International Immunopharmacology 21 (2014) 412–425

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International Immunopharmacology

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Protective effects of certain pharmaceutical compounds against abrininduced cell death in Jurkat cell line

Nandita Saxena ⁎,1, P.V.L.Rao 1,2, A.S.B.Bhaskar 1, Y.D. Bhutia 1

⁎ Corresponding author. Tel.: +91 2390366; fax: +91E-mail address: nan_saxena@yahoo.com (N. Saxena).

1 Division of Pharmacology&Toxicology,Defence ResearcDefence Research Development Organization, Gwalior 4740

2 Present address: RDO-BU Center for Life Sciences, BCoimbatore-641046, India.

http://dx.doi.org/10.1016/j.intimp.2014.05.0281567-5769/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 December 2013Received in revised form 6 May 2014Accepted 22 May 2014Available online 2 June 2014

Keywords:AbrinAntidoteApoptosisNACRibosome inactivating proteinTrolox

Abrin is a plant glycoprotein toxin from the seeds of Abrus precatorius, and shares the structure and propertieswith ricin. Abrin is highly toxic, with an estimated human fatal dose of 0.1–1 μg/kg, causing death after accidentaland intentional poisoning. It is a potent toxin warfare agent. There are no antidotes available for abrinintoxication. It is becoming increasingly important to develop countermeasures for abrin by developing pre-and post-exposure medical therapy. The present study involves the screening of certain pharmaceutical agentsfor their potential to counter abrin toxicity in Jurkat T lymphocytes and the probable mechanism of action ofthe compounds with protective effect.The compounds studied are: Prednisolone, Minocycline, Amifostine, DRDE-07 (amifostine analog), Melatonin,Ebselen, N-Acetyl-L-cysteine (NAC) and Trolox. Among them, only NAC and trolox were found to confersignificant protection in Jurkat cells by restoring antioxidant enzymes depleted by abrin treatment. Abrin alsoshown to increase in stress factor associated proteins SAPK/JNK, c-fos and c-jun levels which were effectivelysuppressed by NAC and trolox. In addition to this, both compounds significantly inhibit abrin induced inflamma-tion and caspase-3 activity.These data suggest that NAC and trolox may serve as potential candidates for management of abrin-inducedpoisoning.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Ribosome-inactivating proteins (RIPs) are a group of proteinsthat share the property of damaging ribosomes in an irreversiblemanner acting catalytically [36]. Examples of plant RIPs includeabrin, ricin, gelonin, momordin, and mistletoe lectin [28]. Abrin is aheterodimeric glycoprotein found in the beans of Abrus precatoriusplant commonly known as jequirity bean or rosary pea. Abrin andricin are structurally and functionally related protein toxins andboth are among the most poisonous substances known, only nextto botulinum toxin. Abrin is a potent biological warfare agent be-cause of its low cost of purification and ease of use, either byaerosolisation as a dry powder or liquid droplets, or by addition tofood and water as a contaminant.

Abrin belongs to the family of type II RIPs that contain 2 subunits.These include a toxic A-chain with RNA N-glycosidase activity and agalactose-binding B-chain with lectin activity. Like ricin from Ricinus

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hDevelopment&Establishment,02, India.harathiar University Campus,

communis, the A-chain of abrin functions via the inhibition of proteinbiosynthesis through depurination of a single adenine residue (A4324)of the 28S ribosomal RNA. In contrast, the B chain of abrin functionsby interacting with the galactose moiety of glycoproteins or glycolipidson the cell membrane and is internalized into cells through receptor-mediated endocytosis [37]. Several reports have documented thatabrin is mitogenic [17], anti-fertility [16], anti-tumoral [23] andimmunopotentiating [30]. In addition to its ability to inhibit proteinsynthesis, abrin is believed to adopt alternative mechanisms to triggerapoptosis [7]. The inactivation of antioxidant proteins, increasedproduction in reactive oxygen species (ROS) have been proposed toplay a role in the abrin-induced cell-death pathway [35]. Abrin triggersapoptosis in caspase dependent manner along with lysosomal mem-brane permeabilization and loss of mitochondrial membrane potential[5]. Abrin and ricin have been shown to induce localized and systemicinflammation [7,10]. Previous studies also reported ricin toxicity tospleen and lymph nodes along with loss of lymphocytes [21]. Still theprecise molecular mechanisms regulating abrin-induced apoptosishave not beenwell defined. However, no antidote has yet been reportedto counteract abrin induced toxicity. Only supportive and symptomatictreatment is the option for medical management of abrin exposure.Considering abrin as a potent warfare agent, it is urgent and importantto develop an effective antidote for abrin.

The present study aims to evaluate mechanism and suitable thera-peutic intervention of abrin-induced toxicity. Since abrin has shown toinduce inflammation, cytotoxicity and oxidative stress, we investigated

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the efficacy of number of compounds having either of thesemechanismof action, namely Prednisolone (anti-inflammatory), Minocycline (anti-inflammatory and anti-oxidant), Amifostine, DRDE-07 [S-2(2-aminoethylamino: amifostine analog) ethyl phenyl sulfide] (both arecyto-protective), Melatonin, Ebselen, N-Acetyl-L-cysteine (NAC) andTrolox (a water soluble vitamin E derivative: 6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-carboxylic acid) (all are antioxidants) usingJurkat (T lymphocyte) cell line.

2. Materials and methods

2.1. Reagents

Propidium Iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), RNase, fluorometric assay kit for caspase-3, in vitro toxicology assay kit for lactic dehydrogenase (LDH) releasemeasurement, Neutral Red based assay kit (NR), Hoechst 33342 and an-tibodies for nuclear factor-kappa B (NF-κB), β-actin were purchasedfrom Sigma Chemical (St. Louis, Missouri, USA). Mito Tracker RedCMXRos was obtained from Molecular Probe (USA). c-Fos antibodywas purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).TBP and c-Jun antibodies were obtained from abcam (USA). Path ScanEnzyme linked Immunosorbent Assay (ELISA) for Phospho SAPK/JNKand PhosphoNF-κBp65were purchased from Cell Signaling Technology(USA). Horseradish peroxidase-conjugated (HRP) secondary antibodies(goat anti-rabbit and goat anti-mouse) were purchased from DAKO(Denmark). All other chemicals were obtained from Sigma ChemicalCo (St Luis, Missouri, and USA) unless otherwise mentioned.

2.2. Isolation of abrin

Abrin was isolated from seeds of the white variety of A. precatoriususing sepharose 6B affinity column chromatography and purified asdescribed in a previous study [19]. The purity and molecular weight ofabrin protein was confirmed by coomassie blue staining and MALDI-TOF (data not shown). The stock protein solution was diluted withphosphate buffered saline (PBS, pH 7.2) to a concentration of 2 mg/ml.

2.3. Evaluation of drugs for their protective efficacy against abrin toxin

Prednisolone, Minocycline, Amifostine, DRDE-07, Melatonin,Ebselen, NAC and Trolox were evaluated for their protective efficacyagainst abrin induced cell death. All compounds were obtainedfrom Sigma-Aldrich (Aldrich, USA) except DRDE-07. DRDE-07 is anamifostine analog and synthesized in the Synthetic Chemistry Divisionof the Establishment. NAC, DRDE-07, Minocyclin and Amifostine weredissolved in phosphate buffer solution (PBS). Prednisolone andMelato-nin were dissolved in ethanol. Ebselen was dissolve in chloroform.Trolox (50 mg/ml) was dissolved in 1 M sodium bicarbonate and dilut-ed to required concentration with PBS. For screening of compoundsagainst abrin, cells were treated with varying concentrations of com-pounds for 1 h prior to abrin treatment. Concentrations of compoundswere established based on previous literature. Cells were also treatedwith respective solvents in which the drugs were dissolved and no tox-icity was observed with any of the solvents when administered alone(data not shown).

2.4. Cell culture

The human leukemic T cell line Jurkat was obtained from NationalCentre for Cell Science, Pune and maintained in RPMI-1640 mediumsupplemented with 10% heat-inactivated fetal bovine serum (FBS),100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at37 °C in a humidified atmosphere containing 5% CO2 and 95% air in anincubator.

2.5. Crystal violet dye exclusion (CVDE) assay

The concentration of abrin that is toxic to 50% of the cells (CC50) wasdetermined from the plot of viability of cells by CVDE assay. Cells weregrown (0.5 × 106 cells/ml) in 24 well tissue culture plates and were ex-posed to various concentration of abrin for 24 h followed by incubationwith 0.25% crystal violet dye. Incorporated dye was solubilized by 33%glacial acetic acid. The CVDE assay evaluates cell density by stainingDNA and the absorbance at 540 nm is proportional to the amount ofcells in each well. For all subsequent time course studies, CC50 of abrinwas used.

2.6. Cytotoxicity assay

The cytotoxic effects of abrin and effect of compounds on abrin tox-icity was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Neutral Red (NR) uptakemethod. For MTT assay, cells were incubated in medium in triplicatein a 24-well plate and then treated with abrin only or pre-treatment oftested compounds followed by abrin exposure for 24 h at 37 °C. MTT(5 mg/ml, 100 μl/well) solution was added to each well and incubatedfor 4 h. Formazan crystals were solubilized with DMSO and absorbancewas measured at 570 nm using a 24-well multiscanner (ChameleonMulti-detection Platform, Finland). For NR assay after 24 h of treatment,0.33% neutral red solution (100 μl) was added to culture and further in-cubated for 4 h. Incorporated dye was solubilized with 1% acetic acid in50%methanol and absorbancewasmeasured at 540 nm. Datawere nor-malized to the measurement from control cultures which were consid-ered 100% survival.

2.7. LDH release assay

LDH activity in the culture media was measured spectro-photometrically at 490 nm according to manufacturer's protocol as anindex of plasma membrane damage and loss of membrane integrity.Enzyme activity was expressed as the percentage of extra cellular LDHactivity of the total LDH activity of the cells.

2.8. DNA fragmentation analysis by fluorescence microscopy

Following abrin treatments, cells were observed for apoptotic nucleiand compared with untreated cells. Briefly, after the treatments, themedia was removed and the cells were washed once with PBS. Thecells were then incubated with 10 μg/ml Hoechst 33342 (used toidentify nuclear fragmentation) in PBS for 30 min at 37 °C. Cells werethen washed with PBS and visualized with fluorescence microscope(Carl Zeiss, Axiomot 2).

2.9. Quantification of hypo-diploid DNA content by flow cytometry

The reduced DNA contents of apoptotic nuclei appeared as a broadhypo-diploid peak. This leads to the occurrence of sub-G1 cell popula-tion with higher DNA content compared to cells in the G1 phase of thecell cycle. Quantitation of hypo-diploid peakwas done by PI staining fol-lowing standard protocol. Briefly, after treatment cells were harvestedand washed with PBS and fixed with 70% ethanol for 30min and storedat −20 °C until assay. At the time of staining fixed cells were washedagain in PBS, and incubated for 1 h in PBS containing 100 μg/ml RNaseand 50 μg/ml PI. The PI fluorescence of individual nuclei was measuredusing a Cyflow space flow cytometer (Partec, Germany) and 20,000events were collected for each sample as described previously [33].The method is based on the binding of PI, a membrane-impermeabledye, to nucleic acid of cells. PI passes through dead cell membranesand intercalates with DNA to forma bright red fluorescent complex. Ap-optotic cells were assumed to have sub-G1 DNA content, and the resultwas expressed as a percentage of total cells counted.

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2.10. Determination of mitochondrial membrane potential

Alteration in mitochondrial membrane potential was observed withMitoTracker® Red CMXRos (Molecular Probes, USA). Briefly, the cells(1 × 106 cells/ml), were pre-treated with compounds for 1 h followedby abrin treatment and then cellswere stainedwith 50nmMitoTracker®Red and incubated at 37 °C for 15min. Then staining solution is replacedwith fresh pre-warmedmedia and observed immediately under fluores-cence microscope (Carl Zeiss, Axiomot 2). Observations were comparedwith abrin treated samples.

2.11. Measurement of intracellular ROS

The intracellular ROS was estimated by fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). The dye diffuses through thecell membrane and hydrolysed by intracellular esterases to non-fluorescent dichloro fluorescein (DCFH), which is oxidized to fluores-cent DCF in the presence of ROS. Jurkat cells were exposed to abrin atCC50 concentration for various time points (30 min–120 min). Aftercompletion of incubation, cells were washed twice with PBS and incu-bated with DCFH-DA for 30 min at a final concentration 5 μM and incu-bated at 37 °C. The fluorescence intensity was measured at differenttime intervals with Ex λ485 nm and Em530λ nm by flow cytometer.Cells were also pretreated with compounds followed by abrin exposureand after treatment image were obtained using fluorescence micro-scope. The DCF fluorescence intensity is proportional to the amount ofROS formed intracellularly.

2.12. Measurement of intracellular glutathione (GSH) and oxidizedglutathione (GSSG) level

Intracellular level of GSH and GSSG were determined by using theHisin and Hilf method [14]. Cells after treatment washed with PBS andcollected in to 0.5 ml 6.5% trichloroacetic acid. 4.5 ml of phosphate-EDTA buffer, pH 8.0 was added to 0.5 ml of trichloroacetic acid contain-ing cells. Cells were homogenized and centrifuged. Then 100 μl of thesupernatant mixed with 1.8 ml phosphate-EDTA buffer pH 8.0 and100 μl solution containing 100 μg of O-phthalaldehyde (OPT). Afterthorough mixing and incubation at room temperature for 15 minfluorescence was read at Em λ 420 nm and Ex λ 350 nm inspectrofluorophotometer (Biotek, USA). The reduced form of GSH wasused as a standard. Results were expressed as a μmol/mg protein. Formeasuring intracellular GSSG after homogenization 100 μl supernatantwas treatedwith 40 μl of 0.04 NN-ethylmaleimide. After 30min incuba-tion at room temperature 860 μl of 0.1 N NaOH and 100 μl (100 μg) ofOPT was added. Reaction mix was again incubated for 15 min at roomtemperature and fluorescence was observed. Results were expressedas nmol GSSG/mg protein. Protein estimation was done by using Bio-Rad protein assay kit.

2.13. Measurement of glutathione reductase (GR) and glutathione-S-transferase (GST) activity

Cells were treated with CC50 concentration of abrin with and with-out 1 h prior treatment of compounds and harvested at 1 h, 4 h, 8 hand 24 h and washed in PBS, Homogenized in buffer (250 nM sucrose,1 mM EDTA, 1 mM DL-Dithiothreitol and 15 nM Tris–HCl, pH 7.4) andsubsequently used for enzyme activity measurement. The activity ofGR and GST were assayed using Sigma GR assay kit (Cat No. GRSA-1KT) and GST assay kit (Cat No. CS0410) as permanufacturer's protocol.

2.14. Measurement of superoxide dismutase (SOD) and catalase activity

After abrin treatment with and without 1 h prior treatment of com-pounds cells were harvested at 1 h, 4 h, 8 h and 24 h and homogenizein cold 20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM

mannitol and 70 mM sucrose and centrifuged. Supernatant was usedto estimate SOD activity by using kit from Calbiochem according tomanufacturer's protocol. Results were expressed as unit/mg protein.For catalase assay, cells were homogenized in cold 50 mM potassiumphosphate, pH 7.0, containing 1mM EDTA and centrifuged. Supernatantwas used to estimate catalase activity by using catalase assaykit (Calbiochem) according to manufacturer protocol. Results wereexpressed as nmol/min/mg protein.

2.15. Real time quantitative PCR

RNA from Jurkat cells were isolated using RNeasy protect mini kit(Qiagen, Germany) according to manufacturer's protocol. RNA wasstored at−80 °C for further use. The quantitative real-timePCRwas car-ried out for the selected gene using gene specific primers fromQuantiTect primer assay kit (Qiagen, Germany) according to previouslydescribed method [33].

2.16. ELISA for tumor necrosis factor-α (TNF-α) and interferon-gamma(IFN-γ)

Cells were pretreated with compounds for 1 h followed by abrintreatment. After 24 h cell free supernatant was stored at −80 °C untilfurther analysis. Cytokine concentration in culture supernatant wasquantitated using DuoSet ELISA kit from R&D, according tomanufacturer's instruction.

2.17. ELISA for Phospho SAPK and Phospho NF-κBp65

After 24 h of treatment cells were harvested and lysed. Lysates werestored at −80 °C until further analysis. The Path Scan ELISA kits wereused to determine Phospho-SAPK/JNK and Phospho NF-κBp65 activityaccording to manufacturer's instruction (Cell Signaling, USA).

2.18. Flurometric assay for active caspase-3

The assay was performed by monitoring cleavage of the caspase-3specific fluorogenic substrate according to the manufacturer's instruc-tions. Briefly, Jurkat cells (1 × 106/ml) were cultured in the absence orpresence of abrin CC50 for 24 h with 1 h prior exposure with com-pounds. The cells were suspended in lysis buffer [10 mM Tris/HCl,pH 7.5/10 mM NaH2PO4/Na2HPO4, pH 7.5/130 mM, NaCl/1% (v/v),Triton X-100]. Lysate (100 μl) was added to 1 ml of reaction buffer(1.4 M NaCl/27 mM, KCl/100 mM, KH2PO4/K2HPO4, pH 7.5), containing100 ng of the fluorogenic substrate Ac-DEVD-AMC and incubated at 37°C for 1 h. Fluorescence intensity was calibrated with standard concen-trations of AMC and measured with excitation at 360 nm and emissionat 440 nm inflourimeter (Biotek, USA). For establishing the specificity ofthe reaction, the caspase-3 inhibitor was used. Caspase-3 activitywas calculated from the slope of the curve and expressed as nmol/AMC/min/mg protein.

2.19. Western blotting analysis

Total protein was isolated from cells and estimated using Bio-Radprotein assay kit. Protein lysate (30 μg/lane) from each samplewas sep-arated on SDS-PAGE and electrophoretically transferred on to a nitrocel-lulose membrane filter, using an electro-blotting apparatus (Bio-Rad).Membranes were incubated in blocking solution containing 5% non-fatdry milk in PBST buffer (PBS buffer containing 0.1% Tween-20) for 1 hat room temperature, followed by incubation for overnight at 4 °C inplatform shaker with various primary antibodies at specified dilutions:c-Jun (dilution 1:1000; detects 36 kDa protein), c-Fos (dilution 1:500,detects 55 kDa protein), NF-κB (1:1000; detects 65 kDa band) andmonoclonal anti-β-actin (Sigma, 1:1000, detects 42 kDa) which wasused as protein loading control. For nuclear protein loading control

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TATA binding protein (TBP, 1:2000; detects 38 kDa protein) was used.All antibodies were diluted in PBST with 5% milk powder. The mem-branes were washed four times in PBST for 15min, followed by incuba-tion for 2 h in HRP conjugated rabbit anti-mouse or goat anti-rabbitsecondary antibody (Dako) used at 1:30,000 dilution. The membraneswere washed again and developed using an enhanced chemilumines-cent detection system (Proteo Qwest™ Chemiluminescent Westernblotting kit, Sigma) according to manufacturer's protocol and theimage was grabbed on Pierce CL-XPosure™ X-ray films.

2.20. Statistical analysis

All data were expressed as a mean± SEM of at least three indepen-dent experiments with triplicate samples. Statistical comparisons be-tween groups were performed using one-way ANOVA followed byDunnett's test or Student's t-test or Student–Newman–Keuls multiplecomparison test. Differences with p value less than 0.05 were taken assignificant.

3. Results

3.1. Abrin induced cytotoxicity in a dose and time dependent manner inJurkat cells

To examine the cytotoxic effect of abrin in Jurkat cells, the cells weretreated with toxin concentrations ranging from 2 to 12 ng/ml for 24 hand the viability was determined by CVDE assay. Cytotoxicity was alsoobserved by measuring mitochondrial dehydrogenase activity by MTTassay, lysosomal activity by NR assay (Fig. 1A). Abrin caused a dose de-pendent decrease in viability with increase in concentration. Based onMTT, CVDE and NR assay CC50 was determined as 2 ng/ml and wasused in subsequent parameter studied. Abrin induced extracellularLDH releasewas also calculated by LDH release assay. Cells were treatedwith 0–5 ng/ml concentration of abrin for 24 h. Abrin from1 ng/ml con-centration showed significant increase in LDH release and reaches to 20fold higher then untreated cells at 5 ng/ml (Fig. 1B). Hoechst 33342staining also showed DNA fragmentation following abrin treatment(Fig. 1C). The cytotoxicity of abrin at 2 ng/ml was also evaluated intime course experiment up to 24 h by PI staining and cells were ana-lyzed for hypo-diploid peak byflow cytometry. The fraction of apoptoticcells was identified as a sub-G1 hypo-diploid population in a DNA histo-gram. Treatment with abrin CC50 was not found to have much effect upto 8 h, but apoptosis was significantly increased at 16 h and 24 h(Fig. 1D).

3.2. Screening of compounds for their potential as an antidote

Different categories of compoundswere evaluated for their potentialas an antidote against abrin toxicity by MTT and NR assay. They arePrednisolone, Minocycline, Amifostine, DRDE-07, Melatonin, Ebselen,NAC and Trolox. For evaluation of protective efficacy, Jurkat cells werepre-treated for 1 h with varying concentrations of compoundsand followed by abrin 2 ng/ml treatment or abrin only for the next24 h. Table 1 summarizes the results of the protective efficacy ofchemoprotectants against abrin toxicity. Prednisolone, Minocycline,DRDE-07 and Melatonin did not offer protection at any doses includedin the study. Amifostine and ebselen increase the viability up to 60–70%. NAC and trolox completely reversed the abrin induced cytotoxicityin Jurkat cells. Both compounds were further evaluated for their mech-anism of action by using various parameters in Jurkat cells.

3.3. Effect of NAC and trolox on abrin induced cytotoxicity

Cells were pretreatedwith varying concentrations of NAC and trolox(200 μg/ml to 1000 μg/ml) for 1 h then exposed to abrin CC50 or abrinonly for 24 h and compared with untreated cells. Viability and

cytotoxicity were assayed by CVDE, MTT and NR assay. All doses ofNACand trolox pretreatment neutralize the effect of abrin induced cyto-toxicity. Interestingly mitochondrial activity was found to be more thancontrol (Fig. 2A and B). To verify whether NAC and trolox inducedmito-chondrial activity is inherent in the cell or is due to increase in cell num-ber, MTT assay and cell counting was donewith same doses of NAC andtrolox alone. There was increase in mitochondrial activity with increasein dose of NAC and trolox but the cell number was not altered (data notshown). Abrin at 2 ng/ml, for 24 h caused a significant increase in extra-cellular LDH release. NAC and trolox 1 h pretreatment lowered the abrininduced extracellular LDH release comparative to basal level (Fig. 3Aand B). LDH release following treatment with NAC or trolox alone wasalso analyzed and no significant release of LDH with NAC, alone, up to1000 μg/ml, and trolox up to 2000 μg/ml (data not shown) was seen.Three doses viz. 200 μg/ml, 400 μg/ml and 600 μg/ml of NAC and troloxwere used for subsequent analyses.

3.4. NAC and trolox reverse abrin induced apoptosis

To further confirm the efficacy of NAC and trolox, 1 h prior treatmentwas done using three doses of NAC and trolox followed by abrin at2 ng/ml concentration for 24 h and cells were stained with PI and ana-lyzed for hypo-diploid peak by flow cytometry. Abrin treatment signif-icantly induced the accumulation of cells in sub-G1 phase (51 ± 2.8%compared to untreated cells) at 24 h. There was dose dependent de-crease in apoptosis when cells were pretreated with NAC and trolox.All three doses of NAC and trolox significantly reduced the apoptosiswhen compared to abrin induced apoptosis. At 600 μg/ml concentrationof NAC and trolox, only 10% cells were entered in sub-G1 phase com-pared to abrin treated (50%) cells suggesting protective efficacy ofNAC and trolox (Fig. 4).

3.5. Abrin altered the mitochondrial membrane potential

The apoptotic cascade in mammalian cells is a multistep process. Inmost cases, it is initiated by loss of integrity of the outer mitochondrialmembrane. Mitochondrialmembrane integrity was checked by stainingwith MitoTracker® Red dye in Jurkat cells with and without NAC andtrolox pretreatment for 1 h (200 μg/ml, 400 μg/ml and 600 μg/ml)followed by abrin exposure for the next 1 h. H2O2 (1 mM) was used asa positive control for mitochondrial membrane dissipation. The cellswithout any treatment showed increased red fluorescence, which cor-responds to the polarized mitochondria. Loss of red labeling signifiesthat abrin disrupts the mitochondrial membrane integrity within 1 h,but all three doses of NAC and trolox pretreatment effectively main-tained it (Fig. 5).

3.6. NAC and trolox effectively block abrin induced ROS accumulation

Since ROS and mitochondrial membrane permeability are linkedphenomena we further estimated ROS levels in a time course experi-ment with 2 ng/ml abrin treatment up to 120 min and the results areshown in Fig. 6A as a histogram by flow cytometry. The intracellularlevel of ROS were increased in a time dependent manner, reaching apeak at 60 min and decreased back to control level at 120 min. The re-sult suggests that increase in ROS levels is an early event in abrin in-duced cytotoxicity. Further cells were pretreated with NAC and troloxfor 1 h followed by 1 h treatment of abrin at 2 ng/ml and visualizedby fluorescentmicroscopy. H2O2 (25 μM)was used as a positive controlfor ROS generation. Typical green fluorescence after abrin or H2O2 treat-ment is indicative of the ROS generation. Abrin pre-incubation of thecells for 1 h with GSH precursor NAC markedly inhibited the abrin in-duced accumulation of ROS. Similarly trolox also effectively suppressthe abrin induced ROS generation (Fig. 6B).

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Fig. 1. Cytotoxic and apoptotic effect of abrin on viability of Jurkat cells. (A) Cytotoxic effect of abrin in Jurkat cells was determined byMTT, CVDE and NR assay. For determination of 50%cytotoxic concentration (CC50), cells were plated on to a 24well plate and treatedwith varying concentrations of abrin for 24 h. The CC50 was calculated as 2 ng/ml. (B) Extracellular LDHactivity in abrin treated cells was observed at 24 h. Abrin induces significant release of LDH starting from 1 ng/ml. (C) Fluorescence micrograph of the cells stained with Hoechst 33342showing the nuclear fragmentation. Jurkat cells were treated with the indicated concentration of abrin for 24 h and labeled respectively as Control, 2 ng/ml, 4 ng/ml, 6 ng/ml (magnifi-cation, ×20); given image is representative of three independent experiments (all microscopic images were recorded from a minimum of 3 areas per sample). (D) Time course effect ofCC50 abrin on percent apoptotic cells was determined by FACS analysis of propidium iodide stained hypo-diploid sub-G1 peak. Percentage of sub G1 fraction is given in each histogram.Given figure is the representative of three independent experiments and labeled respectively as Control, 4 h, 8 h, 16 h and 24 h. Data are themean of three independent experiments eachin triplicates with standard error. ⁎Significantly different from control by Dunnett's test at p b 0.05. For all subsequent experiments CC50 dose, 2 ng/ml was used.

416 N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

3.7. Effect of NAC and trolox on intracellular antioxidants activity(GSH, GSSG, GR, GST, SOD and catalase)

Oxidative stress may occur due to elevation of ROS or down-regulation of antioxidant enzymes or both.We next examined the activ-ity of antioxidants proteins at different time points. For measuring anti-oxidant enzyme activity cells were pre-treated with NAC and trolox

(400 μg/ml) for 1 h followed by abrin CC50 exposure for 1 h, 4 h, 8 hand 24 h and compared with untreated cells. Significant GSH depletionwas observed in abrin treated cells after initial increase at 1 h and 4 h. At1 h both NAC and trolox increases the GSH level but it was less thanabrin induced GSH level. Later NAC at 4 h significantly restored theGSH activity depleted by abrin. Interestingly, trolox showed biphasic re-sponse with initial increase followed by decrease up to 8 h then again

Table 1Effect of compounds on Jurkat cell survival following abrin treatment. The cellswere treatedwith various doses of compounds 1 h prior to abrin treatment at 2 ng/ml for 24 h. Cell viabilitywas determined by MTT assay and NR assay. Given % viability is the maximum cell viability achieved by each compound over CC50 concentration of abrin.

Drugs Concentration (μg/ml) % viability (MTT assay) % viability (NR assay)

Prednisolone 0.001–10 50 ± 1.3% 48 ± 2.1%Minocycline 0.001–10 52 ± 0.01% 50 ± 1.0%Amifostine 0.1–400 70 ± 0.2% 64 ± 2.0%DRDE-07 0.1–200 50 ± 1.0% 49 ± 0.45%Melatonin 0.1–200 57 ± 0.9% 55 ± 2.9%Ebselen 0.01–100 73 ± 0.6% 62 ± 3.2%NAC 100–1000 100 ± 1.3% (200 μg/ml) 100 ± 3.3% (200 μg/ml)Trolox 100–2000 100 ± 0.7% (200 μg/ml) 100 ± 2.5% (200 μg/ml)

All the doses of NAC and trolox (starting from 200 μg/ml) completely reversed the abrin induced cytotoxicity.

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increase at 24 h (Fig. 7A). After abrin treatment GSSG level goes downsignificantly from 4 h. NAC increases the GSSG level at 1 hwhich furthergoes down below control in time dependent manner. Trolox alsoincreased GSSG level at 1 h which further increased up to 8 h anddecreased back to control at 24 h (Fig. 7B).

At the early time point of abrin exposure GRwas unaffected and at 24 hthere was significant decrease. NAC pretreatment significantly enhanced

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*

*

MTTCVDENR

A

B

Fig. 2. NAC and trolox reverse abrin induced cytotoxicity. (A) Pretreatment of the indicat-eddoses of NAC followedby abrin 2 ng/ml treatment for 24 h. Cellswere analyzedbyMTT,CVDE and NR assay. (B) Pretreatment of different doses of trolox followed by abrin2 ng/ml exposure for 24 h. Cells were analyzed by MTT, CVDE and NR assay: both NACand trolox reverse abrin induced cytotoxicity. Data are the mean of three independentexperiments each in triplicates with standard error. ⁎p b 0.05 compared to un-treatedcells.

the GR activity from 1 h andmaintained up to 24 h, similarly trolox also in-creases the GR activity in time dependent manner starting from 8 h withmaximum increase at 24 h (Fig. 7C). Abrin was also found to induce theGST activity at 1 h followed by a significant decrease from 4 h. NACmoder-ately increased the GST activity but trolox significantly enhanced the GSTactivity as early as 1 h and maintained up to 24 h (Fig. 7D).

SOD is an enzyme forming thefirst line of defense against ROS. It cat-alyzes the dismutation reaction of the toxic superoxide radical tomolec-ular oxygen and hydrogen peroxide. Abrin was found to induce the SODactivity at 4 h followed by significant decrease. Both NAC and trolox sig-nificantly increased SODactivity starting from1h andmaintained till 24h (Fig. 7E). Catalase is another enzyme, catalyzes the decomposition ofhydrogen peroxide to water and oxygen. Similar to SOD, abrin wasfound to enhance the catalase activity up to 8 h followed by decrease

L

DH

rel

ease

(U

X10

6 ce

lls/m

l)

0

30

60

90

120LDH Release

LD

H r

elea

se (

UX

106

cells

/ml)

0

20

40

60

80

100

ControlAbrin 2ng/ml

NAC + Ab

C Ab 200 400 600 800 1000

C Ab 200 400 600 800 1000

Trolox + Ab

ControlAbrin 2 ng/ml

Concentration (μg/ml)

Concentration (μg/ml)

*

*

A

B

Fig. 3. NAC and trolox significantly reduced abrin induced extracellular LDH release; cellswere treated with abrin CC50 in the presence or in the absence of NAC or trolox for 24 h.(A) All three doses of NAC could significantly block abrin induced LDH release. (B) Troloxalso reverse abrin induced release and decreased back to normal level. Data are the meanof three independent experiments each in triplicates with standard error. ⁎p b 0.05compared to un-treated cells.

DNA Content

Cel

l Cou

nt

Control Ab 2ng/ml

NAC 200µg/ml+Ab

1.8±0.3% 51±2.8%

NAC 400µg/ml+Ab NAC 600µg/ml+Ab

Trolox 200µg/ml+Ab Trolox 400µg/ml+Ab Trolox 600µg/ml+Ab

27±0.4% 19±1.5% 10±1.0%

22±2.7% 15±1.5% 8±0.7%

∗

∗#

∗# ∗# ∗#

∗# ∗#

Fig. 4. Effect of NAC and trolox on abrin induced apoptosis; cells were incubated with abrin at CC50 concentration for 24 h or pretreated with NAC and trolox (200 μg/ml, 400 μg/ml and600 μg/ml) or left untreated. Apoptosis was determined by FACS analysis of propidium iodide stained hypo-diploid sub-G1 peak. Percentage of sub G1 fraction is given in each histogram.Given figure is representative of three independent experiments. Data shows the mean± SE. ⁎p b 0.05 significantly different from untreated cells; #p b 0.05 significantly differently fromabrin treated cells.

418 N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

at 24 h. BothNAC and troloxwere seen to replenish catalase activity andplay cyto-protective role (Fig. 7F).

3.8. NAC and trolox significantly reduced TNF-α, IFN-γ and NF-κβ levelaugmented by abrin

ROS has been shown to induce the production of inflammatory cyto-kines. TNF-α and IFN-γ are the major pro-inflammatory cytokines ei-ther modulated by oxidative stress or regulate ROS metabolism [39].We next examined the effect of both cytokines with abrin in the pres-ence or absence of NAC and trolox. Incubation with abrin caused 60fold increase in IFN-γ and 15 fold increase in TNF-α mRNA expressionas assessed by RT PCR,while NAC and trolox pre-treatment significantlyreduced the expression of TNF-α, IFN-γ (Fig. 8A).

Consistent with gene expression data abrin treatment also increasesthe protein level of IFN-γ and TNF-α in cell supernatant. Pretreatmentof NAC and trolox could prevent abrin induced inflammation as thecombined treatment of abrin and NAC or trolox caused significant re-duction of secretion of IFN-γ and TNF-α (Fig. 8B).

NF-κB family of proteins is a dimeric transcription factor that regu-lates the expression of large number of genes involved in inflammationand immune response. We also examined the effects of abrin on NF-κBgene modulation. NF-κB is also 2 fold elevated at mRNA level followedby abrin treatment, which was effectively brought to the normal levelwith NAC and trolox pretreatment (Fig. 8A). Since the p65 subunit is

the primary component of activated NF-κB, we studied the phosphory-lated form of NF-κBp65 unit by ELISA (Phospho NF-κBp65) in abrintreated cell lysate with and without NAC and trolox pretreatment.Abrin significantly enhances the phosphorylation of NF-κBp65 andNAC, trolox pretreatment significantly reduces the abrin induced activ-ity of NF-κB. But even a highest dose of NAC and trolox failed to bringnormal level of Phospho NF-κB (Fig. 8C). In non-stimulated cells NF-κB remains bound to the inhibitory protein I-κB and sequesteredin the cytoplasm. However upon stimulation with cytokines such asTNF-α, I-κB degraded and then activated the translocation of NF-κBdimer to the nucleus and its binding to the NF-κB promoter elements,thus driving the expression of large number of genes. Along withPhospho form of NF-κB, we also observe its nuclear localization. It is ev-ident formwestern blot analysis in nuclear and cytosolic lysate, abrin fa-cilitates the nuclear translocation of NF-κB while NAC and troloxappears to block nuclear translocation (Fig. 8D). These data suggestthat the inhibitory effect of NAC and trolox on TNF-αmay be mediatedby down regulation of NF-κB.

3.9. NAC and trolox significantly reverse abrin induced phosphorylation ofSAPK/JNK and c-Fos and c-Jun expression

Accumulating evidence suggests that ROS are not only injurious by-products of cellular metabolism but also essential participants in cellsignaling pathways. Among various signaling pathways, SAPK/JNK and

Control H2O2 Abrin

NAC 200/+Ab NAC 400+Ab NAC 600+Ab

Trolox 200+ Ab Trolox 400+Ab Trolox 600+Ab

Fig. 5. Abrin-induced loss of mitochondrial membrane integrity and protective effect of NAC and trolox in Jurkat cells. To assess the effect of NAC and trolox on abrin induced loss of mi-tochondrial membrane potential, Jurkat cells were pre-treated with the indicated concentration of NAC and trolox (μg/ml) for 1 h followed by exposure to 2 ng/ml abrin or abrin only or1 mM H2O2 for the next 1 h and loaded with mitotracker red to monitor mitochondrial membrane integrity. Visualization of mitochondrial membrane integrity was carried out byfluorescence microscopy (original magnification, ×100). Given image is representative of three independent experiments (all microscopic images were recorded from a minimum of 3areas per sample).

419N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

c-Fos, c-Jun is the component of stress associated pathways and is alsoregulated by ROS [26,39]. The AP-1 transcription factor is mainly com-posed of Jun and Fos andmediates gene regulation in response to path-ological stimuli, including cytokines and stress signals [13]. Abrin at2 ng/ml significantly enhanced gene expression of c-fos and c-junand NAC and trolox at 400 μg/ml concentration decreases the c-fosand c-jun level (Fig. 9A). Similarly at protein level abrin up-regulatethe c-Fos (2.5 fold) and c-Jun (8 fold) expression. The expression levelof c-Fos and c-Jun had gone down significantly in the presence of NACand trolox. However, only higher concentration of NAC (600 μg/ml)could be able to suppress c-Fos expressionwhile all three doses of troloxeffectively inhibited the c-Fos expression. All three doses of NAC andtrolox significantly suppress the abrin induced c-Jun expression(Fig. 9B). The presence of the phosphorylated form of SAPK/JNK incell lysate with abrin treatment with and without 1 h prior treatmentof NAC and trolox was also investigated. Abrin augmented the PhosphoSAPK/JNK level up to 5 fold compared to untreated cells. Furtherpretreatment with NAC and trolox prevented abrin induced activationas the combined treatment of abrin and NAC or trolox caused significantreduction in SAPK/JNK activity compared to abrin alone (Fig. 9C). Downregulation of protein level of c-Fos, c-Jun and Phopsho SAPK/JNK isfound to be more pronounced in trolox as compared to NAC.

3.10. NAC and trolox reduced abrin induced apoptosis by inhibitingcaspase-3 activation

Caspase-3 is the final executor of apoptosis is responsible for thecleavage of the key cellular protein, leading to typical morphologicalchanges observed in cell undergoing apoptosis [32]. Abrin inducedcaspase-3 activity was monitored by fluorimetry. Abrin treatmentcaused 3 fold increases in activated caspase-3 level as assessed by

fluorimetry and NAC or trolox pre-treatment decreased the caspase-3activity compared to abrin but still higher than untreated cells, onlytrolox at 600 μg/ml significantly reduced abrin induced caspase-3 activ-ity and decreased back to comparable level of untreated cells (Fig. 10).

4. Discussion

Abrin toxin is similar to ricin toxin in both molecular structure andthemechanismof action. The two toxinshave been classified as a poten-tial agent for biological warfare and bioterrorism by the Biological andToxin Weapons Convention (BTWC) [6]. Moreover abrin is potentiallya more hazardous terrorist weapon than ricin because the toxicity ofabrin in mice is 75 times higher than that of ricin [37]. Although abrinis not infectious, it is a probable agent for use as a bioweapon becauseof its stability and the relatively easy purification procedure. Thoughthe mechanisms of toxicity of few RIPs at the cellular and molecularlevels have been delineated up to some extent, the development of anantidote has proven elusive [25].

Keeping in view the need of antidote for abrin, we evaluated protec-tive efficacy of certain chemoprotectants. Of the eight compoundsscreened NAC and trolox offered protection and both are antioxidants.NAC is a thiol compound, which provides sulfahydryl groups and actas a precursor of glutathione and ROS scavengers, hence regulatingthe redox status in the cells. Trolox iswater-soluble vitamin E derivativeand peroxyl radical scavenger and rapidly penetrate biologicalmembranes.

The cytotoxicity studies and analysis of hypo-diploid populationconfirmed that the pre-treatment of NAC and trolox could preventabrin induced apoptosis as the combined treatment of abrin and NACor trolox caused significant reduction in apoptosis compared to abrin

Control H2O22 Abrin

NAC 200+ Ab NAC 400+ Ab NAC 600+Ab

Trolox 200+ Ab Trolox 400+ Ab Trolox 600+ Ab

A

B

Treatment Fold ChangeControlAb-30min 1.4+0.3Ab-60min 2.9+0.2∗

Ab-120min 1.3+0.2

DCF fluorescence

Cel

l Cou

nt

___

Fig. 6. Abrin induced ROS generation and effect of NAC or trolox in the presence of abrin: (A) Time course effect of abrin in ROS generation in Jurkat cells by FACScan; filled histogram iscontrol sample. The mean fluorescence intensity (MFI) of DCFDA fluorescence of each cell was plotted. Mean ± SE of fold change in MFI over control at the indicated time point of threeindependent experiments are shown. (B) Jurkat cells were incubated for 1 h with the indicated doses of NAC or trolox, and then for 1 h in the presence of 2 ng/ml abrin or abrin alone.25 μMH2O2 was used as a positive control for ROS accumulation. After which typical image of ROS generation (green DCF fluorescence)were captured by fluorescence microscopy (mag-nification= 20×). Given image is representative of three independent experiments (all microscopic images were recorded from aminimumof 3 areas per sample). Data shows themean± SE. ⁎p b 0.05 significantly different from untreated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

420 N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

GSH: 0.4300 GSH: -- GSH: 1.0600 GSH: 0.8500 GSH: 0.6200 GSH: -- GSH: 0.5400 GSH: 1.0100 GSH: 0.3900 GSH: -- GSH: 0.0700 GSH: 0.9800 GSH: 0.3500 GSH: -- GSH: 0.0800 GSH: 0.7200 GSH: 0.8000

GSH

GSSG

*

*

* *

** *

*

*

**

*

***

*

*

*

* *

*

GR: 5.7600 GR: -- GR: 5.9000 GR: 18.0000 GR: 5.0300 GR: -- GR: 6.8000 GR: 20.8000 GR: 5.2000 GR: -- GR: 7.0000 GR: 20.0000 GR: 8.3000 GR: -- GR: 4.0100 GR: 16.2000 GR: 14.2200

GR

GST

*

*

**

*

*

*

*

*

*

*

*

* *

*

*

Treatment duration (h)

GS

H (

μmo

les/

mg

pro

tein

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1 4 24

nm

ole

s G

SS

G/m

g p

rote

in

0

20

40

60

80

100

8C

1 4 248CTreatment duration (h)

Control Abrin 2ng/ml NAC 400 μg/ml + Ab Trolox 400 μg/ml + Ab

A

BTreatment duration (h)

nm

ole

s N

AD

PH

oxi

dis

ed/

min

/mg

pro

tein

0

5

10

15

20

25

1 4 24

μmo

les/

min

/mg

pro

tein

0.0

0.1

0.2

0.3

0.4

0.5

8C

1 4 248CTreatment duration (h)

Control Abrin 2ng/ml NAC 400 μg/ml + Ab Trolox 400 μg/ml + Ab

C

D

Treatment duration (h)

U/m

g p

rote

in

0.0

0.5

1.0

1.5

2.0

2.5

SOD: 0.5560 SOD: -- SOD: 0.5765 SOD: 1.1310 SOD: 1.6250 SOD: -- SOD: 1.0310 SOD: 1.5250 SOD: 1.9060 SOD: -- SOD: 0.7688 SOD: 1.5500 SOD: 1.7690 SOD: -- SOD: 0.2080 SOD: 1.6180 SOD: 1.9980

1 4 24

nm

ol/m

in/m

g p

rote

in

0.0

0.1

0.2

0.3

0.4

0.5

0.6

8C

1 4 248C

SOD

Catalase

Treatment duration (h)Control Abrin 2ng/ml NAC 400 Trolox 400

E

F

*

*

*

*

*

*

*

*

**

*

* *

*

* *

*

*

*

*

*

**

μg/ml + Ab μg/ml + Ab

421N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

*

*

*

*

**

*

*

* *

D

OD

at

450

nm

0.0

0.5

1.0

1.5

2.0

Control

Abrin 2ng/m

l

NAC 200+Ab

NAC 400+Ab

NAC 600+Ab

Trolox 200+Ab

Trolox 400+Ab

Trolox 600+Ab

Phospho NF-kBp65*

**

*

*

*

*

# #

#

#

#

#

C

NF-κB cytosolic extract

β-actin

NF-κB nuclear extract

TBP

Abrin(CC50) - + + + + + + +NAC(µg/ml) - - 200 400 600 - - -Trolox(µg/ml) - - - - - 200 400 600

mR

NA

(fo

ld c

han

ge)

-10

-5

0

5

10

15

2050

60

70A

Abrin 2ng/ml NAC 400 μg/ml + Ab Trolox 400 μg/ml + Ab

Cyt

oki

ne

(pg

/ml)

0

400

800

1200

1600

2000B

Control

Abrin 2ng/m

l

NAC 200+Ab

NAC 400+Ab

NAC 600+Ab

Trolox 200+Ab

Trolox 400+Ab

Trolox 600+Ab

Concentration (μg/ml)

Concentration (μg/ml)

IFN-γTNF-α

IFN-γTNF-αNF-κB

Fig. 8. NAC and trolox significantly reduced TNF-α, IFN-γ and NF-κβ augmented by abrin. The cells were pre-treated for 1 h with NAC or trolox followed by abrin treatment or only abrintreatment for 24 h. (A) Gene expression profile of TNF-α, IFN-γ and NF-κβ by quantitative real time PCR. The results are presented as fold up-regulation or down-regulation over control.Values are mean ± SE of 3 independent experiments per treatment and relative to RPII mRNA in Jurkat cells; (B) abrin stimulated the secretion of IFN-γ and TNF-α, NAC and troloxpretreatment reversed the abrin induced secretion of cytokines: cytokine release was analyzed by ELISA (C) Phospho NF-κBp65 level by ELISA. (D) Western blotting: NF-κB localizationwas analyzed in cytosolic and nuclear extract. β-actin and TATA binding protein (TBP) served as a loading control for cytoplasm and nuclear extract respectively. ⁎Significantly differentfrom control and #significantly different from abrin treated group at p b 0.05 and by Dunnets's test.

422 N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

only. Protective efficacy of NAC and trolox is further substantiated bydecrease in extracellular LDH release increased by abrin.

The mitochondria play a crucial role in metabolism and coordinatesextrinsic and intrinsic signals for proliferation and cell death. In the pres-ent study abrin is shown to disrupt mitochondrial membrane potentialin as early as 1 h. It is an important early event in the commitment of acell to apoptosis. But NAC and trolox maintain the mitochondrial mem-brane integrity and reverse abrin induced apoptosis.

Abrin also triggered ROS accumulation at early time point in Jurkatcells in agreement with a previous study [35]. In vitro studies have alsodemonstrated that biochemical, cellular and functional disturbancesafter ricin toxin exposure were due to oxidative stress [38]. In our exper-iments abrin induced oxidative stress was significantly inhibited by NAC

Fig. 7. Time course effect of abrin andNACand trolox in the presence of abrin on antioxidant pro(μg/ml) for 1 h followed by exposure to 2 ng/ml abrin or abrin alone. (A) GSH; (B) GSSG; (C) GReach in four replicates. Significantly different from control by Dunnett's test at p ≤ 0.05.

and trolox which is in concurrence with previous study where NAC andtrolox significantly inhibit toxin induced ROS generation [8,35].

Our observation demonstrated that the ROS generation and mito-chondrial membrane disintegrationwas a parallel phenomenon follow-ing abrin treatment, which is suggests that most of the ROS could beproducts of mitochondrial respiration as shown in retinal ganglioncells [22]. Mitochondrial mediated intrinsic pathway of cell death waslong regarded as the ultimate consequence of RIP induced toxicity [2,28,35]. In agreement with these studies, our observation also showedoxidative stress and mitochondrial pathway contribution may be re-sponsible for early sign of abrin toxicity.

Oxidative stress is generally defined as an imbalance that favors theproduction of ROS over antioxidant defenses. Considering the GSH

teins level. Jurkat cellswere pre-treatedwith the indicated concentration of NACand trolox; (D) GST; (E) SOD; (F) catalase. The values are mean± SE of 3 independent experiments

c-fosc-jun

*

*

**

B

Fo

ld c

han

ge

in p

rote

in

exp

ress

ion

ove

r co

ntr

ol

-5

0

5

10c-Fosc-Jun

* * **

Abrin 2ng/m

l

NAC 200+Ab

NAC 400+Ab

NAC 600+Ab

Trolox 200+Ab

Trolox 400+Ab

Trolox 600+Ab*

*

#

#

# #

#

*

* **

*

#

# # #

#

c-Jun

c-Fos

TBP

Abrin(CC50) - + + + + + + +NAC(µg/ml) - - 200 400 600 - - -Trolox(µg/ml) - - - - - 200 400 600

Control

Abrin 2ng/m

l

NAC 200+Ab

NAC 400+Ab

NAC 600+Ab

Trolox 200+Ab

Trolox 400+Ab

Trolox 600+Ab

Phospsho SAPK/JNK

*

*

*

#

# #

# #

#

Concentration (μg/ml)

mR

NA

(fo

ld c

han

ge)

-5

0

5

10

15

20

25

A

OD

at

450

nm

0.0

0.5

1.0

1.5

2.0

C

Abrin 2ng/ml NAC 400 μg/ml + Ab Trolox 400 μg/ml + Ab

Concentration (μg/ml)

Fig. 9. Effect of NAC and trolox on c-Fos, c-Jun and Phospho SAPK/JNK in the presence of abrin (A) mRNA level of c-fos and c-jun expression by quantitative RT-PCR. Gene expression isexpressed as fold up-regulation or down-regulation over control. Values are mean ± SE of four replicates per treatment and normalized to RPII mRNA in Jurkat cells. (B) Western blot:c-Fos and c-Jun expressionwas analyzed in nuclear extract. TBP served as a loading control. Change in c-Fos and c-Jun is comparedwith untreated cell lysate and showed as a bar diagram.(C) Phospho SAPK/JNK level by ELISA. ⁎Significantly different from control and #significantly different from abrin treated group at p b 0.05 and by Dunnets's test.

423N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

depletion as amarker for oxidative damage after abrin exposure, we es-timated the activity of glutathione linked antioxidant enzymes GR, GSTand primary antioxidant enzymes catalase and SOD were estimated. Inthe present study, though there was initial increase in all antioxidantenzymes levels, followed by significant decrease after abrin exposure.GSH is themost abundant redoxmolecule in cells and thus themost im-portant determinant of cellular redox status. The higher rate of GSH de-pletion may be attributed to quenching of ROS generated by abrintreatment. In contrast, other study has shown that glutathione is ex-truded from apoptosis cells in reduced form, before plasma membraneleakage indicating loss of GSH may be the cause of oxidative stress [9].GST is enzyme which prevents the generation of hydrogen peroxideand alkyl hydroperoxides in association with GSH and GR, as well asthe generation of more harmful metabolites such as the hydroxyl radi-cal. In the present study, decreased GST, GR activity in abrin treatedcells can be explained by the consumption of these enzymes during

the detoxification of reactive oxygen metabolites generated due toabrin. Further decrease in GSH content due to abrin exposure can simul-taneously decrease the activities of GST. Among the various antioxidantmolecules, SOD and catalasemutually function as important enzymes inthe elimination of ROS. Decrease in SOD and catalase activities after ini-tial rise may be due to the overproduction of superoxide radical anionsand intracellular hydrogen peroxide, which were eliminated by in-creases SOD and catalase activity by both antioxidants in Jurkat cells. Al-tered antioxidant enzyme levels were also reported to be responsiblefor oxidative stress in mice and rat after ricin treatment [20,31]. Oxida-tive stress in the form of ROS generation in the cells has been shown tonot only affecting cell proliferation and apoptosis but also enhance sig-naling related to inflammation and stress [24,34].

In our present study, suppression of abrin induced TNF-α, IFN-γlevels by both antioxidants confirms, ROS accumulation could be oneof the reasons in inflammatory cytokine production as shown by others

Cas

pas

e-3

acti

vity

(nm

ol/A

MC

/min

/mg

pro

tein

)

0

20

40

60

80

C Abrin 200 400 600 200 400 600Concentration (μg/ml)

ControlAbrin 2 ng/mlNAC+Ab 2ng/mlTrolox+Ab 2 ng/ml

*

*

* **

*

#

# # #

#

#

Fig. 10. Effect of NAC and trolox on caspase-3 activity in abrin treated cells. The cells wereleft untreated or treated with only abrin or pre-treated for 1 h with NAC and trolox andthen incubated with 2 ng/ml of abrin. Caspase-3 activities in cell lysate are estimated byfluorometric assay. An increase in activated caspase-3was observedwith abrin treatment.NAC and trolox significantly reverse the abrin induced caspase-3 activation. ⁎p b 0.05significantly different from untreated cells and #p b 0.05 significantly different fromabrin treated cells.

424 N. Saxena et al. / International Immunopharmacology 21 (2014) 412–425

[27]. NAC and trolox also inhibits nuclear translocation of NF-κB whichcould be translocated to the nucleus due to mitochondrial stress gener-ated by abrin similar to that shown in another study where mitochon-drial stress releases NF-κB dimers from Iκ-B resulting in nucleartranslocation and further up-regulation of target gene [4]. Other possi-bility is antioxidants can only inhibit TNF mediated NF-κB activationby lowering the affinity of TNF receptor to TNF [12].

Our study shows the significant increase in SAPK/JNK activity both atmRNA and protein level. SAPK activation may occur in response to di-rectly by abrin stimuli or by abrin induced pro-inflammatory cytokinesor by ROS accumulation activated as reviewed by Thannickal andFanburg, where ROS generation is responsible for SAPK activationresulting in altered gene expression [39]. In our findings, both antioxi-dants effectively suppress the stress induced by abrin. But trolox wasfound to be more effective compared to NAC as trolox significantly sup-press c-Fos, c-Jun and phosphorylated form of SAPK/JNK.

Caspase-3 is considered to be a final executioner of apoptosis be-cause it generally participates in all major apoptotic pathways. Abrin in-duced mitochondrial pathway of apoptosis has been shown to activatecaspase-3 and DNA damage [29]. Similarly in our study abrin signifi-cantly enhances the caspase-3 activity which was abrogated by NACand trolox. Here, our findings have led us to propose that abrin inducesloss of mitochondrial membrane integrity and ROS accumulationwhichfurther depletes antioxidant enzyme levels and is likely to be criticalstep in the induction of apoptosis. It can be concluded that initial oxida-tive stress may be responsible for all downstream events including in-flammation, SAPK activation and caspase-3 activation [11]. Activationof downstream pathways further generates ROS production and formsa self-sustaining loop and could be responsible for vicious circle of tox-icity. Similar to pancreatic β-cell line MIN6N8 cells where TNF-α andIFN-γ synergistically activate SAPK/JNK andwhich resulted in the accu-mulation of ROS and loss of mitochondrial membrane potential [18].ROS are also thought to play role in the Fas-mediated, extrinsic apopto-tic pathway via JNK-mediated induction of Fas L or Fas R expression [3].In our previous publication Fas pathway has been shown to involve inabrin induced toxicity [33]. It may be possible that NAC and trolox sup-press abrin induced Fas pathway as reported in previous studies [1,15].It could be speculated from our findings that both compounds target atvery initial point of abrin induced oxidative stress and suppresses allsecondary consequences by modifying the signal transduction path-ways. Taken together, our findings suggest that NAC and trolox are

potential therapeutic agents against the abrin toxin and may have im-portant implication for the management of abrin poisoning.

Acknowledgments

This workwas supported by theMinistry of Defence, Government ofIndia and the studywas carried out at DRDE, Gwalior. The authors thankProf (DR)M P Kaushik, Director, DRDE for providing the necessary facil-ities, support and motivation. The authors also thank Dr Pravin Kumar,Head, Pharmacology & Toxicology for his constant support.

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