Subchronic Exposure to Arsenic Decreased Sdha Expression in the Brain of Mice

NeuroToxicology 30 (2009) 538–543

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NeuroToxicology

Subchronic exposure to arsenic decreased Sdha expression in the brain of mice

Yan Hong a, Fengyuan Piao a,*, Yufeng Zhao b, Sheng Li c, Yanyan Wang a, Peng Liu a a Department of Occupational and Environmental Health, Dalian Medical University, No 9 Western Section of Lushun South Road, Dalian, Liaoning 116044, China b Henan University Huaihe Hospital, Kaifeng, Henan 475000, China c Department of Biochemistry, Dalian Medical University, Dalian, Liaoning 116044, China

ARTICLE INFO ABSTRACT

Article history: Exposure of arsenic (As) elevates reactive oxygen species (ROS) level, which is supposed to be a Received 29 December 2008 molecular mechanism of As neurotoxicity. Mitochondria are the major source of ROS. However, the Accepted 29 April 2009 mechanism of the ROS generation induced by As remains unclear. To provide target evidence for Available online 5 May 2009 exploring the molecular mechanism of As-induced neurotoxicity, 8-hydroxy-2-deoxyguanosine (8- OHdG) as an oxidative damage biomarker was examined, and the critical gene expression proﬁles related Keywords: to mitochondrial respiratory chain were analyzed by GeneChip in mice exposed to As2O3 subchronically. Arsenic trioxide Our results showed that immunoreactivity of 8-OHdG increased remarkably. Succinate dehydrogenase Neurotoxicity subunit A (Sdha), ubiquinol-cytochrome c oxidoreductase gene (Uqcr), cytochrome oxidase genes Mitochondria Succinate dehydrogenase subunit A (Sdha) (Cox6a2, Cox17) and ATP Synthase genes (Atp5a1, Atp5g1, Atpif1) were down-regulated in brain cells of Reactive oxygen species (ROS) mice exposed to As. We further analyzed the inﬂuence of As on brain Sdha expression using Western blot method. The quantity of Sdha band and the corresponding succinate dehydrogenase (SDH) activity in the

group exposed to 4 ppm As2O3 signiﬁcantly decreased compared to the 1 ppm or control group, agreeing well with the gene microarray result. These results indicate that subchronic exposure to As induces down-regulation of Sdha expression and inhibition of SDH activity in brain tissue. They also suggest that the Sdha as complex II subunit may be a molecular target for As in mitochondria. Furthermore, the intervening experiment showed that the coadministered antioxidants taurine or vitamin C scavenging ROS in vivo partly rescued Sdha expression. It implies that the increased level of ROS by As may also be a factor in the disrupting Sdha expression in brain tissue of mice exposed to As. Crown Copyright ß 2009 Published by Elsevier Inc. All rights reserved.

1. Introduction task (Nagaraja and Desiraju, 1994), alterations in locomotor behavior and deﬁcits in spatial learning paradigms have been Arsenic (As) is a common environmental contaminant widely observed (Rodrı´guez et al., 2001). These epidemiological and distributed around the world. It enters drinking water supplies experimental studies indicate that the brain is an important target from natural deposits or from agricultural and industrial practices of As. It has been reported that As exposure resulted in marked (Chen et al., 2007). In Argentina, Australia, Bangladesh, Chile, elevation in ROS, causing oxidative DNA damage, severe patho- China, Hungary, India, Mexico, Peru, Thailand and the United States logical changes and even apoptosis in neural cells (Chattopadhyay of America, concentrations higher than the permissible levels have et al., 2002a,b). It implies that ROS is involved in mechanism of As- been reported and deteriorating effects on human health have induced neurotoxicity. been documented (Bienert et al., 2008). It is estimated that 35–77 The central nervous system has the highest metabolic rate million people in Bangladesh alone, have been exposed to As among all organs and depends primarily on oxidative metabolism through contaminated groundwater which is the largest mass as an energy source (Kann and Kova´cs, 2007). Mitochondria play poisoning event in human history by the World Health Organiza- central roles in energy metabolism (Pelicano et al., 2003) and are tion (Smith et al., 2000). Epidemiological studies have demon- the major sites of ROS production as by-products of the electron strated that As causes neurotoxicity including impairments of transport chain (Batandier et al., 2002). During oxidative learning and concentration and deterioration in pattern memory phosphorylation, electrons are delivered through complex I (NADH and switching attention in human (Tsai et al., 2003). In animals dehydrogenase) and complex II (succinate dehydrogenase, SDH) exposed to As, delay in acquisition and extinction of an operant and then to complex III (ubiquinol-cytochrome c oxidoreductase) via ubiquinol. Cytochrome c transfers electrons from complex III to

complex IV (cytochrome oxidase), which reduces O2 to form H2O, * Corresponding author. Tel.: +86 411 86110329; fax: +86 411 86110329. accompanied with ATP production. At the same time, electrons E-mail address: [email protected] (F. Piao). escape from the mitochondria electron transport chain, especially

0161-813X/$ – see front matter. Crown Copyright ß 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2009.04.011 Y. Hong et al. / NeuroToxicology 30 (2009) 538–543 539 at complex I and complex III (Zhang and Gutterman, 2007). They (1:300, 4 8C, overnight) followed by incubation with biotin-labeled À may react with oxygen to from O2 . Mitochondrial complex II is a rabbit anti-mouse IgG for 60 min, and then with fluorescence- key enzymatic complex involved in both the tricarboxylic acid conjugated secondary antibody. The immunostained sections were (TCA) cycle and oxidative phosphorylation as part of the examined under an invert Laser Scan Microscope (LSM 410; Zeiss, mitochondrial respiratory chain (Yankovskaya et al., 2003). A Gottingen, Germany) (Kono et al., 2006). complete lack of succinate dehydrogenase (SDH) activity will hamper electron flow to both respiratory chain complex III and the 2.4. GeneChip ubiquinone pool, resulting in a major oxidative stress (Rustin et al., 2002). In recent years, evidence has been accumulated that 2.4.1. RNA extraction and Affymetrix GeneChip mitochondrion may be the key target organelle of As-neurotoxi- Total RNA was extracted from cerebrum of mice by using the city. However, the molecular mechanisms of As-induced ROS in Trizol reagent (Invitrogen, San Diego, CA, USA) according to the mitochondria are poorly understood. manufacture’s recommendations. The purity of total RNA was In the present study, to provide target evidence for exploring photometrically tested. RNA with a 260:280-nm ratio of 1.8 or the molecular mechanism of As-induced neurotoxicity, expression higher was used to generate biotinylated cRNA target for the of 8-hydroxy-2-deoxyguanosine (8-OHdG) as an oxidative damage Mouse Genome 430 2.0 Araay GeneChip (Affymetrix). All of these biomarker was observed in mice exposed to As2O3 subchronically procedures were carried out as described by Affymetrix. After and the expression profiles of genes related to mitochondrial hybridization, the array was washed, stained with streptavidin respiration chain were studied by microarray analysis. Particularly, phycoerythrin using the Affymetrix GeneChip Fluidies Work- down-regulation of succinate dehydrogenase subunit A (Sdha) and station 450, and scanned on a GeneArrayTM scanner (Affymetrix). SDH activity in mice exposed to As were further examined by Western blot and spectrophotometric assay. Meanwhile, inter- 2.4.2. Bioinformatics analysis vening effect of antioxidants on down-regulation of Sdha protein After the arrays were scanned, the signals generated were was also observed. determined and analyzed by Microarray Suite Version 5.0 software. Single array analyses were used to build databases of 2. Materials and methods gene expression profiles. The transcript was assigned a present call (reliably detected) if user-definable p-value was below 0.04 and an 2.1. Chemicals absent call (not detected) if p-value was above 0.06. The p-value between 0.04 and 0.06 means marginal. On the basis of present All chemicals used were purchased from Sigma Chemical calls, the Signal Log Ratio estimates the magnitude and direction of Company (St. Louis, USA). Mouse monoclonal anti-8-OHdG anti- change of a transcript when two arrays are compared (experiment body and biotin-labeled rabbit anti-mouse IgG were obtained from vs. baseline). Thus, a Signal Log Ratio of 1.0 indicates an increase of Japan Institute for the Control of Aging (Fukuroi, Japan). the transcript level by twofold and À1.0 indicates a decrease by twofold. A Signal Log Ratio of zero would indicate no change. The 2.2. Animals and treatment bioinformation about genes that expressed differentially was obtained by using NetAffy (www.affymetrix.com). Fifty SPF mice (age 9 weeks) weighing 26.3–30.9 g were purchased from Experimental Animal Center, Dalian Medical 2.5. Harvest mitochondria University. The animals were maintained on a standard diet and water ad libitum. They were caged under a 12-h dark–light cycle in Animals on 0, 15th, 30th and 60th days after As exposure were standard conditions of temperature (18–22 8C) and humidity decapitated and their brains were removed rapidly on ice. Fresh (50%). These mice were randomly divided into five groups of 10 mice brains were homogenized in 0.01 M Tris, 0.001 M EDTA, each. Group 1 received drinking water alone as controls. Group 2 0.01 M sucrose and 0.8% NaCl solution (PH 7.4). The homogenate received 1 ppm As2O3. Group 3 received 4 ppm As2O3. Group 4 and was centrifuged at 1,000 Â g for 5 min. The supernatant was group 5 received both of 4 ppm As2O3 and 150 mg/kg taurine or centrifuged at 4,000 Â g for 10 min, and then re-centrifuged 45 mg/kg vitamin C (Vit C), respectively. Arsenic trioxide was given (15,000 Â g for 20 min) to obtain mitochondria pellet. through drinking water for 60 days (Chattopadhyay et al., 2002). Taurine and Vit C were administered by gavation twice a week. The 2.6. Fractionation of the mitochondrial proteins animal experiment was performed in accordance with the Animal Guideline of Dalian Medical University and in agreement with the The mitochondria pellet was dissolved in 10 mM Tris, 150 mM

Ethical Committee of Dalian Medical University. NaCl, 5 mM EDTA Na2, 7 M urea, 2 M thiourea, 1% Triton-100 and 1 mM PMSF (PH 7.5), placed under room temperature for 30 min, 2.3. Immunohistochemistry for 8-OHdG formation and centrifuged for 45 min at 15,000 Â g. The supernatants were collected and stored at À80 8C until analyzed by polyacrylamide Sixty days after exposure, mice were deeply anesthetized by gel electrophoresis (SDS-PAGE). All procedures were done at 4 8Cif intraperitoneal injection of sodium pentobarbital, placed in a not specified (Nagayama et al., 2008). supine position, and the thorax was opened through a bilateral incision. A catheter was inserted into the left ventricle, the right 2.7. Enzymatic activity of mitochondrial SDH atrium was incised, and physiologic saline was infused until the perfusate from the right atrium was bloodless. The saline was The succinate dehydrogenase activity was assayed using the followed by 4% paraformaldehyde. Then, the brain was removed method described by Green and Narahara (1980), with a little and placed in fixative. 3-mm tissue sections were deparaffinized, modification. dehydrated in graded alcohols and then Antigen retrieval was achieved by microwave at 98 8C for 30 min in citrate buffer (Bruder 2.8. Western blot et al., 2007), followed by 1% hydrogen peroxide (15 min) and 5% rabbit serum in buffered solution (15 min). The specimens were Mitochondrial proteins were separated on 10% polyacryla- then incubated with mouse monoclonal anti-8-OHdG antibody mide gels and transferred to PVDF membranes (Millipore, USA). 540 Y. Hong et al. / NeuroToxicology 30 (2009) 538–543

Membranes were blocked in Tris-buffered-saline with Tween (20mMTris–Cl,500mMNaCl,pH7.5,0.05%Tween20) containing 5% bovine serum albumin (BSA) overnight at 4 8C, followed by incubation at 37 8C for 2 h with primary antibodies diluted in TTBS (1:6000 for SDHA, Abcam, USA). After washing with TTBS (three times for 10 min each time), the membranes were incubated in goat anti-mouse horseradish peroxidase conjugate for 1 h at 37 8C. Membranes were then rinsed in TTBS (ﬁve times for 10 min each time). Protein bands were detected by using the enhanced chemiluminescence reagents and exposed to the ﬁlm.

3. Results

3.1. Expression of 8-OHdG in brain tissue of mice exposed to As

8-OHdG immunoreactivity in the brain tissue of mice is shown in Fig. 1. Intensive 8-OHdG immunoreactivity was found in nervous cells of mice exposed to As and it was mainly distributed in nucleus (B). No immunoreactivity of 8-OHdG in brain tissue was observed in controls (A) (Fig. 1).

3.2. The expression proﬁles of genes related to the mitochondrial respiration chain in brains of mice exposed to As

In order to elucidate the inﬂuence of As on brain mitochondrial respiration chain, we picked out genes encoding proteins involved in the pathway and analyzed their different expression levels. Among 11 genes selected, 10 genes were down-regulated, namely, NADH dehydrogenase genes (Ndufab1, Ndufb12, and Ndufb2), succinate dehydrogenase gene (Sdha), ubiquinol- cytochrome c oxidoreductase gene (Uqcr), cytochrome oxidase genes (Cox6a2, Cox17) and ATP Synthase genes (Atp5a1, Atp5g1, Atpif1). NADH dehydrogenase gene (Ndufs4) was up-regulated. From the results of the gene microarray, we Fig. 1. 8-OHdG detection in the brain of mice exposed to As. 8-OHdG formation was found that most genes involved in the respiration chain, analyzed by immunohistochemistry. (A) No 8-OHdG immunoreactivity was including complexes I, II, III, IV, V were transcriptionally observed in the brain of controls. (B) Intensitive immunoreactivity of 8-OHdG repressed (Fig. 2). was observed in the brain of the mice given 4 ppm As2O3.

Fig. 2. As-induced Gene expression changes of mitochondrial respiratory chain in mice brain. Expression proﬁles of the 11 genes in experimental groups (1 and 4 ppm As2O3) were shown in differential expression. Yellow illustrates up-regulation genes in 1 ppm As2O3 group and red illustrates up-regulation genes in 4 ppm As2O3 group. Reseda illustrates down-regulation genes in 1 ppm As2O3 group, dark green illustrates down-regulation genes in 4 ppm As2O3 group. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of the article.) Y. Hong et al. / NeuroToxicology 30 (2009) 538–543 541

Fig. 3. Sdha protein expression changes in the brain of mice exposed to As. (A) Sdha Fig. 5. Analysis of Sdha protein expression in the brain of mice exposed to 4 ppm protein expression were detected by Western blot. b-Actin was used as control. (B) As O on different days after As-intake. (A) Expression level of Sdha protein were Intensity of Sdha protein bands decreased as As concentration increases (compared 2 3 detected by Western blot. b-Actin was used as control. Sdha expression level to control group, ap < 0.01; compared to 1 ppm group, bp < 0.05). decreased in a time-dependent way. (B) Intensity of Sdha protein bands in the brain of mice exposed to As decreased signiﬁcantly on the 60th day compared to that on the 0 day (ap < 0.01). 3.3. Effect of As on expression of Sdha in brains of mice exposed to As

Among the genes involved in mitochondrial respiration chain, taurine or Vit C than that in group given 4 ppm As2O3 alone complex II, succinate dehydrogenase received our attention, since (p < 0.05), suggesting that the two antioxidants may protect Sdha it’s subunit A (Sdha) was found to be down-regulated by threefold expression in mice exposed to As through interfering with ROS in the 4 ppm group compared to control group. We further effects (Fig. 4). analyzed the influence of As on brain Sdha expression using Western blot method. The quantity of Sdha band in the 4 ppm 3.4. Expression level of Sdha protein on different days after As group significantly decreased compared to the 1 ppm or control administration group, agreeing well with the gene microarray result (Fig. 3). On the other hand, we observed also intervention of taurine and Vit C Expression level of Sdha protein in brains of mice was examined as antioxidants in preventing down-regulation of Sdha expression on the 0, 15th, 30th and 60th days after As administration by in brain of mice exposed to As. The Sdha expression was Western blot analysis. The expression level of Sdha protein in the significantly higher in the groups received 4 ppm As2O3 with

Fig. 4. Sdha protein expression in the brain of mice was recovered by Fig. 6. As effect on succinate dehydrogenase activity. (A) After As administration, coadministering antioxidants taurine or Vit C. (A) Expression level of Sdha SDH activity decreased in a dose-dependent manner (compared to control group, protein were detected by western blot. b-Actin was used as control. (B) Intensity of ap < 0.05; compared to 1 ppm group, bp < 0.05). (B) The loss of SDH activity due to Sdha protein bands increased in antioxidant groups received taurine and Vit C As exposure was partly recovered by coadministering taurine or Vit C (compared to (compared to control group, ap < 0.01; compared to taurine group, bp < 0.05; control group, ap < 0.05; compared to taurine group, bp < 0.05; compared to Vit C compared to Vit C group, cp < 0.05). group, cp < 0.05). 542 Y. Hong et al. / NeuroToxicology 30 (2009) 538–543 brain of mice exposed to As decreased in a time-dependent way. Sdha via binding to the promoter region (Piantadosi and Suliman, Especially, its expression level decreased significantly on the 60th 2008). He et al. reported that As stabilizes the transcriptional day compared to that on the 0 day (Fig. 5). function of another Nrf family member Nrf 2 (He et al., 2006). It will be interesting to find whether the down-regulation of Sdha 3.5. Changes of SDH activity in the brain of mice exposed to As expression in brain tissue of mice by As may be due to regulating the transcriptional function of Nrf 1. The activity of SDH in brain mitochondria decreased in the By administering 3-NP to mice, Pandey et al. showed that when groups exposed to As. Especially, the SDH activity was significantly SDH was inhibited, complex I and IV activities were concurrently lower in the group received 4 ppm As2O3 than those in the other decreased to different extents (complex IV activity was reduced groups (p < 0.05) (Fig. 6A). This loss can be partly recovered by more remarkable than complex I) (Pandey et al., 2008). However, coadministering taurine or Vit C (Fig. 6B). in our study, we found SDH (complex II) activity was remarkably reduced by As administration, and complex I and IV activities did 4. Discussion not exhibit noticeable decrease at the protein level. Maybe a different mechanism is involved in As toxicity, compared with As is widely distributed poisonous element and takes neural Pandey’s result. tissue as a major attack target (Sidhu et al., 2006). Acute As Choksi et al. reported that oxidative modifications to mito- exposure affects sensory nerves and long axon neurons resulting in chondrial electron transport chain complex subunits by the nerve axonopathy (Franzblau and Lilis, 1989), while chronic As overproduction of ROS inhibit their activities (Choksi et al., contamination causes deterioration on cognitive development, 2007). It is well known that taurine and Vit C can scavenge ROS psychomotor speed, attention, speech and memory (Tsai et al., and prevent oxidative damage to important biological macro- 2003; Caldero´ n et al., 2001; Rodrı´guez et al., 2003). molecules such as DNA, lipids, and proteins (Kalia and Flora, 2005; In the present study, after 60 days of As exposure, 8-OH-dG, the Kim and Cha, 2009; Mankovskaya et al., 2000; Dawson et al., 1999; product of DNA oxidation, was pervasively found in the brain cells Benedetti et al., 1991). Our results showed that although of mice, indicating that subchronic As intake resulted in oxidative expression of Sdha protein in brain tissue of mice coadministered DNA damage by inducing ROS. Mitochondria are the major sites with taurine or Vit C did not reach the level of Sdha expression in À generating O2 and H2O2 (Liu et al., 2005). The results of GeneChip controls, repression of Sdha by As was partly rescued by these analysis showed that among the analyzed 34,000 genes, 558 and antioxidants. It implies that the increased level of ROS by As may 1131 genes were up or down-regulated respectively in the 1 ppm be also involved in the disrupting expression of Sdha in brain tissue

As2O3 group vs. control group and 1049 or 1721 genes were up and of mice exposed to As. However, there were also contradictory down-regulated respectively in the 4 ppm As2O3 group vs. control reports on protection of taurine against cytotoxicity caused by ROS. group. Especially, among 11 differential expression genes related Navneet et al. investigated free radical scavenging action of taurine to mitochondrial respiration chain, 10 genes were down-regulated, in vitro in isolated mitochondria of brain and found it fails to help implying that As might inhibit aerobic respiration. in scavenging ROS (Navneet et al., 2008). Hence, it is very necessary Recent studies showed that mitochondrial complex II is another to confirm further whether taurine can decrease ROS level induced site of ROS generation (McLennan and Degli Esposti, 2000; Gredilla by As through experiments both in vivo and in vitro. et al., 2001). It is comprised of four subunits (Sdha, Sdhb, Sdhc, and Taken together, As intake inhibits the aerobic respiration in Sdhd). It has been found that defects in Sdha produce bioenergetic brain tissue and represses one of the important members of the deficiency, while defects in Sdhb, Sdhc, and Sdhd induce tumor process—Sdha. However, the definite role of Sdha in ROS formation (Brie`re et al., 2005). Among the respiratory chain genes production and the exact biochemical mechanism of As-induced found down-regulated by As, Sdha is interesting to us. Though it repression of mitochondrial metabolism are not yet fully has been reported that ROS in mitochondria come from complexes uncovered, and require further investigation. I and III (Choksi and Papaconstantinou, 2008), some research groups also claimed that deficiencies of SDH associated with Sdha Conflicts of interest mutations not only cause decreased ATP production, but also promote production of ROS (Rustin et al., 2002). Sdha encodes the None. major catalytical subunit of the SDH (Baysal et al., 2007). Sdha mutation leads to SDH deficiency, which induced to succinate Acknowledgments accumulation (Brie`re et al., 2005). In another study, accumulation of succinate due to SDH inaction was found to cause a reverse This work was supported by the Natural Science Foundation of electron flow from reduced CoQ into complex I generating ROS China (30571584, 30600488) and the project of the Department of (Zoccarato et al., 2008). SDH feeds electrons to the quinone pool, Education of Liaoning province, China (05L113). reducing ubiquinone (Q) to ubiquinol (QH2). The specific redox properties of SDH suggest that Sdha may play a role in preventing References the excess formation of ROS (Rustin et al., 2002). In the present Batandier C, Fontaine E, Ke´ riel C, Leverve XM. Determination of mitochondrial reactive study, the gene expression of Sdha was down-regulated in the oxygen species: methodological aspects. J Cell Mol Med 2002;6:175–87 [PubMed: brain tissue of mice exposed subchronically. The influence of As on 12169203]. Sdha was further analyzed by Western blot. We found that As did Baysal BE, Lawrence EC, Ferrell RE. Sequence variation in human succinate dehydro- decrease the expression of Sdha in a dose-dependent way, agreeing genase genes: evidence for long-term balancing selection on SDHA. BMC Biol 2007;5(12) [PubMed: 17376234]. well with the gene microarray result. SDH activity was reduced Benedetti MS, Russo A, Marrai P, Dostert P. Effects of aging on the content in sulfur- after administering As, consistent with the published results containing amino acids in rat brain. J Neural Transm Gen Sect 1991;86:191–203 (Repetto et al., 1994; Dhar et al., 2005), and this loss could be partly [PubMed: 1777213]. Bienert GP, Thorsen M, Schu¨ ssler MD, Nilsson HR, Wagner A, Tama´s MJ, et al. A recovered by feeding As-treated mice with antioxidants taurine or subgroup of plant aquaporins facilitate the bi-directional diffusion of As(OH)3 and Vit C. These results indicate that subchronic exposure to As induces Sb(OH)3 across membranes. BMC Biol 2008;6(26) [PubMed: 18544156]. down-regulation of Sdha expression and inhibition of SDH activity Brie`re JJ, Favier J, Beńit P, El Ghouzzi V, Lorenzato A, Rabier D, et al. Mitochondrial succinate is instrumental for HIF1alpha nuclear translocation in SDHA-mutant in brain tissue. It has been reported that nuclear respiratory factor fibroblasts under normoxic conditions. Hum Mol Genet 2005;14:3263–9 1 (Nrf 1) is the transcription factor that regulates the expression of [PubMed: 16195397]. Y. Hong et al. / NeuroToxicology 30 (2009) 538–543 543

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