In vitro tests of neurotoxicity

JPM Vol. 29, No. 2 April 1993:69-75

In Vitro Tests of Neurotoxicity E l i z a b e t h M c F a r l a n e A b d u l l a a n d l a i n C. C a m p b e l l

In-vitro Neurotoxicology (E.M.A.), Wellcome, Beckenham; and Department of Neuroscience (1.C.C.), Institute of Psychiatry, London, England, U.K.

This review presents some of the newer techniques in the rapidly advancing area of neurotoxicity testing in vitro. They are not described at length, and more details can be obtained from the cited references. In vitro testing offers the possibility of relatively inexpensive screening of large numbers of pharmaceutical compounds, formulations, and environmental substances. The level of sophistication attained in this field may soon allow much more accurate safety limits to be set, as specific mechanisms of neurotoxicity are elucidated.

Keywords: In vitro neurotoxicity testing; Neurite outgrowth; Tiered tests

Introduction Neurotoxicity testing in vitro must assess the ability of chemicals to 1) cause disruption to the metabolism and function of individual cells and 2) alter their ability to interact within the nervous system. An understanding of the normal functions of the components of the central nervous system (CNS) is a prerequisite for meaningful in vitro tests of neurotoxicity. For example, there are many different and manipulable stages in the processes that comprise neuronal differentiation and function; for example, neurite outgrowth, formation of neuronal network systems and the development of neuronal excitability. The complexity of each of these processes makes them very relevant as model systems for assessing and elucidating modes of neurotoxicity. The larger the number of biochemically defined stages encompassed in the models, the more likely they are to provide tests that will detect the neurotoxic potential of a wide spectrum of different compounds. In addition, they will provide more information on the specific mechanism of action of these substances. The review begins with a discussion of the uniqueness of neurons and the need for specific tests of

A d d r e s s reprint requests to Dr. I. C. Campbell, D e p a r t m e n t of Neuroscience, De Crespigny Park, D e n m a r k Hill, L o n d o n SE5 8AF, U.K. Received August 1992; revised and accepted D e c e m b e r 1992. Journal of Pharmacological and Toxicological Methods 29, 69-75 (1993) © 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

neurotoxicity. This is followed by a section describing sequential or tiered testing; that is, primary screening on cell lines or primary cells for general cellular effects, followed by secondary screens on a more limited series o f compounds for morphological, mechanistic, and/or neurochemical effects on subsets of primary neuronal cells, and/or on complex or organ cultures.

The Uniqueness of Neurons Intact neurons have an extremely large surface to volume area because o f the dendrites and axons and are very responsive physiologically to electrical stimulation, that is, they are excitable. In addition, the neuron is one o f the most metabolically active ceils in the body (Walum, 1990), with complex and highly organized anterograde and retrograde systems for the fast and slow transport of cellular proteins and transmitters. Any toxic insult to such a highly active cell with such a large area of membrane, which is rendered more vulnerable by the diversity of its functional parts (e.g., cell soma, dendrite, synapse), which does not divide, has the potential to be very serious. The neuron is unlike any other cell in the body in the sense that it has an absolute requirement for glucose and cannot metabolize lipids. In addition, neurons share the distinction with the lens of the eye of not requiring insulin for transport of glucose into the cell. Plasticity is another very important specialized

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property of neurons whereby a change in stimulation (in the pattern of signals, transmitters, pressure damage, toxic damage) will lead to permanent functional change in the neuron. An example of this is long-term potentiation (LTP) of synaptic transmission, in which a short burst of intense, electrical stimulation leads to a permanent change in the neurons, including an increase in the strength of synapses, which has long been thought to be involved in memory. New evidence shows that mutant mice lacking the calcium-calmodulin-dependent multifunctional protein kinase II (otCaMKII) have a deficit in spatial memory; hippocampal slices from these mice have greatly impaired LTP (Silva et al., 1992a, b). Other manifestations of the permanent effects of different or toxic stimulation could include neurite outgrowth (stimulation and inhibition), selective expression of a particular group, subgroup, or type of receptor, and activation of genes of different stages of development (e.g., new specialized transmitters). Finally, although neurons do not divide, many undergo apoptosis in the normal course of development: examination of this process may provide an experimental system that could be used to develop an in vitro model of neurotoxicity.

Tiered Tests The general principle of sequential or tiered testing (Table 1) will be discussed first with reference to t h e

Table 1. Tiered or Sequential Testing Primary screens Cell lines, neuroblastomas/gliomas, primary monolayer culture (e.g., chick DRG), rat or chick midbrain micromass culture cytotoxicity Membrane integrity (e.g., neutral red/LDH), mitochondrial enzyme function (e.g., MTT) differentiation effects Microglia to macrophage (CR3 expression), neurite outgrowth (e.g., PC12, chick DRG), protein/gene expression (e.g., c-fos/ c-jun), acute/chronic exposure Secondary screens (limited number of compounds) Reaggregate culture, e.g., Chick Embryo Retinal Culture CREC Tests 1) aggregation (cell-cell interactions, gap junction formation); 2) growth; 3) differentiation (e.g., glutamine synthetase) Mechanistic studies on single compounds Organotypic culture/explant culture Neurochemistry; (many culture systems and synaptosomes), neurotransmitter synthesis (e.g., acetylcholine),; receptor expression (e.g., GABA receptors), Primary cultures of individual neural cell types Electrophysiology on ex-vivo insect neurones Note: Any prior knowledge of a compound can be used to bypass any of these stages.

techniques used; the individual techniques and their particular advantages will be explained in the following sections. Neurotoxicity testing of novel chemicals, for which there is no in vivo data, should start with a primary screen for general cytotoxic and metabolic effects [membrane damage, mitochondrial respiratory function, effiux of preloaded (3H)-2-deoxyglucose (Wallin and Walum, 1992)] and differentiation effects [microglia to macrophages, with concomitant expression of complement CR3 receptor (Grundt and Nyland, 1992)] using neuroblastoma or glioma cell lines or primary neurons such as chick dorsal route ganglion cells. Secondary screens follow and are performed using complex cultures of for example, primary embryonic chick midbrain or primary embryonic rat midbrain, which are more representative of the in vivo population of cells (glial co-culture/regaggregate/micromass/explant culture) than are cell lines or single cell cultures. Measures of cytotoxicity are used such as morphology, neurite outgrowth, ELISAs of neurofilament protein levels (Abdulla and Campbell, 1993a), or B2 level in retinal cells (Ohta et al., 1992). In both primary and secondary screens, both acute and chronic exposure can be studied. Mechanistic investigations can be done on either a single substance or related series of compounds. A more detailed examination of neurochemistry, neurotransmitter synthesis (e.g., acetylcholine) expression of receptors (e.g., GABA receptors), and marker enzymes (e.g., tyrosine hydroxylase or neuronspecific enolase) can be highly informative about specific neurotoxicant effects. Specific neurotoxicological effects can be studied by using either primary neuronal preparations (e.g., insect neurons for electrophysiological studies), cultures of neurons from different brain regions, or synaptosomal preparations. This temporal progression of testing can be short-circuited by either prior in vitro or in vivo data or as a result of structural comparisons to compounds for which the neurotoxicity profile is known. In general, for screening chemicals, the aim is to have reliable tests that are amenable to automation. Assays that can be performed in a 96-well plate are particularly valuable. Test systems that are too laborintensive are likely to be prone to variation and error. Although it is important to attempt to mimic the in vivo situation in vitro and be aware of differences (e.g., hydrophobic chemicals will cross the blood-brain barrier more readily than would hydrophilic chemicals), complexity in an assay must be reproducible to be useful. These criteria may not apply when single compounds are being investigated.

Neurite Outgrowth Neurite outgrowth is uniquely valuable as an in vitro model of neurotoxicity because it is important to use a

E. McFARLANE ABDULLA AND I. C. CAMPBELL IN VITRO TESTS OF NEUROTOXICITY

physiological process which is a general indicator of cellular well being, that is, one that will be affected by the widest possible spectrum of noxious agents. However, because it is a multistage process, it also provides the opportunity to examine the specific effects of different groups of toxic substances and thus may provide a new classification system. Neurite outgrowth can be seen and measured using antibodies to proteins which are specifically increased as it develops (see Abdulla and Campbell, 1993b). The three most important physiological stimulants of neurite outgrowth are nerve growth factor (NGF), extracellular matrix (ECM) proteins, and cell adhesion molecules (CAMs), N-cadherin and N-CAM. In neonatal sensory neurons, NGF causes neurite extension, but in adult neurons it causes increased arborization (branching of neurites) (Yasuda et al., 1990). Neurite outgrowth then can be regarded as a pivotal phenomenon in the health of both developing and mature neurons. ECM molecules, in particular, laminin, (Bozyckzo and Horwitz, 1986), stimulate neurite outgrowth by a protein kinase C- (PKC) dependent pathway. In contrast, N-CAM and N-cadherin, stimulate neurite outgrowth by a G-protein pathway, not initially via PKC. Recently, it has been established that neurotoxic effects are diminished in the presence of certain growth factors (Mattson et al., 1992). This is, potentially, a very important area of research in developmental toxicity, as is the role of signal transduction mechanisms and growth factors in neurite outgrowth and toxicity. The [MA protein, the major component of the amyloid deposition characterizing Alzheimer's disease, has been shown to derive from the amyloid precursor protein (APP). Both soluble and membrane-associated APP (10 -l° M), significantly enhance nerve growth factor-induced neurite length and branching (but not number) in PC 12 cells. APP is half maximally cytotoxic at 5 x 10 - 9 M (Milward et al., 1992). This indicates the importance of neurite outgrowth as a predictive indicator of neurodegenerative changes. N-cadherin causes more rapid, more highly branching neurite outgrowth than laminin, which induces longer, less branched neurite outgrowth, in chick peripheral ganglion cells in culture. This raises the possibility that N-cadherin induces neurite outgrowth like that seen in neonatal sensory neurons stimulated by NGF, and laminin induces neurite outgrowth like that seen in adult neurons stimulated by NGF. N-cadherin, but not N-CAM-induced neurite outgrowth, involves extracellular calcium. Both N-CAM and N-cadherininduced neurite outgrowth involve changes in intracellular-free calcium (and distribution within subcellular compartments). This can be examined using fura 2 staining of intracellular-free calcium. Neurite outgrowth involves the induction of several

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cytoskeletal proteins including neurofilaments, tubulin, actin, MAP 1A, MAP 1B (also known as MAP lx or MAP 1.2 or MAP 5), tau, and chartin MAPs (Wiche et al., 1991), and also integrin alpha-I in PC12 cells (Rossino et al., 1990) and the CAMs, L1 and NCAM (Prentice et al., 1987). Rapid short-term events in neurite outgrowth are the stimulation of the immediate early genes, for example, c-fos, c-jun, NGF1-A, and NGF1-B, which induce transcription factors and proteins (Walicke, 1989). ELISAs can be used to quantify, for example, neurofilament protein levels and other neurite-specific proteins and genes in the presence and absence of drugs. This assay system is particularly valuable as rapid screening of compounds at various dose levels can be performed in 96-well plates. Figure 1 shows neurite outgrowth in PC12 cells grown on polyL-lysine/laminin-coated culture dishes. Parallel computerized image analysis of such preparations allows the direct measurement of neurite outgrowth and for correlations to be made between quantitated changes in proteins/genes and neurite outgrowth. There are, however, a number of problems associated with direct measurement of neurite outgrowth that have led to the quest for an indirect method. Neurite outgrowth is quantified as neurite length in terms of neuronal soma diameter (e.g., 2 × soma) and number of branches. This process is labor-intensive, subject to error, and not amenable to use in simple screening. Another problem is that denser cultures of neuroblastomas produce more neurite outgrowth, however, sparse cultures, which are better for direct measurement, need stimulation with conditioned medium from dense cultures to produce comparable neurite outgrowth.

Glial Co-Culture Glial cells constitute a large percentage of the mammalian CNS and express GABA and glutamate/aspartate receptors and uptake carriers and so on, on their surface. These are linked to second messenger systems like those in neurons, which render them equally vulnerable to toxic effects. Co-culture of neurons and glial cells provides an in vitro model that allows for the sequestration of compounds by glial cells that will alter the direct effect of drugs on neuronal cells. Glial cells have processes that extend to the synapses and are very likely to play a large role in vivo, in signal modulation, by uptake of neurotransmitters. Toxic effects that operate on synaptic transmission will most probably be affected by glial cells.

Complex Culture Systems It is important to examine neural cell differentiation in culture where the mixture of cells being studied is similar to the in vivo situation. These complex systems are potential candidates for robust in vitro models for

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Figure 1. Photograph and annotated diagram of neurite outgrowth. PCI2 rat pheochromocytoma cell line grown with 50 ng/ml NGF on poly-L-lysine/laminincoated plates to facilitate neurite outgrowth.

neurite varicosity

growth

]

"5

neurodevelopmental toxicity assessment. In these systems 1) many cell types of the tissue of origin are present, 2) many developmental processes can be followed, and 3) complex three-dimensional interactions between cells occur. Questions of regional specificity, receptor expression, and various neuronal and glial functions can be addressed: these may play key roles for normal and abnormal development in vivo.

Reaggregate Culture Reaggregate cultures, under constant gyratory motion, have been used extensively in neurotoxicological and teratological studies (Atterwill, 1989). Primary dis-

sociated cells dispersed from CNS cubes or slices, when rotated in suspension, reassociate into small clumps consisting of cells that represent the balance of cell types present in the CNS and achieve a degree of differentiation better than monolayer culture. Acetylcholine, norepinephrine, GABA, and dopamine transmitter systems were found to develop up to a stable plateau, during the first 3 weeks in vitro. The expression of key enzymes, such as tyrosine hydroxylase and neuron-specific enolase, as well as various receptors including ~, A, and K opioid receptors (MonnetTschudi and Honnegger, 1989), underline the differentiation potential of brain aggregates.

E. McFARLANE ABDULLA AND I. C. CAMPBELL IN VITRO TESTS OF NEUROTOXICITY

The Chick Embryo Neuronal Retinal Cell Assay (CERC) (Daston et al., 1991) is a very promising area of developmental neurotoxicity. The neural retinas of incubation day 6.5 of white leghorn chick embryos are dissociated into single cells which are subsequently maintained in a rotating suspension culture. Neural retinal cells form spheroidal aggregates of a consistent size over the first 24 hr in culture (dependent on competent cell-cell interactions). Over the remaining 7 days in culture, the cells continue to divide and differentiate. Each of these developmentally important events, that is, aggregation, growth, and differentiation, is objectively and quantitatively measured as aggregate size and number, aggregate protein content, and glutamine synthetase (a marker of differentiation). The correlation between in vivo data and this assay has so far been high. Test agents differentially affected the different end points in this assay according to their mechanism of action. Chemicals that interfere with intercellular communication, e.g., all trans retinoic acid and TPA, affect aggregation. CdClz and HgC12 diminish growth and chemicals that affect gene expression, inhibit differentiation, for example, bromodeoxyuridine and acitinomycin D.

Micromass Culture Micromass culture uses very high-density primary embryonic rat or chick (Brown and Wiger, 1992) midbrain, grown as small islets (which can be used with limb cells from the same embryo). This system can be used to study whether neural cell functions develop in the presence and absence of toxic substances; among the parameters that can be measured are GABA uptake, ganglioside differentiation, or acetylcholine esterase expression (to be found also in noncholinergic neurons) (G~ihwiler, 1988). Micromass culture differs from reaggregate culture as it adheres to the substratum. For this reason, neurite outgrowth can be also studied in micromass culture. The observation of development of neuronal networks is also a promising area of endeavour; it exploits micromass culture and may prove extremely pertinent to neurotoxicity testing. "~

Hippocampai Slices and Explant Culture Long-term cultures of slices of hippocampal origin derived from infant rat can be examined as a model for neurodevelopmental studies of postgestation stages (G~hwiler, 1988). Their strength lies in the ease of observation and physiological measurements of single cells within the organotypic environment. Explant culture (e.g., fetal rat cerebellar) is another organotypic system that preserves intercellular relationships and

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allows processes, such as myelination, to occur. They are formed when small, thin fragments of neural tissue, and the outgrowth of these cells survive in culture medium. It has been used to examine the toxicity of heavy metals, such as methyl mercury and tin, and also methotrexate (Gilbert et al., 1989), where the demyelination was found to be due to a primary neurotoxic effect.

Electrophysiological Studies on ex vivo and in vitro Insect Neurons Ultimately, it is important to assess the effects of toxins on excitability of neuronal cells. This can be done using electrophysiological techniques with various cultured neuronal preparations. It is thus possible to measure resting and action potentials as well as"single-channel" activity using the "patch-clamp" technique. This method of investigation is extremely sensitive and has achieved a high degree of sophistication due to recent developments in electronics and microcomputers. For example, it is an excellent technique for examining the mode of action of various toxicants on cockroach neurons in culture (Pichon, 1990) and could be extended to other neuronal preparations. Pyrethroid insecticides have been shown (using cockroach neurons in culture) to open sodium channels which differ substantially from normal sodium channels.

Cell Lines Versus Primary Culture There are two basic options for growing neurons in culture; in the first one primary neurons are used. However, these will not divide and will only survive for a limited time. In addition, it is difficult to obtain homogeneous, well-defined, and reproducible cultures. The advantage of primary neurons (particularly fetal and neonatal neurons) is that they extend neurites rapidly with minimal requirement for extracellular matrix protein, neural cell adhesion molecules (N-CAM, N-cadherin), or nerve growth factor. The alternative approach is the use of neuroblastomas, but these are transformed cells and are likely to differ substantially from normal neurons. Cell lines are convenient, and cultures are reproducible and there is a reduction in the use of animals. They are also amenable to longerterm culture for chronicity studies. Cell lines, unlike mature primary neurons, proliferate in culture in the presence of serum. Manipulation with lower concentrations of serum or the addition of nerve growth factor or growth on plastic precoated with either poly-L-lysine and/or extracellular matrix proteins (e.g., laminin) or neuronal cell adhesion molecules encourages them to express differentiated characteristics.

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NGF and fibroblast growth factor (FGF) and other neurotrophic signals in vivo may increase or decrease neuronal susceptibility to toxic insult. This may have important implications in vitro. The relative anoxia that exists in culture also leads to a loss of the differentiated phenotype, although this problem can be overcome by "roller culture techniques" whereby cells are exposed to the air nine times/minute (Daniel and Fish, 1974). Increased oxygen tension can be also achieved using "millipore filter culture," which allows medium to reach both sides of a monolayer. Co-culture of neuronal cells with differentiated astroglial cells or co-culture in mixed cell brain cultures (reaggregate, micromass, and explant) all encourage the expression of the features of the neuron in situ. These features can include adhesion to the substratum, neurite outgrowth, expression of nerve growth factor receptors and integrins, cessation of proliferation and down-regulation of c-myc. The life history of a normal neuron in vivo can be characterized by a temporal progression of transitions that may be conveniently viewed as a discrete series of neurogenic steps through which virtually all cells must pass. These steps include induction, proliferation, migration, restriction and determination, differentiation (expression), the formation of axonal pathways and synaptic connections, and the onset of physiological function. In many parts of the peripheral and central nervous system, roughly one-half of the neurons can be further characterized by their expression of an additional terminal step in the neurogenic process: the occurrence of a cascade of cellular and molecular events leading to regression and ultimate degeneration and cell death.

Diversity of Neurotoxic Agents The most obvious way to study neurotoxicity in vitro is to begin by examining the effects of known neurotoxic agents. It is clear from even a cursory glance at such substances that they are a chemically diverse group. This allows a multipronged approach which may produce a number of discrete common pathways leading to cell damage or cell death, although it is certainly naive to expect only one to emerge. Information on the postulated mode of action of some of these well-known substances is described below. In vitro tests of neurotoxicity that are simple to assay, yet physiologically complex and, therefore, composed of a series of discrete, biochemically definable steps (such as neurite outgrowth) are needed to test for the neurotoxic potential of such a wide range of substances.

tern: some of them are described below. Excitotoxic compounds, such as kainic acid and beta-N-amino-Lalanine (L-BMAA) (from the cycad seed in the island of Guam) cause neuronal damage. L-BMAA is the exogenous non-protein amino acid that causes Guam disease or amyotrophic lateral sclerosis/parkinsonian/dementia (ALS-PD complex) (Spencer et al., 1987). Kainate causes damage by receptor-mediated increases in intracellular-free calcium (Seubert et ai., 1988), and this results in cellular toxicity possibly by a hyperosmotic effect and possibly by a calcium-mediated activation of catabolic enzymes (phospholipases, proteases, endonucleases). Aluminum toxicity has been implicated in Alzheimer's disease because there is 1) increased aluminum in the brain correlated with aging, 2) chemical evidence of aluminum silicate constituents of senile plaques in Alzheimer's diseased brains, and 3) electron probe evidence of aluminum concentrated in neurofibrillary tangles. The pathogenesis has been linked to aluminum utensils and antacids. Against the significance of these findings is the lack of Alzheimer's disease in patients suffering from aluminum intoxication or in aluminum factory workers. Alzheimer's lesions are also seen in Down's syndrome patients. The aluminum seen in the lesions associated with [3-amyloid may be a secondary phenomenon (Glenner, 1989). 1 - Methyl - 4 - phenyl - 1,2, 3, 6 - tetrahydropyridine (MPTP) causes toxicity by generating the l-methyl-4phenylpyridinium ion (MPP +) used in animal models of Parkinson's disease: it is postulated to act by a free radical-mediated mechanism to damage the mitochondrial complex 1 by inhibition of NAD+-Iinked substrates at the same site as rotenone (Ramsay et al., 1986; Schapira, 1990). Organophosphorus esters (used as pesticides) induce direct or indirect inhibition of acetylcholine esterase and, hence, cause excessive activity in cholinergic neurons. In addition, they stimulate calcium-calmodulin kinase 1I (CaM kinase II), an enzyme that phosphorylates various cytoskeletal proteins such as microtubules, neurofilaments, and MAP 2, causing disassembly of these elements and damage to the axon (AbouDonia and Lapadula, 1990). The toxicity of acrylamide and 2,5-hexanedione (the oxidation product of n-hexane) is thought to be due to 1) damage of neurofilaments (caused in the case of 2,5hexanedione by formation of a pyrole adduct with the protein lysyl C-amino group on the neurofilaments) resulting in their accumulation and 2) subsequent axonal swelling (Di Patre and Butcher, 1991).

Disease Models and Mechanisms of Toxicity

In vivo Versus in vitro Neurotoxicity

There are a variety of distinct types of neurotoxic compounds with discrete effects on the nervous sys-

Neurons in culture are different from neurons in vivo for a number of important reasons which must be

E. McFARLANE ABDULLA AND I. C. CAMPBELL IN VITRO TESTS OF NEUROTOXICITY

borne in mind when interpreting data. Primary neurons in culture are metabolically, physiologically, and morphologically stunted because they are not subject to normal excitatory and inhibitory inputs. The susceptibility to toxic insult of these relatively "resting" neurons may then be altered. Artificial "electrical" stimulation could perhaps be partially achieved by depolarization using - 1 5 mM K ÷ (slightly above the normal 5 mM), but this can never really mimic the dynamic electrical state in vivo. This has to be taken into account in experimental models especially as the susceptibility to metabolic poisons may appear to be less. Extrapolations from in vitro results should be done with the knowledge that the selective effects of the blood-brain barrier are absent. Waste disposal of xenobiotics and their metabolites will also differ substantially from the in vivo situation. Limited activation of compounds to more toxic metabolites in vitro may also give a false-negative result. Many in vitro assays incorporate oxidative enzyme conversion systems. Co-culture and complex culture systems will have a better metabolic conversion capacity than some single-cell culture systems. Each in vitro model system has drawbacks and it is thus important to have a battery of tests that can predict neurotoxic potential. The development of increasingly reliable and predictive in vitro neurotoxicity models will reduce the requirement for so much animal testing in both neurotoxicity and teratogenicity.

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