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
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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 secondary antibody (goat received food and water ad libitum. The treatments were anti-mouse and goat anti-rabbit 1:500 Thermo Scientific, made in the biotechnology and toxicology laboratories at the Rockford IL., USA) for two hours at room temperature were University ofTolima. The rats were handled according to performed. After, sections were incubated in Avidin/Biotin Colombian standards (Law 84 of 1989), European Union (1 :250 each; Thermo Scientific, Rockford IL., USA) for two guidelines (86/609/EEC) and the experiments were conducted hours and developed with diaminobenzidine (DAB Sigma upon approval ofthe Local Ethics Committee (Act No. 8 of Aldrich, Sr. Louis, USA) at 11 mg/l S mL PB 0.1 M -H202 June 2, 2010). 0.02 %). The sections were put on slides, covered with cover-
480 - Acta biol. Colomb., 18(3):479-488, septiembre - diciembre de 2013 Neurotoxic Potential ofTrich1orfon to Multiple Sublethal Doses in Wistar Rats
slips and sealed with resinous solution (Shandon Consult vs. Control (*p < 0.05) and highly significant differences Mount®, Kalamazoo, Mi. USA). between T4 and T3 (± ± ±p < 0.001) (Fig. lc). In the PVZ, Photomicrographs were taken by using optic microscope no significant differences between any of the treatments, (Motic Microscopes BA 210 NY, USA) and digital camera neither ofthese with respect to control group, were observed (Moticam 2000 2.0M Pixel, NY, USA) and the digitalized (Fig.ld). images (10X) were taken in the hippocampal CAl, interna! capsule, striatum and the paraventricular zone (PVZ), then Ellect 01 Trichlorlon on GFAP Immunoreactivity analyzed by densitometry with the Fiji-Image J software In the hippocampal CAl area (Fig. 2a, AE), reactivity ofthe (v-lAS - NIH). The brain sections were prepared in parallel GFAP was significantly reduced in the T2, T3 and T4 groups for immunohistochemistry, so that incubation with the relative to the control group (***p < 0.001). Additionally, specific antibodies, the complex avidin / biotin and DAB, T2 vs.Tl (O o o p < 0.001 ), T3 vs. Tl (~
Acta biol. Colomb., 18(3):479-488, septiembre - diciembre de 2013 - 481 Ta piero Y,Ro ndón I, Césp edes A
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Figur e 1. Representative coronal sections of the NeuN im munoreactivity in different bram regi ons of rats exposed to m ultiple doses of TC F. Hipp ocamp al CAl area (Fig. la, A-E, lb), PVZ (Fig. l a, F-], Id ) and lateral striatum body (Fig. la, K-O, l e). T1 : T CF 11 )lgjkg each week for four weeks (A, F, K), T2: T CF 22 )lgjkg each week for fou r weeks (B, G, L), T3: T CF 11 )lgjkg each week for eigh tweeks (C, H, M) an d T4: T CF 22 )lgjkg each week for eight weeks (D, I, N) in companson wit h the control group CTR (E,], O). NeuN marking decreased in a dose- and tim e ofexposure dependent way. n- 16 (lOX). Values are expressed in units of relative density. Selle bar 100 u.
482 - Acta bioL Colomb., 18 (3):479-488 , se pt iem bre - di ciembr e de 20 13 Neurotoxic Potential ofTrichlorfon to Multiple Sublethal Doses in Wistar Rats
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2J . ~ 00 150000 ."=:~ e'E 100000 B . ~ $<1' 50000 8 •• ••• # # # ;; o El ~ ••• o = CIR TI T2 13 14 Figure2. Representarive coronal secrions ofGFAP immunoreactiviry in differenr brain regions ofrats exposed to mulriple doses ofTCF. Hippocampal CAl area (Fig. Za, A-E, 2b), late cal striatum body (Fig. Za, F-], 2e) and PVZ (Fig. Za, K-O, K2). TI: 11 flgfkg TCF eaeh week for four weeks (A, F, K), T2: 22 flgfkg TCF eaeh week for four weeks (B, G, L),13: 11 flgfkg TCF eaeh week for eight weeks (C, H, M) and T4: 22 flgfkgTCF eaeh week for eight weeks (D, 1,N) in comparison wirh the control group CTR (E,], O). GFAP marking decreased in adose and time ofexposure-dependentway. n = 16 (10X). Values are expressed in unirs of relative densiry, Scale bar 100 J-l. phosphorylation ofthe serine hydroxyl group (Aluigi et al., mitters produeing structural and funetional eellular ehanges 2005) and direetly interaet with other moleeules sueh as that interfere with neurotransmission (Yousefpour et al., membrane channels, molecular receptors and neurotrans- 2006). Due to the inhibition ofAChE, triehlorfon has been
Acta biol. Colomb., 18(3):479-488, septiembre - diciembre de 2013 - 483 Tapiero y) Rondón 1) Céspedes A
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Figure 3. Representarive coronal sections of the MBP immunoreacriviry in lateral srriatum body and inrernal capsule of rats exposed to mulriple doses ofTCF. Lateral srriatum body (Fig. 3a and 3b A-E), inrernal capsule (Fig. 3a, F-J and 3c). Tl: 11 J-lglkgTCFeach week for four weeks (A,F), T2: 22 f-lgjkgTCF each weekfor fourweeks (B,G), T3: 11 f-lgjkgTCF each weekforeightweeks (C, H) and T4: 22 f-lgjkgTCF each weekfor 8 weeks (D, 1)compared wirh the control group CTR (E,]). MBP marking increased significant1y in the inrernal capsule mainly by the effect ofTCF in T4. The photographs of allIHC groups correspond to MBP and counrerstained wirh Nissl. n= 16 (10X).Values are expressed in unirs of relative densiry. Scale bar 100 J-l. used as a therapeutic agent againstAlzheimer disease (Liu et shown that metabolites derived from Trichlorfon, like al., 2002; Becker et al., 2009), nevertheless, some studies aware dichlorvos, can induce a neurotoxic effect even greater than about the toxicity ofthis therapy including the inhibition of the starting compound. In addition, chronic exposure to other enzymes affected bythe organophosphate class ofdrugs, trichlorfon affects the cerebral glucose metabolism and may by tolerance of low doses allowing too rapid dose escalation induce an acidosis state (Poindessous-Jazat et al., 1998; Liu et and irreversible enzyme inhibition producingcumulative drug al., 2009). The exposure to trichlorfon decreases the neuronal effects (Rakonczay, 2003; López-Arrieta and Schneider, and glial viability in a dose-dependent manner (Liu et al., 2006; Becker and Greig, 2008; Becker et al., 2009; Becker and 2009), which agree with the findings ofthe present study, Greig, 2010). that evidenced a decrease in immunoreactivity of neurons and astrocytes in CA1 hippocampus and lateral striatum Effect of TCF on Neuronal and Astrocytic Population body; but in PVZ, just astrocyte marker was reduced. It has Several studies have demonstrated the deleterious potential been reported that the decrease in these cell populations is ofOPs on cell populations (Pohanka et al., 2011) including an apoptosis-mediated processes with the high toxic effect neurons and glial cells (Carlson et al., 2000; Yousefpour et al., attributed to the secondary metabolite dichlorvos (Carlson 2006; Flaskos et al., 2007, Liu et al., 2009). Other studies have et al., 2000; Liu et al., 2009).
484 - Acta biol. Colomb., 18(3):479-488, septiembre - diciembre de 2013 Neurotoxic Potential ofTrich1orfon to Multiple Sublethal Doses in Wistar Rats
The OPs induce oxidative stress (Kaur et at., 2007) and cell vacuoles, neuronal degeneration and necrosis, and intraeyto death in animals exposed to trichlorfon orits oxon metabolites plasmic myelin forms (Yousefpour et at., 2006). (Guizzetti et at., 2005). The overproduction offree radicals In another study related with the in vitre effect ofChlorpyrifos involved in the glial activation are typical in c1inical progress (CPF) on glia, itwas established that elicit disruption in glial of neurodegenerative diseases by exposure to toxic (Astiz et development. Furthermore, since astroeytes and oligoden at., 2012) and this can result in apoptotic cell deach, which droeytes (the myelin-forming cells) arise from one common is consistent with a marked reduction in the expression of glial precursor, myelination can be affected by chlorpyrifos neuronal protein immunoreactivity. (Garcia et at., 2002), suggesting that exposure to the CPF is Several studies have been shown the involvement ofastrocytes related to the reduction in the levels of myelin associated in neuroprotection and neurorepair of nervous tissue after glycoprotein (MAG) mRNA, inhibition of DNA synthesis in exposure to the toxins, at blood-brain barrier (BBB) level undifferentiated oligodendrocytes and astrocytes as well as (Giordano et at., 2008; Sofroniew and Vinters, 2010). It has alterations in the expression ofGFAP and MBP (Saulsburyet been described that sublethal doses ofOP decrease protein at., 2009). markers for astrocytes (Garcia et at., 2002) and the total The hyperreactivity of MPB in the present study after TCF number ofglial cells (Roy etal., 2004), that is compatible with exposure shows not only an increase in MBP marker density, this study, where the GFAP protein immunoreactivity was but a distribution ofabnormal forms in comparison with the found signiflcantly reduced in the hippocampus, lateral control group. Because MBP protein was evaluated as a marker striatum body and PVZin all treated groups at both doses of of mature myelin in white matrer-rich areas (intemal capsule TCF (11 and 22 ~g/kg). 5imilarly, exposure to the oxon forms, and striatum body) where there is also a large population of such as chlorpyrifos oxon, significantlydecreases the glutamine radiated astrocytes, is possible that these hyperreactive forms synthetase activity (a marker for astroeytes). This effect may may be involved in the proliferation of oligodendroeytes or be mediated by direct toxicity on astroeytic cells and can astrocytes within a tissue repair process or remyelination. directly interfere with the cellular replication (Qiao et at., Seems that glial cells, astrocytes and oligodendrocytes, are 2001; Flaskos, 2012). essential for neuronal differentiation, myelination, the The TCF and its metabolite dichlorvos easily cross the BBB propagation of synaptic impulses and the maintenance of and inhibir both acetylcholinesterase (AChE) and butyrylcho homeostasis (Saulsbury et at., 2009), thus any disruption of linesterase (BuChE) (Pohanka etal., 2011). In the present these cells can lead to serious functional disturbances. study, the decreased immunoreactivity ofGFAP in the para In mice mutant for the gene encoding the synthesis ofGFAP, ventricular area, which corresponds to a border area in close abnormal myelination in white matter, suggest the involvement contact with the ventricles, the BBB can be seriously com ofGFAP as a link between astrocytic fimction and myelination promised by the decrease in the population of astroeytes. GFAP and MBP have been used as biomarkers in glial Parran etal., (2005) and Song etal., (2004) showed that alterations, suggesting that the effect on glia maycontribute exposure to OPs affects the integrity ofthe BBBand alters their to the late onset ofneuronal damage (Roy et at., 2004) and functionality. can be used as biomarkers ofneurotoxicity induced by OPs. Although c1inical signs in patients with leukoencephalopathy Ellect 01 TCF on Myelination Status induced by organophosphate (LElO), delayed neurotoxicity In this study, the reactivity ofthe MBP protein was increased induced by organophosphates (OPIDN) and chronic neuro by TCF in adose and time dependent manner with higher toxicity induced by ester-type organophosphates has been reactivity to 22 [..lg/kg only after eight weeks ofexposure. This reported, we have not observed any c1inical signs, probably increase in MBP reactivity may be indicative ofmyelination for low doses used in this investigation; maybe it is adose or remyelination processes resultant to tissue damage dependent effect. In the other hand, the aim ofthis studywas induced by the OP, which contrasts with that reported by to evaluate-the effect ofTrichlorfon on neurons, astrocytes Flaskos (2012) who found a decrease in eyclic nucleotide and myelinated tissue in a rat model of brain neurotoxicity phosphohydrolase (a marker for oligodendrocytes). Exposure to multiple and sublethal doses which may help to elucidare to OPs alters eytoskeleton-associated neurofilaments leading the cellular effects that accompany referring syndromes in to the destruction, which in turn leads to destruction ofthe humans and can help in the research oftherapeutic drugs axon as well as myelin sheaths. This process also seems to be and pathophysiological study of neurological diseases by mediated by increased levels ofintracellular calcium (Abou pesticide and other toxic chemicals Donia, 1993; Song etal., 2009). Also, it has been described that OPs, particularly oxon-type, can produce a disruption CONCLUSIONS of neuronal processes by detriment of growth factors and Our results indicate that TCF exposure to low sublethal doses inhibition of its receptors, inducing cell death. At the of 11 and 22 [..lg/kg for four and eight weeks in Wistar rats, ultrastructural level, mitochondrial dilation, disruption of was sufflcient to generate cell damage in neurons and rough endoplasmic reticulum, production oflysosomallipid astrocytes in the hippocampus (CAl), striatum and PVZ,while
Acta biol. Colomb., 18(3):479-488, septiembre - diciembre de 2013 - 485 Tapiero Y, Rondón 1,Céspedes A
MBP hyperreactivity in white matter with anomalous shapes about chronic exposure? Trans R Soc Trop Med Hyg. and changes in the distribution of mature myelin, suggest 2006;100(9):803-806. modification in the remyelination process subsequent to injury Fenske RA, Lu C, Barr O, Needham L. Children's exposure to by TCF. Anti-NeuN, anti-GFAP and anti-MBP antibodies are chlorpyrifos and parathion in an agricultural community proposed as sensitive neurotoxicity biomarkers of OP oxon in central Washington State. Enviren Health Perspect. type exposure or by chronic toxicity to these. 2002;11 O( 5):549-553. Flaskos ], Harris W, Sachana M, Munoz O, Tackj, eta/. The ACKNOWLEDGEMENTS effects of diazinon and cypermethrin on the differen The authors express their gratitude to the Office ofScientific tiation of neuronal and glial cell lines. Toxico! 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