Research Article Oral Administration of Ganoderma lucidum to Lead-Exposed Rats Protects Erythrocytes against Hemolysis: Implicates to Anti-Anemia

Transcription

1 Hindwi Pulishing Corportion Evidence-Bsed Complementry nd Alterntive Medicine Volume 215, Article ID 46373, 8 pges Reserch Article Orl Administrtion of Gnoderm lucidum to Led-Exposed Rts Protects Erythrocytes ginst Hemolysis: Implictes to Anti-Anemi Shhdt Hossin, 1 Sujn Bhowmick, 1 Siful Islm, 1 Liz Rozrio, 1 Srin Jhn, 1 Mehedi Hssn, 1 Mrzn Srkr, 1 Bzlul Krim Choudhury, 2 Sohel Ahmed, 1 nd Hussin Shhjll 1 1 Deprtment of Biochemistry nd Moleculr Biology, Lortory of Alterntive Medicine nd Behviorl Neurosciences, Jhngirngr University, Svr, Dhk 1342, Bngldesh 2 Mnikgnj Medicl College, Mnikgnj, Dhk 18, Bngldesh Correspondence should e ddressed to Shhdt Hossin; shhdt@dhk.net Received 16 April 215; Accepted 12 July 215 Acdemic Editor: Vincenzo De Feo Copyright 215 Shhdt Hossin et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. We studied the effect of chronic orl exposure to led cette (PA) on the sensitivity of RBC to hemolysis nd whether the sensitivity could e decresed y feeding therts with extrct ofmedicinl mushroom Gnoderm lucidum. Three groups of rts, control, PAexposed, nd G. lucidum (Gl)PA, were used. PA (3 mm) ws dministered vi drinking wter nd G. lucidum extrct y gvge t 3 mg/kg BW/dy for 12 weeks. Afterwrds, the rts were killed nd wshed RBCs were sujected to hemolysis in the presence of Fenton s regents. Hemolysis ws determined y estimting the mount of relesed hemogloin. The levels of lipid peroxide (LPO) nd GSH were determined from RBC memrnes nd whole RBCs, respectively. The levels of TNFα nd LPO lso were determined from heptic tissues. The RBCs of PA-exposed rts displyed significntly higher sensitivity to hemolysis thn those of the GlPA rts. The levels of LPO incresed nd GSH decresed in the RBCs, with concomitnt increses in the levels of heptic TNFα nd LPO in the PA-exposed rts. The degree of hemolysis ws significntly low in the RBCs of GlPA rts, concurrently with meliortion of heptic prmeters. Finlly, the study suggests tht PA-induced-hemolysis nd relted oxidtive-toxicity might e minimized y consumption of G. lucidum. 1. Introduction Red lood cell (RBC) primrily trnsports oxygen throughout the ody. In doing so, the RBC hs to compromise with oth endogenous nd exogenous sources of rective oxygen species (ROS) tht dmge nd deteriorte its function. However, the RBC hs n innte defense mechnism, comprising glutthione nd host of ntioxidtive enzymes [1, 2] to void its tremendous wer nd ter. Becuse short life spn RBCs re constntly undergoing turnover nd mking the lood system highly sensitive to environmentlly very poisonous elements such s led (P), the investigtion on the effect of P is of specil significnce in Bngldesh ecuse of the fct tht P poisoning hs een one of the mjor lrming pulic-helth prolems in Bngldesh [3]. RBC serves s the initil receptcle of sored P nd distriutes P throughout the ody, mking it ville to other tissues. P interferes with norml red lood cell formtion y inhiiting importnt enzymes. In ddition, P dmges red lood cell memrnes nd interferes with cell metolism, thus shortening the survivl of ech individul cell [4 6]. Approximtely 99% of the P in lood is ssocited with red lood cells; the remining 1% resides in lood plsm [7, 8]. On the one hnd, erythrocytes detoxify numerous circultory xenoiotics [9], mny of which directly confer oxidtive insults to the erythrocytes [1]; on the other hnd, the erythrocytes themselves re highly vulnerle to oxidtive stress ecuse of the high contents of oxygen nd

2 2 Evidence-Bsed Complementry nd Alterntive Medicine polyunsturted ftty cids in the memrne [11]. All of these fctors mke the RBCs very much susceptile to hemolysis. One of the ims of the P poisoning-relted investigtions should thus e the reduction of susceptiility of RBCs to hemolysis tht ultimtely leds to hemolytic nemi. Gnoderm lucidum is medicinl mushroom nd used in trditionl Chinese medicine, with very rod spectrum of iologicl ctivities nd phrmcologicl functions [12]. G. lucidum is reportedly known to hve nticncer, ntitumor, ntidietic, nd nti-inflmmtory effects [13 15]. Exposure to P cn cuse hypochromic microcytic nemi [16]. This my relte to the fct tht P is sored y iron-soring mchinery, confers competitive inhiition, nd interferes with heme iosynthesis [17]. Anemi in children leds to incresed moridity nd mortlity[18]. Becuse children my e exposed to levels of P which could dversely ffect their helth without exhiiting clinicl symptoms, it is vitl to dopt preventive pproch. Therefore serching for gents cple of reducing the levels of P from the ody could e considered s one of the importnt preventive mesures. Very recently, we hve reported tht the orl dministrtion of G. lucidum extrct prevents prcetmol-induced heptotoxicity in rts [19] nd erythrocyte hemolysis in rts [2], suggesting orl dministrtion of this medicinl mushroom extrct cn ply, t lest prtilly, role in the reduction of P-induced hemolysis nd relted nemi. In this study, we thus investigted whether the chronic dministrtion of P results in n incresed sensitivity of erythrocyte to hemolysis nd, if so, then whether the orl dministrtion of the extrct of G. lucidum decreses it. Also, the liver is the first orgn tht encounters the sored P. Ded erythrocytes re lso recycledintheliver,thusrelesinglltheir(rbcs )contents including toxic P in this orgn. Therefore, the mechnisms of ction of the ntihemolytic effect of G. lucidum nd tht of the effect of P on the heptic tissues were discussed. 2. Mterils nd Methods 2.1. Chemicls. Led cette ws used s test chemicls in the present study. Led cette (PA) of AR grde ws procured from E Merck. TEP (1,1,3,3-tetrethoxypropne), reduced glutthione (GSH), DTNB [5,5 -dithiois (2-nitroenzoic cid)] were purchsed from Sigm Aldrich. TNFα ws from Snt Cruz Biotechnology, CA, USA. Horserdish peroxidse-conjugted nti-rit secondry ntiody ws from Cell Signling Technology. The primry ntiody ntirit TNFα ws from Snt Cruz Biotechnology, CA, USA. ELISA grde BSA ws from Sigm Aldrich. Tetrmethylenzidine is from (Invitrogen) Life Technologies, USA. All other chemicls were of nlyticl grde Animls. Wistr rts otined from niml reeding colony of the icddr,, Dhk, were used in the present study. The nimls were fed on stndrd pellet diet nd mintined under controlled lortory conditions (12 h light: 12 h drk; temperture 25 ± 2 C; reltive humidity 5 ± 1%). The inred second genertion rts (15 weeks old, 18 2 g ody weight, [BW]) were rndomly divided into three groups: the GSH Hemolysis H n=6 RBCs Wshed RBCs RBC memrnes n=8 Led (P) 12 weeks Kill Blood Serum P ssy G. lucidump Liver n=7 Homogentes TNFα LPO Figure 1: Experimentl design. RBCs: red lood cells; GSH: reduced glutthione; H: hemogloin; TNFα: tumor necrosis fctor α. control group (n =6), led cette- (PA-) exposed group (n =8), nd G. lucidum-fed PA-exposed group (GlPA, n = 7)(Figure 1). The PA-exposed group ws orlly fed PA t 3 mm prepred drinking wter. The extrct of G. lucidum dissolved in distilled wter ws orlly dministered t 3 mg/kg BW/dy. The control group ws orlly fed similr volume of the dh 2 O lone. Orl dministrtion of PA nd/or G. lucidum ws continued for 12 weeks. The rts werecredforndkilledinccordncewiththeguidelinesof lortory nimls nd pproved y the Institutionl Animl Ethicl Committee t Jhngirngr University, Svr, Dhk, Bngldesh RBC Preprtion. RBCs were prepred s previously descried y Hshimoto et l. (215) [21]. After deep nesthesi with pentoritl lood from individul rt ws collected from inferior ven cve with heprinized syringe. Hlf of the lood ws used for plsm collection nd the other hlf wsmixedwithlocke ssolution(154mmncl,5.6mmkcl, 2.3 mm CCl 2,1mMMgCl 2,3.6mMNHCO 3,5mMglucose, nd 5 mm HEPES; ph 7.2) nd pelleted t 3 gfor1 minutes in plstic tues. The superntnt ws discrded nd therbcswerewshedthriceyusingthesmelockeuffer solution. The uffy cot nd portion of the upper lyer of the RBCs were removed in ech wsh. The remining RBCs were immeditely sujected to hemolysis nd intrcellulr GSH ssy or used for preprtion erythrocyte ghost memrnes. RBCs were counted y Sysmex XS-i Hemolysis Assy. RBC suspensions [1 7 cells/ml Locke s solution (154 mm NCl, 5.6 mm KCl, 2.3 mm CCl 2,1mM MgCl 2, 3.6 mm NHCO 3, 5 mm glucose, nd 5 mm HEPES; ph 7.2)] from ech rt were sujected to incution for 1 h t 37 CwithfreshlyprepredFenton sregents[h 2 O 2 (45 mm) FeSO 4 (2 mm)]. Then, RBCs were pelleted down y centrifuging the smples t 3 g for 1 min. The superntnt ws spirted nd the extent of hemolysis ws quntified y

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 determining the mounts of relesed hemogloin (H) into the superntnt t 54 nm ginst H stndrd Antihemolytic Effect of α-tocopherol. RBCs [1 7 cells/ml Locke s solution (154 mm NCl, 5.6 mm KCl, 2.3 mm CCl 2, 1 mm MgCl 2, 3.6 mm NHCO 3, 5 mm glucose, nd 5 mm HEPES; ph 7.2)] from the P-nonexposed rts were sujected to Fenton s regents [H 2 O 2 (45mM)FeSO 4 (2 mm)] in the sence or presence of α-tocopherol ( μm). After 1 h of incution t 37 C, the extent of hemolysis ws determined with hemogloin stndrd, s descried ove Erythrocyte Reduced Glutthione (GSH) Assy. Erythrocyte GSH ws mesured ccording to the method of Moron et l. [22]. A prt of the wshed RBCs ws suspended in ice-cold dh 2 O contining 2 mm EDTA. After rief soniction, the suspension ws immeditely treted with 1% trichlorocetic cid (finl concentrtion) nd vortexed for 1 min. Afterwrds, the contents were centrifuged t g for 3 min. Following centrifugtion, μl ofthe superntnt ws mixed with.4 M tris uffer (ph 8.9). The whole solution ws mixed well nd 1 mm DTNB ws dded ndthesorncewsredwithin5minofdditionof DTNB t 412 nm ginst regent lnk with no homogente. For lnk reding, the homogente ws sustituted with distilled wter. The mount of glutthione in the erythrocytes ws expressed s pmol of GSH/1 7 RBCs. 2.7.PreprtionofRBCGhostMemrnes.Wshed RBCs were suspended in 4 volumes of ice-cold 5 mm Tris-HCl uffer (ph 7.), contining 1 mm EDTA, nd centrifuged ( g, 6 minutes, 4 C). Superntnt ws discrded ndwshingwsrepeteduntiltheerythrocytememrnes ppered whitish. The memrnes were stored t 8 C Preprtion of Liver Homogentes. After drwing lood, the liver from ech rt ws seprted, perfused with icecold sline. Afterwrds, 1% liver tissue homogente ws prepred with phosphte uffer ( mm, ph 7.4) contining 1% phenylmethylsulfonyl fluoride (PMSF). The homogente ws centrifuged t g to remove unruptured tissues nd deris nd the resultnt homogente ws ssigned s whole homogente. A prt of the whole homogente ws further spun for 1 h t g to prepre cytosolic frction to mesure TNFα in the liver tissues. The smples were stored t 2 Cuntilnlysis Lipid Peroxide (LPO) of RBC Ghost Memrnes nd Liver Tissues. The sl levels of lipid peroxide (LPO) in the RBC memrnes nd heptic tissues were determined y estimting the thiorituric cid rective sustnces (TBARs), s descried previously [23]. The RBC memrnes or liver tissue whole homogentes (.1 ml) from ech of the rts were dded to.1 ml of 8.1% (w/v) sodium dodecyl sulphte, 2 ml of.4% thiorituric cid in 2% cetic cid (ph 3.5), nd.1 ml distilled wter. Ech tue ws tightly cppedndhetedt95 C for 1 h. After cooling the tues with tp wter, 2 ml of n-utnol-pyridine (15 : 1, v/v) ws dded nd shken vigorously for out 1 minutes. The tues were then centrifuged t 12 gfor1minutestroomtemperture (digitl centrifuge; DSC-1512SD). The sornce of the upper orgnic lyer ws mesured t 532 nm. TEP (1,1,3,3- tetrethoxypropne) ws utilized s stndrd ELISA for Liver Tumor Necrosis Fctor Alph (TNFα). The multiwell plte ws coted with liver cytosolic frction in.1 M sodium icronte, ph 9.6 t 4 Covernight,nd then locked with 1% BSA in tris-uffered sline (TBS). The primry ntiody nti-rit TNFα (Snt Cruz Biotechnology, CA, USA) t 1 : dilutions ws incuted in the plte for overnight period t 4 C. Horserdish peroxidse-coupled nti-rit IgG (Biosource Interntionl, Inc., Cmrillo, CA, USA) ws used s the secondry ntiody nd incuted for 2 h t room temperture efore the ddition of tetrmethylenzidine (Invitrogen, Life Technologies, USA) sustrte to develop color. The rection ws stopped y ddition of.1 N HCl fter incution for 3 min t room temperture. Well coted with only.1 M cronte uffer, ph 9.6 ws used s lnk. The pltes were nlyzed with multiwell plte reder (Er Lisscn II, Mnnheim, Germny) t 45 nm AnlysisofSerumPyAtomicAsorptionSpectrophotometer. The serum from ech rt ws llowed to dry t 12 C until reching constnt weight nd concentrted nitric cid nd hydrogen peroxide (1 : 1 v/v) were dded. The digestion flsks were heted to 13 C until ll the mterils were dissolved nd diluted with doule dh 2 Oppropritely. The element led ws ssyed using Vrin 24 Atomic Asorption Spectrophotometer. The results were expressed s μm In Vitro Antioxidtive Potentil of G. lucidum Extrct. Evlution of in vitro ntioxidnt ctivity of G. lucidum extrct ws performed y determining the (i) DPPH-free rdicl scvenging ility nd (ii) nti-lpo ility, s previously descried from this lortory [24, 25]. For the determintion of in vitro nti-lpo ility of the extrct, homogentes of RBC memrnes from nonexposed rts were divided into control memrnes (n = 5), oxidtive stress-induced (OS, Fenton s regents-incuted) memrnes, nd OSG. lucidum extrct-treted memrnes. The smple mixtures were then incuted t room temperture for 4 hours. Afterwrds, the levels of lipid peroxide (LPO) were determined, s indictor of oxidtive stress, following the methods of Hossin et l. (211; 24) [26, 27]. LPO of RBC memrnes ws clculted s nmol/mg of protein Other In Vitro Methods. Totl polyphenols nd totl flvonoids of the G. lucidum extrct were mesured s previously descried [24]. Liver-function-specific enzymes such s sprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) were mesured y using enzymtic regent kits, s previously descried [2]. Totl protein ws mesured y icinchoninic cid (BCA) method.

4 4 Evidence-Bsed Complementry nd Alterntive Medicine 8 12 (mg H/dL) c As % of positive control (1% SDS) c d e, f P GlP Figure 2: Effect of P on hemolysis. Results re men ± SE (n =6 8), ech with duplicte determintions. Brs with different lphets re significntly different t P <.5. Dt were nlyzed with onewyanova,withfisher splsdforposthoccomprison Sttisticl Anlysis. All the dt were expressed s men ± SE (stndrd error of men). The significnce of difference in mens mong different groups ws determined y one-wy nlysis of vrince (ANOVA), followed y Fisher s PLSD test for post hoc comprisons y using GrphPd prism softwre version 5.. P <.5 ws considered sttisticlly significnt. 3. Results 3.1. Effect of Predministrtion of G. lucidum on Fenton s Regents-Induced Hemolysis of RBCs. The susceptiility of RBCs to the oxidtive stress-induced (Fenton s regents) hemolysis ws mesured in ll erythrocyte smples of the controls, PA-exposed nd G. lucidumpa-fed rts (Figure 2). The RBCs of the PA-exposed rts displyed the highest sensitivity to Fenton s regents-instigted hemolysis. It ws 51% higher (P <.5), s compred to tht of the control. However, the orl dministrtion of G. lucidum extrct to the PA-fed rts significntly reduced the degree of hemolysis in the rts (G. lucidumpa) (Figure2). To understnd whether the ntioxidtive defense ws involved in lowering the extent of hemolysis, the RBCs from the non-p exposed rts were sujected to the oxidtive stress y Fenton s regents in the sence ( μm) or presence of αtocopherol (1 μm). 1% SDS ws used s positive control of hemolysis (Figure 3). Hemolysis dt were normlized to those of the positive control. The levels of hemolysis were decresed with increses in the concentrtions α-tocopherol in the smples, s indicted y the decresed mount of relesed hemogloin in the uffer (Figure 3). 3.2.EffectofOrlAdministrtionofG.lucidumonIntrcellulr Erythrocyte GSH. The orl PA-exposure cused significntdecreseinthelevelsofgshintherbcsofpaexposed rts; however, it ws significntly incresed in the extrct-fed PA-exposed (PAG. lucidum)rts(figure4). FR α-tf 1 μm 2 μm 5 μm μm Figure 3: Effect of α-tocopherol on oxidtive stress (FR: Fenton s regents) induced hemolysis. Results re men ± SE (n = 6 8). Brs with different lphets re significntly different t P <.5. α-tocopherol (α-tf) dose-dependently inhiited the degree of hemolysis, s indicted y the grdul decreses in the levels of relesed hemogloin. 1% SDS ws used s positive control. Dt were nlyzed with one-wy ANOVA, with Fisher s PLSD for post hoc comprison. ( ) indictes sence. () indictes presence. GSH pmol/1 7 RBCs P c GlP Figure 4: Effect of orl dministrtion of G. lucidum on the intrcellulr GSH levels of erythrocytes. Results re men ± SE (n = 6), ech with duplicte determintions. Brs with different lphets re significntly different t P <.5. Dt were nlyzed with onewy ANOVA, with Fisher s PLSD for post hoc comprison Effects of Orl Administrtion of G. lucidum on the Bsl LevelsofLPOintheRBCMemrnesndHepticTissues. The levels of LPO were significntly incresed (y 55%) in the RBC memrnes of the P-exposed rts, s compred to those of the RBCs of control rts. The orl dministrtion of the rts with G. lucidum, however,significntly decresed the levels of LPO, s compred to tht of the PA-exposed rts (Figure 5()). The levels of LPO were lso significntly incresed in the heptic tissues of PA-exposed

5 Evidence-Bsed Complementry nd Alterntive Medicine LPO of RBC memrnes (control s %) LPO of heptic tissues (control s %) P GlP P GlP () () Figure 5: Effects of orl dministrtion of G. lucidum on the levels of lipid peroxide (LPO) of RBC memrnes nd heptic tissues. Results re men ± SE (n =6), ech with duplicte determintions. Brs with different lphets re significntly different t P <.5. Dtwere nlyzed with one-wy ANOVA, with Fisher s PLSD for post hoc comprison. rts (Figure 5()). The orl dministrtion of G. lucidum to the rts, however, reduced the levels to those of the controls (Figure 5()) Effects of Orl Administrtion of G. lucidum on Heptic Tumor Necrosis Fctor α (TNFα). The levels of TNFα were significntly ugmented in the liver tissues of the PAexposed rts, s compred to those of the controls; however, it ws significntly reduced in the G. lucidumpadministered rts (Figure 6) Effects of G. lucidum Extrct on the Plsm P Levels. TheslPlevelsintheplsmofthecontrolrtswere.95 ±.3 μm. The plsm levels of P rose (y >85%) to >1.82 ±.5 μm in the PA-exposed rts, while it dropped significntly (P <.5)intheplsmoftheG. lucidumpa rts (1.43 ±.3 μm) Evlution of Antioxidtive Potentils of G. lucidum Extrct. The extrct of the G. lucidum hd considerle mounts of ntioxidnt phytochemicls, such s totl polyphenols ( 6.6 mg gllic cid equivlent/g extrct) nd totl flvonoids ( 1 mg ctechin equivlent/gm extrct). The G. lucidum extrct possessed significnt DPPH-rdicl scvenging ctivity [IC 5, concentrtion required to scvenge 5% of.4 mm of DPPH, of G. lucidum ws 26 μg/ml extrct nd tht of the vitmin C ws 24 μg/ml]. Incution of RBC memrne smples with Fenton s regents significntly stimulted the oxidtive stress (OS) in the memrnes, s indicted y the incresed levels of lipid peroxide (LPO). The OS-induced increses in the levels of LPO, however, were repressed in the presence of the G. lucidum extrct, Asornce of control s % P GlP Figure 6: Effect of orldministrtion of G. lucidum on the heptic levels of tumor necrosis fctor α (TNFα). Results re men ± SE (n =6 8), ech with duplicte determintions. Brs with different lphets re significntly different t P <.5. Dt were nlyzed with one-wy ANOVA, with Fisher s PLSD for post hoc comprison. thus demonstrting strong nti-lpo ility of this extrct [LPO nmol/mg protein (or in percent): control = 24 ± 1.5 (%); OS = 56±2.5 (235%); OSG. lucidum = 22±1.5 (127%)]. These ntioxidtive properties of the extrct thus suggest potentil therpeutic efficcy of G. lucidum extrct to protect ginst oxidtivestress.thesepropositionsrefurthersupportedy the fct tht the orl P exposure significntly incresed the plsm levels of heptic structurl integrity-relted enzyme mrkers, including ALT nd AST. However, the levels of these enzymes were significntly meliorted upon dministrtion

6 6 Evidence-Bsed Complementry nd Alterntive Medicine of G. lucidum extrct (ALT: control, 36.5 ± 3.5; PA,48.5 ± 4.45; GlPA, 37.4 ± 4.9 U/L) (AST: control, 99.9 ± 1;PA, 121 ± 9.7; GlPA, 66 ± 8.5 U/L). 4. Discussion In the present study, we demonstrte tht P increses susceptiility of erythrocytes to hemolysis nd tht orl dministrtion of G. lucidum reduces the susceptiility to hemolysis. The underlying mechnism(s) of the heightened sensitivity to hemolysis of the RBCs of the P-exposed rts my relte to the fct tht P-exposure mde their erythrocytes more vulnerle to oxidtive stress, which ultimtely rought out the lekge of the ilyer memrnes nd finlly cused n incresed relese of hemogloin in the presence of oxidnt (Fenton s regents). Notly, the hydroxyl rdicl is le to penetrte deep into the lipid hed groups region [28, 29] nd my initite chin rections in the ilyer memrnes. Interestingly, the orl dministrtion of G. lucidum extrct significntly reduced the oxidtive stress nd hence the extent of hemolysis. This finding led us to infer tht the presence of ntioxidnts in the extrct of G. lucidum might hve conferred the oxidtive defense ginst hemolysis y Fenton s regents. To support the proposition tht ntioxidnts hd protected the RBCs ginst hemolysis, we collected RBCs from nonexposed rts, wshed them, nd sujected them to oxidtive stress (y hydroxyl rdicls of Fenton s regents) in the presence of nturl ntioxidnt α-tocopherol (1, 2, 5, nd μm). As expected, αtocopherol dose-dependently inhiited the oxidtive stressinduced hemolysis, thus demonstrting tht the reduction in the degree of hemolysis of the RBCs of the G. lucidump rts might hve, t lest prtilly, een occurring due to the presence of ntioxidnt-like sustnces in the G. lucidum extrct nd/or uilding up of n ntioxidtive defense in their RBCs. De Ros et l. (1954) [3] werethefirstto report the protection y vitmin E ginst nemi due to P toxicity. Cssi et l. (1972) [31] lso reported tht vitmin E deficiency displys incresed propensity for hemolysis ndnemi.thusourresultsofinhiitionofhemolysis in the presence of α-tocopherol re consistent with these studies. P induces two types of nemi: cute high-level P poisoning hs een ssocited with hemolytic nemi; in chronic P poisoning, P induces nemi y oth interfering with erythropoiesis nd diminishing red lood cell survivl [32]. P inhiits severl enzymes tht re criticl to the synthesis of heme. It should e emphsized, however, tht nemi is not n erly mnifesttion of P poisoning nd is evident only when the lood P level is significntly elevted for prolonged periods. Yet, numerous studies hve reported tht P poisoning is ssocited with hemolytic nemi [33, 34]. Both the ntioxidnt nd the thiorituric cid experiments suggested tht RBC hemolysis my e relted to lipid peroxidtion. Although P is not trnsition metl, the ctlysis of peroxidtive rections y P my e mjor contriutor to the toxic effects of this metl [35]. Dose- nd time-dependent increses in peroxides in heptic microsoml memrnes occurred in response to P [36]. GSH is the single most powerful ntioxidnt in our ody. The erythrocyte GSH plys vitl role in mitigting the dmging effects of rective oxygen species (ROS) encountered in the circultion [37] nd produced y continuous oxidtion of hemogloin within the cytosol of the erythrocyte [38, 39]. The whole ody cquires GSH supply primrily through the RBCs; therefore, intrcellulr GSH levels were determined from the whole RBCs. P decresed the levels of intrcellulr GSH. Thus the decresed GSH levels in the P-exposed RBCs might hve resulted from the intrcellulr dptive response (utiliztion) of the RBCs to meliorte P toxicity. Interestingly, the intrcellulr GSH levels of RBCs were incresed y the orl dministrtion of G. lucidum.we gin consider tht the ntioxidnts present in the G. lucidum might hve ttled with the free rdicls derived from oxidtive stress; thus the intrcellulr resources of GSH remined reltively high nd hence plyed meliorting roles ginst hemolysis. Our results re consistent with the reports of Omoowleetl.(214),wherePddecresedthelevelsof GSH [4]. The reduction of the levels of GSH in the RBCs is thus consistent with the rises of the levels of LPO in the Pexposed rts. The increses in the levels of LPO in our Pdexposed rts re thus directly indicting tht Pd-poisoning induces dropping down of the ntioxidtive defense nd leds to hemolysis. Gugliott et l. (212) [41] reported tht the exposure of erythrocytes to P leds to reduction in the verge lifetime of the erythrocytes nd the susequent development of nemi. Though the mechnism(s) is not clerly known, one importnt effect of led toxicity in erythrocytes consists of incresing intrcellulr clcium [C 2 ](i), which in turn cuses ltertions in cell shpe nd volume nd it is ssocited with cellulr rigidity, hemolysis, senescence, nd poptosis [42]. Whtever the mechnism is consistent with the ove reports, we provide evidence tht P increses sensitivity of RBC to hemolysis, leding to nemi, nd tht the orl dministrtion of G. lucidum reduces the sensitivity to hemolysis. Prcticlly, the liver, vi the portl vein, is exposed to enterllysoredpnditisthetissuethtshowsthelrgest repository of led (33%) followed y the kidney cortex nd medull [43]. P induces oxidtion in heptic microsoml memrnes [36]. P lso cuses incresed expression of TNFα in the liver tissues [44]. This leds us to determine the levels of LPO, s indictor of P-induced oxidtive stress, nd proinflmmtory TNFα, in the liver tissues. The incresed levels of LPO nd/or TNFα in the P-exposed rts re thus consistent with these reports. Agin, the orl dministrtion of the G. lucidum significntly meliorted these cell-dmging effects of P in the liver. 5. Conclusion Recently, the frming nd commerciliztion of G. lucidum hve een strted y different privte entrepreneurs in Bngldesh with the ssistnce of its griculturl extension progrms. Herl remedies re reltively effective, chep, nd lmost devoid of side effects, compred to synthetic gents [45]. These findings thus point to the fct tht complementry herl therpies could e good choice here in Bngldesh, where 26% of her 15-million popultion still live under

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 poverty line [46] nd iron deficiency ffects its 5% of ll children nd 7% of ll women [47]. Iron deficiency is the most common cuse of nemi in Bngldesh [48]. Hence the ttrctive therpeutic strtegies focusing on the modultion of P-induced toxicity re wrrnted. Very likely with rsenic toxicity, the led toxicity hs lredy posed severe helth prolems in Bngldesh. Anemi is one of themostwell-knowntoxicheltheffectsssocitedwithp exposure. Vrious mechnisms hve een suggested for Pssocited nemi including interference with iron trnsport, shortening of erythrocyte life spn, nd inhiition of the gloulin synthesis nd the impirment of heme metolism [49, 5]. Our study clerly indictes tht orl dministrtion of G. lucidum extrct significntly meliortes the hemolysis, with concurrent inhiitions of oxidtive stress of the RBC memrnes nd liver tissues. Finlly we suggest tht consumption of the G. lucidum couldeusedsoneofthe prophylctic mens to reduce the ody-urden of toxic P nd P-toxicity-relted erly hemolysis nd hence to comt nemi. Further studies re underwy to understnd the exct mechnism of ction of G. lucidum on P-induced toxicity. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the puliction of this pper. Authors Contriution All uthors contriuted eqully to this pper. Acknowledgment The uthors grtefully cknowledge the contriution of the University Grnt Commission-Higher Eduction Qulity Enhncement Progrm (UGC-HEQEP) for the instrumentl support (CP-358). References [1] R. Gonzles, C. Auclir, E. Voisin, H. Gutero, D. Dhermy, nd P. Boivin, Superoxide dismutse, ctlse, nd glutthione peroxidse in red lood cells from ptients with mlignnt diseses, Cncer Reserch,vol.44,no.9,pp ,1984. [2] E. Ngu, F. J. Chrest, nd J. M. Rifkind, Hydrogenperoxide-induced heme degrdtion in red lood cells: the protective roles of ctlse nd glutthione peroxidse, Biochimic et Biophysic Act,vol.162,no.1 3,pp ,23. [3] R. Kiser, A. K. Henderson, W. R. Dley et l., Blood led levels of primry school children in Dhk, Bngldesh, Environmentl Helth Perspectives,vol.19,no.6,pp ,21. [4] H. Roels, J. P. Buchet, R. Luwerys et l., Impct of ir pollution y led on the heme iosynthetic pthwy in schoolge children, Archives of Environmentl Helth, vol.31,no.6, pp , [5] S. S. Sdikov, A. A. Buglnov, Z. A. Tdzhiev, nd F. Z. Gfurov, Indictors of iron metolism nd cellulr immunity in helthy children nd in those with iron deficiency nemi in reltion to ecologicl conditions, Peditrii, no.8,pp.41 44, 199. [6] P. E. Desilv, Determintion of led in plsm nd studies on its reltionship to led in erythrocytes, British Journl of Industril Medicine,vol.38,no.3,pp ,1981. [7] EPA, Air qulity criteri for led, Tech. Rep. EPA688328F, U.S. Environmentl Protection Agency, Office of Reserch nd Development, Office of Helth nd Environmentl Assessment, Environmentl Criteri nd Assessment Office, Reserch Tringle Prk, NC, USA, [8] Agency for Toxic Sustnces nd Disese Registry (ATSDR), Toxicologicl Profile for Led, US Deprtment of Helth nd Humn Services, Pulic Helth Service, Atlnt, G, USA, 27. [9] S. Pikul, J. Bndorowicz-Pikul, S. Awsthi, nd Y. C. Awsthi, ATP-driven efflux of glutthione S-conjugtes, ntitumor drugs, nd xenoiotics from humn erythrocytes, Biochemicl Archives,vol.12,no.4,pp ,1996. [1] F. Piv-Mrtins, J. Fernndes, S. Roch et l., Effects of olive oil polyphenols on erythrocyte oxidtive dmge, Moleculr Nutrition nd Food Reserch,vol.53,no.5,pp ,29. [11] M. L. A. Sivilotti, Oxidnt stress nd hemolysis of the humn erythrocyte, Toxicologicl Reviews, vol. 23, no. 3, pp , 24. [12] M. S. Shio, Nturl products of the medicinl fungus Gnoderm lucidum: occurrence, iologicl ctivities, nd phrmcologicl functions, Chemicl Record,vol.3,no.3,pp , 23. [13] C.-Y. Hung, J. Y.-F. Chen, J.-E. Wu et l., Ling-zhi polyscchrides potentite cytotoxic effects of nticncer drugs ginst drug-resistnt urothelil crcinom cells, Journl of Agriculturl nd Food Chemistry, vol. 58, no. 15, pp , 21. [14] L. M. Hrhji Trjkovic, S. A. Mijtovic, D. D. Mksimovic- Ivnic et l., Anticncer properties of gnoderm lucidum methnol extrcts in vitro nd in vivo, Nutrition nd Cncer, vol.61,no.5,pp ,29. [15] C.-J. Weng, C.-F. Chu, G.-C. Yen, J.-W. Lio, D.-H. Chen, nd K.-D. Chen, Inhiitory effects of Gnoderm lucidum on tumorigenesis nd metstsis of humn heptom cells in cells nd niml models, JournlofAgriculturlndFoodChemistry, vol. 57, no. 11, pp , 29. [16] S.Turgut,A.Polt,M.Innetl., Interctionetweennemi nd lood levels of iron, zinc, copper, cdmium nd led in children, Indin Journl of Peditrics,vol.74, no.9,pp , 27. [17] R. A. Goyer, Led toxicity: current concerns, Environmentl Helth Perspectives,vol.,pp ,1993. [18] D. Kpur, K. N. Agrwl, nd D. K. Agrwl, Nutritionl nemi nd its control, Indin Journl of Peditrics, vol. 69, no. 7,pp ,22. [19] M. M. Rhmn nd S. Hossin, Preventive effect of Gnoderm lucidum on prcetmol-induced cute heptotoxicity in rts, Journl of Scientific Reserch,vol.5,no.3,pp ,213. [2] N. Ftim, F. A. Selin, M. Hque et l., A polyherl extrct formultion lowers the crdiovsculr disese risk fctors concurrently with systemic oxidtive sttus in normocholesterolemic rts, Glol Journl of Phrmcology,vol.7,no.4,pp , 213. [21] M. Hshimoto, S. Hossin, M. Ktkur, A. Al Mmun, nd O. Shido, The inding of Aβ 1 42 to lipid rfts of RBC is enhnced y dietry docoshexenoic cid in rts: implictes to Alzheimer s disese, Biochimic et Biophysic Act Biomemrnes,vol.1848,no.6,pp ,215.

8 8 Evidence-Bsed Complementry nd Alterntive Medicine [22] M. S. Moron, J. W. Depierre, nd B. Mnnervik, Levels of glutthione, glutthione reductse nd glutthione S-trnsferse ctivities in rt lung nd liver, Biochimic Biophysic Act Generl Sujects,vol.582,no.1,pp.67 78,1979. [23] M. Hshimoto, M. H. Shhdt, T. Shimd et l., Reltionship etween ge-relted increses in rt liver lipid peroxidtion nd ile cnliculr plsm memrne fluidity, Experimentl Gerontology,vol.37,no.1,pp.89 97,21. [24] M. Hque, J. Islm, A. Rhmn, nd S. Hossin, Rphnus stivus meliortes therogeneic lipid profiles in hypercholesterolemic rts nd hypercholesterolemi-ssocited peroxidtive liver dmge, Journl of Advnces in Chemistry, vol.7,no. 3, pp , 214. [25] J. Islm, M. Hque, A. Rhmn, nd S. Hossin, Syzygium cumini (L.) seed extrct protects emryofoetl rins ginst intruterine oxidtive toxicity in rts during hypoxireperfusion injury, Interntionl Journl for Phrmceuticl Reserch Scholrs,vol.3,no.3,pp ,214. [26] S. Hossin, I. H. Chowdhury, M. A. Bsuni et l., Syzygium cumini seed extrct protects the liver ginst lipid peroxidtion with concurrent meliortion of heptic enzymes nd lipid profile of lcoholic rts, Journl of Complementry nd Integrtive Medicine, vol. 8, no. 1, pp. 1 17, 211. [27] H. Shhdt, M. Hshimoto, T. Shimd, nd O. Shido, Synptic plsm memrne-ound cetylcholinesterse ctivity is not ffected y docoshexenoic cid-induced decrese in memrne order, Life Sciences, vol. 74, no. 24, pp , 24. [28] R. M. Cordeiro, Rective oxygen species t phospholipid ilyers: distriution, moility nd permetion, Biochimic et Biophysic Act Biomemrnes, vol.1838,no.1,pp , 214. [29] C.A.Fortier,B.Gun,R.B.Cole,ndM.A.Trr, Covlently ound fluorescent proes s reporters for hydroxyl rdicl penetrtion into liposoml memrnes, Free Rdicl Biology nd Medicine,vol.46,no.1,pp ,29. [3] A. Gmliel, M. Afri, nd A. A. Frimer, Determining rdicl penetrtion of lipid ilyers with new lipophilic spin trps, Free Rdicl Biology nd Medicine,vol.44,no.7,pp ,28. [31] P. I. Csi, J. W. Huswirth, nd P. P. Nir, Biosynthesis of Heme in vitmin E deficiency, Annls of the New York Acdemy of Sciences,vol.23,pp.93 12,1972. [32] A. M. Pope nd D. P. Rll, Environmentl Medicine: Integrting Missing Element into Medicl Eduction, The Ntionl Acdemic Press, Wshington, DC, USA, 241. [33] W. N. Vlentine, D. E. Pgli, K. Fink, nd G. Mdokoro, Led poisoning: ssocition with hemolytic nemi, sophilic stippling, erythrocyte pyrimidine 5 nucleotidse deficiency, nd intrerythrocytic ccumultion of pyrimidines, Journl of Clinicl Investigtion,vol.58,no.4,pp ,1976. [34] M. H. Aly, H. C. Kim, S. W. Renner, A. Boyrsky, M. Kosmin, nd D. E. Pgli, Hemolytic nemi ssocited with led poisoning from shotgun pellets nd the response to Succimer tretment, Americn Journl of Hemtology, vol.44,no.4,pp , [35] B. Bolck, M. Irhim, J. Lu-Hesselmnn, D. Steinritz, nd F. Suhr, Detection of free rdicl rection products nd ctivted signlling molecules s iomrkers of cell dmge in humn kertinocytes upon led exposure, Journl of Moleculr Biomrkers nd Dignosis,vol.5,no.4,p.179,214. [36] L.J.LwtonndW.E.Donldson, Led-inducedtissueftty cid ltertions nd lipid peroxidtion, Biologicl Trce Element Reserch,vol.28,no.2,pp.83 97,1991. [37] I. T. Mk, R. Stfford, nd W. B. Weglicki, Loss of red lood cell glutthione during Mg deficiency: Prevention y vitmin E, D-proprnolol, nd chloroquine, The Americn Journl of Physiology Cell Physiology, vol.267,no.5,pp.c1366 C137, [38] H. S. Hsieh nd E. R. Jffe, The metolism of methemogloin in humn erythrocytes, in The Red Blood Cell, D. M. Surgenor, Ed., vol. 2, pp , Acdemic Press, New York, NY, USA, 2nd edition, [39] C. Rice-Evns, Iron-medited oxidtive stress nd erythrocytes, in Blood Cell Biochemistry. Erythroid Cells, J.R.Hrris, Ed.,vol.1,pp ,PlenumPress,NewYork,NY,USA,199. [4] T. O. Omoowle, A. A. Oygemi, A. S. Akinrinde et l., Filure of recovery from led induced heptoxicity nd disruption of erythrocyte ntioxidnt defence system in Wistr rts, Environmentl Toxicology nd Phrmcology, vol.37,no.3,pp , 214. [41] T. Gugliott, G. De Luc, P. Romno, C. Rigno, A. Scuteri, nd L. Romno, Effects of led chloride on humn erythrocyte memrnes nd on kinetic nion sulphte nd glutthione concentrtions, Cellulr nd Moleculr Biology Letters, vol. 17, no. 4, pp , 212. [42] M. A. Quintnr-Escorz, M. T. González-Mrtínez, I.-O. M. Del Pilr, nd J. V. Clderón-Slins, Oxidtive dmge increses intrcellulr free clcium [C 2 ] ion concentrtion in humn erythrocytes incuted with led, Toxicology in Vitro, vol.24,no.5,pp ,21. [43] S. G. Bruce, Z. Ariev, nd M. G. Igor, Anlysis of led toxicity in humn cells, BMC Genomics,vol.13,rticle 344,212. [44] A.Mudiplli, Ledheptotoxicity&potentilheltheffects, Indin Journl of Medicl Reserch, vol.126,no.6,pp , 27. [45] S. Gupt nd M. Ml, Evlution of hyperglycemi potentil of S. xnthocrpum fruit in norml nd streptozin induced dietic rts, Europen Bulletin of Drug Reserch, vol.13,pp , 25. [46] TIME, After Much Hertrek, Some Good News t Lst for Bngldesh, Time World, July 213, wiki/poverty in Bngldesh#cite note-3. [47] A. Mwniki, Cse study #3-3, iron deficiency in Bngldesh, in Food Policy for Developing Countries: Cse Studies, P. Pinstrup- Andersen nd F. Cheng, Eds., p. 9, 27. [48] R. D. Merrill, A. A. Shmim, H. Ali et l., High prevlence of nemi with lck of iron deficiency mong women in rurl Bngldesh: role for thlssemi nd iron in groundwter, Asi Pcific Journl of Clinicl Nutrition,vol.21,no.3,pp , 212. [49] M. Ichi, K. Tomokuni, nd K. Mori, Erythrocyte nucleotides in led workers, Interntionl Archives of Occuptionl nd Environmentl Helth,vol.63,no.6,pp ,1992. [5] Y.Kim,K.Hrd,S.Ohmori,B.-K.Lee,H.Miur,ndA.Ued, Evlutionofledexposureinworkerstled-cidttery fctory in Kore: With focus on ctivity of erythrocyte pyrimidine 5 -nucleotidse (P5N), Occuptionl nd Environmentl Medicine,vol.52,no.7,pp ,1995.