2011 VERSÃO PUBLICADA Towards a better understanding of Ipomoea asarifolia toxicity - Evidence of the involvement of a leaf lectin

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Toxicon 58 (2011) 502–508 Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Towards a better understanding of Ipomoea asarifolia toxicity: Evidence of the involvement of a leaf lectin H.O. Salles a, I.M. Vasconcelos b, L.F.L. Santos c, H.D. Oliveira b, P.P.C. Costa c, N.R.F. Nascimento c, C.F. Santos c, D.F. Sousa d, A.R.C. Jorge d, D.B. Menezes e, H.S.A. Monteiro d, D.M.F. Gondim b, J.T.A. Oliveira b, * a Embrapa Caprinos e Ovinos,

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  Towards a better understanding of  Ipomoea asarifolia toxicity: Evidence of the involvement of a leaf lectin H.O. Salles a , I.M. Vasconcelos b , L.F.L.Santos c , H.D. Oliveira b , P.P.C. Costa c , N.R.F. Nascimento c ,C.F. Santos c ,D.F. Sousa d , A.R.C. Jorge d , D.B. Menezes e , H.S.A. Monteiro d , D.M.F. Gondim b , J.T.A. Oliveira b , * a Embrapa Caprinos e Ovinos, PO Box 145, 62010-970, Sobral, CE, Brazil b Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, 60451-970, Fortaleza, CE, Brazil c Universidade Estadual do Ceará, Campus do Itaperi, 60740-000, Fortaleza, CE, Brazil d Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, 60430-270, Fortaleza, CE, Brazil e Departamento de Patologia e Medicina Legal, Universidade Federal do Ceará, 60441 – 750, Fortaleza, CE, Brazil a r t i c l e i n f o  Article history: Received 15 April 2011Received in revised form 22 July 2011Accepted 18 August 2011Available online 26 August 2011 Keywords:Ipomoea asarifolia ToxicityLectin a b s t r a c t Natural intoxication of livestock by ingestion of  Ipomoea asarifolia leaves has been reportedto occur widely in Brazil. Previous studies carried out by our research group providedstrong evidence that a lectin could be involved with the toxic properties of  I. asarifolia . Toreinforce this hypothesis, a lectin-enriched fraction (LEF) was isolated from I. asarifolia leaves and its toxic effects were assessed. Leaves of  I. asarifolia were excised from plantsgrowing widely in the fi eld, mechanically wounded and maintained in a chamber at25 Æ 3  C for 72 h in the dark, under near 100% relative humidity. The leaf proteins wereextracted, ammonium sulfate precipitated, chromatographed on DEAE-cellulose andPhenyl-Sepharose to produce LEF that under SDS – PAGE showed a molecular mass of 44.0 kDa and after N-terminal amino acid analysis a primary sequence composed of AGYTPVLDIGAEVLAAGEPY. The in vivo toxicity of LEF assessed by intraorbital injection inmice showed induced severe uncoordinated movements without death. LEF reduced themuscular contraction in a dose depend way and at 29.8 m g/mL (CE 50 ) it produces 50%inhibition of contraction, suggesting that LEF blunts autonomic neurotransmission. Iso-lated rat kidneys were perfused with LEF and no effects on the perfusion pressure or renalvascular resistance were observed, but urinary fl ow and glomerular fi ltration rateincreased. Moreover, the percentage of tubular transport of Na þ , K þ and Cl À decreased.Histological examination of the kidneys perfused with LEF exhibited little alterations.These toxic effects observed above were concomitant with the increase of LEF hemag-glutination activity, which strongly suggest that one of the toxic principles of  I. asarifolia isa lectin present in its leaves. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Ipomoeaasarifolia (Desr.)Roem.&Schult(commonname:salsa or ginger-leaf morning-glory) is a tropical shrubby andquickly growing toxic plant of the Convolvulaceae family.Natural intoxication of livestock with I. asarifolia has beenreported to occur widely in Brazil (Barbosa et al., 2005),particularly in Northeastern. Usually intoxication occurswhen the animals feed on leaves as an alternative source of nutrients during dry seasons owing to food shortage(Medeiros et al., 2003). This intoxication is clinically charac-terized by mild depression, sleepiness, weak tremors of the head and neck muscles or discrete head nodding afterexercise, severe lack of movement coordination, sideway * Corresponding author. Tel.: þ 55 85 3366 9823; fax: þ 55 85 33669789. E-mail address: jtaolive@ufc.br(J.T.A. Oliveira). Contents lists available atSciVerse ScienceDirect Toxicon journal homepage:www.elsevier.com/locate/toxicon 0041-0101/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.toxicon.2011.08.011 Toxicon 58 (2011) 502 – 508  progression and fall, hypermetria, sway while standing andwide based stance (Medeiros et al., 2003).Previously,itwassuggestedthatthesymptomsobservedupon I. asarifolia consumption were due to lysosomalstorage disease (Medeiros et al., 2000) as demonstrated for Ipomoea sericophylla and Ipomoea riedelii (Barbosa et al.,2006). However, no evidence of such disease was foundafter histological or ultrastructural evaluation of tissues ororgans from goats experimentally intoxicated with I. asar-ifolia (Medeiros et al., 2003). In addition, the presence of negligible amount of swainsonine and the absence of calystegines in the samples of  I. asarifolia used in previousexperiments further suggest that the experimental intoxi-cationinducedby I. asarifolia ingoatswasprobably notdueto a storage disease (Medeiros et al., 2003). Actually, thereare few studies on I. asarifolia toxicity and the toxicsubstancesinvolvedareunknown,andtheirmechanismsof action are not yet understood. Nevertheless, experimentalevidence strongly suggested that a lectin present in theleaves of  I. asarifolia could be involved in its toxic effects togoats (Santos, 2001).Lectins are widely distributed in nature and severalhundred of these molecules have been isolated from plants,viruses, bacteria, invertebrates and vertebrates, includingmammals (Kennedy et al., 1995). Lectins are a class of proteins of non-immune srcin, which possess at least onenon-catalyticdomainthatspeci fi callyand reversibly bind tomono- or oligosaccharides (Peumans and Van Damme,1995). A typical lectin has two or more carbohydrate-binding sites, being able to agglutinate cells. Thus they arecommonly designated as agglutinins or hemagglutinins.Based on differences in molecular structures, biochemicalproperties, and carbohydrate-binding speci fi cities, plantlectinsare usuallyconsidered a complex and heterogeneousgroup of proteins with different pharmacological and toxi-cological properties.This study was conducted to isolate a lectin-enrichedfraction (LEF) from the leaves of  I. asarifolia and assess itstoxic effects on various models of study as an attempt toestablish an association between this leaf lectin with theplant toxicity. 2. Materials and methods  2.1. Plant material and chemicals I. asarifolia leaves were collected from naturallygrowing plants at the campus of Federal University of Ceará (UFC), Fortaleza, Brazil. A voucher specimen(registration number 040477) was deposited at PriscoBezerra Herbarium of UFC, where it was botanicallyidenti fi ed. The leaves were collected, washed with tapwater to remove dust and mechanically wounded (fourcircular cuttings of 6 mm diameters equally distributed inthe leaf blade) and maintained in a chamber at 25 Æ 3  Cfor 72 h, in the dark, under near 100% relative humidity.In a previous experiment it was noticed a signi fi cantincrease of the hemagglutination activity upon leaf injury(data will be published elsewhere). After this, the leaveswere powdered in the presence of liquid nitrogen andstored at À 80  C until required. DEAE-cellulose columnwas obtained from Whatman International Ltd., Maid-stone, England; Phenyl-Sepharose 6-Fast Flow columnwas obtained from GE Healthcare, Uppsala, Sweden.Morphine was purchased from Sigma Aldrich Chemical(Saint Louis, MO, USA). The other chemicals were all of analytical grade and obtained from local suppliers.  2.2. Protein extraction and preparation of the lectin-enriched fraction (LEF) The soluble proteins were extracted from the leaf powder with three volumes of 25 mM Tris – HCl, pH 7.5,supplemented with 3% (w/v) polyvinylpolypyrrolidone(PVPP) and 5 mMascorbic acid, for 2 h at 4  C,under gentleshaking.After fi ltrationthroughnyloncloth,the fi ltratewascentrifuged at 10,000 Â  g  for 30 min, at 4  C, and thesupernatant (crude extract) recovered. The crude extractwas precipitated with ammonium sulfate at 30% saturation(176 g/L) and the suspension maintained at 4  C for 12 h.The precipitate obtained (Fraction 0 – 30%, shortly F030)after centrifugation (10,000 Â  g  , 40 min, 4  C) was dialyzedexhaustively against Milli-Q grade water, lyophilized, andsuspended in 25 mM Tris – HCl, pH 7.5. After centrifugation(10,000 Â  g  , 20 min, 4  C), the fraction F030 was submittedto ion-exchange chromatography on a DEAE-cellulosecolumn equilibrated with 25 mM Tris – HCl, pH 7.5. Thethrough fraction was eluted from the column with theequilibrating buffer. The retained material was eluted with25 mM Tris – HCl, pH 7.5, containing 200 mM NaCl, at a fl owrate of 1 mL/min, dialyzed exhaustively against water andlyophilized. Next, it was suspended in 25 mM Tris – HCl, pH7.5, containing 420 mM of ammonium sulfate, centrifuged(10,000 Â  g  , 20 min, 4  C), and the supernatant obtainedchromatographed on a Phenyl-Sepharose 6-Fast Flowcolumn, equilibrated with the above buffer. The proteinfractionobtainedafterelutionwith25 mMTris-HCl,pH7.5,containing 100 mM of ammonium sulfate, at a fl ow rate of 1 mL/min, was dialyzed against Milli-Q grade water andlyophilized. This material represented the lectin-enrichedfraction (LEF) that was characterized and used to assesstoxicity.  2.3. Protein content  It was determined as previously described (Bradford,1976). Absorbance at 280 nm was also used to monitorprotein elution pro fi les during chromatographies.  2.4. Polyacrylamide gel electrophoresis Protein fractions were analyzed by polyacrylamide gelelectrophoresis (15% running gel, 3.5% stacking gel)(Laemmli, 1970). The samples were solubilized in 125 mMTris – HCl buffer, pH 6.8, containing 2.6% (w/v) SDS, 0.5 mMEGTA,0.5 mMEDTA,12.6%(w/v)glycerol.Gelswerestainedwith silver (Blum et al., 1986). Phosphorylase b (97 kDa),bovine serum albumin (66 kDa), ovalbumin (45 kDa),carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa)and a -lactoalbumin (14.4 kDa) were used as molecularmass standards. H.O. Salles et al. / Toxicon 58 (2011) 502 – 508 503   2.5. N-terminal amino acid sequence analysis Following polyacrylamide gel electrophoresis in thepresence of sodium dodecylsulfate (SDS – PAGE), LEF wastransferred to polyvinylidene di fl uoride (PVDF) Hybond-Pmembrane (Amersham Biosciences) following the protocoldescribed byRybicki and Purves (1996)and stained withcoomassie brilliant blue R-250. The protein band corre-sponding to LEF (44 kDa) was excised from the membraneand analyzed by automated Edman degradation, usinga Shimadzu PPSQ-21/23 automated protein sequencer(Shimadzu,Kyoto,Japan).Theaminoacidsequenceobtainedwas compared with other protein sequences deposited inthe SWISS-PROT/TREMBL databases using the FASTA 3 andBLAST programs.  2.6. Hemagglutination assay Hemagglutination activity was measured by a serialdilutionprocedureusinga2%suspensionoftrypsin-treatedrabbit erythrocytes as previously described (Carbonaroet al., 2000) with some modi fi cations. The assay wasdone in polystyrene microtiter U-bottomed 96-well platesand agglutination was visualized after 12 h. One hemag-glutination unit (1HU) was taken as the highest dilutiongiving complete agglutination of trypsin-treated rabbiterythrocytes.  2.7. Hemagglutination inhibition assay Before the hemagglutination assay, two-fold seriallydiluted carbohydrate or glycoprotein samples (25 m L) in150 mM NaCl were incubated for 30 min at 25  C with25 m L of LEF dissolved in 25 mM Tris – HCl, pH 7.5. Theminimal concentration of carbohydrate or glycoprotein inthe fi nal reaction mixture capable of completely inhibiting4 HU was recorded.  2.8. Stability of the hemagglutination activity 2.8.1. Effect of temperature LEF solutions containing 0.0124 mg protein/mL in25 mM Tris – HCl, pH 7.5, were heated at 70, 80, and 90  C,from 5 to 60 min, at 5 min intervals. After cooling to 25  C,the residual hemagglutination activity was assayed.  2.8.2. Effect of dithiotreitol (DTT) LEF solutions containing 0.0124 mg protein/mL in25 mM Tris – HCl, pH 7.5, were incubated for 60 min at25  C, in the presence of the reducing agent DTT at fi nalconcentrations of 5, 10, 50 and 100 mM and the residualhemagglutination activity measured.  2.8.3. Effect of protease treatments LEF (1 mg) was incubated with 500 m L of pepsin(0.02 mg/mL of 100 mM HCl, pH 1.8) at 37  C. After 2 hincubation, two 250 m L aliquots were withdrawal from thereaction mixture and 250 m L of 250 mM Tris – HCl, pH 8.9,were added to adjust pH to 8.0. Then 250 m L of a tryp-sin þ chymotrypsin solution (0.02 mg/mL for each enzymein 250 mM Tris-HCl, pH 8.9) were added to one of thepepsin hydrolysate (250 m L) and incubated for further 3 h,at 37  C. The hemagglutinantion activity was analyzed forthe hydrolyzates of LEF obtained after pepsin and pepsinfollowed by trypsin þ chymotrypsin treatments.  2.9. NMR analysis LEF (5 mg) was dissolved in 0.2 m L of 25 mM Tris – HCl,pH 7.5, containing 0.4 m L of D 2 O. The NMR data wererecorded using a Bruker Avance DPX300 spectrometeroperating in the frequency of  1 H, at 300 MHz, to detectpossible contamination by toxic secondary metabolite(swainsonine and calystegines, for example). The acquisi-tion was made with 256 transients and 5 s relaxation time.  2.10. Biological assays 2.10.1. Intraorbital injection LEF (5 mg/kg body weight) dissolved in 150 mM NaClwas injected intraorbitally in male Swiss mice (15.5 – 20.5 gbody weight) to assess the toxicity in vivo . The animalbehavior was observed for 1 h.  2.10.2. Mouse vas deferens assay The electrically driven mouse vas deferens bioassay wasperformed as described byHenderson et al. (1972), usingSwiss mice (38 – 42 g body weight). Vasa deferentia wereinserted into silver ring electrodes, transferred to organbaths (5 mL capacity) set at 37  C, and attached to forcedisplacement transducers (F-60 Narco Biosystems, Hous-ton, TX, USA) under a loading tension of 300 mg(2.94 Â 10 À 3 N) to record motor responses isometrically.Concentration – response curves were obtained by cumu-lative addition of the crude extract to the bath medium at2.5, 7.5, 25.0, 75.0, 250.0 and 750.0 m g protein/mL or LEF at0.1, 0.3, 1.0, 3.0, 10.0, 30.0, 100.0 and 300.0 m gprotein/mL,both dissolved in Krebs solution. Stimulation of intramuralnerveswascarriedoutatafrequencyof0.1 Hzanddurationof 10 À 3 s at supramaximal voltage (26 V). The motorresponses of each cumulative dose were registered for10 min. After the last dose, the system was washed threetimes with Krebs solution to remove the protein sampletested. Then, morphine (10 m M) was added to the organbath to revert contractions elicited by electrical fi eldstimulation as evidence that they were mainly of neuro-genic srcin.  2.10.3. Kidney perfusion Adult Wistar rats (240 – 280 g body weight) were fastedwith free access to water for 24 h before the experiments.Theanimalswereanaesthetizedwithsodiumpentobarbital(50 mg/kg body weight). The right renal artery was cannu-lated through the upper mesenteric artery, the kidney iso-lated and uninterrupted perfused with modi fi ed Krebs – Henseleit solution (MKHS), pH 7.4, at 37  C, consisting (inmM) of: Na þ 147.0; K þ 5.0; Ca 2 þ 2.5; Mg 2 þ 2.0; Cl À 110.0;HCO 3 À 2.5; SO 42 À 1.0; PO 43 À 1.0. This perfusion system wasassembled according toBowman (1970)andFonteles et al. (1998). Bovine serum albumin (6% w/v, BSA fraction V,Sigma) was added to the modi fi ed MKHS and this solutionwasdialyzedfor48 h,at4  C,toremovecitrate,piruvateand H.O. Salles et al. / Toxicon 58 (2011) 502 – 508 504  lactate(HansonandBallard,1968;Pegg,1971).Next,0.075 gurea, 0.075 g inulin and 0.15 g glucose were added and thepH adjusted to 7.4. This solutionwas gassed with a mixtureof 95% O 2 /5% CO 2 and the temperature stabilized at 37  C.Perfusionpressurewasdeterminedatthetipofthestainlesssteel cannulae with a mercury manometer. Perfusate andurine samples were collected for Na þ , K þ , inulin andosmolaritydetermination.Na þ andK þ concentrationsweredeterminedby fl amephotometry( fl amephotometerModel445; Micronal, Brazil), Cl À using a kit (LABTEST, São Paulo,Brazil) and inulin according toWalser et al. (1955). Sampleosmolality was measured using a WESCOR 5100c vaporpressure osmometer (WESCOR, Needham Heights, MA,USA). Glomerular fi ltration rate (GFR), fractional tubulartransport of sodium (%TNa þ ), potassium (%TK þ ) and chlo-ride(%TCl À ),urinary fl ow(UF)andrenalvascularresistance(RVR) were determined as previously described (Walseret al., 1955). The data were evaluated using analysis of variance followed by Student ’ s t  -test. Results are expressedas mean Æ SEM with the level of signi fi cance set at 5%( P  < 0.05; n ¼ 4). After the experiment, both right and leftkidneys were removed and fi xed in 10% formaldehyde forhistological processing and examination. The experimentsfollowed the methodology recommended by InternationalEthical Standards in animal research and was approved bythe Scienti fi c and Ethical Committee of the Federal Univer-sity of Ceará, Brazil. 3. Results  3.1. Isolation and characterization of LEF  The crude extract of  I. asarifolia leaves injured and keptin the dark for 72 h gave a hemagglutination speci fi cactivity of 650.9 Æ 16.7 HU/mg protein whereas the crudeextractfromuninjuriedleavesnotkeptinthedark(control)gave 416.9 Æ 2.7 HU/mg protein. The increase in activity inwounded\darkenedleaveswasduetothe denovo synthesisof the native leaf lectin because irrespective of woundtreatment the lectin-enriched fraction (LEF) gave the sameN-terminalaminoacidsequence.Thusthestartingmaterialfor LEF production was the crude extract of wounded/darkened leaves.This lectin-enriched fraction (LEF) from I. asarifolia leaves was obtained after protein extraction, ammoniumsulfateprecipitation,DEAE-celluloseandPhenyl-Sepharose6-FastFlowchromatographies,asdetailedin2.2.SDS – PAGEof LEF showed a mainprotein band with relative molecularweight of around 44.0 kDa (Fig. 1,Lane 3). This band was electroblotted onto PVDF membrane and had its N-terminal amino acid sequence determined: AVNLPAGHLSPGGVGNYVVTVGLCTP.LEF had a speci fi c hemagglutination activity of 1118 HU/mg protein. It was inhibited by the glycoprotein fetuin(3.0 Â 10 À 3 mM), as well as by sialic acid (N-acetyl- D -neu-ramic acid, minimum inhibitory concentration of 3.0 mM)(Santos, 2001) which is a component of fetuin. However itwas not inhibited by the simple sugars D -arabinose, L  -fructose, D -galactose, N-acetyl- D -galactosamine, D -glucose,N-acetyl- D -glucosamine, D -mannose, D -xilose, the disac-charides a -lactose, maltose, sacarose, and the trisaccharide D -raf  fi nose, even at high concentration (100 mM), neitherby the glycoproteins BSA and mucin (Santos, 2001).Heat treatment at 70 and 80  C for 60 min reduced LEFagglutination activity against trypsin-treated rabbit eryth-rocytes to 75% and at 90  C it was completely abolishedwithin 10 min (Fig. 2). Treatment of LEF with DTT (5,10, 50 Fig. 1. SDS – PAGE (15% polyacrylamide) of the protein fractions (3 m g)extracted from I. asarifolia leaves. Lane 1, Molecular mass markers; Lane 2,fraction eluted from DEAE-cellulose column with 200 mM NaCl; Lane 3, LEFpreparation eluted from phenyl-sepharose column with 100 mM ammo-nium sulfate. Fig. 2. Thermal stability of LEF. Protein concentration was 0.0124 mg/ml of 25 mM Tris – HCl buffer, pH 7.5. H.O. Salles et al. / Toxicon 58 (2011) 502 – 508 505
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