Mechanism of Rhein-Induced Apoptosis in Rat Primary Hepatocytes

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Mechanism of Rhein induced apoptosis in rat primary hepatocytes: beneficial effect of Cyclosporine A Gati Krushna Panigrahi, Ashish Yadav, Ashish Srivastav, Anurag Tripathi, Sheikh Raisuddin, and Mukul Das Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00063 • Publication Date (Web): 27 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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Chemical Research in Toxicology Rhein induced apoptosis in rat hepatocytes

Mechanism of Rhein induced apoptosis in rat primary hepatocytes: beneficial effect of Cyclosporine A

Gati Krushna Panigrahi1, 2, Ashish Yadav1, Ashish Srivastava1, Anurag Tripathi1, S. Raisuddin2 and Mukul Das1*

1. Food, Drug and Chemical Toxicology Division, Council of Scientific and Industrial Research Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, Uttar Pradesh, India

2. Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi, India

*To whom all correspondence should be addressed E-mail: [email protected] Tel: 091-522-2613786 Fax: 091-522-26282

1 ACS Paragon Plus Environment

Chemical Research in Toxicology

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Rhein induced apoptosis in rat hepatocytes

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Abstract: Association between children death and consumption of Cassia occidentalis (CO) seeds has been established in the past by observational and toxicity studies. Recently, the chemical evidence of this association has been recognized by identification of toxic anthraquinones (aloe-emodin, chrysophanol, emodin, physcion and rhein) in the CO seeds (Panigrahi et al., 2015 Chem Res Toxicol, Accepted). Among all these AQs, earlier studies have shown rhein as the most cytotoxic AQ in hepatocytes. Therefore, the present study was designed to investigate the effect of rhein on rat primary hepatocytes. Results indicated that rhein (50µM) causes apoptosis in rat primary hepatocytes by generation of reactive oxygen species (ROS), increase in intracellular Ca++ , decrease in the mitochondrial membrane potential and depletion of intracellular glutathione (GSH) content. At molecular level rhein induced DNA damage results in over expression of γH2AX protein (2.5 fold) thereby causing enhancement of p53 (4.5 fold) and p21 (3.6 fold) leading to intrinsic pathway mediated apoptosis involving Bax, bcl2, cytochrome c, caspases 3, 9 and poly ADP ribose polymerase (PARP). Further, it was observed that rhein induced ROS generation was also involved in the modulation of signaling molecules like MAPK kinases including ERK1/2, p38 and JNK; and mitochondrial energetics proteins including complex II-V, p-AMPK and Sirt-1. Among the different protective agents, it was shown that cyclosporine A (CSA) at 100 nM concentrations was the most effective agent to prevent apoptosis in hepatocytes by interfering in various metabolic pathways which were found to be altered by rhein. Key words: Rhein, Apoptosis, Primary hepatocyte, Cyclosporine A, Mitochondrial energetics



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1. Introduction Accidental poisoning of Cassia occidentalis (CO) seeds is known to be the causative agent for hepatomyoencephalopathy (HME) in several parts of India.

1-3

Recently, the toxic

anthraquinones (AQs) (aloe-emodin, chrysophanol, emodin, physcion and rhein) were identified in the CO seeds. 4 Subsequently, these AQs were detected in the serum of accidental poisoning study cases as well as in the experimental rats exposed to CO seeds, suggesting that the toxicity may be due to these AQs. 4 The cytotoxicity of the above five AQs revealed that rhein is most toxic compound in CO seeds. Additionally, the highest concentration of rhein in serum as compared to other AQs suggested rhein to be the main etiological agent in HME. 4 Free AQs (AQ aglycones); including rhein undergo a different metabolic fate following oral administration. 5 Lipophilicity of AQ aglycones facilitate absorption through small intestine and thereby transported to liver. Therefore, the ability of free AQs to be absorbed through the small intestine and enter into systemic circulation appears to determine the toxic potential of these compounds; in contrast AQ glycosides bypass the enterohepatic recirculation. 5 In general, quinones exhibit the toxicity either by redox cycling or nucleophilic addition reaction with biomolecules. 6 Since, rhein has a quinone moiety in its structure, it is likely that the mechanism of toxicity may be due to the above two mechanisms.

7-9

Regarding the first

mechanism, recently we have demonstrated the high protein binding affinity of rhein to mediate toxicity in rat primary hepatocytes and HepG2 cells.

10

Earlier, rhein has been reported to

interfere with a number of mitochondrial functions including inhibition of oxidation of NAD or FAD linked substrates and oxidative phosphorylation. 11-13 In addition, anti-neoplastic activity of rhein has also been reported. 14-16 Furthermore, rhein has been found to alter the cytoskeleton and thereby affect the plasma membrane and intracellular membranes.

17

So far, most of the studies


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on rhein have been performed on either sub cellular fractions or pure enzyme systems. Although, few of the studies have been carried out in rat primary hepatocytes,

8-9

11-12, 18

none of these

studies have addressed the underlying molecular mechanisms of the rhein toxicity. For the prevention of HME, the mode of action or the mechanism of toxicity of these AQs (e.g. rhein) of CO seeds needs to be known in detail. Hence, the present investigation was aimed to study the molecular mechanisms involved in rhein induced hepatocyte toxicity. In this study, some of the key physiological processes/metabolic pathways including apoptosis, MAPK pathways and mitochondrial energetics were targeted, as the involvement of these pathways were observed during the transcriptional analysis of hepatic tissue of rats following CO seeds exposure in our earlier study. 19

Furthermore, several protective agents were used in this study to investigate the attenuation of

rhein induced toxicity, so that they can be exploited for remedial measures against CO poisoning. 2. Material and Methods 2.1. Chemicals and reagents Rhein, collagen type IV, collagenase, ethylene glycol tetra acetic acid (EGTA), hydroxyethyl piperazineethanesulfonic acid (HEPES), RPMI-1640, sodium pyruvate, L-glutamine, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red, 2,7dichloro fluorescein diacetate (DCFH-DA), hoechst 33258, propidium iodide (PI), 5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1 dye), fura-2 pentakis (acetoxymethyl) ester (Fura 2AM), reduced glutathione (GSH), dithio-bis (2-nitrobenzoic acid) (DTNB), 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), N-acetyl cysteine (NAC), L-carnitine, α-tocopherol, diphenyleneiodonium sulfate

(DPI),

cyclosporine

A

(CSA),

ethylene

glycol

tetraacetic

acid

(EGTA),



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phenylmethylsulfonyl fluoride (PMSF), sodiumdodecyl sulphate (SDS), acrylamide, bisacrylamide, tris-HCl, bromophenol blue, Tween20, β-mercaptoethanol and protease inhibitor cocktail were purchased from Sigma Aldrich Chemical Co. (St Louis, MO). Foetal bovine serum (FBS) and antibiotic-antimycotic solution were procured from Gibco Life Technologies (Grand Island, NY). Annexin V-FITC reagent and PVDF membrane were purchased from BD Bioscience (San Diego, CA) and Millipore (Billerica, MA) respectively. All the other chemicals and solvents used were of the highest purity available from commercial sources. 2.2. Rat primary hepatocytes culture Male wistar rats (150-200 g) used for primary hepatocyte isolation were procured from the breeding colony of CSIR-Indian Institute of Toxicology Research, Lucknow, India. This study was approved by Institutional Animal Ethics Committee of CSIR-IITR with reference number ITRC/IAEC/16/2009. Hepatocytes were isolated from the liver of overnight fasted male wistar rat (150-200g) according to the two step collagenase perfusion as described earlier with minor modifications. 20 In brief, liver perfusion was done initially by injecting calcium and magnesiumfree HEPES buffer containing EGTA (0.4 g/L) at 80-100 mL/min for 10-15 min. The liver was then perfused with 0.5g/L collagenase solution at 50-70 mL/min for 10 min. After perfusion, the liver capsule was incised, the fibrous tissue was discarded and cell suspension was harvested. The released cells were filtered through 100 µm cell strainer and washed three times with incomplete media (RPMI-1640) with concomitant centrifugations (50 g x 3min). The hepatocyte viability greater than 95% as determined by trypan blue exclusion was used for culture. Hepatocytes were maintained in RPMI-1640 media supplemented with 10% FBS and 1% of penicillin-streptomycin solution, 1mM sodium pyruvate, 2mM glutamine under an atmosphere of 5% CO2; 95% air in an incubator (Eppendorf, Hamburg, Germany) with controlled humidity at


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370C. After 6 hrs, the media was changed to remove the unattached cells and the adhered cells along with media were exposed to different concentrations of rhein. 2.3. Cell viability assays following rhein exposure in rat primary hepatocytes 2.3.1. MTT assay MTT dye uptake in primary hepatocytes in the presence and absence of rhein was determined by the method described earlier.

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In brief, a desired number of cells (5x104cells/well) in 200µl

media were dispensed in a 98 well plate (pre treated with 0.01% rat tail collagen) on day 1, followed by exposure to different concentrations of rhein on day 2. After 24 hr of treatment, 20 µl MTT (5 mg/ml PBS) was added to each well. The plates were further incubated for 2 hr. After completion of the incubation time, the media was decanted and the formazone formed was dissolved in 100 µl of DMSO. Finally, the absorbance was taken at 550 and 660 nm after 10 min on a micro plate reader (Synergy HT; BIO-TEK International, Winooski, VT). The percentage viability was calculated by comparing the absorbance (δA) of control and treated cells. 2.3.2. Neutral red uptake assay The neutral red uptake (NRU) assay was carried out according to the method described earlier to determine the accumulation of neutral red dye in the lysosome of viable cells.

22

In brief,

5x104cells/well were seeded in a 98 well plate (pre treated with 0.01% rat tail collagen) on day 1 followed by exposure with different concentrations of rhein on the next day. After 24hr of treatment, the media was removed from the wells and washed with PBS, followed by addition of 100 µl NR medium [media and NR stock solution (4mg/ml PBS) in a ratio of 100:1, v/v]. The plates were then incubated at 370c for 2 hr. After incubation, the NR medium was removed followed by washing with PBS. Finally, 100 µl of NR destaining solution (50% ethanol, 1% acetic acid and 49% distilled water) was added to each well with proper mixing. The



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accumulation of NR dye in the lysosome was detected by reading the absorbance at 540 and 660 nm after 5 min in a micro plate reader (Synergy HT; BIO-TEK International, Winooski, VT). The percentage viability was calculated by comparing the absorbance (δA) of control and treated cells. 2.4. Reactive oxygen species measurement The fluorescent probe DCFH-DA was used to measure ROS generation in hepatocytes by the method described earlier.

23

After entering the cell, the diacetate bond of the DCFH-DA is

cleaved by esterases leaving DCFH, which is oxidized to DCF by the oxidants. The fluorescence of DCF is taken as an indicator of ROS production in the cell. In brief, both control and treated hepatocytes (in 96 well black bottom plates pre treated with 0.01% rat tail collagen)) were incubated with DCFH-DA (5 µg/ml) for 30 min at 370C. Subsequently, the cells were washed with PBS and fluorescence was read in a microplate reader (FLUOstar Omega-BMG, Cary, NC) at an excitation and emission of 485 and 520 nm, respectively. The values were expressed as fluorescence/104cells. 2.5. Assessment of mitochondrial membrane potential (MMP) MMP in hepatocytes was assessed following the method described earlier,

24

using JC-1 as the

fluorescent probe. At low membrane potentials (180mV) this exists as a red-fluorescing dimer (J-aggregate). At 488 nm excitation, the shift in red to green fluorescence emission measures the change in MMP. In brief, both control and treated hepatocytes were incubated with JC-1 (2.5 µg/ml) for 30 min at 370C in CO2 incubator. Cells were then washed twice with PBS and the change in MMP was assessed by comparing the ratio of red and green fluorescence at 590 nm (red)/527 nm (green) using a microplate reader (FLUOstar Omega-BMG, Cary, NC). Finally, the


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percentage decreases of MMP in treated groups were calculated by comparing the fluorescence of control. 2.6. Measurement of glutathione (GSH) GSH content in rat primary hepatocytes in the presence and absence of different concentration of rhein was measured according to the earlier described method with minor modifications.

25

In

brief, 50 µl of cell lysate was diluted with 50 µl of 100 mM phosphate buffer containing 1 mm EDTA. Subsequently, 100 µl of reaction buffer (295 mM DTNB in 10 ml of phosphate buffer) was added and read at 412 nm. Different concentration of GSH (1-100µM) was employed to obtain a standard curve. Finally, reduced GSH was expressed as µM GSH/104 cells. 2.7. Intracellular calcium measurement Intracellular calcium in the control and treated hepatocytes was measured using Fura-2AM dye, which specifically binds to free calcium, following the method described earlier with some modifications.

26

Briefly, 1×104cells were suspended in PBS and Fura- 2AM (4 µM) was added

30 min prior to the addition of rhein and incubated at 37◦C in dark. After addition of 50 µM rhein, fluorescence was monitored for every 15 min up to 120 min in a microplate reader (FLUOstar Omega-BMG, Cary, NC). The values were expressed as relative Fura- 2AM fluorescence units. 2.8. Fluorographic monitoring of apoptosis Apoptosis related nuclear condensation in rhein treated hepatocytes was observed using bisbenzimide (hoechst 33258) fluorochrome following the earlier described method.

27

In brief,

rat primary hepatocytes (control and treated with rhein for 24 hrs) were fixed in 4% paraformaldehyde for 10 min in a 6 well plate (1×106 cells/ well). Subsequently, cells were stained with hoechst 33258 (2 µg/ml PBS) for 15 min followed by washing and mounting in a



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solution of 90% glycerol (pH 5.5). Apoptosis associated nuclear alterations were examined at a wavelength of Ex/Em-350/460 nm using Nikon Eclipse Ti-S inverted fluorescent microscope equipped with Nikon Digital Sight Ds-Ri1 CCD camera and NIS Elements BR imaging software (Nikon, Kanagawa, Japan). For quantitative purpose, the number of hoechst 33258 positive cells were counted in each image and expressed relative to the total number of nuclei present. 2.9. Optimization of doses of protective agents for rhein induced cytotoxicity in hepatocytes In the present study, anti apoptotic or protective agents including NAC (precursor of GSH), αtocopherol (protect against oxidative damage), L-carnitine (mitochondrial fatty acid transporter), BAPTA-AM (intracellular calcium chelater), DPI (inhibitor of cytochrome p450 reductase) and CSA (Inhibitor of release of cytochrome c from mitochondria by blocking the mitochondrial permeability transition pore

28

) were used to investigate the modulation of rhein induced

apoptosis in rat primary hepatocytes. Prior to their use, the optimum concentration showing maximum efficacy was evaluated through cell viability assays (MTT and NRU) as described earlier in section 2.3. The effect of optimum concentration of L-carnitine, NAC, α-tocopherol, cyclosporine A, BAPTA-AM and DPI to prevent rhein induced cell death was examined by cell viability assays (MTT and NRU). The rat primary hepatocytes were pre incubated for 30min with the optimum concentration of protective agents (L-carnitine: 100 µM, tocopherol: 100 µM, NAC: 100 µM, cyclosporine A: 100 nM, BAPTA-AM: 20 µM and DPI: 100 nM). Subsequently, cells were treated with rhein (50 µM) and the cell viability was assessed after 24 hr according to the method described in section 2.3. 2.10. Preparation of whole cell extracts of rat primary hepatocytes The whole cell extracts of rat primary hepatocytes were prepared following the earlier described method with minor modification. 29 Briefly, primary hepatocytes from different treatment groups


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[control, rhein (50 µM), rhein (50 µM) + L-carnitine (100 µM), rhein (50 µM) + NAC (100 µM), rhein (50 µM) + α-tocopherol (100 µM), rhein (50 µM) + CSA (100 nM)] were lysed and homogenized in radio immunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl (pH 7.5), 1% nonidet P-40, 0.25% sodium deoxycholate, 0.25% SDS, 150 mM NaCl, 1 mM EGTA, 0.2 mM PMSF] supplemented with Protease Inhibitor Cocktail (10:1, v/v) using Ultra Turrax Polytron (Janke & Kunkel, IKA-Labortechnik, Staufen, Germany) followed by 60 min incubation on ice with an intermittent pulse at every 15 sec. The extracts were centrifuged at 14,000 rpm for 20 min and the supernatants were stored at -800C. The protein was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific, Rockford, IL) following manufacturer’s instructions and the samples were used for western blot studies. 2.11. Western blot analysis Forty microgram proteins were resolved on 10% SDS-polyacrylamide gel and electro transferred on polyvinylidene fluoride (PVDF) membranes. The blotted membranes were blocked with 5% BSA in PBS containing 0.1% Tween 20 (blocking solution) for 2 hr at room temperature. Subsequently, blocking solution was removed and the membranes were incubated overnight with the appropriate dilution of respective primary antibodies. The detail specifications of the primary antibodies used in the present study are mentioned supplementary material (Table S1). After completion of the incubation, the membranes were further incubated with appropriate HRPconjugated secondary antibodies (anti rabbit/anti mouse/ anti goat) specific to the primary antibodies as mentioned in Table S1. Bound secondary antibodies were detected by enhanced chemiluminescence using Super signal west femto maximum sensitivity substrate (Thermo scientific, Waltham, MA) following manufacturer’s instructions. All the blots were stripped and



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reprobed with β-actin to ensure equal loading of protein. The optical density of the protein bands was quantified using Image J software. 30 2.12. Fluorographic monitoring of early apoptotic, late apoptotic and necrotic cells Qualitative and quantitative analysis of early apoptotic, late apoptotic and necrotic cells using three marker molecules (Annexin V, Hoechst 33342 and propidium iodide) were carried out following the method described earlier with minor modification.

31

In brief, rat primary

hepatocytes cultured on collagen coated six well plates were washed twice with PBSD+ [phosphate-buffered saline solution (PBS) containing CaCl2 (1.2 mM) and MgCl2. 6H2O (340 µM)]. Subsequently, cells were stained with 2% Annexin V, 3 µg/ml hoechst 33258 and 1 µg/ml propidium iodide in Annexin V binding buffer (140 mM NaCl, 5 mM CaCl2 and 10 mM HEPES) for 15 min at room temperature. Thereafter, plates were rinsed three times with PBSD+ and subjected to fluorescence microscopy using Nikon Eclipse Ti-S inverted fluorescent microscope equipped with Nikon Digital Sight Ds-Ri1 CCD camera and NIS Elements BR imaging software (Nikon, Kanagawa, Japan). The excitation and emission wave length for each markers were set as follow; Annexin V (Ex 488, Em 530), Hoechst 33258 (Ex 350, Em 461) and propidium iodide (Ex 488, Em 610). At least three images per plate were taken (original magnification X 60). The number of cells positive for the concerned marker was counted in each image and was expressed relative to the total number of nuclei present. 3. Results 3.1. Dose and time dependant response of rhein exposure on the viability of rat primary hepatocyte In the present study, MTT and NRU assays were employed to evaluate the cytotoxicity of different concentration of rhein in rat primary hepatocytes. Experiments were carried out using six different concentration of rhein (0, 12.5, 25, 50, 100 and 200 µM) at four different time 12 ACS Paragon Plus Environment

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points (6, 12, 24 and 48 hr). The cytotoxicity profiles obtained from MTT and NRU assay are depicted in Figure 1A and 1B, respectively. From both assays, it is evident that rhein at a minimum concentration of 50 µM, significantly (P

Mechanism of Rhein-Induced Apoptosis in Rat Primary Hepatocytes

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