Biol Trace Elem Res DOI 10.1007/s12011-015-0284-9

Biochemical and Molecular Alterations Following Arsenic-Induced Oxidative Stress and Mitochondrial Dysfunction in Rat Brain Chandra Prakash & Manisha Soni & Vijay Kumar

Received: 26 December 2014 / Accepted: 24 February 2015 # Springer Science+Business Media New York 2015

Abstract Oxidative stress is associated with the generation of reactive oxygen species (ROS), which is supposed to be one of the mechanisms of arsenic-induced neurodegeneration. Mitochondria, being the major source of ROS generation may present an important target of arsenic-mediated neurotoxicity. Hence, we planned the study to elucidate the possible biochemical and molecular alterations induced by arsenic exposure in rat brain mitochondria. Chronic sodium arsenite treatment (25 ppm for 12 weeks) resulted in decreased activity of mitochondrial complexes I, II, and IV followed by increased ROS generation. There was decrease in mitochondrial superoxide dismutase (MnSOD) activity in arsenic-treated rat brain further showing increased superoxide radical generation in mitochondria. The decrease in MnSOD activity might be responsible for the increased protein and lipid oxidation as observed in our study. Protein and messenger RNA (mRNA) levels of MnSOD and mitochondrial uncoupling protein 2 (UCP-2) were downregulated suggesting decreased removal of ROS in rat brain. Fourier transform infrared (FTIR) spectroscopy analysis revealed significant decrease in amide A, amide I, amide II, and Olefinic=CH stretching band area suggesting molecular alteration in proteins and lipids after arsenic treatment. The results of present study indicate that arsenicinduced disturbed mitochondrial metabolism, decreased removal of ROS, decrease in protein synthesis, and altered membrane lipid polarity and fluidity may be responsible for the mitochondrial oxidative damage in rat brain that may further be implicated as contributing factor in arsenic-induced neurodegeneration.

C. Prakash : M. Soni : V. Kumar (*) Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, Haryana, India e-mail: [email protected]

Keywords Arsenic . Mitochondria . Oxidative stress . FTIR . MnSOD . Neurodegeneration

Introduction Arsenic is present ubiquitously in the environment affecting the population around the world. Arsenic enters drinking water through natural deposits and agricultural and industrial practices [1]. In Southeast Asia and several other countries, millions of people are exposed to arsenic due to consumption of drinking water found to have arsenic concentrations higher than the permissible levels [2–4]. It is estimated that more than 35–77 million people in Bangladesh alone have been exposed to arsenic through contaminated ground water [5]. In West Bengal, India, more than 26 million people are drinking heavily arsenic contaminated ground water [6]. Adverse health effects of arsenic exposure depend on the dose and time of exposure [7]. Long-term exposure to arsenic through groundwater and occupational sources results in a plethora of dermatological and non-dermatological health effects [8, 9]. Epidemiological studies have shown learning and memory impairments in humans and deficits in spatial learning paradigms in animals [10–12], indicating that brain is an important target of arsenic toxicity. Arsenic can cross the blood brain barrier and accumulate in different regions of the brain suggesting its role in neurological diseases [13, 14]. Arsenic exposure has been associated with reactive oxygen species (ROS)-mediated oxidative stress generation, eliciting oxidative DNA damage, pathological changes, and neural apoptosis [4, 15, 16]. It implies that ROS generation may be involved in the mechanism of arsenic-induced neurotoxicity [17]. Mitochondria play pivotal role in energy metabolism and are the major site of ROS generation through electron transport chain (ETC) [18, 19]. Toxic conditions involving oxidative stress

Prakash et al.

may lead to impairment of mitochondrial functions and increase ROS generation which is major contributing factors towards various human disorders and neurodegenerative diseases [20]. Oxidative stress also leads to modification and accumulation of oxidized proteins as observed in various cellular dysfunctions [20]. The mitochondrial proteins and lipids represent main target of oxidative modification, and their accumulation may play an important role in the progression of neurodegeneration. Mitochondrial superoxide dismutase (MnSOD) and mitochondrial uncoupling protein 2 (UCP-2) are major proteins involved in the protection of cells against oxidative damage to mitochondria by ROS. MnSOD is inducible and has the potential to protect neurons by its superoxide dismutating activity. Disruption of the MnSOD gene in mice produces early postnatal lethality [21] while MnSOD over expression protects mitochondrial functions and block apoptosis [22]. Like MnSOD, UCP-2 is also expressed widely in various neurodegenerative diseases and plays important role in mitochondrial protection [23, 24]. Oxidative stress has been the most widely accepted mechanism of arsenic-induced neurotoxicity; however, its relation to mitochondrial function has not been studied in detail. Moreover, the metabolic study like Fourier transform infrared (FTIR) may be used as indicator of structural and functional biophysical changes occurring in arsenic neurotoxicity. Hence, we planned this study to provide new insights into the molecular mechanisms of mitochondrial dysfunctions under arsenic-induced oxidative stress.

Materials and Methods Chemicals Sodium arsenite, 2′,7′ dichlorofluorescin diacetate (DCF-DA) and 3,3′,5,5′-tetramethylbenzidine (TMB) were from SigmaAldrich, St Louis, USA. Acrylamide/bis-acrylamide, ammonium persulfate, N,N,N′,N′-tetramethylenediamine (TEMED), 2,4-dinitrophenylhydrazine (DNPH), Tris (hydroxymethyl) aminomethane, agarose, sodium dodecyl sulfate (SDS), and glycine were from Sisco Research Laboratory, Mumbai, India. Primary polyclonal antibodies antiMnSOD, UCP-2, β-actin, and HRP-labeled IgG were from Santa Cruz Biotechnology, CA, USA. Nitrocellulose membrane was from Whatman Gmbh, Dassel, Germany. Glass redistilled water was used throughout the present investigation. Animals and Their Use Male albino rats (Wistar strain) in the weight range of 120– 150 g were procured from the approved animal house source.

The animals were housed in polypropylene cages and kept in well ventilated rooms. Animals were provided standard rat pellet diet and water ad libitum. Ethical clearance for killing of animals was duly obtained from the Institute Animal Ethical Committee. Experimental Design Total 24 rats were divided into following two groups: Control Group In this group, rats were administered an equal volume of distilled water (vehicle) as administered to the rats of the sodium arsenite treated group. Arsenic-Treated Group Rats received sodium arsenite (25 ppm, intragastrically) dissolved in distilled water for 12 weeks. The dose was given once a day and 6 days a week. The 25 ppm solution of sodium arsenite contained approximately 14.2 ppm arsenic. During arsenic treatment period, rats were monitored for any change in body weight, dietary intake, as well as morphological symptoms. After the completion of treatment, rats were fasted overnight to preserve metabolic levels at basal rate, anesthetized and sacrificed by decapitation by following the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals, India. The whole brain was isolated and rinsed in ice-cold physiological saline. Brain Arsenic Estimation The content of brain arsenic was determined as described previously [25] using atomic absorption spectrophotometer (AAS, ECIL-4141, India) at 193.7 nm wavelength and 10 mA current. Briefly, 200 mg of brain tissue was wet digested by adding concentrated nitric acid in a water bath at 85 °C for 3 h. After digestion, each sample was diluted to 50 ml with a 2 % solution of nitric acid. A calibration curve was constructed by adding known amounts of arsenic standard to calculate arsenic levels in brain regions. Results were expressed in μg/g of wet tissue weight. Preparation of Mitochondria Rat brain mitochondria were isolated using method of Bermann and Hasting [26]. Briefly, the brain tissue was homogenized in glass Dounce homogenizer containing five volumes of isolation buffer with 1 mM EGTA, 215 mM mannitol, 75 mM sucrose, 0.1 % BSA, 20 mM HEPES. The tissue homogenate was spun at 13,000×g for 5 min at 4 °C. The resulting pellet was resuspended in 0.5 ml of isolation buffer and spun again at 13,000×g for 10 min in order to pellet the mitochondria. The pellet was washed in the appropriate buffer

Biochemical and Molecular Alterations in Arsenic-Induced Neurotoxicity in Rats

containing EGTA, centrifuged at 13,000×g for 10 min, and resuspended in the buffer without EGTA. Mitochondrial Complexes Activity Mitochondrial complexes activity was assayed as described previously [27]. For complex I activity assay, the reaction mixture contained 0.2 M glycyl glycine buffer pH 8.5, 6 mM NADH in 2 mM glycyl glycine buffer, 10.5 mM cytochrome c, and requisite amount of solubilized mitochondrial sample. Enzyme activity was calculated on the basis of absorbance index of cytochrome c (reduced-oxidized), 19.2 mM−1 cm−1 and results were expressed as nmole NADH oxidized/min/mg protein. Complex II activity was assayed by using reaction mixture containing 0.2 M phosphate buffer pH 7.8, 1 % BSA, 0.6 M succinic acid, 0.03 M potassium ferricyanide, and sample. Results were expressed as nmoles of succinate oxidized/min/mg protein. For the measurement of activity of complex IV, the assay mixture contained 0.3 mM reduced cytochrome c in 75 mM phosphate buffer and solubilized mitochondrial sample. Results were expressed as nmoles of cytochrome c oxidized/min/mg protein. ROS Generation Mitochondrial ROS generation was assessed using cell permeable dye DCF-DA. Briefly, equal amount of mitochondrial protein was incubated with fluorescent dye for 15 min in dark. In the presence of a proper oxidant, dichlorodihydrofluorescein is oxidized to the highly fluorescent 2′,7′-dichlorofluorescein. The fluorescence was quantified using fluorimeter (excitation 488 nm, emission 525 nm). Mitochondrial Superoxide Dismutase Assay MnSOD activity was measured as method of MacMillanCrow et al. [28]. MnSOD activity in total solubilized mitochondrial extract was measured by the cytochrome c reduction method in the presence of 1 mM potassium cyanide to inhibit both Cu-Zn SOD and extra cellular SOD. The amount of enzyme required to produce 50 % inhibition was considered as 1 U of MnSOD activity, and results were expressed as U/mg protein. Quantification of Protein Carbonyl and Lipid Peroxidation Quantization of protein carbonyl as an index of protein oxidation in mitochondrial fraction was determined after derivatization with DNPH using protein carbonyl assay kit (Cayman, Chemicals, USA). The amount of protein carbonyl was calculated on the basis of molar extinction coefficient of DNPH (0.022 μM−1 cm−1). The results were expressed as nmol carbonyl/mg protein.

The lipid peroxidation was assayed by the method of Wills [29]. Briefly, mitochondrial sample (0.5 ml) was diluted to 1.0 ml using Tris–HCl buffer (0.1 M, pH 7.4) and 1.0 ml of TCA (10 %, w/v) was added after 2 h incubation. The reaction mixture was centrifuged at 500 rpm for 10 min. To 1.5 ml supernatant, 1.5 ml TBA (0.67 %w/v) was added and color was developed by placing the tubes in a boiling water bath. The results were expressed as nmol MDA/mg protein. Fourier Transforms Infrared (FTIR) Analysis Brain tissue samples were prepared as described by Akkas et al. [30]. A small and equal amount of brain tissue was lyophilized over night to remove the water. The dried samples were ground to obtain brain powder. The brain powder was mixed with potassium bromide (KBr) at ratio of 1:100. The mixture was then subjected to a pressure of 1100 kg/cm2 to produce KBr pellets. Pellets of the same thickness were prepared by taking the same amount of sample and applying the same pressure. FTIR spectra of the region 4000–400 cm−1 were recorded at the room temperature on an FTIR spectrometer (Alpha, Bruker Optics, Ettlingen, Germany). The spectra were analyzed in two different regions of 3800–2700 cm−1 and 1800–1400 cm−1 using ORIGIN® 8.6 software (Origin Lab Corporation, Massachusetts, USA). RNA Extraction and Semiquantitative RT-PCR Total RNA was extracted from rat brain using the RNAsure® mini kit (Nucleopore) according to manufacturer’s instructions. RNA was reverse transcribed in a total volume of 20 μl using RevertAidTM cDNA synthesis kit (Fermentas, Germany). Complementary DNA (cDNA) products were subjected to semiquantitative RT-PCR analysis on a gradient thermal cycler (PEQLAB, Germany). For RT-PCR analysis, cDNA was amplified using following primers: MnSOD forward (5′-ACAGGCCTTATTCCACTGCT-3′) and reverse (5′CTACAAAACACCCACCACGG-3′) and UCP-2 forward (5′-AGACCATTGCACGA GAGGAA-3′) and reverse (5′AGAAGTGAAGTGGCAAGGGA-3′). β-actin, which was used as internal control, was amplified with primers forward (5′-TTGCCCTAGACTTCGAGCAA-3′) and reverse (5′AGACTTACAGTGTGGCCTCC-3′). PCR was then carried out for 30 cycles consisting 1 min each for 94 °C (denaturation), 60 °C (annealing), and 72 °C (elongation), and final extension was done at 72 °C for 10 min. Western Blotting Brain tissue was homogenized and mitochondria were isolated. The samples containing 80 μg protein were subjected to 12 % SDS-PAGE followed by transfer to a nitrocellulose membrane. The membranes were blocked with 5 % non-fat

Prakash et al.

dried milk in phosphate buffer saline (PBS) for 2 h at room temperature followed by incubation for 4 h with primary antibodies of MnSOD, UCP-2, and β-actin at 1:1000 dilution. After incubation, the membrane was washed twice with PBS followed by PBST for 15 min each. The membrane was then incubated with corresponding horse-radish peroxidase-conjugated secondary antibodies (1:10,000) at 37 °C for 1 h. After washing, immunoreactive proteins were visualized by TMB. The densitometry analysis of the protein bands was carried out using Image J software (http://imagej.nih.gov/ij) to compare the relative expression of proteins. Protein Determination Protein content in each sample was determined by the method of Lowry et al. [31] using bovine serum albumin as standard. Statistical Analysis The data was analyzed using Student t test. A p value