TOXICOLOGICAL SCIENCES 113(1), 95–115 (2010)

doi:10.1093/toxsci/kfp175

Advance Access publication August 3, 2009

mRNA Expression is a Relevant Tool to Identify DevelopmentalNeurotoxicants Using an In Vitro Approach

Helena T. Hogberg,*,† Agnieszka Kinsner-Ovaskainen,* Sandra Coecke,* Thomas Hartung,‡ and Anna K. Bal-Price*,1

*In-Vitro Methods Unit, European Centre for the Validation of Alternative Methods, Institute for Health and Consumer Protection, European Commission Joint

Research Centre, 21026 Ispra (VA), Italy; †Department of Physiology, The Wenner-Gren Institute, Stockholm University, Sweden; and ‡The Johns Hopkins

University, School of Public Health, Center for Alternatives to Animal Testing, Baltimore, Maryland 21205

1 To whom correspondence should be addressed at In-Vitro Methods Unit/ECVAM, Institute for Health and Consumer Protection, European Commission, Joint

Research Centre, TP 580, Via Fermi 1, 21026 Ispra (VA), Italy. Fax: þ39-0332-78-5336. E-mail: anna.price@jrc.it.

Received May 8, 2009; accepted July 17, 2009

So far, only a few industrial chemicals have been identified as

developmental neurotoxicants. Because the current developmental

neurotoxicity (DNT) guideline (Organisation for Economic Co-

operation and Development TG 426) is based entirely on in vivostudies that are both time consuming and costly, there is a need to

develop alternative in vitro methods for initial screening to

prioritize chemicals for further DNT testing. In this study, gene

expression at the mRNA level was evaluated to determine whether

this could be a suitable endpoint to detect potential developmental

neurotoxicants. Primary cultures of rat cerebellar granule cells

(CGCs) were exposed to well known (developmental) neuro-

toxicants (methyl mercury chloride, lead chloride, valproic acid,

and tri-methyl tin chloride) for different time periods. A significant

downregulation of the mRNA level for the neuronal markers (NF-

68, NF-200, N-methyl D-aspartate glutamate receptor, and gamma-

amino butyric acid receptor) was observed after exposure to methyl

mercury chloride, valproic acid, and tri-methyl tin chloride.

Moreover, a significant increase of the neural precursor marker

nestin mRNA was also observed. The mRNA expression of the

astrocytic markers (glial fibrillary acidic protein [GFAP] and

S100b) was unchanged. In contrast, exposure to lead chloride

significantly decreased the mRNA level of the astrocytic marker

GFAP, whereas the neuronal markers were less affected. These

results suggest that gene expression could be used as a sensitive tool

for the initial identification of DNT effects induced by different

mechanisms of toxicity in both cell types (neuronal and glial) and at

various stages of cell development and maturation.

Key Words: gene expression; developmental neurotoxicity;

primary cell culture.

Evidence indicates that exposure to environmental chemicals

could have an impact on children’s health and development.

The developing central nervous system (CNS) of fetus and

children is particularly susceptible to chemically induced

damage compared with the brain of adults due to the different

pharmacokinetic factors, diminished defence mechanisms, or

the fact that the developing nervous system undergoes a highly

complex series of ontogenetic processes which are vulnerable

to chemical perturbation (Rice and Barone, 2000; Rodier,

1994, 1995). In fact, it has been demonstrated that chemicals

are toxic to the developing CNS at much lower doses than

those affecting the adult CNS (Claudio et al., 2000; Eriksson,

1997; Tilson, 2000).

It is now generally accepted that neurodevelopmental

disorders such as mental retardation, attention deficit, or autism

could be linked to exposure to industrial chemicals during early

fetal development (Boyle et al., 1994; Grandjean and Landrigan,

2006). To protect children, it is necessary to have relevant tools

that identify chemicals with DNT potential in a reliable way.

Once these DNT chemicals are identified, regulatory decisions

may be taken in order to restrict their use and to control exposure

as, for example in the case of lead (Silbergeld, 1997).

So far, induced developmental neurotoxicity (DNT) has

been evaluated in conventional animal-based toxicological

studies. However, only a few industrial chemicals (e.g., lead,

methyl mercury, arsenic, toluene, polychlorinated biphenyls)

are actually recognized as developmental neurotoxicants due to

the lack of studies (Grandjean and Landrigan, 2006). The

scarcity of DNT data is mainly due to the fact that there are no

general requirements for pesticides or other chemicals to be

tested for DNT effects prior to their registration and use

(Claudio et al., 2000). DNT testing is recommended only for

chemicals that have been found to ‘‘trigger’’ certain criteria in

other regulatory tests. However, the value of such trigger

concept is questionable due to the lack of data. Additionally,

for regulatory requirements, evaluation is based on DNT

testing guideline [Organisation for Economic Co-operation and

Development (OECD, 2007)] that refers only to in vivo animal

studies. Such studies are complex, time consuming, expensive,

and are not suitable for testing large numbers of chemicals. In

the present study, an in vitro approach has been used to

determine whether a primary culture of rat cerebellar granule

cells (CGCs) could serve as a relevant model for the initial

� The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For permissions, please email: journals.permissions@oxfordjournals.org

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identification of possible DNT chemicals. Primary cultures of

CGCs were used as a suitable model for DNT studies because

the crucial stages of neurodevelopment (proliferation, migra-

tion, differentiation, morphological, and functional maturation)

are present (Altman, 1987; Hogberg et al., 2009). The model is

also well characterized and has been widely used in diverse

DNT studies (Hogberg et al., 2009; Qiu et al., 1998; Radio and

Mundy, 2008; Sass et al., 2001). Some of the important key

events during the development of the CNS that could be targets

for toxic effects are the coordinated windows of specific

cellular processes such as neuronal and glial proliferation,

differentiation, migration, neurite outgrowth, synaptogenesis,

myelination, and programmed cell death (Cowan et al., 1997;

Sanes et al., 2005). Using primary cultures of CGCs, this

present study was to evaluate whether mRNA expression (real-

time PCR measurements) could be used as a relevant tool to

identify DNT critical processes affected by the exposure to well

known DNT metals such as methyl mercury chloride (Amin-

Zaki et al., 1981; Grandjean et al., 1997; Harada, 1995; Marsh

et al., 1987), lead chloride (Landrigan et al., 1975; Needleman

et al., 1979), and tri-methyl tin chloride (Feldman et al., 1993;

Kreyberg et al., 1992; Ruppert et al., 1983) as well as the drug

valproic acid (Christianson et al., 1994; Williams and Hersh,

1997; Williams et al., 2001).

The expression of genes specific for different developmental

stages, such as neuronal differentiation (NF-68 and NF-200)

and functional maturation (N-methyl D-aspartate glutamate

[NMDA] and gamma-amino butyric acid [GABAA] receptors),

proliferation, and differentiation of astrocytes (glial fibrillary

acidic protein [GFAP] and S100b) as well as the presence of

neural precursor cells (nestin and SRY-box containing gene 10

[Sox10]) was studied.

Already during fetal development, many metals, if present in

maternal blood, can easily cross the placenta as it is not an

effective barrier against environmental chemicals. It has been

shown that the mercury concentration in umbilical cord blood

can be significantly higher than in the maternal blood

(Sakamoto et al., 2004). This could affect various develop-

mental processes (Clarkson, 1997; Hassett-Sipple et al., 1997;

Pendergrass et al., 1997) leading to behavioral dysfunctions

associated with autism (Bernard et al., 2001). Like mercury,

lead also crosses the human placenta and accumulates in fetal

tissue during gestation (David et al., 1972). When the use of

lead additives in petrol was discontinued, the concentration of

lead in children’s blood dramatically decreased (by 90%).

However, this did not completely solve the problem as even

low levels of exposure to lead seem to cause surprisingly

significant functional damage to children’s CNS (Lanphear

et al., 2005).

DNT effects can also be induced by the antiepileptic drug

valproic acid. Besides its teratogenic effects (Koch et al., 1992;

Robert and Rosa, 1983; Sugimoto et al., 1983) valproic acid

can induce delayed neurological symptoms in infants exposed

to it prenatally (Christianson et al., 1994; Williams and Hersh,

1997; Williams et al., 2001). Taking into consideration that all

existing neurotoxicants could have strong DNT effects, tri-

methyl tin chloride has been evaluated as an example of

a neurotoxicant that is not yet recognized as a DNT compound.

The obtained results suggest that by using a primary culture of

CGCs, gene expression can be used as a sensitive endpoint to

identify which cell types (neuronal or glial) and which stages of

neuronal development (proliferation, differentiation, or matu-

ration) are affected by the chemicals studied.

MATERIALS AND METHODS

Chemicals and reagents. Reagents for cell culture were purchased from

Gibco Invitrogen (Milan, Italy); Dulbecco’s Modified Eagle Medium (DMEM),

fetal bovine serum, horse serum, L-glutamine, gentamicin, versene, 4-(2-

Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), and from Sigma-

Aldrich (Milan, Italy); poly-L-lysine, D (þ)-glucose and potassium chloride.

Primary culture of rat CGCs. The primary cultures of CGCs were

prepared from 7-day-old Wistar rat pups as described previously (Kinsner et al.,

2005). The cerebella were dissociated in versene solution (1:5000) and plated at

0.25 3 106 cells/cm2 in 12- or 96-well plates (Costar, Pero, Italy) coated with

poly-L-lysine (0.01% diluted 1:10 [vol/vol] in sterile MilliQ water). Cultures

were maintained in DMEM supplemented with 5% heat inactivated horse

serum, 5% heat inactivated fetal bovine serum, 13mM glucose, 0.5mM HEPES

buffer, 2mM L-glutamine, 25mM KCl, and 10 lg/ml gentamicin. Cells were

maintained at 37�C in a humidified atmosphere of 5% CO2. The medium of

CGCs was not changed throughout the whole experimental period as these cells

have to be cultured in self-conditioned medium. Cell samples from control

(nontreated) and treated cultures were prepared at 1, 4, 8, and 12 days in vitro(DIV) for real-time PCR analysis of mRNA expression (for aspirin at 1, 4, and

12 DIV).

Tested chemicals. Four neurotoxic substances (methyl mercury chloride,

lead chloride, valproic acid, and tri-methyl tin chloride) and one non-neurotoxic

substance (aspirin) were studied. All chemicals were purchased from Sigma-

Aldrich (Milan, Italy). To prepare the stock solutions toxicants were dissolved

in culture medium or dimethyl sulfoxide (DMSO).

Chemical treatments of CGCs. The concentrations of tested chemicals

were chosen based on preliminary range-finding experiments, where wide

ranges of concentrations have been tested using the Alamar Blue (AB)

(resazurin, Sigma, Milan, Italy) cell viability assay. In final experiments, three

noncytotoxic concentrations were selected or concentrations below IC20 values

based on the AB assay results were studied. In the case of chemicals dissolved in

DMSO, a noncytotoxic concentration (0.5% [vol/vol]) of DMSO was used and

it was constant in all wells of the test plates, independently from the studied

chemical concentrations. Twenty-four hours after isolation, the neuronal cultures

were exposed to the chemicals for up to 12 DIV, to cover critical developmental

processes at various stages of cell maturation. To determine whether the

presence of the chemicals influenced the selected gene expression, cell samples

were prepared for real-time PCR analysis after exposures of 1, 4, 8, and 12 DIV

to methyl mercury chloride, lead chloride, valproic acid, and tri-methyl tin

chloride and after exposures of 1, 4, and 12 DIV to aspirin (negative control).

Assessment of cell viability using AB. Cell viability was determined after

exposure to the selected chemicals at 4, 8, and 12 DIV using the AB (resazurin)

assay (O’Brien et al., 2000). The blue colored indicator dye resazurin is

reduced into fluorescent resorufin by red-ox reactions in viable cells. Because

the CGCs are considered to be mature after at least 8 DIV (Privat et al., 1974)

these time points were chosen for the cell viability test to ensure that the

process of cell maturation is covered. Resazurin (10 ll of 100lM stock) in

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Hank’s Buffered Salt Solution was added directly to the 96-well plates, without

removing the medium (100 ll). The plates were incubated for 2 h at 37�C, 5%

CO2. After incubation the fluorescence of the resazurin metabolite, resorufin was

measured at 530 nm/590 nm (excitation/emission) in a multiwell fluorometric

reader (Fluoroskan Ascent, Labsystem, Helsinki, Finland). The results were

expressed as a percentage of the mean value for the untreated cultures. Three

noncytotoxic concentrations (based on the AB assay results) were also evaluated

by cell counting to distinguish between neuro- and glia toxicity.

Assessment of neuronal and glial (astrocytes and microglia) viability by

cell counting. The viability of CGCs cultures were evaluated by immuno-

fluorescent staining using a fluorescent microscope (Olympus IX70, Hamburg,

Germany) taking into consideration a typical cell morphology observed in

phase-contrast microscope. The cell-specific protein staining was performed for

GFAP (astrocytes marker) and fluorescein isothiocyanate (FITC)-labeled

isolectin B4 (microglia marker) costained with Hoechst 33342 that permitted

quantification of the astrocytes and microglia. The cells not stained for either

GFAP or isolectin but showing characteristic neuronal morphology (phase-

contrast microscopy) were considered as neurons.

Live neurons, astrocytes, and microglia were counted in four microscopic

fields (around 150 cells per field) in each well (two wells per treatment) and

expressed as the total number of cells at 4, 8, and 12 DIV. Each experiment was

repeated at least three times. To assess the cell viability, the total number of live

cells was quantified and compared with the total number of each cell type in

nontreated control cultures.

RNA purification, reverse transcription, and quantitative real-time

PCR. Cell samples for analysis of mRNA expression were lysed and total

RNA extraction was performed according to the manufacturer’s protocol of

RNeasy Mini Kit (Qiagen, Milan, Italy). Any contaminating with DNA was

removed by digestion using an RNase-free DNase set (Qiagen). RNA

concentration and protein contamination were assessed spectrophotometrically

(Biophotometer; Eppendorf, Milan, Italy). Reverse transcription was performed

as follows: 500 ng RNA was incubated with 2.5mM PCR Nucleotide Mix

(Promega, Milan Andorra, Italy) and 12.5 lg/ml random primers (Promega) for

5 min at 65�C using a Perkin-Elmer Geneamp PCR system 9600. Subsequently,

2 units/ll RNaseOut inhibitor (Invitrogen), 10 units/ll moloney murine

leukemia virus (M-MLV) reverse transcriptase (Promega) were added with the

respective M-MLV buffer (Promega) and the samples were incubated for 10

min at 25�C for annealing, 60 min at 37�C for cDNA synthesis and 15 min at

70�C for inactivation of enzymes.

An AbiPrism 7000 sequence detector system in conjunction with TaqMan

Universal PCR Master Mix and TaqMan Real-Time PCR Assays-on-Demand

(Applera Italia, Monza, Italy) was used for investigating the gene expression

and the house keeping gene according to the manufacturer’s protocol. The

primers used were 18S ribosomal RNA (18S rRNA, Hs99999901_s1) (TaqMan

Gene Expression Assays ID), nestin (Nes, Rn00564394_m1), Sox10

(Rn00569909_m1), neurofilament, light polypeptide 68 kDa (Nfl,

Rn00582365_m1), neurofilament, heavy polypeptide 200 kDa (Nefh,

Rn00709325_m1), ionotropic glutamate receptor N-methyl D-aspartate 1

(GRIN1, Rn00433800_m1), gamma-aminobutyric acid A receptor delta

(Gabrd, Rn01517015_g1), glial fibrillary acidic protein (Gfap,

Rn00566603_m1), and S100 protein, beta polypeptide (S100b,

Rn00566139_m1). Relative RNA quantification was performed using the

comparative CT method, normalizing the data to a standard calibrator (a

mixture of samples from the different time points of the cell proliferation and

differentiation), and to the 18S rRNA content (Livak and Schmittgen, 2001).

Immunocytochemistry. CGCs cultures for immunocytochemistry were

fixed for 20 min with 4% paraformaldehyde in PBS in room temperature at DIV

12. The cells were permeabilized for 15 min with 0.1% TritonX100 and were

followed by a blocking step (10% goat serum) for 2 h at room temperature.

Primary antibodies (all from Sigma) diluted in 1% goat serum in PBS against

GFAP (mouse monoclonal 1:800), nestin (rabbit, 1:200), and NF-200 (rabbit,

1:1000) were applied to the cells over night at 4�C. Subsequently, the

secondary antibodies, goat anti-mouse IgG Alexa 546 (1:1000) and IgG Alexa

488 (1:1000) (Gibco Invitrogen) were applied. Cell nuclei were stained by

Hoechst 33342 (10 lg/ml) purchased from Molecular Probes Europe (Leiden,

The Netherlands). Microglia cells were visualized with FITC-labeled isolectin

B4 (10 ng/ml) (from Sigma) for 2 h at room temperature. Controls for specific

immunostaining were performed by omitting the primary antibodies from the

procedure. All stained cultures were examined by fluorescent microscopy

(Olympus IX70, Hamburg, Germany).

Statistical analysis. The GraphPad Prism 4.0 (GraphPad software, San

Diego, CA) program was used for statistical analyses. All data given are the

means of three independent experiments performed in six replicates (cell

viability assay), or duplicates (counting and real-time PCR analysis) ± SEM.

One-way ANOVA was performed to assess differences between treated and

control in the AB and counting cell based on immunocytochemistry assays. For

the statistical analysis of the real-time PCR experiments, differences between 1

DIV against the other time points studied were assessed by one-way ANOVA

and differences between treated and nontreated by two-way ANOVA. All data

were log-transformed to achieve Gaussian distribution. Statistical significance

was indicated as follows *p < 0.05, **p < 0.01, and ***p < 0.001 (treated vs.

control); þp < 0.05 and þþp < 0.01 (DIV 4, 8, and 12 vs. DIV 1).

RESULTS

Assessment of Cytotoxicity Using AB and Cell Counting toDifferentiate Neuronal from Glial Toxicity

Characterization of the control cultures. Primary cultures

of CGCs were prepared from 7-day-old rats because the

development of the cerebellum takes place postnatally, between

P0 and P15 (Altman and Bayer, 1978). The control, mixed

neuronal-glial cultures showed well differentiated neurons with

an extensive neuritic network which developed with time, as

observed with phase-contrast microscopy (Fig. 1A).

The mean cell count obtained in control cultures at 4 DIV

was as follow: 93 ± 3% neurons, 4 ± 0.3% astrocytes and 3 ±0.1% microglia. At 8 DIV, the control cultures consisted of

78 ± 3% neurons, 18 ± 0.8% astrocytes and 4 ± 0.3% microglia

and at 12 DIV of 79 ± 3% neurons, 18 ± 0.9% astrocytes and

3 ± 0.2% microglia (Figs. 1B-E). In control cultures,

proliferation of astrocytes and microglia was observed over

time, whereas the amount of neurons remained the same

(postmitotic cells), implicating an increase in the total number

of cells. Because the total number of cells in the control culture

from different DIVs is adjusted to 100%, the percentage of

neurons will decrease over the time.

Characterization of the cultures exposed to selected DNTchemicals. Initially, cytotoxicity was assessed using AB staining

(data not shown) after exposure to developmental toxicants

(methyl mercury chloride, lead chloride, valproic acid) and

a neurotoxic compound (tri-methyl tin chloride) as well as to the

non-neurotoxic chemical, aspirin, as a negative control. After 24 h

in culture (to allow the cells to attach and recover from the isolation

procedure), a wide range of concentrations for each chemical were

tested at different time points (4, 8, and 12 DIV). However, cell

viability assays that are based on mitochondrial activity such as AB

(or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium-

bromid) do not distinguish between neuronal or glial cell

toxicity. For this reason, immunocytochemistry staining for

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GFAP (astrocyte marker) and costaining with FITC-isolectin

B4 (microglia marker) and Hoechst 33343 (cell nuclei) were

used to count the viable cells. Hoechst 33343 staining (binds to

DNA) and the protein cell-specific immunostaining together

with a typical cell morphology (phase-contrast microscopy

image) permitted the different cell types to be distinguished

and quantified (Fig. 2). The cells that stained for Hoechst

33343 but did not stain for GFAP or FITC-isolectin B4 were

considered neurons (Fig. 2).

Based on the AB results a dose-dependent curve was

established and three noncytotoxic concentrations or concen-

trations around IC20 values were chosen for the cell counting

experiments. In these experiments, primary cultures of CGCs

were exposed to the same chemicals as in the AB cytotoxicity

studies (methyl mercury chloride, lead chloride, tri-methyl tin

chloride, valproic acid, and aspirin) and then the cells were

counted at 4, 8, and 12 DIV to distinguish between neuronal

and glial cell death. In the case of exposure to aspirin

FIG. 1. (A) Characteristic morphology of CGCs in control cultures at 1, 4, 8, and 12 DIV. Counting of alive cells (neurons, astrocytes, and microglia) in

control (untreated) cultures and after exposure to (B) methyl mercury chloride (0.5, 5, 50nM), (C) lead chloride (1, 5, 10lM), (D) valproic acid (130, 200, 300lM),

and (E) tri-methyl tin chloride (0.5, 1, 2lM) for various time periods (4, 8, and 12 DIV). The process of morphological cell maturation was observed using phase-

contrast microscopy. (A) Initially, neurons with various cell body shapes and few neurites (1 DIV) progressively differentiated into characteristic neuronal

morphology with outgrowth of neuritis (4 DIV) creating dense neuronal network over the time (8–12 DIV). The cell counting was assessed based on

immunocytochemistry for GFAP (astrocytes marker), costained for FITC-B4 isolectin (microglia marker) and Hoechst 33342. Exposure to (B) methyl mercury

chloride (50nM, at 4, 8, and 12 DIV), (D) valproic acid (130–300lM, at 12 DIV), and (E) tri-methyl tin chloride (2lM, at 8 and 12 DIV) significantly decreased the

numbers of neurons. In addition (B) methyl mercury chloride (50nM, at 8 and 12 DIV) and (E) tri-methyl tin chloride (2lM, at 8 and 12 DIV) treatment induced

a proliferation of astrocytes and microglia. Exposure to (C) lead chloride (5 and 10lM, at 8 and 12 DIV) significantly decreased the amount of astrocytes, whereas

the number of neurons was unchanged.

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(non-neurotoxic substance), cell counting was only performed

at 12 DIV.

Neurons and Glial Cells (Astrocytes and Microglia) areAffected Differently by Exposure to the Selected Chemicals

Effects of methyl mercury chloride. Treatment with the

highest concentration of methyl mercury chloride (50nM)

induced significant neuronal cell death already at 4 DIV (by

48 ± 7%; p < 0.01), when compared with the control cultures,

whereas the astrocytes and microglia remained at the same

levels as assessed by cell counting (Fig. 1B). Over time the

percentage of neurons compared with the control, decreased

even more; at 8 DIV by 83 ± 5% (p < 0.01) and at 12 DIV by

82 ± 2% (p < 0.01) (Fig. 1B). Simultaneously the number of

astrocytes and microglia significantly increased at 8 DIV

(astrocytes; 222 ± 15%, p < 0.01 and microglia; 249 ± 33%,

p < 0.01) to reach 226 ± 33% of astrocytes (p < 0.01) and

400 ± 33% of microglia (p < 0.01) at 12 DIV (Fig. 1B).

However, at the lower concentrations of methyl mercury (0.5

and 5nM) no changes were observed (Fig. 1B).

The results showed that counting cells is a more reliable

method than AB staining in these mixed cultures (neurons and

glial cells) because the AB assay did not detect any cell death

even though up to 82% of the neurons were dead after the

exposure to methyl mercury chloride (50nM at DIV 12). This

was probably due to the fact that neuronal cell death triggered

glial proliferation (both astrocytes and microglia) and because

the AB assay is based on mitochondrial activity, newly

proliferated glial cells compensated for the neuronal damage.

This is an important disadvantage of cytotoxicity assays that

should be taken into consideration when cell death is evaluated

in mixed populations of cells.

Interestingly, after exposure to methyl mercury chloride

(50nM) the immunocytochemistry staining for GFAP (astro-

cyte marker) showed the presence of mainly type one

astrocytes (flat cell body, Fig. 2B), whereas in the control

cultures mainly type two astrocytes (stellate cell body shape)

were observed (Fig. 2A). This effect could be due to type one

and type two astrocytes having different sensitivity to methyl

mercury chloride induced toxicity.

Effects of lead chloride. The opposite effect to methyl

mercury chloride was observed after exposure to lead

chloride, as a significant reduction in astrocytes was

observed (Fig. 1C). The decrease in the number of astrocytes

could already be detected at 4 DIV by 44 ± 8% (p < 0.01) at

the highest concentration (10lM). At 8 DIV, a further

decrease in the number of astrocytes was observed (by 59 ±5%, p < 0.01) at the lower concentration of 5lM followed by

73 ± 4% (p < 0.01) of astrocytic cell death at 12 DIV. This

decrease became even more pronounced at the higher

concentration of lead chloride (10lM) (Fig. 1C) as assessed

by cell counting based on immunocytochemistry staining

(Fig. 2C)

FIG. 2. Representative phase-contrast microscopy images and immu-

nocytochemistry (of the same field) for GFAP (red, astrocytes), costained

for FITC-B4 isolectin (green, microglia), and Hoechst 33342 (blue, nuclei)

in (A) control (untreated) culture and after exposure to (B) methyl mercury

chloride (50nM), (C) lead chloride (10lM), (D) valproic acid (300lM), and

(E) tri-methyl tin chloride (2lM) for 12 DIV. (B) Note the reduction of

neurons (arrows) and proliferation of type1 astrocytes (red) after exposure

to methyl mercury chloride when compared with (A) control, (C) astrocytic

cell death (red) after exposure to lead chloride, (D) neuronal cell death

(arrows) after exposure to valproic acid, and (E) proliferation of microglia

(green) and neuronal cell death (arrows) after tri-methyl tin chloride

exposure.

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The number of neurons and microglia remained the same

as in the control cultures (Figs. 1C and 2C). Astrocytes

have been reported to accumulate and be more sensitive

than neurons to lead exposure (Lindahl et al., 1999;

Tiffany-Castiglioni et al., 1989; Zurich et al., 2002)

confirming our results, where only astrocytic cell death

was observed.

Effects of valproic acid. Treatment with valproic acid,

a drug used for epilepsy both in children and pregnant

women did not affect the cell viability of neurons or glia

after 4 DIV (Fig. 1D). However, at 8 DIV a significant

proliferation of astrocytes was detected compared with the

control for all concentrations (130lM, 163 ± 7%, p < 0.01;

200lM, 183 ± 14%, p < 0.01 and 300lM, 157 ± 24%, p <0.05) (Fig. 1D). At 12 DIV only exposure to the lowest

concentration (130lM) showed a slightly higher amount of

astrocytes compared with the control (121 ± 5%, p < 0.01)

(Fig. 1D). At the same time, all concentrations of valproic

acid induced a significant reduction in the number of

neurons (130lM, by 30 ± 5%, p < 0.01; 200lM, by 30 ±4%, p < 0.01 and 300lM, by 37 ± 4%, p < 0.05) (Fig. 1D).

Cell counting based on immunocytochemistry staining for

GFAP (an astrocyte marker) and costaining with FITC-

isolectin B4 (a microglia marker) (Fig. 2D) was necessary

as even at the highest concentration of valproic acid

(300lM, 37% reduction of neurons), AB assay did not

show any cytotoxicity (Fig. 1D). These findings demon-

strate again that a simple cytotoxicity assay such as AB was

not effective in detecting neuronal cell death in a mixed

neuronal-glial culture.

Effects of tri-methyl tin chloride. Treatment with the

highly neurotoxic chemical tri-methyl tin chloride (2lM,

IC20 as measured with AB) induced a selective proliferation

of microglia already at 4 DIV (169 ± 21% of control, p <0.01) with no effect on neurons or astrocytes (Fig. 1E). At 8

DIV the same concentration of tri-methyl tin chloride induced

a significant decrease in neurons (by 76 ± 1%, p < 0.01),

whereas the astrocytes and the microglia number increased by

119 ± 6% (p < 0.05) and 410 ± 63% (p < 0.01), respectively

(Fig. 1E). The reduction of neurons (by 71 ± 4% of, p < 0.05)

and increased proliferation of astrocytes (169 ± 11%, p <0.01) and microglia (322 ± 37%, p < 0.01) remained until 12

DIV (Fig. 1E) as visualized by immunocytochemistry for

GFAP and costaining with FITC-isolectin B4 and

Hoechst33343 (Fig. 2E). The increase in microglial cell

number after exposure to tri-methyl tin chloride has also been

observed by others (Eskes et al., 2003; Monnet-Tschudi et al.,1995).

Effects of aspirin (negative control). Exposure to the non-

neurotoxic drug aspirin up to 0.5mM did not cause any toxicity

as no changes were observed in the amount of neurons,

astrocytes or microglia (data not shown).

Evaluation of Gene Expression to Determine WhetherNeuronal and Glial Differentiation and Maturation isAffected by the Exposure to Toxicants

In order to detect toxic effects, induced by the studied

chemicals, on the critical DNT neuronal and glial processes

such as cell proliferation, differentiation, and morphological

and functional maturation, gene expression of cell-specific

markers at various stages of brain development was evaluated.

Primary cultures of CGCs were exposed to noncytotoxic

concentrations of methyl mercury chloride, lead chloride,

valproic acid, tri-methyl tin chloride, and aspirin (negative

control) after 24 h in culture. Samples for RT-PCR analysis

were prepared from control and treated CGCs cultures at 1, 4,

8, and 12 DIV and were quantified against the content of the

housekeeping gene 18S rRNA, which did not show any

statistical differences between the various time points of

sampling or after chemical exposure (data not shown). The

mRNA expression at 1 DIV was normalized to 1 for each gene.

Expression of Nestin and Sox10 mRNA (Markers of NeuralProgenitor Cells) was Regulated Differently afterExposure to Various Chemicals

Characterization of the control cultures. To cover the early

developmental stage such as proliferation of progenitor cells,

two markers specific for neural precursor cells, nestin, and

Sox10, were studied. Nestin is a cytoskeletal intermediate

filament protein expressed mainly in neural precursor cells.

During differentiation and maturation into neurons or glial cells

this protein is substituted by cell-specific intermediate

filaments, for example, neurofilaments (neurons), GFAP

(astrocytes), or vimentin (oligodendrocytes) (Lee and Cole,

2000). Nestin has also been reported to be re-expressed in

activated astrocytes after brain injury or neuronal damage and

has been recognized as a sensitive marker for reactive

astrocytes in the CNS (Chen et al., 2002; Clarke et al., 1994;

Rutka et al., 1999). Additionally, the transcription factor Sox10

was studied as it is expressed mainly in neural crest cells

(Cheng et al., 2000; Pevny and Lovell-Badge, 1997) but also

partly expressed in mature glial cells (Kuhlbrodt et al., 1998).

Sox10 has been reported to be of particular importance for glial

differentiation both in the peripheral and CNS (Kuhlbrodt

et al., 1998; Pevny and Placzek, 2005; Stolt et al., 2002).

Interestingly, both precursor markers were expressed in

control (nontreated) CGC cultures isolated from postnatal rat

cerebellum. A relatively stable expression of nestin mRNA was

observed in the control cultures between 1 and 12 DIV (Fig. 3)

as confirmed also by immunocytochemistry staining (Fig. 9E).

At the same time (8–12 DIV) the transcription factor Sox10

was approximately three to fourfold upregulated (Fig. 4, p <0.01). Additional studies are needed to assess whether the

observed increase of Sox10 could be due to proliferation of

precursor cells, higher expression of protein per cell or re-

expression in some populations of glia.

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Effects of methyl mercury chloride. Already at 4 DIV,

exposure to 50nM methyl mercury chloride caused a significant

upregulation of the mRNA levels of nestin (232 ± 89%, p <0.001). The mRNA continued to be upregulated until 8 DIV

(257 ± 49%, p < 0.001) followed by a decrease to the level of

the control cultures at 12 DIV (Fig. 3A). The upregulation

of nestin observed at 8 DIV could be due either to proliferation

of precursor cells or re-expression in activated astrocytes.

Counting of specific cell types supports the first suggestion

because at 8 DIV increased glial proliferation was detected

(Figs. 1B and 2B). However, the clear neuronal damage as seen

both by cell counting (Figs. 1B and 2B) and RT-PCR analysis

(Figs. 5A-7A) could also induce the activation of astrocytes.

In contrast, the mRNA expression of the precursor marker

Sox10 was significantly downregulated by 50nM methyl

mercury chloride exposure at 8 DIV (by 45 ± 8%, p < 0.05)

and at 12 DIV (by 85 ± 3%, p < 0.001) (Fig. 4A). Further

studies are needed to clarify which mechanisms are behind the

observed increase in nestin mRNA levels and the decrease in

mRNA levels of Sox10 after the exposure to methyl mercury

chloride.

Effects of tri-methyl tin chloride. Exposure to tri-methyl tin

chloride (2lM) caused a significant increase in nestin mRNA

levels at 12 DIV (56 ± 9%, p < 0.001) (Fig. 3D), whereas the

same exposure decreased the mRNA levels of Sox10 at 8 and

12 DIV (by 48 ± 14% and 98 ± 1%, p < 0.001, respectively)

(Fig. 4D). Data obtained from the cell counting assessment

suggests that increased levels of nestin mRNA could be due to

the proliferation of astrocytes (Fig. 1D) especially because

neuronal damage was observed (Figs. 1E, 2E, 5D-7D).

Effects of valproic acid and lead chloride. After exposure

to valproic acid, mRNA expression of nestin was significantly

upregulated in a concentration dependent way, with the highest

FIG. 3. Changes in the mRNA level of the neural precursor cell marker nestin in primary culture of CGCs after chemical exposure to (A) methyl mercury

chloride (0.5, 5, 50nM), (B) lead chloride (1, 5, 10lM), (C) valproic acid (130, 200, 300lM), and (D) tri-methyl tin chloride (0.5, 1, 2lM) for various time periods

(1, 4, 8 and 12 DIV). The mRNA level of nestin was increasing at 12 DIV in control (untreated) cultures (open circle). Note the significant upregulation after

exposure to (A) methyl mercury chloride (50nM at 4 and 8 DIV), (C) valproic acid (130–300lM, at 8 and 12 DIV), and (D) tri-methyl tin chloride (2lM at 12

DIV). Gene expression levels were normalized to the standard calibrator, the housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. Data represent

mean ± SEM of three independent experiments performed in duplicates. *p < 0.05, **p < 0.01, ***p < 0.001 comparing with control (untreated) culture.þp < 0.05, þþp < 0.01 comparing 4–12 DIV of control culture to 1 DIV.

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change observed after the treatment with 300lM at 8 DIV

(180 ± 45%, p < 0.001) and 12 DIV (378 ± 26%, p < 0.001)

(Fig. 3C). The Sox10 mRNA levels were not altered at any

time point (Fig. 4C). The upregulation of nestin could again be

caused by re-expression in activated astrocytes or increased

proliferation of astrocytes, as observed by cell counting (Fig. 1D).

Lead chloride exposure did not significantly change the

mRNA expression of either nestin or Sox10 at any time point

(Figs. 3B and 4B).

Markers of the Neuronal Cytoskeleton Proteins (NF-68 andNF-200) were Downregulated by Exposure to MethylMercury Chloride, Valproic Acid, and Tri-Methyl TinChloride, but not by Lead Chloride

Characterization of the control cultures. To evaluate the

chemical effects on neuronal morphological maturation, two

cell skeleton proteins, neurofilaments 68 and 200 (NF-68 and

NF-200) were studied. NF-68 is the first neurofilament to be

expressed and covers the initial neurite outgrowth in the

immature neurons, whereas NF-200 is the last neurofilament to

be expressed and represents the later stages of the neuronal

morphological maturation (Carden et al., 1987). In control

(nontreated) primary cultures of CGCs, the expression of NF-

68 mRNA was already at its highest level at 4 DIV (Fig. 5),

whereas the peak expression of NF-200 mRNA in most

cultures was later, at 8 DIV (Fig. 6).

Effects of methyl mercury chloride. Treatment with methyl

mercury chloride at the highest concentration (50nM) signif-

icantly downregulated the mRNA levels of NF-68 at 4 DIV (by

74 ± 6%; p < 0.01) (Fig. 5A). The decrease was also consistent

at 8 and 12 DIV by 84 ± 7% (p < 0.001) and 89 ± 5% (p <0.001) reduction, respectively. NF-200 was affected in a similar

way, as 50nM of methyl mercury chloride significantly

decreased the mRNA by 76 ± 5% at 4 DIV (p < 0.01), by

FIG. 4. Quantification of Sox10 mRNA levels (marker of neural crest cells) determined by RT-PCR in primary culture of CGCs exposed to (A) methyl

mercury chloride (0.5, 5, 50nM), (B) lead chloride (1, 5, 10lM), (C) valproic acid (130, 200, 300lM), and (D) tri-methyl tin chloride (0.5, 1, 2lM) for various time

periods (1, 4, 8, and 12 DIV). In control (untreated) cultures (open circle) a significant increase was observed in the mRNA level of Sox10 at 12 DIV. The mRNA

expression of Sox10 was significantly downregulated after exposure to (A) methyl mercury chloride (50nM, at 8 and 12 DIV) and (D) tri-methyl tin chloride (2lM,

at 8 and 12 DIV). Gene expression levels were normalized to the housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. Data represent mean ± SEM

of three independent experiments performed in duplicates. *p < 0.05, **p < 0.01, ***p < 0.001 comparing with control (untreated) culture. þp < 0.05, þþp < 0.01

comparing 4–12 DIV of control culture to 1 DIV.

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88 ± 6% at 8 DIV (p < 0.001) and by 95 ± 2% at 12 DIV (p <0.001) (Fig. 6A). These results are due to neuronal cell death as

confirmed by cell counting (Figs. 1B and 2B) and NF-200

immunostaining (Fig. 6E).

Effects of tri-methyl tin chloride. Similar effects to methyl

mercury chloride were observed after exposure to the highest

concentration of tri-methyl tin chloride (2lM) where the

mRNA level of NF-68 was significantly decreased by 48 ± 3%

at 4 DIV (p < 0.001), by 70 ± 6% at 8 DIV (p < 0.001) and by

91 ± 3% at 12 DIV (p < 0.001) (Fig. 5D) when compared with

the control cultures. The same concentration of tri-methyl tin

chloride (2lM) also decreased the mRNA level of NF-200 by

58 ± 2% at 4 DIV (p < 0.01), by 76 ± 5% at 8 DIV (p < 0.001)

and by 88 ± 2% at 12 DIV (p < 0.001) (Fig. 6D) observed also

at the protein level by NF-200 immunostaining (Fig. 6E).

These results confirmed that gene expression is a sensitive

endpoint for toxicity assessment because the effects on the

mRNA levels of both neurofilaments were already observed at

4 DIV (Figs. 4 and 5) before the neuronal cell death was

detected by cell counting at 8 DIV (Fig. 1E).

Effects of valproic acid and lead chloride. Treatment with

valproic acid downregulated the mRNA level of NF-68 at the

lowest concentration (130lM) at 4 DIV and it became

significant after prolonged exposure at 12 DIV (by 77 ± 6%,

p < 0.001) (Fig. 5C). At the same time (12 DIV) this

concentration of valproic acid decreased the mRNA levels of

NF-200 by 56 ± 13% (p < 0.001) (Fig. 6C) and the protein

level by NF-200 immunostaining (Fig. 6E) when compared

with the control cultures.

In the case of lead chloride exposure no changes in the

mRNA expression of NF-68 were observed but interestingly,

a significant increase of the NF-200 mRNA after 4 DIV (120 ±27%, p < 0.01) (Fig. 6B) was determined and with time it was

still steadily increasing (until 12 DIV).

FIG. 5. Effects of (A) methyl mercury chloride (0.5, 5, 50nM), (B) lead chloride (1, 5, 10lM), (C) valproic acid (130, 200, 300lM) and (D) tri-methyl tin

chloride (0.5, 1, 2lM) on mRNA levels of the earlier neuronal marker NF-68 in CGCs cultures quantified by real-time PCR for 1, 4, 8, and 12 DIV. The peak

expression of the NF-68 mRNA was between 4 and 8 DIV in control (untreated) cultures (open circle). Note the significant downregulation of the mRNA level of

NF-68 after exposure to (A) methyl mercury chloride (50nM, at 4–12 DIV), (C) valproic acid (130–300lM, at 12 DIV), and (D) tri-methyl tin chloride (2lM, at 4–

12 DIV). Interestingly, (B) lead chloride exposure did not induce any changes of the mRNA level of NF-68. Gene expression levels were normalized to the

housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. Data represent mean ± SEM of three independent experiments performed in duplicates. *p <

0.05, **p < 0.01, ***p < 0.001 comparing to control (untreated) culture. þp < 0.05, þþp < 0.01 comparing 4–12 DIV of control culture with 1 DIV.

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The results obtained suggest that the gene expression of

cytoskeleton proteins is more sensitive to methyl mercury

chloride and tri-methyl tin chloride exposure than to valproic

acid or lead chloride. In the case of methyl mercury chloride

and tri-methyl tin chloride, much lower concentrations resulted

in downregulation of both neurofilaments at mRNA level

FIG. 6. Changes in mRNA level of the later neuronal marker NF-200 quantified by RT-PCR in primary culture of CGCs exposed to (A) methyl mercury

chloride (0.5, 5, 50nM), (B) lead chloride (1, 5, 10lM), (C) valproic acid (130, 200, 300lM) and (D) tri-methyl tin chloride (0.5, 1, 2lM) for 1, 4, 8, and 12 DIV.

In the control (untreated) cultures (open circle) the peak expression of the NF-200 mRNA was between 8 and 12 DIV. Treatment with (A) methyl mercury chloride

(50nM, at 4–12 DIV), (C) valproic acid (130–300lM, at 12 DIV) and (D) tri-methyl tin chloride (2lM, at 4–12 DIV) induced statistically significant

downregulation of NF-200 mRNA expression. In contrast, exposure to (B) lead chloride (10lM, at 4 DIV) induced significant upregulation. Gene expression levels

were normalized to the housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. Data represent mean ± SEM of three independent experiments

performed in duplicates. (E) Immunocytochemistry of NF-200 (green) and GFAP (red) (costained with Hoechst 33342, blue) was performed for control and treated

cultures (methyl mercury chloride 50nM, valproic acid 300lM and tri-methyl tin chloride 2lM) at 12 DIV. *p < 0.05, **p < 0.01, ***p < 0.001, comparing with

control (untreated) culture. þp < 0.05, þþp < 0.01 comparing 4–12 DIV of control culture to 1 DIV.

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(50nM and 2lM, respectively) when compared with valproic

acid (130lM) or lead chloride (no effect or increase).

Gene Expression of NMDA and GABAA Receptors was eitherDown- or Upregulated after Exposure to StudiedChemicals

Characterization of the control cultures. Important markers

of neuronal functional maturation are the subunits of the main

neuronal excitatory and inhibitory receptors, NMDA-R and

GABAA-R. In this study, the subunit 1 of the NMDA-R, with the

peak expression of mRNA at 4 DIV in control (untreated) cultures

(Fig. 7) and the delta subunit of GABAA-R receptor with the

highest expression of mRNA at 8 and 12 DIV in control cultures

were evaluated (Fig. 8). The delta subunit of the GABAA receptor

has been shown to be most abundant in the granular layer of the

cerebellum and immunostaining for this subunit correlates well

with the expression pattern of mRNA (Benke et al., 1991).

Effects of methyl mercury chloride. The highest concen-

tration of methyl mercury chloride (50nM) significantly

decreased the mRNA levels of the subunits of both NMDA

and GABAA receptors. In the case of the NMDA-R, the

following reductions were observed: at 8 DIV by 86 ± 6% (p <0.001) and at 12 DIV by 90 ± 5% (p < 0.001) (Fig. 7A). The

gene expression for the GABAA subunit delta was already

dramatically downregulated at 4 DIV by 76 ± 7% (p < 0.05), at

8 DIV by 85 ± 6% (p < 0.001) and at 12 DIV by 93 ± 3% (p <0.001) (Fig. 8A). These significant decreases in mRNA levels

for both receptor subunits were probably due to neuronal cell

death, as detected by counting of the neurons (Fig. 1B).

Effects of valproic acid and tri-methyl tin chloride. Inter-

estingly, both valproic acid and tri-methyl tin chloride exposure

affected the mRNA levels of the GABAA receptor earlier than

the NMDA receptor. Exposure to 130lM valproic acid

significantly downregulated the GABAA mRNA levels at

FIG. 7. Quantification of the NMDA-R mRNA level by RT-PCR, determined in primary culture of CGCs exposed to (A) methyl mercury chloride (0.5, 5,

50nM), (B) lead chloride (1, 5, 10lM), (C) valproic acid (130, 200, 300lM) and (D) tri-methyl tin chloride (0.5, 1, 2lM) for 1, 4, 8, and 12 DIV. The peak

expression of the NMDA-R mRNA in control (untreated) cultures (open circle) was around 4 DIV. Note the significant downregulation of the mRNA level after

exposure to (A) methyl mercury chloride (50nM, at 8 and12 DIV), (C) valproic acid (130–300lM, at 12 DIV), and (D) tri-methyl tin chloride (2lM, at 8 and 12

DIV), whereas (B) lead chloride exposure (10lM, at 12 DIV) significantly upregulated the mRNA of the NMDA-R. Gene expression levels were normalized to the

housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. Data represent mean ± SEM of three independent experiments performed in duplicates. *p <

0.05, **p < 0.01, ***p < 0.001 comparing to control (untreated) culture. þp < 0.05, þþp < 0.01 comparing 4–12 DIV of control culture with 1 DIV.

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8 DIV by 50 ± 3% (p < 0.001) and at 12 DIV by 73 ± 7% (p <0.001) (Fig. 8C), whereas mRNA levels of the NMDA receptor

were downregulated only after prolonged exposure, at 12 DIV

(by 71 ± 7%, p < 0.001) (Fig. 7C). Valproic acid is shown to

increase the release of the GABA neurotransmitter (Cunning-

ham et al., 2003; Owens and Nemeroff, 2003) which

consequently could cause downregulation of the GABAA

receptor expression observed in our studies.

Tri-methyl tin chloride exposure significantly decreased the

mRNA levels of the GABAA receptor only at the highest

concentration (2lM) both at 4 DIV (by 52 ± 2%, p < 0.001), 8

DIV (by 77 ± 5%, p < 0.001) and 12 DIV (by 44 ± 10%, p <0.001) (Fig. 8D), whereas the same concentration only down-

regulated the mRNA of NMDA receptor at 8 DIV (by 49 ± 7%,

p < 0.001) and 12 DIV (by 80 ± 5%, p < 0.001) (Fig. 7D).

Effects of lead chloride. In contrast to valproic acid and tri-

methyl tin chloride, exposure to lead chloride caused an

increase in the mRNA levels of both the GABAA and the

NMDA receptor. Interestingly, this increase was already

observed at 4 DIV for mRNA levels of GABAA receptor (by

197 ± 87%, p < 0.01) (Fig. 8B), whereas the increase in the

mRNA levels of NMDA was observed later, at 12 DIV (by

74 ± 16%, p < 0.01) (Fig. 7B).

mRNA Expression of the Astrocytic Cell Skeleton ProteinGFAP was Only Affected by Lead Chloride Exposure

Characterization of the control cultures. Astrocytes are of

great importance in the development of the CNS because of

their ability to guide the neurons to the right position, provide

protection for neurons by releasing various factors and by

metabolic and trophic support (Goritz et al., 2002; Hatten and

Liem, 1981; Pfrieger and Barres, 1997; Wang et al., 1994).

Chemical interference with any of these astrocytic processes

might affect the neuronal development and function. For these

reasons the glial toxicity was evaluated by investigating two

specific markers for mature astrocytes; the cell skeleton protein

FIG. 8. Changes in the mRNA level of the GABAA-R in primary cultures of CGCs exposed to (A) methyl mercury chloride (0.5, 5, 50nM), (B) lead chloride

(1, 5, 10lM), (C) valproic acid (130, 200, 300lM), and (D) tri-methyl tin chloride (0.5, 1, 2lM) for 1, 4, 8, and 12 DIV. In control (untreated) cultures (open circle)

a significant increase was observed in the mRNA level of GABAA-R over the time. The exposure to (A) methyl mercury chloride (50nM, at 4–12 DIV), (C)

valproic acid (130–300lM, at 8 and 12 DIV), and (D) tri-methyl tin chloride (2lM, at 4–12 DIV) significant downregulated the mRNA, whereas (B) lead chloride

exposure (10lM, at 4 DIV) significantly upregulated the mRNA of the GABAA-R. Gene expression levels were normalized to the housekeeping gene 18S rRNA

and the mRNA expression at 1 DIV. Data represent mean ± SEM of three independent experiments performed in duplicates. *p < 0.05, **p < 0.01, ***p < 0.001

comparing to control (untreated) culture. þp < 0.05, þþp < 0.01, comparing 4–12 DIV of control culture with 1 DIV.

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GFAP and the calcium- zinc-binding protein S100 beta

(S100b) that are recognized as being astrocytic markers

(Heizmann and Cox, 1998; Yang et al., 1996). A continuous

increase in the mRNA levels of GFAP and S100b was

observed over time in the control (nontreated) cultures

indicating a proliferation and maturation of astrocytes (Figs.

9 and 10). The proliferation of astrocytes was also confirmed

by immunocytochemistry for GFAP (Fig. 9E).

Effects of lead chloride. The only studied chemical that

induced mRNA changes of the astrocytic cell skeleton protein

GFAP was lead chloride. The mRNA levels were significantly

downregulated at both 4 DIV (5lM by 62 ± 8% and 10lM by

54 ± 11%, p < 0.01) and at 12 DIV (5lM by 51 ± 11% and

10lM by 42 ± 13%, p < 0.05) (Fig. 9B). These results are

confirmed by the data obtained from cell counting experi-

ments where astrocytic cell death was observed at studied

time points (Figs. 1C and 2C). The data obtained are in line

with previous studies where lead was shown to be pre-

dominantly glia toxic as accumulation of lead was observed in

glial cells (Lindahl et al., 1999; Tiffany-Castiglion and Qian,

2001; Zurich et al., 2002). There were no significant changes

FIG. 9. Effects of (A) methyl mercury chloride (0.5, 5, 50nM), (B) lead chloride (1, 5, 10lM), (C) valproic acid (130, 200, 300lM) and (D) tri-methyl tin

chloride (0.5, 1, 2lM) on mRNA levels of the astrocytic marker GFAP in CGC cultures quantified by real-time PCR for 1, 4, 8, and 12 DIV. In control (untreated)

cultures (open circle) the mRNA expression of GFAP was significantly increasing over the time. Note the significant downregulation of the GFAP mRNA level

after exposure to (B) lead chloride (5 and10lM, at 4 and 12 DIV). No changes were observed for (A) methyl mercury chloride, (C) valproic acid, and (D) tri-

methyl tin chloride exposure. Gene expression levels were normalized to the housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. (E)

Immunocytochemistry of GFAP (red) and nestin (green) (costained with Hoechst 33342, blue) was performed for control cultures at 1, 4, and 12 DIV. Data

represent mean ± SEM of three independent experiments performed in duplicates. *p < 0.05, **p < 0.01, ***p < 0.001 comparing to control (untreated) culture.þp < 0.05, þþp < 0.01 comparing 4–12 DIV of control culture with 1 DIV.

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in the mRNA levels for S100b after exposure to any studied

chemicals (Fig. 10).

Effects of aspirin (negative control). Exposure to aspirin up

to 0.5mM did not induce any changes in the mRNA levels of

any studied genes (Fig. 11) suggesting that gene expression

changes was indeed able to detect specific neuronal or glial

toxic effects.

DISCUSSION

In this study we have shown that a change in the mRNA

level of genes involved in key developmental processes such as

proliferation, differentiation and maturation of both cell types,

neuronal and glial, provides a relevant endpoint for the

detection of chemicals with potential to induce DNT effects.

After exposure of primary cultures of CGCs to several

chemicals with well known developmental neurotoxic effects

(methyl mercury chloride, lead chloride, and valproic acid) or

neurotoxic effects (tri-methyl tin chloride) diverse responses

were observed in neurons and glial cells, already at very low

concentrations. Indeed, methyl mercury chloride, valproic acid,

and tri-methyl tin chloride mainly affected the neuronal

markers (neurofilaments and receptors), whereas lead chloride

affected the astrocytic marker (GFAP). Furthermore, measure-

ment of gene expression could detect more specific effects as

valproic acid and tri-methyl tin chloride decreased the mRNA

level for the inhibitory GABAA receptor before any changes

could be observed for the mRNA of the excitatory NMDA

receptor. At the same time aspirin (non-neurotoxic drug,

negative control) did not induce any changes in the mRNA

level of the selected genes.

The chemicals tested are of major concern because most of

them have been proven to have an impact on children’s health.

Human exposure to methyl mercury is primarily via consump-

tion of contaminated fish and mercury-containing fungicide in

seed grain (Ip et al., 2004; World Health Organization, 1990).

In the case of children a major source of mercury is thimerosal,

a preservative added to many vaccines (Ip et al., 2004). In

addition to the high exposure risk, methyl mercury passes both

FIG. 10. No changes were observed after exposure to (A) methyl mercury chloride (0.5, 5, 50nM), (B) lead chloride (1, 5, 10lM), (C) valproic acid (130, 200,

300lM), and (D) tri-methyl tin chloride (0.5, 1, 2lM) on the mRNA of the astrocytic marker S100b in CGC cultures quantified by real-time PCR for 1, 4, 8, and 12

DIV. In control (untreated) cultures (open circle) a significant increase was observed in the mRNA level of S100b over the time. Gene expression levels were

normalized to the housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. Data represent mean ± SEM of three independent experiments performed in

duplicates. þp < 0.05, þþp < 0.01 comparing 4–12 DIV of control culture with 1 DIV.

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the blood-brain and the placenta barrier (The National Research

Control (NRC), 2000; Sakamoto et al., 2004). Moreover, the

toxicological effects of methyl mercury, and its affinity to fetal

hemoglobin are higher than for the pregnant woman giving

25% higher levels in the fetal blood than in the mother (Amin-

Zaki et al., 1976). Furthermore, several studies have reported

that relatively low concentrations of methyl mercury can result

in neurotoxic effects such as cognitive and motor dysfunctions

(Amin-Zaki et al., 1981; Dolbec et al., 2000; Grandjean et al.,1997; Lebel et al., 1998; NRC, 2000). In the United States,

approximately 500,000 children are born every year with blood

mercury levels above 5.8 lg/l (26.6lM), a level that has been

associated with cognitive deficits (Trasande et al., 2005). In

fact, the threshold of methyl mercury concentrations in the

brain resulting in clinical effects has been suggested to be as

low as 0.3 ppm (1.5lM) (Kjellstrom et al., 1986). In our

studies using gene expression as a tool for toxicity assessment

we could detect DNT effects already at the very low

concentration of 50nM methyl mercury chloride. Interestingly

enough the granule cells within the cerebellar cortex seem to be

particulary vulnerable to methyl mercury toxicity (Atchison

and Hare, 1994) and for this reason we consider the primary

culture of CGCs at this particular window to be a relevant and

reliable model to predict DNT effects. Methyl mercury is

particularly neurotoxic as it predominantly accumulates in

neurons (Kaur et al., 2006). Glial cells are typically spared

from direct damage caused by methyl mercury although

reactive gliosis might occur (Atchison and Hare, 1994;

Moller-Madsen, 1990; Schionning and Moller-Madsen, 1992;

Tiffany-Castiglion and Qian, 2001). Indeed the mRNA levels

of neuronal markers such as NF-68, NF-200, NMDA-R, and

GABAA-R were already downregulated at 4 DIV (Figs. 5A,

6A, 7A, 8A), whereas the mRNA of the astrocytic markers

(GFAP and S100B) (Figs. 9A, 10A) remained unchanged,

indicating that neurons are more sensitive to methyl mercury

induced toxicity than glial cells. However, we also observed an

increase in nestin mRNA expression which could be a marker

of either neural precursor cell proliferation or glia proliferation

(Fig. 3A). Indeed in all performed experiments the increase in

the expression of nestin mRNA was followed by increased glia

proliferation possibly in response to neuronal cell death (Fig.

1B). Regarding the increased proliferation of astrocytes

(probably reactive astrogliosis) and microglia (microgliosis)

induced by exposure to methyl mercury in our mixed neuronal-

glial cultures similar responses have also been observed in

humans and animals at wide ranges of methyl mercury

concentrations (Davis et al., 1994; Eto et al., 1999; Roda

et al., 2008).

In contrast to methyl mercury, lead selectively accumulates in

glia (Lindahl et al., 1999; Tiffany-Castiglioni et al., 1989; Zurich

et al., 2002), whereas neuronal differentiation as seen by neurite

initiation and axon branching are only affected by high lead

concentrations (Liu et al., 2000). Similar results were obtained in

our studies at the level of gene expression because the main

effect observed was a decrease in the mRNA level of the

astrocytic marker GFAP (Fig. 9B). The mRNA of the neuronal

markers was only partly affected as increased mRNA level of

NF-200 (Fig. 6B), NMDA-R (Fig. 7B), and GABAA-R (Fig.

8B) were seen. During development of the CNS the neuronal

connections between cells are formed in far greater numbers than

necessary and are later slowly reduced to the required level.

Interestingly, lead has been proposed to interfere with the

process of selective pruning of neuronal connections leading to

a pathological increase in the number of synapses (Goldstein,

1992). If it is true this could explain the increased mRNA levels

for both NFs and neuronal receptors observed in our study.

However, because neuronal developmental processes such as

neurite outgrowth, migration, synaptic elaboration and cell–cell

interactions require glial cells (Goritz et al., 2002; Hatten and

Liem, 1981; Pfrieger and Barres, 1997; Wang et al., 1994),

disruption in the maturation of astrocytes would most likely also

alter neuronal maturation, affecting the formation of the network

and cellular interactions. Indeed, human exposure to lead is

associated with numerous adverse effects in the CNS, including

destruction of the blood–brain barrier, loss of neurons, and

gliosis, which results in a variety of neurological disturbances

particularly observed in young children (Finkelstein et al., 1998;

Hu et al., 2006). Moreover, lead has been reported to pass the

placenta barrier (Foltinova et al., 2007; Lauwerys et al., 1978).

Despite the efforts to reduce lead levels in the ecosystem, for

example, use of lead-free gasoline, which have significantly

reduced median blood lead concentrations (Silbergeld, 1997),

the risks associated with environmental exposure still remain

because of the long biological life time of lead (Cory-Slechta,

1990). Although most governmental agencies, including the

US Environmental Protection Agency and Centers for Disease

Control (1991), have defined the highest acceptable blood lead

level in children as 10 lg/dl (0.48lM), impaired cognitive

functions have been observed by several research groups

already at levels as low as 5 lg/dl (0.24lM) (Bellinger, 2008;

Chiodo et al., 2007; Jusko et al., 2008; Surkan et al., 2007). It

is important to have sensitive tools that identify DNT effects at

low concentrations and gene expression could be one of these

especially when incorporated with other relevant endpoints.

A reliable in vitro testing strategy should be built up and

initially be used for the prioritization of the compounds with

DNT potential. This approach would decrease the number of

chemicals further tested using OECD DNT Test Guideline 426

that is complex and time consuming, as it is entirely based on

in vivo testing.

In these studies, mRNA expression determination also

confirmed that the antiepileptic drug valproic acid, which is

recognized as teratogenic should also be considered as a DNT

chemical. Indeed, in our studies we already observed the

neurotoxic effects of valproic acid after prolonged exposure at

concentrations between 0.13 and 0.3mM. These concentrations

caused significant down regulation of the mRNA of neuronal

markers such as NF-68 (Fig. 5C), NF-200 (Fig. 6C), NMDA-R

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FIG. 11. No changes were observed after exposure to aspirin at any studied concentration (100, 250, 500lM) on the mRNA level of (A) nestin, (B) Sox10, (C)

NF-68, (D) NF-200, (E) NMDA-R, (F) GABAA-R, (G) GFAP, and (H) S100b in CGC cultures quantified by real-time PCR for 4 and 12 DIV. Gene expression

levels were normalized to the housekeeping gene 18S rRNA and the mRNA expression at 1 DIV. Data represent mean ± SEM of three independent experiments

performed in duplicates.

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(Fig. 7C), and GABAA-R (Fig. 8C). Interestingly, the mRNA

level of GABAA-R was the first significant decrease to be

observed before the expression of the NMDA-R was affected.

This could be due to the main mechanism of valproic acid

toxicity because it modifies the release of the inhibitory

neurotransmitter GABA, although both effects, reduced

(Cunningham et al., 2003) and increased (Loscher and Vetter,

1985; Owens and Nemeroff, 2003) levels of GABA have been

reported. However, an increase or decrease in GABA concen-

tration is likely to mainly affect the expression of the GABA

receptors but might also be followed by more prolonged

variations in other neurotransmitters functions, in particular

those belonging to the aminergic group (Tringali et al., 2004)

such as glutaminergic functions. Any chemical that changes the

expression of either inhibitory GABA or excitatory NMDA

receptors, as in the case of valproic acid, could affect the general

neuronal activity of the brain followed by the changes in activity-

dependent processes such as dendritic arborization and synapse

formation (Luthi et al., 2001).

Furthermore, in the same experiments, after exposure to

valproic acid, upregulation in the mRNA expression of the

precursor marker nestin was observed in a concentration

dependent way (Fig. 3C) suggesting an activation of glial cells

(Brook et al., 1999; Kalman and Ajtai, 2001; Lin et al., 1995),

or proliferation of neural precursor cells or/and astrocytes

(Messam et al., 2000; Wei et al., 2002) triggered possibly by

neuronal cell death. These effects in CGCs were induced by the

concentrations of valproic acid (0.13–0.3mM) which were

within the range of the therapeutic levels found in plasma of

both adults and children (0.2–1.3mM) (Ingels et al., 2002;

Klotz and Schweizer, 1980; Lundberg et al., 1982).

In addition to our own results it has already been shown in

recent studies (Jergil et al., 2009) that valproic acid induced

deregulation of genes associated with neural tube defects

clearly suggesting that it is a strong developmental neuro-

toxicant. Additionally it has been shown that valproic acid not

only has the possibility to pass the placenta barrier but also

accumulates in fetal blood at higher concentrations due to

increased levels of albumin in fetal blood compared with

maternal (Dickinson et al., 1979; Nau et al., 1984). There are

several reports of malformations and developmental abnormal-

ities in the CNS caused by valproic acid (Christianson et al.,1994; Williams and Hersh, 1997; Williams et al., 2001), such

as increased risk of autism, increased serotonin levels and

problems in social behavior associated with prenatal exposure

to valproic acid, and thus its use during pregnancy is not

recommended (Koch et al., 1992).

Many neurotoxic chemicals could also have strong effects on

the developing CNS, however due to the lack of studies only

five compounds are considered as DNT chemicals (methyl

mercury, lead, arsenic, toluene, polychlorinated biphenyls)

affecting human brain development (Grandjean and Landrigan,

2006). In the case of tri-methyl tin, this has not yet been

reported to be a DNT toxin, however, there are several cases in

which neurotoxic symptoms such as aggressive behavior,

depression, disorientation, seizures, hearing loss and severe

memory loss (Feldman et al., 1993; Kreyberg et al., 1992) have

been reported after acute accidental exposure. Nevertheless, it

has been suggested that the neurotoxicity of tri-methyl tin

could be more prominent if exposure occurs during the period

of nervous system development (Jenkins et al., 2004) and DNT

effects have been observed in animal studies (Noland et al.,1982; Ruppert et al., 1983; Stanton et al., 1991). The primary

human contact occurs due to accidental poisoning incidents but

there is a concern that some people without known acute

exposure to organic tin still have detectable levels in their

blood (Whalen et al., 1999) and urine (Braman and Tompkins,

1979). The risks of exposure are through consumption of sea

food (Hong et al., 2002), drinking water carried by polyvinyl

chloride (PVC) pipes (Sadiki and Williams, 1999) or beverages

stored or transported in PVC containers (Liu and Jiang, 2002).

The plasma level in a case of acute tri-methyl tin poisoning

with neurotoxic symptoms was 327.5 lg/l (1.64lM) (Yoo

et al., 2007). In these studies CGCs were exposed to the range

of 0.5–2lM tri-methyl tin chloride, however, the neurotoxic

effects were observed at 2lM. This concentration of tri-methyl

tin clearly affected neurons because the mRNA expression of

all neuronal markers (NF-68, NF-200, NMDA-R, and

GABAA-R) was significantly down-regulated (Figs. 5D, 6D,

7D, 8D). Additionally, a significant increase in the number of

microglia (~4-fold at 8 DIV) followed by increased pro-

liferation of astrocytes (~1.7-fold at 12 DIV) was observed

(Fig. 1E). The increased proliferation of astrocytes was also

supported by the upregulation of nestin mRNA expression

(Fig. 3D) that is recognized as a sensitive marker of astrocyte

activation (Clarke et al., 1994; Rutka et al., 1999). It is possible

that the tri-methyl tin chloride induced neurotoxicity in our cell

culture model could be mediated by activated microglia and

astrocytes. Indeed, it is well known that once activated, glia

secrete a variety of proinflammatory and neurotoxic factors

such as cytokines and free radicals (Bal-Price and Brown,

2001). Similar mechanisms of neurotoxicity induced by tri-

methyl tin (mediated by glia activation) have been already

suggested in previous studies (El Fawal and O’Callaghan,

2008; Eskes et al., 2003; Maier et al., 1995; McCann et al.,1996; Monnet-Tschudi et al., 1995). Taken together, the

mechanisms of toxicity identified in the in vitro model by

mRNA markers mirror our understanding of their (develop-

mental) neurotoxicity. In order to be able to test a large number

of chemicals for their potential to induce DNT effects, it has

been proposed to use an in vitro testing strategy for an initial

prioritization based on a battery of different alternative models

and endpoints (Bal-Price et al., 2008; Coecke et al., 2007; Lein

et al., 2007). Such a simplified in vitro approach could be

useful in identifying chemicals for further testing. We have

demonstrated in these studies together with a previous study on

pesticides (Hogberg et al., 2009) that CGCs cultures and gene

expression endpoints are promising in vitro tools for initial

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DNT chemical screening. However, more neurotoxic (and non-

neurotoxic) chemicals should be tested to define, if possible,

the characteristic gene expression profile for DNT compounds.

Such gene expression profile specific for DNT substances

could be incorporated together with other endpoints, for

example, the key molecular targets or toxicity pathways to

obtain a mode of action specific for potential DNT toxicants.

Such a testing strategy could speed up the process of DNT

chemical assessment for regulatory purposes leading to their

restricted use and a tighter control of children’s exposure to

potential DNT compounds.

ACKNOWLEDGMENTS

We would like to acknowledge Prof. Jan Nedergaard and

Barbara Cannon for scientific contribution and Dr Joanne

Gartlon, Steven Price, and Claire Thomas for the scientific and

linguistic editing of the text.

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