Protective Effect of Taurine Against Potassium Bromate-Induced Hemoglobin Oxidation, Oxidative Stress, and Impairment of Antioxidant Defense System in Blood Mir Kaisar Ahmad, Riaz Mahmood Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, U.P., India

Received 16 January 2014; revised 23 August 2014; accepted 23 August 2014 ABSTRACT: Potassium bromate (KBrO3) is widely used as a food-additive and is a major water disinfection by-product. KBrO3 causes severe toxicity in humans and experimental animals. Bromate is considered a probable human carcinogen and a complete carcinogen in animals. We have investigated the potential role of taurine in protecting against KBrO3-induced oxidative stress in rat blood. Animals were given taurine for 5 days prior to KBrO3 and then sacrificed. Blood was collected and used to prepare hemolysates and plasma, which were then used for the analysis of several biochemical parameters. Administration of single oral dose of KBrO3 alone induced hepato- and nephro-toxicity as evident by elevated marker levels in plasma. Lipid peroxidation and protein oxidation were increased both in plasma and erythrocytes, suggesting the induction of oxidative stress. KBrO3 increased methemoglobin, nitric oxide, and hydrogen peroxide levels. It also altered the activities of the major antioxidant enzymes and lowered the antioxidant power of blood. Administration of taurine, prior to treatment with KBrO3, resulted in significant attenuation in all these parameters but the administration of taurine alone had no effect. These results show that taurine is effective in mitigating the oxidative insult induced in rat blood by C 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. KBrO3. V Keywords: blood; lipid peroxidation; methemoglobin; oxidative stress; potassium bromate; taurine

INTRODUCTION

Potassium bromate (KBrO3) is widely used as a food additive. It is used as a flour improver in bakeries giving strength and elasticity to the dough during the baking process while also promoting the rise of bread. The resulting bread tends to be strong and spongy with fine crumb structure. Bromate also promotes gluten development in dough. KBrO3 is used in beer making, in cheese produc-

Correspondence to: R. Mahmood; e-mail: [email protected] Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22045

tion and is commonly added to fish paste products. It is also used in pharmaceutical and cosmetic industries and is a constituent of cold wave hair solutions (IARC, 1986). Bromate is also the major by-product generated during ozonation of surface water and is frequently detected in tap, and even bottled water. Exposure to KBrO3 results in multiorgan toxicity and kidney is considered to be the primary target organ of this compound (Kurokawa et al., 1990; EPA, 2001). KBrO3 is also carcinogenic and chronic exposure causes renal cell carcinomas, thyroid, and mesothelioma tumors in experimental animals. Bromate is now considered a probable human carcinogen and a complete carcinogen in animals.

C 2014 Wiley Periodicals, Inc. V

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The toxic effects of KBrO3 are attributed to its ability to induce oxidative stress (OS) leading to enhanced production of reactive oxygen species (ROS) which cause oxidative damage to essential macromolecules (EPA, 2001; Bao et al., 2008). The ROS are widely thought to be generated in the cell because of reduction of KBrO3 to bromide by intracellular reductants. KBrO3 has been shown to induce oxidative modification of lipids and proteins in several animal tissues (Kurokawa et al., 1990; EPA, 2001; Ahmad et al., 2014; Ahmad and Mahmood, 2012; Ahmad et al., 2012, 2013). Supporting the involvement of ROS in bromate action, several antioxidants (AO) have been shown to ameliorate the bromate-induced renal toxicity (Bao et al., 2008; Stasiak et al., 2010; Khan et al., 2012).The generation of OS and ROS are also thought to contribute to the carcinogenicity and mutagenicity of KBrO3. Taurine (2-aminoethane sulfonic acid) is the major intracellular free beta-amino acid and is one of the few known naturally occurring sulfonic acids. It is present in various foods like eggs, milk, and is especially abundant in seafood and meat; it is found in high concentrations in most animal tissues (Huxtable, 1992). Taurine has many fundamental biological roles such as conjugation of bile acids, membrane stabilization, neuromodulation, osmoregulation, and modulation of calcium signaling (Bidri and Choay, 2003). It has been shown to be essential for the development and survival of mammalian cells, particularly those of the cere-

bellum, retina, and kidney (Huxtable, 1992). Taurine is also an AO and is a potent scavenger of the hydroxyl radical suggesting that it may be useful in treating oxygen radical pathophysiology (Ripps and Shen, 2012). The AO activity exhibited by taurine has been shown to protect various body organs from OS because of heavy metals, drugs, and chemicals (Sinha et al., 2008; Das et al., 2009; Manna et al., 2009; Alam et al., 2011; Roy and Sil, 2012; Ozden et al., 2013). We have previously shown that a single oral dose of KBrO3 induces OS and alters the redox status of rat blood (Ahmad and Mahmood, 2012). In the present work, we have explored the use of taurine in attenuating bromate-induced hemotoxicity using rats as the animal model. This was done in view of the effectiveness of taurine in mitigating toxicities involving ROS and OS and the fact that there is no information regarding its potential beneficial role in reducing toxicity of bromate or related compounds (iodate, chlorate, etc.). The aim of this work was to examine if orally administered taurine could be used to attenuate KBrO3-induced toxicity in animals. Our results show for the first time the effectiveness of taurine in protecting blood from KBrO3-induced hemoglobin (Hb) oxidation, OS, and impairment of AO power.

MATERIALS AND METHODS Animal Protocol

Abbreviations AO CAT DPPH FRAP G6PD GPx GSH H2O2 Hb KBrO3 LPO MetHb MetHbR NADP1 and NADPH NO OS ROS SOD VC

anti-oxidant catalase 2,2-diphenyl-1-picrylhydrazyl ferric reducing ability of plasma glucose 6-phosphate dehydrogenase glutathione peroxidise reduced glutathione hydrogen peroxide hemoglobin potassium bromate lipid peroxidation methemoglobin methemoglobin reductase oxidized and reduced nicotinamide adenine dinucleotide nitric oxide oxidative stress reactive oxygen species superoxide dismutase vitamin C

Environmental Toxicology DOI 10.1002/tox

Adult male rats of Wistar strain weighing 150– 200 g were used in all the experiments. Animal experiments were conducted according to the guidelines of Ministry of Environment and Forests, Government of India, approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) vide registration no. 714/02/a/CPCSEA. The study was approved by the Institutional Animals Ethic Committee (IAEC) that monitors the research involving animals. All animals were stabilized for one week prior to the experiment on standard pellet rat diet with free access to water. Solutions of taurine and KBrO3 were prepared in drinking water and they were given orally (by gavage), rather than intraperitoneally or subcutaneously, to the animals to simulate real life exposure and intake of these agents. The doses of taurine and KBrO3 administered to animals were those which have been previously used

TAURINE PROTECTS BLOOD FROM BROMATE-INDUCED OXIDATIVE STRESS

by us and other workers (Das et al., 2009; Manna et al., 2009; Ahmad and Mahmood, 2012). Animals were randomly divided into four groups with six rats in each group: 1. Control: Animals were given suitable volume of drinking water by gavage. 2. KBrO3 alone: Animals were given a single oral dose of KBrO3 at 100 mg/kg body weight. 3. Taurine alone: Animals were orally given taurine at 100 mg/kg body weight/day for five consecutive days. 4. Taurine1KBrO3: Animals were first given taurine for five days at 100 mg/kg body weight/day. Then 6 h after the last dose of taurine they were given a single dose of KBrO3 at 100 mg/kg body weight.

The animals were sacrificed 48 h after the above treatments under light ether anaesthesia. This time interval was selected as our previous work has shown that KBrO3-induced changes were maximum 48 h after its administration (Ahmad and Mahmood, 2012). The experiment was planned such that all animals were sacrificed on the same day. All animals had free access to water and food throughout the duration of the experiment. Blood was collected in tubes following cardiac puncture and mixed with glucose–citric acid–trisodium citrate as anticoagulant. Isolation of Erythrocytes and Preparation of Hemolysates

Blood was centrifuged at 2500 rpm for 10 min at 4 C in a clinical centrifuge and the plasma so obtained was quickly frozen. The erythrocyte pellet was washed thrice with phosphate buffered saline (10 mM phosphate buffer, 0.9% NaCl, pH 7.2) and the cells were lysed with 10 volumes of distilled water for 2 h at 4 C. The samples were centrifuged at 3000 rpm for 10 min at 4 C and the supernatants (hemolysates) were used for the analysis of several biochemical parameters. Hemoglobin and Methemoglobin Levels, Methemoglobin Reductase Activity, and Protein Determination

Hemoglobin (Hb) concentration in hemolysates was determined by the Drabkins reagent using kits from Crest Biosystems (Goa, India) and protein concentration in plasma was determined by the Folin-phenol reagent using bovine serum albumin as standard. Methemoglobin (MetHb) levels were determined from the absorbance of hemolysates at 540, 576, and 630 nm and expressed as MetHb 3 1024 M. Methemoglobin reductase (MetHbR) activity was assayed after incubation of hemoly-

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sates with reduced nicotinamide adenine dinucleotide (NADH) and 2,6-dicholorophenolindophenol. Urea Nitrogen, Creatinine, and Inorganic Phosphate Levels

Urea nitrogen was determined in plasma by the diacetyl monoxime method using a kit from Span Diagnostics, India. Creatinine and inorganic phosphate levels were determined in deproteinized plasma samples. Creatinine was determined by using saturated picric acid and inorganic phosphate by using ammonium molybdate and ferrous sulfate. Aspartate Aminotransferase, Alanine Aminotransferase, and Glucose Levels

Activities of aspartate aminotransferase and alanine aminotransferase were assayed in plasma by reaction with 2,4-dinitrophenyl hydrazine and glucose by reaction with 4-amino antipyrene using kits from Span Diagnostics (Surat, India). Vitamin C, Xanthine Oxidase, and Nitric Oxide Levels

Vitamin C (VC) was determined in plasma by reaction with DTC (2,4-dinitrophenylhydrazine, thiourea, and copper) reagent (Omaye et al., 1979). Xanthine oxidase was assayed in plasma by following the change in absorbance at 292 nm after addition of the substrate xanthine (Bergmeyer et al., 1974). Nitric oxide (NO) was measured in plasma and hemolysates by using the Greiss reagent (Miranda et al., 2001). Carbonyl Content, Malonaldehyde, Total Sulfhydryl Groups, Reduced Glutathione, and Hydrogen Peroxide Levels

Carbonyl content of proteins was determined after reaction with 2,4-dinitrophenyl hydrazine (Levine et al., 1990). Malonaldehyde, a product of LPO, was measured as thiobarbituric acid reactive substance in plasma and hemolysates (Buege and Aust, 1978). Total sulfhydryl groups and glutathione (GSH) were determined from the yellow color produced after reaction with 5,50 -dithiobis-2-nitrobenzoic acid (Sedlak and Lindsay, 1968). Hydrogen peroxide (H2O2) levels were determined using xylenol orange in presence of 100 mM sorbitol (Gay and Gebicki, 2000).

Environmental Toxicology DOI 10.1002/tox

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Fig. 1. Effect of taurine and KBrO3 on (A) MetHb levels and (B) MetHbR activity in hemolysates. MetHb levels and MetHbR activity were determined spectrophotometrically as described under Materials and Methods. Results are mean 6 SEM for six different samples. *Significantly different at p < 0.05 from control. C: control; T: taurine alone; PB: KBrO3 alone; T1PB: taurine1KBrO3; MetHb: methemoglobin; MetHbR: methemoglobin reductase.

Assay of Enzymes Involved in Maintaining AO Status of Cell

of NADPH to NADP1 at 340 nm in the presence of oxidized glutathione.

The enzymes of AO defence mechanism were assayed as described by (Manna et al., 2008). Briefly, Cu–Zn superoxide dismutase (SOD) was assayed by following the inhibition of autooxidation of pyrogallol at 420 nm and catalase (CAT) from the conversion of H2O2 to water at 240 nm. Glucose 6-phosphate dehydrogenase (G6PD) was assayed by the formation of NADPH and glutathione reductase from the conversion of NADPH to nicotinamide adenine dinucleotide phosphate (NADP1) at 340 nm during the reduction of oxidized glutathione to GSH. GlutathioneS-transferase activity was determined using 1chloro-2,4-dinitrobenzene as substrate and thioredoxin reductase by following the yellow color formed at 410 nm upon reduction of 5,5’-dithiobis-2-nitrobenzoic acid by NADPH. Glutathione peroxidase (GPx) was assayed from the conversion

Ferric Reducing Ability of Plasma and 2,2-Diphenyl-1-Picrylhydrazy Assays

AO power of plasma and hemolysates was determined by the ferric reducing ability of plasma (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays (Janaszewska and Bartosz, 2002). Statistical Analysis

All data are expressed as mean 6 standard error of mean. Analysis of variance was used in combination with Post-Hoc tests using GraphPad InStat 3.0. (USA) to evaluate the data by comparing the results of treatment groups to control group. All differences of p < 0.05 were considered significantly different. All experiments were done three times to document reproducibility.

TABLE I. Effect of taurine and KBrO3 on some plasma parameters

ALT AST Creatinine Urea nitrogen Glucose Pi VC

Control

KBrO3 alone

Taurine alone

Taurine1KBrO3

12.19 6 1.04 16.78 6 1.54 0.39 6 0.04 14.28 6 1.25 148.81 6 8.41 40.21 6 2.11 3.01 6 0.14

101.12 6 8.14* 127.13 6 10.04* 1.36 6 0.13* 34.46 6 1.89* 88.25 6 5.24* 12.01 6 1.01* 0.88 6 0.02*

11.18 6 0.98 14.92 6 1.14 0.37 6 0.03 12.44 6 0.74 146.95 6 9.17 41.04 6 2.24 3.42 6 0.17

19.46 6 1.97* 21.24 6 2.04* 0.47 6 0.05* 19.75 6 1.07* 120.45 6 7.84* 34.17 6 2.84* 2.57 6 0.11*

Results are mean 6 SEM for six different preparations. ALT and AST are in IU/L. Glucose, urea nitrogen, and creatinine levels are in mg/ 100 mL. Pi and VC are in mg/mL. *Significantly different from control at p < 0.05. ALT, alanine aminotransferase; AST, aspartate aminotransferase; Pi, inorganic phosphate; VC, vitamin C.

Environmental Toxicology DOI 10.1002/tox

TAURINE PROTECTS BLOOD FROM BROMATE-INDUCED OXIDATIVE STRESS

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Fig. 2. Effect of taurine and KBrO3 on some parameters of oxidative stress in plasma (A and B) and hemolysates (C and D). Results are mean 6 SEM for six different preparations. *Significantly different from control at p < 0.05. MDA: malonaldehyde; SH: sulfhydryl; GSH: reduced glutathione; C: control; T: taurine alone; PB: KBrO3 alone; T1PB: taurine1KBrO3.

RESULTS

The protective effect of taurine on renal toxicity induced by single oral dose of KBrO3 was examined. There were four groups of animals in each experiment: untreated control, KBrO3 alone, tau-

rine alone, and the taurine1KBrO3 group in which animals were given taurine prior to the administration of KBrO3. The effect of KBrO3, alone and in combination with taurine, was determined on parameters of OS, hepato- and nephro-toxicity as well as AO enzymes and the AO status.

Fig. 3. Effect of taurine and KBrO3 on (A) hydrogen peroxide and (B) nitric oxide levels in plasma and hemolysates. Results are mean 6 SEM for six different preparations. *Significantly different from control at p < 0.05. H2O2: hydrogen peroxide; NO: nitric oxide; C: control; T: taurine alone; PB: KBrO3 alone; T1PB: taurine1KBrO3.

Environmental Toxicology DOI 10.1002/tox

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Fig. 4. Effect of taurine and KBrO3 on the activities of some anti-oxidant enzymes in hemolysates. Results are mean 6 SEM for six different preparations. Specific activities of CAT, GPx, GST, GR, TR, and G6PD are in nmoles/mg Hb/min. Specific activity of SOD is in U/mg Hb (One unit is the amount which causes 50% inhibition of pyrogallol oxidation in a reaction volume of 3 mL). *Significantly different from control at p < 0.05. CAT: catalase; SOD: Cu-Zn superoxide dismutase; GPx: glutathione peroxidase; GR: glutathione reductase; GST: glutathione-S-transferase; TR: thioredoxin reductase; G6PD: glucose 6-phosphate dehydrogenase; C: control; T: taurine alone; PB: KBrO3 alone; T1PB: taurine1KBrO3.

MetHb Levels and MetHbR

The MetHb levels were determined in the hemolysates. MetHb is formed when the iron of Hb in ferrous form is oxidized to the ferric form. There was a significant increase in the MetHb levels (1.5 folds) in the KBrO3-treated group when compared to control [Fig. 1(A)]. The increase in MetHb levels was accompanied by a concomitant increase in MetHbR activity [Fig. 1(B)]. However, administration of taurine to animals prior to KBrO3 treatment significantly abrogated the KBrO3-induced alterations in MetHb levels and MetHbR activity. Plasma Parameters

The effect of taurine on KBrO3 induced changes in some plasma parameters was studied. Significant alterations in creatinine (4-fold increase), urea nitrogen (3.5-fold increase), glucose (1.5-fold decrease), and inorganic phosphate (3.5-fold decrease) levels were seen after treatment with KBrO3 alone compared to the control group show-

ing the induction of nephrotoxicity (Table I). There was also a significant increase in aspartate aminotransferase (7.5 folds) and alanine aminotransferase (8.5 folds) activities suggestive of hepatotoxicty in animals. Treatment of rats with KBrO3 led to decrease in VC levels (3.3 folds) also. Administration of taurine alone did not have any effect on these parameters which were similar to control values; thus taurine itself does not alter the function of blood. Administration of taurine to animals prior to KBrO3 treatment resulted in significant attenuation in the KBrO3-induced alterations in all these plasma parameters (Table I). Thus pretreatment of animals with taurine protects them from KBrO3-induced nephrotoxicity and hepatotoxicity. Carbonyl Content, Malonaldehyde, Total Sulfhydryl Groups, GSH, and H2O2 Levels

Administration of KBrO3 alone greatly enhanced both LPO and protein oxidation as reflected in elevated levels of malonaldehyde and carbonyls in

TABLE II. Effect of taurine and KBrO3 on the activities of some pro- and anti-oxidant enzymes in plasma

CAT SOD GPx XO

Control

KBrO3 alone

Taurine alone

Taurine1KBrO3

69.71 6 4.12 66.01 6 3.44 11.28 6 1.67 1.04 6 0.01

27.24 6 2.14* 154.75 6 9.47* 3.24 6 10.04* 9.85 6 0.14*

70.01 6 3.97 67.14 6 4.11 12.01 6 1.14 1.11 6 3.17

58.09 6 2.97* 75.68 6 5.11* 8.85 6 0.74* 4.67 6 0.22*

Results are mean 6 SEM for six different preparations. Specific activities of XO, CAT, and GPx are in nmoles/mg protein/min and SOD is in U/mg protein (One unit of SOD is the amount which causes 50% inhibition of pyrogallol oxidation in a reaction volume of 3 mL). *Significantly different from control at p < 0.05. CAT, catalase; SOD, superoxide dismutase; GPx, glutathione peroxidase; XO, xanthine oxidase.

Environmental Toxicology DOI 10.1002/tox

TAURINE PROTECTS BLOOD FROM BROMATE-INDUCED OXIDATIVE STRESS

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TABLE III. Effect of taurine and KBrO3 on the anti-oxidant power of plasma and hemolysates

FRAP Plasma Hemolysate DPPH Plasma Hemolysate

Control

KBrO3 alone

Taurine alone

Taurine1KBrO3

4.22 6 0.14 3.19 6 0.04

1.74 6 0.09* 1.58 6 0.07*

4.31 6 0.22 3.22 6 0.05

3.57 6 0.13* 2.89 6 0.08*

78.94 6 3.71 64.12 6 2.42

48.01 6 2.18* 30.11 6 1.97*

80.11 6 1.19 65.02 6 1.47

70.17 6 3.21* 59.87 6 2.17*

Results are mean 6 SEM for six different preparations. FRAP results are in units/ mg protein for plasma and units/ mg Hb for hemolysate; DPPH results are as percent quenching of the DPPH radical. *Significantly different from control at p < 0.05. FRAP, ferric reducing ability of plasma; DPPH, 2,2-diphenyl-1-picrylhydrazyl.

plasma and hemolysates when compared to the control group [Figs. 2(A,C)]. It also resulted in significant reduction in total sulfhydryl and GSH content [Figs. 2(B,D)]. NO levels were significantly enhanced both in plasma (2.6 folds) and hemolysates (4 folds) of KBrO3-treated rats. There was also a marked increase in level of H2O2 in both hemolysates (2.2 folds) and plasma (1.9-fold) of KBrO3-treated animals (Fig. 3). However, these KBrO3-induced changes were significantly attenuated by administration of taurine prior to treatment with KBrO3. Treatment with taurine alone did not significantly alter any of these parameters (Figs. 2 and 3). The results indicate marked protection by taurine against KBrO3-induced OS in blood. Activity of Some Enzymes Involved in Maintaining AO Status

Maintenance of normal cellular functions largely depends on the efficiency of the defence mechanisms against free-radical mediated OS and AO enzymes are considered to be a major line of cellular defence. KBrO3 administration significantly altered the activities of AO enzymes. The activity of SOD increased while that of CAT and GPx decreased in hemolysates [Fig. 4(A)]. The activity of glutathione-S-transferase (4.8 folds) increased, while G6PD (3 folds), glutathione reductase (2.6 folds), and thioredoxin reductase (2.2 folds) were significantly reduced in hemolysates [Fig. 4(B)]. Oral administration of KBrO3 also increased the activities of SOD and xanthine oxidase and decreased CAT and GPx in the plasma (Table II). These changes were abrogated by pre-treatment with taurine which, when given alone, had no effect on the activities of these enzymes.

AO Power and Hemolysates of Plasma

The intracellular AO power of plasma and hemolysates was assayed by FRAP and DPPH reduction assays (Table III). The DPPH assay showed that free radical scavenging activity was less both in plasma (1.6 folds) and hemolysates from KBrO3treated animals (2.1 folds) as compared to control. There was also a significant decrease in the ferric reducing activity in plasma (2.4 folds) and hemolysates (2 folds) confirming the fact that that the AO power of both plasma and erythrocytes is greatly compromised upon exposure of animals to KBrO3. However, pre-treatment with taurine significantly attenuated the alterations induced by KBrO3 while the administration of taurine alone had no effect on these parameters. DISCUSSION

Erythrocytes are terminally differentiated cells where most of the biosynthetic pathways are absent. They are among the first cells to be exposed to a xenobiotic regardless of the route of exposure (inhalation, ingestion, or applied on skin). Because of their role as oxygen carriers, erythrocytes are in constant danger posed by intracellular oxygen radicals and also from external sources. In addition, the high content of polyunsaturated lipids and transition metals makes them especially vulnerable to oxidative damage. As a consequence, erythrocytes have a well-developed AO system which builds up an efficient defence against the cytotoxic effects of ROS. Because of their simple structure erythrocytes have been extensively used as a cellular model to study oxidative damage by a variety of agents.

Environmental Toxicology DOI 10.1002/tox

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Fig. 5. Summary of KBrO3 effects on rat blood. KBrO3 enters the cell where it is reduced to KBr by intracellular reductants. This process generates ROS and RNS which then induce oxidative and nitrosative stress in the blood. The ROS and RNS induce depletion of anti-oxidants like GSH and vitamin C and alter the activities of AO enzymes. This results in redox imbalance in the blood and oxidation of proteins, lipids, and hemoglobin. The consequences are increased cell injury. Taurine protects the cell either by inhibiting the intracellular reduction of KBrO3 to KBr or, more likely, the quenching of free radicals and ROS/ RNS by its well-documented AO property. AO: antioxidant; Hb: hemoglobin; KBr: potassium bromide; KBrO3: potassium bromate; RNS: reactive nitrogen species; ROS: reactive oxygen species; T: taurine.

We have previously reported that KBrO3 induces OS in the blood of rats and alters the AO defence mechanism (Ahmad and Mahmood, 2012). In this work we have examined the role of pre-treatment with taurine in attenuating the effects of KBrO3 in rat blood. Taurine was selected as it is a dietary compound which is found in several foods and exhibits protective effects against certain environmental agents because of its AO and anticarcinogenic properties (Alam et al., 2011; Das et al., 2012; Ozden et al., 2013). The use of dietary compounds like taurine offers great potential as they are part of normal diet and foods rich in such compounds can be safely taken by persons who are at risk of exposure to toxicants. Several reports have shown that AO in food are not only efficient, but also have relatively fewer harmful side effects. For this reason chemoprevention using natural compounds (as opposed to synthetic ones) to block, reverse, or prevent toxicities, has gained immense attention in recent years. Natural AO have become increasingly important as therapeutic agents against disorders involving OS

Environmental Toxicology DOI 10.1002/tox

such as Alzheimer’s, cancer, and diabetes (Obrenovich et al., 2011). A typical pattern of tissue toxicity was seen in KBrO3-treated animals as indicated by increased alanine aminotransferase, aspartate aminotransferase, creatinine, urea nitrogen levels accompanied by massive glucosuria and phosphaturia. Similar results have been reported previously by us and other workers also (Bao et al., 2008; Ahmad and Mahmood, 2012). The changes in these parameters were reversed by prior administration of taurine. These protective effects can be attributed to the fact that taurine is able to revert the toxicantinduced changes in liver and kidney (Seabra and Timbrell, 1997; Tabassum et al., 2007). Increased production of ROS and free radicals has been implicated in mediating KBrO3-induced toxicity (Ahmad et al., 2014). To assess the functional aspects the activities of various AO enzymes were determined after administration of KBrO3. Significant alterations were observed in the activities of CAT, GPx, and SOD, the major enzymes involved in quenching the ROS and reducing OS in the cell. The activity of SOD increased significantly while CAT and GPx were greatly reduced in both plasma and hemolysates. The activity of G6PD, the major enzyme responsible for the generation of NADPH in erythrocytes, was reduced upon treatment with KBrO3. Lowered NADPH levels will result in decrease in CAT activity, which contains four NADPH bound per tetrameric enzyme molecule that serve to protect it from inactivation by the substrate H2O2. Reduced levels of NADPH will also decrease the activity of glutathione reductase and thioredoxin reductase, as both use NADPH provided by G6PD. The reduced activity of glutathione reductase will, in turn, lead to lower levels of GSH, a necessary cofactor for the detoxification of H2O2 and organic peroxides by GPx. As both CAT and GPx are the major enzymes responsible for detoxication of peroxides in erythrocytes, their reduced activities result in elevated levels of H2O2, an ROS that causes extensive oxidative modification of biomolecules. The reduced activities of CAT, glutathione reductase, GPx, and thioredoxin reductase must have overcome the enhancement in the activity of glutathione-S-transferase (which is involved in the detoxification of drugs and poisons by conjugating them with GSH) and SOD. Administration of KBrO3 also enhanced the activity of xanthine oxidase, a pro-oxidant enzyme, and decreased the VC levels significantly as also shown in earlier studies (Bao et al., 2008; Ahmad and Mahmood, 2012).

TAURINE PROTECTS BLOOD FROM BROMATE-INDUCED OXIDATIVE STRESS

As erythrocytes are unable to synthesize new proteins, the alterations in enzyme activities seen here is not because of changes in their levels but rather by enzymatic activation or inactivation/inhibition. Treatment with taurine prior to KBrO3 administration greatly attenuated the activities of AO enzymes, possibly by membrane stabilization or by stimulating the elimination of excess ROS produced by KBrO3 and preventing them from causing any further oxidative damage (Timbrell et al., 1995; Birdsall, 1998). Administration of KBrO3 also increased LPO and protein carbonyls and decreased total sulfhydryl content suggesting increased formation of ROS in the erythrocytes (Ahmad et al., 2014). However, these bromate-induced alterations were attenuated by administration of taurine which could have inhibited the KBrO3-induced generation of free radicals and ROS. A similar protective effect of taurine against oxidative damage induced in blood, especially erythrocytes, by various toxicants has been previously reported by other workers (Sinha et al., 2008; Roy and Sil, 2012). Administration of KBrO3 increased the NO levels, thereby inducing nitrosative stress. NO is known to react with superoxide radicals to form the damaging peroxynitrite, a reactive nitrogen species. One reason for the increased activity of SOD might be to reduce the superoxide anions available for reaction with NO so that peroxynitrite is not produced. The increased nitrosative stress coupled with OS makes the cells quite vulnerable to toxic insult and hence compromises the AO power of blood (Ahmad and Mahmood, 2012). There was significant elevation in MetHb levels in erythrocytes from KBrO3-treated animals probably because of the increased levels of NO and H2O2, both of which stimulate the oxidation of ferrous ion in oxyHb to form MetHb. The activity of MetHbR, the enzyme which converts MetHb back to Hb, was also increased indicating that the higher MetHb levels were not because of inhibition of this enzyme. The increased activity of MetHbR must have been an adaptive response to the higher MetHb formed upon exposure to KBrO3. However, administration of taurine prior to KBrO3 significantly attenuated the observed changes, thereby protecting erythrocytes from oxidative damage. The mechanism by which taurine protects against the KBrO3-induced changes is probably because of its antioxidant property. Taurine protects the impairment in the AO power by reducing the levels of pro-oxidant NO and H2O2 and restor-

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ing the levels of nonenzymatic antioxidants GSH and VC which are major endogenous AO that counterbalance free radical mediated changes. GSH is especially important in this regard as it is abundant in the blood (present in millimolar concentrations) and protects the cell by maintaining the redox homeostasis, quenching free radicals produced by xenobiotics and taking part in detoxification reactions. Taurine administration also showed normalization of the activities of major AO enzymes. The strengthening of the nonenzymatic and enzymatic defence systems results in improved AO power and lower levels of oxidation of lipids and proteins in erythrocytes from taurine1KBrO3-treated group compared to the KBrO3 alone group. Taurine is a commonly ingredient of “energy drinks” which can contain large amounts of this amino acid (upto several grams) per serving. Two major reasons have been proposed for adding taurine in these drinks. One, it is thought to improve the cardiovascular function and circulation of blood in the body (Schaffer et al., 2012). Two, it helps the skeletal muscles to function, especially during strenuous exercise, by lowering exhaustion levels and improving endurance. This is attributed to the cytoprotective role of taurine in muscle injury by reducing the exercise-induced ROS and OS (Dawson et al., 2002; Schaffer et al., 2012). Our results suggest another way by which taurine can be beneficial is by reducing the build up of oxidants in the blood because of arduous exercise and increasing its oxygen carrying capacity by inhibiting conversion of Hb to MetHb, which is inactive as an oxygen carrier. In conclusion our results clearly demonstrate that pre-treatment of rats with taurine protects the blood from the oxidative and nitrosative stress produced by oral administration of single dose of KBrO3. It strengthens the enzymatic and nonenzymatic AO system of the erythrocytes and also prevents oxidation of Hb to MetHb. The protective effects of taurine reported in this study are summarized in Figure 5. The protection by taurine against KBrO3 can be attributed to its intrinsic biochemical and natural AO properties. These findings show the importance of taurine on the functions of blood, especially erythrocytes, and provide an insight into the mechanisms by which the bromate-induced tissue toxicity can be attenuated. However, more detailed studies with varying doses of taurine and KBrO3 need to be done to delineate the exact molecular events of the beneficial effects of this sulfonic amino acid.

Environmental Toxicology DOI 10.1002/tox

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AHMAD AND MAHMOOD

CONFLICT OF INTEREST

The authors declare there is no conflict of interest in this work. We thank the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for the award of Senior Research Fellowship (to MKA). Financial support to the department from the University Grants Commission (SAP-DRS scheme) is gratefully acknowledged. We are thankful to Mr. Mohd. Fareed and Dr. Ashreeb Naqshbandi for their help in this study.

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Environmental Toxicology DOI 10.1002/tox

Protective effect of taurine against potassium bromate-induced hemoglobin oxidation, oxidative stress, and impairment of antioxidant defense system in blood.

Potassium bromate (KBrO3 ) is widely used as a food-additive and is a major water disinfection by-product. KBrO3 causes severe toxicity in humans and ...
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