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Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway
Jyotirmoy Ghosh 1, Joydeep Das 1, Prasenjit Manna, Parames C. Sil ⁎
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, West Bengal, India

a r t i c l e i n f o

Article history:
Received 3 June 2009
Revised 3 July 2009
Accepted 7 July 2009
Available online 17 July 2009

Keywords:
Arsenic
Cardiac oxidative stress NF-κB and MAPK Apoptosis
Taurine Cardioprotection
a b s t r a c t

Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-κB (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-κB and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-κB activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-κB activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection against As-induced cardiovascular burden.
© 2009 Elsevier Inc. All rights reserved.

Introduction

Arsenic (As), one of the most notoriously poisonous metalloid, is ubiquitous in the environment. Although the majority of humans are chronically exposed to low levels of As through ingestion of food and inhalation in the ambient air, As-contaminated ground water is an unavoidable source of this poisoning and a considerable percentage of the total populations of the world are suffering from As related organ dysfunctions because of drinking this poisonous water. Once absorbed, As redistributes itself to nearly entire organ systems of the body including heart (Ratnaike, 2003; Chang et al., 2007; Manna

Abbreviations: CAT, catalase; FRAP, Ferric Reducing/Antioxidant Power; GSH, glutathione; GSSG, glutathione disulfide; GST, glutathione S-transferase; GPx, glu- tathione peroxidase; GR, glutathione reductase; HDL, high-density lipoprotein; MDA, malonaldehyde; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinases; NF-κB, nuclear factor kappa B; ROS, reactive oxygen species; NaAsO2, sodium arsenite; SOD, superoxide dismutase.
⁎ Corresponding author. Fax: +9133 2355 3886.
E-mail addresses: [email protected], [email protected] (P.C. Sil).
1 Both authors contributed equally to the study.
et al., 2007; Manna et al., 2008; Das et al., 2009a; 2009b). It causes lipid peroxidation, and the oxidation of proteins, enzymes as well as DNA and DNA adducts (Yamauchi et al., 2004). Thus the genotoxic effects of arsenic may be connected with an inhibition of DNA repair as well as the induction of oxidative stress (Liu et al., 2001).
Although emerging evidence supports the role of free radical- mediated oxidative stress in the pathophysiology of As-mediated organ injury and cell death, very little is known about the mechanism of As-induced cardiac abnormalities and the use of antioxidants as the possible preventive or curative agents that can be targeted as therapeutic tools in future. Taurine (2-aminoethanesulfonic acid) is a derivative of the sulphur-containing amino acid, cysteine and is present in many tissues of mammals with high concentrations. It is neither metabolized nor incorporated into cellular proteins, suggest- ing that it could be an important need for the cellular cytosolic function (Huxtable, 1992; Sole and Jeejeebhoy, 2000). A number of recent studies showed that taurine has a combination of effects on ion channels, transporters, and enzymes, leading to modulation of intracellular Ca2+ levels (Sole and Jeejeebhoy, 2000; Yan-Jun et al., 2006; Holloway et al., 1999; Satoh, 2001). In addition, this con- ditionally essential amino acid has been considered to have potent

antioxidant properties under various pathophysiological situations (Huxtable, 1992; Sole and Jeejeebhoy, 2000; Biasetti and Dawson, 2002; Sinha et al., 2007). The present study was, therefore, designed and carried out to investigate the probable mechanism of As-induced myocardial dysfunction and the beneficial role of taurine using both in vivo and in vitro working models. The results of the present study could clarify the role of this important bioactive molecule in the prevention of As-induced cardiotoxicity, and may shed light on a possible solution to the serious cardiac complications arising due to drinking As-contaminated water and other unavoidable ways of its exposures in humans.

Materials and methods

Chemicals. Taurine (2-aminoethane sulfonic acid), anti-caspase-3, cleaved caspase-3, ERK1/2 and phosphorylated ERK1/2 antibodies were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). NF-κB (p65 sub unit), anti-phosphorylated p65, p38 and phosphorylated p38 antibodies were purchased from Cell signaling technology (Beverly, MA, USA). All other antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Sodium arsenite (NaAsO2) was bought from Sisco research laboratory (Mumbai, India).

Animals. Male albino rats of Wistar strain weighing approximately 120–130 g (4 weeks of age) were acclimatized under laboratory conditions for two weeks prior to the experiments. All the experiments with animals were carried out according to the guidelines of the institutional animal ethical committee.

Determination of dose and time for As-induced cardiac dysfunctions in vivo. To establish the dose of As necessary for cardiac damage, rats were randomly allocated into seven groups each consisting of six rats and they were treated as follows. First group served as normal control (received only water as vehicle). Remaining six groups were treated with six different doses of NaAsO2 orally (0.5 mg, 1 mg, 1.5 mg, 2 mg,
2.5 mg and 3 mg/kg body weight in distilled water for 5 weeks).
To find out the time needed for As-induced cardiac damage in rats, experiments were carried out with four groups of animals consisting six animals in each group. NaAsO2 was administered orally at a dose of 2 mg/kg body weight for 1, 3, 5 and 7 weeks respectively.
Twenty-four hours after the final dose of NaAsO2 intoxication, all rats were euthanized and catalase (CAT) activity was measured in the heart homogenates.

Determination of dose and time dependent activity of taurine in vivo.
To determine the beneficial role of taurine, rats were randomly distributed into eight groups each consisting of six animals. First two groups were served as normal control (received only water as
vehicle) and toxin control (received NaAsO2 at a dose of 2 mg/kg body weight for 5 weeks) respectively. Remaining six groups of animals were treated with six different doses of taurine (10 mg, 20 mg, 30 mg, 40 mg, 50 mg and 60 mg/kg body weight for two weeks) followed by NaAsO2 intoxication (2 mg/kg body weight for 5 weeks).
To determine the time dependent effects of taurine, experiments were carried out with six groups of animals consisting six animals in each group. Taurine was administered at a dose of 50 mg/kg body weight for 0.5, 1, 1.5, 2, 2.5 and 3 weeks respectively prior to NaAsO2 intoxication (2 mg/kg body weight for 5 weeks).
Twenty-four hours after the final dose of taurine, all rats were euthanized and FRAP activity was measured in the heart homogenates of all experimental rats.

Experimental design for in vivo treatments. Experimental design needed for the present in vivo study has been summarised in Fig. 1. Brieﬂy, 6 week old rats were randomly assigned to three groups. Rats in “Normal group” received only water as vehicle. Rats in “Toxin treated group” received NaAsO2 orally at a dose of 2 mg/kg body weight in distilled water for 5 weeks. For protective group, rats received taurine orally at a dose of 50 mg/kg body weight in distilled water for 2 weeks followed by NaAsO2 administration at a dose of 2 mg/kg body weight in distilled water for 5 weeks.

Harvest of serum and cardiac samples. The arsenic treated rats were euthanized at day 36th after the first dose of As and other rats were euthanized at day 50th. The body weight (taken before) and heart weight were measured and compared between groups. Blood samples were drawn from the caudal vena cava, collected in test tubes containing heparin solution, and centrifuged at 1500 g for 10 min to obtain serum. The cardiac tissues were either fixed in 10% formalin for histopathologic examinations or stored at −80 °C till
later analysis.

Preparation of cardiac tissue homogenates. The hearts were minced, washed, and homogenized in a Dounce glass homogenizer in 10 mM HEPES-KOH/1 mM EGTA buffer (pH 7.5) containing 250 mM sucrose and supplemented with protease and phosphatase inhibitors. The homogenates were spun down for 10 min at 2000 g to discard the myofilaments at 4 °C. The supernatant was collected and used for the in vivo experiments.

Biochemical analyses. Specific markers related to cardiac dysfunction
e.g. total cholesterol and HDL cholesterol levels in the sera were estimated by using standard kits. The lipid peroxidation was estimated according to the method of Esterbauer and Cheeseman (1990). LDH activity was determined according to the method of Kornberg (1955).

Fig. 1. Schematic diagram of in vivo experimental protocol.

Ferric Reducing/Antioxidant Power (FRAP), activities of antioxidant enzymes (SOD, CAT, GST, GR, GPx) and cellular metabolites levels (GSH and GSSG) in the heart tissue were determined following the method as described by Ghosh et al. (2008).

Cardiomyocyte isolation and in vitro experimental protocol. Primary cultures of neonatal cardiomyocytes were prepared according to the procedure described by Sil et al. (1998). After 4 days of culture, these preparations contained N 95% cardiomyocytes. Cardiomyocytes were then treated with taurine (25 mM), arsenic (5 μM) and taurine coupled with arsenic (1 h after) for 24 h and harvested at 4 °C for further molecular and biochemical analyses.

Assessment of cardiomyocyte viability. Viability of cardiomyocytes was determined by MTT assay. Brieﬂy, 250 μL of MTT solution (300 mg/mL) was added to the culture medium (200 μL in each well) of cardiomyocytes cultured in 24-well plates 1 h before the end of 24 h treatment and incubated at 37 °C for 30 min. After incubation supernatants were discarded and 200 μL of dimethyl sulfoxide was added and mixed thoroughly to dissolve the crystals. Absorbance was taken at 570 nm and 630 nm.

Measurement of intracellular ROS production. Brieﬂy, cardiomyocytes were incubated with DCF-DA (10 mM) for 1 h at 37 °C in the dark. After treatment, the cells were immediately washed and resuspended in PBS. Intracellular ROS production was detected using the ﬂuorescent intensity of the oxidant sensitive probe 2,7 dichlorodihydroﬂuorescein diacetate (H2DCFDA). The ﬂuorescence emitted was measured at 525 nm.

Flow cytometric analysis of cardiomyocyte apoptosis. Cardiomyocytes were washed with PBS, centrifuged at 800 g for 6 min, resuspended in ice-cold 70% ethanol/PBS, centrifuged at 800 g for a further 6 min, and resuspended in PBS. Cells were then incubated with propidium iodide (PI) and FITC-labelled Annexin V for 30 min at 37 °C. Excess PI and Annexin V were then washed off; cells were fixed and then stained cells were analysed by ﬂow cytometry using FACS Calibur (Becton Dickinson, Mountain View, CA) equipped with 488 nm argon laser light source; 515 nm band pass filter for FITC-ﬂuorescence and 623 nm band pass filter for PI-ﬂuorescence using CellQuest software. A dot plot of PI-ﬂuorescence (y-axis) versus FITC-ﬂuorescence (x-axis) has been prepared.

Immunoblotting. An equal amount of protein (50 μg) from each sample was resolved by 10% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked at room temperature for 2 h in blocking buffer containing 5% non-fat dry milk to prevent non specific binding and then incubated with anti-p-38 (1:1000 dilution), anti- ERK1/2 (1:1000 dilution), anti-p-JNK (1:1000 dilution), anti-PARP (1:1000 dilution), anti-NF-κB (p65) (1:250 dilution), anti-IKK-α/β (1:1000 dilution), anti-Bad (1:1000 dilution), anti-Bax (1:1000 dilution), anti-Bcl-2 (1:1000 dilution), anti-Bcl-xL (1:1000 dilution), anti-caspase-3 and anti-cleaved caspase-3 (1:100 dilution) primary antibodies separately at 4 °C overnight. The membranes were washed in TBST (50 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20) for 30 min and incubated with appropriate HRP conjugated secondary antibody (1:2000 dilution) for 2 h at room temperature and developed by the HRP substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) system (Bangalore Genei, India).

Preparation of rat heart nuclear extract. Nuclear proteins were extracted from the frozen heart samples according to the method of Valen et al. (2000). Brieﬂy, hearts were homogenized and lysis buffer was added. After incubation on ice, nuclei were collected by means of centrifugation for 1 min at 8000 g. The pellet was washed with 20 mM KCl buffer, centrifuged again, and resuspended in 20 mM KCl buffer.
An equal part 600 mM KCl was added to the pellet and kept on ice for 30 min. After centrifugation for 15 min at 8000 g, the supernatant containing nuclear proteins was obtained. This nuclear extract was subjected to immunoblotting as described above to determine the activity of NF-κB in vivo.

Isolation of mitochondria and determination of mitochondrial membrane potential (Δψm). Mitochondria were isolated from the heart tissue of experimental rats. Brieﬂy, the supernatant, as obtained in the Preparation of cardiac tissue homogenates section, was overlaid on 0.75 M sucrose in HEPES buffer and centrifuged for 30 min at 10,000 g. The supernatant was discarded and the mito- chondria pellet were resuspended in HEPES buffer and recentrifuged

Fig. 2. Panel A: Dose and time dependent effect of NaAsO2 on CAT activity. Closed circle: CAT activity in normal rats. Closed square: CAT activity in NaAsO2 intoxicated rats for 1 week at a dose of 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg and 3 mg/kg body weight. Blank circle: CAT activity in NaAsO2 intoxicated rats for 3 weeks at above-mentioned doses. Closed triangle: CAT activity in NaAsO2 intoxicated rats for 5 weeks at above-mentioned doses. Closed star: CAT activity in NaAsO2 intoxicated rats for 7 weeks at above- mentioned doses. Panel B: Dose and time dependent effect of taurine on intracellular antioxidant power against arsenic-induced cardiac toxicity of the experimental rats. Cont: antioxidant power in normal rats, As: antioxidant power in NaAsO2 treated rats, TAU-10 +As, TAU-20 +As, TAU-30 +As, TAU-40 +As, TAU-50 +As, TAU-60 +As:
antioxidant power in taurine (TAU) treated rats for 2 weeks at a dose of 10, 20, 30, 40, 50 and 60 mg/kg body weight prior to NaAsO2 administration; TAU-0.5W +As, TAU- 1W+As, TAU-1.5W+As, TAU-2W+As, TAU-2.5W+As, TAU-3W+As: antioxidant
power in taurine (TAU) treated rats for 0.5, 1, 1.5, 2, 2.5 and 3 weeks respectively at a dose of 50 mg/kg body weight prior to As administration. Each column represents mean
±SD, n =6. “a” indicates the significant difference between the normal control and toxin treated groups and “b” indicates the significant difference between the toxin treated and taurine treated groups. (Pa b 0.05, Pb b 0.05).

for 10 min at 10,000 g. This supernatant was also discarded and the
final mitochondrial pellet was resuspended in PBS. It was stored at
−80 °C until use. Analytic ﬂow cytometric measurements for the membrane potential (Δψm) of isolated mitochondria were performed
using a FACScan ﬂow cytometer with an argon laser excitation at 488 nm (Korichneva et al., 2003). Mitochondrial membrane potential (Δψm) was estimated on the basis of cell retention of the ﬂuorescent cationic probe rhodamine 123.

Fig. 3. Effect on heart weight, body weight, and cellular GSH, GSSG content of arsenic treated rats. Panel A: absolute body weight, panel B: absolute heart weight, panel C: heart weight to body weight ratio, panel D: cellular GSH content, panel E: cellular GSH to GSSG ratio. Cont: normal rats; As: rats treated with As and TAU +As: rats treated with taurine prior to As exposure. Each column represents mean±SD, n =6. “a” indicates the significant difference between the normal control and toxin treated groups and “b” indicates the significant difference between the toxin treated and taurine treated groups. (Pa b 0.05, Pb b 0.05).

Table 1
Effect of As and taurine on the activities of the antioxidant enzymes in cardiac tissue.

Name of the antioxidant enzymes Activities of the antioxidant enzymes
Normal control Toxin control TAU +As
SOD (unit/mg protein) 107.3 ± 5.19 52.61 ± 3.22a 98.21 ± 4.55b
CAT (μmol/min/mg protein) 48.21 ± 2.84 19.61 ± 1.13a 37.92 ± 1.68b,c
GST (μmol/min/mg protein) 1.98 ± 0.1 0.75 ± 0.05a 1.65 ± 0.08b
GR (nmol/min/mg protein) 108.5 ± 5.14 52.71 ± 2.35a 96.77 ± 4.86b
GPx (nmol/min/mg protein) 169.2 ± 8.58 89.98 ± 4.85a 147.3 ± 7.65b
Values are expressed as mean ±SD, for 6 animals in each group. “a” values differs significantly from normal control (Pa b 0.05); “b” values differ significantly from toxin control (Pb b 0.05); “c” values significantly differ from normal control (Pc b 0.05).

Assay of cytochrome C release. The cytosolic cytochrome C release in heart tissue was measured with the cytochrome C enzyme immunometric assay kit (Minneapolis, MN).

Measurement of cytosolic free calcium in cardiomyocytes. Cardiomyocytes (as isolated in the Cardiomyocyte isolation and in vitro experimental protocol section) were harvested in physiological salt solution (PSS, pH 7.4) containing 145 mmol/L NaCl, 3.6 mmol/L KCl, 10 mmol/L HEPES,
1.8 mmol/L CaCl2,1 mmol/L MgCl2 and 5 mmol/L D-glucose. The cells were loaded with fura-2 (5 μM, 35 min, 37 °C) working solution and washed twice with fresh PSS (Grynkiewicz et al., 1985). Fluorescent calcium ions were detected at λex =340 and 380 nm, and λem =510 nm. The concentration of cytosolic free calcium was then obtained from the values of F, Fmax and Fmin by using the following formula: (Ca2+)cyt =Kd (F
−Fmin)/(Fmax−F); where Kd is the association constant of calcium with
fura 2 and set at 224 nm at physiological pH. Fmax and Fmin values were determined by the addition of 20 μL Triton X-100 (10%) and 40 μL EGTA (400 mM), respectively.

Histological studies. Hearts from the normal and experimental rats were fixed in 10% buffered formalin and were processed for paraffin sectioning. Sections of about 5 μm thickness were stained with haematoxylin and eosin to study the histology of hearts of all experimental rats.

Statistical analysis. All the values are expressed as mean±S.D. (n = 6). Significant differences between the groups were determined with SPSS 10.0 software (SPSS Inc., Chicago, IL, USA) for Windows using one-way analysis of variance (ANOVA) and the group means were compared by Duncan’s Multiple Range Test (DMRT). A difference was considered significant at the p b 0.05 level.

Results

Dose and time dependent effect of arsenic

As the first step of determining the dose and time necessary for As to induce cardiotoxicity, we carried out dose and time dependent assays using CAT activity in heart. As evidenced from Fig. 2A, As intoxication decreased the CAT activity linearly up to a dose of 2 mg/kg body weight for 5 weeks. This dose and time were, therefore, chosen as the optimum dose and time of NaAsO2 throughout the study.

Dose and time dependent study of taurine

We used the FRAP assay to determine the dose and time necessary for taurine for the protection of cardiac function against As-induced oxidative damages. Experimental results suggest that As exposure decreased the intracellular Ferric Reducing/Antioxidant Power and that could be prevented by the pre-treatment with taurine up to a dose of 50 mg/kg body weight for two weeks (Fig. 2B).
Taurine suppressed the deleterious effects of arsenic on the growth of body and heart

Our studies showed that As treated rats developed early physical lethargy. These animals also gained less body weight and heart weight compared to control animals (Figs. 3A, B). Taurine pre-treatment, however, compensated this deficiency of the body weight and heart weight loss, although the heart weight to body weight ratios of these groups remained practically unaltered, suggesting that the growth- impeding effect of As could be blocked by the counteracting action of taurine.

As-induced alterations in biochemical parameters are attenuated by taurine

In oxidative stress-induced organ pathophysiology, intracellular antioxidant enzymes are considered to be the first line of cellular defense as these enzymes protect biological macromolecules like DNA and proteins from oxidative damage. We, therefore, determined the activities of antioxidant enzymes and other cellular metabolites in the heart tissues of the experimental animals. We observed that As intoxication reduced the activities of the antioxidant enzymes (Table 1), level of GSH and GSH/GSSG ratio (Figs. 3D, E). Results showed that taurine supplementation could prevent the As-induced alterations of these parameters. In addition, As intoxication caused a significant enhancement in total cholesterol, MDA and LDH levels whereas decreased HDL cholesterol level. Treatment with taurine could, however, maintain these levels almost close to normal (Table 2).

Taurine ameliorated As-induced cytotoxicity and cardiomyocyte apoptosis

To determine how does As induce cardiotoxicity and whether taurine can prevent it from this pathophysiology, viability of cardiomyocytes was assessed with an MTT assay. As shown in Figs. 4A, B, As (5 μM) significantly reduced cardiomyocyte viability to 60% of the control value, whereas treatment with 25 mM taurine maintained the viability at N 90%, indicating the cytotoxic effect of arsenic and the cytoprotective effect of taurine in cardiomyocytes. A higher concentration of taurine (up to 40 mM) provided no additional benefit to cell viability, so the dose of 25 mM was selected for subsequent in vitro experiments.
To evaluate whether the impact of arsenic and taurine on cardiomyocyte viability involves the process of cell apoptosis, cardiomyocytes of all groups were assessed by ﬂow cytometric analysis. Flowcytometric data (Fig. 4C) revealed that, in comparison to control, As intoxicated cardiomyocyte showed maximum Annexin V-FITC-binding (45.68%), but very little PI staining (0.4%) indicating that majority of the cells were apoptotic. In taurine treated cardiomyocytes, the number of apoptotic cells was significantly low (12.9%) indicating that taurine protected cardiomyocytes from As- induced apoptosis.

Table 2
Effect of As and taurine on the heart specific biochemical parameters.

Name of biochemical parameters Levels of the biochemical parameters
Normal control Toxin control TAU +As
Total cholesterol (mg/dL) 168.4 ± 8.23 267.6 ± 12.45a 198.26 ± 9.68b
HDL cholesterol (mg/dL) 59.15 ± 2.57 28.37 ± 1.55a 48.89 ± 2.32b
MDA (nmol/mg protein) 16.71 ± 0.96 35.66 ± 2.11a 26.7 ± 31.25b,c
LDH (U/L) 0.343 ± 0.018 0.483 ± 0.052a 0.368 ± 0.031b
Values are expressed as mean ±SD, for 6 animals in each group. “a” values differ significantly from normal control (Pa b 0.05); “b” values differ significantly from toxin control (Pb b 0.05); “c” values differ significantly from normal control (Pc b 0.05).

Fig. 4. Impact of As and taurine on cardiomyocyte viability and cardiomyocyte apoptosis. Panel A: Dose dependent effect of As on cell viability; panel B: dose dependent effect of taurine on As treated cardiomyocytes; Cont: cell viability in normal myocytes; As-1, As-3, As-4, As-5, As-6 and As-7: cell viability in As treated myocytes for 24 h at a dose of 1, 3, 4, 5, 6 and 7 μM; TAU-10, TAU-20, TAU-25, TAU-30, and TAU-40: cell viability level in myocytes treated with taurine (1 h prior to As addition) and As for 24 h at a dose of 10, 20, 25, 30 and 40 mM. “a” indicates the significant difference between the normal control and toxin treated cells, “b” indicates the significant difference between toxin control and taurine treated cells. Each column represents mean ±SD, n =6; ( Pa b 0.05, Pb b 0.05). Panel C: Percent distribution of apoptotic and necrotic cells. Cell distribution analysed using Annexin V binding and PI uptake. The FITC and PI-ﬂuorescence measured using ﬂow cytometer with FL-1 and FL-2 filters, respectively. Results expressed as dot plot representing one of the six independent experiments. Panel D: Western blot analysis of the effects of As, taurine and taurine +As on caspase-3 activation and PARP cleavage in cardiomyocytes. β-actin was used as an internal control. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and As treated groups, “b” indicates the significant difference between the As treated and taurine alone treated groups (TAU), “c” indicates the significant difference between the As treated and taurine pre-treated groups (TAU +As). (Pa b 0.05, Pb b 0.05, Pc b 0.05).

Fig. 5. Panel A: Western blot analysis of the effects of As (5 μM), taurine (25 mM) and taurine +As on IKKα, IKKβ, and NF-κB phosphorylation in cardiomyocytes. β-actin was used as an internal control. Panel B: Western blot analysis of the effects of PS-1145 on As-induced IKKα and IKKβ phosphorylation in cardiomyocytes. Cardiomyocytes were pre-treated with 25 μM of PS1145 (IKK inhibitor) for 15 min and then treated with As (5 μM) for 24 h. Panel C: Western blot analysis of the effects of IKK inhibition on caspase-3 activation and PARP cleavage in cardiomyocytes. Cardiomyocytes were pre-treated with 25 μM of PS1145 for 15 min and then treated with As (5 μM) and taurine (25 mM, added 1 h prior to As treatment) for 24 h. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and As treated groups, “b” indicates the significant difference between the As treated and taurine/PS1145 alone treated groups (TAU/PS1145), “c” indicates the significant difference between the As treated and taurine/PS1145 pre-treated groups (TAU +As)/(PS1145 +As). (Pa b 0.05, Pb b 0.05, Pc b 0.05).

We also studied myocytes apoptosis by determining caspase-3 activation and PARP cleavage. The activation of caspase-3 by proteolytic processing of pro-caspase-3 into 17 and 12 kDa subunits serves as an early marker of apoptosis in various cell types. The 17 kDa cleaved caspase-3 fragment was detected in As exposed cardiomyo- cytes. Taurine treatment, however, prevented the As-induced frag- mentation of caspase-3 although taurine alone did not show any effect on caspase-3 activation (Fig. 4D). In order to confirm caspase-3 activation, we monitored the cleavage of its substrate, the nuclear enzyme PARP. In As treated groups, caspase-3-induced proteolysis of 116 kDa-native PARP molecule was detected as a 89 kDa fragment in immunoblots although no such band was detected in taurine treated myocytes.

Phosphorylation of IKKα, IKKβ, and NF-κB

The NF-κB pathway is a key component of the cellular response to a variety of extracellular stimuli. So, we checked whether As plays any role on this transcription factor and if any alteration comes out, whether taurine could prevent it. We, therefore, examined the phosphorylation of IKKα, IKKβ, and NF-κB in all sets of cardiomyo- cytes used in this study. As evidenced from the results, As exposure caused a significant increase in IKKα, IKKβ, and NFκB phosphorylation compared to the respective control group. Treatment with taurine alone did not cause any change in these parameters, whereas As- induced increase in IKKα, IKKβ, and NF-κB phosphorylation was prevented by taurine treatment (Fig. 5A).

Inhibition of NF-κB pathway

To check whether IKK inhibitor, PS-1145 actually inhibited As- induced IKK phosphorylation, cardiomyocytes were pre-incubated with PS-1145 (25 mM) for 15 min followed by treatment with As and IKK phosphorylation was determined. Treatment with PS-1145 significantly inhibited As-induced IKKα and IKKβ phosphorylation (Figs. 5B, C). In order to study the role of NF-κB pathway in As-induced cardiomyocyte apoptosis, we measured the caspase-3 activation and
PARP cleavage after inhibiting IKK with PS-1145. In the presence of IKK inhibitior, As exposure did not cause cardiomyocytes apoptosis, as no change was observed in caspase-3 activation and PARP cleavage. Taurine treatment alone as well as along with As did not show any change in caspase-3 activity and PARP cleavage (Fig. 5C) in the presence of IKK inhibitior. When the cells were treated with PS-1145 alone, we did not observe any effect on caspase-3 activation as well as PARP cleavage (data not shown).

Activation of MAP kinase by As

MAPKs are the known mediators of cell death due to apoptosis under various pathophysiological conditions (Feuerstein and Young, 2000). Intracellular oxidative-stress stimuli can activate both NF-κB and MAP kinase modules (Schulze-Osthoff et al., 1997). So we examined whether the MAPKs are also involved in As-induced NF-κB activation (Fig. 6). We determined the protein contents of different MAPKs in As intoxicated hearts and cardiomyocytes. Results showed a marked increase in protein content of phosphorylated p38 MAPK (2.5 fold) and p-JNK (2.1 fold) in the heart tissue as well as 4.1 fold and 3 fold up-regulation in cardiomyocytes respectively, without any change in total protein content of these kinases. In contrast a less marked increment of pERK was only noted in both the heart tissue (1.2 fold) and myocytes (1.4 fold). Our results indicate that As effectively activates p38 and JNK MAPKs in addition to NF-κB. From the data shown in Fig. 6, it is evident that the increase in protein contents of phosphorylated MAPKs could be prevented by taurine treatment.

Effects of p38 and JNK MAPK inhibition on As-induced apoptosis

In this study, to investigate the involvement of p38 and JNK MAPKs in taurine modulation of As-induced NF-κB pathway and caspase-3 activation in cardiomyocytes, we pre-treated the cardio- myocytes for 15 min with SP600125, SB203580 and then studied the effects of As and taurine on IKK and NF-κB activation. Results showed that p38 and JNK inhibitors blocked the As-induced NF-κB

Fig. 6. Western blot analysis for protein content of different mitogen-activated protein kinases (MAPKs) in response to As and taurine treatment, both in heart and cardiomyocytes. Cardiomyocytes were treated with As (5 μM) and taurine (25 mM, added 1 h prior to As treatment) for 24 h. Panel A: Phosphorylated JNK and total JNK, panel B: phosphorylated p38 and total p38, panel C: phosphorylated ERK1/2 and total ERK1/2. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and As treated groups, “b” indicates the significant difference between the As treated and taurine pre-treated groups (TAU +As). (Pa b 0.05, Pb b 0.05).

activation with a similar trend to the results of taurine treatment. These results suggest that MAPK activity essentially contributes to NF-κB activation possibly through the mechanisms involving activation of IKKs (Fig. 7A).
Fig. 7B shows the results of the experiments aimed at quantifying the roles of NF-κB, p38 and JNK MAPK in As treated cardiomyocytes and, at the same time, providing information about the mechanism(s) underlying the beneficial effect of taurine in As-induced cardiac pathophysiology. Studies showed that combined effects of PS-1145, SB203580 and SP600125 significantly decreased the oxidative insults in As-induced cardiomyocytes as judged by the ROS production measurements. When taurine treatment was performed in the presence of these inhibitors, there was a further reduction in ROS production, implicating the involvement of p38, JNK MAPKs signaling pathway in protection by taurine treatment.

Taurine mitigated the pro-apoptotic effects of As on Bcl-2 family proteins

Since the process of apoptosis is considered to be regulated by a complex interplay of pro-apoptotic (Bax, Bad) and anti-apoptotic (Bcl- 2, Bcl-xL) mitochondrial membrane proteins as well as the activation of effector caspases, the status of these targets was also investigated in As intoxicated heart and cardiomyocytes in the absence and presence of taurine supplementation. Immunoblotting studies demonstrated that As down-regulated the anti-apoptotic (Bcl-2, Bcl-xL) and up- regulated the pro-apoptotic (Bax, Bad) Bcl-2 family proteins in cardiomyocytes as well as in the heart tissue (Fig. 8A). Taurine might, however, protect the myocardium by preventing the altera- tions of these proteins.
Effect of taurine against As-induced mitochondrion-dependent cell death

Loss of mitochondrial membrane potential (Δψm), release of cytochrome C from mitochondria and subsequent activation of caspase 3 represent a key step in the mitochondrion-dependent apoptotic cell death pathway. To determine whether taurine exerts its anti-apoptotic action against As-induced apoptotic death via above mechanism, we measured the mitochondrial membrane potential (Δψm) and cytosolic cytochrome C level in the heart tissue. The results showed that As administration significantly reduced the mitochon- drial membrane potential (Fig. 8B) and enhanced the concentration of cytosolic cytochrome C (Fig. 8D). Treatment with taurine prior to the As administration could, however, significantly inhibit As-induced alterations of these parameters.

Effects on intracellular [Ca2+] concentration

Calcium ions are secondary messengers in numerous signaling pathways especially in cardiac myocytes. It is evident that alteration in calcium homeostasis by pathophysiological stimuli plays a very important role in the pathogenesis of cell injury and death. So, we measured the intracellular [Ca2+] in cardiomyocyte. As intoxication increased intracellular [Ca2+] in cardiomyocyte whereas taurine pre- treatment could prevent this increase in calcium level (Fig. 8C).

Histological assessment

Fig. 9 illustrates histological assessments of different cardiac segments of experimental animals. As administration caused

Fig. 7. Panel A: Western blot analysis of the effects of SB203580 and SP600125 on NF-κB phosphorylation and phospho IKKα/β in cardiomyocytes treated with As (5 μM) and taurine (25 mM). Cardiomyocytes were pre-treated with 10 μM SB203580 and SP600125 for 15 min then treated with As (5 μM) and taurine (25 mM, added 1 h prior to As treatment) for 24 h. β-actin was used as an internal control. Panel B: The intracellular ROS production was detected by DCF-DA method in cardiomyocytes in the absence (As) and presence (TAU
+As) of taurine as well as in the presence or absence of PS-1145, SB203580 and SP600125. “a” indicates the significant difference between the normal control and toxin treated cells, “b” indicates the significant difference between toxin control and other groups. Each column represents mean±SD, n =6. ( Pa b 0.05, Pb b 0.05).

Fig. 8. Panel A: Western blot analysis of Bcl-2 family proteins in the heart and cardiomyocytes. Cardiomyocytes were treated with As (5 μM) and taurine (25 mM, added 1 h prior to As treatment) for 24 h. Panel B: In vivo mitochondrial membrane potential (Δψm) by ﬂow cytometry analysis (represented both by histogram plot and the dot plot), panel C: intracellular calcium [Ca2+] level in cardiomyocytes, panel D: in vivo cytosolic cytochrome C level. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and As treated groups, “b” indicates the significant difference between the As treated and taurine pre-treated groups. (Pa b 0.05, Pb b 0.05).

disorganization of normal radiating pattern of cell plates in the heart. Treatment with taurine before As intoxication reduced such changes and kept the organ almost similar to that of normal.
Discussion

Increasing evidence suggests that oxidative stress is a major stimulus of cellular apoptosis in many cardiovascular abnormalities. Signaling pathways related to this pathophysiological processes not only initiate and maintain the phenotypic alterations in cardiac structure and function, these pathways also play a critical role in affecting the decision of cardiac cells either to survive or to undergo apoptosis.
Antioxidant defense machineries in our body operate for scaven- ging ROS to prevent the oxidative stress. Among the different antioxidant molecules, SOD and CAT mutually function as important enzymes in the elimination of ROS. Reduction in cardiac SOD activity in arsenic exposed animals may be due to the over production of super oxide radical anions (Yamanaka et al., 1991). In order to remove excess free radicals from the system, GST and GPx utilize GSH during their course of reactions. Decrease in GSH content due to arsenic toxicity simultaneously decreased the activities of GST as well as GPx with a concomitant decrease in the activity of GSH regenerating enzyme, GR. Earlier studies showed that GSH depletion and simultaneous GSSG accumulation occur in the heart during oxidative stress. This pathophysiology, probably caused by increased cellular demand of GSH, leads to impaired cell function because of the disturbed re-dox status in the heart (Hill and Singal, 1997; Forgione et al., 2002; Li et al., 2003). In the present study we found that taurine supplementation completely prevented the As-induced reduction in the activities of the antioxidant enzymes and GSH levels while protecting glutathione re- dox ratio in the cardiac tissue of the experimental animals suggesting a novel protective action of taurine in conditions of increased cardiac oxidative stress.
Among many transcription factors involved in the intracellular
signaling pathways, NF-κB is known to be a rapidly induced stress- responsive one and is exquisitely sensitive to arsenic-induced cellular oxidative status, cell transformation, and apoptosis (Bode and Dong, 2000, 2002). Although involvement of NF-κB in As-induced toxicity has been reported in many other cells and organs, very little is known about its role in As-induced cardiac pathophysiology. Our results clearly demonstrated that As exposure leads to a significant increase in NF-κB phosphorylation in cardiomyocytes. The mechanism of NF- κB activation involves up-regulation of IKK, which leads to the degradation of IκB and in turn translocation of NF-κB to the nucleus and its activation. We just checked the activity of NF-κB using nuclear extracts from the heart tissue (in vivo data not shown) and the subsequent studies with various inhibitors on NF-κB and other signaling molecules were carried out in vitro (using myocytes) as it was difficult to conduct those studies on whole animals by treating them with the inhibitors. Results suggest that As exposure leads to a significant increase in IKK phosphorylation in myocytes. Furthermore, when the cardiomyocytes were pre-exposed to PS-1145, a specific IKK inhibitor, As-induced increase in cardiomyocyte apoptosis was blocked, as no activation of caspase-3 and PARP cleavage was observed, which implies that NF-κB pathway is responsible for As- induced cardiomyocyte apoptosis. PS-1145 itself had no effect on caspase-3 activation and PARP cleavage. The present study also showed that taurine treatment prevented As-induced activation of NF-κB in cardiomyocytes.
It has been reported that NF-κB can serve as a target of MAP kinases (p38, ERK1/2 and JNK) (Cowan and Storey, 2003; Yang et al., 2003), belonging to a class of re-dox-signaling molecules sharing a common Thr–X–Tyr site in the activation loop and Thr–Tyr phosphor- ylation for the initiation of kinase activity. They have been shown to play important roles in the pathogenesis of many cardiac diseases such as hypertrophy and heart failure (Bueno and Molkentin, 2002; Li et al., 2002). The role of these kinases in the signal transduction mechanisms of apoptosis in ischemic and failing hearts has also been reported earlier (Feuerstein and Young, 2000). So, in order to

Fig. 9. Haematoxylin and eosin stained cardiac section of (panel A) normal rats’ heart (×100), (panel B) arsenic treated heart section (×100) and (panel C) taurine pre-treated cardiac section (× 100). Arrows indicate the abnormal ultra structural changes (as evidenced by the disorganization of normal radiating pattern of cell plates) in the heart.

understand the underlying mechanisms of apoptosis in As intoxicated heart (and cardiomyocytes) and the beneficial role of taurine in this cardiac pathophysiology, we investigated the changes in the levels of ERK1/2, p38 and JNK by immunoblot analyses. We observed a marked increase in protein content of phosphorylated p38 MAPK and JNK without any change in total protein content of these kinases in As intoxicated hearts and cardiomyocytes although more prominent changes were observed in cardiomyocytes compared to cardiac tissue. A marginal increment of pERKs was also noted in both the heart tissue and myocytes. In the present study, cardiomyocytes treated with p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125, suppressed the As-induced NF-κB activation. Thus, decreased NF-κB activity by these specific inhibitors strongly suggests the involvement of p38 and JNK MAPK in the As-induced NF-κB activation. Results clearly showed that As associated activation of p38 and JNK MAPK signaling pathways in NF-κB activation via IKK pathway in cardiomyocytes and taurine treatment prevented this As-induced cardiac pathophysiology.
Apoptosis has been shown to play an important role in the pathogenesis of heart failure of various etiologies like chronic pressure overload, ischemia–reperfusion injury, congestive heart failure and so on. The balance between the up and down-regulations of the members of pro-apoptotic (Bax, Bad) and anti-apoptotic (Bcl-xl, Bcl- 2) family proteins determines the fate of the cells either to undergo apoptosis or to survive in pathophysiology. Besides, Bcl-2 family proteins are upstream regulators of mitochondrial membrane poten- tial (Δψm) and release of cytochrome C. We, therefore, checked whether As could depolarize Δψm, enhance the release of cytochrome
C and inﬂuence the equilibrium of Bcl-2 family proteins in the heart and cardiomyocytes, while taurine could prevent these alterations. Immunoblotting studies demonstrated that As down-regulated the anti-apoptotic (Bcl-2, Bcl-xl) and up-regulated the pro-apoptotic (Bax, Bad) Bcl-2 family proteins in cardiomyocytes and in heart tissue (Fig. 8A) whereas taurine treatment effectively repressed these As- evoked pro-apoptotic events. We also observed that As intoxication caused pronounced disruption of the mitochondrial membrane potential (Fig. 8B) and subsequently release of cytochrome C in cardiac tissue. However taurine treatment effectively suppressed the disruption of mitochondrial membrane potential (Δψm) and cyto- chrome C release thereby indicating its role as an anti-apoptotic agent in As-induced cardiac pathophysiology.
Histological examination of the cardiac segment reveals that arsenic treatment caused abnormal ultra structural changes in the cardiac tissue. However, pre-treatment with taurine could prevent this change and could also maintain the ultra structure almost similar to that of normal control.
In conclusion, results of our study strongly suggest that As not only increased phosphorylation of p38 and JNK MAPK, but also led to NF-κB activation via IKK pathway. This activation and phosphorylation of p38 and JNK MAPK pathways are required for As-induced NF-κB activa- tion. This interaction may be a cross-talk site between the NF-κB signaling pathways with MAPK related other signaling pathways. Taurine treatment mitigated the activation of this pathway and blocked the apoptotic signaling cascade. In other words, taurine supplementation represents a promising approach for the protection

of heart tissue in As-induced cardiac injury and cell death. To the best of our knowledge, this study is probably the first to show that taurine could exert its beneficial role in As-induced cardiac pathophysiology. With this benefit and absence of any adverse effect, taurine supplementation in regular diet could provide a new approach for the reduction of the serious cardiac complication due to arsenic poisoning.

Acknowledgment

The authors are grateful to Mr. Prasanta Pal for excellent technical assistance for the study.

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