JOURNAL OF NEUROCHEMISTRY

| 2011 | 117 | 3847

doi: 10.1111/j.1471-4159.2010.07162.x

State Key Laboratory of Natural and Biomimetic Drugs and Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, China

Abstract Introduction of Gadolinium (Gd) to the nervous system is linked to the development of neurotoxicity involving both oxidative and endoplasmic reticulum (ER) stress. Gd levels (0.220 lM) in the form of gadolinium trichloride (GdCl3) cause neurotoxicity in vitro. We investigated the signaling pathways in primary cultured rat cortical neurons and tested whether GdCl3 induced oxidative and ER stress. Results showed that Gd-induced neural cell death followed a rapid accumulation of intracellular reactive oxygen species. In addition, Gd exposure resulted in spliced X-box binding protein 1 mRNA and increased expression of binding immunoglobulin protein, thus activating transcription factor 4 (ATF4), ATF6, and C/EBP homologous

protein mRNA. Up-regulated expression of binding immunoglobulin protein is a hallmark of ER stress and C/EBP homologous protein is an ER stress-related pro-apoptotic transcription factor. Activation of ER stress downstream substrates, inositol-requiring kinase 1 and ATF6, was also observed in Gd-treated cells. The neurotoxic effects of Gd were blocked by the antioxidant N-acetylcysteine. Results demonstrated that Gd-induced cytotoxicity in neurons occurs via oxidative injury and ER stress-related signal transduction, thus offering new insight into the neurotoxicology of gadolinium. Keywords: endoplasmic reticulum stress, gadolinium, neurotoxicity, oxidative stress. J. Neurochem. (2011) 117, 3847.

Gadolinium (Gd), a member of the lanthanide family, has been used extensively in various industrial and medical applications. Gd-chelated derivatives have been widely used in clinical practice as a contrast medium for magnetic resonance imaging (Adding et al. 2006). A Gd derivative, Motexan gadolinium, is currently undergoing phase III clinical trials as a treatment for lung cancer, specically targeting brain metastases (Gelsomina et al. 2001). Human exposure to Gd and its derivatives is signicantly increasing, posing a high risk of accumulation and retention. Recently, nephrogenic systemic brosis, a fatal disease, was reported in patients with severe kidney failure who were administered Gd-based contrast agents (Perazella and Rodby 2007; Abraham et al. 2008). Experimental studies demonstrated severe behavioral changes, including neurological alterations and seizure phenomena, following intrathecal administration of 515 lmol Gd-based contrast agents/g rat brain (Toney et al. 2001). Other lanthanides, such as lanthanum (La) and ytterbium, have been shown to be neurotoxic in rats by penetrating the bloodbrain barrier and accumulating in the cerebral cortex, hippocampus, and cerebellum (Feng et al. 2006; Yang et al. 2006). Although in vivo release of toxic-free Gd ions (Gd3+) through transmetalation and tissue retention have been proposed as

the genesis of the toxic effects (Marckmann et al. 2006; Broome 2008), the mechanisms by which Gd induces neuronal toxicity remain poorly understood. Increasing evidence suggests oxidative stress as the underlying mechanism in a variety of metal-induced cytotoxicity (Yang et al. 2004; Milatovic et al. 2007).
Received June 2, 2010; revised manuscript received December 15, 2010; accepted December 20, 2010. Address correspondence and reprint requests to Qing Xia and Xiaoda Yang, State Key Laboratory of Natural and Biomimetic Drugs and Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China. E-mails: xqing@bjmu.edu.cn and xyang@bjmu.edu.cn Abbreviations used: ATF, activating transcription factor; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; CLSM, laser-scanning confocal microscope; DCFH-DA, 2,7-dichloruorescein-diacetate; EBSS, Earles balanced salt solution; ER, endoplasmic reticulum; GdCl3, gadolinium trichloride; GRP78, glucose-regulated protein 78; IRE1, inositol-requiring kinase 1; LDH, lactate dehydrogenase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NAC, N-acetylcysteine; p-eIF2, phosphorylated a-subunit of translation initiation factor 2; PERK, protein kinase regulated by RNA-like ER-associated kinase; ROS, reactive oxygen species; TBST, tris-balanced saline Tween-20; TRPC, transient receptor potential channel; UPR, unfolded protein responses; XBP1, X-box binding protein1.

38

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

Gd-induced OS triggers ER stress | 39

Promoted production of free radicals, including reactive oxygen species (ROS) and nitric oxide, as a result of upstream redox, causes organelle dysfunction in cells (Holtz et al. 2006). The endoplasmic reticulum (ER), a eukaryote organelle, is responsible for synthesizing, folding, and transporting nascent proteins (Marciniak and Ron 2006; Nakayama et al. 2008). Previous studies have shown that the ER plays an important role in neuronal death and pathogenesis, including neurological and neurodegenerative diseases (Tajiri et al. 2004; Korhonen et al. 2005). Various cell toxins trigger ER signaling events, which occur as ER stress and subsequent unfolded protein responses (UPR). In mammalian cells, various signaling pathways are involved in the regulation of UPR. ER-resident signaling molecules, including activating transcription factor 6 (ATF6), inositol-requiring kinase 1 (IRE1), protein kinase regulated by RNA-like ER-associated kinase (PERK), and binding immunoglobulin protein (BiP) (also known as glucose-regulated protein 78, GRP78), were activated in response to ER stress (Kudo et al. 2008). Activation of ATF6 and IRE1, increases transcription of ER chaperones and genes (such as BiP/GRP78), and IRE1-initiated X-box binding protein 1 (XBP1) mRNA splicing, which protects neurons from injury (Hayashi et al. 2003; Rungtawan et al. 2004). As a protein serine/threonine kinase, PERK undergoes transphosphorylation in response to ER stress and phosphorylates the a-subunit of translation initiation factor 2 (eIF2a), which results in inhibition of translation initiation. Consequently, PERK alleviates ER stress by reducing the production of unfolded/misfolded proteins. However, severe ER stress triggers cell apoptosis, initiated by ER-localized caspases (caspase 12) and CCAAT/ enhancer binding protein homologous protein (CHOP, also called Gadd153) (Benavides et al. 2005; Smith and Deshmukh 2007). Recent studies demonstrated that ER is an important target of free radicals in neurons exposed to neurotoxic metals (Yu et al. 2006). Gadolinium, as well as other lanthanides, exhibit various biological effects (Fricker 2006) and was previously reported to induce ROS generation in human hepatic cells (Liu et al. 2003). ER stress may possibly be directly involved in the toxicity of Gd to neurons because ER is vulnerable to the effects of ROS (Holtz et al. 2006), and preliminary studies demonstrated apparent ER stress in rat brain cortex upon injection of gadolinium trichloride (GdCl3) into the caudate nucleus (Fig. S1). Therefore, we investigated the role of ER stress in Gd-induced neurotoxicity relating to ROS.

non-radioactive cytotoxicity assay kit and AccessQickTM RT-PCR system were purchased from Promega (Madison, WI, USA); GdCl3, Thapsigargin (Tg) and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA); antibodies against BiP/GRP78 and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against CHOP, IRE1, ATF6, and phosphorylated eIF2a (p-eIF2a) were obtained from Cell Signaling Technology (Danvers, MA, USA). Primary cell culture and treatment Primary cortical neuronal cultures were prepared as previously described (Jiang et al. 2007). The brains were quickly removed from 18-day old embryonic Sprague Dawley rats, and whole cerebral neocortices were isolated. Tissues were incubated in 0.25% trypsin for 15 min at 37C and then triturated with a Pasteur pipette. The clumps were removed by ltering. The dissociated cortical neurons were cultured in poly-D-lysine (5 lg/mL)-coated wells in Dulbeccos modied Eagles medium (10% fetal bovine serum, 100 U/mL penicillin, 5 mM HEPES, and 500 ng/mL insulin) at 37C and 5% CO2. Cytosine arabinoside (10 lg/mL) was added to the culture medium 3 days after plating to reduce the number of astrocytes, and the medium was changed twice per week. The experiments were performed after cells were maintained in culture for 9 days. Cells were washed twice with Earles balanced salt solution (EBSS; containing 116 mM NaCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5.4 mM KCl, 1 mM NaH2PO4, 14.7 mM NaHCO3, 5.5 mM glucose and 10 mM HEPES at pH 7.4), and then exposed to different concentrations of Gd (diluted in EBSS) for varying periods of time as indicated. Cadmium (Cd) was used as a positive control for neurotoxicity (Lopez et al. 2003) and 8 lM Tg was used as positive control for ER stress. Cell viability and lactate dehydrogenase (LDH) release assay Cell viability was measured via a colorimetric assay based on reduction of [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sufo-phenyl)-2H-tetrazolium, inner salt; MTS] using Cell Titer 96AQueous MTS reagent. The LDH released into the culture medium was quantied using a Cytotox 96 Non-radioactive Cytotoxicity Assay kit. Formazan product was measured using a micro-plate reader (Thermo Fisher Scientic Inc., Waltham, MA, USA) at 492 nm. The results were presented as percentages of the sample values over the control. Measurement of intracellular Ca2+ levels A uorescent probe (Fluo-3 AM) was used to detect changes of intracellular Ca2+ levels. To detect immediate changes in cellular Ca2+ levels, the cells were pre-incubated with 5 lM Fluo-3 AM at 37C for 30 min. The cells were then washed with EBSS and exposed to different concentrations of Gd (0.2, 2, and 20 lM). Next, 5 lM KCl and 8 lM Tg were used as positive controls for intracellular Ca2+ homeostasis. Upon addition of Gd, changes in intracellular Ca2+ levels were monitored for 20 min on a laserscanning confocal microscope (CLSM, Leica TCSNT; Leica Co., Wetzlar, Germany) equipped with 488-nm excitation and 526-nm emission. Fluorescent intensity represented intracellular Ca2+ levels. Data were analyzed using Leica TCSNT software. To measure changes in intracellular Ca2+ levels caused by exposure to Gd, cells were rst treated with varying concentrations

Materials and methods

Materials Dulbeccos modied Eagles medium and fetal bovine serum were purchased from Gibco BRL (Gaithersburg, MD, USA); Flu3-AM and 2,7-dichloruorescein-diacetate (DCFH-DA) were obtained from Invitrogen (Paisley, UK); Titer 96AQueous MTS reagent, Cytotox 96

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

40 | Q. Xia et al.

Primer sets XBP1 BiP ATF4 ATF6 CHOP b-actin

Nucleotide sequences of primers 5-acacgcttggggatggatgc-3 5-ccatgggaagatgttctggg-3 5-gtttgctgaggaagacaaaaagctc-3 5-cacttccatagagtttgctgataattg-3 5-cctgactctgctgcttatattactctaac-3 5-actccaggtgggtcataaggtttg-3 5-ggatttgatgccttgggagtcagac-3 5-atttttttctttggagtcagtccat-3 5-cctgaaagcagaaaccggtc-3 5-cctcataccaggcttccagc-3 5-tcctccctggagaagagctac -3 5-tcctgcttgctgatccacat-3

Product length (bp) 171 272 160 163 101 383

Cycles 30 30 30 30 35 30

Annealing Temperature (C) 60 60 60 60 55 55

Table 1 PCR primers, amplication product length, and PCR conditions

of Gd for 90 min. The cells were then washed twice with EBSS and incubated for 30 min with 5 lM Fluo-3 AM at 37C. After another three EBSS washings, intracellular Ca2+ levels were observed using a CLSM. Measurement of intracellular ROS levels 2,7-Dichloruorescein-diacetate was used to monitor ROS levels. Primary cultured cortical neurons were treated with Gd at different concentrations (0.2, 2, and 20 lM) for 90 min, washed twice, and then incubated with 5 lM DCFH-DA at 37C for 30 min. After three washings with phosphate buffered saline, the cells were observed on a CLSM (488-nm excitation and 526-nm emission). Fluorescent intensity corresponds to levels of intracellular ROS. Data were analyzed using Leica TCSNT software. To detect continuous changes in ROS levels, cells were preincubated with 5 lM DCFH-DA at 37C for 30 min. After washing with EBSS, the cells were monitored for  25 min on a CLSM. Reagents (20 lM Gd, 5 lM H2O2, or 20 lM Cd) were added to the culture dish after baselines were established (5 min). Fluorescent intensity corresponds to levels of intracellular ROS. Data were analyzed using Leica TCSNT software. RT-PCR analysis To determine XBP1 mRNA splicing and mRNA expression levels of BiP, ATF4, ATF6, CHOP, and b-actin mRNA, RT-PCR was performed as previously described (Shen et al. 2008). Total RNA was harvested immediately after the cells were exposed to different concentrations of Gd for the respective periods of time. RT-PCR analysis was performed in accordance with the AccessQick RTPCR System (Promega) manufacturer instructions. PCR was conducted for 30 cycles (94C for 30 s; 58C for 30 s; and 72C for 1 min in nal cycle). Primer sequences are presented in Table 1. The number of cycles for each product was determined from preliminary kinetic studies. PCR products were electrophoresed in a 2.0% agarose gel and visualized using ethidium bromide. Band density was measured using United-Bio GK-330C Plus densitometer software (United-Bio, Tokyo, Japan). The ratio of target cDNA to b-actin was used as an estimate of mRNA abundance.

Western blot analysis Cell lysates were prepared using NP-40 lysis buffer (Li and Lee 1991) after neuron cells were exposed to Gd for 90 min. Proteins were separated on 12% acrylamide sodium dodecyl sulfatepolyacrylamide gel electrophoresis and electroblotted to a polyvinylidene diuoride immunoblotting membrane. The membrane was blocked with 5% non-fat milk in TBST (Trisbalanced saline Tween-20: 50 mM Tris-HCl; 150 mM NaCl, and 0.02% Tween-20, pH 7.5) for 1 h, then incubated overnight at 4C with primary antibodies (rabbit polyclonal antibodies against GRP78 and ATF6, or mouse polyclonal antibodies against CHOP, IRE1, and p-eIF2a) diluted in TBST (1 : 1000). This was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (1 : 6000) for 1 h at 37C. The membrane was washed three times with TBST buffer between each step. The bands were visualized using an enhanced chemiluminescent detection kit (Applygen Technologies, Beijing, China). Optical band densities were quantied using Gel-Pro Analyzer (Media Cybernetics, Inc., Bethesda, MD, USA). Statistical analysis Statistical signicance for multiple comparisons was determined by Students t-test using SPSS16.0 software (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically signicant. Group sizes for the various experiments varied (n = 57) and are described in the corresponding gures. Error bars in the graphs indicate SD.

Results
Gadolinium decreases cell viability of neurons The effects of Gd treatment on neural death/cell viability were estimated by measuring MTS production and LDH release into culture medium. Results showed that incubation of neurons with Gd decreased cell viability (Fig. 1a) and increased LDH release into the culture medium (Fig. 1b) in both time- and concentration-dependent treatments. The immunocytochemistry experiment shows that the purity of primary cultured neurons is above 95% (Fig. S1).

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

Gd-induced OS triggers ER stress | 41

(a)

(a)

(b)

(b)

Fig. 1 Quantitative analyses of primary neural cell death by MTS and LDH assays. Primary cultured rat neurons incubated with EBSS contains 0.2, 2, and 20 lM of GdCl3 for 30, 60, 90, and 180 min. Cell death was measured by MTS and LDH assays as described. (a) MTS assay of cell viability and (b) LDH release assays. Values represent mean SD in separate experiments (n = 5). *p < 0.05, **p < 0.01 compared with the control group.

(c)

Gadolinium disrupts intracellular Ca2+ homeostasis The low-afnity uorescent Ca2+ probe Fluo 3-AM was used to detect changes in intracellular Ca2+ levels following exposure of neurons to Gd. Gd (0.2, 2, or 20 lM) did not induce changes in intracellular Ca2+ levels within 20 min (Fig. 2a) in contrast to rapid elevations of Ca2+ levels upon treatment with KCl and Tg. However, exposure to Gd for 90 min resulted in signicantly elevated intracellular Ca2+ levels (Fig. 2b and c). Gadolinium causes a rapid elevation in intracellular ROS levels Previously, Gd was shown to induce rapid elevation of intracellular ROS in human liver cell line (Liu et al. 2003). Intracellular ROS production was observed on a CLSM using a DCFH-DA probe. Increased intracellular ROS levels in cortical neuronal cultures were observed with increased incubation time and Gd concentrations. The production of intracellular ROS was modest after treatment with 20 lM Gd for 20 min (Fig. 3a), and rapidly increased after 30 min up to 120 min (Fig. 3b). A concentration-dependent increase in intracellular ROS was also observed after exposure of neurons to 0.2, 2, and 20 lM Gd for 90 min (Fig. 3c and d).

Fig. 2 Changes in intracellular Ca2+ levels. (a) Real-time laser confocal microscopy of intracellular Ca2+ within 20 min of loading with Flu3-AM following addition of 0.2, 2, and 20 lM Gd, 5 lM KCl, or 8 lM Tg. (b) Imaging of cells exposed to 20 lM Gd, or 20 lM Cd and 8 lM Tg for 90 min. (c) Quantication of uorescence intensity of (b). Data represent mean SD (n = 7). *p < 0.05, **p < 0.01 compared with the control group.

Gadolinium promotes expression of ER stress markers The ER is a primary target in various neuronal disorders (Paschen 2003). ER stress has been proposed to be involved in ROS-induced cell death (Hayashi et al. 2005). In this article, a RT-PCR assay was utilized to estimate the expression levels of ER stress-related genes. Results revealed expression of unspliced and spliced XBP1 in cells following treatment with varying concentrations of Gd with varying exposure durations (Fig. 4a) and altered expression levels of

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

42 | Q. Xia et al.

(a)

(c)

(b)

(d)

Fig. 3 Gadolinium rapidly elevates intracellular ROS levels in primary cultured cortical neurons. (a) Real-time laser confocal microscopy of ROS generation in neurons loaded with DCFH-DA following treatment with 20 lM Gd within 20 min. (b) Generation of ROS in neurons treated with 20 lM Gd for varying periods of time. (c) Imaging of intracellular

ROS in neurons treated with Gd (0.2, 2, and 20 lM) for 90 min. (d) Quantication of uorescent intensity from (c). Cells exposed to 20 lM Cd or 5 lM H2O2 served as the positive control for ROS production. Data represent mean SD (n = 7). *p < 0.05, **p < 0.01 compared with the control group.

other ER signaling genes, such as BiP, ATF4, ATF6, and CHOP (Fig. 4b). Expression of the ER membrane-anchored protein BiP/ GRP78 and other ER stress-associated proteins (CHOP, ATF6, and IRE1) were measured via western blot analysis following exposure of neurons to0.2, 2 and 20 lM of Gd for 90 min. Tg, as positive control for ER stress, signicantly increased BiP/GRP78 expression. BiP/GRP78 expression increased intensively in cells treated with 2 lM Gd and higher (Fig. 5). In addition, spliced ATF6, increased CHOP, PERK, and p-eIF2a expression (Fig. 5) in neural cells were observed upon Gd treatment.

20 lM Gd for various time periods. Pre-treatment with NAC also blocked ROS production, reduced expression of GRP78 and CHOP, and increased neuronal viability (Fig. 7).

Discussion
Endoplasmic reticulum stress has been involved in pathogenesis of neurodegenerative diseases, such as Parkinsons disease (Schwab et al. 1996), Alzheimers disease (Gibson and Zhang 2001), and cerebral ischemia (DeGracia and Montie 2004). ER stress and oxidative stress also have been reported playing vital role in heavy metal-induced neurotoxicity (Erikson et al. 2004; Biagioli et al. 2008). Our previous study has revealed that oxidative stress was involved in the neurotoxicity of Gadolinium (Feng et al. 2010, 2011; Xia et al. 2010). Meanwhile, in another related research, we reported that Gadolinium could trigger ER stress in primary cultured rat cortical astrocytes (Feng et al. 2010, 2011). Therefore, it is meaningful to investigate the potential mechanisms between oxidative stress and ER stress underlying this toxicity. Results demonstrated that Gd induced cell death in both time- and dose-dependent trials (Fig. 1). Exposure of cells to Gd caused rapid accumulation of intracellular ROS (Fig. 3), which led to the elevation of cellular Ca2+ levels (Fig. 2) and

N-acetylcysteine decreases ER stress and cell death To investigate the correlation between cellular ROS, ER stress, and cell death, the effects of the antioxidant NAC was studied. Results indicated that NAC (100 lM) signicantly blocked production of ROS (Fig. 6a) and completely blocked neuronal death (Fig. 6b) induced by 20 lM Gd. Meanwhile, expression of ER stress-associated proteins BiP/GRP78, IRE1, ATF6, and CHOP decreased upon NAC treatment, indicating the association of ROS with ER stress in Gdinduced neuronal death. To exclude the direct interaction between Gd and NAC, neuron cells were pre-treated with NAC and then exposed to

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

Gd-induced OS triggers ER stress | 43

(a)

(b)

Fig. 4 Expression of ER stress-associated mRNA in neurons treated with Gd. (a) Spliced XBP1 (XBP1s) in cells treated with 20 lM Gd for varying times, or with 0.2, 2, and 20 lM Gd for 90 min. (b) Expression of BiP, ATF4, ATF6, and CHOP mRNA in neurons treated with 0.2, 2, and 20 lM Gd. Exposure of cells to 8 lM thapsigargin (Tg) served as the positive control for ER stress.

production of ER stress makers (Figs 4 and 5). Meanwhile, interestingly, increased intracellular ROS production, ER stress, and even cell death induced by Gd treatment were blocked by NAC, suggesting an elevated cellular oxidative stress is the initial phase of Gd-induced neural cell death (Figs 6 and 7). Therefore, Gd causes neural cell death by elevating cellular ROS and ER stress (illustrated in Scheme 1). Endoplasmic reticulum stress was reported as the initial state of neuronal cell death (Reijonen et al. 2008; Miyake and Nagai 2009; Nishitsuji et al. 2009). Recent study suggested that enhanced production of the ER molecular chaperone GRP78 protects neuronal cells against death after glutamine- and oxidative insult-induced ER stress (Yu et al. 1999). The interaction of neurotoxic metal ions with GRP78 is deleterious to neuronal cells. Previously, we found that the Gd analog La resulted in increased expression of GRP78 in HepG2 cells (Shen et al. 2008). This study suggests the UPR signaling induced by Gd includes: (i) the IRE1-XBP1 pathway, indicated by spliced XBP1 mRNA (Fig. 4) and increased expression of GRP78 upon Gd treatment (Fig. 5). (ii) The ATF6 pathway, indicated by spliced ATF6 (Fig. 5). Upon activation, ATF6 is cleaved by proteases in the Golgi complex and leads to expression of ER chaperones (Yoshida et al. 2001). (iii) The PERK-eIF2a pathway, indicated by elevated levels of p-eIF2a and ATF4 (Fig. 5). PERK

Fig. 5 Western blot analysis of Gd-induced ER stress in primary cultured rat cortical neurons. (a) Expression of BiP/GRP78, CHOP, ATF6, IRE1a, PERK, and p-eIF2a derived from neurons treated with different concentrations of GdCl3 for 90 min. (b) Densitometry measurement of relative expression of BiP/GRP78, CHOP, IRE1a, PERK, p-eIF2a, and ATF6 normalized to that of b-actin. Data represent mean SD from three independent experiments. *p < 0.05, **p < 0.01 compared with the normal control group. Thapsigargin (Tg, 8 lM) served as the positive control of ER stress.

phosphorylates the a-subunit of eIF2a, thus inducing ATF4 translation while transiently blocking most protein translation (Bertolotti et al. 2000). Furthermore, we did in vivo study of Gd on CNS, western blot analysis and immunoorescent results suggested increased expression of Bip/GRP78, ATF6 and CHOP after injection of GdCl3 in rat caudate nucleus (Fig. S2). As an early response, Gd induced elevation of ROS in neurons (Fig. 3). This effect is similar to its lanthanide analogs. Apoptosis in hepatocytes, induced by Gd, La, and ytterbium, is closely associated with mitochondrial alterations and elevated ROS levels (Liu et al. 2003). La causes a decrease in antioxidant levels in the brain of rats (Charis et al. 2009). Similarly, ROS is also important in the neurotoxicity of other metals, such as Pb, Al, and Cd (Reijonen et al. 2008; Miyake and Nagai 2009). Previously, it was suggested that oxidative stress impairs disulde bond formation, leading to protein folding, and, subsequently, to ER stress and response (Rutkowski and Kaufman 2004). As shown in Figs 6 and 7, Gd-induced ROS is the major reason for ER stress of neurons, conrmed by the inhibitory effect of NAC.

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

44 | Q. Xia et al.

(a)

(c)

(b)

Fig. 6 Antioxidant NAC protects cells from death by decreasing ROS production and ER stress in primary cultured cortical neurons induced by 20 lM Gd. (a) Gd-induced production of cellular ROS is decreased by NAC treatment in primary cultured neurons. (b) NAC prevents cell

death induced by Gd. (c) Gd-induced expression of ER stress-related proteins is blocked by NAC. Data represent mean SD from three independent experiments. *p < 0.05, **p < 0.01 compared with the normal control group.

(a)

(c)

(b)

Fig. 7 Pre-treatment of NAC blocks Gd-induced ER stress by decreasing ROS production and protecting neurons from death. (a) Neuronal production of intracellular ROS induced by 90-min exposure to 20 lM Gd is decreased following 30-min pre-treatment with NAC (Pre-NAC + Gd), as well as cells treated with Gd and NAC together

(NAC + Gd). (b) Cell death induced by 20 lM Gd is inhibited when cells are pre-treated with 100 lM NAC for 30 min. (c) Pre-treatment of NAC blocks Gd-induced, ER stress-associated characteristics in neurons. Data represent mean SD from three independent experiments. *p < 0.05, **p < 0.01 compared with the normal control group.

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

Gd-induced OS triggers ER stress | 45

and Tahti 2004; Niu et al. 2005). Since Gd3+ does not have redox activity, does not bind to thiol(SH) groups, and does not exist in a form similar to tetrahydroxoaluminate (the active form of Al3+) (Dill et al. 1987), the mechanism of promotion of ROS by Gd is different from that of other metals and therefore warrants further investigation. In conclusion, Gd treatment causes elevated cellular ROS, inducing UPR in neurons. Gd-induced oxidative stress and subsequent activation of CHOP signaling ultimately cause neuronal death. However, this mechanism is blocked by antioxidants, such as NAC. This study may offer new insight into the toxicology of Gd on neurons.

Acknowledgement
Scheme 1 Potential mechanisms by which Gd induced neural cell death.

This study was supported by the National Natural Science Foundation of China (20901005 and 20637010).

Since ER is an intracellular Ca2+ source, severe cellular disruption could lead to ER stress and a subsequent change in Ca2+ concentration (Biagioli et al. 2008; Shen et al. 2008). As shown in Fig. 2, Gd only causes signicant elevation of Ca2+ after 90 min incubation (Fig. 2), which is responsible for the elevation of cellular ROS (within 30 min, Fig. 3). Considering the ER calcium pump is sensitive to oxidative damage (Cabiscol and Levine 1995), signicant increases in cellular Ca2+ level following 90-min rather than 20-min exposure to Gd (Fig. 2) indicate other cellular events, such as rapidly increasing cellular ROS levels (Fig. 3), which may be involved in Gd-induced intracellular Ca2+ disruption. Recently, Nicolas et al.s research has also pointed out that oxidative stress can partially deplete ER calcium stores, but ER Ca2+ release alone is not sufcient to explain ER stress activation during oxidative stress (Dejeans et al. 2010). Another study of our group has found that Gd elevates the calcium level via promotion of an inux of extracellular Ca2+ (Feng et al. 2010, 2011). The mechanisms of how rare earth metal ions. such as La3+ and Gd3+ are not conrmed, Jung et al. (2003) reported that micromoles of La3+ and Gd3+ were found to exert potentiating effects on the transient receptor potential channel (TRPC) subfamilies TRPC4 and TRPC5, which are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. Yu et al. also found La3+ can activate L-type Ca2+ and induce inux of Ca2+ (Yu et al. 2006). Therefore, Gd-induced increase in calcium level might be the result of inux of extracellular Ca2+ as well as release of ER calcium stores. The mechanism by which Gd promoted ROS production remains unknown. La3+ was shown to promote mitochondrial ROS production (Dong et al. 2009). Various toxic metals, such as Al3+ and Cd2+, induce ROS production by causing mitochondrial dysfunction (Liu et al. 2003; Toimela

Supporting information
Additional supporting information may be found in the online version of this article: Figure S1. Immunocytochemistry analysis of primary cultured cortical neurons. Figure S2. In vivo study of Gd on central nervous system, rats were injected of GdCl3 in caudate nucleus. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing les) should be addressed to the authors.

References
Abraham J. L., Thakral C., Skov L., Rossen K. and Marckman P. (2008) Dermal inorganic gadolinium concentrations: evidence for in vivo transmetallation and long-term persistence in nephrogenic systemic brosis. Br. J. Dermatol. 158, 273280. Adding L. C., Gerard L. B. and Lars E. G. (2006) Basic experimental studies and clinical aspects of gadolinium salts and chelates. Cardiovasc. Drug Rev. 19, 4156. Benavides A., Pastor D., Santos P., Tranque P. and Calvo S. (2005) CHOP plays a pivotal role in the astrocyte death induced by oxygen and glucose deprivation. Glia 52, 261275. Bertolotti A., Zhang Y., Hendershot L., Hardling H. and Ron D. (2000) Dynamic interaction of BiP and the ER stress transducers in the unfolded protein response. Nat. Cell Biol. 2, 326332. Biagioli M., Pifferi S., Ragghianti M., Bucci S., Rizzuto R. and Pinton P. (2008) Endoplasmic reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis. Cell Calcium 43, 184195. Broome D. R. (2008) Nephrogenic systemic brosis associated with gadolinium based contrast agents: a summary of the medical literature reporting. Eur. J. Radiol. 66, 230234. Cabiscol E. and Levine R. L. (1995) Carbonic anhydrase III. Oxidative modication in vivo and loss of phosphatase activity during aging. J. Biol. Chem. 270, 1474214747.

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

46 | Q. Xia et al.

Charis L., Apostolos Z., Stamatios T., Hussam A. H., Foteini A., Elena G., Zois M., Nikolina S. and Stylianos T. (2009) The neuroprotective role of L-cysteine towards the effects of short-term exposure to lanthanum on the adult rat brain antioxidant status and the activities of acetylcholinesterase, (Na+, K+)- and Mg2+-ATPase. Biometals 22, 329335. DeGracia D. J. and Montie H. L. (2004) Cerebral ischemia and the unfolded protein response. J. Neurochem. 91, 18. Dejeans N., Tajeddine N., Beck R., Verrax J., Taper H., Gailly P. and Calderon P. B. (2010) Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells. Biochem. Pharmacol. 79, 12211230. Dill E. T., Holden M. J. and Colombini M. (1987) Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum. J. Membr. Biol. 99, 187196. Dong S., Zhao Y., Liu H., Yang X. and Wang K. (2009) Duality of effect of La3+ on mitochondrial permeability transition pore depending on the concentration. Biometals 22, 917926. Erikson K. M., Dobson A. W., Dorman D. C. and Aschner M. (2004) Manganese exposure and induced oxidative stress in the rat brain. Sci.Total Environ. 334335, 409416. Feng L., Xiao H., He X., Li Z., Li F., Liu N., Zhao Y., Huang Y., Zhang Z. and Chai Z. (2006) Neurotoxicological consequence of long-term exposure to lanthanum. Toxicol. Lett. 165, 112 120. Feng X. D., Xia Q., Yuan L., Yang X. D. and Wang K. (2010) Impaired mitochondrial function and oxidative stress in rat cortical neurons: implications for gadolinium-induced neurotoxicity. Neurotoxicology 31, 391398. Feng X. D., Xia Q., Yuan L., Huang H. F., Yang X. D. and Wang K. (2011) Gadolinium triggers unfolded protein responses (UPRs) in primary cultured rat cortical astrocytes via promotion of an inux of extracellular Ca(2 + ). Cell Biol. Toxicol. 27, 12. Fricker S. P. (2006) The therapeutic application of lanthanides. Chem. Soc. Rev. 35, 524533. Gelsomina D. S., Patrizia C., Roberto P. et al. (2001) Gadolinium in human glioblastoma cells for gadolinium neutron capture therapy. Cancer Res. 61, 42724277. Gibson G. E. and Zhang H. (2001) Abnormalities in oxidative processes in non-neuronal tissues from patients with Alzheimers disease. J. Alzheimers Dis. 3, 329338. Hayashi T., Saito A., Okuno S., Ferrand-Drake M. and Chan P. H. (2003) Induction of GRP78 by ischemic preconditioning reduces endoplasmic reticulum stress and prevents delayed neuronal cell death. J. Cereb. Blood Flow Metab. 23, 949961. Hayashi T., Saito A., Okuno S., Ferrand-Drake M., Dodd R. L. and Chan P. H. (2005) Damage to the endoplasmic reticulum and activation of apoptotic machinery by oxidative stress in ischemic neurons. J. Cereb. Blood Flow. Metab. 25, 4153. Holtz W. A., Turetzky J. M., Jong Y. J. and OMalley K. L. (2006) Oxidative stress-triggered unfolded protein response is upstream of intrinsic cell death evoked by parkinsonian mimetics. J. Neurochem. 99, 5469. Jiang G. C. T., Tidwell K., McLaughlin B. A., Cai J., Gupta R. C., Milatovic D., Nass R. and Aschner M. (2007) Neurotoxic potential of depleted uranium effects in primary cortical neuron cultures and in Caenorhabditis elegans. Toxicol. Sci. 99, 553 565. Jung S., Muhle A., Schaefer M., Strotmann R., Schultz G. and Plant T. D. (2003) Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth. J. Biol. Chem. 278, 35623571. Korhonen L., Hansson I., Kukkonen J. P., Brannvall K., Kobayashi M., Takamatsu K. and Lindholm D. (2005) Hippocalcin protects

against caspase-12-induced and age-dependent neuronal degeneration. Mol. Cell. Neurosci. 28, 8595. Kudo T., Kanemoto S., Hara H., Morimoto N., Morihara T., Kimura R., Tabira T., Imaizumi K. and Takeda M. (2008) A molecular chaperone inducer protects neurons from ER stress. Cell Death Differ. 15, 364375. Li X. A. and Lee A. S. (1991) Competitive inhibition of a set of endoplasmic reticulum protein genes (GRP78, GRP94, and ERp72) retards cell growth and lowers viability after ionophore treatment. Mol. Cell. Biol. 11, 34463453. Liu H., Yuan L., Yang X. and Wang K. (2003) La3+, Gd3+ and Yb3+ induced changes in mitochondrial structure, membrane permeability, cytochrome c release and intracellular ROS level. Chem. Biol. Interact. 146, 2737. Lopez E., Figueroa S., Oset-Gasque M. J. and Gonzalez M. P. (2003) Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in culture. Br. J. Pharmacol. 138, 901911. Marciniak S. J. and Ron D. (2006) Endoplasmic reticulum stress signaling in disease. Physiol. Rev. 86, 11331149. Marckmann P., Skov L., Rossen K., Dupont A., Damholt M. B., Heaf J. G. and Thomsen H. S. (2006) Nephrogenic systemic brosis: suspected causative role of gadodiamide used for contrast-enhanced magnetic resonance imaging. J. Am. Soc. Nephrol. 17, 23592362. Milatovic D., Yin Z., Gupta R. C., Sidoryk M., Albrecht J., Aschner J. L. and Aschner M. (2007) Manganese induces oxidative impairment in cultured rat astrocytes. Toxicol. Sci. 98, 198205. Miyake K. and Nagai K. (2009) Inhibition of alpha-mannosidase attenuates endoplasmic reticulum stress induced neuronal cell death. Neurotoxicology 30, 144150. Nakayama H., Hamada M., Fujikake N., Nagai Y., Zhao J., Hatano O., Shimoke K., Isosaki M., Yoshizumi M. and Ikeuchi T. (2008) ER stress is the initial response to polyglutamine toxicity in PC12 cells. Biochem. Biophys. Res. Commun. 377, 550555. Nishitsuji K., Tomiyama T., Ishibashi K., Ito K., Teraoka R., Lambert M. P., Klein W. L. and Mori H. (2009) The E693Delta mutation in amyloid precursor protein increases intracellular accumulation of amyloid beta oligomers and causes endoplasmic reticulum stressinduced apoptosis in cultured cells. Am. J. Pathol. 174, 957969. Niu P. Y., Niu Q., Zhang Q. L., Wang L. P., He S. E., Wu T. C., Conti P., Di Gioacchino M. and Boscolo P. (2005) Aluminum impairs rat neural cell mitochondria in vitro. Int. J. Immunopathol. Pharmacol. 18, 683689. Paschen W. (2003) Endoplasmic reticulum: a primary target in various acute disorders and degenerative diseases of the brain. Cell Calcium 34, 365383. Perazella M. A. and Rodby R. A. (2007) Gadlinium use in patients with kidney disease: a cause of concern. Semin. Dial. 20, 179185. Reijonen S., Putkonen N., Nrremlle A., Lindholm D. and Korhonen L. (2008) Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins. Exp. Cell Res. 314, 950960. Rungtawan S., Suzanne J., Kazutoshi M. and Joseph W. B. (2004) XBP1: a link between the unfolded protein response,lipid biosynthesis, and biogenesis of the endoplasmic reticulum. J. Cell Biol. 167, 3541. Rutkowski D. T. and Kaufman R. J. (2004) A trip to the ER: coping with stress. Trends Cell Biol. 14, 2028. Schwab C., Steele J. C. and McGeer P. L. (1996) Neurobrillary tangles of Guam parkinson-dementia are associated with reactive microglia and complement proteins. Brain Res. 707, 196205. Shen C., Li Z., Yang X. and Wang K. (2008) La3+ binds to BiP/GRP78 and induces unfolded protein response in HepG2 cells. Chem. Biol. Interact. 176, 196203.

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847

Gd-induced OS triggers ER stress | 47

Smith M. I. and Deshmukh M. (2007) Endoplasmic reticulum stressinduced apoptosis requires bax for commitment and Apaf-1 for execution in primary neurons. Cell Death Differ. 14, 10111019. Tajiri S., Oyadomari S., Yano S., Morioka M., Gotoh T., Hamada J. I., Ushio Y. and Mori M. (2004) Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ. 11, 403415. Toimela T. and Tahti H. (2004) Mitochondrial viability and apoptosis induced by aluminum, mercuric mercury and methylmercury in cell lines of neural origin. Arch. Toxicol. 78, 565574. Toney G. M., Chavez H. A. and Ibarra R. (2001) Acute and subacute physiological and histological studies of the central nervous system after intrathecal gadolinium injection in the anesthetized rat. Invest. Radiol. 36, 3340. Xia Q., Feng X. D., Yuan L., Yang X. D. and Wang K. (2010) Brainderived neurotrophic factor protects neurons from GdCl3-induced impairment in neuron-astrocyte co-cultures. Sci. China Chem. 53, 21932199. Yang S. J., Lee J. E., Lee K. H., Huh J. W., Choi S. Y. and Cho S. W. (2004) Opposed regulation of aluminum-induced apoptosis

by glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor in rat brains. Mol. Brain Res. 127, 146 149. Yang W., Zhang P., Liu J. and Xue Y. (2006) Effect of long-term intake of Y3+ in drinking water on gene expression in brains of rats. J. Rare Earths 24, 369373. Yoshida H., Matsui T., Yamamoto A., Okada T. and Mori K. (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881891. Yu Z., Luo H., Fu W. and Mattson M. P. (1999) The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis. Exp. Neurol. 155, 302314. Yu S., Hu J., Yang X., Wang K. and Qian Z. M. (2006) La3+-induced extracellular signal-regulated kinase (ERK) signaling via a metalsensing mechanism linking proliferation and apoptosis in NIH 3T3 cells. Biochemistry 45, 1121711225.

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 117, 3847