Food and Chemical Toxicology 82 (2015) 27–35
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells Jong-Hang Chen a, Chin-Cheng Chou b,* a b
Institute of Cellular and System Medicine, National Health Research Institutes, No. 35, Keyan Rd., Miaoli 350, Taiwan School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan
A R T I C L E
I N F O
Article history: Received 3 September 2014 Accepted 29 April 2015 Available online 7 May 2015 Keywords: Acrylamide Cellular differentiation Neuroblastoma Glioblastoma
A B S T R A C T
This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Acrylamide (ACR), a neurotoxin causing ataxia, skeletal muscle weakness and numbness of the hands and legs in animals and humans (Exon, 2006), has been applied in many industries and laboratories, and can be found in many food and tobacco products after heating (Kutting et al., 2009; Rice, 2005; SNFA, 2002; Sunayama et al., 2010; FAO/WHO, 2002). The mean intake of ACR for humans is 10– 40 μg/day (Wilson et al., 2012), which is higher than the lifelong intake of 0.08 μg/kg body weight/day known to result in an additional cancer risk of 1 out of 10,000 people (NSFA, 2002). Therefore, toxicological concerns regarding ACR intake go beyond occupational settings into daily long-term and low-dose human exposure. ACR causes central-peripheral neuropathy (LoPachin et al., 2002, 2004). Axons are the primary site at which ACR causes axonopathy after impairment of the neurotransmitter by increasing the number of vesicles in the synapses, swelling the distal nerve terminal axon, and filling it with neurofilaments (LoPachin et al., 2002). The disturbance of kinesin-related motor protein has been proposed to occur
* Corresponding author. School of Veterinary Medicine, National Taiwan University, No. 1, Section 4, Roosevelt Rd., Taipei 106, Taiwan. Tel.: +886 2 3366 1292; fax: +886 2 2363 0495. E-mail address: [email protected] (C.-C. Chou). http://dx.doi.org/10.1016/j.fct.2015.04.030 0278-6915/© 2015 Elsevier Ltd. All rights reserved.
in ACR-induced axonal toxicity (Exon, 2006; LoPachin, 2005). Microtubule-associated proteins (MAPs) are important for neurite outgrowth during neurogenesis and extension development of glia cells (Couchie et al., 1985; Gonzalez-Billault et al., 2004). Dysfunction of MAP1b and MAP2c causes microtubules to be structurally unstable and leads to neurodegenerative diseases (Gonzalez-Billault et al., 2004). ACR exposure has been reported to affect the distribution of MAP1 and MAP2 proteins in different brain areas of rats (Chauhan et al., 1993), and the relative binding affinity of ACR with MAP proteins has been reported to be greater than that of ACR with tubulin (Lapadula et al., 1989). Moreover, signal pathways of the Janus kinase-signal transducer and activator of transcription (JAKSTAT) and the mitogen-activated protein kinase (MAPK) are critical for various cellular events involved with cellular differentiation (both neurogenesis and astrogliogenesis), inflammation, apoptosis, proliferation, and cell survival (Douglas, 2012; Kyriakis and Avruch, 2012; Rawlings et al., 2004). Although ACR affects the distribution of rat brain MAPs, the effects of MAP expressions and JAK-STAT signal pathways, which are related to differentiation in neurogenesis and astrogliogenesis, have not been clarified. Human neuroblastoma cells (SH-SY5Y) can differentiate into neurons in the presence of retinoic acid (RA) (Hartley et al., 1997). In addition, glioblastoma cells can differentiate into neuronal lineage cells in the presence of butyric acid (BA) (Sidiropoulou et al., 2009). Different concentrations (0, 2.5, 5 and 10 μM) of RA and BA were
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used to stimulate SH-SY5Y and U-1240 MG cells for 72 h, which resulted in cellular differentiation in a dose-dependent manner. Significantly different morphologies were observed in 10 μM RAstimulated SH-SY5Y and 10 μM BA-stimulated U-1240 MG cells (data not shown). This study showed that RA-induced neurogenesis of SHSY5Y cells and butyric acid (BA)-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and that MAPs expression and JAKSTAT signaling were down-regulated. 2. Materials and methods 2.1. Chemicals and reagents ACR, ammonium persulfate (APS), BA, ethanol, glycine, methanol, NaCl, phosphatase inhibitor, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protease inhibitor, and RA were from Sigma-Aldrich (St Louis, MO). Dimethyl sulfoxide (DMSO), KCl, tetramethylethylenediamine (TEMED), and Triton X-100 were from J.T. Baker (Phillipsburg, NJ). Protein markers and protein dyes were from Fermentas (Vilnius, Lithuania), and SDS, tris-base, and acrylamide/bis (37.5:1) were from AMRESCO (Solon, OH). Non-fat milk was from Anchor (Auckland, NZ), caffeine was from Calbiochem (Gibbstown, NJ), and polyvinylidene fluoride (PVDF), enhanced chemiluminescence (ECL) substrate, and Na2HPO4 were from Merck KGaA (Darmstadt, Germany). Anti-p38 antibody was from Abcam (Abcam, Cambridgeshire, UK), and anti-GAPDH, MAP1b, MAP2, neurofilament-L (NF-L), glial fibrillary acidic protein (GFAP), JAK1 antibody, and rabbit IgG antibody (HRP) were from GeneTex (Irvine, CA). Anti-extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) antibody were from Cell Signaling Technology (Beverly, MA). All of the cell culture reagents and saline buffers were purchased from Gibco (Rockville, MD). 2.2. Cell culture Human neuroblastoma cell line SH-SY5Y was purchased from the American Type Culture Collection (Manassas, VA, USA) and was grown in 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s nutrient F12 medium (DMEM/F12) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Human astrocytoma cell line U-1240 MG was obtained from The Ohio State University (Columbus, OH). U-1240 MG cells were grown in minimum essential medium (MEM) supplemented with 10% (v/v) calf serum (CS), 100 units/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained at 37 °C in a 5% CO2, 100% humidified atmosphere. 2.3. Cell differentiation and morphological observation For differentiation studies, the SH-SY5Y cells were transferred into differentiation medium (DMEM/F12 (1:1) medium supplemented with 1% (v/v) FBS, 100 units/ mL penicillin, and 100 μg/mL streptomycin) with different concentrations of RA (0, 2.5, 5, and 10 μM) for 72 h. The U-1240 MG cells were transferred into differentiation medium (MEM medium supplemented with 1% (v/v) CS, 100 units/mL penicillin, and 100 μg/mL streptomycin) with different concentrations of BA (0, 2.5, 5, and 10 μM) for 72 h. All cells were maintained at 37 °C in a 5% CO2, 100% humidified atmosphere. Differentiation of SH-SY5Y and U-1240 MG cells with or without ACR treatment was observed using an inverted microscope (IX71; Olympus, Tokyo, Japan). 2.4. Cell treatments ACR stock solution (1 M) was prepared with double-distilled water (ddH2O) and sterilized through 0.22 μm filters. Appropriate ACR concentrations were prepared with culture medium of differentiation in the petri dish. Cells were then treated with ACR and cultured at 37 °C with 5% CO2 for 72 h according to our previous study (Chen et al., 2009). For the determination the blocking effects of signal upstream of ACRimpairing the phosphorylation of MAPKs, U-1240 MG cells were pretreated with 0, 0.25, 0.5, and 1 mM caffeine for 30 min and then with 2 mM ACR for another 48 h. 2.5. Cell proliferation analysis Cell viability was analyzed with an MTT assay according to our previous protocols (Chen et al., 2010). In brief, cells were placed in 96 well microtiter plate at a density of 1000 cells per well in a final volume of 100 μL of culture medium for 1 day prior to the experiment. Cells after ACR treatment were immediately incubated with 10 μL MTT solution (0.5 mg/mL) for 3 h at 37 °C, and the medium was then removed and 100 μL DMSO was added for 5 min at room temperature. Cell viability was quantified by ELISA reader (Spectra MAXM5 photometry, Molecular Devices, Sunnyvale, CA) at OD595 nm. 2.6. Western blot analysis Protein expressions were measured with Western blot analysis, which was modified from our previous protocol (Chen et al., 2009, 2010). Cells were harvested in
lysis buffer (Sigma, St Louis, MO) and left on ice for 20 min and then were centrifuged (8000 × g for 30 min at 4 °C). The proteins in the supernatant were measured by Thermo BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). The supernatant was diluted to 15–30 μg of protein/mL with loading buffer (Fermentas, Vilnius, Lithuania) and ddH2O to 20 μL. Samples were heated to 92 °C for 5 min to denature the proteins. Proteins were separated by electrophoresis on a 10% (v/v) polyacrylamide gel at 60 V for 30 min and 100 V for 1 h, transferred onto a polyvinylidene fluoride membrane at 100 mA for 1 h at 4 °C, and then immunoblotted with antiactin, GAPDH, MAP1b, MAP2, NF-L, GFAP, JAK1, ERK, JNK, and p38 antibody (with 1:10,000, 1:10,000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:2000, and 1:1000 dilution, respectively) purified from rabbit serum at 4 °C overnight. Immunoreactive bands were detected using the horseradish peroxidase-conjugated antirabbit IgG and enhanced by Geliance 600 Imaging System (J&H Technology Co., Ltd). Bandwidths were quantified by MultiGauge V1.1 (Fujifilm, Stamford, CT). 2.7. Statistical analysis Data were presented as means ± standard deviation. Each experiment was performed in triplicate at least three times independently. The statistical significance of differences among groups was performed with one-way analysis of variance (ANOVA), followed by Duncan’s test using SSPS (ver 17.0; Chicago, IL). The differences were considered statistically significant when p < 0.05.
3. Results 3.1. Acrylamide inhibits RA-induced neurite outgrowth and cell proliferation of SH-SY5Y cells As shown in Figure 1A, after 10 μM RA stimulation, SH-SY5Y cells developed a round cell body and an extended axon-like neurite. Under co-exposure to the RA and different concentrations (0, 0.1, 0.5, 1, and 2 mM) of ACR, 0.5 mM ACR caused the SH-SY5Y cells to exhibit shorter neurite morphology, while 1 and 2 mM ACR resulted in cells with no neurite morphological extensions (Fig. 1A). Moreover, Western blot analysis found that expression levels of NFL, a neurofilament subunit expressed in mature neurons, were increased after RA treatment and decreased under ACR co-treatment in SH-ST5Y cells (Fig. 1B). MTT assay showed that different concentrations (0, 2.5, 5, and 10 μM) of RA treatment significantly induced SH-SY5Y cell proliferation at 24, 48, and 72 h when compared with the control (0 h) (Fig. 2A). Cell proliferation of SHSY5Y cells at 72 h did not show statistically significant differences as a result of different concentrations (0, 2.5, 5, and 10 μM) of RA treatment (Fig. 2A). However, induction of SH-SY5Y cell proliferation by 10 μM RA at 72 h was significantly decreased in 0.5, 1, and 2 mM ACR co-treatment when compared with the control (0 mM) (Fig. 2B). These results indicated that neurite outgrowth of SHSY5Y cells was inhibited by ACR and that proliferation of SH-SY5Y cells was attenuated by ACR treatment in a time- and dosedependent manner. 3.2. Acrylamide inhibits BA-induced extension development and cell proliferation of U-1240 MG cells As shown in Figure 3A, the round cell bodies of U-1240 MG cells were differentiated into an axon-like extended neurite after 10 μM BA stimulation for 72 h. ACR exposure interfered with BA-induced U-1240 MG cell development such that the cells developed shorter extensions after exposure to 0.5 and 1 mM ACR, while no cell extensions were developed after exposure to 2 mM ACR. GFAP, a marker for glia cell differentiation, was analyzed by Western blot for U-1240 MG cells, which were co-treated with BA and different concentrations of ACR (Fig. 3B). Relative levels of GFAP in U-1240 MG cells increased after 10 μM BA treatment but decreased significantly in BA and ACR co-treatment groups. As shown in Figure 4A, different concentrations (0, 2.5, 5, and 10 μM) of BA treatment induced U-1240 MG cell proliferation significantly at 24, 48, and 72 h when compared with the control (0 h). There were no statistically significant differences in U-1240 MG cell proliferation resulting from
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NF-L GAPDH Fig. 1. ACR inhibited the RA-induced neurite outgrowth of SH-SY5Y cells. SH-SY5Y cell neurite outgrowth was induced by treatment with 10 μM RA for 72 h and was compared with the control (the control cells were grown in the cell-cultured medium without RA) (A). 10 μM RA induced the neurite outgrowth of SH-SY5Y cells, which were inhibited by different concentrations (0.1, 0.5, 1, and 2 mM) of ACR for 72 h (A). The scale bar is 200 μm. Protein expression of NF-L was analyzed by Western blot in the RA (10 μM) co-treatment with different concentrations (0.1, 0.5, 1, and 2 mM) of ACR at 72 h (B). ACR, acrylamide; RA, retinoic acid; NF-L, neurofilament protein-L.
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Fig. 2. ACR inhibited RA-induced SH-SY5Y cell proliferation. Cell proliferation was analyzed via MTT assay. SH-SH5Y cell proliferation was induced by different concentrations (0, 2.5, 5, and 10 μM) of RA for 72 h (A). ACR inhibited RA (10 μM)-induced SH-SY5Y cell proliferation in a time (0, 24, 48, and 72 h)- and concentration (0, 0.1, 0.5, 1, and 2 mM)-dependent manner (B). Results are presented as the mean ± SD from three independent experiments. Statistically significant differences with the controls (0 h or 0 μM) are shown as *p < 0.05, **p < 0.01, ***p < 0.001. ACR, acrylamide; RA, retinoic acid.
treatment with different concentrations of BA (Fig. 4A). Induction of U-1240 MG cell proliferation by 10 μM BA at 72 h was significantly decreased only in 2 mM ACR co-treatment when compared with the control (0 mM) (Fig. 4B). These results revealed that cell extension and proliferation of U-1240 MG cells were inhibited after ACR treatment.
3.3. Acrylamide inhibits MAPs expression and JAK-STAT signaling of cellular differentiation in SH-SY5Y and U-1240 MG cells The biochemical indicators for morphologic changes were detected through Western blot analysis with different antibodies against MAP1b and MAP2c. The results showed that SH-SY5Y cells stimulated with 10 μM RA exhibited a 21% increase in MAP1b expression and a 123% increase in MAP2c expression (Fig. 5A), while U-1240 MG cells stimulated with 10 μM BA exhibited a 16% increase in MAP1b expression and a 52% increase in MAP2c expression (Fig. 5B), respectively. When co-treated with 2 mM ACR, the expressions of MAP1b and MAP2c in SH-SY5Y cells (Fig. 5A) were reduced by 39% and 64%, respectively, while the expressions of MAP1b and MAP2c in U-1240 MG cells (Fig. 5B) were reduced by 52% and 57%, respectively. In terms of JAK-STAT signaling, JAK1 expression (Fig. 5) was increased by 93% in 10 μM RA-stimulated SH-SY5Y cells and by 108% in 10 μM BA-stimulated U-1240 MG cells. Furthermore, JAK1 expression in SH-SY5Y cells was decreased by 68% when the cells were co-treated with 2 mM ACR and 10 μM RA (Fig. 5A), while JAK1 expression in U-1240 MG cells was decreased by 74% when the cells were co-treated with 10 μM BA and 2 mM ACR (Fig. 5B). These results revealed that MAPs expression and JAK-STAT signaling were involved in ACR-inhibited differentiation in SH-SY5Y and U-1240 MG cells.
3.4. Acrylamide inhibits phosphorylation of MAPKs in U-1240 MG cells The influences of MAPKs in ACR-treated neuroblastoma cells (SHSY5Y) have also been reported in Okuno et al. (2006), but no previous studies have reported on their influences in ACR-treated glioblastoma cells. Therefore, the protein expressions of ERK, JNK, and p38 of MAPKs in cultured U-1240 MG cells after exposure to 2 mM ACR for 48 h were measured by Western blot. As shown in Figure 6, phosphorylation of ERK and JNK, but not the total amount of ERK and JNK, was reduced after 2 mM ACR treatment. Furthermore, caffeine – an inhibitor of ATM/ATR used to block the upstream portion of the ACR-induced DNA-damaging signal pathway – successfully reversed the suppression of phosphorylation of ERK and JNK caused by 2 mM ACR treatment. These results demonstrated that caffeine attenuated ACR-inhibited phosphorylation of MAPKs in U-1240 MG cells.
4. Discussion The results of the present study suggested that cellular differentiation in human neuroblastoma and glioblastoma cells is inhibited after ACR exposure and that down-regulation of the JAK-STAT signal pathway may further explain the ACR-induced neurodegeneration. In a neuronal system, astrocytes maintain the survival of neuronal cells by glutamate uptake, calcium control, and glia scar formation (Sofroniew and Vinters, 2010). Astrocytes play a role in neurodegenerative diseases of the CNS (Miller, 2005; Xie et al., 2004), and GFAP accumulation is a hallmark of astrogliosis, which represents glia-increasing activation during CNS damage (Eng and Ghirnikar, 1994). Sriram et al. (2004) reported evidence that
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GFAP GAPDH Fig. 3. ACR inhibited BA-induced extension of U-1240 MG cells. Extension of U-1240 MG cells was induced by 10 μM BA for 72 h treatment, which was compared with the control (the control cells were grown in the cell-cultured medium without BA) (A).10 μM BA-induced extension of U-1240 MG cells was inhibited by different concentrations (0.1, 0.5, 1, and 2 mM) of ACR co-treatment for 72 h (A). The scale bar is 200 μm. Protein expression of GFAP was analyzed by Western blot in the BA (10 μM) cotreatment with different concentrations (0.1, 0.5, 1, and 2 mM) of ACR at 72 h (B). ACR, acrylamide; BA, butyric acid; GFAP, glial fibrillary acidic protein.
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Fig. 4. ACR inhibited cell proliferation in BA-induced U-1240 MG cells. Cell proliferation was analyzed via MTT assay. Different concentrations (0, 2.5, 5, and 10 μM) of BA treatment for 72 h induced U-1240 MG cell proliferation (A). ACR inhibited BA (10 μM)-induced U-1240 MG cell proliferation in a time (0, 24, 48, and 72 h)- and concentration (0, 0.1, 0.5, 1, and 2 mM)-dependent manner (B). Results are presented as the mean ± SD from three independent experiments. Statistically significant differences with the controls (0 h or 0 μM) are shown as *p < 0.05, **p < 0.01, ***p < 0.001. ACR, acrylamide; BA, butyric acid.
JAK-STAT signaling is related to the accumulation of GFAP in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration or astrogliosis. In this study, JAK1 expression was increased during the induction of differentiation and decreased under ACR co-treatment in both SH-SY5Y and U-1240 MG cells (Fig. 5). Therefore, down-regulation of the JAK-STAT signal pathway was involved in the ACR-inhibited differentiation of human neuronal and glial cells in vitro. Among the MAPKs, ERK showed functional differences in SHSY5Y and U-1240 MG cells under ACR-induced toxicity. ERK, JNK, and the p38 kinases are members of the MAPK pathway that regulate cellular proliferation, differentiation, and apoptosis of the neuronal developmental systems (Cargnello and Roux, 2011; Zhang and Liu, 2002). The crosstalk between MAPK and JAK-STAT signal cascades regulates cellular functional events by stimulation of cytokines and growth factors (Eulenfeld et al., 2012). Okuno et al. (2006) reported that the ERK pathway causes phosphorylation of p53 at Ser15 and the cytotoxicity of ACR in SH-SY5Y cells without RA-induced differentiation. Our previous studies showed that ACR induced cytotoxicity in U-1240 MG cells through the phosphorylation of p53 at Ser15 during ATM/ATR signaling (Chen et al., 2010). The current study further demonstrated that the phosphorylation of ERK and JNK were decreased by ACR exposure in U-1240 MG cells without BA-induced differentiation. These results suggested that the MAPK pathway is important in the regulation of ACR-induced cytotoxicity, and ERK presented functional differences in SH-SY5Y and U-1240 MG cells after ACR exposure. This study implies that ACR may affect early neurological development when the daily intake of ACR is high. ACR is an environmental neurotoxin and probable human carcinogen (Rice, 2005). Food-borne ACR can be produced during food processing via the Maillard reaction, especially during the processing of food con-
taining asparagine and glucose (Friedman, 2003; Tareke et al., 2002). Investigators have previously suggested that pregnant women and breast-feeding mothers avoid ACR-containing food to lessen the possibility of contributing to ACR-induced toxicity in their children as well as the possibility of fetal exposure to ACR via the exchange of blood flow in the placenta (Sorgel et al., 2002; FAO/WHO, 2005). The toxic effects of ACR on neurons have been investigated intensively, including the reduction of cell viability, the induction of apoptosis and p53 phosphorylation (Okuno et al., 2006; Sumizawa and Igisu, 2007), the formation of perikaryal inclusion bodies (Hartley et al., 1997), and the neurodegeneration of signal transduction (Yuzo et al., 2001). ACR-induced DNA breaks will increase p21 expression and G0/G1 cell-cycle arrest by inhibiting cyclin D1/Cdk4/6 kinase activity, which in turn causes cellular mitochondria collapse and apoptosis (Chen et al., 2010, 2013). Our study further suggested that ACR impairs the differentiation efficiency of human neurons and glia cells and that the interactions of neurons and glia cells in the neuronal system may be affected by ACR-induced neurotoxicity. Thus, ACR-induced toxicity in a fetus or during childhood could hinder early neuronal system development, a potential effect which is worthy of further investigations in vivo. In addition, it should be pointed out that this is a mechanistic study that used an in vitro system, and thus outlines the effects of ACR on cellular differentiation. Previous studies have reported that diet (including smoking) is the main source of environmental ACR exposure in humans and that, according to LC-MS/MS assay, the halflife of ACR is 2.2–7 hours in various bodily fluids (Sorgel et al., 2002; Boettcher et al., 2006). Thus, it would be difficult to accumulate ACR via a normal human diet to cause effects similar to those of the current high-dose study. However, ACR is chemically reactive toward nucleophiles (including amino and thiol groups in amino acids and proteins) and reacts with DNA to generate adducts with ring
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Fig. 5. ACR induced dose-dependent inhibition of protein expression in biochemical and signal transduction relative to differentiation in SH-SY5Y and U-1240 MG cells. Protein expressions of MAP1b, MAP2c, and JAK1 in cells treated with different concentrations of ACR (0.1, 0.5, 1, and 2 mM) were analyzed by Western blot. Panel A shows RA (10 μM)-treated SH-SY5Y cells treated with or without ACR for 72 h. Panel B shows BA (10 μM)-treated U-1240 MG cells treated with or without ACR for 72 h. Results are presented as the mean ± SD from three independent experiments. Statistically significant differences are shown as *p < 0.05, **p < 0.01, and ***p < 0.001. ACR, acrylamide; RA, retinoic acid; BA, butyric acid; MAP1b/2c, microtubule-associated protein 1b/2c; JAK1, Janus kinase 1.
nitrogen atoms and extra-nuclear amino groups of adenine and guanine (Klaunig, 2008; Rice, 2005). These DNA adducts cause depurinating lesions, and the repair of these lesions can also lead to the formation of DNA breaks (Friedberg et al., 1995; Martins et al., 2007). In a study that followed human subjects for a period of 13.3 years, a positive association between dietary acrylamide and renal cell cancer risk was found (Hogervorst et al., 2008), and increasing incidences of endometrial, ovarian, and renal cancer with increased dietary ACR intake have also been reported (Hogervorst
et al., 2009). Moreover, ACR-induced neurological symptoms followed by neuropathy have been reported in a cohort study of occupationally exposed workers (He et al., 1989). This study strongly indicated that accumulative effect of ACR might exist and significantly high levels of ACR exposure also might exist at least in occupationally exposed workers. In addition, our previous studies demonstrated that different concentrations (0.5–2 mM) of ACR exposure (0–72 h) could lead to DNA damage, apoptosis and cytotoxicity effects of U-1240 MG and/or SH-SY5Y cells in a
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Fig. 6. ACR inhibited the MAPK pathway in U-1240 MG cells. Protein expressions of MAPKs (ERK, JNK, and p38) were analyzed by Western blot. Caffeine was used as ATM/ATR inhibitor to block the upstream signal of the ACR-induced DNAdamaging signal pathway. The ACR (2 mM)-treated U-1240 MG cells were cotreated with different concentrations (0, 0.25, 0.5, and 1 mM) of caffeine for 48 h. ACR, acrylamide; ERK, extracellular-signal-regulated kinases; JNK, c-Jun N-terminal kinases.
time- and dose-dependent manner (Chen et al., 2009, 2010, 2013). The present study further indicates that ACR inhibits cellular differentiation of U-1240 MG and SH-SY5Y cells. Taken together, the aforementioned findings suggest that mechanistic studies involving in vitro systems should be conducted in order to better understand ACR-induced toxicity. In conclusion, the findings of the current study suggest that ACR impairs the differentiation efficiency of neuronal (SH-SY5Y) and glial (U-1240 MG) cells. RA-induced neurogenesis of SH-SY5Y and BAinduced astrogliogenesis of U-1240 MG cells were attenuated by ACR, and these effects were associated with the down-regulation of MAP expression and JAK-STAT signaling. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This study was supported by a grant from National Science Council (NSC 99-2313-B-002 -029) of Taiwan. We thanked Miss YanShiu Wang for her excellent technical assistance. References Boettcher, M.I., Bolt, H.M., Angerer, J., 2006. Acrylamide exposure via the diet: influence of fasting on urinary mercapturic acid metabolite excretion in humans. Arch. Toxicol. 80, 817–819.
Cargnello, M., Roux, P.P., 2011. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol. Mol. Biol. Rev. 75, 50–83. Chauhan, N.B., Spencer, P.S., Sabri, M.I., 1993. Effect of acrylamide on the distribution of microtubule-associated proteins (MAP1 and MAP2) in selected regions of rat brain. Mol. Chem. Neuropathol. 18, 225–245. Chen, J.H., Wu, K.Y., Chiu, I.M., Tsou, T.C., Chou, C.C., 2009. Acrylamide-induced astrogliotic and apoptotic responses in human astrocytoma cells. Toxicol. In Vitro 23, 855–861. Chen, J.H., Tsou, T.C., Chiu, I.M., Chou, C.C., 2010. Proliferation inhibition, DNA damage, and cell-cycle arrest of human astrocytoma cells after acrylamide exposure. Chem. Res. Toxicol. 23, 1449–1458. Chen, J.H., Yang, C.H., Wang, Y.S., Lee, J.G., Cheng, C.H., Chou, C.C., 2013. Acrylamideinduced mitochondria collapse and apoptosis in human astrocytoma cells. Food Chem. Toxicol. 51, 446–452. Couchie, D., Fages, C., Bridoux, A.M., Rolland, B., Tardy, M., Nunez, J., 1985. Microtubule-associated proteins and in vitro astrocyte differentiation. J. Cell Biol. 101, 2095–2103. Douglas, A.H., 2012. The JAK/STAT pathway. Cold Spring Harb. Perspect. Biol. 4, a011205. Eng, L.F., Ghirnikar, R.S., 1994. GFAP and astrogliosis. Brain Pathol. 4, 229–237. Eulenfeld, R., Dittrich, A., Khouri, C., Müller, P.J., Mütze, B., Wolf, A., et al., 2012. Interleukin-6 signalling: more than JAKS and STATs. Eur. J. Cell Biol. 91, 486–495. Exon, J.H., 2006. A review of the toxicology of acrylamide. J. Toxicol. Environ. Health B Crit. Rev. 9, 397–412. FAO/WHO, 2002. Consultation on the health implications of acrylamide in food. Summary Report
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells Jong-Hang Chen a, Chin-Cheng Chou b,* a b
Institute of Cellular and System Medicine, National Health Research Institutes, No. 35, Keyan Rd., Miaoli 350, Taiwan School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan
A R T I C L E
I N F O
Article history: Received 3 September 2014 Accepted 29 April 2015 Available online 7 May 2015 Keywords: Acrylamide Cellular differentiation Neuroblastoma Glioblastoma
A B S T R A C T
This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Acrylamide (ACR), a neurotoxin causing ataxia, skeletal muscle weakness and numbness of the hands and legs in animals and humans (Exon, 2006), has been applied in many industries and laboratories, and can be found in many food and tobacco products after heating (Kutting et al., 2009; Rice, 2005; SNFA, 2002; Sunayama et al., 2010; FAO/WHO, 2002). The mean intake of ACR for humans is 10– 40 μg/day (Wilson et al., 2012), which is higher than the lifelong intake of 0.08 μg/kg body weight/day known to result in an additional cancer risk of 1 out of 10,000 people (NSFA, 2002). Therefore, toxicological concerns regarding ACR intake go beyond occupational settings into daily long-term and low-dose human exposure. ACR causes central-peripheral neuropathy (LoPachin et al., 2002, 2004). Axons are the primary site at which ACR causes axonopathy after impairment of the neurotransmitter by increasing the number of vesicles in the synapses, swelling the distal nerve terminal axon, and filling it with neurofilaments (LoPachin et al., 2002). The disturbance of kinesin-related motor protein has been proposed to occur
* Corresponding author. School of Veterinary Medicine, National Taiwan University, No. 1, Section 4, Roosevelt Rd., Taipei 106, Taiwan. Tel.: +886 2 3366 1292; fax: +886 2 2363 0495. E-mail address: [email protected] (C.-C. Chou). http://dx.doi.org/10.1016/j.fct.2015.04.030 0278-6915/© 2015 Elsevier Ltd. All rights reserved.
in ACR-induced axonal toxicity (Exon, 2006; LoPachin, 2005). Microtubule-associated proteins (MAPs) are important for neurite outgrowth during neurogenesis and extension development of glia cells (Couchie et al., 1985; Gonzalez-Billault et al., 2004). Dysfunction of MAP1b and MAP2c causes microtubules to be structurally unstable and leads to neurodegenerative diseases (Gonzalez-Billault et al., 2004). ACR exposure has been reported to affect the distribution of MAP1 and MAP2 proteins in different brain areas of rats (Chauhan et al., 1993), and the relative binding affinity of ACR with MAP proteins has been reported to be greater than that of ACR with tubulin (Lapadula et al., 1989). Moreover, signal pathways of the Janus kinase-signal transducer and activator of transcription (JAKSTAT) and the mitogen-activated protein kinase (MAPK) are critical for various cellular events involved with cellular differentiation (both neurogenesis and astrogliogenesis), inflammation, apoptosis, proliferation, and cell survival (Douglas, 2012; Kyriakis and Avruch, 2012; Rawlings et al., 2004). Although ACR affects the distribution of rat brain MAPs, the effects of MAP expressions and JAK-STAT signal pathways, which are related to differentiation in neurogenesis and astrogliogenesis, have not been clarified. Human neuroblastoma cells (SH-SY5Y) can differentiate into neurons in the presence of retinoic acid (RA) (Hartley et al., 1997). In addition, glioblastoma cells can differentiate into neuronal lineage cells in the presence of butyric acid (BA) (Sidiropoulou et al., 2009). Different concentrations (0, 2.5, 5 and 10 μM) of RA and BA were
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used to stimulate SH-SY5Y and U-1240 MG cells for 72 h, which resulted in cellular differentiation in a dose-dependent manner. Significantly different morphologies were observed in 10 μM RAstimulated SH-SY5Y and 10 μM BA-stimulated U-1240 MG cells (data not shown). This study showed that RA-induced neurogenesis of SHSY5Y cells and butyric acid (BA)-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and that MAPs expression and JAKSTAT signaling were down-regulated. 2. Materials and methods 2.1. Chemicals and reagents ACR, ammonium persulfate (APS), BA, ethanol, glycine, methanol, NaCl, phosphatase inhibitor, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protease inhibitor, and RA were from Sigma-Aldrich (St Louis, MO). Dimethyl sulfoxide (DMSO), KCl, tetramethylethylenediamine (TEMED), and Triton X-100 were from J.T. Baker (Phillipsburg, NJ). Protein markers and protein dyes were from Fermentas (Vilnius, Lithuania), and SDS, tris-base, and acrylamide/bis (37.5:1) were from AMRESCO (Solon, OH). Non-fat milk was from Anchor (Auckland, NZ), caffeine was from Calbiochem (Gibbstown, NJ), and polyvinylidene fluoride (PVDF), enhanced chemiluminescence (ECL) substrate, and Na2HPO4 were from Merck KGaA (Darmstadt, Germany). Anti-p38 antibody was from Abcam (Abcam, Cambridgeshire, UK), and anti-GAPDH, MAP1b, MAP2, neurofilament-L (NF-L), glial fibrillary acidic protein (GFAP), JAK1 antibody, and rabbit IgG antibody (HRP) were from GeneTex (Irvine, CA). Anti-extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) antibody were from Cell Signaling Technology (Beverly, MA). All of the cell culture reagents and saline buffers were purchased from Gibco (Rockville, MD). 2.2. Cell culture Human neuroblastoma cell line SH-SY5Y was purchased from the American Type Culture Collection (Manassas, VA, USA) and was grown in 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s nutrient F12 medium (DMEM/F12) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Human astrocytoma cell line U-1240 MG was obtained from The Ohio State University (Columbus, OH). U-1240 MG cells were grown in minimum essential medium (MEM) supplemented with 10% (v/v) calf serum (CS), 100 units/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained at 37 °C in a 5% CO2, 100% humidified atmosphere. 2.3. Cell differentiation and morphological observation For differentiation studies, the SH-SY5Y cells were transferred into differentiation medium (DMEM/F12 (1:1) medium supplemented with 1% (v/v) FBS, 100 units/ mL penicillin, and 100 μg/mL streptomycin) with different concentrations of RA (0, 2.5, 5, and 10 μM) for 72 h. The U-1240 MG cells were transferred into differentiation medium (MEM medium supplemented with 1% (v/v) CS, 100 units/mL penicillin, and 100 μg/mL streptomycin) with different concentrations of BA (0, 2.5, 5, and 10 μM) for 72 h. All cells were maintained at 37 °C in a 5% CO2, 100% humidified atmosphere. Differentiation of SH-SY5Y and U-1240 MG cells with or without ACR treatment was observed using an inverted microscope (IX71; Olympus, Tokyo, Japan). 2.4. Cell treatments ACR stock solution (1 M) was prepared with double-distilled water (ddH2O) and sterilized through 0.22 μm filters. Appropriate ACR concentrations were prepared with culture medium of differentiation in the petri dish. Cells were then treated with ACR and cultured at 37 °C with 5% CO2 for 72 h according to our previous study (Chen et al., 2009). For the determination the blocking effects of signal upstream of ACRimpairing the phosphorylation of MAPKs, U-1240 MG cells were pretreated with 0, 0.25, 0.5, and 1 mM caffeine for 30 min and then with 2 mM ACR for another 48 h. 2.5. Cell proliferation analysis Cell viability was analyzed with an MTT assay according to our previous protocols (Chen et al., 2010). In brief, cells were placed in 96 well microtiter plate at a density of 1000 cells per well in a final volume of 100 μL of culture medium for 1 day prior to the experiment. Cells after ACR treatment were immediately incubated with 10 μL MTT solution (0.5 mg/mL) for 3 h at 37 °C, and the medium was then removed and 100 μL DMSO was added for 5 min at room temperature. Cell viability was quantified by ELISA reader (Spectra MAXM5 photometry, Molecular Devices, Sunnyvale, CA) at OD595 nm. 2.6. Western blot analysis Protein expressions were measured with Western blot analysis, which was modified from our previous protocol (Chen et al., 2009, 2010). Cells were harvested in
lysis buffer (Sigma, St Louis, MO) and left on ice for 20 min and then were centrifuged (8000 × g for 30 min at 4 °C). The proteins in the supernatant were measured by Thermo BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). The supernatant was diluted to 15–30 μg of protein/mL with loading buffer (Fermentas, Vilnius, Lithuania) and ddH2O to 20 μL. Samples were heated to 92 °C for 5 min to denature the proteins. Proteins were separated by electrophoresis on a 10% (v/v) polyacrylamide gel at 60 V for 30 min and 100 V for 1 h, transferred onto a polyvinylidene fluoride membrane at 100 mA for 1 h at 4 °C, and then immunoblotted with antiactin, GAPDH, MAP1b, MAP2, NF-L, GFAP, JAK1, ERK, JNK, and p38 antibody (with 1:10,000, 1:10,000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:2000, and 1:1000 dilution, respectively) purified from rabbit serum at 4 °C overnight. Immunoreactive bands were detected using the horseradish peroxidase-conjugated antirabbit IgG and enhanced by Geliance 600 Imaging System (J&H Technology Co., Ltd). Bandwidths were quantified by MultiGauge V1.1 (Fujifilm, Stamford, CT). 2.7. Statistical analysis Data were presented as means ± standard deviation. Each experiment was performed in triplicate at least three times independently. The statistical significance of differences among groups was performed with one-way analysis of variance (ANOVA), followed by Duncan’s test using SSPS (ver 17.0; Chicago, IL). The differences were considered statistically significant when p < 0.05.
3. Results 3.1. Acrylamide inhibits RA-induced neurite outgrowth and cell proliferation of SH-SY5Y cells As shown in Figure 1A, after 10 μM RA stimulation, SH-SY5Y cells developed a round cell body and an extended axon-like neurite. Under co-exposure to the RA and different concentrations (0, 0.1, 0.5, 1, and 2 mM) of ACR, 0.5 mM ACR caused the SH-SY5Y cells to exhibit shorter neurite morphology, while 1 and 2 mM ACR resulted in cells with no neurite morphological extensions (Fig. 1A). Moreover, Western blot analysis found that expression levels of NFL, a neurofilament subunit expressed in mature neurons, were increased after RA treatment and decreased under ACR co-treatment in SH-ST5Y cells (Fig. 1B). MTT assay showed that different concentrations (0, 2.5, 5, and 10 μM) of RA treatment significantly induced SH-SY5Y cell proliferation at 24, 48, and 72 h when compared with the control (0 h) (Fig. 2A). Cell proliferation of SHSY5Y cells at 72 h did not show statistically significant differences as a result of different concentrations (0, 2.5, 5, and 10 μM) of RA treatment (Fig. 2A). However, induction of SH-SY5Y cell proliferation by 10 μM RA at 72 h was significantly decreased in 0.5, 1, and 2 mM ACR co-treatment when compared with the control (0 mM) (Fig. 2B). These results indicated that neurite outgrowth of SHSY5Y cells was inhibited by ACR and that proliferation of SH-SY5Y cells was attenuated by ACR treatment in a time- and dosedependent manner. 3.2. Acrylamide inhibits BA-induced extension development and cell proliferation of U-1240 MG cells As shown in Figure 3A, the round cell bodies of U-1240 MG cells were differentiated into an axon-like extended neurite after 10 μM BA stimulation for 72 h. ACR exposure interfered with BA-induced U-1240 MG cell development such that the cells developed shorter extensions after exposure to 0.5 and 1 mM ACR, while no cell extensions were developed after exposure to 2 mM ACR. GFAP, a marker for glia cell differentiation, was analyzed by Western blot for U-1240 MG cells, which were co-treated with BA and different concentrations of ACR (Fig. 3B). Relative levels of GFAP in U-1240 MG cells increased after 10 μM BA treatment but decreased significantly in BA and ACR co-treatment groups. As shown in Figure 4A, different concentrations (0, 2.5, 5, and 10 μM) of BA treatment induced U-1240 MG cell proliferation significantly at 24, 48, and 72 h when compared with the control (0 h). There were no statistically significant differences in U-1240 MG cell proliferation resulting from
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NF-L GAPDH Fig. 1. ACR inhibited the RA-induced neurite outgrowth of SH-SY5Y cells. SH-SY5Y cell neurite outgrowth was induced by treatment with 10 μM RA for 72 h and was compared with the control (the control cells were grown in the cell-cultured medium without RA) (A). 10 μM RA induced the neurite outgrowth of SH-SY5Y cells, which were inhibited by different concentrations (0.1, 0.5, 1, and 2 mM) of ACR for 72 h (A). The scale bar is 200 μm. Protein expression of NF-L was analyzed by Western blot in the RA (10 μM) co-treatment with different concentrations (0.1, 0.5, 1, and 2 mM) of ACR at 72 h (B). ACR, acrylamide; RA, retinoic acid; NF-L, neurofilament protein-L.
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Fig. 2. ACR inhibited RA-induced SH-SY5Y cell proliferation. Cell proliferation was analyzed via MTT assay. SH-SH5Y cell proliferation was induced by different concentrations (0, 2.5, 5, and 10 μM) of RA for 72 h (A). ACR inhibited RA (10 μM)-induced SH-SY5Y cell proliferation in a time (0, 24, 48, and 72 h)- and concentration (0, 0.1, 0.5, 1, and 2 mM)-dependent manner (B). Results are presented as the mean ± SD from three independent experiments. Statistically significant differences with the controls (0 h or 0 μM) are shown as *p < 0.05, **p < 0.01, ***p < 0.001. ACR, acrylamide; RA, retinoic acid.
treatment with different concentrations of BA (Fig. 4A). Induction of U-1240 MG cell proliferation by 10 μM BA at 72 h was significantly decreased only in 2 mM ACR co-treatment when compared with the control (0 mM) (Fig. 4B). These results revealed that cell extension and proliferation of U-1240 MG cells were inhibited after ACR treatment.
3.3. Acrylamide inhibits MAPs expression and JAK-STAT signaling of cellular differentiation in SH-SY5Y and U-1240 MG cells The biochemical indicators for morphologic changes were detected through Western blot analysis with different antibodies against MAP1b and MAP2c. The results showed that SH-SY5Y cells stimulated with 10 μM RA exhibited a 21% increase in MAP1b expression and a 123% increase in MAP2c expression (Fig. 5A), while U-1240 MG cells stimulated with 10 μM BA exhibited a 16% increase in MAP1b expression and a 52% increase in MAP2c expression (Fig. 5B), respectively. When co-treated with 2 mM ACR, the expressions of MAP1b and MAP2c in SH-SY5Y cells (Fig. 5A) were reduced by 39% and 64%, respectively, while the expressions of MAP1b and MAP2c in U-1240 MG cells (Fig. 5B) were reduced by 52% and 57%, respectively. In terms of JAK-STAT signaling, JAK1 expression (Fig. 5) was increased by 93% in 10 μM RA-stimulated SH-SY5Y cells and by 108% in 10 μM BA-stimulated U-1240 MG cells. Furthermore, JAK1 expression in SH-SY5Y cells was decreased by 68% when the cells were co-treated with 2 mM ACR and 10 μM RA (Fig. 5A), while JAK1 expression in U-1240 MG cells was decreased by 74% when the cells were co-treated with 10 μM BA and 2 mM ACR (Fig. 5B). These results revealed that MAPs expression and JAK-STAT signaling were involved in ACR-inhibited differentiation in SH-SY5Y and U-1240 MG cells.
3.4. Acrylamide inhibits phosphorylation of MAPKs in U-1240 MG cells The influences of MAPKs in ACR-treated neuroblastoma cells (SHSY5Y) have also been reported in Okuno et al. (2006), but no previous studies have reported on their influences in ACR-treated glioblastoma cells. Therefore, the protein expressions of ERK, JNK, and p38 of MAPKs in cultured U-1240 MG cells after exposure to 2 mM ACR for 48 h were measured by Western blot. As shown in Figure 6, phosphorylation of ERK and JNK, but not the total amount of ERK and JNK, was reduced after 2 mM ACR treatment. Furthermore, caffeine – an inhibitor of ATM/ATR used to block the upstream portion of the ACR-induced DNA-damaging signal pathway – successfully reversed the suppression of phosphorylation of ERK and JNK caused by 2 mM ACR treatment. These results demonstrated that caffeine attenuated ACR-inhibited phosphorylation of MAPKs in U-1240 MG cells.
4. Discussion The results of the present study suggested that cellular differentiation in human neuroblastoma and glioblastoma cells is inhibited after ACR exposure and that down-regulation of the JAK-STAT signal pathway may further explain the ACR-induced neurodegeneration. In a neuronal system, astrocytes maintain the survival of neuronal cells by glutamate uptake, calcium control, and glia scar formation (Sofroniew and Vinters, 2010). Astrocytes play a role in neurodegenerative diseases of the CNS (Miller, 2005; Xie et al., 2004), and GFAP accumulation is a hallmark of astrogliosis, which represents glia-increasing activation during CNS damage (Eng and Ghirnikar, 1994). Sriram et al. (2004) reported evidence that
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GFAP GAPDH Fig. 3. ACR inhibited BA-induced extension of U-1240 MG cells. Extension of U-1240 MG cells was induced by 10 μM BA for 72 h treatment, which was compared with the control (the control cells were grown in the cell-cultured medium without BA) (A).10 μM BA-induced extension of U-1240 MG cells was inhibited by different concentrations (0.1, 0.5, 1, and 2 mM) of ACR co-treatment for 72 h (A). The scale bar is 200 μm. Protein expression of GFAP was analyzed by Western blot in the BA (10 μM) cotreatment with different concentrations (0.1, 0.5, 1, and 2 mM) of ACR at 72 h (B). ACR, acrylamide; BA, butyric acid; GFAP, glial fibrillary acidic protein.
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Fig. 4. ACR inhibited cell proliferation in BA-induced U-1240 MG cells. Cell proliferation was analyzed via MTT assay. Different concentrations (0, 2.5, 5, and 10 μM) of BA treatment for 72 h induced U-1240 MG cell proliferation (A). ACR inhibited BA (10 μM)-induced U-1240 MG cell proliferation in a time (0, 24, 48, and 72 h)- and concentration (0, 0.1, 0.5, 1, and 2 mM)-dependent manner (B). Results are presented as the mean ± SD from three independent experiments. Statistically significant differences with the controls (0 h or 0 μM) are shown as *p < 0.05, **p < 0.01, ***p < 0.001. ACR, acrylamide; BA, butyric acid.
JAK-STAT signaling is related to the accumulation of GFAP in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration or astrogliosis. In this study, JAK1 expression was increased during the induction of differentiation and decreased under ACR co-treatment in both SH-SY5Y and U-1240 MG cells (Fig. 5). Therefore, down-regulation of the JAK-STAT signal pathway was involved in the ACR-inhibited differentiation of human neuronal and glial cells in vitro. Among the MAPKs, ERK showed functional differences in SHSY5Y and U-1240 MG cells under ACR-induced toxicity. ERK, JNK, and the p38 kinases are members of the MAPK pathway that regulate cellular proliferation, differentiation, and apoptosis of the neuronal developmental systems (Cargnello and Roux, 2011; Zhang and Liu, 2002). The crosstalk between MAPK and JAK-STAT signal cascades regulates cellular functional events by stimulation of cytokines and growth factors (Eulenfeld et al., 2012). Okuno et al. (2006) reported that the ERK pathway causes phosphorylation of p53 at Ser15 and the cytotoxicity of ACR in SH-SY5Y cells without RA-induced differentiation. Our previous studies showed that ACR induced cytotoxicity in U-1240 MG cells through the phosphorylation of p53 at Ser15 during ATM/ATR signaling (Chen et al., 2010). The current study further demonstrated that the phosphorylation of ERK and JNK were decreased by ACR exposure in U-1240 MG cells without BA-induced differentiation. These results suggested that the MAPK pathway is important in the regulation of ACR-induced cytotoxicity, and ERK presented functional differences in SH-SY5Y and U-1240 MG cells after ACR exposure. This study implies that ACR may affect early neurological development when the daily intake of ACR is high. ACR is an environmental neurotoxin and probable human carcinogen (Rice, 2005). Food-borne ACR can be produced during food processing via the Maillard reaction, especially during the processing of food con-
taining asparagine and glucose (Friedman, 2003; Tareke et al., 2002). Investigators have previously suggested that pregnant women and breast-feeding mothers avoid ACR-containing food to lessen the possibility of contributing to ACR-induced toxicity in their children as well as the possibility of fetal exposure to ACR via the exchange of blood flow in the placenta (Sorgel et al., 2002; FAO/WHO, 2005). The toxic effects of ACR on neurons have been investigated intensively, including the reduction of cell viability, the induction of apoptosis and p53 phosphorylation (Okuno et al., 2006; Sumizawa and Igisu, 2007), the formation of perikaryal inclusion bodies (Hartley et al., 1997), and the neurodegeneration of signal transduction (Yuzo et al., 2001). ACR-induced DNA breaks will increase p21 expression and G0/G1 cell-cycle arrest by inhibiting cyclin D1/Cdk4/6 kinase activity, which in turn causes cellular mitochondria collapse and apoptosis (Chen et al., 2010, 2013). Our study further suggested that ACR impairs the differentiation efficiency of human neurons and glia cells and that the interactions of neurons and glia cells in the neuronal system may be affected by ACR-induced neurotoxicity. Thus, ACR-induced toxicity in a fetus or during childhood could hinder early neuronal system development, a potential effect which is worthy of further investigations in vivo. In addition, it should be pointed out that this is a mechanistic study that used an in vitro system, and thus outlines the effects of ACR on cellular differentiation. Previous studies have reported that diet (including smoking) is the main source of environmental ACR exposure in humans and that, according to LC-MS/MS assay, the halflife of ACR is 2.2–7 hours in various bodily fluids (Sorgel et al., 2002; Boettcher et al., 2006). Thus, it would be difficult to accumulate ACR via a normal human diet to cause effects similar to those of the current high-dose study. However, ACR is chemically reactive toward nucleophiles (including amino and thiol groups in amino acids and proteins) and reacts with DNA to generate adducts with ring
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Fig. 5. ACR induced dose-dependent inhibition of protein expression in biochemical and signal transduction relative to differentiation in SH-SY5Y and U-1240 MG cells. Protein expressions of MAP1b, MAP2c, and JAK1 in cells treated with different concentrations of ACR (0.1, 0.5, 1, and 2 mM) were analyzed by Western blot. Panel A shows RA (10 μM)-treated SH-SY5Y cells treated with or without ACR for 72 h. Panel B shows BA (10 μM)-treated U-1240 MG cells treated with or without ACR for 72 h. Results are presented as the mean ± SD from three independent experiments. Statistically significant differences are shown as *p < 0.05, **p < 0.01, and ***p < 0.001. ACR, acrylamide; RA, retinoic acid; BA, butyric acid; MAP1b/2c, microtubule-associated protein 1b/2c; JAK1, Janus kinase 1.
nitrogen atoms and extra-nuclear amino groups of adenine and guanine (Klaunig, 2008; Rice, 2005). These DNA adducts cause depurinating lesions, and the repair of these lesions can also lead to the formation of DNA breaks (Friedberg et al., 1995; Martins et al., 2007). In a study that followed human subjects for a period of 13.3 years, a positive association between dietary acrylamide and renal cell cancer risk was found (Hogervorst et al., 2008), and increasing incidences of endometrial, ovarian, and renal cancer with increased dietary ACR intake have also been reported (Hogervorst
et al., 2009). Moreover, ACR-induced neurological symptoms followed by neuropathy have been reported in a cohort study of occupationally exposed workers (He et al., 1989). This study strongly indicated that accumulative effect of ACR might exist and significantly high levels of ACR exposure also might exist at least in occupationally exposed workers. In addition, our previous studies demonstrated that different concentrations (0.5–2 mM) of ACR exposure (0–72 h) could lead to DNA damage, apoptosis and cytotoxicity effects of U-1240 MG and/or SH-SY5Y cells in a
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Fig. 6. ACR inhibited the MAPK pathway in U-1240 MG cells. Protein expressions of MAPKs (ERK, JNK, and p38) were analyzed by Western blot. Caffeine was used as ATM/ATR inhibitor to block the upstream signal of the ACR-induced DNAdamaging signal pathway. The ACR (2 mM)-treated U-1240 MG cells were cotreated with different concentrations (0, 0.25, 0.5, and 1 mM) of caffeine for 48 h. ACR, acrylamide; ERK, extracellular-signal-regulated kinases; JNK, c-Jun N-terminal kinases.
time- and dose-dependent manner (Chen et al., 2009, 2010, 2013). The present study further indicates that ACR inhibits cellular differentiation of U-1240 MG and SH-SY5Y cells. Taken together, the aforementioned findings suggest that mechanistic studies involving in vitro systems should be conducted in order to better understand ACR-induced toxicity. In conclusion, the findings of the current study suggest that ACR impairs the differentiation efficiency of neuronal (SH-SY5Y) and glial (U-1240 MG) cells. RA-induced neurogenesis of SH-SY5Y and BAinduced astrogliogenesis of U-1240 MG cells were attenuated by ACR, and these effects were associated with the down-regulation of MAP expression and JAK-STAT signaling. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This study was supported by a grant from National Science Council (NSC 99-2313-B-002 -029) of Taiwan. We thanked Miss YanShiu Wang for her excellent technical assistance. References Boettcher, M.I., Bolt, H.M., Angerer, J., 2006. Acrylamide exposure via the diet: influence of fasting on urinary mercapturic acid metabolite excretion in humans. Arch. Toxicol. 80, 817–819.
Cargnello, M., Roux, P.P., 2011. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol. Mol. Biol. Rev. 75, 50–83. Chauhan, N.B., Spencer, P.S., Sabri, M.I., 1993. Effect of acrylamide on the distribution of microtubule-associated proteins (MAP1 and MAP2) in selected regions of rat brain. Mol. Chem. Neuropathol. 18, 225–245. Chen, J.H., Wu, K.Y., Chiu, I.M., Tsou, T.C., Chou, C.C., 2009. Acrylamide-induced astrogliotic and apoptotic responses in human astrocytoma cells. Toxicol. In Vitro 23, 855–861. Chen, J.H., Tsou, T.C., Chiu, I.M., Chou, C.C., 2010. Proliferation inhibition, DNA damage, and cell-cycle arrest of human astrocytoma cells after acrylamide exposure. Chem. Res. Toxicol. 23, 1449–1458. Chen, J.H., Yang, C.H., Wang, Y.S., Lee, J.G., Cheng, C.H., Chou, C.C., 2013. Acrylamideinduced mitochondria collapse and apoptosis in human astrocytoma cells. Food Chem. Toxicol. 51, 446–452. Couchie, D., Fages, C., Bridoux, A.M., Rolland, B., Tardy, M., Nunez, J., 1985. Microtubule-associated proteins and in vitro astrocyte differentiation. J. Cell Biol. 101, 2095–2103. Douglas, A.H., 2012. The JAK/STAT pathway. Cold Spring Harb. Perspect. Biol. 4, a011205. Eng, L.F., Ghirnikar, R.S., 1994. GFAP and astrogliosis. Brain Pathol. 4, 229–237. Eulenfeld, R., Dittrich, A., Khouri, C., Müller, P.J., Mütze, B., Wolf, A., et al., 2012. Interleukin-6 signalling: more than JAKS and STATs. Eur. J. Cell Biol. 91, 486–495. Exon, J.H., 2006. A review of the toxicology of acrylamide. J. Toxicol. Environ. Health B Crit. Rev. 9, 397–412. FAO/WHO, 2002. Consultation on the health implications of acrylamide in food. Summary Report
