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Chemico-Biological Interactions 183 (2010) 34–39

Contents lists available at ScienceDirect

Chemico-Biological Interactions

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tereospecificity in the cytotoxic action of hexachlorocyclohexane isomers

nup Srivastavaa,∗, T. Shivanandappab

Department of Pathology, Center for Free Radical Biology, 901, 19th St. S., Rm #347, University of Alabama at Birmingham, Birmingham, AL 35294, USADepartment of Food Protectants and Infestation Control, Central Food Technological Research Institute, Mysore 570020, Karnataka, India

r t i c l e i n f o

rticle history:eceived 31 July 2009eceived in revised form0 September 2009ccepted 30 September 2009vailable online 8 October 2009

a b s t r a c t

Hexachlorocyclohexane (HCH) is a highly recalcitrant organochlorine insecticide known for its chronictoxicity. In spite of many isolated studies a clear mechanism of cytotoxic action of HCH and the struc-ture–toxicity relationship of its isomers is not well understood. We have investigated the toxicity of HCHisomers and its mechanism in Ehrlich Ascites tumor (EAT) cells. Our studies show differential cytotoxi-city of HCH isomers (�, �, �, and �), � isomer being most toxic and � the least. HCH-induced cell deathwas associated with induction of reactive oxygen species (ROS) formation, lipid peroxidation (LPO), and

eywords:exachlorocyclohexanexidative stressa+,K+-ATPasea2+,Mg2+-ATPaseADPH oxidase

depletion of glutathione (GSH). The increase in oxidative stress was linked with increased NAD(P)H oxi-dase activity. HCH inhibited Na+,K+-ATPase, which could be involved in raising the intracellular calciumand increased Ca2+,Mg2+-ATPase activity. HCH lead to apoptotic as well as necrotic cell death as it wasmarked by increased caspase-3 activity and lactate dehydrogenase (LDH) leakage, respectively. Based onthe results it is concluded that the HCH isomers inflict differential cytotoxicity which was highest by �and lowest by �. Further, this study demonstrates for the first time a clear link between Na+,K+-ATPase,

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. Introduction

Hexachlorocyclohexane (HCH), an organochlorine pesticide, haseen widely used in agriculture, public health, and in ectoparasiteontrol [1]. The technical HCH is a mixture of atleast five distincttereoisomers namely alpha, beta, gamma, delta, and epsilon. Ofhese only the gamma isomer also called “lindane” is the mainnsecticidal component [2]. At acute doses, HCH induces neurotoxicffects such as convulsive seizures and increased neuronal activity3], enhanced transmitter release [4], alterations in the activities ofcetylcholinesterase [5], Na+,K+-ATPase [6] and Mg2+-ATPase [7].t subchronic exposure, HCH is reported to induce changes in neu-otransmitter levels [8]. Organochlorine pesticides including HCHnduce oxidative stress in neural tissues of rat [9]. Involvement ofOS has been postulated as a possible mechanism for HCH toxicity10].

The biological activities of HCH isomers depend on theirtereoisomeric structure. Their solubility in phospholipids has theollowing order � > � > � > � [11]. This is similar to the order of

egree of inhibition of activities of phosphatidyl inositol synthasend other membrane-associated enzymes [12]. Pharmocologicaltudies in mammals have shown that � and � isomers are neurode-ressants [13]. � and � isomers have higher inhibiting properties

∗ Corresponding author. Tel.: +1 205 975 9576; fax: +1 205 975 7447.E-mail address: anup@uab.edu (A. Srivastava).

009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2009.09.026

ss in HCH-induced cytotoxicity.© 2009 Elsevier Ireland Ltd. All rights reserved.

for GABA-mediated chloride channels in the brain than the � and� isomers [14]. HCH isomers differentially stimulate expression ofhsp70 in larval tissues of Drosophila melanogaster, the highest by �than � and weakly by � and � [15]. HCH isomers exert cytotoxicaction by stimulating release of calcium from dantrolene-sensitivestores by � and in contrast from dantrolene-insensitive stores by �[16]. �, �, and � HCHs but not �-HCH are potent stimuli for the pro-duction of superoxide anion and the release of calcium in humanpolymorphonuclear leuckocytes [17].

In the present view of differences in the toxic mechanismsand lack of clear understanding of molecular events in the cyto-toxicity of HCH isomers, this study deals with the comparativestructure–toxicity relationship of the four isomers of HCH, viz., �, �,�, and � (Fig. 1), in Ehrlich Ascites tumor (EAT) cells and their pos-sible mode of action Epsilon isomer was not included in this studybecause of its lack of availability (minor constituent of technicalHCH, <0.6%).

2. Materials and methods

2.1. Chemicals

Adenosine triphosphate (ATP), oubain, trypan blue, thio-barbituric acid, bovine serum albumin (BSA), FURA-2AM, 2,7-dichlorofluorescein diacetate (DCFH-DA), caspase-3 assay kit,tetraethoxy propane were purchased from Sigma Chemical Co. (St.Louis, MO, USA). Nitroblue tetrazolium (NBT), trichloroacetic acid

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TCA), NAD, NAD(P)H, DMSO and other chemicals were purchasedrom Sisco Research Laboratories, Mumbai, India. HCH isomerspurity >98%) were kindly supplied by Hindustan Insecticides Ltd.,

umbai.

.2. EAT cells

Ehrlich Ascites tumor (EAT) cells were cultured in the peri-oneum of male Swiss albino mice [18]. After harvesting, cells wereuspended in Hanks balanced salt solution (HBSS) with 0.1% dex-rose and 0.4% bovine serum albumin. Cells were used for thexperiments within 6 h after harvesting and experiments wereone for only 60 min to understand the molecular mechanism ofcute toxicity. EAT cells were chosen because they offer a goododel to study ROS induction and consequent oxidative stress by

enobiotics (our unpublished work).

.3. Cytotoxicity

EAT cells (10 × 106) suspended in 1 ml HBSS were treated withCH isomers (dissolved in DMSO) at various concentrations and

ncubated for 60 min in shaking water bath at 37 ◦C. Parallel controlncubations were done with the solvent (DMSO, 20 �l/ml) also. Athe end of incubation, an aliquot of cells was taken for viabilityssay by trypan blue exclusion method [19]. DMSO was not toxico cells at the concentrations used and did not interfere with otherssays performed.

.4. Lactate dehydrogenase (LDH) leakage

After incubation in the presence of HCH isomers, cells were cen-rifuged and the supernatant was assayed for LDH activity withactate as the substrate [20].

.5. Reactive oxygen species (ROS)

The formation of intracellular ROS was measured using 2,7-ichlorofluorescein diacetate (DCFH-DA). DCFH-DA enters cellsnd is hydrolyzed to membrane-impermeant dichlorofluorescein,hich reacts with ROS to form the highly fluorescent dichloroflu-

rescein (DCF). EAT cells (10 × 106) were loaded with DCFH-DA

5 �M) in HBSS for 30 min at 37 ◦C, washed, and then followed byreatment with HCH isomers (1 mM) for 60 min. Fluorescence ofhe generated DCF was then observed using a spectrofluorometert excitation and emission wavelengths of 485 and 535 nm, respec-ively. The amount of ROS formed was expressed as fluorescencenits/10 × 106 cells.

ological Interactions 183 (2010) 34–39 35

2.6. Lipid peroxidation (LPO)

After incubation with HCH isomers (1 mM), the cells were cen-trifuged and the cell pellet was washed in saline (2×) and boiledin TCA (5.5%) and TBA (0.34%) for 15 min, cooled and centrifuged.Fluorescence of the supernatant was measured in a fluorescencespectrophotometer at excitation and emission wavelengths of 532and 553 nm, respectively [21]. Lipid peroxidation was quantifiedby the amount of malondialdehyde (MDA) formed which was cal-culated using a standard curve prepared with tetraethoxypropane.

2.7. Glutathione (GSH)

Cells (10 × 106) suspended in 1 ml of HBSS were treated withHCH isomers (1 mM) and incubated for 60 min in a shaking waterbath at 37 ◦C. After incubation the cells were washed with phys-iological saline, homogenized in 1 ml tris–HCl buffer (0.1 M, pH7.4), centrifuged at 2000 × g at 4 ◦C for 5 min and glutathione wasestimated (using GSH standard) in the deproteinized (5% TCA)supernatant by Ellman’s reagent [22] and represented as nmole/mgprotein.

2.8. NAD(P)H oxidase activity (NOX)

Cells (10 × 106) suspended in HBSS were incubated with HCHisomers (1 mM), washed with physiological saline and homog-enized in tris–HCl buffer (0.1 M, pH 7.4). The homogenate wascentrifuged at 2000 × g at 4 ◦C for 10 min and the supernatant wasused for estimating the NAD(P)H oxidase activity, modified fromSanner et al. [23]. Reduction of nitro blue tetrazolium (NBT, 0.2 mM)by the enzyme using NAD(P)H (3 �M) as the substrate in phosphatebuffer (0.1 M, pH 7.4) was monitored at 560 nm, and the values areexpressed as �A/min/mg protein.

2.9. ATPase activity

Cells (10 × 106) suspended in HBSS were incubated with HCHisomers (1 mM), washed with physiological saline and homog-enized in tris–HCl buffer (0.1 M, pH 7.4). The homogenate wascentrifuged at 2000 × g at 4 ◦C for 10 min and the supernatant wasused for estimating the ATPase activity. For assaying Ca2+,Mg2+-ATPase activity the reaction mixture contained tris–HCl buffer(30 mM, pH 8.0), MgCl2 (2 mM), EGTA (1 mM), NaCl (0.6 M) and KCl(0.3 M) with CaCl2 to give a final concentration of 5 �M in a final vol-ume of 1 ml and preincubated at 37 ◦C for 10 min. The reaction wasstarted by adding ATP (5 mM), incubated for 30 min, and stopped byadding TCA (10%) followed by centrifugation at 2000 × g at 4 ◦C for10 min. Phosphate content in the supernatant was estimated [24].The Na+,K+-ATPase activity corresponded to the activity inhibitedby 1 mM ouabain [25]. The ATPase activities were represented byamount of inorganic phosphate formed per mg protein.

2.10. Intracellular free Ca2+

The intracellular calcium concentration, i[Ca2+], was measuredusing fura-2AM. Fura-2AM crosses cell membranes and onceinside the cell, the acetoxymethyl groups are removed by cellu-lar esterases to generate Fura-2, the fluorescent calcium indicator.EAT cells were incubated in calcium free HBSS with 5 �M fura-2AM

at 37 ◦C for 30 min in shaking water bath. After washing (2×) withcalcium free HBSS, the cells were suspended in complete HBSS andtreated with HCH isomers (1 mM) for 60 min in shaking water bath.Fluorescence was measured with spectrofluorometer at an emis-sion wavelength of 500 nm for dual excitation wavelength at 340and 380 nm [26]. The i[Ca2+] was expressed as nmole/10 × 106 cells.

3 ico-Biological Interactions 183 (2010) 34–39

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.11. Caspase-3 activity

Cells (10 × 106) after treatment with HCH isomers (1 mM) wereysed in caspase assay buffer containing 50 mM HEPES (pH 7.5),00 mM NaCl, 2 mM EDTA, 0.1% CHAPS, 10% sucrose and 5 mM DTT.he caspase-3 activity was measured using a kit (Caspase-3 Assayit, Sigma–Aldrich, St. Louis, MO, USA), according to the manufac-

urer’s protocol. The caspase-3 fluorometric assay is based on theydrolysis of acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarinAc-DEVD-AMC) by caspase-3, resulting in the release of the fluo-escent 7-amino-4-methylcoumarin (AMC) [27]. The excitation andmission wavelengths of AMC are 360 and 460 nm, respectively.he concentration of the AMC released can be calculated from aalibration curve prepared with AMC standards. The activity wasxpressed as nanomoles AMC released per minute per milliliter ofhe cell lysate.

Protein content was measured by the method of Lowry et al.28] using BSA as the standard.

.12. Statistical analysis

All the data are expressed as mean ± S.E. of three separatexperiments performed in duplicate and significant difference wasetermined by Duncan’s multiple range test (DMRT) using the com-uter programme Excel and Statistica software (1999).

. Results

.1. Cytotoxicity

EAT cells treated with HCH isomers showed differential cyto-oxicity. The order of cytotoxicity of HCH isomers was � > � > � � �t equimolar concentration. The LC50 values of �, �, �, and � were.64, 1.34, 1.77, and >8 mM, respectively (Fig. 2). The LDH leakagerom EAT cells in the media post-HCH treatment correlated withhe cytotoxicity. For further understanding the mechanism of acuteytotoxicity of HCH isomers equimolar concentration (1 mM) wassed.

.2. ROS generation

HCH isomers (at equimolar concentration) caused generationf ROS in EAT cells which was in the order � > � > � > � (Table 1).imilar results were obtained with NBT reduction assay (data nothown).

able 1ifferential effect of HCH isomers (1 mM) in EAT cells.

Parameter studied Control �-HCH

Lactate dehydrogenase leakagea 0.023a ± 0.0009 0.045c ± 0.002Reactive oxygen species generationb 22.0a ± 1.3 40.8c ± 3.7Lipid peroxidationc 25.6a ± 1.3 45.3b ± 2.6GSH contentd 345.0a ± 10.8 235.2b ± 10.9NAD(P)H oxidase activitye 0.104a ± 0.0051 0.222b ± 0.008Na+,K+-ATPase activityf 486.3a ± 13.1 386.0c ± 13.2Ca2+,Mg2+-ATPase activityg 547.3a ± 16.3 738.5b ± 18.2Intracellular Ca2+h 362.8a ± 18.1 634.2b ± 28.2Caspase 3 activityi 140.6a ± 14.2 350.0b ± 15.9

umbers with different suffix letters differ significantly at p < 0.05.a NADH (nmol/10 �l supernatant).b Fluorescence units/10 × 106 cells.c MDA (nmol/10 × 106 cells).d GSH (nmol/mg protein).e �A560 nm/min/mg protein.f Pi (ng)/mg protein.g Pi (ng)/mg protein.h nmol/10 × 106 cells.i AMC (nmol/min/ml).

Fig. 2. Differential toxicity of HCH isomers in EAT cells.

3.3. LPO

HCH isomers (at equimolar concentration) induced LPO exceptfor the � isomer. LPO induction was highest by � isomer followedequally by � and � isomers (Table 1).

3.4. GSH

GSH content reduced after HCH isomer exposure in EAT cells.The order of decrease in GSH by HCH isomers at equimolar con-centration was in the order � > � = �, with � isomer not causing anysignificant decrease (Table 1).

3.5. NAD(P)H oxidase

NADPH oxidase activity increased after HCH isomer exposurein EAT cells. The increase in activity by HCH isomers at equimolarconcentration was in the order � > � = �, with � isomer not showingany effect (Table 1).

3.6. Na+,K+-ATPase

HCH isomers inhibited the Na+,K+-ATPase, and the inhibition bythe isomers was in the following order � > � > � > � at equimolarconcentration (Table 1).

�-HCH �-HCH �-HCH

0 0.032b ± 0.0019 0.070d ± 0.0017 0.133e ± 0.006526.4b ± 2.0 59.8d ± 5.2 137.5e ± 6.827.3a ± 1.5 43.6b ± 2.4 106.9c ± 4.4

334.6a ± 10.7 218.4b ± 7.9 153.8c ± 10.43 0.115a ± 0.0086 0.233b ± 0.0086 0.311c ± 0.0094

454.9b ± 15.5 273.1d ± 11.2 43.3e ± 5.5560.3a ± 18.9 762.1b ± 24.1 914.4c ± 30.7365.1a ± 23.9 865.4c ± 36.7 1232.7d ± 48.7158.1a ± 15.5 460.6c ± 18.7 753.0d ± 26.6

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.7. Ca2+,Mg2+-ATPase

Ca2+,Mg2+-ATPase activity increased after HCH isomer exposuren EAT cells. The increase in activity by HCH isomers at equimolaroncentration was in the order � > � = �, with � isomer not showingny effect (Table 1).

.8. Intracellular calcium (i[Ca2+])

There was a raise in the intracellular calcium levels after treat-ent with HCH isomers except the � isomer. HCH isomers raised

he i[Ca2+] in the following order � > � > � at equimolar concentra-ion (Table 1).

.9. Caspase-3 activity

Caspase-3 activity increased after HCH isomer exposure in EATells. The increase in activity by HCH isomers at equimolar concen-ration was in the order � > � > �, with � isomer not showing anyignificant effect (Table 1).

. Discussion

In vitro studies on cell cultures offer a good model system totudy the molecular mechanism of xenobiotic-induced cell death.CH is known to induce cell injury/death in mammalian cells suchs neuroactive (PC-12) cells, cerebellar granule neurons and alve-lar macrophages [16,29]. Our cytotoxicity studies show that theytotoxicity of HCH is stereospecific. The comparison of LC50 valuesf HCH isomers shows following order � > � > � � � at equimolaroncentration, which is in agreement with earlier reports [30].

HCH isomers inhibited Na+,K+-ATPase which followed the samerend as that of cytotoxicity at equimolar concentration. Our earliertudy demonstrates a correlation between cytotoxic action of HCHnd inhibition of Na+,K+-ATPase in EAT cells [10]. Several studiesave shown in vitro inhibition of Na+,K+-ATPase by organochlorineesticides. Srivastava et al. [6] have reported marked inhibition ofa+,K+-ATPase in the testicular plasma membrane of rats adminis-

ered with HCH. The primary role of Na+,K+-ATPase is to maintainhe homeostasis of Na+ and K+ ions at the expense of ATP in eukary-tic cells. The ionic gradients formed by the Na+,K+-ATPase areritical in regulating osmotic balance, cell volume, cytoplasmicH (through the Na+/H+ exchanger), Ca2+ levels (by the action ofa+/Ca2+ exchanger), and Na+-coupled transport of nutrients andmino acids into the cells. It is reported that inhibition of Na+,K+-TPase causes rise in intracellular calcium levels [31]. Probably

t is the inhibition of sodium pump that alters the sodium gra-ient and influences the activity of the Na+/Ca2+-exchanger, thus

eading to increased cytosolic Ca2+ [32]. A rise in i[Ca2+] occurshrough Ca2+ influx across the plasma membrane and/or througha2+ release from intracellular stores which leads to the activa-ion of Ca2+ dependent cellular signaling. In this study we observedncreased i[Ca2+] levels by HCH isomer treatment which corre-ponds to the cytotoxicity pattern of the isomers. HCH isomersave been shown to increase i[Ca2+] levels in neurohybridomaells [33]. It has also been shown that �-HCH causes rise in i[Ca2+]y dantrolene-sensitive pools but �-HCH does it by dantrolene-

nsensitive pools [16].HCH isomers, except �, increased ROS production through

ncreased activity of NADPH oxidase (NOX). It is reported that lin-

ane stimulates the production of superoxide anion by altering thectivity of NOX in macrophages [17]. It has been shown that lindaneffects NOX activity by acting on intracellular calcium homeostasis34]. Lindane treatment resulting in a large increase in intracy-osolic calcium levels can activate PKC which directly stimulates

ological Interactions 183 (2010) 34–39 37

NOX and ROS production [35]. NOX enzymes may be activatedthrough Ca2+ signaling. However, the reverse situation also occurs.A ROS-dependent regulation of heterologously expressed neuronalP/Q-type voltage dependent Ca2+ channels has been reported [36].ROS can also induce a rise in i[Ca2+] through Ca2+ release fromintracellular stores [37]. The Ca2+ release channels of the ryan-odine receptor family, which possess reactive cysteine residues, arehighly sensitive to oxidation by ROS [38]. Activation of such Ca2+

release channels has been demonstrated for NOX-dependent ROSgeneration [39]. Further, NOX-derived ROS increase neuronal Ca2+

influx by increasing the opening of voltage-dependent L-type Ca2+

channels [40].Oxidative modifications of proteins involved in the control

of intracellular Ca2+ homeostasis leads to cytotoxic increasesof the i[Ca2+] [41]. ROS modulate the activity of Ca2+-ATPasepumps [42] in a bimodal fashion. The ROS-dependent Ca2+-ATPasepump activation involves post-translational processing, namely,S-glutathiolation [43]. An induction in the activity of Ca2+,Mg2+-ATPase was observed post-HCH isomer treatment (except with �)which may be indicative of ROS-mediated modification. At higherHCH concentration there was complete inhibition of Ca2+,Mg2+-ATPase (data not shown) which may be explained by a strongeroxidative stress which leads to an irreversible oxidation of thiolsand thereby to enzyme inhibition [37].

Endogenous antioxidant, GSH, is an abundant and ubiquitouslow-molecular weight thiol with putative roles in many cellularprocesses, such as amino acid transport, synthesis of proteins andmetabolism of xenobiotics, carcinogens and ROS. Low concentra-tions of GSH and increased LPO have been used as an index ofincreased oxidative stress [44]. HCH isomers, except �, depletedGSH and increased LPO in cells contributing to the oxidative stress.We have earlier shown in EAT cells that HCH causes reduced antiox-idant status by depletion of GSH and decreased SOD and catalaseactivities and increased LPO [10]. The mechanism by which HCHisomers cause lipid peroxidation may involve cytochrome P 450-dependent microsomal metabolism in which a reactive metabolitesuch as pentachlorocyclohexene (PCCH) is produced apart from theROS generated [45].

The cytotoxicity induced by HCH isomers was found to be byapoptosis, through the induction caspase-3, as well as by necro-sis which was evidenced by LDH leakage. Kang et al. [29] haveshown that lindane induces apoptosis in HL-60 cells, which wasfound to be related to increased i[Ca2+], mainly through intracellu-lar Ca2+ stores. Recent evidence demonstrates that increased i[Ca2+]can induce either apoptotic or necrotic cell death [46]. ROS gen-eration has also been shown to be associated with the inductionof apoptosis in neuronal and other cell types [47]. Lindane cyto-toxicity is reported to be associated with high i[Ca2+] and ROS[48]. We have earlier shown that ROS scavengers, quercetin andellagic acid, can only partially prevent HCH-induced cytotoxicity[10,49]. Na+,K+-ATPase may be the perpetrator of HCH cytotoxicityas it can lead to increased i[Ca2+] and ROS. It has been shown thatouabain (Na+,K+-ATPase inhibitor)-induced elevation of both ROSand cytosolic [Ca2+] in SH-SY5Y cells synergistically triggers theapoptotic events through cytochrome C release and activation ofcaspase-3 [32]. But at higher ROS concentrations, hydrogen perox-ide can inhibit caspases and thereby lead to a switch from apoptosisto necrosis [50].

Based on the results it can be concluded that the differentialcytotoxicity of HCH isomers, highest by � and lowest by �, involvesinhibition of Na+,K+-ATPase and oxidative stress (Fig. 3). Inhibi-

tion of Na+,K+-ATPase (cell surface) may be a primary event inHCH toxicity. Induction of NOX activity and consequent genera-tion of ROS by HCH leads to oxidative stress which is marked bydepletion of GSH and increased LPO. Inhibition of Na+,K+-ATPaseand increased oxidative stress leads to raised i[Ca2+], which in turn

38 A. Srivastava, T. Shivanandappa / Chemico-Bio

Fig. 3. HCH-induced cytotoxicity: HCH causes cytotoxicity by increased ROS pro-duction (respiratory burst), inhibiting the Na+,K+-ATPase, and raised intracellularcaNS

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alcium levels. Increased oxidative stress and intracellular calcium can then lead topoptotic cell death independently or concomitantly. HCH, Hexachlorocyclohexane;OX, NAD(P)H oxidase; ROS, reactive oxygen species; i[Ca2+], intracellular calcium;OD, superoxide dismutase; CAT, catalase; LPO, lipid peroxidation.

an lead to increased ROS production. The increase in i[Ca2+] andOS leads independently or synergistically to apoptotic/necroticell death by HCH. Even though several aspects of HCH toxicityre known we have for the first time demonstrated a clear associ-tion between Na+,K+-ATPase, i[Ca2+] level and oxidative stress inCH-induced cytotoxicity. We suggest that the cytotoxicity patternf HCH isomers could be dependent on their membrane solubilityhich leads to differential degree of disruption of the plasma mem-

rane bound signaling cascade and thereby inducing differentialytotoxicity.

onflict of interest

The authors declare that there are no conflicts of interest.

cknowledgements

This work was carried out at Central Food Technologicalesearch Institute, Mysore, India. The authors wish to thank theirector of the institute for his keen interest in this study. The firstuthor acknowledges the CSIR, New Delhi for awarding the researchellowship. This work was supported by the financial assistance ofepartment of Biotechnology, Govt. of India.

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