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Dec 3, 2013 - Arsenic induced hepatic mitochondrial toxicity in rats and its amelioration by diallyl trisulfide. S. Miltonprabu and NC. Sumedha. Department of ...
Just Accepted by Toxicology Mechanisms and Methods Arsenic induced hepatic mitochondrial toxicity in rats and its amelioration by diallyl trisulfide S. Miltonprabu and NC. Sumedha doi: 10.3109/15376516.2013.869778

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Abstract The present investigation was aimed to investigate the possible protective role of Diallyl trisulfide (DATS) against Arsenic (As) induced hepatic mitochondrial toxicity in rats. Mitochondria were isolated from the liver tissue of rats from all the groups. Lipid profile, lipid peroxidation, antioxidant enzyme activities, hepatic function enzymes, mitochondrial swelling, cytochrome-c-oxidase activity, mitochondrial Ca+-ATPase and Na+/K+-ATPase activity, mitochondrial calcium content and mitochondrial enzyme activities were measured. Short-term arsenic exposure (5 mg/kg body weight/day for 28 days) caused liver damage as evidenced by changes in activities of liver enzymes. The effects of arsenic were coupled with enhanced ROS generation, mitochondrial swelling, inhibition of cytochrome-c-oxidase, complex –I mediated electron transfer, decreased Ca2+-ATPase and Na+/K+-ATPase activity, a reduction in mitochondrial calcium content, changes in indices of hepatic mitochondrial oxidative stress, significant increase in mitochondrial lipid peroxidation products and alterations in mitochondrial lipid profile. Significant decreases in mitochondrial antioxidants and tricarboxylic acid cycle enzymes were also found in the liver mitochondria of As-induced hepatic mitochondrial toxicity in rats. Arsenic also increased hepatic caspase-3 activity and DNA fragmentation. All these apoptosis related molecular changes caused by arsenic could be alleviated by supplementation with diallyl trisulfide, which likely suggests a protective role against arsenic-induced hepatotoxic changes and hepatic mitochondrial toxicity. The protective effect of DATS on the liver mitochondria was evidenced by altering all the changes induced by arsenic. Free radical scavenging and metal chelating activities of DATS may be the mechanism, responsible for the protective action against As induced mitochondrial damage in liver.

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Arsenic induced hepatic mitochondrial toxicity in rats and its amelioration by diallyl trisulfide S. Miltonprabu and NC. Sumedha Department of Zoology, Faculty of Science,

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Annamalai University, Annamalainagar – 608002, Tamilnadu, India

*

Corresponding author:

Dr. S. Miltonprabu Assistant Professor Department of Zoology Faculty of Science, Annamalai University, Annamalai Nagar – 608 002. Tamil Nadu, India. Tel: +91 04144 – 237094 Fax: +91 04144 – 238282 Email: [email protected]

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Abstract The present investigation was aimed to investigate the possible protective role of Diallyl trisulfide (DATS) against Arsenic (As) induced hepatic mitochondrial toxicity in rats. Mitochondria were isolated from the liver tissue of rats from all the groups. Lipid profile, lipid

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peroxidation, antioxidant enzyme activities, hepatic function enzymes, mitochondrial swelling, cytochrome-c-oxidase activity, mitochondrial Ca+-ATPase and Na+/K+-ATPase activity, mitochondrial calcium content and mitochondrial enzyme activities were measured. Short-term arsenic exposure (5 mg/kg body weight/day for 28 days) caused liver damage as evidenced by changes in activities of liver enzymes. The effects of arsenic were coupled with enhanced ROS generation, mitochondrial swelling, inhibition of cytochrome-c-oxidase, complex –I mediated electron transfer, decreased Ca2+-ATPase and Na+/K+-ATPase activity, a reduction in mitochondrial calcium content, changes in indices of hepatic mitochondrial oxidative stress, significant increase in mitochondrial lipid peroxidation products and alterations in mitochondrial lipid profile. Significant decreases in mitochondrial antioxidants and tricarboxylic acid cycle enzymes were also found in the liver mitochondria of As-induced hepatic mitochondrial toxicity in rats. Arsenic also increased hepatic caspase-3 activity and DNA fragmentation. All these apoptosis related molecular changes caused by arsenic could be alleviated by supplementation with diallyl trisulfide, which likely suggests a protective role against arsenic-induced hepatotoxic changes and hepatic mitochondrial toxicity. The protective effect of DATS on the liver mitochondria was evidenced by altering all the changes induced by arsenic. Free radical scavenging and metal chelating activities of DATS may be the mechanism, responsible for the protective action against As induced mitochondrial damage in liver. Keywords: Arsenic, Diallyl trisulfide, Liver, Mitochondria, Rats, Oxidative stress

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INTRODUCTION Arsenic (As) is an omnipresent environmental pollutant and a natural drinking water contaminant (NRC, 2001). Occurrence of arsenic contamination through drinking water has been reported in many parts of countries like Bangladesh, India (West Bengal) and China (Inner

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Mongolia) (Parvez et al., 2006, Guha Mazumder, 2005). Chronic exposure of this high priority hazardous metal has been linked with a myriad of possible health effects, including, hypertension, cardiovascular disease, pulmonary disease, reproductive and neurological dysfunctions and malignancies of skin and internal organs (Waalkes et al., 2004; Gopalkrishnan and Rao, 2006; Santra et al., 2007). The liver has long been identified as a major target organ of arsenic exposure. Its importance as an organ for arsenic biotransformation is well established (Santra et al., 1999), which shows cascade of enzymatic reactions. Arsenate is reduced to arsenite in a reaction thought to be dependent on glutathione (GSH) or other endogenous reductants. The exact molecular mechanisms by which arsenic produces hepatotoxicity in vivo are unknown, but conceivably reflect differential damage to a number of cellular organelle systems and their biochemical functions. In fact, oxidative stress has been implicated in arsenic-induced cytotoxicity and genotoxicity and a positive correlation between arsenic-induced nitric oxide (NO) production and oxidative stress has been reported earlier (Lyn et al., 1998). Furthermore, it has been claimed that generation of reactive oxidants mediated oxidative stress during arsenic metabolism can play an important role in arsenic-induced hepatic injury (Miltonprabu and Muthumani, 2012). Previous in vitro studies have indicated that mitochondria are highly sensitive to damage from arsenicals. Prolonged oral exposure to arsenic was linked to mark ultrastructural and biochemical changes in hepatocyte mitochondria (Fowler et al., 1977). In addition,

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mitochondria, as an important source of reactive oxygen species (ROS) (Raha and Robinson, 2000), has been reported to be a primary target in arsenic-induced genotoxic responses (Liu et al., 2005). Arsenic directly inhibits complex I of the mitochondrial electron transport chain, which results in mitochondrial permeability transition (MPT), coupled with generation of ROS

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and thiol oxidation (Letak et al., 2005). Such observation finds its support from an earlier study which claimed that arsenic-induced apoptosis is associated with condensation of the mitochondrial matrix and disruption of mitochondrial transmembrane potentials (Cai, et al., 2000). Owing to increased health problems associated with oral exposure of inorganic arsenic, there has been a replenished interest in understanding the metabolism and toxicity of the compounds of this metalloid. Methylation of arsenic to monomethyl arsinic acid (MMA) and dimethyl arsenic acid (DMA) catalyzed by methyltransferases in the presence of glutathione has been believed by many to be the major mechanism for detoxifying inorganic arsenic although there exist considerable differences between species and individuals (Vahter, 1999). It is well ascertained that mitochondria are the major site of utilization of oxygen and many of the mitochondrial enzymes contain essential sulfhydryl groups. In addition, since the inner and outer mitochondrial membranes contain unsaturated lipids, mitochondria are more susceptible to arsenic attack as well as by the free radicals produced by it than other organelles (Ramanathan et al., 2003). Although injury of mitochondria causing cellular dysfunction is well known and even necrosis, only recently it has been reported that mitochondria are critical in mediating apoptotic signal transduction (Brenner and Kroemer, 2000). Further, it has been reported that arsenic disrupts mitochondrial membrane potential and increases reactive oxygen species (ROS) production, leading to cytochrome c release, a key

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component of early apoptosis, which indicates possible dysfunction of mitochondria (Larochette et al., 1999). Humans exposed to dietary arsenic for extended periods were observed to develop pronounced liver damage, thus identifying this organ as a major target for arsenic toxicity. In vitro studies previously demonstrated that mitochondria are highly sensitive to damage from

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arsenicals. These organelles, which have a number of cellular functions, are known to accumulate arsenic actively and to possess an arsenylated binding component, which is contemplated to be associated with the electron transport chain (Fowler et al., 1977). It is also well established that, mitochondria, an important source of ROS (Lenaz, 2001) are a primary target in arsenic induced toxicity. The health benefits of garlic sulfides on human health to treat a variety of ailments have long been realized. Structure–function studies demonstrate that the polysulfide DATS is more potent than the other two-organosulfur compounds in the modulation of the antioxidant and phase II enzymes, and effectively lessens alcohol induced liver injury (Zeng et al., 2008). Although the pharmacological effects of DATS have been extensively studied, there is no direct report about the effects of DATS on As-induced liver damage and mitochondrial dysfunction in the current literature. In view of the potent antioxidant capacity of DATS and the role of ROS in the etiology of mitochondrial toxicity by As, the protective effects of DATS on As-induced liver injury are worthy to be studied. Thus, we investigated the ability of DATS, to prevent short-term arsenic-induced mitochondrial damage. Studies were carried out to evaluate the effect of DATS on liver mitochondrial oxidative and antioxidative markers as well as hepatic DNA damage and alterations to the serum hepatic markers of hepatotoxicity in response to short-term arsenic exposure. Moreover, the ability of DATS to attenuate the effects of arsenic on mitochondrial

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function and DNA damage will provide insight into whether defects in mitochondrial and cytogenetic functions are linked to the development of early stages of arsenic mediated hepatic injury. MATERIALS AND METHODS

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Reagents Arsenic was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Diallyl trisulfide (DATS) was purchased from Lukang Cisen Pharmaceutical Co., Ltd. (Shangdong, China). Bovine serum albumin (BSA), N-(2-Hydroxyethyl)-piperazine-N0 -(2-ethanesulfonic acid) hemisodium salt (HEPES), ethidium bromide, thiobarbituric acid (TBA), Tris-3-(N-morpholino) propane sulfonic acid (MOPS), succinate-Tris, Tris-Phosphate (Pi), ethyleneglycol-bis (aminoethylether)-tetraacetic acid (EGTA), rotenone, menadione, mannitol, Bradford reagent, kit for Mammalian Genomic DNA isolation, Cytochrome c oxidase and Sigma diagnostics (I) Pvt. Ltd., Baroda, India, and Caspase-3 assay, all were obtained from Sigma Aldrich Chemical Co. (St. Louis, MO). Reduced nicotinamide adenine dinucleotide (NADH) and sucrose were purchased from Himedia Laboratories Pvt. Ltd. (Mumbai, India). All other analytical grade chemicals were purchased from E. Merck (India). Animals Male albino rats weighing 170–190 g were used in this study. They were maintained in an environmentally controlled animal house (temperature 24º - 38ºC) with 12 h light/dark schedule and free access to deionized drinking water. The animal treatment and protocol employed were approved by the Institutional Animal Ethics Committee, Annamalai University (Registration Number: 885/2012/CPCSEA).

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Experimental Design For experiments, 24 rats were randomly selected and divided into four groups consisting of six rats in each group: group I-Control rats were orally administered with normal saline and corn oil for 28 days, group II- rats were orally administered with As (5 mg/kg.BW) in normal saline for

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28 days, group III- rats were orally Pre-administered with DATS (80mg/kg.BW) 90min before the As (5 mg/kg.BW) intoxication and group IV- were orally Pre-administered with Vitamin ‘C’ (100mg/kg.BW) 90min before the As (5 mg/kg.BW) intoxication . The animals from all the groups were provided with a control diet composed of 71% carbohydrate, 18% protein, 7% fat, and 4% salt mixture (Chanda et al., 1996) with free access to deionized drinking water. Preparation of serum After the last treatment, rats were fasted overnight and all the rats were anesthetized with pentobarbital sodium (35 mg/kg, IP) and euthanized by cervical decapitation. Blood was collected in tubes containing ethylene diamine tetraacetate (EDTA). The plasma was obtained after centrifugation (2000 x g for 20 min at 4°C) and used for the assay of hepatic marker enzymes. Liver tissues were excised immediately and rinsed in ice-chilled physiological saline. Isolation of Hepatic Mitochondria The mitochondria were isolated by the method of Cain and Skilleter (1987). Briefly, liver was minced in 20 mL buffer (0.25 M sucrose, 5 mM Tris-HCl and pH 7.4), homogenized, and then centrifuged at 460×g for 10 min at 4ºC. The retained supernatant was centrifuged at 12,500×g for 7 min. The mitochondrial layer was removed, resuspended, and repelleted at 12,500×g for 7 min. The resultant mitochondrial pellet was then washed and resuspended in the same buffer and was preserved at −70ºC until analyses were made. Fresh mitochondria were used for each experiment. The mitochondria isolated were characterized by respiratory ratio (Fleischer et al.,

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1967). Rotenone titration experiments were also done to substantiate complex I assay. Submitochondrial particles (SMP) were prepared from isolated mitochondria by sonicating (at 4ºC) mitochondrial suspension at 60% frequency 3 times for 10 s each, with an interval of 2 min using a probe sonicator (Dr Heilscher Gmbtt Co., Germany).

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Estimation of Hepatic mitochondrial oxidative stress markers The role of nitric oxide synthase (NOS) was indirectly assessed by estimating the amount of NO production. Nitric oxide (NO) decomposes rapidly in aerated solutions to form stable nitrite/nitrate products. In our study, nitrite accumulation was estimated by Griess reaction (Raso et al., 1999) and was used as an index of NO production. The amount of nitrite in the sample (µmolar unit) was calculated from a sodium nitrite standard curve. The role of lipid peroxidation was assessed by studying the level of formation of thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation. Quantitative measurement of lipid peroxidation was performed following the thiobarbituric acid (TBA) test (Wills, 1987). The amount of TBARS formed was quantitated spectrophotometrically and the results were expressed as TBARS nmol/mg protein. The levels lipid hydroperoxides (LOOH) in the hepatic mitochondria were estimated by the methods of Jiang, et al. (1992) respectively. Hydroxyl radical formation was assessed by monitoring the hydroxylation of salicylate by Fe- ascorbate - H2O system (Tripathi and Pandey, 1999). Absorbance at 510 nm was measured in a UV-Double Beam Spectrophotometer (Shimadzu 160A). The amount of hydroxyl radical in the sample (µmolar unit) was calculated from a standard curve using 2, 3 dihydroxybenzoate (2, 3 DHB), a hydroxylation product of salicylate and an indicator of hydroxyl radical formation.

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Assay of Hepatic Mitochondrial Antioxidant Defense Markers Superoxide Dismutase (SOD) was assayed according to the method of Misra and Fridovich (1972). The change in absorbance due to the conversion of epinephrine to adrenochrome can be markedly inhibited by the presence of SOD. The reaction was initiated by the addition of

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epinephrine and the increase in absorbance at 480 nm was measured in a UV-Double Beam Spectrophotometer (Shimadzu 160A). The unit of enzyme activity is defined, as the enzyme required giving 50% inhibition of auto oxidation of epinephrine. Catalase (CAT) was assayed by the method of Cohen, et al. (1970). The enzyme-catalyzed decomposition of H2O was measured at 480 nm in a UV Double Beam Spectrophotometer (Shimadzu 160A). Glutathione (GSH) was estimated according to the method of Ellman (1959). The change in absorbance of DTNB (5, 5’ dithiobis 2-nitrobenzoic acid) was read at 412 nm in a UV-Double Beam spectrophotometer (Shimadzu 160A). Glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were assayed by the methods of Rotruck, et al. (1973) and Habig and Jakoby (1981). Analysis of Hepatic Function markers in serum The activities of serum aspartate aminotransferase (E.C.2.6.1.1), alanine aminotransferase (E.C. 2.6.1.2), alkaline phosphatase (E.C.3.1.3.1) and acid phosphatase (E.C.3.1.3.2) were assayed by commercially available diagnostic kits (Sigma diagnostics (I) Pvt. Ltd., Baroda, India). Estimation of mitochondrial lipids From the mitochondrial fraction, the lipids were extracted by the method of Folch, et al. (1957). The concentration of cholesterol in the mitochondrial lipid fraction was estimated by the method of Zlatkis, et al. (1953). The concentration of free fatty acid (FFA) in the mitochondrial lipid fraction was estimated by the method of Falholt, et al. (1973). The levels of triglycerides in the mitochondrial lipid fraction were estimated by a reagent kit from Accurex Bio Pvt. Ltd, Mumbai.

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Phospholipid content in the mitochondrial lipid fraction was estimated by the method of Zilversmit and Davis (1950). Measurement of Mitochondrial Permeability Transition Mitochondrial swelling as an indicator of mitochondrial permeability transition (MPT) was

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estimated by measuring the decrease in absorbance at 540 nm. The changes of mitochondrial MPT were measured by following the methods of Petit, et al. (1998). For the detection of mitochondrial swelling, mitochondria were resuspended in the buffer (400 mM mannitol, 10 mM KH2PO4, and 5 mg/ml BSA, and 50 mM Tris-HCl, pH 7.2, 4 º C) at a concentration of 100 µg protein/10 µl for 30 min. For determination of mitochondrial MPT, 1 mg mitochondrial protein was suspended in test buffer [200 mM sucrose, 10 mM Tris-3-(N-morpholino) propane sulfonic acid (MOPS), pH 7.4, 5 mM succinate-Tris, 1 mM Tris-Phosphate (Pi), 10 µM ethyleneglycolbis (aminoethylether)-tetraacetic acid (EGTA)-Tris, 2 µM rotenone, 1 µg/mL oligomycin], and the changes of absorbance at 540 nm were monitored before and after the addition of 150 µM CaCl2 using UV-Double Beam Spectrophotometer (Shimadzu 160A). Cytochrome c Oxidase Activity Cytochrome c oxidase activity was assayed with a colorimetric assay kit purchased from SigmaAldrich Chemical Co. (St. Louis, MO). Absorbance was measured by UV- Double Beam Spectrophotometer (Shimadzu, 160A) at 550 nm. Cytochrome c oxidase activity was expressed as U/mL. Estimation of Mitochondrial Ca2+ -ATPase and Na+/K+ATPase Activity The activity of mitochondrial Ca2+-ATPase was studied by the method of Rorive and Kleinzellar (1974). Phosphate liberated during Ca2+ –ATPase activity was estimated by the method of Lowry

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and Lopez (1946). Mitochondrial Na+/K+-ATPase (Bonting, 1970) was also estimated in liver mitochondria isolate. Estimation of Mitochondrial Calcium Content Atomic Absorption Spectrometer (Perkin Elmer, Analyst 200) was used to estimate the calcium

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content of mitochondrial samples by using the method of Zydowo, et al. (1985) described elsewhere. Hepatic Protein Extraction and Caspase-3 Activity Estimation To measure caspase-3 activity, the liver was homogenized in 25 mM/L N-(2-Hydroxyethyl)piperazine-N0355 -(2-ethanesulfonic acid) hemisodium salt (HEPES) buffer (pH-7.4) containing 5 mM/L ethylenediaminetetra acetic acid (EDTA), 2 mmol/L dithiothreitol, and 0.1% 3-[(3Cholamidopropyl)

dimethylammonio]-1-propanesulfonate

hydrate

(CHAPS).

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centrifugation at 20,000 x g for 30 min (Zhou et al., 2001), the diluted supernatant fluid was assayed for caspase- 3 activity by using 96-well plate format of caspase-3 assay kit (Sigma Chemicals, USA). p-Nitroaniline was used as the standard. Cleavage of the specific activity was expressed as nitroaniline produced in µmol/min/mL. Estimation of Protein Protein in liver homogenate was estimated by using Bradford reagent (Sigma Chemicals, St. Louis, MO). Assay of liver mitochondrial enzymes The activities of isocitrate dehydrogenase (ICD), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), α-ketoglutarate dehydrogenase (α-KGDH) and NADP were estimated according to the standard procedures of King (1965), Slater and Bonner (1952), Mehler, Kornberg, et al., (1948), Reed and Mukherjee (1969), and Minakami, et al. 1962 respectively.

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Detection of mitochondrial membrane potential (∆Ψm)

The mitochondrial membrane potential (∆Ψm) was detected by monitoring the fluorescence quenching of Rh 123 dynamically (Emaus et al., 1986). Fluorescence with excitation at 503 nm

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and emission at 527nm was detected in a reaction buffer (containing 250 mM sucrose, 2 mM HEPES, 0.5 mM KH2PO4, 4.2 mM sodium succinate, pH 7.4, and 0.3µm Rh 123) using F-4500 FL SPECTROPHOTOMETER (Hitachi High- Technologies Corporation). Then mitochondria was added to 0.5 mg/ml in the buffer and incubated for 3 min. The fluorescence was recorded again, and the alteration of the ∆Ψm was detected by the decrease of fluorescence. Hepatic DNA isolation and agarose gel electrophoresis Cellular DNA was isolated by GenElute Mammalian Genomic DNA Miniprep kit from Sigma Chemicals, St. Louis, MO, USA. Isolated DNA was quantified (absorbance at 260 nm) spectrophotometrically, and samples of 5µg of hepatic cells were electrophorsed in TAE buffer in a 1.5% agarose gel containing 0.5µg/ml ethidium bromide by constant voltage mode electrophoresis (100 V). Gels were visualized under UV illumination and were documented using gel documentation system (Bio-Rad, NSW, Australia). Complex І-mediated electron transfer assay The effect of As on NADH dehydrogenase (complex І)-mediated electron transfer was studied using NADH as the substrate, menadione as electron acceptor and rotenone as the standard inhibitor. The reaction mixture contained phosphate buffer 0.1 M (pH 8.0) containing 200 µM menadione and 150 µM NADH. The mixture was taken in a spectrophotometric cuvette and the temperature was maintained at 30 ºC. To this, SMP (100 µg) from each experimental rats was added and mixed immediately and quickly observed for change in the absorbance at 340 nm for

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8 min using a Lambda 25, Perkin-Elmer spectrophotometer (Paul et al., 2007; Schulte and Weiss, 1995). Determination of oxygen consumption by mitochondria The mitochondria suspension to be assayed was placed in a sealed chamber that was exposed to

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the surface of Clark-type oxygen electrode and stirred with a flea. Oxygen consumption of the freshly isolated liver mitochondria was measured in respiration buffer, pH 7.2 (125 mM sucrose, 50 mM KCl, 5 mM HEPES, 2 mM KH2PO4 and 1 mM MgCl2), in the presence of different substrate, like NADH for complex I (Paul et. al., 2007). The oxygen consumption was monitored as a drop in the % oxygen content of the buffer. Estimation of mitochondrial ROS generation One of the widespread methods in detecting ROS production by mitochondria is based on 2´ 7´dichloroflourescein (H2DCFDA) oxidation (Betainder et al., 2002; Paul et al., 2007; Zheng et al., 2005). H2DCFDA, an uncharged, cell-permeable fluorescent probe readily diffuses into cells and gets hydrolyzed by intracellular esterases to yield H2DCF, which is trapped inside the cell. Then it is oxidized from the nonfluorescent form to a highly fluorescent compound dichlorofluorescein by hydrogen peroxide (H2O2) or other low-molecular-weight peroxides produced in the cells. Thus, the fluorescence intensity is proportional to the amount to the H2O2 produced by the cell. For normal mitochondria, the reaction mixture contained a respiration buffer (pH 7.2), and mitochondria (500 µg/ ml) incubated with or without As for 8 min. To this mixture, 20 µM H2DCFDA, 0.4 mM NADH was added and mixed properly and the fluorescence change (λex=488 nm and λcm=540 nm) was observed for 8 min using a Perkin-Elmer fluorimeter. The difference in fluorescence intensity was expressed as % arbitrary fluorescence unit (AFU).

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Statistical Analysis Results were expressed as mean ± S.E of number of experiments. The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 13.0 (SPSS, Cary, NC, USA) and DMRT was use to obtained individual comparison. A value of p