxyurea Imminodiproprionitrile (IDPN) Lindane LSD Maneb Methadone Methanol Methimazole (methylimidazole) Methoxyethanol, 2Methylazoxymethanol Monosodium glutamate Naloxone

Naltrexone Nicotine Parathion PCBs Permethrin Phenylacetate Phenylalanine Phthalates Propylthiouracil Salicylate Tellurium Thalidomide Toluene Triamcinolone Tributyltin chloride Trichlorfon Trichloroethylene Triethyllead Triethyltin Trimethyltin Trypan blue Urethane Vincristine

Fig. 5. The priority list of selected chemicals to be considered for DNT evaluation (based on the recommendations from the DNT2 meeting, November 12-14, 2008, Reston, Virginia).

The implementation of alternative DNT tests, as a standalone or as part of an integrated test strategy, where a battery of mechanistic assays are incorporated, would accelerate the process of testing by delivering mechanistic data on chemically induced toxicity, particularly as many chemicals are missing efficient DNT data. However, before alternative assays can be incorporated into an intelligent testing strategy for human DNT evaluation, they will have to undergo a validation process to prove that they are neuronal and glial specific, robust and preferably amenable to highthroughput screening. Some recommendations when developing and evaluating alternative testing methods for DNT studies have been suggested (DNT2 meeting, November 12-14, 2008, Reston, Virginia). First the test method should incorporate endpoints that reflect the key cellular processes of human neurodevelopment. Furthermore, the endpoints should measure specific responses and be able to determine changes in both directions, e.g. increases and decreases. Selectivity of the endpoints should be evaluated by comparing different assays (e.g. cytotoxicity vs. neuronal/glial specific endpoints) and by using positive control chemicals that alter the endpoint by known mechanisms. A training set of chemicals with DNT concern was identified to be taken into consideration for in vitro and in vivo studies. These should be tested to evaluate the specificity and sensitivity of the developed test methods (Fig. 5). Sensitivity is defined as the proportion of active substances that are correctly identified, while specificity is defined as the proportion of inactive substances that are correctly identified. Consequently, also a number of non-neurotoxic chemicals (negative controls) need to be correctly evaluated by the test method. Since the alternative models and endpoints presented in this thesis have shown capacity for prediction of human DNT effects, they might be of great value and hopefully they will play an important role during the process of regulatory decision making in the near future.

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8. SUMMARY AND CONCLUSIONS

Since the existing DNT guidelines are costly, complex and time consuming they are not suitable for chemical highthroughput screening. Still, many chemicals should be tested for DNT to better protect children’s health. Because, at present, in vivo-based DNT testing cannot be replaced by in vitro approaches, incorporation of alternative testing as a part of an intelligent testing strategy could at least refine and speed up the process of evaluation (Coecke et al., 2007; Lein et al., 2007). For these purposes investments in alternative models and endpoints are greatly needed. In this thesis, two different in vitro models, cerebellar granule cells (CGCs) and cortical primary neuronal cultures, have been characterised to evaluate their suitability for DNT testing. Furthermore, two emerging technologies (gene expression and electrical activity) have been studied and identified as promising tools for in vitro DNT assessment. Paper I characterises the rat primary CGCs during development in culture and identifies several important cellular key processes such as proliferation, differentiation and maturation present in this in vitro model. Moreover, the study concludes that gene expression is a sensitive endpoint to evaluate DNT effects after exposure to diverse pesticides with different toxic mechanisms. In addition, the measurements of mRNA levels of the selected markers allowed us to assess which cell type (neuronal or glial) and which stage of neuronal or glial development (proliferation, differentiation or maturation) was affected by the exposure to pesticides. In paper II the same model (CGCs) is exposed to known (developmental) neurotoxic chemicals and the effects are evaluated using the same tool as in paper I (mRNA expression) to further assess the robustness and relevance of the applied approach. Interestingly, the chemically induced toxicity, as identified by mRNA expression, is observed at a much lower concentration than found in human plasma, suggesting that gene expression could be used as a (more) sensitive DNT endpoint. Furthermore, the paper shows the importance of working with mixed neuronal-glial cultures in DNT studies as the toxicity of some chemicals can only be detected by one specific cell type. Paper III investigates the shellfish toxin domoic acid (a potential DNT compound) and compares the toxic effects in mature and immature CGC cultures using gene expression as a tool to evaluate toxicity. The mature cultures are more vulnerable to domoic acid exposure than the immature, as the effects in the mature cultures were observed at a lower concentration and at an earlier time point. Moreover, the neurotoxic effects seem to be mediated mainly by the AMPA/KA receptor, as the competitive antagonist NBQX completely inhibits the observed toxic effects detected at the mRNA level. Interestingly, the domoic acid toxicity, as measured by the mRNA level of the astrocytic markers (GFAP and S100β), could also be prevented by the NMDA receptor antagonist APV indicating different mechanisms of domoic acid induced toxicity in astrocytes and neurons. Additionally, the inhibitory neurotransmitter GABA could prevent the toxicity in the mature cultures but not in the immature, suggesting that the expression of both glutamate and GABA receptors could be of critical importance for the domoic acid induced mechanisms of toxicity. Paper IV presents electrical activity measurement as a completely new endpoint to detect DNT effects. Here, to mimic environmental exposure, we investigated long term treatment with low concentrations of domoic acid. We measured how it influenced the spontaneous neuronal activity, as well as, the response of the neurotransmitter GABA and the GABAA receptor antagonist (bicuculline). Indeed, long term treatment at low concentrations, which did not induce any acute toxicity in mature cultures, significantly increased the basal spontaneous activity. Such effects could have serious consequences for overall neuronal brain activity leading to various pathologies. Moreover, all the cultures (non-treated and treated with domoic acid) responded in the same way to the neurotransmitter GABA (decreased activity). The effect of bicuculline on domoic acid treated cultures differed from the control as bicuculline in the control experiments increased the activity above the spontaneous one, while the activity of the domoic acid treated cultures remained at the same level as the basal spontaneous one.

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In conclusion, CGCs and cortical neuronal cultures are relevant models for DNT testing as key processes during brain development are present. Furthermore, gene expression is a promising endpoint that can detect chemicals with DNT potential at low concentrations induced by various mechanisms of toxicity. The measurement of electrical activity using MEAs is a promising, neuronal specific tool for DNT assessment. However, more chemicals with both neurotoxic and non-neurotoxic effects need to be tested before this endpoint can be routinely applied. These endpoints (gene expression and electrical activity measurement) applied to various in vitro models in combination with other assays could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by prioritising chemicals with DNT potential for further testing.

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9. ACKNOWLEDGEMENTS

First of all I would like to express my gratitude to ECVAM (Institute for Health and Consumer Protection at the European Commission’s Joint Research Center) in Ispra where the experiments for this thesis were performed. I would like to start with thanking my supervisor at ECVAM, Italy Dr. Anna Price for all her enthusiasm and support, especially during our late evening writing sessions. It has been a pleasure to work with you and I have learned a lot. It has been an experience I will always appreciate. I am very grateful to Dr. Sandra Coecke and Professor Thomas Hartung who welcomed me in the neurotoxicity group of ECVAM and always have given me support and advices. A special thanks to Professor Barbara Cannon, my supervisor at Stockholm University, Sweden for your guidance and support. I would also like to thank Professors Jan Nedergaard and Anders Jacobsson, and Docent Tore Bengtsson for interesting discussions. Also thanks to the rest of the people at the Department of Physiology at Stockholm University. I would like to thank all my colleagues who have helped me with either the experimental work or the writing, especially the neurotoxicity group: Dr. Agnieszka Kinsner, Dr. Lena Buzanska and Dr. Erwin van Vliet. I would also like to thank Roberta Brustio, Gerry Bowe, Dr. Lars Hareng, Dr. Nina Hasiwa, Dr. Sebastian Hoffmann, Dr. Daniele Ferrario, Efrat Forti, Dr. Siggi Morath, Dr. Antonio Novellino, Dr. Cristian Pellizzer, Dr. Tomek Sobanski, Dr. Tina Stummann, Claire Thomas and Dr. Maurice Whelan for their help in the lab, advice, writing and statistical analysis. Also thanks to the rest of my colleagues at ECVAM. I am very grateful to Dr. Susan Salovaara and David Horby for the reading and corrections of my thesis. I am very impressed by your eagerness and enthusiasm for reading my thesis. Thank you, it really made a difference. I would like to thank the first people that believed in me as a student and scientist: Bertil Hjälmgren, Docent Tore Bengtsson, Dr. Peteris Alberts and Professor Erik Walum. Without your encouragement I would never have taken the step to do a PhD. On the private side I have many people to thank. First I would like to thank the ones in Sweden. I would like to start with my closest friends “värsta brudarna”, Petra, Malin, Tessan och Sara. Thanks for always supporting me and making me feel special. Without you life would be empty. Hedda and Reneé you are great friends, always so positive and full of joy, which I really need sometimes. Lotta, thanks for being my link to the University and for helping me out with everything. But most of all thank you for being a fantastic friend. It was a great pleasure having you there during my visits to the University.

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Thanks to Olle, Natacha and Robert for always making me feel welcome at the department. Benita, thanks for all the fun singing and performances, we should continue in US. Monika and Lena thanks for spreading so much fun for everybody around you, and especially for the support to my wonderful mother. To all my dear friends in Italy. Efrat and Susan my wonderful friends, you have helped me through so many difficult times. I could not have done this without you and I am so happy to have you in my life. Nina, Corvin, Tex, Lola, Riita and Touko, thanks for being like a family to me here in Italy. I truly enjoy your company. I am grateful to you that have been supporting me, especially this last hard period: Nina H, Riita, Efrat, Susan, Aga, Niina K, Chantra, Nina R, Jenny, Iris, Jacob, Björn, Fransisco, Gustav S, Floriana, Anna and Sandra. I am so lucky having you as friends. My dear karaoke friends Nina, Riita, Taina, Aga, Susan, Coleen, Petteri, Juha, Dave, Marko and Björn thanks for giving me the pleasure of spending so many nights and mornings with ABBA and Queen. I will miss it very much. My wonderful floorball team and the coaches Gustav and Jacob, it was so much fun to play with you. Also thanks to the Sunday floorball people, it was a great way to end the week. Thank you Fesil for all the nice football evenings with good company and hot curries. Jean-Christophe without you my thesis would not look this brilliant. Thank you for making me happy and changing my life. Finally my fantastic complex family. Thanks to my stepmother Eva-Maria and my extra brother Marcus and sister Emelie, you will always have a place in my heart. My sweet grandparents, I admire you a lot. You are simply the best. My uncles, aunts, cousins, parents’ cousins and all the ones close to you. It is great to have such a big family. Family van Vliet, thanks for letting me be a part of your family and for all the encouragement you have given me. My dearest little brother, your belief in me made me start to study at the University. You and Joy are more important to me than you can ever imagine. My extra parents Anita and Kjelle, thanks for coming into our family. You truly belong with us. Most of all I want to thank my father Bengt and my mother Kicki. Thanks for your endless support and love. I love you so much. Finally, whilst working on this thesis my thoughts have been on the well being of our future generations, especially the lights of my life, Noah and Hampus, two of the children I want us to be able to protect in the future. Perhaps this thesis can help us find the way. Thank you all. 49

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