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Effects of diazinon on the lymphocytic cholinergic system of Nile tilapia fish (Oreochromis niloticus)
Veterinary Immunology and Immunopathology 176 (2016) 58–63
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Effects of diazinon on the lymphocytic cholinergic system of Nile tilapia fish (Oreochromis niloticus) G.A. Toledo-Ibarra a , K.J.G. Díaz-Resendiz a , L. Pavón-Romero b , A.E. Rojas-García c , I.M Medina-Díaz c , M.I. Girón-Pérez a,∗ a
Laboratorio de Inmunotoxicología, Universidad Autónoma de Nayarit, Secretaría de Investigación y Posgrado, Laboratorio de Inmunotoxicología, Boulevard Tepic-Xalisco s/n, Cd de la Cultura Amado Nervo, C.P. 63190, Tepic Nayarit, Mexico b Departmento de Psicoimmunología, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, Calzada México-Xochimilco 101, Col. San Lorenzo Huipulco, Tlalpan, 14370 Mexico City, DF, Mexico c Laboratorio de Contaminación y Toxicología Ambiental, Universidad Autónoma de Nayarit, Secretaría de Investigación y Posgrado, Laboratorio de Inmunotoxicología, Boulevard Tepic-Xalisco s/n, Cd de la Cu ltura Amado Nervo, C.P. 63190, Tepic Nayarit, Mexico
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Article history: Received 11 June 2015 Received in revised form 2 May 2016 Accepted 20 May 2016 Keywords: Acetylcholine Acetylcholinesterase Immunotoxicity Organophosphorus pesticides Diazinon Nile tilapia fish
a b s t r a c t Fish rearing under intensive farming conditions can be easily disturbed by pesticides, substances that have immunotoxic properties and may predispose to infections. Organophosphorus pesticides (OPs) are widely used in agricultural activities; however, the mechanism of immunotoxicity of these substances is unclear. The aim of this study was to evaluate the effect of diazinon pesticides (OPs) on the cholinergic system of immune cells as a possible target of OP immunotoxicity. We evaluated ACh levels and cholinergic (nicotinic and muscarinic) receptor concentration. Additionally, AChE activity was evaluated in mononuclear cells of Nile tilapia (Oreochromis niloticus), a freshwater fish mostly cultivated in tropical regions around the world. The obtained results indicate that acute exposure to diazinon induces an increase in ACh concentration and a decrease in nAChR and mAChR concentrations and AChE activity in fish immune cells, This suggests that the non-neuronal lymphocytic cholinergic system may be the main target in the mechanism of OP immunotoxicity. This study contributes to the understanding of the mechanisms of immunotoxicity of pollutants and may help to take actions for animal health improvement. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture consists in the rearing of aquatic organisms such as fishes, molluscs, crustaceans and plants. In this context, fishes are by far the main class of organisms that are produced by means of this activity. Indeed, the fish production through aquaculture has surpassed the production through fisheries. One of the most farmed freshwater fish species in the world is the tilapia (Oreochromis niloticus). According to data from the FAO, the production of this type of fish in 2011 was 4027 million tons (FAO, 2012). Disease incidence in tilapia farming, in optimum farming densities and conditions, is not frequent. Nevertheless, there are factors that can compromise the health of these organisms and the innocuousness of the product for human consumption. Among these, we can mention pathogenic organisms and environment pollutants such as pesticides (Harms et al., 2003). In the case of pesticides,
∗ Corresponding author. E-mail address: ivan [email protected] (M.I. Girón-Pérez). http://dx.doi.org/10.1016/j.vetimm.2016.05.010 0165-2427/© 2016 Elsevier B.V. All rights reserved.
these substances can penetrate aquatic ecosystems as a consequence of rain, soil leaching or for being carelessly discharged directly into bodies of water (Fanta et al., 2003; Kavitha and Venkateswara Rao, 2009). Furthermore, in some countries, pesticides are used directly in aquaculture for the elimination of ectoparasites (Khoshbavar-Rostami et al., 2006). However, there is evidence that pesticides can induce an immunotoxic effect; hence the health of fishes exposed to these substances can be compromised (Harford et al., 2005), and even cause death, which has a serious effect in aquaculture production (Khoshbavar-Rostami et al., 2006; Pirarat et al., 2006). Diazinon (O,O-diethyl-O-[2-isopropyl-4-methyl-6pyrimidinyl] phosphorothioate) is one of the most used organophosphorus pesticides (OPs) in agricultural (control of agriculture, livestock and aquaculture pests) and domestic activities (NRA, 2000; Khoshbavar-Rostami et al., 2006). OPs are powerful neurotoxins since they inhibit the activity of the acetylcholinesterase (AChE) enzyme. Diazinon is metabolized to diazoxon, a substance that phosphorylates the hydroxyl group of serines in the active site of AChE. This phosphorylation disrupts the
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Fig. 1. Activity of ChEt and AChE in spleen mononuclear cells of fish (n = 7) exposed to diazinon for 6 h (a), 12 h (b) and 24 h (c). Results are expressed as mean ± S.D.; one-way ANOVA and Tukey tests with 95% confidence were used.
enzyme´ıs ability to breakdown the acetylcholine neurotransmitter (ACh), resulting in an excessive accumulation of ACh, which overstimulates the muscarinic (mAChR) and nicotinic (nAChR) receptors, located in the post-synaptic cells. This way, inhibition of AChE induces uncontrolled nervous impulses that manifest as changes in behavior (movement diminishment, breathing capacity, predator avoidance, prey selection and reproduction loss) and causes death (Fanta et al., 2003; Barbieri and Alves Ferreira, 2011; Tilton et al., 2011; Costa, 2006). In addition to the neurotoxic effects, OPs can cause immunotoxic effects; however, there is little information regarding the possible immunotoxic mechanisms of these substances. It has been reported that OPs can cause direct effects such as the inhibition of serine hydrolases, immunological complement system, oxidative damage, alteration of signal transduction pathways, proliferation and differentiation of cells of the immune system (Galloway and Handy, 2003; Li, 2007). In this regard, our research group has reported that sublethal concentrations of diazinon cause a decrease in lymphocyte proliferative capacity, phagocytic activity and relative spleen weight in the Nile tilapia (Oreochromis niloticus). Also, it induces an increase in the respiratory burst of phagocytes and plasma IgM concentration (Girón-Pérez et al., 2007, 2009). Apart from the possible direct immunotoxic mechanisms, it has also been suggested that an indirect mechanism can be related with the interruption of neuroimmune communication, altering particularly the cholinergic neuronal system and its influence on the immune response (Galloway and Handy, 2003; Girón-Pérez et al., 2008). However, it has been recently proven that, in mammalian models, neurons are not the only cells that express ACh.
Other types of tissue produce this neurotransmitter and, in this context, it has been denominated the non-neuronal or extra neuronal cholinergic system. In this regard, it has been proven that immune system cells of mammals possess all the biochemical and molecular components to generate de novo ACh, a molecule that could play an important role in the regulation of the immune system (Kawashima and Fujii, 2000). Therefore, the lymphocytic cholinergic system could be the target for OPs in the immunotoxicity phenomenon (Charoenying et al., 2011). Data from our research group have shown that in vitro exposure to diazinon and diazoxon did not alter the proliferative capacity of tilapia lymphocytes, even though these substances induced an increase in the concentration of ACh, molecule that caused a significant diminishment in lymphoproliferation (Girón-Pérez et al., 2008). The objective of this paper was to evaluate the concentration of ACh and muscarinic (mAChR) and nicotinic (nAChR) cholinergic receptors, as well as AChE activity in mononuclear cells of Nile tilapia (O. niloticus) exposed to diazinon in vivo, in order to better understand the immunotoxic mechanisms of OPs on fish and vertebrates in general.
2. Materials and methods 2.1. Animals Male Nile tilapias (273 ± 43 g y 20 ± 3 cm) were obtained from a local fishery. The fish were maintained in a 400 L tank. During the acclimation period (4-weeks), the fish were fed with commercial
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Fig. 2. Concentration of ACh in spleen mononuclear cells of fish (n = 7) exposed in vivo to diazinon for 6 h (a), 12 h (b) and 24 h (c). Results are expressed as mean ± S.D.; one-way ANOVA and Tukey tests with 95% confidence were used. Bars with different letters indicate statistically significant differences (p < 0.05) between groups.
feed at a day-rate of 3% of fish body weight. Water temperature was maintained at 26 ± 2 ◦ C and dissolved oxygen values were 6.0 mg/L. 2.2. Experimental design
according to the Bradford method (1976), using bovine serum albumin as a standard. 2.4. Cholinesterases activities
Previous to the exposition bioassays, fish were acclimated in a 30 L glass aquarium for 24 h (1 fish/tank). In order to decrease stress, temperature and aeration were kept constant. In addition, organisms were not fed during this period in order to avoid prandial effects and to prevent deposition of stool during the bioassay. Once the acclimation process was finished, fish (n = 7) were exposed to 0.97, 1.95 and 3.91 ppm (1/8, 1/4 and 1/2 of the previously reported CL50 value at 96 h) (Girón-Pérez et al., 2007) of a commercial formulation of diazinon (25% active ingredient), for 6, 12 and 24 h. The bioassays were performed statically (with no water replacement. Then, organisms were euthanized by submersion in an ice bath and the spleens were immediately dissected. A control group was established with organisms that were maintained in the same conditions but without pesticide
Total cholinesterase activity (ChEt) was calculated by homogenizing a volume of mononuclear cells (0.1 mg de protein/mL) and mixing them with 1 mM acetylcholine iodide (ASChI) and 10 nM 5,5-O-dithiobis(2-nitrobenzoicacid) (DTNB); these substances were used as substrate and indicator, respectively. The absorbance was determined at 405 nm. The enzymatic activity was calculated from the absorbance difference (Abst2 –Abst1 ), where t1 = absorbance at the beginning of the reaction, while t2 = absorbance obtained 20 min after the reaction was initiated (Ellman et al., 1961). In order to evaluate the specific activity of AChE (omitting pseudo-cholinesterase activity), before the addition of ASChl and DTNB, the mononuclear cell homogenate was mixed with 1.0 mM iso-OMPA (selective inhibitor of BChE) and incubated at room temperature for 30 min. Subsequently, enzymatic activity was determined as described previously.
2.3. Sample preparation
2.5. Acetylcholine levels
The spleen was obtained and grinded with a syringe in PBS buffer (pH 7.2). The mononuclear cells were separated by a density gradient using Hystopaque-1077. Mononuclear cells were collected and washed at 3500 rpm/30 min, the cell pellet was resuspended in PBS and mechanically homogenized with a Polytron at 5000 rpm/30 seg. Protein concentration was determined from the homogenate
Ach concentration was performed with a fluorometric commercial kit (Amplex Red Invitrogen). A standard curve was obtained (0.5–100 M ACh) following the kit´ıs instructions. Then, a volume of the mononuclear cell homogenate (0.1 mg/mL protein) was mixed with 100 L of working solution (Horseradish peroxidase: 2 U/mL; choline oxidase: 0.2 U/mL; acetylcholinesterase: 1 U/mL).
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Fig. 3. Concentration of nAChR (a) and mAChR (b) in spleen mononuclear cells of fish exposed in vivo at 0.97, 1.95 and 3.91 ppm of diazinon. Each bar represents receptor concentration in random samples of organisms exposed to pesticide for 6, 12 and 24 h (n = 7). Results are expressed as mean ± S.D.; one-way ANOVA and Tukey tests with 95% confidence were used. Bars with different letters indicate statistically significant differences (p < 0.05) versus the control group.
The mix (200 L) was incubated for 60 min at room temperature and in the dark. ACh concentration was determined by a fluorometric microplate reader at 530 nm excitation and 590 nm emission. Background fluorescence was eliminated by subtracting the values derived from the non-acetylcholine control and the ACh values in the samples were determined through a standard curve. 2.6. Quantification of nAChR and mAChR Quantification of nAChR and mAChR in the mononuclear cells was performed with the fish nicotinic (N-ACHR) and muscarinic (M-ACHR) acetylcholine receptors ELISA kits (MyBiosource, Inc). Quantification of receptors was performed in random samples of fish exposed to 6, 12 and 24 h to each concentration of the pesticide (n = 7/per group). 50 L of the mononuclear cell homogenate were placed in wells with an antibody coating of anti-nAChR or mAChR. Then, 100 L of antibody-HRP were added to each well. The mix was incubated for 60 min at 37 ◦ C in the dark. After that, stopping solution was added and optical density was determined at 450 nm nAChR and mAChR concentration was calculated through a standard curve of the receptor (62.5–2000 pg/mL).
3.2. Acetylcholine concentration The concentration of ACh in mononuclear cells of fish exposed to diazinon for 6 and 12 h increased slightly with respect to the control group; however, there was no significant difference in the concentration of the neurotransmitter between the groups of fish exposed to the pesticide and the control group (Fig. 2a and b). On the other hand, the concentration of ACh in mononuclear cells of fishes exposed to 1.95 and 3.91 ppm diazinon for 24 h increased significantly (38.47 ± 7.33 M and 35.52 ± 9.03 M, respectively) compared to the control group (23.55 ± 6.03 M) (p < 0.05) (Fig. 2c). 3.3. Nicotinic and muscarinic acetylcholine receptors The results show that concentration of nAChR decreased significantly (p < 0.05) in cells of fish exposed to 3.91 ppm diazinon (352.6 ± 55.5 pg/mL), compared to the control group (568.5 ± 116.6 pg/mL). The same effect was observed when evaluating the concentration of mAChR, which diminished significantly (p < 0.05) in mononuclear cells of fish exposed to 3.91 ppm (480.0 ± 404.9 pg/mL) compared to the control group (1590 ± 711.5 pg/mL) (Fig. 3).
2.7. Statistical analyses Mean ± SD was determined in each case. Data were analyzed by one-way ANOVA and Tukey test using SigmaStat® (ver. 3.5. statistical software). The statistical difference was determined with a level of p < 0.05. 3. Results 3.1. Cholinesterase activity ChEt and AChE activities were evaluated in spleen mononuclear cells of Nile tilapia exposed to diazinon and in the non-exposed group. Activities of ChEt and AChE in these cells are shown in Fig. 1. Results indicate that exposure to diazinon at the three evaluated concentrations induces a decrease in enzyme activity After 6 and 12 h of pesticide exposure, both ChEt and AChE activities decreased significantly (p < 0.05) with all assayed concentrations (Fig. 1a and b); while at 24 h of exposure, a significant decrease was only detected at a concentration of 1.95 and 3.91 ppm of diazinon, even the activity of AChE was not detected in mononuclear cells of fish exposed to these concentrations (Fig. 1c).
4. Discussion The global demand on animal protein for human consumption has driven the development of intensive aquaculture farming. Nevertheless, the exponential growth of aquaculture can be limited due to the increase in the organisms´ı disease susceptibility. This susceptibility is often induced by the presence of pesticides, substances that are used indiscriminately in agricultural activities and can induce alterations in the immune response of organisms such as fish (Nardocci et al., 2014). Immunotoxicity mechanisms of OPs are not clear. However, it has been reported that OPs are capable of inhibiting serine hydrolases, which can directly influence immune system function. On the other hand, the alteration of components and immune functions has also been related with the sequence and intensity of phosphorylation and dephosphorylation of protein kinases, processes that are vital for the modulation of the immune response (Li, 2007). Furthermore, other researchers have suggested an implication of OPs in apoptosis, a mechanism that involves the activation of caspases and the death of the cell in a programmed way (Saleh et al., 2003).
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In contrast, the disruption of the neuroimmune communication, particularly the cholinergic system, is another possible mechanism for the immunotoxicity of OPs (Girón-Pérez et al., 2008). In addition, extra-neuronal cholinergic activity has been reported in mammalian lymphocytes (Kawashima and Fujii, 2003). However, up to this date, reports on fish lymphocytic cholinergic system are scarce. This system could be closely related with the immunotoxicity mechanism of OPs. In the present study, the activity of cholinergic system enzymes was determined. The obtained results show a decrease in the activity of ChEt and AChE in mononuclear cells of fish exposed to diazinon. These results are in agreement with previous studies, where the enzymatic activity on different tissues has been evaluated. In this regard, Guimarães et al. (2007) reported a significant decrease in the muscle AChE activity of Nile tilapia (O. niloticus) exposed to 0.25 ppm of trichlorfon from 8 to 96 h. Likewise, Durmaz et al. (2006) reported a dose-dependent inhibition of AChE in tissues such as gills, muscle, kidney and alimentary tract of O. niloticus exposed to sublethal concentrations of diazinon (0.1, 1 and 2 ppm). Results from our research group have shown that in Nile tilapia exposed in vivo to 3.91 and 7.83 ppm of diazinon, for 96 h, AChE activity is significantly reduced in spleen (Girón-Pérez et al., 2007). Data on the modulation of cholinesterase enzymes by OPs in immune system cells are scarce. In this context, Charoenying et al. (2011) showed that in the MOLT3 cell line, the exposure to paraoxon (100, 500 and 1000 M) for 48 h induces an increase in the expression of the N-AChE region (N-terminus acetylcholinesterase). The biological meaning of the AChE alteration can be related with what Da Silva et al. (2011) published. In this study, they reported a significant increase in the expression of AChE in lymphocytes during events of acute infections. In contrast, the results of the present work show the presence of ACh in mononuclear cells of Nile tilapia. Up to this moment, reports on the presence of this neurotransmitter in lower vertebrates such as fish are scarce. However, in cell lines and mammalian models, this neurotransmitter has been widely studied. In T-cell lines, such as MOLT-3, HSB-2 and CEM, ACh has been detected at levels of 251.5 ± 34.9, 36.2 ± 3.5 and 12.6 ± 0.8 pmol/106 cells, respectively (Kawashima and Fujii, 2003). Rinner et al. (1998), also determined the levels of ACh in lymphocytes from thymus (1521 ± 270 pg/106 cells), spleen (1340 ± 311 pg/106 cells) and rat peripheral blood lymphocytes (1148 ± 182 pg/106 cells). In addition to the presence of ACh in mammalian lymphocytes, the cholinergic components necessary for the ACh synthesis have also been detected in previous studies. This way, ACh seems to be an extremely relevant molecule for the activation of T cells, since its synthesis increases during the antigen presentation and seems to be regulated by PKC and PKA, as well as Ca2+ flow changes (Kawashima and Fujii, 2003). In the present study, we also determined the AChRs concentrations in spleen mononuclear cells of Nile tilapia. Our results show a significant decrease of these molecules in fish exposed to 3.91 ppm of diazinon compared to the control group. The latter is in line with what was reported by Charoenying et al. (2011), who showed that mAChR expression in MOLT-3 cells are modulated by the exposure to paraoxon, the main metabolite of the organophosphate pesticide parathion. The biological function of these receptors, apart from being related with the activation of lymphocytes, seems to be related with the production of pro-inflammatory cytokines and antibody synthesis. It has also been reported that the stimulation of nAChR induces a depolarization and membrane excitation due to the rapid Na+ , K+ and Ca2+ permeability increase (Kawashima et al., 2012). The data obtained in the present study reveal the presence of cholinergic components in Nile tilapia mononuclear cells. Furthermore, it is shown that these components are altered by the exposure to diazinon, a widely used pesticide in agricultural activ-
ities. Overall, our results indicate that AChE activity is decreased in fish mononuclear cells acutely exposed to sublethal concentrations of diazinon. This induces an increase in ACh concentration and a decrease in nAChR and mAChR concentrations The present data suggest that the increase in ACh concentration induces down-regulation mechanisms, which can be responsible for the decrease in the response of lymphocytes to antigenic stimulation. In this sense, our research group has reported that in vitro pre-exposure of lymphocytes to ACh decreases the response capacity of these cells to mitogenic stimuli (Girón-Pérez et al., 2008). In mammals, it has been suggested that lymphocyte cholinergic receptors induce an immediate intracellular signaling cascade involving diverse molecules such as c-Fos, which in turn alters the levels of second messengers. The activation of cholinergic receptors can act up-stream of the signaling cascades, causing a disturbance in cellular homeostasis and, eventually, apoptosis (Charoenying et al., 2011). 5. Conclusion This is the first report where the lymphocytic cholinergic system is evaluated as a target of the immunotoxic effect of OPs in aquatic organisms. Results of this research indicate that the immunotoxicity mechanism of OPs such as diazinon involves components of the lymphocytes’ own cholinergic system. However, further research on the effect of this type of pollutants on the immune system of fish is necessary. The results of this study support the accumulating evidence on the immunotoxic effect of OPs. These substances can alter fish phisiology and are potential immunosuppressants that favor susceptibility to infections and have a negative effect on aquaculture production. Conflict of interests There is no conflict of interests, and the authors declare that they have no direct relationship with the previously mentioned commercial entities or any other related one. Acknowledgments This work was funded by a grant from the financial resources of SEP-CONACyT-Mexico for Basic Research (Project no. 2012179508). We gratefully acknowledge Gabriela González de Pablos for reading the manuscript, correcting English language and style. The first two authors are postgraduate students of Biological and Agricultural Sciences (Posgrado en Ciencias Biológico Agropecuarias CBAP) of the State University of Nayarit (Universidad Autónoma de Nayarit, México). References Barbieri, E., Alves Ferreira, L.A., 2011. Effects of the organophosphate pesticide Folidol 600® on the freshwater fish, Nile tilapia (Oreochromis niloticus). Pestic. Biochem. Phys. 99, 209–214. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Charoenying, T., Suriyo, T., Thiantanawat, A., Chaiyaroj, S.C., Parkpian, P., Satayavivad, J., 2011. Effects of paraoxon on neuronal and lymphocytic cholinergic systems. Environ. Toxicol. Phar. 31, 119–128. Costa, L., 2006. Current issues in organophosphate toxicology. Clin. Chim. Acta 366, 1–13. Da Silva, A.S., Monteiro, S.G., Gonc¸alves, J.F., Spanevello, R., Schmatz, R., Oliveira, C.B., Costa, M.M., Franc¸a, R.T., Jaques, J.A., Schetinger, M.R., Mazzanti, C.M., Lopes, S.T., 2011. Trypanosoma evansi: immune response and acetylcholinesterase activity in lymphocytes from infected rats. Exp. Parasitol. 127 (2), 475–480. Durmaz, H., Sevgiler, H., Üner, N., 2006. Tissue-specific antioxidative and neurotoxic responses to diazinon in Oreochromis niloticus. Pestic. Biochem. Phys. 84, 215–226.
G.A. Toledo-Ibarra et al. / Veterinary Immunology and Immunopathology 176 (2016) 58–63 Ellman, G.L., Courtney, D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. FAO, 2012. El Estado Mundial de la Pesca y la Acuicultura, Last Access: October 2014. http://www.fao.org/docrep/016/i2727s/i2727s00.htm. Fanta, E., Sant’Anna Rios, F., Romão, S., Casagrande Vianna, A.C., Freiberger, S., 2003. Histopathology of the fish Corydoras paleatus contaminated with sublethal levels of organophosphorus in water and food. Ecotox. Environ. Saf. 54, 119–130. Galloway, T., Handy, R., 2003. Immunotoxicity of organophosphorous pesticides. Ecotoxicology 12, 345–363. Girón-Pérez, M.I., Santerre, A., Gonzalez-Jaime, F., Casas-Solis, J., Hernández-Coronado, M., Peregrina-Sandoval, J., Takemura, A., Zaitseva, G., 2007. Immunotoxicity and hepatic function evaluation in Nile tilapia (Oreochromis niloticus) exposed to diazinón. Fish Shellfish Immunol. 23, 760–769. Girón-Pérez, M.I., Zaitseva, G., Casas-Solis, J., Santerre, A., 2008. Effects of diazinon and diazoxon on the lymphoproliferation rate of splenocytes from Nile tilapia (Oreochromis niloticus): the immunosuppresive effect could involve an increase in acetylcholine levels. Fish Shellfish Immunol. 25, 517–521. Girón-Pérez, M.I., Velázquez-Fernández, J., Díaz-Resendiz, K., Díaz-Salas, F., Canto-Montero, C., Medina-Díaz, I., Robledo-Marenco, M., Rojas-García, A., Zaitseva, G., 2009. Immunologic parameters evaluations in Nile tilapia (Oreochromis niloticus) exposed to sublethal concentrations of diazinon. Fish Shellfish Immunol. 27, 383–385. Guimarães, A.T.B., Silva de Assis, H.C., Boeger, W., 2007. The effect of trichlorfon on acetylcholinesterase activity and histopathology of cultivated fish Oreochromis niloticus. Ecotoxicol. Environ. Saf. 68, 57–62. Harford, A.J., O’Halloran, K., Wright, P.F.A., 2005. The effects of in vitro pesticide exposures on the phagocytic function of four native Australian freshwater fish. Aquat. Toxicol. 75, 330–342. Harms, C.A., Howard, K.E., Wolf, J.C., Smith, S.A., Kennedy-Stoskopf, S., 2003. Transforming growth factor- response to mycobacterial infection in striped bass Morone saxatilis and hybrid tilapia Oreochromis spp. Vet. Immunol. Immunopathol. 9, 155–163. Kavitha, P., Venkateswara Rao, J., 2009. Sub-lethal effects of profenofos on tissue-specific antioxidative responses in a Euryhyaline fish, Oreochromis mossambicus. Ecotoxicol. Environ. Saf. 72, 1727–1733.
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Kawashima, K., Fujii, T., 2000. Extraneuronal cholinergic system in lymphocytes. Pharmacol. Ther. 86 (1), 29–48. Kawashima, K., Fujii, T., 2003. The lymphocytic cholinergic system and its contribution to the regulation of immune activity. Life Sci. 74 (6), 675–696. Kawashima, K., Fujii, T., Moriwaki, Y., Misawa, H., 2012. Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function. Life Sci. 91, 1027–1032. Khoshbavar-Rostami, H.A., Soltani, M., Hassan, H.M.D., 2006. Immune response of great sturgeon (Huso huso) subjected to long-term exposure to sublethal concentration of the organophosphate, diazinon. Aquaculture 256, 88–94. Li, Q., 2007. New mechanism of organophosphorus pesticide-induced immunotoxicity. J. Nippon Med. Sch. 74, 92–105. NRA (National Registration Authority), 2000. The NRA Review of Diazinon. National Registration Authority for Agricultural and Veterinary chemicals, Canberra, Australia, Last Access: October 2014. http://apvma.gov.au/sites/ default/files/diazinon-phase-5-chemistry.pdf. Nardocci, G., Navarro, C., Cortés, P.P., Imarai, M., Montoya, M., Valenzuela, B., Jara, ˜ P., Acuna-Castillo, C., Fernández, R., 2014. Neuroendocrine mechanisms for immune system regulation during stress in fish. Fish Shellfish Immunol. 40, 531–538. Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., Endo, M., 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet. Immunol. Immunopathol. 113, 339–347. Rinner, I., Kawashima, K., Schauenstein, K., 1998. Rat lymphocytes produce and secrete acetylcholine in dependence of differentiation and activation. J. Neuroimmunol. 81 (1–2), 31–37. Saleh, A.M., Vijayasarathy, C., Masoud, L., Kumar, L., Shahin, A., Kambal, A., 2003. Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways. Toxicol. Appl. Pharm. 190, 47–57. Tilton, F.A., Tilton, S.C., Bammler, T.K., Beyer, R.P., Stapleton, P.L., Scholz, N.L., Gallagher, E.P., 2011. Transcriptional impact of organophosphate and metal mixtures on olfaction: copper dominates the chlorpyrifos-induced response in adult zebrafish. Aquat. Toxicol. 102, 205–215.
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Effects of diazinon on the lymphocytic cholinergic system of Nile tilapia fish (Oreochromis niloticus) G.A. Toledo-Ibarra a , K.J.G. Díaz-Resendiz a , L. Pavón-Romero b , A.E. Rojas-García c , I.M Medina-Díaz c , M.I. Girón-Pérez a,∗ a
Laboratorio de Inmunotoxicología, Universidad Autónoma de Nayarit, Secretaría de Investigación y Posgrado, Laboratorio de Inmunotoxicología, Boulevard Tepic-Xalisco s/n, Cd de la Cultura Amado Nervo, C.P. 63190, Tepic Nayarit, Mexico b Departmento de Psicoimmunología, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, Calzada México-Xochimilco 101, Col. San Lorenzo Huipulco, Tlalpan, 14370 Mexico City, DF, Mexico c Laboratorio de Contaminación y Toxicología Ambiental, Universidad Autónoma de Nayarit, Secretaría de Investigación y Posgrado, Laboratorio de Inmunotoxicología, Boulevard Tepic-Xalisco s/n, Cd de la Cu ltura Amado Nervo, C.P. 63190, Tepic Nayarit, Mexico
a r t i c l e
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Article history: Received 11 June 2015 Received in revised form 2 May 2016 Accepted 20 May 2016 Keywords: Acetylcholine Acetylcholinesterase Immunotoxicity Organophosphorus pesticides Diazinon Nile tilapia fish
a b s t r a c t Fish rearing under intensive farming conditions can be easily disturbed by pesticides, substances that have immunotoxic properties and may predispose to infections. Organophosphorus pesticides (OPs) are widely used in agricultural activities; however, the mechanism of immunotoxicity of these substances is unclear. The aim of this study was to evaluate the effect of diazinon pesticides (OPs) on the cholinergic system of immune cells as a possible target of OP immunotoxicity. We evaluated ACh levels and cholinergic (nicotinic and muscarinic) receptor concentration. Additionally, AChE activity was evaluated in mononuclear cells of Nile tilapia (Oreochromis niloticus), a freshwater fish mostly cultivated in tropical regions around the world. The obtained results indicate that acute exposure to diazinon induces an increase in ACh concentration and a decrease in nAChR and mAChR concentrations and AChE activity in fish immune cells, This suggests that the non-neuronal lymphocytic cholinergic system may be the main target in the mechanism of OP immunotoxicity. This study contributes to the understanding of the mechanisms of immunotoxicity of pollutants and may help to take actions for animal health improvement. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture consists in the rearing of aquatic organisms such as fishes, molluscs, crustaceans and plants. In this context, fishes are by far the main class of organisms that are produced by means of this activity. Indeed, the fish production through aquaculture has surpassed the production through fisheries. One of the most farmed freshwater fish species in the world is the tilapia (Oreochromis niloticus). According to data from the FAO, the production of this type of fish in 2011 was 4027 million tons (FAO, 2012). Disease incidence in tilapia farming, in optimum farming densities and conditions, is not frequent. Nevertheless, there are factors that can compromise the health of these organisms and the innocuousness of the product for human consumption. Among these, we can mention pathogenic organisms and environment pollutants such as pesticides (Harms et al., 2003). In the case of pesticides,
∗ Corresponding author. E-mail address: ivan [email protected] (M.I. Girón-Pérez). http://dx.doi.org/10.1016/j.vetimm.2016.05.010 0165-2427/© 2016 Elsevier B.V. All rights reserved.
these substances can penetrate aquatic ecosystems as a consequence of rain, soil leaching or for being carelessly discharged directly into bodies of water (Fanta et al., 2003; Kavitha and Venkateswara Rao, 2009). Furthermore, in some countries, pesticides are used directly in aquaculture for the elimination of ectoparasites (Khoshbavar-Rostami et al., 2006). However, there is evidence that pesticides can induce an immunotoxic effect; hence the health of fishes exposed to these substances can be compromised (Harford et al., 2005), and even cause death, which has a serious effect in aquaculture production (Khoshbavar-Rostami et al., 2006; Pirarat et al., 2006). Diazinon (O,O-diethyl-O-[2-isopropyl-4-methyl-6pyrimidinyl] phosphorothioate) is one of the most used organophosphorus pesticides (OPs) in agricultural (control of agriculture, livestock and aquaculture pests) and domestic activities (NRA, 2000; Khoshbavar-Rostami et al., 2006). OPs are powerful neurotoxins since they inhibit the activity of the acetylcholinesterase (AChE) enzyme. Diazinon is metabolized to diazoxon, a substance that phosphorylates the hydroxyl group of serines in the active site of AChE. This phosphorylation disrupts the
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Fig. 1. Activity of ChEt and AChE in spleen mononuclear cells of fish (n = 7) exposed to diazinon for 6 h (a), 12 h (b) and 24 h (c). Results are expressed as mean ± S.D.; one-way ANOVA and Tukey tests with 95% confidence were used.
enzyme´ıs ability to breakdown the acetylcholine neurotransmitter (ACh), resulting in an excessive accumulation of ACh, which overstimulates the muscarinic (mAChR) and nicotinic (nAChR) receptors, located in the post-synaptic cells. This way, inhibition of AChE induces uncontrolled nervous impulses that manifest as changes in behavior (movement diminishment, breathing capacity, predator avoidance, prey selection and reproduction loss) and causes death (Fanta et al., 2003; Barbieri and Alves Ferreira, 2011; Tilton et al., 2011; Costa, 2006). In addition to the neurotoxic effects, OPs can cause immunotoxic effects; however, there is little information regarding the possible immunotoxic mechanisms of these substances. It has been reported that OPs can cause direct effects such as the inhibition of serine hydrolases, immunological complement system, oxidative damage, alteration of signal transduction pathways, proliferation and differentiation of cells of the immune system (Galloway and Handy, 2003; Li, 2007). In this regard, our research group has reported that sublethal concentrations of diazinon cause a decrease in lymphocyte proliferative capacity, phagocytic activity and relative spleen weight in the Nile tilapia (Oreochromis niloticus). Also, it induces an increase in the respiratory burst of phagocytes and plasma IgM concentration (Girón-Pérez et al., 2007, 2009). Apart from the possible direct immunotoxic mechanisms, it has also been suggested that an indirect mechanism can be related with the interruption of neuroimmune communication, altering particularly the cholinergic neuronal system and its influence on the immune response (Galloway and Handy, 2003; Girón-Pérez et al., 2008). However, it has been recently proven that, in mammalian models, neurons are not the only cells that express ACh.
Other types of tissue produce this neurotransmitter and, in this context, it has been denominated the non-neuronal or extra neuronal cholinergic system. In this regard, it has been proven that immune system cells of mammals possess all the biochemical and molecular components to generate de novo ACh, a molecule that could play an important role in the regulation of the immune system (Kawashima and Fujii, 2000). Therefore, the lymphocytic cholinergic system could be the target for OPs in the immunotoxicity phenomenon (Charoenying et al., 2011). Data from our research group have shown that in vitro exposure to diazinon and diazoxon did not alter the proliferative capacity of tilapia lymphocytes, even though these substances induced an increase in the concentration of ACh, molecule that caused a significant diminishment in lymphoproliferation (Girón-Pérez et al., 2008). The objective of this paper was to evaluate the concentration of ACh and muscarinic (mAChR) and nicotinic (nAChR) cholinergic receptors, as well as AChE activity in mononuclear cells of Nile tilapia (O. niloticus) exposed to diazinon in vivo, in order to better understand the immunotoxic mechanisms of OPs on fish and vertebrates in general.
2. Materials and methods 2.1. Animals Male Nile tilapias (273 ± 43 g y 20 ± 3 cm) were obtained from a local fishery. The fish were maintained in a 400 L tank. During the acclimation period (4-weeks), the fish were fed with commercial
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Fig. 2. Concentration of ACh in spleen mononuclear cells of fish (n = 7) exposed in vivo to diazinon for 6 h (a), 12 h (b) and 24 h (c). Results are expressed as mean ± S.D.; one-way ANOVA and Tukey tests with 95% confidence were used. Bars with different letters indicate statistically significant differences (p < 0.05) between groups.
feed at a day-rate of 3% of fish body weight. Water temperature was maintained at 26 ± 2 ◦ C and dissolved oxygen values were 6.0 mg/L. 2.2. Experimental design
according to the Bradford method (1976), using bovine serum albumin as a standard. 2.4. Cholinesterases activities
Previous to the exposition bioassays, fish were acclimated in a 30 L glass aquarium for 24 h (1 fish/tank). In order to decrease stress, temperature and aeration were kept constant. In addition, organisms were not fed during this period in order to avoid prandial effects and to prevent deposition of stool during the bioassay. Once the acclimation process was finished, fish (n = 7) were exposed to 0.97, 1.95 and 3.91 ppm (1/8, 1/4 and 1/2 of the previously reported CL50 value at 96 h) (Girón-Pérez et al., 2007) of a commercial formulation of diazinon (25% active ingredient), for 6, 12 and 24 h. The bioassays were performed statically (with no water replacement. Then, organisms were euthanized by submersion in an ice bath and the spleens were immediately dissected. A control group was established with organisms that were maintained in the same conditions but without pesticide
Total cholinesterase activity (ChEt) was calculated by homogenizing a volume of mononuclear cells (0.1 mg de protein/mL) and mixing them with 1 mM acetylcholine iodide (ASChI) and 10 nM 5,5-O-dithiobis(2-nitrobenzoicacid) (DTNB); these substances were used as substrate and indicator, respectively. The absorbance was determined at 405 nm. The enzymatic activity was calculated from the absorbance difference (Abst2 –Abst1 ), where t1 = absorbance at the beginning of the reaction, while t2 = absorbance obtained 20 min after the reaction was initiated (Ellman et al., 1961). In order to evaluate the specific activity of AChE (omitting pseudo-cholinesterase activity), before the addition of ASChl and DTNB, the mononuclear cell homogenate was mixed with 1.0 mM iso-OMPA (selective inhibitor of BChE) and incubated at room temperature for 30 min. Subsequently, enzymatic activity was determined as described previously.
2.3. Sample preparation
2.5. Acetylcholine levels
The spleen was obtained and grinded with a syringe in PBS buffer (pH 7.2). The mononuclear cells were separated by a density gradient using Hystopaque-1077. Mononuclear cells were collected and washed at 3500 rpm/30 min, the cell pellet was resuspended in PBS and mechanically homogenized with a Polytron at 5000 rpm/30 seg. Protein concentration was determined from the homogenate
Ach concentration was performed with a fluorometric commercial kit (Amplex Red Invitrogen). A standard curve was obtained (0.5–100 M ACh) following the kit´ıs instructions. Then, a volume of the mononuclear cell homogenate (0.1 mg/mL protein) was mixed with 100 L of working solution (Horseradish peroxidase: 2 U/mL; choline oxidase: 0.2 U/mL; acetylcholinesterase: 1 U/mL).
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Fig. 3. Concentration of nAChR (a) and mAChR (b) in spleen mononuclear cells of fish exposed in vivo at 0.97, 1.95 and 3.91 ppm of diazinon. Each bar represents receptor concentration in random samples of organisms exposed to pesticide for 6, 12 and 24 h (n = 7). Results are expressed as mean ± S.D.; one-way ANOVA and Tukey tests with 95% confidence were used. Bars with different letters indicate statistically significant differences (p < 0.05) versus the control group.
The mix (200 L) was incubated for 60 min at room temperature and in the dark. ACh concentration was determined by a fluorometric microplate reader at 530 nm excitation and 590 nm emission. Background fluorescence was eliminated by subtracting the values derived from the non-acetylcholine control and the ACh values in the samples were determined through a standard curve. 2.6. Quantification of nAChR and mAChR Quantification of nAChR and mAChR in the mononuclear cells was performed with the fish nicotinic (N-ACHR) and muscarinic (M-ACHR) acetylcholine receptors ELISA kits (MyBiosource, Inc). Quantification of receptors was performed in random samples of fish exposed to 6, 12 and 24 h to each concentration of the pesticide (n = 7/per group). 50 L of the mononuclear cell homogenate were placed in wells with an antibody coating of anti-nAChR or mAChR. Then, 100 L of antibody-HRP were added to each well. The mix was incubated for 60 min at 37 ◦ C in the dark. After that, stopping solution was added and optical density was determined at 450 nm nAChR and mAChR concentration was calculated through a standard curve of the receptor (62.5–2000 pg/mL).
3.2. Acetylcholine concentration The concentration of ACh in mononuclear cells of fish exposed to diazinon for 6 and 12 h increased slightly with respect to the control group; however, there was no significant difference in the concentration of the neurotransmitter between the groups of fish exposed to the pesticide and the control group (Fig. 2a and b). On the other hand, the concentration of ACh in mononuclear cells of fishes exposed to 1.95 and 3.91 ppm diazinon for 24 h increased significantly (38.47 ± 7.33 M and 35.52 ± 9.03 M, respectively) compared to the control group (23.55 ± 6.03 M) (p < 0.05) (Fig. 2c). 3.3. Nicotinic and muscarinic acetylcholine receptors The results show that concentration of nAChR decreased significantly (p < 0.05) in cells of fish exposed to 3.91 ppm diazinon (352.6 ± 55.5 pg/mL), compared to the control group (568.5 ± 116.6 pg/mL). The same effect was observed when evaluating the concentration of mAChR, which diminished significantly (p < 0.05) in mononuclear cells of fish exposed to 3.91 ppm (480.0 ± 404.9 pg/mL) compared to the control group (1590 ± 711.5 pg/mL) (Fig. 3).
2.7. Statistical analyses Mean ± SD was determined in each case. Data were analyzed by one-way ANOVA and Tukey test using SigmaStat® (ver. 3.5. statistical software). The statistical difference was determined with a level of p < 0.05. 3. Results 3.1. Cholinesterase activity ChEt and AChE activities were evaluated in spleen mononuclear cells of Nile tilapia exposed to diazinon and in the non-exposed group. Activities of ChEt and AChE in these cells are shown in Fig. 1. Results indicate that exposure to diazinon at the three evaluated concentrations induces a decrease in enzyme activity After 6 and 12 h of pesticide exposure, both ChEt and AChE activities decreased significantly (p < 0.05) with all assayed concentrations (Fig. 1a and b); while at 24 h of exposure, a significant decrease was only detected at a concentration of 1.95 and 3.91 ppm of diazinon, even the activity of AChE was not detected in mononuclear cells of fish exposed to these concentrations (Fig. 1c).
4. Discussion The global demand on animal protein for human consumption has driven the development of intensive aquaculture farming. Nevertheless, the exponential growth of aquaculture can be limited due to the increase in the organisms´ı disease susceptibility. This susceptibility is often induced by the presence of pesticides, substances that are used indiscriminately in agricultural activities and can induce alterations in the immune response of organisms such as fish (Nardocci et al., 2014). Immunotoxicity mechanisms of OPs are not clear. However, it has been reported that OPs are capable of inhibiting serine hydrolases, which can directly influence immune system function. On the other hand, the alteration of components and immune functions has also been related with the sequence and intensity of phosphorylation and dephosphorylation of protein kinases, processes that are vital for the modulation of the immune response (Li, 2007). Furthermore, other researchers have suggested an implication of OPs in apoptosis, a mechanism that involves the activation of caspases and the death of the cell in a programmed way (Saleh et al., 2003).
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In contrast, the disruption of the neuroimmune communication, particularly the cholinergic system, is another possible mechanism for the immunotoxicity of OPs (Girón-Pérez et al., 2008). In addition, extra-neuronal cholinergic activity has been reported in mammalian lymphocytes (Kawashima and Fujii, 2003). However, up to this date, reports on fish lymphocytic cholinergic system are scarce. This system could be closely related with the immunotoxicity mechanism of OPs. In the present study, the activity of cholinergic system enzymes was determined. The obtained results show a decrease in the activity of ChEt and AChE in mononuclear cells of fish exposed to diazinon. These results are in agreement with previous studies, where the enzymatic activity on different tissues has been evaluated. In this regard, Guimarães et al. (2007) reported a significant decrease in the muscle AChE activity of Nile tilapia (O. niloticus) exposed to 0.25 ppm of trichlorfon from 8 to 96 h. Likewise, Durmaz et al. (2006) reported a dose-dependent inhibition of AChE in tissues such as gills, muscle, kidney and alimentary tract of O. niloticus exposed to sublethal concentrations of diazinon (0.1, 1 and 2 ppm). Results from our research group have shown that in Nile tilapia exposed in vivo to 3.91 and 7.83 ppm of diazinon, for 96 h, AChE activity is significantly reduced in spleen (Girón-Pérez et al., 2007). Data on the modulation of cholinesterase enzymes by OPs in immune system cells are scarce. In this context, Charoenying et al. (2011) showed that in the MOLT3 cell line, the exposure to paraoxon (100, 500 and 1000 M) for 48 h induces an increase in the expression of the N-AChE region (N-terminus acetylcholinesterase). The biological meaning of the AChE alteration can be related with what Da Silva et al. (2011) published. In this study, they reported a significant increase in the expression of AChE in lymphocytes during events of acute infections. In contrast, the results of the present work show the presence of ACh in mononuclear cells of Nile tilapia. Up to this moment, reports on the presence of this neurotransmitter in lower vertebrates such as fish are scarce. However, in cell lines and mammalian models, this neurotransmitter has been widely studied. In T-cell lines, such as MOLT-3, HSB-2 and CEM, ACh has been detected at levels of 251.5 ± 34.9, 36.2 ± 3.5 and 12.6 ± 0.8 pmol/106 cells, respectively (Kawashima and Fujii, 2003). Rinner et al. (1998), also determined the levels of ACh in lymphocytes from thymus (1521 ± 270 pg/106 cells), spleen (1340 ± 311 pg/106 cells) and rat peripheral blood lymphocytes (1148 ± 182 pg/106 cells). In addition to the presence of ACh in mammalian lymphocytes, the cholinergic components necessary for the ACh synthesis have also been detected in previous studies. This way, ACh seems to be an extremely relevant molecule for the activation of T cells, since its synthesis increases during the antigen presentation and seems to be regulated by PKC and PKA, as well as Ca2+ flow changes (Kawashima and Fujii, 2003). In the present study, we also determined the AChRs concentrations in spleen mononuclear cells of Nile tilapia. Our results show a significant decrease of these molecules in fish exposed to 3.91 ppm of diazinon compared to the control group. The latter is in line with what was reported by Charoenying et al. (2011), who showed that mAChR expression in MOLT-3 cells are modulated by the exposure to paraoxon, the main metabolite of the organophosphate pesticide parathion. The biological function of these receptors, apart from being related with the activation of lymphocytes, seems to be related with the production of pro-inflammatory cytokines and antibody synthesis. It has also been reported that the stimulation of nAChR induces a depolarization and membrane excitation due to the rapid Na+ , K+ and Ca2+ permeability increase (Kawashima et al., 2012). The data obtained in the present study reveal the presence of cholinergic components in Nile tilapia mononuclear cells. Furthermore, it is shown that these components are altered by the exposure to diazinon, a widely used pesticide in agricultural activ-
ities. Overall, our results indicate that AChE activity is decreased in fish mononuclear cells acutely exposed to sublethal concentrations of diazinon. This induces an increase in ACh concentration and a decrease in nAChR and mAChR concentrations The present data suggest that the increase in ACh concentration induces down-regulation mechanisms, which can be responsible for the decrease in the response of lymphocytes to antigenic stimulation. In this sense, our research group has reported that in vitro pre-exposure of lymphocytes to ACh decreases the response capacity of these cells to mitogenic stimuli (Girón-Pérez et al., 2008). In mammals, it has been suggested that lymphocyte cholinergic receptors induce an immediate intracellular signaling cascade involving diverse molecules such as c-Fos, which in turn alters the levels of second messengers. The activation of cholinergic receptors can act up-stream of the signaling cascades, causing a disturbance in cellular homeostasis and, eventually, apoptosis (Charoenying et al., 2011). 5. Conclusion This is the first report where the lymphocytic cholinergic system is evaluated as a target of the immunotoxic effect of OPs in aquatic organisms. Results of this research indicate that the immunotoxicity mechanism of OPs such as diazinon involves components of the lymphocytes’ own cholinergic system. However, further research on the effect of this type of pollutants on the immune system of fish is necessary. The results of this study support the accumulating evidence on the immunotoxic effect of OPs. These substances can alter fish phisiology and are potential immunosuppressants that favor susceptibility to infections and have a negative effect on aquaculture production. Conflict of interests There is no conflict of interests, and the authors declare that they have no direct relationship with the previously mentioned commercial entities or any other related one. Acknowledgments This work was funded by a grant from the financial resources of SEP-CONACyT-Mexico for Basic Research (Project no. 2012179508). We gratefully acknowledge Gabriela González de Pablos for reading the manuscript, correcting English language and style. The first two authors are postgraduate students of Biological and Agricultural Sciences (Posgrado en Ciencias Biológico Agropecuarias CBAP) of the State University of Nayarit (Universidad Autónoma de Nayarit, México). References Barbieri, E., Alves Ferreira, L.A., 2011. Effects of the organophosphate pesticide Folidol 600® on the freshwater fish, Nile tilapia (Oreochromis niloticus). Pestic. Biochem. Phys. 99, 209–214. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Charoenying, T., Suriyo, T., Thiantanawat, A., Chaiyaroj, S.C., Parkpian, P., Satayavivad, J., 2011. Effects of paraoxon on neuronal and lymphocytic cholinergic systems. Environ. Toxicol. Phar. 31, 119–128. Costa, L., 2006. Current issues in organophosphate toxicology. Clin. Chim. Acta 366, 1–13. Da Silva, A.S., Monteiro, S.G., Gonc¸alves, J.F., Spanevello, R., Schmatz, R., Oliveira, C.B., Costa, M.M., Franc¸a, R.T., Jaques, J.A., Schetinger, M.R., Mazzanti, C.M., Lopes, S.T., 2011. Trypanosoma evansi: immune response and acetylcholinesterase activity in lymphocytes from infected rats. Exp. Parasitol. 127 (2), 475–480. Durmaz, H., Sevgiler, H., Üner, N., 2006. Tissue-specific antioxidative and neurotoxic responses to diazinon in Oreochromis niloticus. Pestic. Biochem. Phys. 84, 215–226.
G.A. Toledo-Ibarra et al. / Veterinary Immunology and Immunopathology 176 (2016) 58–63 Ellman, G.L., Courtney, D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. FAO, 2012. El Estado Mundial de la Pesca y la Acuicultura, Last Access: October 2014. http://www.fao.org/docrep/016/i2727s/i2727s00.htm. Fanta, E., Sant’Anna Rios, F., Romão, S., Casagrande Vianna, A.C., Freiberger, S., 2003. Histopathology of the fish Corydoras paleatus contaminated with sublethal levels of organophosphorus in water and food. Ecotox. Environ. Saf. 54, 119–130. Galloway, T., Handy, R., 2003. Immunotoxicity of organophosphorous pesticides. Ecotoxicology 12, 345–363. Girón-Pérez, M.I., Santerre, A., Gonzalez-Jaime, F., Casas-Solis, J., Hernández-Coronado, M., Peregrina-Sandoval, J., Takemura, A., Zaitseva, G., 2007. Immunotoxicity and hepatic function evaluation in Nile tilapia (Oreochromis niloticus) exposed to diazinón. Fish Shellfish Immunol. 23, 760–769. Girón-Pérez, M.I., Zaitseva, G., Casas-Solis, J., Santerre, A., 2008. Effects of diazinon and diazoxon on the lymphoproliferation rate of splenocytes from Nile tilapia (Oreochromis niloticus): the immunosuppresive effect could involve an increase in acetylcholine levels. Fish Shellfish Immunol. 25, 517–521. Girón-Pérez, M.I., Velázquez-Fernández, J., Díaz-Resendiz, K., Díaz-Salas, F., Canto-Montero, C., Medina-Díaz, I., Robledo-Marenco, M., Rojas-García, A., Zaitseva, G., 2009. Immunologic parameters evaluations in Nile tilapia (Oreochromis niloticus) exposed to sublethal concentrations of diazinon. Fish Shellfish Immunol. 27, 383–385. Guimarães, A.T.B., Silva de Assis, H.C., Boeger, W., 2007. The effect of trichlorfon on acetylcholinesterase activity and histopathology of cultivated fish Oreochromis niloticus. Ecotoxicol. Environ. Saf. 68, 57–62. Harford, A.J., O’Halloran, K., Wright, P.F.A., 2005. The effects of in vitro pesticide exposures on the phagocytic function of four native Australian freshwater fish. Aquat. Toxicol. 75, 330–342. Harms, C.A., Howard, K.E., Wolf, J.C., Smith, S.A., Kennedy-Stoskopf, S., 2003. Transforming growth factor- response to mycobacterial infection in striped bass Morone saxatilis and hybrid tilapia Oreochromis spp. Vet. Immunol. Immunopathol. 9, 155–163. Kavitha, P., Venkateswara Rao, J., 2009. Sub-lethal effects of profenofos on tissue-specific antioxidative responses in a Euryhyaline fish, Oreochromis mossambicus. Ecotoxicol. Environ. Saf. 72, 1727–1733.
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Kawashima, K., Fujii, T., 2000. Extraneuronal cholinergic system in lymphocytes. Pharmacol. Ther. 86 (1), 29–48. Kawashima, K., Fujii, T., 2003. The lymphocytic cholinergic system and its contribution to the regulation of immune activity. Life Sci. 74 (6), 675–696. Kawashima, K., Fujii, T., Moriwaki, Y., Misawa, H., 2012. Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function. Life Sci. 91, 1027–1032. Khoshbavar-Rostami, H.A., Soltani, M., Hassan, H.M.D., 2006. Immune response of great sturgeon (Huso huso) subjected to long-term exposure to sublethal concentration of the organophosphate, diazinon. Aquaculture 256, 88–94. Li, Q., 2007. New mechanism of organophosphorus pesticide-induced immunotoxicity. J. Nippon Med. Sch. 74, 92–105. NRA (National Registration Authority), 2000. The NRA Review of Diazinon. National Registration Authority for Agricultural and Veterinary chemicals, Canberra, Australia, Last Access: October 2014. http://apvma.gov.au/sites/ default/files/diazinon-phase-5-chemistry.pdf. Nardocci, G., Navarro, C., Cortés, P.P., Imarai, M., Montoya, M., Valenzuela, B., Jara, ˜ P., Acuna-Castillo, C., Fernández, R., 2014. Neuroendocrine mechanisms for immune system regulation during stress in fish. Fish Shellfish Immunol. 40, 531–538. Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., Endo, M., 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet. Immunol. Immunopathol. 113, 339–347. Rinner, I., Kawashima, K., Schauenstein, K., 1998. Rat lymphocytes produce and secrete acetylcholine in dependence of differentiation and activation. J. Neuroimmunol. 81 (1–2), 31–37. Saleh, A.M., Vijayasarathy, C., Masoud, L., Kumar, L., Shahin, A., Kambal, A., 2003. Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways. Toxicol. Appl. Pharm. 190, 47–57. Tilton, F.A., Tilton, S.C., Bammler, T.K., Beyer, R.P., Stapleton, P.L., Scholz, N.L., Gallagher, E.P., 2011. Transcriptional impact of organophosphate and metal mixtures on olfaction: copper dominates the chlorpyrifos-induced response in adult zebrafish. Aquat. Toxicol. 102, 205–215.