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Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

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Atorvastatin induced hepatic oxidative stress and apoptotic damage via MAPKs, mitochondria, calpain and caspase12 dependent pathways

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Sankhadeep Pal, Manoranjan Ghosh, Shatadal Ghosh, Sudip Bhattacharyya, Parames C. Sil* Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata, 700054, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 December 2014 Received in revised form 25 May 2015 Accepted 26 May 2015 Available online xxx

Atorvastatin (ATO), a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, is used widely for the treatment of hypercholesterolemia and hypertriglyceridemia. Application of this drug has now been made somehow limited because of ATO associated several acute and chronic side effects. The present study has been carried out to investigate the dose-dependent hepatic tissue toxicity in ATO induced oxidative impairment and cell death in mice. Administration of ATO enhanced ALT, ALP level, increased reactive oxygen species (ROS) production and altered the pro oxidant-antioxidant status of liver by reducing intracellular GSH level, anti-oxidant enzymes activities and increasing intracellular lipid peroxidation. Our experimental evidence suggests that ATO markedly decreased mitochondrial membrane potential, disturbed the Bcl-2 family protein balance, enhanced cytochrome c release in the cytosol, increased the levels of Apaf1, caspase-9, -3, cleaved PARP protein and ultimately led to apoptotic cell death. Besides, ATO distinctly increased the phosphorylation of p38, JNK, and ERK MAPKs, enhanced Caspase12 and calpain level. Histological studies also support the dose-dependent toxic effect of ATO in these organs pathophysiology. These results reveal that ATO induces hepatic tissue toxicity via MAPKs, mitochondria and ER dependent signaling pathway, in which calcium ions and ROS act as the pivotal mediators of the apoptotic signaling. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Atorvastatin ROS Oxidative stress Apoptosis

1. Introduction Atorvastatin (ATO), a highly substituted pyrrole, is an inhibitor of HMG-CoA reductase (HMGRI) that decreases low-density lipoprotein (LDL) and cholesterol in humans. ATO belongs to a group of drugs known as statins. Statins are specific and potent inhibitors of LDL cholesterol because it blocks the action of HMG-CoA reductase enzyme in the liver at the rate-limiting step of cholesterol biosynthesis (Tobert, 2003; Endo, 2004; Jacobson, 2008). Statins also reduce the progression of atherosclerosis by inhibiting monocyte activation, enhancing the metalloprotease synthesis in the vessel wall and the production of pro-inflammatory cytokines interleukin (IL)-6, tumor necrosis factor, TNF-a and IL-1b (Ferro et al., 2000; Solheim et al., 2001). Thus, it is used in the prevention and treatment of cardiovascular disease and the various other

Abbreviations: ATO, atorvastatin; GSH, glutathione; GSSG, glutathione disulfide; GPx, glutathione peroxidase; GST, glutathione S-transferase; GR, glutathione reductase; ROS, reactive oxygen species; SOD, superoxide dismutase. * Corresponding author. Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Calcutta, 700054 West Bengal, India. E-mail addresses: [email protected], [email protected] (P.C. Sil).

pathophysiologies of the circulatory system (Topol, 2004; Wang et al., 2008). Moreover, statins inhibit inflammatory responses in different models of autoimmune disease, such as collagen- and complete Freund's adjuvant (CFA)-induced arthritis and experimental encephalomyelitis (Aktas et al., 2003; Barsante et al., 2005). These drugs also act as an anti-hypernociceptive agent n et al., 2006). (Santodomingo-Garzo Mild-to-moderate elevations in liver transaminases are the most commonly seen side effects of statin treatment in clinical practice (Bays, 2006). Other hepatic side effects of ATO include hepatitis, cholestatic jaundice, cirrhosis, fulminant hepatic necrosis, and liver failure. Another side effect of atorvastatin is myalgia, defined as muscle aches or weakness in the absence of blood creatine kinase elevation (Thompson et al., 2003; Bruckert et al., 2005; Draeger et al., 2006; Buettner et al., 2008). In rare cases, potentially life-threatening statin-induced rhabdomyolysis, a condition characterized by acute muscle damage, may occur. These changes also accompanied by the elevation of creatine kinase levels, myoglobinuria, proteinuria, and possible renal failure (Launay-Vacher et al., 2005). ATO also causes neurological diseases such as dizziness, drowsiness, fatigue, cranial nerve dysfunction, tremor, vertigo,

http://dx.doi.org/10.1016/j.fct.2015.05.016 0278-6915/© 2015 Elsevier Ltd. All rights reserved.

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memory loss, declined cognitive function; paresthesias, peripheral neuropathy and peripheral nerve palsy (Sakaeda et al., 2011). The objective of this study was to investigate the mechanism of ATO-induced hepatic tissue toxicity in dose-dependent manner. The hepatic tissue toxicity caused by ATO was evaluated by measuring the activities of liver-specific biomarkers and other oxidative stressrelated parameters. In this particular study, we investigated mitochondrion-dependent intrinsic pathway, role of MAPKs and ER stress in ATO induced hepatotoxicity. In addition, ROS production in liver tissue homogenates as well as in mitochondria has been measured. Histological studies have been performed to provide further support of toxic behavior of ATO. Besides, DNA fragmentation analysis was also carried out to find out the cell death pathways. The present study, for the first time, would be expected to provide some important information and mechanistic insight in ATO induced hepatic tissue damage in a dose-dependent manner. 2. Materials and methods 2.1. Materials Atorvastatin was purchased from Ranbaxy Laboratories Limited; New Delhi, India. Kits for the measurement of ALT and ALP were purchased from Span diagnostic Ltd., India. 1-chloro-2,4dinitrobenzene (CDNB), dimethyl sulphoxide (DMSO), 5,50 -dithiobis (2-nitrobenzoic acid) [DTNB, (Ellman's reagent)], N-ethylmaleimide (NEM), nicotinamide adenine dinucleotide reduced disodium salt (NADH), nitro blue tetrazolium chloride (NBT), oxidized glutathione (GSSG), phenazine methosulphate (PMS), reduced glutathione (GSH), thiobarbituric acid (TBA), were bought from Sisco research laboratory, India. 2.2. Animals Healthy adult male albino mice of Swiss strain, age 6e8 weeks, weighing between 20 and 25 g, were purchased from CNCI, Kolkata, India. The animals were acclimatized under laboratory conditions for a fortnight before starting experiments and fed standard palate diet every day. All the experiments with animals were carried out according to the guidelines of the institutional animal ethical committee (IAEC), Bose Institute, Kolkata (the permit number is: IAEC/BI/3(I) cert./2010) and full details of the study was approved by both IAEC and CPCSEA (committee for the purpose of control and supervision on experiments on animals), Ministry of Environment & Forests, New Delhi, India (the permit number is: 1796/PO/ERe/S/ 14/CPCSEA).

Group 5: Drug treated [animals received 30 mg/kg/day body weight ATO during the experimental period of 8 weeks, through gavage]. Group 6: Drug treated [animals received 50 mg/kg/day body weight ATO during the experimental period of 8 weeks, through gavage]. The animals were sacrificed on day 3 after the last administration of ATO exposure for morphological and cellular studies. Livers were quickly dissected out, cleaned of accessory tissues in normal saline and processed as need. 2.4. Assessment of markers enzymes The leakage of marker enzymes like ALT, ALP, etc. are considered as the important indicators of hepato-cellular membrane damage and are associated cell viability. Activities of these enzymes were estimated by using the standard kit (Span diagnostic Ltd., India, and Code no for ALP- 75 MB 100e40, for ALT- 76 MB 101e50). After appropriate experimental procedures, blood samples were drawn from the caudal vena cava, collected in test tubes, centrifuged at 1500 g for 10 min to obtain serum and their activities were estimated using the kits as mentioned above. 2.5. Measurement of ROS production and other oxidative stressrelated parameters Liver samples were homogenized (1:4, w/v) in ice-cold 0.1 M phosphate buffer (pH 7.4) containing 2 mM EDTA. The homogenate was centrifuged at 10,000 g for 30 min at 4  C. The supernatant has been collected and further centrifuged at 105,000 g for 60 min. The supernatant was collected and used for the experiments referred to “cytosolic fraction”. The protein content of the cytosolic fraction was measured by the method of Bradford (1976). Lipid peroxidation and protein carbonylation were estimated according to the methods of Esterbauer and Cheeseman (1990) and Uchida and Stadtman (1993) respectively. Intracellular ROS production was estimated by following the method of LeBel and Bondy (1990). Briefly, 100 ml of tissue homogenates were incubated with the assay media (20 mM triseHCl, 130 mM KCl, 5 mM MgCl2, 20 mM NaH2PO4, 30 mM glucose and 5 mM DCFDA) at 37  C for 15 min. The formation of DCF was measured at the excitation wavelength of 488 nm by fluorimeter. Cellular metabolites levels like GSH, GSSG and activities of antioxidant enzymes (SOD, CAT, GST, GR, GPX) in

2.3. Experimental design for dose dependent study in vivo treatment The animals were randomly divided into six groups of six mice each for the dose-dependent study and they were treated as follows. Group 1: Normal control [animals received only water as a vehicle for 8 weeks through gavage, once daily]. Group 2: Drug treated [animals received 1 mg/kg/day body weight ATO during the experimental period of 8 weeks, through gavage]. Group 3: Drug treated [animals received 5 mg/kg/day body weight ATO during the experimental period of 8 weeks, through gavage]. Group 4: Drug treated [animals received 10 mg/kg/day body weight ATO during the experimental period of 8 weeks, through gavage].

Fig. 1. Survival curve for the experimental animals. Total number of animals taken for each group was six.

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Fig. 2. Panel A-B. Dose-dependent effect of ATO on ALP and ALT level in the serum of experimental mice respectively. Cont: normal; ATO-1, 5, 10, 30, 50 indicate ATO exposed group at a dose of 1 mg, 5 mg, 10 mg, 30 mg, and 50 mg/kg body weight ATO during the experimental period of 8 weeks, through daily gavage Each bar represents mean ± SEM, n ¼ 6. “a” indicates the significant difference between the normal control and ATO exposed groups, “b” indicates the significant difference between ATO-1 group and other ATO exposed groups, “c” indicates the significant difference between the ATO-5 and other ATO exposed groups, “d” indicates the significant difference between the ATO-10 and other ATO exposed groups, (pa < 0.05).

Fig. 3. Panel A Dose-dependent effect of ATO on the ROS production in hepatic tissue of the experimental mice. Cont: normal; ATO-1, 5, 10, 30, 50 indicate ATO exposed group at a dose of 1 mg, 5 mg, 10 mg, 30 mg, and 50 mg/kg body weight ATO during the experimental period of 8 weeks, through daily gavage. Panel B. Each column represents mean ± SEM, n ¼ 6. “a” indicates the significant difference between the normal control and ATO exposed groups, “b” indicates the significant difference between ATO-1 group and other ATO exposed groups, “c” indicates the significant difference between the ATO-5 and other ATO exposed groups, (pa < 0.05).

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the hepatic tissue have been determined following the method as described by Das et al. (2009a); Das et al. (2009b).

laser excitation at 485and 530 nm band pass filter using DCFDA as a probe (Eruslanov et al., 2010). FITC-A counts represent Rhodamine counts.

2.6. Measurement of mitochondrial ROS in experimental hepatic tissue

2.7. Histological studies

For the determination of mitochondrial ROS, mitochondria were isolated from all sets of experimental hepatic tissue. ROS production was measured using FACScan flow cytometer with an argon

Liver tissue from the normal and experimental mice was fixed in 10% buffered formalin and has been processed for paraffin sectioning. Sections of about five mm thickness were stained with

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Fig. 4. Panel A-D. Dose-dependent effect of ATO on GSH, GSSG level, lipid peroxidation and protein carbonylation in the liver tissue of mice respectively. Cont: Normal ATO-1, 5, 10, 30, 50 indicate ATO exposed group at a dose of 1 mg, 5 mg, 10 mg, 30 mg, and 50 mg/kg body weight ATO during the experimental period of 8 weeks, through daily gavage. Each column represents mean ± SEM, n ¼ 6. “a” indicates the significant difference between the normal control and ATO exposed groups “b” indicates the significant difference between ATO-1 group and other ATO exposed groups, “c” indicates the significant difference between the ATO-5 and other ATO exposed groups, “d” indicates the significant difference between the ATO-10 and other ATO exposed groups, “e” indicates the significant difference between the ATO-30 and ATO-50 exposed group, (pa < 0.05).

haematoxylin and eosin. Histological slide of the experimental liver was observed under light microscope. 2.8. TUNEL staining Paraffin-embedded hepatic tissue sections (5 mm) were warmed 30 min (64  C), deparaffinized and rehydrated. Terminal transferase-mediated dUTP nick end-labeling of nuclei was performed by using APO-BrdU TUNEL Assay kit (A-23210; Molecular Probes, Eugene, OR) following the manufacturer's protocol. 2.9. Determination of mitochondrial membrane potential (Djm) Analytic flow cytometric measurements for the membrane potential (Djm) of isolated mitochondria were performed using

an FACScan flow cytometer with an argon laser excitation at 488 nm and 525 nm band pass filter as described by Chandrasekaran et al. (2009). Briefly, liver tissue was minced, homogenized in ice-cold isolation buffer containing 10 mL TrisMOPS [0.1 M; pH7.4], 20 mL sucrose [1 M], and 1 mL EGTA-Tris buffer [0.1 M; pH 7.4]. The tissue homogenates were centrifuged at 600 g at 4  C for 10 min; supernatant was collected and again centrifuged at 7000 g at 4  C for 10 min. The pellet was collected, and washed once with isolation buffer. The centrifugation steps were repeated twice. After discarding the supernatant, pellet was suspended in 1 mL of isolation buffer (Pal et al., 2015). Mitochondrial membrane potential (Djm) was measured using the fluorescent cationic probe rhodamine 123 (Mingatto et al., 2003). FITC-A counts represent Rhodamine counts. The data was analyzed by Cell Quest software.

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Fig. 5. Panel A-E. Dose-dependent effect of ATO on the activities of the antioxidant enzymes like SOD, CAT, GST, GR and GPX in the liver tissue of mice. Cont: Normal ATO-1, 5, 10, 30, 50 indicate ATO exposed group at a dose of 1 mg, 5 mg, 10 mg, 30 mg, and 50 mg/kg body weight ATO during the experimental period of 8 weeks, through daily gavage. Each column represents mean ± SEM, n ¼ 6. “a” indicates the significant difference between the normal control and ATO exposed groups, “b” indicates the significant difference between ATO-1 group and other ATO exposed groups, “c” indicates the significant difference between the ATO-5 and other ATO exposed groups, “d” indicates the significant difference between the ATO-10 and other ATO exposed groups, “e” indicates the significant difference between the ATO-30 and ATO-50 exposed group, (pa < 0.05).

2.10. Immunoblotting For immunoblotting, cytosolic samples containing 50 mg proteins was subjected to 10-% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 2 h in blocking buffer containing 5% non-fat dry milk and then incubated separately with anti-Bcl2, anti-Bad, anti-apaf1, anticleaved caspase 9, anti-cleaved PARP, anti-caspase 3, anticaspase12, anti-calpain, anti-cytrochrome c, anti-JNK, anti-ERK

and anti-p38 (1:1000 dilution each) at 4  C overnight. Anti b actin (1:3000) was used as a loading control. The membranes were washed in TBST (50 mmol/L TriseHCl, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20) for 30 min and incubated with appropriate ALP conjugated secondary antibody (1:2000 dilution) for 2 h at room temperature and developed by the NBT and BCIP solution with the help of substrate buffer (Ghosh et al., 2010; Manna et al., 2012; Das et al., 2012). All antibodies were purchased from Cell-Signaling Technology (CST), USA. Normalization of the band intensities of the

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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 < 0.05 level. 3. Results 3.1. Survival curve of animals at the administered doses of the ATO A survival curve of animals at the different doses of ATO (1 mg/ kg body wt/day to 50 mg/kg body wt/day) has been shown in the Fig. 1. 3.2. Effect on serum marker enzymes Fig. 6. Effect of ATO on the mitochondrial ROS of the liver tissue of normal and experimental mice. Cont: normal; ATO-1, 5, 10, 30, 50 indicate ATO exposed group at a dose of 1 mg, 5 mg, 10 mg, 30 mg, and 50 mg/kg body weight ATO during the experimental period of 8 weeks, through daily gavage. Mitochondrial ROS was measured using DCF-DA as a probe by flow cytometer with FL-1 filter. FITC-A counts represent Rhodamine counts. Results represent one of the six independent experiments.

Fig. 2A and B illustrate the activities of ALP and ALT respectively in normal and experimental groups of mice. It has been observed that ATO administration increased the activities of ALP and ALT dose-dependently compared to the normal. There was also a significant change of ALP and ALT activities between the different doses of atorvastatin.

western blot components were carried out by densitometric analysis.

3.3. Effects on intracellular ROS production

2.11. Assessment of cytochrome C in the cytosolic as well as in the mitochondrial fraction

Excess ROS production plays a vital role in ATO-induced organ pathophysiology. Fig. 3 showed an enhanced intracellular dosedependent ROS production in the hepatic tissue of ATO administrated mice compared to normal controls.

Depletion in mitochondrial membrane potential causes release of cytochrome C and initiates mitochondrial dependent cell death pathway. The expression of cytochrome C both in the cytosolic and mitochondrial fraction were investigated by immunoblotting using anti cytochrome C antibody (1:1000 dilution) purchased from CellSignaling Technology (CST), USA. 2.12. Statistical analysis All the values are expressed as mean ± SEM (n ¼ 6). Significant differences between the groups were determined with SPSS 10.0 software (SPSS Inc., Chicago, IL, USA) for Windows using one-way

3.4. Effect on glutathione levels Glutathione, an important antioxidant, prevents damage to important cellular components caused by free radicals and peroxides (ROS). GSH level was significantly decreased dose-dependently due to ATO exposure along with the increased level of GSSG compared with normal group. But there was no significant change of GSH between two consecutive doses of ATO administration except ATO-10 and ATO-30 doses. Whereas in case of GSSG there also a sharp significant difference between two consecutive doses of ATO administration. (Fig. 4A & B).

Fig. 7. Dose-dependent effect of ATO on histology of the liver tissue of mice. Cont: normal; ATO-1, 5, 10, 30, 50 indicate ATO exposed group at a dose of 1 mg, 5 mg, 10 mg, 30 mg, and 50 mg/kg body weight ATO during the experimental period of 8 weeks, through daily gavage. The magnification of the picture is 40.

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Fig. 8. TUNEL assay of the liver tissue of mice. Normal: liver section from normal animals; ATO-1, 5, 10, 30, 50 indicate ATO exposed group at a dose of 1 mg, 5 mg, 10 mg, 30 mg, and 50 mg/kg body weight ATO during the experimental period of 8 weeks, through daily gavage. The magnification of the picture is 40.

Fig. 9. Effect of ATO on the mitochondrial membrane potential (Djm) of the liver tissue of normal and experimental mice. Mitochondrial membrane potential (Djm) was measured using a fluorescent cationic probe rhodamine-123 by flow cytometer with FL-1 filter. FITC-A counts represent Rhodamine counts. Results represent one of the six independent experiments.

3.5. Effect on lipid peroxidation and protein carbonyl content Lipid peroxidation and protein carbonylation are considered to be the two important parameters of oxidative stress. Lipid peroxidation refers to the oxidative degradation of lipids in cell membranes which cause cell damage. Fig. 4C and D represent the changes of lipid peroxidation and protein carbonylation in the normal and experimental hepatic tissue respectively. ATO intoxication increased the levels of MDA (lipid peroxidation end product) dose-dependently compared with normal. But there was no

significant change of MDA between two consecutive doses of ATO administration except ATO-5 and ATO-10 doses. The extent of protein carbonylation has also been significantly increased dosedependently due to ATO exposure compared to normal. 3.6. Effect on antioxidant enzymes Equilibrium between antioxidant enzymes plays an important role in the elimination of free radicals and the simultaneous decrease in oxidative stress. The activities of antioxidant enzymes

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Fig. 10. Effect of ATO on Bad, Bcl-2, cytochrome c, caspase 9, Apaf1, caspases 3 and cleaved PARP by western blots and densitometric analysis of liver tissue in dose dependent manner. b actin was used as an internal control. Data represent the mean ± SEM of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and ATO-exposed liver tissue; (pa < 0.05).

were significantly reduced in ATO exposed hepatic tissue dose dependently. Significant changes of antioxidant enzymes activity between different doses of ATO have also been shown in Fig. 5AeE.

organelle to the cytosol and nucleus. ATO increased the level of mitochondrial ROS in dose-dependent manner. Here it is worth mentioning that the FITC-A counts represent Rhodamine counts (Fig. 6).

3.7. Effects on mitochondrial ROS production 3.8. Histological assessment The ROS production by mammalian mitochondria is important because it causes oxidative damage in various pathological conditions and contributes to retrograde redox signaling from the

Hepatic lobular architecture was clear and intact without any abnormalities in the liver section of the normal group. The most

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Fig. 11. Panel A-B. Effect of ATO on p38, JNK, ERK, calpain and caspase 12 by western blots and densitometric analysis of liver tissue in dose dependent manner. b actin was used as an internal control. Data represent the mean ± SEM of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and ATO-exposed liver tissue; (pa < 0.05).

easily recognizable feature of the apoptotic cells is the condensed nuclear chromatin that either appears as a darkly staining single nucleus or a fragmented nucleus. The apoptotic cells may also be seen within vacuoles (with an apparently empty space surrounding the apoptotic bodies within the vacuole). In our study, the histological picture showed that liver tissue was damaged in ATO administrated mice in a dose-dependent manner (Fig. 7).

3.9. ATO-induced cell death To investigate the nature of ATO-induced cell death, TUNEL assay was performed. Administration of ATO caused apoptotic cell death as indicated with increasing number of TUNEL positive nucleus in TUNEL assay (Fig. 8).

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Fig. 12. Schematic diagram of the ATO induced hepatic tissue toxicity in dose-dependent manner.

Disruption of mitochondrial membrane, reduction ofmitochondrial membrane potential (Djm) and subsequent release of cytochrome c in the cytosol is a well-known biomarker of oxidative stress-induced cell death via mitochondria-dependent pathway (Fig. 9). Apoptotic nature of ATO-induced hepatic tissue toxicity and cell death were studied in terms of the disruption of mitochondrial membrane potential (FITC-A counts represent Rhodamine counts), caspase-3 activation and PARP cleavage. In addition, it is also known that Bcl-2 family proteins are upstream regulator of mitochondrial events and play critical roles in mitochondrial-mediated cell death. The Bax/Bcl-2 ratio drew particular interest in this type of study because of their significance in the mitochondrial mediated cellular functions. ATO caused a dose-dependent decrease in Bcl-2 expression and increase in that of Bax leading to the increased ratio of Bax over Bcl2 and subsequent cell death via mitochondrial dependent pathway (Fig. 10). In addition, ATO exposure increased cytochrome C content in the cytosol (with a concomitant decrease of the same in the mitochondria), caspase 3 activity and PARP cleavage in the liver.

3.10. Activation of MAP kinase by ATO MAPKs are the known mediators of cell death due to apoptosis under various pathophysiological conditions. So we examined whether the MAPKs are also involved in ATO-induced cell death. We determined the protein contents of different MAPKs in ATO exposed liver and found that protein expression of JNK, ERK and p38 were increased in a dose-dependent manner compared with normal (Fig. 11A).

3.11. Activation of Ca-mediated pathway by ATO Calpain, a cytosolic calcium-activated neutral cysteine endopeptidase, may play an important role in hepatic pathophysiology. Caspase-12 is specifically activated in cells subjected to ER stress. It has been observed that ATO exposure increased the level of calpain and Caspase-12 dose-dependently in the hepatic tissue compared to the normal (Fig. 11B).

Please cite this article in press as: Pal, S., et al., Atorvastatin induced hepatic oxidative stress and apoptotic damage via MAPKs, mitochondria, calpain and caspase12 dependent pathways, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.016

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4. Discussion Present study demonstrated the toxic behavior ATO in the hepatic tissue of the experimental animals. We observed that ATO administration caused oxidative stress and hepatic tissue damage dose-dependently. ROS are known to play a major role in ATO induced oxidative stress (Qi et al., 2013; Bouitbir et al., 2012). It also contributes to the mitochondrial damage, oxidative injury of enzymes and lipid membranes, and causes DNA damage in living cells (Das et al., 2008). Besides, it is responsible for nuclear chromatin condensation and apoptosis (Eruslanov et al., 2010; Miller et al., 2004; Bras et al., 2005; Pelicano et al., 2003; Balaban et al., 2005). In the present study, hepatic tissue damage was detected by measuring the activities of the membrane leakage enzymes, ALT and ALP. We observed that ATO exposure increased the activities of membrane leakage enzymes in a dose-dependent manner. Lipid peroxidation and protein carbonylation are the additional indicators of hepatic oxidative injury. Lipid peroxidation is responsible for the damage of cellular membranes (Sinha et al., 2007). Antioxidant molecules in our body operate for scavenging ROS and preventing oxidative stress. Thiol based intracellular antioxidant GSH scavenges free radicals, ROS and other oxidant species (Dolphin, 1989), detoxifies various xenobiotics and consequently converts itself to its oxidized form, GSSG. Thus, decreased GSH level associated with increased GSSG level results in the depletion of GSH/GSSG ratio (Ghosh et al., 2014). In the present study, results suggest that the oxidative stress plays an important role in ATO-induced liver tissue damage as evidenced from dosedependent decrease in antioxidant enzyme activities, GSH level and GSH/GSSG ratio. ATO-induced liver injury also supported by the histological assessments, TUNEL assay, nuclear chromatin condensation and cell apoptosis (Bras et al., 2005; Pelicano et al., 2003). 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 ATO toxicity simultaneously decreased the activities of GST as well as GPx with a concomitant decrease in the activity of GSH regenerating enzyme, GR. GSH with its eSH group functions as a catalyst in disulfide exchange reaction. It functions by scavenging free radicals as well as detoxifying various xenobiotics and consequently converts itself to its oxidized form, glutathione disulfide (GSSG) (Jones, 2002). In the present study following ATO intoxication, this functions were greatly impaired as indicated by a significant decrease in the levels of total thiols and GSH along with increase in the level of its metabolite GSSG. Apoptosis, a self-destructive mechanism, is known to be a delicately controlled programmed cell death pathway. Earlier investigations suggest that the disruption of mitochondrial membrane and reduction of mitochondrial membrane potential (Djm) is well-known biomarkers of oxidative stress-induced apoptotic cell death (Keeble and Gilmore, 2007). Some pro-apoptotic and antiapoptotic proteins in the Bcl-2 family and caspases are considered as executors of the apoptotic pathways (Hengartner, 2000; Roy et al., 2009). We observed that ATO significantly up-regulated pro-apoptotic Bax and down-regulated anti-apoptotic Bcl-2 proteins and increased cytochrome c release in the cytosol dosedependently. Reduction of the mitochondrial membrane potential, activation of caspases (caspase 3 and caspase 9) and subsequent modulation of PARP cleavage from its full-length form (116 kDa) to the cleaved form (84 kDa) in response to ATO in hepatic tissue have also been detected dose-dependently in the same exposure. MAPKs, a family of serine/threonine kinases, are activated by dual phosphorylation on serine and threonine residues. They play important roles in intracellular transcriptional and post-

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translational responses. ROS production causes sustained MAPKs activation which has been studied intensively as a possible mechanism for cell proliferation, differentiation, survival and apoptosis (Pearson et al., 2001; Festa et al., 2011; Das et al., 2012; Ghosh et al., 2015). Our present data demonstrated that ROS may serve as a link between mitochondrial dysfunction and MAPKs. We observed that ATO exposure up-regulated the expression of phospho-JNK, p38 and ERK 1/2 dose-dependently. The activation of MAPKs was suggested to be a critical component in the ATO-induced apoptotic process. Calpains and caspases 12 are two members of cysteine protease families that play important roles in regulating pathological cell death. Calpain may be responsible for cleaving procaspase-12, a caspase localized in ER to generate active caspase-12. Furthermore, caspase-12-deficient cells are resistant to inducers of ER stress, suggesting that caspase-12 is responsible in ER stress-induced apoptosis (Nakagawa and Yuan, 2000; Nakagawa et al., 2000; Rashid and Sil, 2015a; Rashid and Sil, 2015b). Caspase-12 translocates from ER to cytosol and then directly cleaves pro-caspase-9, which, in turn, activates caspase 3 that leads to cell death (Morishima et al., 2002). In our present study, ATO exposure dosedependently increased the level of both calpain and Caspase-12 proteins in the hepatic tissue. Finally, results of our present study suggest that ATO caused liver tissue dysfunction as well as hepatic cell death via oxidative stress-induced cell signaling pathways including the signals from the mitochondria, caspases and calpain in a dose-dependent manner. ATO administration caused oxidative stress and increased ROS in hepatic tissue damage. This ROS are known to play a major role in ATO induced oxidative stress. ROS significantly upregulated pro-apoptotic Bax and down-regulated anti-apoptotic Bcl-2 proteins and increased cytochrome C release in the cytosol as well as mitochondria. Reduction of the mitochondrial membrane potential, activation of caspases (caspase 3 and caspase 9) and subsequent modulation of PARP cleavage in response to ATO in hepatic tissue have also been detected dose-dependently in the same exposure. ROS may serve as a link between mitochondrial dysfunction and MAPKs. ROS are responsible for up-regulating the expression of phospho-JNK, p38, ERK 1/2 MAPKs proteins and calpain and caspase 12 protein expression (Fig. 12). In conclusion, it can be said that the observed changes are associated with the ATOinduced liver damage and identify the novel effects as shown in this research. Disclosure statement The authors have declared that no conflict of interest exists. Uncited reference Slater, 1984. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.05.016. References n, T., Cunha, T.,M., Verri Jr., W.A., Vale rio, D., Parada, C.,A., Santodomingo-Garzo Poole, S., Ferreira, S.,H., Cunha, F.,Q., 2006. Atorvastatin inhibits inflammatory hypernociception. Br. J. Pharmacol 149, 14e22. Aktas, O., Waiczies, S., Smorodchenko, A., Dorr, J., Seeger, B., Prozorovski, T., 2003. Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1 cells through atorvastatin. J. Exp. Med. 197, 725e733.

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