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Short communication Clinical and pathological effects of Calotropis procera exposure in sheepand rats Joseney Maia de Lima, Francisco Joelson Correia de Freitas, Raimundo Neilson Lima Amorim,Antônio Carlos Lopes Câmara, Jael Soares Batista, Benito Soto-Blanco * Department of Animal Sciences, Universidade Federal Rural do Semi-Árido (UFERSA), BR 110 Km 47, Mossoró, RN 59625-900, Brazil a r t i c l e i n f o Article history: Received 16 August 2010Received in revised form 20 October 2010Accepted 9 November 2010Available online 16 November 2010 Keywords: Poisonous plants Calotropis procera CardiotoxicityHeart lesionsSmall ruminants a b s t r a c t This study aimed to describe the toxic effects resulting from the administration of Calo-tropis procera (Aiton) W. T. Aiton latex to rats and C. procera leaves to sheep. We studiedmale sheep that received C. procera leaves bygavage. Twenty male rats were separated into5 groups and were subjected to an intra-peritoneal injection of fresh C. procera latex(without carrier solvent) at 1.0, 0.6, 0.3 or 0.1 ml of latex/kg of body weight, and controlanimals were injected with 0.9% NaCl. All rats were treated with the highest dose, but noneof the rats from the other groups, died. The histological lesions were restricted to ratsdosed with 1.0 ml of latex/kg body weight and included multi-focal coagulation necrosis of cardiac bers and vacuolized hepatocytes. Subsequently, three groups of two sheep weretreated with (1) a single dose of 30 g/kg, (2) a single dose of 60 g/kg or (3) 60 g/kg per dayfor 10 consecutive days. Exposure to the C. procera leaves was responsible for tachycardiaand transitory cardiac arrhythmias in sheep from all groups. Gross pathological analysis of sheep dosed with 60 g/kg per day for 10 days revealed mild ascites, exudates on thetrachea, pulmonary edema, mild hemorrhage in the liver, hydropericardium, accid heart,ulcers on the abomasum and kidneys presenting pale juxtamedullary cortex. The histo-logical ndings of the rat and sheep studies were similar and included multi-focal coag-ulation necrosis of cardiac bers and vacuolized hepatocytes. In conclusion, our ndingsindicate that C. procera is a cardiotoxic and hepatotoxic plant. 2010 Elsevier Ltd. All rights reserved. Calotropis procera (Aiton) W. T. Aiton is an invasive alienweed from the Asclepiadaceae family and is verycommonly found in the semi-arid northeastern region of Brazil. Hay made from C. procera has been considereda good animal food because it contains high levels of crudeprotein content and is highly digestible. However, lambsfed with C. procera hay present impaired weight gain(Madrugaetal., 2008).Furthermore,incidentalingestionof fresh C. procera leaves has been suggested as toxic to manyruminants by several farmers from the Brazilian semi-aridregion. These observations are supported by a few studiesthat have reported toxic effects promoted by C. procera latex (Mahmoud et al.,1979b; Pahwa and Chatterjee,1988;Singhal and Kumar, 2009) and leaves (Mahmoud et al.,1979a). This study aimed to describe the toxic effects of administration of C. procera leaves to sheep and C. procera latex to rats.Leaves and latex from C. procera (Aiton) W. T. Aiton(Apocynaceae) were collected immediately before use.Only mature leaves without any sign of lesion were used.Latex was collected by breakage of the stem and directput in a glass vial without solvent. The experiments andplant collection were performed near Mossoró city, RN, * Corresponding author. Tel.: þ 55 84 33151920; fax: þ 55 84 33151770. E-mail address:
[email protected] (B. Soto-Blanco). Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon 0041-0101/$ – see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.toxicon.2010.11.007 Toxicon 57 (2011) 183 – 185 northeastern Brazil (5 11 0 15 00 S and 37 20 0 39 00 W) at an alti-tude of 16 m above sea level. The climate in this region ischaracterized as semi-arid. The mean annual temperaturein this region is 27.4 C, and the mean annual rainfall andmean relative humidity are 674 mm and 68.9%,respectively.Adult male Wistar rats (weights of about 150 g) wereobtained from the Animal Sciences Department, Uni-versidadeFederalRuraldoSemi-Árido,Mossoró,RN,Brazil.Commercial food rations (Labina, Purina, São Lourenço daMata, PE, Brazil) and tap water were provided to theanimals ad libitum .Theanimalroomwasmaintainedat22 – 24 C with a 12-h light/dark cycle. Twenty male rats wereseparated into ve groups (four animals/group) and weretreated with intra-peritoneal injection of fresh C. procera latex (without carrier solvent) at 1.0, 0.6, 0.3 or 0.1 ml of latex/kg of body weight, and control animals were injectedwith 0.9% NaCl. The rats were monitored closely for 48 h.Dead rats were necropsied for pathological study. Duringthe necropsy, fragments of the heart, liver, kidneys, lungsand spleen were collected and xed in 10% formalin. Theparaf n-embedded sections were stained with hematox-ylin and eosin (H&E).Intact male sheep, weighing 12 – 19 kg, were exposed to C.procera leavesbygavage.Threegroupsoftwosheepweretreated with (1) a single dose of 30 g/kg, (2) a single dose of 60 g/kg or (3) 60 g/kg per day for 10 consecutive days,administered by gavage in a single dose per day. The plantmaterial was prepared by chopping the leaves in a blenderwith the lowest amount of water possible (approximately750 ml). All animals wereclosely monitoredforanyclinicaldisturbance. The two sheep dosed for 10 consecutive dayswere euthanized 24 h after the nal dose for pathologicalstudy. After they were sacri ced, the sheep were necrop-sied, and samples from the liver, kidney, lungs, heart,spleen, rumen, omasum, abomasum and intestines werecollected, xed, and stored in 10% buffered formalin forhistopathological examination. The paraf n-embeddedsections were stained with H&E.All of the rats dosed with 1.0 ml of latex/kg body weightshowed severe lethargy beginning5 – 8 min afterdosing anddied within 2 h. No death or clinical signs of toxicity wereobservedintheratsfromcontrolgroupanddosedwith0,0.1,0.3or0.6mloflatex/kgbodyweight.Nomacroscopiclesionswere found in the necropsies of dead rats. The histologicallesions were restricted to rats dosed with 1.0 ml of latex/kgbody weight. Microscopic lesions in the hearts appearedas bers separated by edematous uid, and the ratsexhibited subendocardic hemorrhages, multi-focal coagula-tion necroses of the muscular bers evidenced by granularappearance of the sarcoplasm, distinct eosinophilic cyto-plasm lacking transverse striations and presenting pyknoticor absent nuclei (Fig.1). In ltration of mononuclear in am-matorycells was observed between the cardiac bers. Somemuscle bers presented basophilic granulation and prom-inent vacuolization of the sarcoplasm (Fig. 2). The liversshowed diffuse vacuolization of the hepatocyte cytoplasm,marked sinusoidal congestion and small hemosiderindeposits in the parenchymal hepatocytes.The administration of C. procera leaves to sheep from allgroups was responsible for tachycardia and transitorycardiac arrhythmias at auscultation 4 h after dosing. Thenecroscopic examination of sheep dosed with 60 g/kg perday for 10 days revealed mild ascites, exudates on thetrachea, pulmonary edema, mild hemorrhage in the liver,hydropericardium, accid heart, ulcers on the omasum andkidneys presenting a pale juxtamedullary cortex. Thehistological examination of livers and hearts from thesheep revealed similarlesionstothoseobservedin therats,but the intensity of these lesions varied from mild tomoderate. Congestion was observed in the kidneys andlungs. No lesions were found in the spleen, rumen,omasum, abomasum or intestine samples from thesesheep.Our results demonstrate that C. procera is a cardiotoxicplant. The lesions promoted by exposure to C. procera latexand fresh leaves were different from those observed inother studies (Mahmoud et al., 1979a, 1979b; Pahwa and Chatterjee, 1988; Singhal and Kumar, 2009). The lesions promoted by C. procera exposure in sheep and goats inearlier studies included hemorrhage in the heart, necrosis Fig.1. Coagulationnecrosisoftheheartmuscle bersofa C.procera -dosedrat,evidencedbygranular-appearingsarcoplasm,distincteosinophiliccytoplasm,lack of transverse striations and pyknotic or absent nuclei (HE, 40x). Fig. 2. Heart from a rat dosed with C. procera, showing prominent vacuo-lization of the sarcoplasm (HE, 40x). J.M. de Lima et al. / Toxicon 57 (2011) 183 – 185 184 of hepatocytes and renal convoluted tubular cells(Mahmoud et al.,1979a,1979b). Two previous studies with rodents observed lesions in the liverand kidneys, but thesestudies did not evaluate the heart (Pahwa and Chatterjee,1988; Singhal and Kumar, 2009). It is known that the Brazilian species Nerium oleander , Cryptostegia venusta , Ateleia glazioviana , Tetrapterys acuti- folia , Tetrapterys multiglandulosa and Senna occidentalis promote direct heart disruption in ruminants (Barros et al.,1999; Stigger et al., 2001; Riet-Correa et al., 2005; Soto- Blanco et al., 2006; Barbosa et al., 2008; Pedroso et al.,2009; Nunes et al., in press). Poisoning by S. occidentalis istypically not acute and is characterized by cardiomyopathyand degeneration of skeletal muscular bers (Barros et al.,1999). The species A. glazioviana , T. acutifolia and T. multi- glandulosa can be responsible for cardiotoxicity and abor-tions (Stigger et al., 2001; Riet-Correa et al., 2005). The clinical and pathological features of S. occidentalis , A. gla- zioviana , T. acutifolia and T. multiglandulosa poisonings areverydifferent fromthoseobservedin C. procera poisonings. N. oleander and C. venusta are potent cardiotoxic plants.Experimental administration of N. oleander to sheeprevealed myocardial degeneration and necrosis associatedwith severe hemorrhage and in ltration of mononuclearin ammatorycells (Aslani et al., 2004). Microscopic lesions in cattle poisoned by N. oleander were revealed as coagu-lation necroses of individual cardiac bers or small groupsof bers, characterized by enhanced cytoplasmic eosino-philia and pyknotic nuclei (Pedroso et al., 2009). Theprimary microscopic lesions found in goats dosed with N.oleander (Barbosa et al., 2008) or C. venusta (Nunes et al., inpress) were degeneration and multi-focal necroses of thecardiac muscle bers. Thus, the pathological lesions of C. procera poisoning are very similar to those observed inpoisoning by C. venusta and N. oleander .Phytochemical studies have revealed that C. procera contains a mixture of cardenolides, including procerageninand 2 00 -oxovoruscharin (Akhtar et al., 1992; Hanna et al.,1999, 2002; Van Quaquebeke et al., 2005). Cardenolides are cardiac-active compounds that inhibit the cellularmembrane Na þ /K þ ATPase, resulting in an electrolyticdisturbance that affects the electrical conductivity of theheart ( Joubert, 1989; Aslani et al., 2004). In conclusion, our results indicate that C. procera isa cardiotoxic and hepatotoxic poisonous plant, and thesafetyofitsuseinanimalfeedshouldbecarefullyevaluated. Acknowledgment The research received nancial support from the Insti-tuto Nacional de Ciência e Tecnologia para o Controle dasIntoxicações por Plantas, CNPq, Grant 573534/2008-0. Con ict of interest statement The authors declare that there is no con ict of interest. 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