Acta Biológica Colombiana ISSN: 0120-548X [email protected] Universidad Nacional de Colombia Sede Bogotá Colombia TAPIERO HERNÁNDEZ, YACSON; RONDÓN BARRAGÁN, IANG; CÉSPEDES RUBIO, ANGEL NEUROTOXIC POTENTIAL OF TRICHLORFON TO MULTIPLE SUBLETHAL DOSES IN WISTAR RATS Acta Biológica Colombiana, vol. 18, núm. 3, septiembre-diciembre, 2013, pp. 479-487 Universidad Nacional de Colombia Sede Bogotá Bogotá, Colombia Available in: http://www.redalyc.org/articulo.oa?id=319029232007 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative . UNIVE RSIDAD ~ • NACIONAL .; DECOLOMBIA , SE DE 1I 0 G O T Á ACTA BIOLÓGICA COLOMBIANA f ACUlT AD DE CIENCIAS DEPAAT.uolEI'(f() DE8101.OOiA. Artículo de investigación NEUROTOXIC POTENTIAL OF TRICHLORFON TO MULTIPLE SUBLETHAL DOSES IN WISTAR RATS Potencial neurotóxico del Triclorfón a dosis múltiples subletales en ratas wistar YACSON TAPI ERO HERNÁNDEZ1, Est. MVZ; IANG RONDÓN BARRAGÁN1, M.5c.; ANGEL CÉSPEDES RUBIO', Ph. D. 1 Grou p for Research in Ne urodege nera tive o isease.Toxico logy Laboratory (33 L-1 01 ), De pa rt ment ofAn imal Health, Fac uIty ofVeteri nary Medicine and Zootecnia,Universidad del Tolima. A.A. 546 lbagué, Colombia. isro ndon S'u t.edu.co, yacsontapiero@hotmail,com, b iorned icineresearch<&yahoo.es Corresponding author: Angel Céspedes, bio med icineresearch@ya hoo ,es, aecesp ed @ut ,ed u ca Presentado el30 de abril de 2013, aceptado el30 de mayo de 2013, fecha de reenvfo el14 de septiembre de 2013. Citation/ Citar este articulo como: TAPIERO Y, RONDÓN 1, CÉSPEDES A. Neurotoxic potential oftrichlorfon to multiple sublethal doses in wistar rats. Acrabiol. Colomb.18(3):479-488. ABSTRACT The organophosphates used for pest control induce sensory, motor and psychiatric disturbances alter chronic exposure. The ester type is the cause ofthe intermediate syndrome and delayed neuropathy, in which the white and gray matter in the brain are severely affected.The aim of this study was to evaluate the effect of multiple sublethal doses ofTrichlorfon on neurons, astrocytes and myelinated tissue in a rat model ofbrain neurotoxicity. Trichlorfon (metrifonate) was administered to adult Wistar rats at doses of 11 or 22 I-lg/kg by oral gavage every seven days for four or eight weeks (four experimental groups) and a control group (placebo). One week alter the last dose, animals were euthanized and the brains perfused, removed and cut into coronal segments of50 p m ofthickness by using a vibratome. The sections were analyzed by immunohistochemistry, using markers of neuronal survival, astrocytic reactivity and the myelin basic protein. Neuronal and astrocytic reactivity were significantiy reduced in Trichlorfon-treated animals relative to controls, whereas myelin reactivity was significantly increased, with abnormal distribution of myelin in white matter. The results suggest a neurotoxic damage ofTrichlorfon on neuronal and astrocyte functional balance and abnormal myelin formation consequent to the cell damage. Keywords: astrocytes, myelin, neurons, organophosphates, toxicology. RESUMEN Los organofosforados usados para control de plagas inducen trastornos sensoriales, motores y psiquiátricos por exposición crónica, siendo los de tipo éster, causa del síndrome intermedio yde la neuropatía retardada, que afectan severamente la sustancia blanca y gris del cerebro. El objetivo del presente trabajo fue evaluar el efecto del organofosforado Triclorfón sobre neuronas, astrocitos y tejido mielinizado en un modelo murino de neurotoxicidad encefálica a dosis múltiples subletales. Se suministró a ratas Wistar, triclorfón (metrifonato) a dosis de 11 y 22 I-lg/kg mediante sondaje esofagogástrico, cada siete días durante cuatro y ocho semanas en cuatro grupos experimentales y un grupo control (placebo). Una semana después de la última dosis, los animales fueron sacrificados y los cerebros pe-fundidos, extraídos y cortados en segmentos coronales de 50 urn de grosor mediante vibrátomo. Los cortes fueron analizados por inmunohistoquímica, usando marcadores de supervivencia neuronal, de reactividad astrocitaria y de la proteína base mielina. La reactividad neuronal y astrocitaria se redujo significativamente en los animales tratados con triclorfón en relación a los controles, mientras la reactividad de la mielina se incrementó significativamente, con distribución anormal en la sustancia blanca. Los resultados sugieren un daño neurotóxico del Triclorfón sobre el equilibrio Acta biol. Colomb., 18(3):479-488, sep tiembre - d iciembre de 20 13 - 479 Tapiero Y, Rondón 1,Céspedes A funcional neuronal y astrocitario, con formaciones anómalas Experimental Design de mielina consecuente al daño celular. The rats were distributed into four experimental groups T'l , T2, T3, T4 (n = 16) with four rats per group and a control Palabras clave: astrocitos, mielina, neuronas, organofos­ group (n ~ 8). Trichlorfon (Dimethylphosphonate of2,2,2­ forados, toxicología. trichloro-l-hydroxyethyl) powder 97 % (Bayer ®) was pre­ pared in solution at 1:1000 (1 mg/mL) in corn oil asvehicle. INTRODUCTION The rats ofthe groups Tl and T3 received a weekly dose of Inappropriate use of pesticides is common in developing 11 pgj kg ofTCF for four or eight weeks, respectively. The countries and approximately 3,000,000 cases of acute animals ofthe groups T2 and T4, received a weekly dose of toxicosis are reported every year, with 220,000 deaths TCF (22 pgjkg) for four and eight weeks, respectively. The (Fenske et at., 2002;Jaga and Dharmani, 2003; Barguil-Díaz calculated doses were administered by gavage and the rats et at., 2012). The uncontrolled use of organophosphates ofcontrol group received the same volume ofTCF-free corn (OPs) affects ecosystems and organisms that are not direct oil (1 mL) via the same administration route, frequency and target ofits action (Yavuzetal., 2005; De Silva et at., 2006). duration that the experimental groups. Doses were esta­ OPs have been used in industry as antioxidants and blished from the acceptable daily intake (ADI Acceptable plasticizers, as agriculture and household insecticides, and Daily Intake) reported bythe WHO (Lu, 1995) fortrichlorfon in the production of neurotoxic gases for warfare (Carod­ (ADI ~ 0.011 mgjkg weight) and after a previous test with 4 Artal and Speck-Martins, 1999; Jaga and Dharmani, 2003). dose (SS - 5.5 - 0.55 and 0.055 mgjkg weight) to establish OPs are inhibitors ofplasma and eryth roeyte cholinesterase which ofthem did not triggered TCF cholinergic syndrome. activity Gaga and Dharmani, 2003); however, trichlorfon The average lethal dose reported as LOSO in rats is 450-650 (TCF), chlorpyrifos (CPF) and other OPs, produce toxicity mgjkg (Karademir-Catalgol et at., 2007). upon metabolic transformation into "oxons", which are less stable and up to three times more active as AChE inhibitors Extraction and Preparation 01 Brain Tissue than the original compounds (Monnet-Tschudi et at., 2000). Seven days after the last treatment, the animals were anesthe­ While acute toxicity ofpesticides has been well documented, tized with sodium pentobarbital 60 mg/kg (Penthal 6.48 %, effects after chronic exposureare yet unknown (De Silva et at., Invet, SAl and xylazine 10 mgjkg 2 % Bayer SAl intraperito­ 2006). However, they have been reported to result in syndromes neally (i.p.) The brains were perfused intracardiallywith NaCl such as leukoencephalopathy induced by organophosphate 0.9 %N (200 mL) using aortic advance at moderate positive (LElO), delayed neurotoxicity induced by organophosphates pressure and subsequently fixed with paraformaldehyde (OPIDN) and chronic neurotoxicity induced byester-type (PFA) 4% (200 mL). The brainswere extracted and post-fixed organophosphates (OPICN) (Carod-Artal and SpeckMartins, (PFA 4 % at 4 °C/24 hours) for subsequent cutting into 50 1999; Abou-Donia, 2003). Kamanyire and Karalliedde (2004) urn coronal sections (Vibratome 1500) and conservation in describe the final stage of neuropathy caused by OPs after a cryopreservative. seven-ten days of exposure, which persisted by four weeks after eight weeks ofcontinuous exposure, presumably caused Immunohistochemistry by inhibition ofNeuropathyTarget Esterase (NTE) in a murine Immunohistochemistrywas carried out following the protocol model of subchronic exposure (Aiuto et at., 1993; Moretto described in Current Protocols in Neuroscience (Volpicelli­ and l otti, 1998; Rayand Richards, 2001). Daley and Levey, 2003) with modifications as follows: The aim ofthis study was to evaluate the effect ofmultiple Inhibition ofendogenous peroxidase (Methanol:PBS 1:1 - 1 % sublethal doses ofTrichlorfon on neurons, astrocytes and H202), washed with PB 0.1 M, pre-incubation (PB 0.1 M ­ myelinated tissue in the brain ofWistar rats. Triton 1OOX 3 % - BSA 1 %) for 60 minutes and incubation at 4 "C overnight in the primary antibodies (anti-NeuN A60 MATERIALS AND METHODS 1:1000 Millipore Corporation, Billerica, USA, anti-GFAP Animals 1:500 Sigrna-Aldrich, St. Louis, USA and anti-MBP 1:100 Twenty male Wistar rats with an average weight of 200 ± Sigrna-Aldrich.Sr. Louis, USA) prepared in buffer (PB 0.1 M­ 10 g were used. Animals, from the vivarium ofthe University Triton 1OOX 0.3 % and BSA 0.3%). Consecutively, washes of Tolima, were kept on dark/light cycle (12:12-h) and with PB 0.1 M and incubation in