quercetin in vesicular delivery systems: evaluation in combating arsenic-induced acute liver...
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Chemico-Biological Interactions 186 (2010) 61–71
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Chemico-Biological Interactions
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uercetin in vesicular delivery systems: Evaluation in combatingrsenic-induced acute liver toxicity associated gene expression in rat model
ebasree Ghosha, Swarupa Ghosha, Sibani Sarkara, Aparajita Ghosha, Nirmalendu Dasa,∗,rishna Das Sahab, Ardhendu K. Mandala,∗∗
Biomembrane Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, IndiaInfectious Diseases and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India
r t i c l e i n f o
rticle history:eceived 27 January 2010eceived in revised form 26 March 2010ccepted 29 March 2010vailable online 4 April 2010
eywords:rseniceactive oxygen speciesxidative attackibrosisene expressioniposomal or nanocapsulated QC
a b s t r a c t
Arsenic, the environmental toxicant causes oxidative damage to liver and produces hepatic fibrosis. Thetheme of our study was to evaluate the therapeutic efficacy of liposomal and nanocapsulated herbalpolyphenolic antioxidant Quercetin (QC) in combating arsenic induced hepatic oxidative stress, fibrosisassociated upregulation of its gene expression and plasma TGF ß (transforming growth factor ß) in ratmodel.
A single dose of Arsenic (sodium arsenite-NaAsO2, 13 mg/kg b.wt) in oral route causes the generationof reactive oxygen species (ROS), arsenic accumulation in liver, hepatotoxicity and decrease in hepaticplasma membrane microviscosity and antioxidant enzyme levels in liver. Arsenic causes fibrosis asso-ciated elevation of its gene expression in liver, plasma TGF ß (from normal value 75.2 ± 8.67 ng/ml to196.2 ± 12.07 ng/ml) and release of cytochrome c in cytoplasm. Among the two vesicular delivery sys-tems formulated with QC, polylactide nanocapsules showed a promising result compared to liposomal
delivery system in controlling arsenic induced alteration of those parameters. A single dose of 0.5 mlof nanocapsulated QC suspension (QC 2.71 mg/kg b.wt) when injected to rats 1 h after arsenic adminis-tration orally protects liver from arsenic induced deterioration of antioxidant levels as well as oxidativestress associated gene expression of liver. Histopathological examination also confirmed the pathologicalimprovement in liver. Nanocapsulated plant origin flavonoidal compound may be a potent formulation incombating arsenic induced upregulation of gene expression of liver fibrosis through a complete protectionn hep
against oxidative attack iAbbreviations: QC, quercetin; NaAsO2, sodium arsenite; ROS, reactive oxygenpecies; 4-HP, 4-hydroxy-proline; O2
•− , superoxide; •OH, hydroxyl radical; ROO• ,eroxyl radicals; H2O2, hydrogen peroxide; GPx, glutathione peroxidase; GR, glu-athione reductase; GST, glutathione-S-transferase; G6PDH, glucose-6-phosphateehydrogenase; SOD, superoxide dismutase; PE, phosphatidyl ethanolamine; DPH,iphenyl hexatriene; AST, serum aspartate transaminase; AP, alkaline phosphatase;M-H2DCFDA, 5-(and-6)-chloromethyl-2′ ,7′-dichlorodihydro-fluorescein diacetatecetyl ester; TGF ß, transforming growth factor beta.∗ Corresponding author at: Indian Institute of Chemical Biology, 4, Raja S.C. Mul-
ick Road, Jadavpur, Kolkata 700032, India. Tel.: +91 33 2499 5825/5715;ax: +91 33 2473 5197/2472 3967.∗∗ Corresponding author. Present address: Graduate Centre for Toxicology, Uni-ersity of Kentucky, 232 Bosomworth, Lexington, KY 40536-0001, United States.el.: +1 8592574054; fax: +1 8593231059.
E-mail addresses: [email protected] (D. Ghosh),[email protected] (S. Ghosh), sibani [email protected] (S. Sarkar),parajita [email protected] (A. Ghosh), [email protected] (N. Das),rdhendu [email protected] (A.K. Mandal).
009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.03.048
atic cells of rat liver.© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Arsenic is one of the most important global environmental toxi-cants and its exposure in humans comes mainly from consumptionof drinking water contaminated with inorganic arsenic [1].
Acute exposure to arsenite has been reported to induce deathand/or multiple organ failure with symptoms including nau-sea, vomiting, diarrhoea, gastrointestinal haemorrhage, cerebraledema, tachycardia, dysrhythmias and hypovolemic shock.
Arsenic induced hepatic injury is known to be exerted throughexcess production of reactive oxygen species, namely superoxide(O2
•−), hydroxyl (•OH), and peroxyl (ROO•) radicals and hydrogenperoxide (H2O2) [2]. The harmful expressions of arsenic are pri-marily due to an imbalance between pro-oxidant and antioxidanthomeostasis in physiological system and also due to its fascina-
tion to bind sulfhydryl groups of proteins and thiols of glutathione(GSH). Arsenic initiates a rapid depletion of antioxidant enzymessuch as glutathione peroxidase (GPx), glutathione reductase (GR),glutathione-S-transferase (GST), glucose-6-phosphate dehydroge-nase (G6PDH), superoxide dismutase (SOD) and catalase [3].
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Arsenic-generated oxidative insult eventually leads to fatty livernd hepatic fibrosis in rats as well as human [4,5]. Peroxidativeamage of membrane lipids plays an important role in free radical-
nduced excess collagen deposition and fibrogenesis. Molecularvaluation of pro-collagen gene expression of arsenic induced liveroxicity leading to fibrogenesis is very important for providing per-pective on therapy development. Upregulation of transformingrowth factor ß (TGF ß) has been implicated in the pathogenesis ofbrosis in rats [6]. TGF ß stimulate fibroblast and produce collagennd inhibit collagen degradation. Arsenic exposure also acceler-tes type I pro-collagen mRNA expression, responsible for liverbrogenesis [7]. However, exact mechanism(s) by which arsenic
nduces hepatic fibrosis still remains unclear. The critical eventn hepatic fibrosis is the activation of lipocytes (stellate, fat stor-ng, or Ito cell), the main source of extracellular matrix in fibrosisormation [8]. All these effects have been verified in rats. Rats,nlike other mammals, retain the dimethyl arsenic acid, one ofhe key intermediate metabolites of NaAsO2 in the blood. Stud-es were performed in this report on rats because this species isnown to be superior to others in forming monomethyl arsenouscid, the most toxic metabolic intermediates of NaAsO2. Withinh after NaAsO2 injection (50 �mol/kg b.wt, i.v.), rats excreted0% of the injected arsenite dose, whereas mice excreted only1% of the dose [9]. Thus arsenic metabolic rate is high in ratompared to mice. Monomethyl arsenous acid is produced as inter-ediate during arsenic metabolism in rats as well as human [10].onomethyl arsenous acid (MMAsIII) has been found in the urine of
8% of humans chronically consuming arsenic-contaminated watern West Bengal, India [11].
Quercetin, a polyphenolic flavonoid, present in large amounts inegetable, fruits, tea and olive oil, has been used to serve as a stressrotectant for its ability to interact with free radicals involved inxidative damage and progression of fibrogenesis in liver [12,13].
But simple flavonoidal compound therapy is not an effectivepproach to counter the oxidative damage, for poor availability ofntioxidant to interact with hepatocellular membranes. For opti-um efficacy, antioxidant should be targeted to either hepatocytes
r Kupffer cells of liver. To maximize the therapeutic effect and min-mize systemic toxicity, the sufficient dose of antioxidant shoulde specifically delivered to a target with minimal exposure to non-arget cells [14].
Hence, it is essential to develop a delivery system for vector-ng antioxidants to hepatic cells [15]. In this context, liposomesre accepted as a potent drug vehicle not only for increasing thefficacy of the drug and decreasing the toxicity of the same butlso due to the feasibility of incorporating both hydrophobic andydrophilic substances, can possess a surface charge, have bio-ompatible nature and have non immunogenic property in theiological system. Nanoparticles are accepted as effective drug car-iers because of several important technological advantages overiposomes, e.g. long shelf life, high carrier capacity and feasibilityf variable routes of administration including oral route.
The aim of our work is to optimise the dose of flavonoidal com-ound QC in liposome or nanocapsule drug delivery system and toompare their biological effectiveness in combating acute arsenitenduced hepatocellular oxidative damage related to upregulation ofytokine TGF ß and fibrosis associated gene expression in rat liver.
. Materials and methods
.1. Materials
Poly-d-l-lactide, phosphatidyl ethanolamine (PE), diphenylexatriene (DPH), Triton x-100, glutathione reductase, quercetinere purchased from Sigma Chemicals (St. Louis, MO, USA). Sodium
nteractions 186 (2010) 61–71
arsenite from E. Merck, Germany (Suprapure, A.R. Grade) was usedfor experimental purpose. Chloramine-T, Fast Green FCF and SiriusRose BB were acquired from Loba-Chemie and Fluka respectivelywhereas chloroform and methanol were from E. Merck (India) Ltd.All other reagents were of analytical grade.
2.2. Preparation of liposomal QC
Multilamellar liposomes were prepared with PhosphatidylEthanolamine, Cholesterol, Dicetyl Phosphate (DCP) and Quercetin(QC) in molar ratio 7:1:1:1 [16]. In short PE, Cholesterol, DCP andQC are dissolved in chloroform and methanol mixture (2:1, v/v) ina round bottom flask. The lipid film was made by drying the organicsolvents and was desiccated over night. The thin film was swollen inPBS (pH 7.2) for 1 h and sonicated in a probe type sonicator for 60 s.The suspension was centrifuged (10,000 × g) for 1 h and washedtwice in PBS.
To estimate incorporation into liposomes, the pellet was dis-solved in methanol and an aliquot of 100 �l was taken and diluted to1 ml with PBS. The O.D. was measured at 369 nm. The total amountof quercetin (Dm = 0.03825 l mol−1 cm−1) in the liposomal form wascalculated from the concentration of the drug in the dissolved pel-let divided by the total amount of drug added during the liposomepreparation. The incorporation of quercetin in the liposome was89.9%.
2.3. Preparation of nanocapsules
In brief, 18 mg polylactide was dissolved in 10 ml ofdichloromethane. Phosphatidyl ethanolamine (26 mg) dissolved in250 �l of isopropylmyristate along with quercetin (4.5 mg) and wasadded to dichloromethane phase. The organic phase was addedslowly into 50 ml of 0.025 M PBS (pH 7.2) containing 0.4% of thenon-ionic surfactant (Tween 80), with moderate magnetic stirringfor approximately 3 h and the traces left, if any, removed by passinggaseous nitrogen for 5 min. The suspension was ultracentrifuged at105,000 × g in Sorval RC 5B Plus using the rotor Sorval T-865 for1 h. The pellet of nanocapsules was washed with PBS and collectedand re-suspended in 2 ml PBS [17].
To estimate the intercalation of nanocapsules, the pellets werethen dissolved in 2 ml of dichloromethane and kept for 3 days at4 ◦C. The O.D. was measured at �max 369 nm and % of incorpora-tion was calculated by following the above procedures. The percentintercalation of QC in nanocapsule was found to be 45% of the addedamount.
2.4. Determination of size of quercetin-encapsulatedliposomes/nanocapsules
The size determination of QC-encapsulated liposomes andnanocapsules was done through dynamic light scattering using amean laser wavelength of 632.2 nm and a 90◦-angle measurement.The average diameters of the liposomes and nanocapsulated QCwere measured to be 200 and 100 nm, respectively.
2.5. Animals and treatment
Adult male Swiss Albino rats, each weighing 120–150 g wereacclimatized to conditions in the laboratory (26–28 ◦C, 60–80%relative humidity, 12 h light/dark cycle) for 10 days prior to thecommencement of the treatment during which they received food
(commercial pelleted rat chow, purchased from Hindustan UnileverLimited, Maharashtra, India) and drinking water ad libitum. Foodand water were checked to be arsenic free before giving to ani-mals (mentioned under Section 2.13). Animals were randomlyselected for groups and arsenic and drug were administered as
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er individual body weight of rat. Rats were divided into sevenroups (seven cages) with five animals in each group. Cages weref polycarbonate type with husk type bedding. Rats in the nor-al group were fed a single oral gavage dose of physiological
aline (5 ml/kg b.wt). Nanocapsulated QC (2.71 mg/kg b.wt) sus-ension (5 ml/kg b.wt) was injected into tail vein of each rat ofecond normal group. Rats in the arsenic treated groups were fedith a single oral gavage dose of NaAsO2 (13 mg/kg b.wt). Only
rsenic fed group of animals were kept as control without any drugreatment. Free QC (2.71 mg QC/kg b.wt) suspension (5 ml/kg b.wt)f 0.2% Tween 80 was injected into tail vein of second arsenicreated group of rats (1 h after NaAsO2 treatment). Empty nanocap-ules (5 ml/kg b.wt suspension), liposomal QC and nanocapsulesntercalated with QC (5 ml/kg b.wt suspension containing 2.71 mgC/kg b.wt) were injected in the tail vein of rats in other threersenic fed groups (1 h after NaAsO2 treatment). All the rats usedn this study received proper care and handling in compliance withnimal Ethics Committee, India, Registration No.147/99/CPC SEA,
ndia. QC uptake by liver was estimated after 2 h of free QC, liposo-al and nanoencapsulated QC treatment.
.6. General procedures
Twenty-four hours after NaAsO2 administration the rats werenaesthetized with ether and blood was collected by cardiac punc-ure [18]. One part of the blood was taken for serum and the otherart for plasma. Serum and plasma were made. Serum was iso-
ated by following standard protocol. Blood was centrifuged at000 × g for 10 min at 4 ◦C to assay the activity of serum enzymes.lasma was made by adding the anticoagulant heparin followed byentrifugation. Serum aspartate transaminase (AST), alkaline phos-hatase (AP) [19,20] and serum urea and creatinine (mod.Berthelotethod and alkaline picrate method) were determined using a
tandard kit manufactured by Coral Clinical Systems; India. Thelasma was used for TGF ß measurement. After collection of blood,ll rats were decapitated and their livers were isolated promptlynd washed with cold physiological saline. A part of the organ wasmmediately fixed in Bouin’s fixative and processed for histologicalxamination. The other part was kept at −80 ◦C for further experi-ents. Liver histochemistry for collagen and liver tissue histologyere studied by microscopic examination [21].
.7. Different enzyme activities
A portion of the liver was homogenized in 0.25 M sucrose solu-ion. The homogenate was centrifuged at 8200 × g for 10 min. usingSorvall SS34 rotor. The supernatant obtained was again spun at05,000 × g for 1 h in an OTD-50B Sorvall ultracentrifuge (4 ◦C). Theupernatant from the second centrifugation was collected as theytosolic fraction of liver cells.
The assay of superoxide dismutase (EC1.15.1.1) in cytosolic frac-ions of liver homogenate was estimated by following the methodsf Beyer and Fridovech, 1987 using spectrophotometer with slightodifications. The activity of SOD was expressed in unit by assum-
ng that the activity of the enzyme as one unit which inhibited thenitial rate of reduction of 10 mM ferricytochrome C by 50% detectedn Rayleigh UV 2601 double beam spectrophotometer [22].
A part of the cytosolic fraction was used for the assay of cata-ase activity spectrophotometrically by the method of Moragon etl. [23] with slight modifications. The reaction mixture contained
odium phosphate buffer (0.05 M, pH 7.0), 50 mM−1 H2O2, and0 �l of enzyme extract in a 3-ml volume. The activity was assayedy monitoring the decrease in absorbance at 240 nm as a conse-uence of H2O2 consumption and enzyme activity expressed asmount of H2O2 decomposed per minute per milligram of protein.nteractions 186 (2010) 61–71 63
GPx activity from cytosolic fraction of liver was measured spec-trophotometrically by the method of Sarkar and Das [24]. Cytosolcontaining the enzyme source was mixed with 0.25 M potassiumphosphate buffer, 25 mM EDTA, glutathione reductase, 40 mM glu-tathione GSH), and 20 mM NADPH. The activity was determinedand expressed as �mol NADPH oxidized/min/protein.
GR was assayed by the method of Castro et al. [25] with slightmodifications. A 3-ml mixture contained 100 mM phosphate buffer(pH 7), 1 mM GSSG, 1 mM EDTA, and 0.1 mM NADPH. The rateof NADPH oxidation was followed by monitoring the decrease inabsorbance at 340 nm with a recording spectrophotometer. Theactivity was expressed as �mol of NADPH oxidation/min/mg pro-tein.
GST activity was determined spectrophotometrically using 1-Chloro-2-4-dinitrobenzene (CDNB) by the method of Maiti andChatterjee [26] using a spectrophotometer. The rate of formation ofCDNB–GSH complex was monitored at 340 nm and used to expressthe enzyme activity.
G6PDH activity was determined using a Sigma Diagnostics Kit bythe method of Iimuro et al. using a spectrophotometer with slightmodifications [27]. The rate of formation of NADPH is proportionalto G6PDH activity and was measured spectrophotometrically as anincrease in absorbance at 340 nm. One unit of G6PDH activity wasdefined as 1 �M NADPH produced/min.
2.8. Measurement of ROS level
Intracellular ROS level was measured in liver tissue [28].Briefly, liver tissue was homogenized (10%) in PBS (pH 7.2).The homogenized cells (0.4 mg/ml) were loaded with thecell permeant probe CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate acetyl ester) (2 �M) for15 min at 30 ◦C in dark, and fluorescence was monitored. H2DCFDA,an uncharged, cell-permeable fluorescent probe readily diffusesinto cells and gets hydrolyzed by intracellular esterases to yieldH2DCF, which is trapped inside the cell. Then it is oxidizedfrom the non-fluorescent form to a highly fluorescent compounddichlorofluorescein primarily by hydroxyl radical (•OH), hydrogenperoxide (H2O2) or other low-molecular-weight peroxides pro-duced in the cells. Thus the fluorescence intensity is proportionalto the amount to the ROS produced by the cells. Fluorescence wasmeasured through a spectrofluorometer (LS 3B, Perkin Elmer, USA)by using 499 nm as excitation and 520 nm as emission wavelengths.Data were expressed as relative fluorescence intensity taking thevalue for normal as 100%.
2.9. Lipid peroxidation assay
Lipid peroxidation in the whole liver membrane was deter-mined by measuring the amount of conjugated diene by themethod of Mandal et al. using a spectrophotometer [14]. Lipidswere extracted from liver homogenate in a chloroform–methanolmixture (2:1, v/v). The extract was evaporated to dryness undernitrogen atmosphere at 25 ◦C, and redissolved in n-cyclohexane.Lipids in cyclohexane solvent were assayed at 234 nm and theresults were expressed as �mol of lipohydroperoxide/mg proteinby using ∈m of 2.52 × 104 M−1 Cm−1. Total proteins was measuredby the method of Lowry et al. [29].
2.10. Quantitation of quercetin level in liver
Quantitation of QC in liver homogenate was performed spec-trophotometrically by the method of Mandal and Das [30]. Liverhomogenates of normal and experimental rats were diluted withan equal volume of absolute ethanol containing 1 �gm/ml buty-lated hydroxyanisole. One mililitre of n-heptane was added and
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he whole suspension was vortexed. This sample was centrifugedt 1000 rpm for 5 min at 48 ◦C. The heptane layer was removednd another 1 ml of fresh heptane was added. The sample wasentrifuged as earlier until three volumes of heptane had beendded. These volumes were combined and dried under nitrogentmosphere. The residue was dissolved in 0.2 ml of methanol and0 ml was injected onto a high performance liquid chromatographyHPLC) ODS-2 column and quantitation of QC in liver homogenateetected spectrophotometrically at 370 nm.
.11. Fluorescence depolarization measurements of the fluidity ofiver cell membrane
The fluorescence depolarization, associated with the hydropho-ic fluorescence probe diphenyl hexatriene DPH, was estimated
n hepatic cell membrane by spectrofluorimeter by the method ofandal and Das [30]. The fluorescence depolarization, associatedith the hydrophobic fluorescence probe DPH, was used to moni-
or the changes in the fluidity of the lipid matrix accompanying theel to liquid crystalline phase transition. Plasma membrane frac-ions of hepatic cells were incubated at 378 ◦C by the addition ofPH dissolved in tetrahydrofuran (DPH/lipid molar ratio 1:500).he excitation and the emission maxima were kept at 365 and30 nm, respectively. The fluorescence anisotropy was calculatedy using the equation
= I|| − I⊥I|| + 2I⊥
here I|| and I⊥ are the fluorescence intensities in parallel and per-endicular to the direction of polarization of the excited light. Theicro viscosity parameters [(r0/r) − 1]−1 were calculated in each
ase, knowing the maximal limiting fluorescence anisotropy r0,hich for DPH is 0.362.
.12. Estimation of hepatic 4-hydroxyproline (4-HP) and collagen
4-HP and collagen were estimated following the Mandal etl. using a spectrophotometer [31]. The liver was cut into smallieces and homogenized in sufficient deionized water to yield 10%omogenate (w/v). Aliquots (2 ml) of the homogenate were addedo an equal volume of 12 N HCl and hydrolyzed in Teflon-cappedials at 105 ◦C for 18 h. 4-Hydroxyproline (4-HP) levels from thoseydrolysates were measured (Sarin et al. [5]). The absorbance at58 nm was read, and values were plotted against a standard graphsing known amounts of 4-HP.
Hepatic collagen was determined by selective binding of Siriusose BB and Fast Green FCF to collagen and non collagen compo-ents, respectively. When the sections are stained with both dyesissolved in aqueous saturated picric acid, they are eluted readilyith NaOH; simultaneously, the absorbance obtained at 550 and
25 nm can be used to determine the amount of collagen and totalrotein, respectively.
.13. Determination of total arsenic content
Arsenic in drinking water given to the animals in the cages wasetermined by the following method. 1 ml of water was taken. Itas treated with 6 ml of 10% HCl, 1 ml of 5% KI, 1 ml of 5% ascorbic
cid and 1 ml of conc HCl. It was allowed to stand at room temper-ture for 45 min. Reading was taken using As3+ as standard usingow injection hydride generation atomic absorption spectrometer
Perkin Elmer, AA 700) fitted with a graphite furnace. Arsenic quan-ification in rodent chow was done following the method of Mandalt al. [31], Flora et al. [32]. Food pellets were digested with acid mix-ure containing nitric acid, sulfuric acid, and perchloric acid in theatio of 6:1:1 over a regulated heater. After the digestion, the acidnteractions 186 (2010) 61–71
mixture was evaporated with occasional addition of triple distilledwater, and the solution thus obtained was employed for estimationof arsenic content. Estimation was carried out using flow injec-tion hydride generation atomic absorption spectrometer (PerkinElmer, AA 700) fitted with a graphite furnace. The detection limitfor arsenic is (1–10) ppb.
Total arsenic accumulation in liver homogenate was also mea-sured following the method of Mandal et al. [31] by flow injectionatomic absorption spectrometer (spectra AA 30/40; Varian, Inc.,Palo Alto, CA) fitted with a graphite furnace. Subcellular fractionswere digested with acid mixture prior to quantification.
2.14. Reduced glutathione (GSH) level
Glutathione level of a part of tissue homogenate was determinedby the method of Mandal and Das [30] with the help of a spec-trophotometer by using tetrachloroacetic acid with EDTA as proteinprecipitating reagent. The mixture was allowed to stand for 5 minprior to centrifugation for 10 min at 200 × g at 4 ◦C. The mixturewas then transferred to a new set of test tubes and 0.3 M phos-phate buffer and Ellmen Reagent (5,5′ dithiobis-2 nitrobenzoic acidin 1% Na-citrate) were added. After completion of the total reaction,solutions were read at 412 nm. Absorbance values were comparedwith a standard curve generated from known GSH concentrationto evaluate liver homogenate GSH levels.
2.15. Western blot analysis
Liver tissues were homogenized (1:10, w/v) in 10 mM Tris–HCl,pH 7.4, 150 mM NaCl, containing freshly added protease inhibitorsand cytosols were prepared by centrifugation at 15,000 × g for10 min at 4 ◦C. Protein concentrations were determined [29].SDS/PAGE was performed by subjecting 30 �g total protein underreducing conditions on 10% polyacrylamide gels, followed by elec-trophoretic transfer to PVDF membrane (Sigma) at 15 V for 20 minby using Semi dry (Bio-Rad) transblot apparatus. Membrane wasblocked in 4% BSA in PBS (overnight) at 4 ◦C, followed by incuba-tion with the primary rabbit anti-cytochrome c antibody (1:100)in blocking solution (0.2% Tween 20 in PBS and 2% BSA) for 3 h atroom temperature. After 5 washes with 0.2% Tween20 in PBS, themembrane was incubated in secondary alkaline phosphatase con-jugated anti-rabbit goat IgG antibody (1:1000) for 1 h 30 min. After5 washes with 0.2% T-20 in PBS and 2% BSA, proteins were visualizedby the development of colour using Sigma premixed BCIP/NBT sub-strate solution. Their density was analyzed with Image J softwaresystem.
2.16. Silver staining
Silver staining of total proteins in liver cytosolic fraction sub-jected to SDS PAGE gel was done [33].
2.17. RNA extraction and RT-PCR analysis
The total RNA of liver tissue was extracted by SV Total RNAIsolation System of Promega. It was treated with RNase-freeDNase1 (Promega) and 2.5 �g of RNA was reverse transcribed intocDNA using the Promega Kit at a final volume of 20 �l. �-Actinwas used as an internal reference gene. The sense and anti-sense primers for �-actin were 5′-ACATCTGCTGGAAGGTGGAC-3′
and 5′-GGTACCACCATGTACCCAGG-3′ (the expected product size
163 bp), respectively [34]. The sense and anti-sense primers fortype I pro-collagen �1 (I): 5′-CACCCTCAAGAGCCTGAGTC-3′ and 5′-GTTCGGGCTGATGTACCAGT-3′ (the expected product size 253 bp)[34]. The RT-PCR reaction was carried on with Access PromegaRT-PCR Kit. The mixture was heated at 42 ◦C for 60 min in an
D. Ghosh et al. / Chemico-Biological Interactions 186 (2010) 61–71 65
Fig. 1. Values of arsenic levels in liver tissue from normal, sodium arseniteand nanocapsulated QC treated rats. The groups are Normal (A), Nor-mal + Nanoencapsulated QC treated (B), Arsenite treated (C), C + Empty nanocap-sCsa
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Fig. 2. Values of GSSG/GSH ratio in liver tissue from normal, sodiumarsenite and nanocapsulated QC treated rats. The groups are Normal (A),Normal + Nanoencapsulated QC treated (B), Arsenite treated (C), C + Empty nanocap-
peroxidation levels in the whole liver tissue of rats
Normal rats treated with nanocapsulated QC showed a decreasein ROS generation and conjugated diene level when compared tonormal rats. A single oral application of NaAsO2 (13 mg/kg b.wt)
Fig. 3. Microviscosity ([r0/r − 1]−1) of rat liver cell membrane from normal,
ule treated (D), C + Free QC treated (E), C + Liposomal QC treated (F) and+ Nanoencapsulated QC treated (G). Values are mean ± S.E. of five rats. *P < 0.001
ignificantly different from normal. #P < 0.001 significantly different from sodiumrsenite treated rats.
ppendorf PCR system Master Cycler. Type I pro-collagen �1 (I)nd �-actin were amplified in 40 sequential cycles, the programas denaturation (94 ◦C, 50 s), annealing (49 ◦C, 45 s), and exten-
ion (72 ◦C, 1 min). The PCR product was fractionated in 2% agaroseel electrophoresis containing ethidium bromide (EB) 0.5 mg/l andhotographed under UV transilluminator. Their density was semi-uantitatively analyzed with Image J software system.
.18. Measurement of plasma TGF ß1
Plasma TGF ß1 was measured using an ELISA kit (R&D Systems,atalog MB 100B).
.19. Statistical analysis
Statistical analysis was performed with one-way ANOVA withost hoc Tukey’s test. The software used was version 15.0; SPSS Inc:hicago, IL. In all instances, P < 0.05 was considered as the minimum
evel of significance.
. Results
.1. Nanocapsulated QC on NaAsO2-induced accumulation ofnorganic arsenic in rat liver
A single oral application of NaAsO2 (13 mg/kg b.wt) induced aubstantial amount of accumulation of inorganic arsenic in liverells. No significant protections were observed in rats treated withmpty nanocapsules or free QC 1 h after arsenic consumption. Lipo-omal QC treatment significantly reduced arsenic level in liver.owever maximum reduction was achieved through nanocapsu-
ated QC treatment. No detectable amount of arsenic was presentn the liver homogenate of normal and nanocapsulated QC treatedats not receiving arsenic (Fig. 1). This signifies that arsenic accu-ulation in the arsenic administered rats is solely due to the oral
avage dosing of arsenic and not due to any contamination fromood and drinking water given to rats.
.2. Nanocapsulated QC on NaAsO2-induced GSSG/GSH ratio
evels in the liver tissue of ratsThe GSSG/GSH ratio, an important marker of oxidative stress,ecreased in normal rats treated with nanocapsulated QC asompared to normal. A single oral application of NaAsO2
sule treated (D), C + Free QC treated (E), C + Liposomal QC treated (F) andC + Nanoencapsulated QC treated (G). Values are mean ± S.E. of five rats. *P < 0.001significantly different from normal. #P < 0.001 significantly different from sodiumarsenite treated rats.
(13 mg/kg b.wt) induced considerable increase in this ratio. Theratio was significantly reduced in rats treated with liposomal QCbut maximum reduction was observed in case of rats treatedwith nanocapsulated QC. However no noticeable reduction wasobserved in case of rats treated with empty nanocapsules or freeQC (Fig. 2).
3.3. Nanocapsulated QC on NaAsO2-induced membranemicroviscosity in the liver tissue of rats
A single oral dose of arsenic (13 mg/kg b.wt) induced a signif-icant decrease in the membrane microviscosity of liver tissue ascompared with the rats of the normal group and normal rats treatedwith nanocapsulated QC. Free QC treated and empty nanocap-sules treated groups also showed a significant decrease in themembrane microviscosity similar to the arsenic fed control group.However liposomal QC treated rats showed a significant eleva-tion but maximum increase was observed in the rats treated withnanocapsulated QC (Fig. 3).
3.4. Nanocapsulated QC on NaAsO2-induced ROS and lipid
sodium arsenite and nanocapsulated QC treated rats. The groups are Normal(A), Normal + Nanoencapsulated QC treated (B), Arsenite treated (C), C + Emptynanocapsule treated (D), C + Free QC treated (E), C + Liposomal QC treated (F) andC + Nanoencapsulated QC treated (G). Values are mean ± S.E. of five rats. *P < 0.001significantly different from normal, **P and #P < 0.001 significantly different fromsodium arsenite treated rats.
66 D. Ghosh et al. / Chemico-Biological I
Table 1Effect of QC in free, liposomal and nanoencapsulated forms on the changes in thegeneration of reactive oxygen species (ROS) and lipid peroxidation levels in rat liverby the induction of sodium arsenite.
Groups DCF-fluorescence(% of normal)
Lipohydroperoxide(�mol/mg protein)
Normal (A) 100 ± 6.32 1.45 ± 0.01(A) + Nanoencapsulated QC treated 103 ± 2.73 1.36 ± 0.06NaAsO2-treated (B) 300 ± 15.30* 2.75 ± 0.04*(B) + Empty nanocapsule treated 290 ± 14.72* 2.63 ± 0.02*(B) + Free QC treated 270 ± 13.55* 2.60 ± 0.03*(B) + Liposomal QC treated 200 ± 10.02# 2.20 ± 0.04#
(B) + Nanoencapsulated QC treated 110 ± 7.11# 1.50 ± 0.01#
Spectrofluorimetric determination of DCF fluorescence as a measure of ROS gener-ation in normal and experimental liver tissues. Results are expressed as % of normalcells, n = 5; mean ± S.E. *P < 0.001 represent significant differences from normal val-ues (A). #P < 0.001 represent significant differences from sodium arsenite treated (B)values.F*s
itnQjo
3
isnfstt
3c
sc
Rats when treated with a single dose of NaAsO developed
F1t(w
or Lipohydroperoxide levels, results are expressed as mean ± S.E. of five rats.P < 0.001 significantly different from normal. #P < 0.001 significantly different fromodium arsenite treated (B).
nduced a marked increase in those levels. No significant reduc-ion was observed in the case of rats treated with free QC or emptyanocapsules 1 h after arsenic consumption. Although liposomalC treatment induced a significant reduction of the ROS and con-
ugated diene levels, maximum reduction was observed in the casef rats treated with nanocapsulated QC (Table 1).
.5. QC in nanocapsules on NaAsO2-induced antioxidant enzymes
Normal rats injected with nanocapsulated QC showed anncrease of different antioxidant enzyme activities in liver cells. Aingle oral application of NaAsO2 (13 mg/kg b.wt) resulted in a sig-ificant depletion of those enzyme levels. No significant protection
rom arsenic insult was provided with free QC or empty nanocap-ules. Although liposomal QC treatment checked the depletion ofhose enzyme levels, nanoencapsulated QC treatment preventedhe depletion completely in liver cells (Table 2).
.6. QC in nanocapsules on NaAsO2-induced hepatic 4-HP and
ollagen contents in rat liverNormal rats injected with nanocapsulated QC showed nouch change in the hepatic 4-HP and collagen contents whenompared to normal. A single oral application of NaAsO2
ig. 4. (a1) Silver staining of total protein in cytosolic fraction subjected to SDS-PAGE. Lah after arsenite intake (C), Lane 4: Cyt c marker. (a2) The effect of nanocapsulated QC on C
he expression of the Cyt c protein. Lane 1: normal (A), Lane 2: arsenite treated (B), Lanea3) Densitometric analysis of Cyt c bands obtained in Western blot: normal (A), Arseniteere statistically significant from normal with *P < 0.001 and from NaAsO2 treated rats w
nteractions 186 (2010) 61–71
(13 mg/kg b.wt) induced a marked increase in those levels (Table 3).But this increase was not inhibited much by the treatment offree QC or empty nanocapsules whereas QC in liposomes andnanocapsules injection inhibited partially and completely the col-lagen protein deposition and 4-HP increase respectively in ratliver.
3.7. QC-entrapped nanocapsules on NaAsO2-mediatedhepatocellular and nephro toxicity
Normal rats injected with nanocapsulated QC showed similarresults with the normal group. Rats treated with a single doseof NaAsO2 developed significant hepatic and nephro damages asobserved from elevated AST, AP and urea, creatinine in serum. Asingle injection (i.v.) of empty nanocapsules or free QC exertedno significant protection against NaAsO2-induced liver and nephrotoxicity although liposomal QC provided a significant depletion ofthose levels. The degree of protection was observed close to normallevels when arsenic fed groups were injected with nanocapsulatedQC (Table 4).
3.8. Quantification of quercetin in liver homogenate
In Table 5, QC levels were expressed per mg protein. By deter-mining the amount of protein present in total liver homogenate,the amount of uptake of QC and QC entrapped in liposomesand nanocapsules by liver were calculated. Normal rats injectedwith nanocapsulated QC showed the highest uptake of 91.32% inthe liver as compared with the other experimental groups. Only25.30% of the injected dose was detected in the liver when freeQC was injected. Arsenic fed rats injected with nanoencapsulatedQC showed the second highest uptake in the liver which was83.92% whereas the uptake of liposomal QC in the liver was only49.98%.
3.9. Nanocapsulated QC against NaAsO2-induced release ofcytochrome c in rat liver cytosolic fraction
254.77% increment of Cyt c release in cytosol above the level of nor-mal group of rats as detected by Western blot. NanoencapsulatedQC decreased the release to 53.19% with respect to the NaAsO2treated control group of rats (Fig. 4a3).
ne 1: normal (A), Lane 2: arsenite treated (B), Lane 3: nanoencapsulated QC treatedyt c protein expression in liver cytosolic fraction. Western blot was used to analyze3: nanoencapsulated QC treated 1 h after arsenite intake (C), Lane 4: Cyt c marker.treated (B) and Nanoencapsulated QC treated 1 h after arsenite intake (C). Results
ith #P < 0.001.
D. Ghosh et al. / Chemico-Biological Interactions 186 (2010) 61–71 67
Tab
le2
Effe
ctof
QC
infr
ee,l
ipos
omal
and
nan
oen
cap
sula
ted
form
son
the
chan
ges
inSO
D,c
atal
ase,
GPx
,GR
,GST
and
G6P
DH
acti
viti
esin
rat
live
rby
the
ind
uct
ion
ofso
diu
mar
sen
ite.
Gro
up
sSO
D(U
/mg
pro
tein
)C
atal
ase
(�m
olH
2O
2re
du
ced
/m
in/m
gp
rote
in)
GPx
(�m
olN
AD
PHox
idat
ion
/m
in/m
gp
rote
in)
GR
(�m
olN
AD
PHox
idat
ion
/m
in/m
gp
rote
in)
GST
(nm
olp
rod
uce
d/
min
/mg
pro
tein
)G
6PD
H(n
mol
NA
DP
red
uce
d/
min
/mg
pro
tein
)
Nor
mal
(A)
66.2
0±
2.09
8.67
±0.
4510
.06
±1.
1232
.16
±1.
7910
7.35
±1.
8013
.02
±1.
28(A
)+N
anoe
nca
psu
late
dQ
Ctr
eate
d71
.81
±3.
419.
32±
0.31
11.6
8±
2.17
33.7
2±
2.18
110.
81±
2.18
15.9
2±
2.11
NaA
sO2
trea
ted
(B)
23.6
5±
0.57
*3.
33±
0.12
*4.
06±
0.13
*11
.24
±0.
88*
53.0
4±
0.53
*4.
67±
0.27
*
(B)+
Emp
tyn
anoc
apsu
letr
eate
d24
.34
±0.
70*
3.56
±0.
09*
5.25
±0.
33*
13.4
4±
0.99
*56
.84
±0.
64*
5.45
±0.
35*
(B)+
Free
QC
trea
ted
26.7
3±
1.20
*4.
65±
0.23
*6.
62±
0.66
*16
.27
±1.
23*
63.1
2±
0.71
*7.
36±
0.77
*
(B)+
Lip
osom
alQ
Ctr
eate
d42
.68
±1.
51#
5.49
±0.
33#
7.28
±0.
72#
21.4
5±
1.44
#72
.66
±1.
04#
9.13
±0.
88#
(B)+
Nan
oen
cap
sula
ted
QC
trea
ted
59.9
8±
1.80
#8.
33±
0.48
#9.
68±
0.84
#30
.96
±1.
68#
96.1
7±
1.66
#12
.47
±1.
15#
Res
ult
sar
eex
pre
ssed
asm
ean
±S.
E.of
five
rats
.*
P<
0.00
1si
gnifi
can
tly
dif
fere
nt
from
nor
mal
.#
P<
0.00
1si
gnifi
can
tly
dif
fere
nt
from
sod
ium
arse
nit
etr
eate
d(B
).
Table 3Effect of free QC, liposomal and nanoencapsulated QC treatments on 4-HP and col-lagen levels in liver following NaAsO2 treatment.
Groups Hepatic 4-HP(�g protein)
Hepatic collagen(�g/mg protein)
Normal (A) 21.96 ± 0.99 12.08 ± 1.32(A) + Nanoencapsulated QC treated 19.62 ± 0.78 11.24 ± 0.92NaAsO2-treated (B) 85.63 ± 4.46* 21.69 ± 1.75*
(B) + Empty nanocapsule treated 82.48 ± 3.73* 20.13 ± 1.67*
(B) + Free QC treated 74.67 ± 3.08* 17.35 ± 1.55*
(B) + Liposomal QC treated 58.54 ± 2.81# 15.27 ± 1.36#
(B) + Nanoencapsulated QC treated 22.45 ± 1.44# 11.98 ± 1.0#
Results are expressed as mean ± S.E. of five rats.* P < 0.001 significantly different from normal for hepatic 4-HP and collagen.# P < 0.001 significantly different from NaAsO2-treated (B) for hepatic 4-HP and
collagen.
3.10. Nanocapsulated QC against NaAsO2-induced type Ipro-collagen ˛1 (I) expression in rat liver
RT–PCR analysis revealed that a single oral application ofNaAsO2 (13 mg/kg b.wt) induced 85.7% increment of type I pro-collagen �1 (I) mRNA expression in comparison to normal ratswhereas nanoencapsulated QC reduced it to 49.24% in contrast toNaAsO2 treated rats (Fig. 5a2).
3.11. Nanocapsulated QC treatment on NaAsO2-inducedupregulation of TGFß concentration in rat blood plasma
Fig. 6 shows the blood TGF ß concentration of both thecontrol and experimental animals exposed to arsenic. Oral feed-ing of NaAsO2 ((13 mg/kg b.wt) caused a significant increaseof TGF ß in blood plasma (From normal 75.2 ± 8.67 ng/ml to196.2 ± 12.07 ng/ml). Free QC treatment resulted no significantchange in TGF ß level in blood plasma. But nanocapsulated QC treat-ment caused a substantial decrease in TGF ß level in blood plasmaof NaAsO2 fed rats (87.6 ± 7.23 ng/ml).
3.12. Pathomorphology and histochemistry of the liver
Haematoxylin–eosin-stained liver sections of normal ratsshowed (Fig. 7a) the cords of normal hepatocytes, normal look-ing sinusoids lined by Kupffer cells. But the positive histologicalchanges in the areas of hepatocellular and fatty metamorphosis,few focal areas of necrosis, Kupffer cell hyperplasia and localizedfibrosis in the periportal region resulted from a single oral applica-tion of NaAsO2 (Fig. 7b). No such alterations appeared in the caseof NaAsO2 intoxicated animals injected with nanoencapsulated QC(Fig. 7c).
Liver section of normal rats stained with Van Gieson exhibitedvery small amount of collagen in the periportal region (Fig. 7d).But periportal region showed mild to moderate amount of col-lagen tissues when fed with a single intake of NaAsO2 (Fig. 7e).The maximum protection from the deposition of collagen contentswas observed when rats were treated with nanoencapsulated QC(Fig. 7f).
4. Discussion
Arsenic generates ROS such as •OH, O2•−, H2O2 which cause
oxidative damage [35] by increased lipid peroxidation and thioldepletion, and oxidative damage, in turn, initiates gene expres-
sion and liver fibrogenesis [4] as evident by our results (Figs. 1–4and Table 2). ROS interacts with a number of components ofcell including lipid, protein, DNA, carbohydrate, thiols, and otherlow-molecular-weight antioxidants causing ultimate oxidation ofmacromolecules and ultimately leading to pathogenesis.
68 D. Ghosh et al. / Chemico-Biological Interactions 186 (2010) 61–71
Table 4Effect of liposomal and nanoencapsulated QC injections (i.v.) on blood serum biochemical parameters in NaAsO2-induced hepatocellular injury.
Groups AP (U/l) AST (IU/l) Urea (g/l) Creatinine (mg/l)
Normal (A) 270 ± 10.92 33.86 ± 1.72 0.46 ± 0.03 13.56 ± 1.45(A) + Nanoencapsulated QC treated 269 ± 13.71 31.48 ± 2.33 0.45 ± 0.02 12.91 ± 2.17NaAsO2-treated (B) 768 ± 21.03* 98.71 ± 5.07* 1.31 ± 0.06* 46.80 ± 4.63*
(B) + Empty nanocapsule treated 741 ± 14.54* 95.62 ± 4.33* 1.24 ± 0.05* 44.60 ± 3.66*
(B) + Free QC treated 724 ± 8.55* 89.71 ± 3.64* 1.18 ± 0.05* 40.17 ± 4.34*
(B) + Liposomal QC treated 510.25 ± 7.20# 65.23 ± 3.03# 0.69 ± 0.05# 25.12 ± 3.26#
(B) + Nanoencapsulated QC treated 292.44 ± 4.60# 36.43 ± 2.20# 0.47 ± 0.04# 13.91 ± 1.63#
Results are expressed as mean ± S.E. of five rats.* P < 0.001 significantly different from normal.# P < 0.001 significantly different from NaAsO2-treated (B).
Table 5Quercetin level in liver homogenate from rats treated with QC (in Tween 80), liposomal and nanoencapsulated QC.
Groups QC concentration in liver homogenate (nM/mg protein) Injected QC in whole liver (%)
Normal (A) 0.04 ± 0.02* –(A) + Nanoencapsulated QC treated 2.96 ± 0.49 91.32(B) + Empty nanocapsule treated 0.06 ± 0.04* –(B) + Free QC treated 0.82 ± 0.13* 25.30(B) + Liposomal QC treated 1.62 ± 0.25*,# 49.98(B) + Nanoencapsulated QC treated 2.72 ± 0.33*,# 83.92
R ed QC
tdoat(bpieaob
tyEtlm
FbL�f
ats received 2.71 mg/kg b.wt intravenous injection of free QC and nanoencapsulat* Values expressed as mean ± S.E. (n = 5).# Value is significantly different (P < 0.001) from free QC.
Fibrosis indicates the critical stage of liver injury, where it mayurn to cirrhosis, and even to liver cancer [36]. Hepatic fibrosis is aiseased state characterized by exuberant synthesis and depositionf collagen in the extracellular matrix. Fibrogenesis is expressed byn increase in the hepatic hydroxyproline level [37]. Our observa-ions indicate that acute arsenic toxicity by a single dose of NaAsO213 mg/kg b.wt) in oral route could initiate fibrogenesis in rat livery an indication of increment of 4-HP and overexpression of type Iro-collagen mRNA (Table 2 and Fig. 5). TGF ß have been implicated
n the induction of synthesis and accumulation of collagen in thextracellular matrix [38]. But a moderate increase of pro-collagennd TGF ß levels and an inadequate deposition and accumulationf collagen in the extracellular matrix in our observations could note a positive indication of liver fibrosis.
Liver fibrosis is a disease where the rate of synthesis of scarissue increases with a rapid imbalance in fibrogenesis and fibrol-sis in liver. Prolonged treatment of arsenic causes liver fibrosis.
arly fibrosis is difficult to diagnose because it is often asymp-omatic. Toxic manifestation of arsenic starts with the induction ofactic acid production because of an imbalance in cellular energyetabolism and the enol acid thus produced when utilized by
ig. 5. (a1) Effect of nanoencapsulated QC on the gene expression of typeI pro-collagen oromide. Lanes 1, 2 and 3: �1 (I) pro-collagen of normal, arsenite treated and nanoencapsanes 5, 6 and 7: � actin of normal, arsenite treated and nanoencapsulated QC treated 1 h a1 (I) pro-collagen mRNA expression in: Normal (A), Arsenite treated (B) and nanoencaps
rom normal with *P < 0.001 and from NaAsO2 treated rats with #P < 0.001.
.
cells significantly increase the intracellular proline pool along withcollagen synthesis by stimulating the activity of Prolyl hydroxy-lase.
A greater number of drugs including arsenic specific antidoteshave been tested to reduce hepatic damage or necrosis and toinhibit liver fibrogenesis. However, none of them is liver specific[39]. Quercetin, a polyphenolic flavonoidal compound has beensuggested in preventing the development of hepatic fibrosis [12]. Itis also known to reduce toxicant induced liver damage [13]. Lipo-somal and nanoencapsulated QC with a high incorporation rate inhepatocytes have been formulated by us and tested in reducingNaAsO2-induced liver fibrogenesis and hepatocellular damage. Weobserved that QC in liposomes and nanocapsules interacts with tar-get cells at a much faster rate than that of free components. Clinicaltrial of the herbal flavonoidal compound against toxicant inducedtissue damage is not possible because of its insoluble property.Quercetin in vesicle based drug delivery systems is expected to
be an ideal formulation to protect cells against toxicant inducednecrosis in cellular level.Arsenic toxicity was reported as an inhibitory effect on cellularrespiration at the level of mitochondria [40]. In our observations,
f liver tissue. RT-PCR product 6 �l was run on 2% agarose gel stained with ethidiumulated QC treated 1 h after arsenite administration of rat liver. Lane 4: PCR marker,fter arsenite administration of rat liver, respectively. (a2) Densitometric analysis ofulated QC treated 1 h after arsenite intake (C). Results were statistically significant
D. Ghosh et al. / Chemico-Biological I
Fig. 6. Effect of nanoencapsulated QC treatment on TGF ß concentration inplasma of rats. Normal (A), NaAsO2 treated (B), (B) + Free QC treated (C),(B) + Nanoencapsulated QC treated (D). Values are mean ± S.E. of five rats. *P < 0.001significantly different from normal. #P < 0.001 significantly different from sodiumarsenite treated rats.
Fig. 7. Histopathological examination of eosin–haematoxylin stained liver section of normnormal, (b) NaAsO2-treated, (c) NaAsO2 + nanoencapsulated QC (2.71 mg/kg b.wt) treateexperimental rats with magnification ×400. (d) Physiological saline-treated normal, (eindicates initiation of fibrosis, ↓↓ indicates fattymetamorphosis, ↓↓↓ indicates necrosis.
nteractions 186 (2010) 61–71 69
the increased arsenic depositions in whole liver cells from NaAsO2treated rats generate more ROS which in turn produce more lipohy-droperoxides and ultimately upregulates gene expression (Table 1and Fig. 5). When arsenic accumulation in liver cell is preventedby nanoencapsulated QC treatment, hepatocellular levels of ROS,lipohydroperoxide and collagen gene expression are maximallyreduced (Table 1 and Fig. 5). The GSSG/GSH ratio increased sub-stantially by the induction of arsenic. On the other hand membranemicroviscosity decreased considerably by arsenic induction. Butvesicular QC treatments prevented the alteration of the ratio andmembrane microviscosity confirming the protective role of QCagainst arsenic induced cell damage.
We observed that liver injury is accompanied by the accu-mulation of arsenic with impaired activity and depletion ofhepatocellular antioxidant status in NaAsO2 treated rats. Maximumreduction of liver injury with an improvement of antioxidant sta-tus and marked reduction of arsenic content in liver is noticed bythe treatment of QC in nanocapsules compared to liposomal QC
treatment (Fig. 1 and Table 2).Our findings indicate that nanoencapsulated QC results in max-imum prevention of arsenic deposition and protects liver fromNaAsO2-induced collagen deposition and fibrosis initiation in liver
al and experimental rats with magnification ×400. (a) Physiological saline-treatedd. Histochemical examination of Van Gieson stained liver section of normal and) NaAsO2-treated, (f) NaAsO2 + nanoencapsulated QC (2.71 mg/kg b.wt) treated. ↓
7 gical I
(tQtcQriaarab
[C1ert
Tvpscunrph
mmbib(
rciwibmbanl
5
ctaNtrnmißm
[
[
[
[
[
[
[
[
[
[
[
0 D. Ghosh et al. / Chemico-Biolo
Figs. 1 and 5). Administration of QC in nanocapsules to rats protectshose animals from arsenic induced acute liver toxicity while freeC does not. QC in nanocapsules may be more protective compared
o liposome as enhanced intracellular accumulation of QC in hepaticells is noticed only when rats are treated with nanoencapsulatedC (Table 5). A single oral dose of NaAsO2 (13 mg/kg b.wt) to rats
esults in impairment of antioxidant status with a marked increasen arsenic content in liver. Our observation suggests that the mech-nism of the protective effect of liposomal or nanoencapsulated QCgainst arsenic induced liver injury can be related primarily to theeduction of arsenic accumulation in liver and it may be because ofrsenic chelation or/and inhibition of liver arsenic entry as observedy Aherne and Brien’O [41].
Release of cytochrome c in cytosol indicates cellular damage42]. Our results indicate that NaAsO2 induces an increase of 17 kDayt c release in cytosol whereas nanoencapsulated QC treatmenth after NaAsO2 treatment decreases the release significantly. Thisvent suggests that targeted QC in hepatocytes can check the Cyt celease in cytosol favouring the protective role of QC against hepa-ocellular damage.
Our results indicate that arsenite induces an upregulation ofGF ß in plasma of rats and nanocapsulated QC treatment pre-ents it (Fig. 6). Nanoencapsulated QC treatment downregulatesro-collagen gene expression with a prevention of collagen depo-ition in hepatic cells. The mechanism how nanoencapsulated QControls arsenic induced pro-collagen gene expression and upreg-lation of TGF ß is not known, however, it may be presumed thatanocapsule mediated targeting of QC to hepatic cells may be aeason for better accumulation of QC in liver and then preventsro-collagen mRNA activation with a control of TGF ß synthesis inepatic tissue.
The levels of AST and AP in blood plasma increase by the treat-ent of NaAsO2. Targeting of QC prevents NaAsO2-induced hepaticembrane damage and thus the leakage of AST and AP in the
lood circulation is prevented (Table 4). The protective role of QCn nanocapsules against NaAsO2-induced hepatotoxicity has alsoeen examined by our histopathological and histochemical analysisFig. 7).
Whatever the exact mechanism is, judging from the efficiency ineducing oxidative damage, protection from fibrogenesis initiation,ontrolling of TGF ß synthesis and pro-collagen gene expressionn liver and capacity to reduce overall toxicity, quercetin could
ell be tried in the clinics and best result could be expected whent could be treated in nanoencapsulated form. Obviously, thoseiocompatible and biodegradable polymeric nanocapsules haveuch longer circulation time in blood and they remain unaffected
y circulating lipases, protecting the drug from bio-environmentnd play a key role to deliver QC in liver by following a mecha-ism of common colloidal particles clearance from circulation by
iver.
. Conclusion
The approach of delivering a nontoxic herb origin polyphenolicompound QC to liver might be useful in therapeutic applicationo prevent NaAsO2-induced acute liver toxicity. Further studiesre planned to perform with chronic and subchronic dosages ofaAsO2 to substanitiate our observation that QC might be useful in
herapeutic approach in combating NaAsO2-induced fibrogenesisesulted from chronic arsenic exposure. Application of polylactide
anocapsulated QC containing a very low dose of the compounday be recommended as a potent therapeutic approach for arsenicnduced hepatic fibrosis gene expression and upregulation of TGFin plasma through a complete protection of liver against arsenicediated oxidative attack.
[
nteractions 186 (2010) 61–71
Conflict of interest statement
None.
Acknowledgements
This work has been supported financially by Council of Sci-entific and Industrial Research (CSIR), Govt. of India, New Delhiand Indian Council of Medical Research (ICMR) (Ad-hoc Projectnumber 58/17/2003-BMS), Govt. of India. The authors acknowl-edge Dr. Bikramjit Roychoudhury for his cooperation in WesternBlotting.
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