Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y

    Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y K. Attoff, D. Kertika, J. Lundqvist, S. Oredsson, A. Forsby PII: DOI: Reference:

S0887-2333(16)30109-6 doi: 10.1016/j.tiv.2016.05.014 TIV 3789

To appear in: Received date: Revised date: Accepted date:

2 December 2015 8 May 2016 26 May 2016

Please cite this article as: Attoff, K., Kertika, D., Lundqvist, J., Oredsson, S., Forsby, A., Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y, (2016), doi: 10.1016/j.tiv.2016.05.014

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Acrylamide affects proliferation and differentiation

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neuroblastoma cell line SH-SY5Y

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of the neural progenitor cell line C17.2 and the

K Attoffa, MSc, [email protected], D Kertikaa, MSc, [email protected], J

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Forsbya,c, PhD, [email protected]

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Lundqvista, MSc, [email protected], S Oredssonb, PhD , [email protected], A

Department of Neurochemistry, Stockholm University, Stockholm, 106 91

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Department of Biology, Lund University, Lund, 223 62

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Swedish Toxicology Science Center (Swetox), Södertälje

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a)

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Corresponding author: Kristina Attoff, Svante Arrhenius väg 16B, plan 4. SE-106 91, Stockholm. Phone number +468-161576, [email protected],

Abbreviations; BDNF, brain-derived neurotrophic factor; BrdUrd, 5-bromo-2’-deoxyuridine; BSA, bovine serum albumin; DMEM/F12, Dulbecco’s modified Eagles medium:Nutrient mixture F-12; DMSO, dimethyl sulfoxide; GFAP, glial fibrillary acidic protein; MTT, 3-[4,5dimethylthiazol- 2-yl]-2,5 diphenyl-tetrazolium bromide; NGF, nerve growth factor; PBS, phosphate-buffered saline; PI, propidium iodide; TBP, TATA-box binding protein.

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ACCEPTED MANUSCRIPT Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses

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the placenta barrier, the fetus is also being exposed resulting in a risk for developmental

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neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma

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cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the

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number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10 fM attenuated the

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differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10 pM. Acrylamide significantly reduced

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the number of neurons starting at 1 μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten μM of acrylamide also reduced the

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expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide. Keywords: Developmental neurotoxicity, acrylamide, neural progenitor cells, SH-SY5Y, C17.2, differentiation

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Introduction

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Acrylamide is a water-soluble, vinyl monomer that has various applications in the chemical

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industry for water management, dye synthesis, in textile additives and in paint softeners as well

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as in the cosmetic industry (IARC, 1994). Acrylamide can also be generated from food components during heat treatment as a result of the Maillard reaction between amino acids

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(mainly asparagine) and reducing sugars or reactive carbonyls (Mottram et al., 2002). Most people are exposed to acrylamide through food consumption and environmental pollutants

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(Kutting et al., 2009). Studies on exposed human populations and laboratory animals have shown that acrylamide can be absorbed orally, dermally, and by inhalation (Dearfield et al.,

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1988, Sumner et al., 2003). Regardless of the absorption route, acrylamide is distributed throughout the body. The tissues that are the most susceptible to acrylamide exposure include

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liver, kidney, smooth and skeletal muscles and the nervous system (Miller et al., 1982). Animal studies have shown that the polymeric form of acrylamide is non-toxic but the monomeric form induces neurotoxicity, reproductive toxicity, genotoxicity and carcinogenicity (Chapin et al., 1995, Dearfield et al., 1995). However, only acrylamide-induced neurotoxicity has been documented in occupationally exposed populations in humans, indicating implications in human health (Kjuus et al., 2004). Acrylamide has been recognized as a neurotoxic substance since the 1950s when it was realized that occupational exposure could give rise to cumulative neurotoxicity characterized by ataxia, skeletal muscle weakness, cognitive impairment, and numbness of the extremities (Garland and Patterson, 1967). It has been shown in animals, as well as in tissue- and cell cultures that the monomeric form of acrylamide gives rise to similar neurotoxic effects as seen in exposed human populations

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ACCEPTED MANUSCRIPT (McCollister et al., 1964, Miller and Spencer, 1985). It has been suggested that the acrylamideinduced neurotoxicity is mediated through axonopathy in both central and peripheral nervous

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system caused by initial distal nerve terminal damage and subsequent retrograde axon

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degeneration (LoPachin, 2002).

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Acrylamide crosses the placental barrier. Hence, the risk for the fetus to be exposed in utero is obvious. Acrylamide has also been detected in breast milk, indicating that acrylamide exposure

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may continue during infancy (Sörgel et al., 2002, von Stedingk et al., 2011). Prenatal and

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perinatal exposure to acrylamide decreases the average horizontal motor activity and auditory startle response in exposed rats (Wise et al., 1995). Furthermore, acrylamide disrupts the

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development of cerebellum and induces lipid peroxidation and oxidative stress in the

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developing medulla oblongata in rats (Allam et al., 2013).

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The number of children being diagnosed with learning and neurodevelopmental disorders has increased over the past three decades (US EPA, 2015). Hence, there is an emerging need to develop and validate robust methods that are capable to alert for chemicals that may induce neurodevelopmental toxicity. Also, many neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis, are in fact believed to be neurodevelopmental diseases because they share common pathophysiological cascades involving oxidative stress and impaired hippocampal neurogenesis in mice (Park et al., 2010). Since acrylamide has a nerve terminal specific site of action and can compete for the cysteine binding sites of nucleophilic scavengers, it has been suggested that neuropathogenic processes could be accelerated by acrylamide (LoPachin et al., 2008). Here, we have studied features of developmental neurotoxicity of acrylamide using mechanistic endpoints during proliferation

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ACCEPTED MANUSCRIPT and different stages of neuronal differentiation in the murine neural progenitor cell line C17.2

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and the human neuroblastoma cell line SH-SY5Y.

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Materials and methods

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Chemicals and reagents

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Acrylamide (99.9% purity) (A9099), putrescine dihydrochloride (P5780), progesterone (P8783), sodium selenite (S5261), bovine insulin (I1882), all-trans-retinoic acid (R2625),

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dimethyl sulfoxide (DMSO) (D4540), bovine serum albumin (BSA) (A2153), NP-40 and agar were purchased from Sigma Aldrich (Sweden). Recombinant mouse β-nerve growth factor

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(NGF) (1156-NG) and recombinant human brain-derived neurotrophic factor (BDNF) (248-

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BD) were purchased from R&D systems (United Kingdom). Apo-transferrin from bovine

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plasma (02152334) was purchased from MP Biomedicals. GIBCO® phosphate-buffered saline (PBS), GIBCO® Trypsin/ EDTA (0.05/0.02%), GIBCO® Dulbecco’s modified Eagles medium (DMEM), GIBCO® Dulbecco’s modified Eagles medium:Nutrient mixture F-12 (DMEM/F12), GIBCO® minimum essential medium (MEM), GIBCO® MEM non-essential amino acid solution, horse serum, fetal calf serum, GIBCO® L-glutamine, GIBCO® Penicillin, 10000 U/ml and streptomycin, 10000 μg/ml, RevertAid

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H Minus First Strand cDNA Synthesis Kit

(K1621), RiboRuler High Range RNA Ladder, 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT), Horseradish Peroxidase-conjugated Anti-Goat IgG (31402), DRAQ5, Anti-rabbit antibody conjugated with Alexa Fluor 488, SouthernBiotech Fluoromount-GTM and Maxima® SYBR Green/Fluorescein qPCR Master Mix were purchased from ThermoFisher Scientific (Sweden). RNA extraction kit RNeasy Plus Mini Kit was purchased from Qiagen. All primers for the Real-Time Reverse Transcription quantitative 5

ACCEPTED MANUSCRIPT Polymerase Chain Reaction (RT-qPCR) were purchased from Eurofins MWG (Germany). All plastic ware used for cell culturing were purchased from Corning Inc. Antibodies against glial

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fibrillary acidic protein (GFAP) (ab7260) for western blot and βIII-tubulin (ab18207) for

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western blot and immunofluorescence were purchased from Abcam (United Kingdom).

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Antibody against GFAP for immunofluorescence (Z033429-2) was purchased from Dako (Denmark). Antibody against β-actin (sc-1616) was purchased from Santa Cruz Biotechnology

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Inc (USA). Horseradish peroxidase-conjugated anti-rabbit IgG (NA934V) was purchased from GE Healthcare Life Sciences (Sweden). Bio-Rad protein assay (500-0006) was purchased from

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Bio-Rad. [Methyl-3H]-thymidine (NET027A001MC), Filtermat A (1450-421) and Ultima Gold XR (6013111) were purchased from PerkinElmer (USA). Scintillation vials (KART933) were

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purchased from VWR International (USA). 5-Bromo-2’-deoxyuridine (BrdUrd) and propidium iodide (PI) were purchased from Sigma, (USA). The antibody against BrdUrd (M744) and the

Cell lines

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secondary-FITC-conjugated antibody (F3213) were purchased from Dako patts (Denmark).

Chemicals can give rise to different toxicological effects depending on what cell model they are tested in. Therefore, we chose to study the effect of acrylamide on neuronal differentiation as indication of developmental neurotoxicity in two different cell lines. The murine neural progenitor cell line C17.2 (Snyder et al., 1992) was a generous gift from Professor Sandra Ceccatelli (Karolinska Institute, Stockholm, Sweden) with permission of Professor Evan Snyder (Harvard Medical School, Boston, USA). The C17.2 cell line is a multipotent

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ACCEPTED MANUSCRIPT progenitor cell line that can differentiate into neurons and glia cells as a mixed cell population (Lundqvist et al., 2013).

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The human neuroblastoma SH-SY5Y cell line (Biedler et al., 1978) has been shown to be a

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very useful model system to study neuronal differentiation and function (Agholme et al.,

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2010). The SH-SY5Y cells develop into a neuronal-like phenotype upon differentiation with

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all-trans-retinoic acid (Påhlman et al., 1990).

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Cell culturing

For routine cultures, the C17.2 cells were seeded at a density of 1.27 x 103 cells/cm2 in cell

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culture Petri dishes. They were cultured in DMEM supplemented with 5% horse serum, 10%

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fetal calf serum, 2 mM L-glutamine, 100 U penicillin/mL and 100 μg streptomycin/mL. The

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confluent cells were detached every 3rd and a half day using 0.05/0.02% trypsin/EDTA and seeded in a new cell culture Petri dish at the original density. For differentiation studies, the C17.2 cells were seeded in routine culture medium (see individual experiment for density). Twenty-four hours after seeding the medium was changed to DMEM/F-12 medium supplemented with 1 mM L-glutamine, 100 U penicillin/mL, 100 μg streptomycin/mL, modified N2 supplements (to a final concentration of 5 μg/mL bovine insulin, 20 nM progesterone, 30 nM sodium selenite, 100 μg/mL bovine apo-transferrin, and 100 μM putrescine dihydrochloride), 10 ng/mL NGF and 10 ng/mL BDNF. The differentiation medium was changed every 3rd day for the duration of the differentiation. For routine cultures, the SH-SY5Y cells were seeded at a density of 27 x 103 cells/cm2 in cell culture flasks. They were cultured in MEM supplemented with 10% fetal calf serum, 1% nonessential amino acids, 2 mM L-glutamine, 100 μg streptomycin/mL, 100 U penicillin/mL. The 7

ACCEPTED MANUSCRIPT confluent cells were detached once a week using 0.05/0.02% trypsin/EDTA and seeded in a new cell culture flask at the original density. For differentiation studies, the SH-SY5Y cells

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were seeded in routine culture medium (see individual experiment for density). Twenty-four

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hours after seeding the medium was changed to DMEM/F-12 medium supplemented with 1

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mM L-glutamine, 100 U penicillin/mL, 100 μg streptomycin/mL, modified N2 supplements as described above and 1 µM of all-trans-retinoic acid. The differentiation medium was changed

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every 3rd day for the duration of the differentiation. Both cell lines were kept in a humidified

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atmosphere of 5% CO2 in air at 37oC.

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Acrylamide exposure

Acrylamide was dissolved in the culture medium used for the different experiments. Before

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addition to the cells, the acrylamide stock solution was sterile-filtered through a 0.2 µm filter and then diluted to different concentrations ranging from 1 fM to 2 mM. Cell cultures exposed to medium without acrylamide were used as a control. For all experiments, the cells were exposed to acrylamide 24 hours after seeding, together with the change of the culture medium (change to differentiation medium for the differentiation studies and replacement to fresh routine culture medium for proliferation studies). Acrylamide was added at every change of medium throughout the duration of the experiment. Fresh medium without acrylamide was added to control cells. A fresh stock solution of acrylamide was prepared right before addition to the cells for each exposure time. Exposure time and concentrations differs between the two cell lines. Since the C17.2 cells are multipotent progenitor cells and hence, display a more immature starting point than the SH-

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ACCEPTED MANUSCRIPT SY5Y cells, the C17.2 cells were differentiated for 6 and 10 days while the SH-SY5Y cells were only differentiated for 3 and 6 days. The two time periods of differentiation for each cell

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line were chosen to reflect two different time windows of neuronal development.

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Growth curve experiments

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The C17.2 cells were seeded in routine culture medium in 10 cm diameter cell culture Petri dishes at a density of 1.27 x 103 cells/cm2. The SH-SY5Y cells were seeded in routine culture

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medium at a density of 27 x 103 cells/cm2 in cell culture Petri dishes with a diameter of 6 cm. For both cell lines, the cells were exposed to 10, 100 and 1000 µM of acrylamide for 24, 48

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and 72 hours. The cells were detached using 0.05/0.02% trypsin/EDTA and counted in a

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Bürker chamber using a phase contrast microscope.

5-Bromo-2’-deoxyuridine cell proliferation assay The C17.2 cells were seeded in routine culture medium at a density of 1.27 x 103 cells/cm2 in cell culture Petri dishes with a diameter of 6 cm. The SH-SY5Y cells were seeded in routine culture medium at a density of 27 x 103 cells/cm2 in cell culture Petri dishes with a diameter of 6 cm. For both cell lines, the medium was changed 24 hours after seeding and the cells were simultaneously exposed to acrylamide concentrations ranging between 1-1000 µM. The C17.2 cells were exposed for 3 days and the SH-SY5Y cells were exposed for 3 and 6 days. The C17.2 cells were exposed for 3 days since they cannot be kept in culture for a longer period of time without spontaneously differentiating. One hour before harvesting, BrdUrd was added to a

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ACCEPTED MANUSCRIPT final concentration of 5 µM. The cells were detached using 0.05/0.02% trypsin/EDTA, centrifuged for 5 minutes at 4°C at 600g and the pellet was resuspended in 1 mL of 70% ice-

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cold ethanol and stored at -20°C until further analysis. The cells were prepared for analysis of

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DNA and BrdUrd contents according to Fredlund et al. (1994). In short, the cells were

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incubated in a pepsin solution to isolate cell nuclei and partially degrade chromatin-associated proteins. To allow access of the primary BrdUrd antibody to BrdUrd incorporated into DNA,

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the DNA was then partially denatured by incubating the cells in 2 M HCl. A secondary FITCconjugated antibody was used to detect the primary antibody. The double-stranded regions of

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DNA were then stained with PI. The nuclei were analyzed with respect to DNA content (red fluorescence from PI) and BrdUrd (green fluorescence from FITC) in a BD Accuri C6 flow

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cytometer (BD Biosciences) and analyzed using the MultiCycle® software (Phoenix Flow Systems). After gating of nuclei in a PI fluorescence area versus height cytogram to exclude

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doublets, the DNA versus BrdUrd cytogram was displayed. Three regions were set in the cytogram: one around the total population, one around the BrdUrd negative nuclei and one around the BrdUrd positive nuclei. The data were used to calculate the percentage of BrdUrd positive cells for each sample.

Flow cytometric analysis of Sub-G1 region of the DNA histogram The C17.2 cells were seeded in routine culture medium at a density of 1.27 x 103 cells/cm2 in cell culture Petri dishes with a diameter of 6 cm. The SH-SY5Y cells were seeded in routine culture medium at a density of 27 x 103 cells/cm2 in cell culture Petri dishes with a diameter of 6 cm. For both cell lines, the medium was changed 24 hours after seeding and the cells were

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ACCEPTED MANUSCRIPT simultaneously exposed to acrylamide concentrations ranging between 1-1000 µM. The C17.2 cells were exposed for 3 days and the SH-SY5Y cells were exposed for 3 and 6 days. The

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C17.2 cells were exposed for 3 days only since they cannot be kept in culture for a longer

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period of time without spontaneously differentiating. The cells were detached using

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0.05/0.02% trypsin/EDTA and pooled with detached cells floating in the medium. The cells were centrifuged for 5 minutes at 4°C at 600g and the pellet was resuspended in 1 mL of 70%

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ice-cold ethanol and stored at -20°C until further analysis. The cells were washed in 10 mL of PBS, pelleted and resuspended in PBS containing 100 µg/mL PI, 0.6% NP-40 and 100 µg/mL

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ribonuclease A. The cell nuclei were analyzed in a BD Accuri C6 flow cytometer (BD Biosciences) and analyzed using the MultiCycle® software (Phoenix Flow Systems). After

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gating of nuclei in a PI fluorescence area versus height cytogram to exclude doublets, the DNA histogram including the sub-G1 region with signals from dead cells was displayed. A region

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was set around all signals in the histogram and another region around the sub-G1/G0 signals i.e. not including the proper DNA histogram of G1/G0, S, and G2 cells. M phase cells are not included in the assay since they do not contain a nuclear membrane and are removed by the PI staining solution when the cell membrane is disrupted. The data were then used to calculate the percentage sub-G1 signals, which is a representation of cell death.

Number of viable cells The number of viable cells was measured using the MTT assay (Mosmann, 1983). For all experiments, the cells were seeded in 96-well plates. For viability studies in undifferentiated neural progenitor cells, the C17.2 cells were seeded at a density of 1.27 x 103 cells/cm2 and the

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ACCEPTED MANUSCRIPT SH-SY5Y cells were seeded at a density of 27 x 103 cells/cm2. Acrylamide was added 24 hours after seeding to a final concentration between 1-2000 µM. The C17.2 cells were exposed for 3

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days and the SH-SY5Y cells were exposed for 3 and 6 days. The C17.2 cells were exposed for

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3 days only since they cannot be kept in culture for a longer period of time without

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spontaneously differentiating.

To examine the number of viable cells after exposure to differentiating cells, C17.2 cells were

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seeded at a density of 3.75 x 103 cells/cm2 and SH-SY5Y cells were seeded at a density of 12.5

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x 103 cells/cm2. Acrylamide was added in differentiation medium 24 hours after seeding, in a range between 1 nM and 1 mM for C17.2 cultures and from 10 fM to 1 mM for SH-SY5Y

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cultures. The C17.2 cells were exposed for 6 and 10 days and the SH-SY5Y cells were exposed

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for 3 and 6 days during differentiation. After the desired exposure time, 10 μL of 0.5 mg/mL MTT solution in PBS were added to each well. The plate was incubated in a humidified

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atmosphere of 5% CO2 in air at 37oC for 1 hour. After 1 hour, the medium was carefully removed and the formazan crystals were dissolved in 100 µL of DMSO while placed on a shaker. The formazan dye product was quantified with SunriseTM microplate absorbance reader (TECAN) at 540 nm.

Determination of protein level The Lowry assay (Lowry et al., 1951) with some small modifications was used to measure the protein level, reflecting the amount of SH-SY5Y cells per well in control cells or after exposure to acrylamide during differentiation. The SH-SY5Y cells were seeded and treated in exactly the same way as for the MTT assay until the protein determination. After 3 or 6 days of

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ACCEPTED MANUSCRIPT exposure, the medium was removed and the cells were washed with 100 µL of PBS. Afterwards, the cells were lysed in 70 µL of 0.1 M NaOH. Fifty μL of cell lysate from each

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well were transferred to a 96-well plate for protein determination. On the day of the

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measurement, 2 ml of equal volume of 1% CuSO4 and 2% Na2C4H4O6 (w/v) were added to 2 g

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of Na2CO3 and 0.4 g of NaOH in 100 mL Milli-Q® water. From this solution, 200 μL were added to each well. The plate was carefully shaken for exactly 10 minutes. Fifty μL of Folin’s

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reagent (diluted 1:4 in Milli-Q® water) were added to each well and carefully shaken on a plate shaker for exactly 30 minutes in darkness. The absorbance was measured at 690 nm using a

H-Thymidine incorporation proliferation assay

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SunriseTM microplate absorbance reader (TECAN).

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The SH-SY5Y cells were seeded and treated in exactly the same way as for the MTT and the Lowry assay. Twenty-four hours before harvesting, the cells were incubated with 1 µCi/well of 2 Ci/mmol, 1 mCi/mL [methyl-3H]-thymidine. After 3 or 6 days of exposure, the medium was removed and the cells were washed 2 times with 100 µL of PBS. Afterwards, the cells were lysed in 100 µL of hypotonic buffer containing NP-40 for 30 minutes on ice during gentle shaking. The lysates were harvested on to a glass fiber filter, washed with PBS and left to dry at room temperature. Once dry, the filters were collected in scintillation vials and covered with 3 ml of scintillation fluid. The samples were analyzed using a Packard tri-carb liquid scintillation analyzer (PACKARD).

Real-time reverse transcription polymerase chain reaction 13

ACCEPTED MANUSCRIPT The C17.2 cells were seeded in 6 cm diameter culture dishes at a density of 3.7 x 103 cells/cm2. Twenty-four hours after seeding, the medium was changed to differentiation medium. At the

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same time, 10 or 100 µM of acrylamide was added to the exposed cells. The medium was

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changed every 3 days. After 6 and 10 days the cells were harvested by trypsinization and cell

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pellets were collected. Cells were centrifuged for 5 minutes at 500 g and the pellet was stored at -80oC until mRNA extraction. On the day of the experiment, the cells were lysed and mRNA

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was extracted using the Qiagen RNeasy Plus Mini Kit according to manufacturer’s instructions. The mRNA concentration in each sample was determined by using a

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NanoPhotometerTM P-class (IMPLEN GmbH). To assess RNA integrity, samples were prepared together with sample buffer from the RiboRuler High Range RNA ladder kit and

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separated on a 1% agarose gel containing 18% of a 37% formaldehyde solution for approximately 2 hours at 110V. The gel was photographed using ChemiDocTM XRS+ (Bio-

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Rad) and the results were analyzed by using the Image Lab 3.0 software program. Two μg of each sample were reverse transcribed into cDNA using oligo (dT)18 primer and RevertAidTM H Minus First Strand cDNA Synthesis Kit. Real-time qPCR reactions were carried out as described before (Lundqvist et al., 2013) in an iCycler iQ5TM machine (Bio-Rad) using Maxima® SYBR Green/Fluorescein qPCR Master Mix. The samples were normalized against the reference gene TATA box binding protein (TBP). The following primer sequences were used;

TBP

5’-GAATTGTACCGCAGCTTCAAAA-3’

AGTGCAATGGTCTTTAGGTCAAGTT-3’

(reverse),

(forward)

and

βIII-tubulin

5’5’-

CAGGCCCGACAACTTTATCT -3’ (forward) and 5’-CTCTTTCCGCACGACATCTA-3’ (reverse),

GFAP

5’-GATCGCCACCTACAGGAAAT-3’

GGATCTGGAGGTTGGAGAAA-3’ (reverse).

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(forward)

and

5’-

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Western blot The C17.2 cells were seeded in 10 cm diameter culture dishes at a density of 3.7 x 103

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cells/cm2. Twenty-four hours after seeding, the medium was changed to differentiation

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medium. At the same time, 10 or 100 µM of acrylamide was added to the exposed cells. The

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medium was changed every 3 days. The SH-SY5Y cells were seeded in 10 cm diameter culture dishes at a density of 12.5 x 103 cells/cm2. Twenty-four hours after seeding, the medium was

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changed to differentiation medium. At the same time, acrylamide in ranging concentrations between 1 pM-1000 µM was added to the exposed cells. As for the C17.2 cells, the medium

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was changed every 3 days. After 6 and 10 days for the C17.2 cells and 3 and 6 days for the

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SH-SY5Y cells, the cells were lysed in a hypotonic buffer containing NP-40 and 20-30 μg of

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the protein lysate, determined by the Bradford assay according to manufacturer’s instructions, were separated in a 10% SDS- polyacrylamide gel. The proteins were transferred to nitrocellulose membranes and incubated overnight at 4oC with rabbit anti-βIII-tubulin (1:5000) or rabbit anti-GFAP (1:1000) primary antibodies. Horseradish peroxidase-conjugated antirabbit IgG (1:50000 for GFAP and 1:3000 for βΙΙΙ-tubulin) was used as secondary antibody. Goat anti-β-actin (1:5000) was used as a loading control for C17.2. Horseradish peroxidaseconjugated anti-goat IgG (1:3000) was used as secondary antibody for β-actin. The membranes were developed using ChemiDocTM XRS+ image system (Bio- Rad) and the results were analyzed by using the Image Lab 3.0 software program (Bio-Rad).

Immunofluorescence microscopy

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ACCEPTED MANUSCRIPT The C17.2 cells were seeded on glass coverslips (VWR collection, VWR International, Stockholm, Sweden) in 24-well plates at a density of 1.47 x 103 cells/cm2. Twenty-four hours

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after seeding, the medium was changed to differentiation medium. At the same time, 10 or 100

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µM of acrylamide was added to the exposed cells. The medium was changed every 3 days.

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After 6 and 10 days, the medium was removed and the cells were fixed in ice cold 100% MeOH for 5 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS for 10 min

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and blocked for 10 min in AbDil (0.1% Triton- X 100 in buffer for immunofluorescence staining of cytoskeletal proteins [10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl2, and 2 mM

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EGTA, 0.32 M sucrose] supplemented with 2% BSA and 0.1% azide dilution). Abdil was stored at 4°C and the sucrose was added on the day of the experiment. The cells were

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incubated overnight at 4oC with primary antibodies rabbit, i.e. anti-GFAP (1:100) or rabbit anti-βΙΙΙ tubulin (1:1000) in AbDil. Anti-rabbit conjugated with AlexaFluor488 (1:2500) in

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PBS 2% BSA was used as a secondary antibody and nuclei were stained with DRAQ5 (1:10000) in AbDil. The coverslips were mounted on slides with SouthernBiotech Fluoromount-GTM and left to dry overnight. The immunofluorescence was visualized with a fluorescence microscope Leica DM/IRBE 2. Images were taken with Hamamatsu Orca ER CCD camera and analyzed by Micro-Manager (Edelstein et al., 2010) and ImageJ software program.

Ratio of different cell populations The immunofluorescence images were used to calculate the ratio of βIII-tubulin positive cells and the βIII-tubulin negative cells, i.e. the progenitor cells and the astrocytes. For each image,

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ACCEPTED MANUSCRIPT the total number of DRAQ5 stained nuclei and the number of βIII-tubulin positive cells was calculated. The remaining cells were considered as the βIII-tubulin negative cells. Two images

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were depicted for each biological replicate and the mean value for each situation was

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calculated. The mean value was considered as one n. The number of nuclei counted for each

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picture was between 100-200, depending on the concentration of acrylamide exposure. The

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statistical analysis for this experiment was based on n=3, i.e. 3 individual experiments.

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Morphological evaluation

C17.2 cells were seeded and treated as for the western blot experiment. After 6 and 10 days the

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cells were photographed using a phase contrast microscope (Olympus). Microscopy images

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were captured at 200x magnification using a CCD camera (Olympus DP50).

Neurite outgrowth

C17.2 cells were seeded in 6-well plates at a density of 1.47 x 103 cells/cm2. Twenty-four hours after seeding, the medium was changed to differentiation medium. At the same time, acrylamide was added to the exposed cells in a range from 100 nM to 100 µM. SH-SY5Y cells were seeded in 6-well plates at a density of 4.8 x 103 cells/cm2. Twenty-four hours after seeding, the medium was changed to differentiation medium. At the same time, acrylamide was added to the exposed cells in a range from 1 pM to 100 µM. The medium was changed every 3 days for both cells lines. Each seed out consisted of biological duplicates (i.e. two wells per situation). After 3 and 6 days for the SH-SY5Y cells and 6 and 10 days for the C17.2

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ACCEPTED MANUSCRIPT cells, the cells were photographed using a phase contrast microscope (Olympus). Microscopy images were captured at 150x magnification using a CCD camera (Olympus DP50). Cell

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bodies and neurites that were longer than the diameter of the cell body were counted and the

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number of neurites per cell was estimated as previously described (Nordin-Andersson et al.,

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1998). For the C17.2 cell line, the images were also used to determine the percentage of neurons in the cell culture. Two pictures were taken for each biological replicate and the mean

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value for each situation was calculated. The mean value was considered as one n. The number of cell bodies counted for each picture was between 60-120, depending on the concentration of

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acrylamide exposure. The statistical analyzes for this experiment was based on n=3, i.e. 3

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Statistical analysis

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individual experiments, each performed in duplicates.

GraphPad Prism 6.0 was used for statistical analysis of the data. Results were analyzed using one- or two-way ANOVA followed by Fischer’s LSD test, *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 as compared to control.

Results Acrylamide affects proliferation of neural progenitor and neuroblastoma cells During the initial phase of brain development there is an expansion of neural progenitor cells. Studying the proliferation of undifferentiated neuronal and glial precursors can be used as a

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ACCEPTED MANUSCRIPT model to mimic the expansion of progenitor cells and hence, the proliferative stage of neural development (Jaaro-Peled et al., 2009). Figure 1A shows that, during 48 hours of exposure to

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1000 µM of acrylamide, the proliferation was significantly reduced in the undifferentiated

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neural progenitor cells C17.2. In the undifferentiated neuroblastoma SH-SY5Y cells, exposure

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to 1000µM of acrylamide reduced the number of viable cells to the extent of being even fewer cells than were initially seeded in the cell culture dishes (Figure 2A). After 72 hours of

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exposure to 100 µM of acrylamide, the number of undifferentiated C17.2 cells was significantly reduced. The number of viable cells in the two undifferentiated cell lines was

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measured with the MTT assay after 3 days of acrylamide exposure (Figures 1B and 2B). Both cell lines showed that 750 µM of acrylamide significantly reduced the number of viable cells at

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this time point. For the SH-SY5Y cells, which can be grown for a longer time without spontaneously differentiating or undergoing apoptosis, the viability was also measured after 6

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days of exposure. Prolonged exposure showed a significant reduction in the number of viable cells, i.e. decreased formazan formation, already at 250 µM of acrylamide in comparison with control cells. For both cell lines, the reduction in cell number and cell viability during acrylamide exposure was due to a combination of both reduced cell proliferation and increased cell death (seen in Figure 1C for the C17.2 cell line and in Figures 2C and 2D for the SHSY5Y cell line). For both cell lines, cell death (analyzed as sub-G1) seems to occur at lower concentrations as compared to the attenuated cell proliferation, i.e. reduction in BrdUrd positive cells. These results show that acrylamide affects cell proliferation and induce cell death in undifferentiated neural progenitor cells and neuroblasts. It also pinpoints the fact that the toxicity of acrylamide is correlated to duration of exposure. The cell cycle phases were

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ACCEPTED MANUSCRIPT analyzed after PI staining but did not reveal any significant changes. However, there was a

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slight indication of a G0/G1-block after acrylamide exposure (data not shown).

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Acrylamide affects neuronal differentiation and the viability of differentiating nerve cells

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Both cell lines were exposed to a wide range of acrylamide concentrations throughout 6 and 10

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days of differentiation for the C17.2 neural progenitor cells and for 3 and 6 days of differentiation for the neuroblastoma SH-SY5Y cells. The different durations of exposure were

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used to reflect different exposure windows during neurodevelopment. The amount of viable cells was measured after the same time points using the MTT assay. Acrylamide significantly

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reduced the number of viable C17.2 cells during differentiation in a time- and concentration-

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dependent manner, starting at 100 µM during 6 days of exposure and at 10 µM during 10 days

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of exposure (Figure 3). The number of viable SH-SY5Y cells significantly increased during 6 days of exposure to 10 pM-10 µM of acrylamide, followed by a decrease during 3 and 6 days of exposure to 100 µM or higher concentration (Figure 4A). To confirm that the increase in MTT reduction reflected the amount of viable cells as a result of increased cell proliferation rather than increased metabolic rate, the 3H-thymidine incorporation proliferation assay was performed (Figure 4B). The assay followed the same trend as the MTT viability assay and displayed a significant increase in 3H-thymidine incorporation for concentrations between 1 fM-10 μM, followed by a decrease in 3H-thymidine incorporation at higher concentrations. The total cellular protein concentration was determined in the same experimental setup. The cellular protein concentrations followed the same trend as the MTT viability assay and the 3Hthymidine incorporation assay (Figure 4C). Taken together, these results manifests that very low concentrations of acrylamide increased the number of cells due to increased proliferation 20

ACCEPTED MANUSCRIPT and that higher concentration than 500 µM induced cytotoxicity and results in a decrease in

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cell number in comparison to unexposed control cells.

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Acrylamide changes the expression of biomarkers for neuronal and glial cell

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differentiation in C17.2 cells

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One way of studying neuronal and glial differentiation is to study the expression levels of mRNA and protein biomarkers that are significant for differentiation. βIII-Tubulin is a

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biomarker for semi-mature neurons (Menezes and Luskin, 1994) and it was selected as the biomarker for neuronal differentiation. Glial fibrillary acidic protein is the most commonly

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used biomarker for astrocytes (Bramanti et al., 2010) hence it was used as indicator of

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astrocytes in the differentiated C17.2 cell cultures (Lundqvist et al., 2013).

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Exposure to 100 µM of acrylamide for 10 days slightly reduced the mRNA level and significantly reduced the protein level of βIII-tubulin (Figures 5A and 5B). The same trend was seen for GFAP, where 100 µM of acrylamide exposure for 10 days significantly reduced the mRNA and protein levels (Figures 5C and 5D). No significant change in mRNA or protein expression of neither βIII-tubulin, nor GFAP was observed after 6 days of exposure (Figure 5). The attenuated biomarker expression was also confirmed by immunofluorescence microscopy. By looking at the immunofluorescence of βIII-tubulin-positive cells after exposure to 10 µM and 100 µM acrylamide, it was clear that the population was reduced (Figure 6A-C). This was confirmed by calculating the ratios for the two different cell populations i.e. βIII-tubulin positive cells representing the neuronal cell population and βIII-tubulin negative cells representing the progenitor and the astrocytic cell populations (Figure 6K). The fraction of

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ACCEPTED MANUSCRIPT βIII-tubulin positive cells decreased while the fraction of βIII-tubulin negative cells increased. The number of neurons was significantly reduced after exposure to 1 μM of acrylamide (Figure

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6J). Interestingly, although the number of neurons decreased, the numbers of neurites per cell

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was not affected by acrylamide exposure in the C17.2 cell line (Figure 6J). The phase contrast

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images (Figure 6G-I) further display that the cell cultures consisted of two distinctly different morphological phenotypes. The neurons grew on top of the astrocytes and it was possible to

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see that acrylamide exposure reduced the fraction of neurons in differentiating C17.2 cells. Acrylamide exposure also reduced the fluorescence intensity for the GFAP-positive cell

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population (Figure 6D-F). Thus, acrylamide reduced the levels of both the neuronal and the

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glial biomarker during differentiation of the multipotent neural progenitor C17.2 cells.

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Acrylamide impairs neurite outgrowth and βIII-tubulin expression in differentiating SHSY5Y neuroblastoma cells

The SH-SY5Y cell line is a commonly used cell model for studying neurite degeneration and neurite outgrowth (Nordin-Andersson et al., 2003). In this experiment, the SH-SY5Y cells were exposed to acrylamide during differentiation, and after 3 and 6 days the number of neurites per cell was evaluated. The number of neurites was significantly reduced after 6 days of exposure to 10 pM of acrylamide and by 100 nM after 3 days of exposure (Figure 7). Taken together, acrylamide impairs neurite outgrowth in the SH-SY5Y cell line during differentiation in a time- and concentration-dependent manner. To further investigate how the differentiation process was affected by acrylamide exposure we measured the level of βIII-tubulin with western blot. As mentioned above, βIII-tubulin is a biomarker for semi-mature neurons.

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ACCEPTED MANUSCRIPT Acrylamide exposure slightly reduced the level of βIII-tubulin in differentiating SH-SY5Y

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cells after 3 and 6 days of differentiation (Figure 7J).

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Discussion

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People are exposed to acrylamide throughout their lifespan, starting in utero. After birth, the exposure to acrylamide continues via the breast milk, intake of food and drinks, but also from

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the environment. In this study, concomitantly with several earlier studies (reviewed by (LoPachin, 2004)), it was shown that the neurotoxic effects of acrylamide are concentration-

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dependent and also accumulate over time. It seems like it is not the accumulation of acrylamide

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itself, but rather the accumulation of damages over prolonged exposure time that leads to

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neurotoxicity (Crofton et al., 1996). With this in mind, it raises the question about the developmental consequences of prenatal acrylamide exposure and what the tolerated daily intake of acrylamide is during pregnancy. In the WHO report from 2005, it was estimated that the average consumption of acrylamide was 0.8-3 μg acrylamide/kg/day for adults. However, the consumption in children was estimated to be 2–3 times higher when expressed as a body weight ratio (World Health Organization, 2005). Aside from the fact that children eat higher amount of food relative to their size, foodstuff containing relatively higher levels of acrylamide is also preferred by children e.g. cereal, French fries and potato chips (Cengiz and Gündüz, 2013). In this study we show that acrylamide-induced effects on early phases of neurodevelopment, such as the expansion of neural progenitor cells and neuroblasts, but also during the differentiation process affecting biomarkers for neural differentiation and axonal outgrowth. We show that acrylamide impaired neural differentiation in the SH-SY5Y cell line 23

ACCEPTED MANUSCRIPT at nine orders of magnitude and in the C17.2 cell line at two orders of magnitude lower concentration than previously reported for other neural cell systems (Chen and Chou, 2015,

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Sisnaiske et al., 2014). The previous studies displayed significant effects of acrylamide on

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developmental endpoints at 0.5 mM, i.e. close to the cytotoxic concentration of acrylamide,

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which has been reported to be around 1 mM (Nordin-Andersson et al., 2003, Cody et al., 2013). It also raises a question about the physical relevance of the previously tested

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concentrations. There are only few reports on the plasma level of acrylamide in the general population, but a study on exposed workers showed that the unexposed control group had a

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concentration of free acrylamide in plasma of approximately 1 µM (Calleman et al., 1994). With the placental transfer of acrylamide being around 20% (Sörgel et al., 2002), a rough

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estimation indicates that the fetus might be exposed to an acrylamide concentration around 0.2 µM. Our results show that in one of the cell lines, SH-SY5Y, acrylamide induced significant

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effects on differentiation starting at 10 fM, which is seven orders of magnitude lower than the estimated plasma concentration of free acrylamide in the fetus. For the undifferentiated cells, acrylamide reduced the number of viable cells in both cell lines. By performing assays for both proliferation and cell death, we could conclude that the reduction in viability seen from the MTT assay was caused by a combination of cell death and reduced proliferation in both cell lines (Figures 1 and 2). However, it seems like cell death occurs at lower concentrations than what affects cell proliferation. Comparing the two cell lines, it seems like the neural progenitor cells are slightly more sensitive to acrylamide exposure after 3 days of exposure, showing significant increase in cell death starting at 500 μM. As mentioned above, the physiological relevance of these concentrations can be questioned. However, one should keep in mind that the exposure scenario is far shorter in vitro 24

ACCEPTED MANUSCRIPT than the chronic exposure that the fetus is exposed to in the uterus and that acrylamide toxicity is closely linked to the duration of exposure.

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The SH-SY5Y cells undergo differentiation and become post-mitotic when exposed to all-

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trans-retinoic acid (Påhlman et al., 1990). The MTT reduction assay and the protein

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concentration were measured after exposing the SH-SY5Y cells to acrylamide during differentiation (Figure 4). Both assays showed that low concentrations of acrylamide (1 pM-10

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μM) increased the number of viable cells as well as the protein concentration. The 3H-

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thymidine incorporation assay was performed with the same experimental setup as the two previous assays to see if the increase in MTT reduction and protein concentration was due to

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increased proliferation. The 3H-thymidine incorporation assay confirmed that the SH-SY5Y

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cells, although exposed to all-trans-retinoic acid, increased the proliferation compared to

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control when exposed to low concentrations of acrylamide (Figure 4B). In the control situation, the differentiation process in the SH-SY5Y cells begins at the start of all-retinoic-acid exposure. However, the entire cell population is not synchronized in the cell cycle. Hence, some cells enroll the differentiation process before others. Acrylamide seems to attenuate the differentiation process into post-mitotic neurons, resulting in increased proliferation giving an increased number of cells. In conclusion, concentrations lower than 10 µM did not seem to display any cytotoxicity but attenuated the differentiation process in neurons. No increase in the number of cells during exposure to low concentrations of acrylamide as compared to control cells was observed in the C17.2 cell line (Figure 3). This may be explained by several observations. Firstly, the C17.2 cell line consisted of several different cell populations. The proliferation of a population of the progenitor cells, probably astrocyte precursors, proceeded during differentiation even in the control situation. Since there were fewer neurons than 25

ACCEPTED MANUSCRIPT astrocytes in the cultures, an increase in proliferation in this smaller cell population (neurons) would be harder to detect above the background noise. Secondly, the two cell lines were

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differentiated via different pathways. The attenuation of differentiation in the SH-SY5Y cells

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might be a result from acrylamide interfering with the all-trans-retinoic acid signaling and

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hence, would not affect the differentiation of C17.2 cells in the same way. Acrylamide have been shown in previous studies to change the expression and status of different MAP kinases

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(Duan et al., 2015). All-trans-retinoic acid signals through retinoic acid receptors and retinoid X receptors in retinoic acid mediated transcription. A subset of retinoic acid receptors signals

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through MAP kinases (Piskunov and Rochette-Egly, 2012) and acrylamide interference with these kinases could result in aberrant transcription of differentiation related genes. Thirdly, the

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C17.2 cell cultures consisted of a mixed culture of neurons and astrocytes. Astrocytes have a pivotal role in the function of neurons. Astrocytes possess some metabolic capacity that may

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affect the concentration of acrylamide (Sagara and Sugita, 2001). Furthermore, astrocytes synthesize and secrete a wide range of cytokines, neurotrophic factors, growth factors, proteoglycans, extracellular matrix proteins and cholesterol that are involved in neuronal proliferation, differentiation, synaptogenesis and survival (Blondel et al., 2000). These factors might further stimulate the differentiation process and compensate for the acrylamide exposure. However, the number of neurons decreased in the C17.2 cell cultures during acrylamide exposure. Although the neurotoxic concentrations were not as low as for the SHSY5Y cells, there was attenuated differentiation of the neuronal precursor population also in the C17.2 cell culture starting at 1 μM of acrylamide exposure (Figure 6J,K). For both cell lines, higher concentrations, starting at 10 µM for C17.2 cells and 100 µM for SH-SY5Y cells, reduced the number of viable cells and showed signs of cytotoxicity. Again, the differentiating

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ACCEPTED MANUSCRIPT C17.2 and SH-SY5Y cell models were more sensitive to acrylamide than other neuronal cell models, here regarding cytotoxicity (Chen et al., 2014, Walum and Flint, 1993).

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Axonal outgrowth was affected by acrylamide in the SH-SY5Y cell line. There was a reduction

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in the number of neurites per cell in the SH-SY5Y cells, starting at a concentration of 10 pM of

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acrylamide (Figure 7), which is an indication of a less differentiated neuronal cell culture. These results further strengthen the observation of increased proliferation and attenuated

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differentiation seen in Figure 4. The inhibition or delay of axonal outgrowth might be due to

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conjugation of acrylamide to neurofilaments and components of the microtubular system that are important for proper axonal function (Tandrup and Jakobsen, 2002). Furthermore, it could

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also be a consequence of the multiple possible conjugation-targets of acrylamide in the nerve

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terminal (Barber et al., 2007). The attenuated neuronal differentiation in the SH-SY5Y cell line

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was also observed when looking at the levels of βIII-tubulin (Figure 7J), which was slightly reduced during acrylamide exposure. The fact that there was less difference seen after 6 days of differentiation compared to 3 days might be explained by the fact that βIII-tubulin is a biomarker for semi-mature neurons, and is not as significantly expressed in more mature cultures.

Both the βIII-tubulin-positive cell population and the GFAP-positive cell population were reduced by 100 µM of acrylamide exposure during differentiation. The reduced mRNA expression of both biomarkers indicates that acrylamide did not only change the protein levels of these biomarkers, but also had an effect at the transcriptional level of gene expression (Figures 5A and 5C). It should be noted, though, that 100 µM acrylamide generated approximately 15% reduced number of viable cells. Acrylamide exposure decreased the number of neurite-bearing C17.2 progenitor cells when looking at the immunofluorescence 27

ACCEPTED MANUSCRIPT staining with βIII-tubulin and also the phase contrast images (Figure 6). However, the number of neurites per cell was not affected by acrylamide exposure. One explanation between the

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differences seen by the two cell lines might be that the astrocytes in the mixed C17.2 cell

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culture can buffer some of the neurotoxicity, e.g. by providing other conjugation-sites for

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acrylamide such as glutathione (Kurebayashi and Ohno, 2006). Also, the neurons in the C17.2 cell culture grew on top of the astrocytes providing neurotrophic support, whereas the SH-

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SY5Y cells grew directly on the tissue culture treated plastic. One should also mention that the two different cell lines are derived from two different species, giving rise to possible

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interspecies differences. The two cell lines also derived from two different origins of the nerve system; the neural progenitor cells were isolated from cerebellum and the neuroblastoma cells

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derived from the neural crest. As mentioned above, their differentiation processes might differ

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in the sense of what receptors and pathways are involved. In conclusion, our results indicate that acrylamide affected both proliferation and differentiation in two different neuronal cell models. The fact that acrylamide seemed to attenuate differentiation at low concentrations raises questions about possible developmental neurotoxicity. The incidence of children being diagnosed with disorders that are associated with impaired cognition is increasing. A report from the National Academy of Sciences states that 28% of all major developmental disorders in children are linked entirely or partly to environmental exposures (National Research Council, 2000). Three percent of these being a direct result of environmental exposure to chemicals and the other 25% arise from interactions between environmental factors and specific genetic factors, making that particular individual susceptible (Grandjean and Landrigan, 2006). The present study suggests and the differences between the two cell lines should be further evaluated in regards to their sensitivities to 28

ACCEPTED MANUSCRIPT acrylamide. The difference might be able to provide some insight to the toxicological

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mechanism of acrylamide.

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Funding

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The study was financed by grants from the Swedish Research Council (K2013-79X-2137305-

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3) and Carolina LePrince with the "Kalenderflickorna" and associated sponsors.

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Acknowledgments

The authors are very grateful to Dr. Ricardo Figueroa for the support using the beta

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scintillation counter and with fluorescence microscopy. The authors would also like to thank Ewa Dahlberg for the technical assistance in the BrdUrd and DNA sample preparation procedure.

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Figure captions

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Figure 1. The effect of acrylamide on proliferation and the number of viable cells in

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undifferentiated C17.2. A) Number of C17.2 cells after exposure to 10, 100 or 1000 µM

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acrylamide. The arrow indicates the start of exposure. The data are presented as the mean of 5 independent experiments performed in duplicates. B) Viability of the C17.2 cells after 3 days

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of acrylamide exposure at different concentrations. The data are presented as the mean of 4 independent experiments performed in hexaplicates. C) Cell proliferation displayed as BrdUrd

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positive cells and cell death displayed as Sub-G1 region after 3 days of acrylamide exposure at

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different concentrations. The data are presented as the mean of 3 independent experiments.

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Results were analyzed using one-way ANOVA followed by Fischer’s LSD test. A-C) Bars represent the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to control (cells exposed to medium without acrylamide).

Figure 2. The effect of acrylamide on proliferation and the number of viable cells in undifferentiated SH-SY5Y. A) Number of SH-SY5Y cells after exposure to 10, 100 or 1000 µM acrylamide. The arrow indicates the time of exposure. The data are presented as the mean of 5 independent experiments performed in duplicates. B) Viability of the SH-SY5Y cells after 3 or 6 days of acrylamide exposure. The data are presented as the mean of 4 independent experiments performed in hexaplicates. C) and D) Cell proliferation displayed as BrdUrd positive cells and cell death displayed as Sub-G1 region after 3 and 6 days respectively, of

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Fischer’s LSD test. A-D) Bars represent the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

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compared to control (cells exposed to medium without acrylamide).

Figure 3. The effect of acrylamide on the number of viable C17.2 cells after 6 and 10 days of

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differentiation determined by the MTT reduction assay. Data are presented as the mean of 5 independent experiments performed in hexaplicates. Results were analyzed using two-way

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ANOVA followed by Fischer’s LSD test. The bars represent the mean ± SEM. *p ≤ 0.05, **p

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≤ 0.01, ***p ≤ 0.001 compared to control (cells exposed to medium without acrylamide).

Figure 4. The effect of acrylamide on the number of viable cells and proliferation of SH-SY5Y

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cells during 3 and 6 days of differentiation. A) Cell viability determined by the MTT reduction assay. B) 3H-thymidine incorporation proliferation assay. C) Protein determination by the Lowry assay. For all experiments, the data are presented as the mean of 3-5 independent experiments each performed in hexaplicates. Results were analyzed using two-way ANOVA followed by Fischer’s LSD test. The bars represent the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to control (cells exposed to medium without acrylamide).

Figure 5. The effect of acrylamide on the expression levels of biomarkers for neurons and astrocytes in the C17.2 cell line. The cells were exposed to either 10 µM or 100 µM of acrylamide during the differentiation for 6 or 10 days. A) mRNA level of β-III tubulin during differentiation and acrylamide exposure. B) mRNA level of GFAP during differentiation and

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Representative western blot staining for β-III tubulin, GFAP and the loading control β-actin.

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The data from mRNA expression levels were normalized against the reference gene TBP and

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are presented as the mean of 4 independent experiments performed in duplicates. The data from the protein levels were normalized to β-actin and presented as the mean of 3 independent

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experiments performed in duplicates. Results were analyzed using one- or two-way ANOVA followed by Fischer’s LSD test. Bars represent the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01,

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compared to control (cells exposed to medium without acrylamide).

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Figure 6. The effect of acrylamide on neurons and astrocytes in differentiating C17.2 cells.

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The cells were exposed to either 10 µM or 100 µM of acrylamide during differentiation for 10 days. Immunofluorescence images of the level of β-III tubulin (green) A) in control cells, B) cells exposed to 10 µM and C) cells exposed to 100 µM. Immunofluorescence images of the level of GFAP (green) in D) control cells, E) cells exposed to 10 µM and F) cells exposed to 100 µM. Nuclei were stained in blue with DRAQ5. Phase contrast image of G) control cells, H) cells exposed to 10 µM and I) cells exposed to 100 µM of acrylamide. The scale bars represent 50 μm in all images. J) Number of neurons and number of neurites per cell after 10 days of differentiation and acrylamide exposure. K) Ratios of the different cell populations after 10 days of differentiation and acrylamide exposure. Statistical significance is based on comparison to the control for each respective cell population. The data are presented as the mean of 3 independent experiments performed in duplicates. Results were analyzed using two-

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**p ≤ 0.01, ***p ≤ 0.001 compared to control (cells exposed to medium without acrylamide).

Figure 7. The effect of acrylamide on the neurite outgrowth in differentiating SH-SY5Y cells.

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Cells differentiated for 3 days during exposure A) without acrylamide, B) with 1 µM of

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acrylamide, C) with 10 µM of acrylamide or D) with 100 µM of acrylamide. Cells differentiated for 6 days during exposure E) without acrylamide, F) with 1 µM of acrylamide,

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G) with 10 µM of acrylamide or H) with 100 µM of acrylamide. The scale bars represent 50 μm in all images. I) Number of neurites per cell after 3 and 6 days of differentiation and

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acrylamide exposure. The data are presented as the mean of 3 independent experiments, each

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seed out consisted of biological duplicates. Bars represent the mean of each biological

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duplicate ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to control (cells exposed to medium without acrylamide). J) Protein levels of β-III tubulin during differentiation and acrylamide exposure. The data is presented as the mean of 4 independent experiments. Results were analyzed using two-way ANOVA followed by Fischer’s LSD test. Bars represent the mean ± SEM. *p ≤ 0.05, *p ≤ 0.05, **p ≤ 0.01, compared to control (cells exposed to medium without acrylamide).

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Acrylamide attenuated differentiation of neurons and astrocytes in C17.2 cells.

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Picomolar concentrations of acrylamide attenuated differentiation in SH-SY5Y cells.

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Acrylamide reduced proliferation of undifferentiated C17.2 and SH-SY5Y cells.

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