Arch Toxicol (2014) 88:769–780 DOI 10.1007/s00204-013-1174-6

ORGAN TOXICITY AND MECHANISMS

Cytotoxic effects of acrylamide in nerve growth factor or fibroblast growth factor 1‑induced neurite outgrowth in PC12 cells Jong‑Hang Chen · Don‑Ching Lee · Ing‑Ming Chiu 

Received: 4 November 2013 / Accepted: 20 November 2013 / Published online: 7 December 2013 © Springer-Verlag Berlin Heidelberg 2013

Abstract  Acrylamide is a neurological and reproductive toxicant in humans and laboratory animals; however, the neuron developmental toxicity of acrylamide remains unclear. The aims of this study are to investigate the cytotoxicity and neurite outgrowth inhibition of acrylamide in nerve growth factor (NGF)- or fibroblast growth factor 1 (FGF1)-mediated neural development of PC12 cells. MTS assay showed that acrylamide treatment suppresses NGF- or FGF1-induced PC12 cell proliferation in a time- and dosedependent manner. Quantification of neurite outgrowth demonstrated that 0.5 mM acrylamide treatment resulted in significant decrease in differentiation of NGF- or FGF1stimulated PC12 cells. This decrease is accompanied with the reduced expression of growth-associated protein-43, a neuronal marker. Moreover, relative levels of pERK, pAKT, pSTAT3 and pCREB were increased within 5–10 min when PC12 cells were treated with NGF or FGF1. Acrylamide (0.5 mM) decreases the NGF-induced activation of AKT–CREB but not ERK–STAT3 within 20 min. Similarly, acrylamide (0.5 mM) decreases the FGF1-induced activation of AKT–CREB within 20 min. In contrast to the NGF treatment, the ERK–STAT3 activation that was induced by FGF1 was slightly reduced by 0.5 mM acrylamide. We further showed that PI3K inhibitor (LY294002), but not MEK inhibitor (U0126), could synergize with acrylamide (0.5 mM) to reduce the cell viability and neurite outgrowth in NGFor FGF1-stimulated PC12 cells. Moreover, acrylamide J.-H. Chen · D.-C. Lee · I.-M. Chiu (*)  Institute of Cellular and System Medicine, National Health Research Institutes, 35, Keyan Rd, Miaoli 350, Taiwan e-mail: [email protected] I.-M. Chiu  Department of Internal Medicine, The Ohio State University, 480 Medical Center Drive, Columbus, OH 43210, USA

(0.5 mM) increased reactive oxygen species (ROS) activities in NGF- or FGF1-stimulated PC12 cells. This increase was reversed by Trolox (an ROS scavenging agent) co-treatment. Together, our findings reveal that NGF- or FGF1-stimulation of the neuronal differentiation of PC12 cells is attenuated by acrylamide through the inhibition of PI3K–AKT–CREB signaling, along with the production of ROS. Keywords  Neurite outgrowth · Acrylamide · NGF · FGF1 · PC12 cells

Introduction Acrylamide, a water-soluble vinyl monomer, is an environment neurotoxin that has been recognized as a neurological and reproductive toxicant in humans and laboratory animals. It is not only used in many applications of industry and laboratories but also has been found in food processed at high temperatures and in tobacco smoke (Kutting et al. 2009; Rice 2005; SNFA 2002; Sunayama et al. 2010; FAO/WHO 2002). He et al. (1989) reported that short-term occupational exposure to acrylamide induced the following symptoms: week legs, loss of toe reflexes and sensations and numb hands and feet; long-term exposure resulted in more severe symptoms including cerebellar dysfunction followed by neuropathy. In recent years, scientists reported that the average daily intake of acrylamide for adults is approximately 0.5 μg/kg body weight; children may have two to three times more intake than adults (Garey et al. 2005; Konings et al. 2003; Svensson et al. 2003). Wilson et al. (2012) also reported that the mean intake of acrylamide for humans is 10–40 μg/day. Furthermore, the Scientific Committee of the Norwegian Food Control Authority in 2002 estimated an additional carcinogenic risk of 1 per 10,000 exposed people at a lifelong intake

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of 0.08 μg/kg body weight/day. Rodríguez-Ramiro et al. (2011) also showed that low doses of acrylamide can act as a toxicant when administered in long-term exposure to human Caco-2 cells. Therefore, acrylamide bring about a risk factor of toxicity not only in human occupational exposure but also in human daily long-term and low-dose exposure. Exposure of acrylamide in human and laboratory animals causes ataxia and hindlimb skeletal muscle weakness (Lehning et al. 2003). LoPachin et al. (2002, 2004) reported that acrylamide produced a central–peripheral neuropathy in laboratory animals, including rats and monkeys, as well as in human, and the nerve terminals are the initial sites for lesion with axonopathy as a conditional effect related to long-term, low-dose intoxication. The toxic effects of acrylamide on neurons have been investigated, including the reduction in cell proliferation, induction of apoptosis, phosphorylation of p53 and extracellular signal regulated kinase (ERK), formation of perikaryal inclusion bodies and transduction of neurodegeneration-related signals (Hartley et al. 1997; Jiang et al. 2007; Keith and Schreiber 1995; Koyama et al. 2006; Okuno et al. 2006; Smith et al. 2001). Acrylamide has been identified in breast milk and can cross the human placenta, resulting in deficit in development and motor coordination before weaning (Sogel et al. 2002). However, the neuron developmental toxicity of acrylamide remains unclear. PC12 cells, a rat adrenal pheochromocytoma cell line that can be differentiated into sympathetic-like neurons, are a widely used model system for studies of promoting neurogenesis, neural development, neuronal survival and functional maintenance of neurons by neurotrophic factors such as nerve growth factor (NGF) and fibroblast growth factor 1 (FGF1) (Greene 1978; Greene et al. 1987; Lin et al. 2009; Rydel and Greene 1987; Vaudry et al. 2002). Previous studies reported that levels of neurotrophic factors are changed in some neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis (Lai et al. 2011; Levy et al. 2005). The neurotrophic factors of NGF and FGF1 are known to activate pathways involving MEK–ERK and PI3K–AKT, resulting in the induction of neurite outgrowth in PC12 cells (Lai et al. 2011; Lin et al. 2009; Wang et al. 2011). Here, we show that NGF- or FGF1-stimulation of the neuronal differentiation of PC12 cells is attenuated by acrylamide through the inhibition of PI3K–AKT–CREB signaling, along with the production of reactive oxygen species (ROS).

Arch Toxicol (2014) 88:769–780

(St. Louis, MO, USA); 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), U0126 and LY294002 were from Promega (Madison, WI, USA); Tris (base) was from J.T. Baker (Phillipsburg, NJ, USA); skim milk powder was from Anchor (Auckland, NZ). All cell culture reagents and saline buffers were from Gibco (Rockville, MD, USA); rat tail collagen I was from BD Biosciences (Franklin Lakes, NJ, USA); human NGF was from PeproTech Inc. (Rocky Hill, NJ, USA); human FGF1 was from Chingen Inc. (Dublin, OH, USA). An antibody against growth-associated protein-43 (GAP-43) was from Millipore (Bedford, MA, USA); phospho-AKT (pAKT; Ser473), phospho-ERK (pERK), phospho-STAT3 (pSTAT3; Ser727) and phospho-cAMP-response element binding protein (pCREB; Ser133) were from Cell Signaling Technology (Danvers, MA, USA); β-actin was from Abcam (Cambridgeshire, UK). Cell culture PC12 cells, the rat adrenal pheochromocytoma cell line, were purchased from American Type Culture Collection. Cells were maintained on the collagen-coated plates (0.1 mg/ml rat tail collagen in 0.02 N acetic acid) and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % horse serum (HS), 5 % fetal bovine serum (FBS), 1 % l-glutamine and1 % penicillin/streptomycin. All cells were cultured at 37 °C with 5 % CO2. Cell treatments

Materials and methods

Acrylamide stock solution (1 M) was prepared with water and filter-sterilized. Cell media containing different concentrations of acrylamide (0, 0.1 and 0.5 mM) were prepared and added to PC12 cells. The treated cells were cultured at 37 °C with 5 % CO2 for different time periods (0, 24, 48, 72 and 96 h). To determine the involvement of ERK and AKT phosphorylation in regulating acrylamideaffected NGF- or FGF1-induced signaling, cells were pretreated with 10 μM U0126 (MEK inhibitor) and 20 μM LY294002 (PI3K inhibitor) for 1 h and were then treated with 50 ng/ml NGF or 200 ng/ml FGF1 with or without 0.5 mM acrylamide for another 96 h at 37 °C with 5 % CO2. To determine the involvement of ROS in regulating acrylamide-inhibited NGF- or FGF1-induced cell proliferation, cells were pretreated with 1 mM Trolox (an ROS scavenging agent) for 30 min and were then treated with 50 ng/ml NGF or 200 ng/ml FGF1 with or without 0.5 mM acrylamide for another 96 h at 37 °C with 5 % CO2.

Reagents and antibodies

Cell proliferation assay

Acrylamide, 2,7-dichlorodihydrofluorescein diacetate (DCFHDA) and Trolox were purchased from Sigma-Aldrich

Cell proliferation was evaluated by MTS assay; 1 × 103 cells per well were plated in a 96-well microtiter plate

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Arch Toxicol (2014) 88:769–780

with 100 μl culture medium containing NGF (0, 12.5, 25 and 50 ng/ml) or FGF1 (0, 50, 100 and 200 ng/ml). These cells were treated with different acrylamide concentration (0, 0.1 and 0.5 mM) for different time period (0, 24, 48, 72 and 96 h) at 37 °C with 5 % CO2. After treatment, the cells were then added 20 μl MTS reagent in each well for 2 h. The enzymatic reduction in MTS to formazan was quantified by Spectramax 190 photometer (Molecular Devices, Sunnyvale, CA, USA) at 490 nm. To determine the involvement of ERK and AKT phosphorylation in regulating acrylamide-affected NGF- or FGF1-induced signaling, MTS assay was performed after cells co-treated with MEK1/2 and PI3K inhibitor for 96 h at 37 °C with 5 % CO2. To determine the involvement of ROS in regulating acrylamide-inhibited NGF- or FGF1-induced cell proliferation, MTS assay was performed after cells co-treated with Trolox for 96 h at 37 °C with 5 % CO2. Neurite outgrowth analysis In PC12 cells, neurite outgrowth, morphological analysis and quantification of neurite-bearing cells were carried out as previous studies (Lai et al. 2011; Wang et al. 2011). PC12 cells were plated on collagen I-coated dishes in the normal serum medium for 24 h. For neuronal differentiation, NGF was treated with 0, 12.5, 25 or 50 ng/ml in lowserum medium (DMEM containing 2 % HS, 1 % FBS, 1 % l-Gln and 1 % AA) to co-operate with 0, 0.1 and 0.5 mM acrylamide, respectively; FGF1 was treated with 0, 50, 100 or 200 ng/ml in low-serum medium (DMEM containing 2 % HS, 1 % FBS, 1 % l-Gln and 1 % AA) to co-operate with 0, 0.1 and 0.5 mM acrylamide, respectively; 50, 100 and 200 ng/ml FGF1 were, respectively, mixed with 5, 10 and 20 μg/ml heparin in low-serum medium before adding to the cells. These cells were cultured for 48 h in NGFtreated cells or for 96 h in FGF1-treated cells at 37 °C with 5 % CO2. The definition of PC12 cell morphological differentiation is that the length of neuritis should be at least twice the diameter of the cell body. Images were taken using microscope (Olympus IX71, Tokyo, Japan).

sulfate–polyacrylamide gel (SDS−PAGE) and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories). Thereafter, membranes were blocked in 5 % skimmed milk or 5 % bovine serum albumin (Bio Basic, Markham Ontario, Canada) blocking buffer for 30 min at room temperature. The membranes were then incubated with specific primary antibodies in either 5 % skimmed milk or 5 % bovine serum albumin blocking buffer followed by incubation with corresponding HRP-conjugated secondary antibodies. Protein levels were detected with a Western lightning kit (PerkinElmer, Boston, MA, USA) with β-actin as an internal control. Determination of ROS production The intracellular accumulation of ROS was monitored by ELISA Reader using DCHFDA (Jang and Surh 2001). This dye is a stable compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield dichlorofluorescein (DCHF). Hydrogen peroxide or low molecular weight hydroperoxides produced by cells oxidize DCHF to the highly fluorescent compound, 2,7-dichlorofluorescein (DCF). Thus, the fluorescence intensity is proportional to the amount of peroxide produced by cells. After treated with ACR and ACR with Trolox simultaneously, cells were washed twice with PBS, and 10 μM DCHFDA was added for another 30 min incubation at 37 °C. After PBS washing, the intracellular ROS accumulation was detected by ELISA Reader (infinite M200, TECAM, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Statistical analysis The data are expressed as mean ± SD. The statistical significance was determined by the one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test by a statistical package for the social science 13.0 software (SPSS, Chicago, IL, USA). The differences were considered statistically significant when p