2 pathway, apoptosis and senescence in Nile tilapia (Oreochromis niloticus) splenocytes

Fish & Shellfish Immunology 31 (2011) 1291e1296

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Effects of low concentration of endosulfan on proliferation, ERK1/2 pathway, apoptosis and senescence in Nile tilapia (Oreochromis niloticus) splenocytes Martha Cecilia Tellez-Bañuelos, Pablo C. Ortiz-Lazareno, Anne Santerre, Josefina Casas-Solis, Alejandro Bravo-Cuellar, Galina Zaitseva* Universidad de Guadalajara, Mexico

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 July 2011 Received in revised form 10 September 2011 Accepted 3 October 2011 Available online 13 October 2011

Endosulfan is a potent organochlorinated pesticide that is known to induce side effects in aquatic organisms, including Oreochromis niloticus (Nile tilapia). It has been previously shown that endosulfan induces oxidative stress and non-specific activation of splenic macrophages and exacerbated serum interleukin-2 synthesis in Nile tilapia. Endosulfan may promote proliferation of T cells through MAP kinase (MAPK) activated signal transductions. The ERK family of MAPKs includes ERK1 and ERK2. Phosphorylated ERK1/2 (pERK1/2) molecules are involved in many aspects of cellular survival, and are important for apoptosis or oxidative stress-induced senescence. In order to study the mechanisms by which endosulfan affects fish health, the present study was aimed at evaluating the in vitro effects of this insecticide on proliferation, the ERK1/2 pathway, apoptosis and cell senescence in splenocytes from Nile tilapia. Lymphoproliferation was evaluated by colorimetric method using the WST-1 assay. Flow cytometry was used to assess pERK1/2, apoptosis and senescence, using Annexin V-FITC and b-galactosidase respectively. Experimental data showed that exposure to 7 mg mL
1 of endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA þ ionomycin. Splenocytes exposed to endosulfan for 15e180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence. In conclusion, exposure of immune cells to a low concentration of endosulfan deregulates their function and may facilitate the development of multiple diseases. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Oreochromis niloticus Endosulfan pERK1/2 Apoptosis Cell senescence

Endosulfan (CAS 115-29-7) is a potent organochlorinated pesticide from the cyclodiene group and is widely used to control insects in cultivated areas. Organochlorides are characterized by the presence of a covalent bond between carbon and chloride atoms. This special chemical feature is not common in nature, thus living organisms have not developed mechanisms to cope with it. Moreover, this pesticide is highly lipophilic, thus it bioaccumulates and biomagnifies along the alimentary chain [1]. Because of its stability in water and its accumulation in tissue, endosulfan induces toxic effects in target and non-target organisms, even at low doses. The high toxicity of endosulfan has been demonstrated in fish such as rainbow trout (Oncorhynchus mykiss) and cat fish (Ictalurus

* Corresponding author. Biología Celular y Molecular, Universidad de Guadalajara, Carretera a Nogales Km 15.5, 45110 Zapopan, Jalisco, Mexico. Tel./fax: þ52 33 3673 8375. E-mail address: [email protected] (G. Zaitseva). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.10.003

punctatus) [2]. Our research group recently reported that, in Nile tilapia (Oreochromis niloticus), exposure to endosulfan, in vivo, at low sub lethal concentration and over a short period of time, induces non-specific activation and oxidative stress in splenic macrophages from juvenile fish [3] as well as an exacerbated interleukin-2 (IL-2) synthesis [4]. IL-2 is an immunomodulatory cytokine that promotes proliferation, activation and differentiation of T cells and is required for immunoglobulin synthesis by B cells [5]. In fish, T cells are found in the thymus, head kidney and spleen, and represent approximately 75% of circulating lymphocytes [6]. Lymphocyte activation generates MAP kinase (MAPK) activated signal transductions, from the membrane to the nuclei, which coordinate biochemical pathways and activate transcription factors. Amongst the four main groups of MAPK described so far, the ERK family is regulated by extracellular signals and includes ERK1 and ERK2 kinases. ERK1/2 molecules translocate to the nuclei and activate the Elk-1 protein which in turn phosphorylates c-Fos, in order to activate the AP-1 transcription factor [7]. In humans, the

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Raf/MEK/ERK pathway is involved in many aspects of cell mobility, proliferation, differentiation and survival [8]. This pathway is highly conserved in poikilothermic organisms [9]. Endosulfan, at micro molar concentration, is able to induce the expression of phosphorylated ERK1/2 molecules in the human HaCaT keratinocyte cell line [10]. The other two known MAPK pathways, which involve the p38 and JNK families of MAPK, are involved in promoting inflammation and apoptosis [11]. Apoptosis is an important cellular mechanism used to maintain healthy cell populations; it is a vital component of embryonic development and of the immune system, as well as essential for xenobiotic-induced cell death. Deficient apoptosis (too low or too high) has been shown to be involved in many pathologies, including neurodegenerative diseases, autoimmunity and cancer [12]. Antherieu et al. (2007) reported that the pre-exposure of HaCaT cells to similar concentrations of endosulfan, for 24 h, decreased staurosporine-induced apoptosis [13]. Damaged cells that escape apoptosis generally undergo senescence. Cellular senescence is induced in proliferating cells within a short period of time by oxidative stress. This phenomenon is known as premature senescence; these cells are no longer capable of dividing, yet remain metabolically active [14]. Cell senescence is characterized by a wide variety of parameters, including changes in gene expression patterns, mitochondrial dysfunction, accumulation of lipofuscin (an age-related pigment), deregulation of the production of reactive oxygen species (ROS), and activity of bgalactosidase at near neutral pH (senescence-associated b-galactosidase activity) [15]. Senescence is the result of complex genetic interactions [16] and, Nowak and Kingsford (2003) also reported that environmental contaminants such as organochlorides induce cell senescence in aquatic organisms [17]. The biological effects of endosulfan may differ depending on the organism, cell type, concentration and exposure period. Thus, in order to study the mechanisms by which endosulfan affects fish health, the present study was aimed at evaluating the in vitro effects of this insecticide, at an experimental concentration equivalent to levels previously reported in environmental research [18]. In the experiments the following chemicals and antibodies were used: chemotherapeutic drugs etoposide (Etop) and adriamycin (ADR) as positive controls were obtained from LemeryÒ Laboratories, México. Endosulfan (mixture of endosulfan a and b isomers (7:3)) was purchased from Chem Service Inc.Ô (West Chester, PA, USA). Cell proliferation reagent WST-1 (4-[3-(4-Iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) was purchased from Roche Molecular Biochemicals. Baf A1, histopaque 1077, trypan blue, fetal calf serum (FCS), pervanadate (a tyrosine phosphatase inhibitor), propidium iodate (PI), ionomycin, and phorbol myristate acetate (PMA) were obtained from SigmaeAldrich CoÔ (St. Louis, MO, USA). PMA used at a 200 ng mL
1 working dilution dissolved in RPMI-1640 with 4 mM glutamine (Gibco-Invitrogen, Grand Island, NY, USA) and 100 mg mL
1 gentamycin sulfate supplemented with 10% FCS (RPMI-S). Ionomycin was used at a 20 mg mL
1 working dilution dissolved in RPMI-S. Alexa-fluorÒ 488 mouse anti-rabbit-ERK1/2 (pT202/pY204) was obtained from BD Biosciences Pharmingen (Franklin Lakes, NJ, USA) and for isotype control a goat anti-rabbit IgG - FITC (sc2012) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). C12FDG (5-dodecanoylaminofluorescein di-b-D-galactopyranoside) [D2893], Annexin V-FITC were obtained from Gibco-Invitrogen Inc. For cell isolation male Nile tilapias (approximately 150e200 g) were used, fish were maintained individually in 40-L glass aquarium, at 27  1  C and constant aeration, until sacrifice under optimum conditions parameters (chlorine free and continuously aerated tap-water was used, aquarium pH ranged from 7.0 to 7.3 units, dissolved oxygen varied from 4.2 to 4.9 mg L
1, total

ammonia was lower than 0.01 mg L
1). For each experiment 9 fish were anesthetized with clove oil at 100 mg L
1 and sacrificed by decapitation and the spleens were carefully dissected aseptically [19]. In order to evaluate the direct effect of endosulfan on immune cells, spleens of non-exposed healthy tilapia were obtained. The spleens were then passed through a 100 mm nylon mesh and cells were suspended in PBS 1. Red blood cells were separated from the cell suspension, using a modification of the method described by Harford et al. (2005), by density gradient centrifugation: 4 mL of cell suspension was layered over 4 mL of histopaque 1077 and centrifuged at 1200  g for 30 min at 20  C [20]. The immune cells were collected from the upper histopaque layer and washed twice with PBS 1. For each cell suspension, cell number and viability were evaluated using trypan blue dye exclusion method at 0.4% (w/ v). Cell concentration was further adjusted with RPMI-S as required for each assay. In all experiments, splenocytes were incubated in microplates at 28  2  C and 5% of CO2 in a humid incubator (NuAire HEPAIII, Plymouth, MN, USA). In order to evaluate the direct effect of endosulfan on spleen cells proliferation the WST-1 assay was used, this colorimetric assay is based on the cleavage of the tetrazolium salt WST-1 to a formazan-class dye by mitochondrial succinate-tetrazolium reductase from viable cells. Cell suspension containing 1  106 viable cells mL
1 resuspended in RPMI-S was dispensed into 24 flat bottom well culture plates (Costar 3524) in duplicates for each experimental group, and incubated for 72 h at 28  2  C and 5% of CO2 in a humid incubator. In the first group the cells (100 mL per well) were pre-exposed for 4 h to 7 mg mL
1 of endosulfan (equivalent to 7 ppm ¼ 17.2 mM), before the mitogenic stimulation with an equal volume of PMA and ionomycin (to reach a final concentration of 100 ng mL
1 of PMA and 1 mg mL
1 of ionomycin). Cells from the second group were stimulated with these mitogenic substances without endosulfan and control cells were cultivated only with RPMI-S. Then, cells were resuspended in RPMI-S and 300 mL of each experimental group were transferred to 96 well-plates (in triplicates) and 20 mL of WST-1 was added directly to each well. After a 4 h period of incubation (at 28  2  C and 5% of CO2), plate was shaken for 1 min and cell proliferation was determined by using a multi-mode microplate reader (Synergy HT, Biotek) to measure absorbance at 450 nm. Stimulation index was determined as: SI ¼ A1A
1 0 ; where A1 is the absorbance of the stimulated sample and A0 is the absorbance of the sample without stimulation. For measurement of pERK1/2 expression in spleen cells from Nile tilapia flow cytometry was used according to the procedure of Chow et al. (2001) [21]. Briefly, to determine pERK1/2, splenocytes (100 mL per well from the suspension at 1  106 viable cells mL
1) were incubated for 15 min, 180 min, 24 h, 48 h and 72 h into 24 well-plates (Costar 3524), in duplicates for each experimental group: endosulfan (E) 7 mg mL
1, pervanadate (P) 0.1 M, P þ E, and control cells, with RPMI-S. Then cells were harvested by centrifugation 1200  g, 6 min, 24  C, resuspended in a 5 mL polypropylene tube with 100 mL of IntraPrep1 (Immunotech, Coulter Company, CA, USA), incubated for 15 min at room temperature and washed twice with 2.5 mL of PBS 1 and finally harvested by centrifugation 1200  g, 6 min, 24  C. Cells were resuspended in 100 mL of IntraPrep2 for 20 min at 4  C protected from light, then incubated with 10 mL of conjugated Alexa-fluorÒ 488 mouse anti-rabbit-ERK1/ 2 (pT202/pY204) and 20 mL of a goat anti-rabbit IgG - FITC (sc2012) was used (1:25) as an isotype control and labeled for 30 min at room temperature, covered from light. Finally, samples were washed and resuspended in 500 mL of PBS 1 with 0.5% formaldehyde and analyzed using a flow cytometer (Cell Sorter BD FACS Aria, San Jose, USA) with laser excitation at 488 nm. The flow

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cytometer was set to collect 10,000 events. We used PI (0.5 mg mL
1) prior to acquisition for exclusion of dead cells; gates were set around viable lymphocytes based on their forward/sideward light scatter pattern. An appropriate isotype control was used to adjust for background fluorescence. Data were processed with the FACSDiva Software (BD Bioscience, San Jose, USA). The results in the figures and table are expressed as a percentage of the untreated control value. Quantification of apoptotic splenocytes by flow cytometry was done by using the fluorescein isothiocyanate conjugated monoclonal Annexin V-FITC apoptosis Kit, according to the manufacturer’s instructions. Annexin V-FITC is a sensitive method for the detection of apoptotic cells at an early stage, well before DNA fragmentation occurs. Untreated and treated (with 7 mg mL
1 endosulfan (E), 10 mg mL
1 etoposide (Etop) and E þ Etop) cell cultures of tilapia splenocytes were analyzed. Briefly, 1  106 cells mL
1 were cultivated (100 mL) into 24 well-plates in duplicates, for each experimental group, with three different incubation periods (24 h, 48 h and 72 h). Cells were harvested by centrifugation 1200  g, 6 min, 4  C. Then 100 mL binding Annexin V buffer, 2 mL Annexin V conjugated to FITC and 2 mL PI buffer were added to the cell bottom, cells were incubated for another 15 min at room temperature, protected from light. Finally the cells were resuspended in 300 mL 1 binding buffer (Annexin B Buffer) before being analyzed by flow cytometry (EPIC XL-MCL, Beckman Coulter, Kreffen, Germany) with laser excitation at 488 nm. The flow cytometer was set to collect 10,000 events. Annexin V-FITC-negative and PI-negative cells were considered live cells. Cells positive for Annexin V-FITC but negative for PI were considered to be in early apoptosis. Cells positive for both Annexin V-FITC and PI were considered to be undergoing late apoptosis. Data were processed with the system II software package (Beckman Coulter, CA, USA). Senescence was determined by flow cytometry by using the Senescence Detection Kit (which detects senescence-associated b-galactosidase activity (SA-b-gal) present in senescent cells), according to the manufacturer’s instructions (Molecular Probes, Inc., OR, USA). The experimental groups were: endosulfan treated (7 mg mL
1) (E), 1 mM ADR (a control drug used to induce senescence [22]), E þ ADR, and untreated control cells. Briefly 1  106 cells mL
1 were cultured into 24 well-plates (100 mL) in duplicate for different time periods (24 h, 48 h and 72 h) at 4  C. Then cells were centrifuged 1200  g for 6 min at 24  C in polypropylene tubes, washed twice with PBS 1 and incubated for 30 min with 200 mL RPMI-S, into 96 well-plates at 28  2  C and 5% of CO2 in a humid incubator. Afterward, 50 nM of Baf A1 was added and cells were incubated for 1 h, then 10 mM of C12FDG (fluorogenic glycosidase substrates) was added and the mixture was incubated for 1 h. Finally, the cells were harvested, washed twice with PBS 1 and resuspended in 200 mL PBS 1, before being analyzed by flow cytometry (Cell Sorter BD FACS Aria, San Jose, USA) as described above. Statistical analysis was carried out utilizing one-way ANOVA and Tukey’s test for all pair wise comparison of the mean responses (mean  standard deviation) of the different experimental groups using Statgraphics 5.1 software. The significance level was set at p < 0.05. Our results showed that exposure to E per se increased significantly cellular proliferation, but the lymphoproliferative response to the mitogenic stimulus of immune cells pre-exposed to E, decreased significantly in comparison with the control group (p < 0.05) (Fig. 1). Table 1 illustrates E activated pERK1/2, given as % of expression of pERK1/2. The activation reached a plateau at 15 min and maintained it up to 180 min, with levels of expression of pERK1/2 of 11.18  0.85 and 11.84  0.78% respectively. Control splenocytes

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Fig. 1. Proliferative response of Nile tilapia splenocytes’ pre-exposed in vitro to endosulfan (E). The WST-1 cell proliferation reagent was used to evaluate the proliferative response of splenocytes from Nile tilapia pre-exposed to E alone or stimulated with PMA þ ionomycin (M) or pre-exposed to E and stimulated with M (E þ M). Data are shown as stimulation indices and compared using one-way ANOVA and Tukey’s test  SD. * The level of significance was set at p < 0.05.

non-exposed to E showed levels in the percentage of pERK1/2 of 6.10  0.14%, at 15 min and 1.06  0.42%, at 180 min which are lower than E exposed cells. Pervanadate (P) is a positive control and induced hyperphosphorylation (63.78  1.98% positive cells at 15 min and 60.78  2.69%, at 180 min). The cells that received both E and P presented higher levels of pERK1/2 expression compared to E alone at 15 and 180 min, but lower than P alone however at 48 and 72 h of exposure no significant difference was observed between E þ P group and P alone, but E þ P presented higher levels of pERK1/2 than E alone. Fig. 2 shows a representative example of an independent experiment, in which endosulfan can be seen to mediate a decrease in etoposide (Etop)-induced apoptosis. In the untreated control group the percentage of apoptosis was minor (4.87  3.75%, 2.93  2.63% and 7.46  7.00% at 24, 48 and 72 h respectively); the presence of E in immune cell cultures induced no significant changes in the percentages of apoptosis at 24 h (10.28  5.99%), 48 h (4.25  3.78%) and 72 h (6.45  5.44). The presence of Etop alone in cell cultures induced higher values of early apoptosis than in untreated control cells: 21.75  13.83%, 13.25  7.30% and 11.83  7.82% (at 24 h, 48 h and 72 h, respectively). However, when the cells were incubated with both E and Etop the values of early apoptosis decreased (10.25  6.52%, 6.40  4.88% and 6.65  4.48%

Table 1 Kinetics of the expression of phosphorylated ERK1/2 (pERK1/2) in spleen cells from Nile tilapia exposed in vitro to endosulfan. Time-course analysis of pERK1/2 in splenocytes from Nile tilapia exposed in vitro to 7 mg mL
1 of endosulfan (E), pervanadate (P), endosulfan and pervanadate (E þ P), and non-exposed control. Data are presented as percentage of total cells that positively expressed pERK1/2. Background fluorescence with isotype control staining was minor to 1.7% and this was subtracted out of percentage in all groups. Data show that endosulfan activates pERK1/2 expression with a plateau reached after 15 min of exposure and maintained up to 180 min. No significant changes were observed between 24, 48 and 72 h of exposure to E compared with control. One-way ANOVA and Tukey’s test  SD were used. Time

Control

15 min 180 min 24 h 48 h 72 h

6.10 1.06 2.43 2.48 2.06

    

0.14 0.42 0.95 1.01 1.76

Endosulfan (E) 11.18 11.84 2.70 3.60 2.35

    

0.85 0.78 0.94 0.18 0.72

Pervanadate (P) 63.78 60.87 57.76 30.79 30.80

    

1.98 2.69 4.24 2.18 3.17

EþP 20.56 20.60 40.43 29.70 31.37

    

1.84 4.10 8.33 3.79 3.59

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Fig. 2. Evaluation of apoptosis in spleen cells from Nile tilapia exposed in vitro to endosulfan. Fig. 2 shows representative doteplot graphics of an independent experiment, based on the use of flow cytometry and the Annexin V-FITC kit, demonstrating apoptosis in spleen cells from Nile tilapia exposed in vitro to endosulfan. Apoptotic cells can be observed in the lower right square.

at 24 h, 48 h and 72 h, respectively). Statistical significance is not reached; however, a remarkable tendency of endosulfan to protect against etoposide-induced apoptosis can be observed. Fig. 3 shows that untreated cells exhibit minimal senescence at all time groups. Endosulfan did not induce significant senescence in immune cells at 24 h of exposure. However, significant increases in senescent cell number were observed in E treated cells at 48 h (senescence ¼ 14. 02  1.78%) and at 72 h (senescence ¼ 16.85  0.07%) these values were more than 2 times higher than the untreated control cells (p < 0.05). We also observed that E greatly increases (over 2 fold, p < 0.05) the senescence induced by ADR. The percentage of senescence found in E þ ADR treated cultures of spleen cells was 32.72  6.45%, 41.85  1.38 and 33.44  5.30 for 24, 48 and 72 h respectively. Thus, E, ADR or their combination (E þ ADR) are capable of inducing profound changes in senescence in splenocytes from Nile tilapia. The present work describes the first investigation into the response of immune cells from Nile tilapia with in vitro exposure to the environmental pollutant endosulfan, a potent insecticide known to affect the health of non-target organisms through several mechanisms of action, particularly through the inhibition of GABA associated chloride channels and the stimulation of estrogenic receptors (ER) [23e25]. The two types of ER, alfa and beta, are present on the surface of immunocompetent cells [26], thus their function can be affected by exposure to endosulfan. It has been demonstrated that teleost fish, such as tilapia, exposed in vivo to sub lethal doses of endosulfan (7 mg L
1) present exacerbated innate and specific immune responses [3,4]. The

present in vitro study shows that a low concentration of endosulfan (7 mg mL
1) can, per se, induce proliferation of splenic lymphocytes from Nile tilapia (Fig. 1). These experimental data coincide with Harford et al. (2005) [20], who observed an increase in the percentage of lymphocytes in the head kidney of rainbow trout (Melanotaenia fluviatilis) exposed to 10 mg L
1 of endosulfan suggesting that it is a generalized effect of endosulfan and not of one specie. In vitro experiments indicate that endosulfan acts as a xenoestrogen [27], and may induce the non-specific activation of lymphocytes through its binding to ER-a. This binding could in turn provoke significant changes in the intracellular calcium concentration [28], and activate the ERK1/2 cascade as observed in the present work and reported by Ledirac et al. (2005) in HaCat cells [10]. In a related study, in vivo endosulfan treatment has also been found to increase IL-2 synthesis in tilapia [4], which also may explain the earlier findings as to the increase in splenocyte proliferation. However, we observed that splenocytes pre-exposed to endosulfan did not respond to mitogenic stimuli with PMA (a polyclonal mitogen which facilitates the activation of the MAP kinase pathway, a key event for cell proliferation) in combination with ionomycin (an ionophore widely used to increase intracellular levels of calcium). Several authors report data similar to the ones presented here, in mammals as well as fish, which confirms that exposure to organochlorinated pesticides decreases the blastogenic capacity of lymphocytes to respond to mitogenic or antigenic stimulation [29,30]. This decrease in the proliferative response is probably due to the double stimuli of these cells using the same pathway. It has

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Fig. 3. Endosulfan (E) induces senescence in spleen cells from Nile tilapia. Induction of senescence in Nile tilapia splenocytes exposed to 7 mg mL
1 of E for 24, 48 and 72 h. Data are presented as percentage of b-galactosidase activity in experimental versus control groups and are compared using one-way ANOVA and Tukey’s test  SD. The level of significance was set at p < 0.05. It can be seen that endosulfan inhibits etoposide-induced apoptosis.

previously been reported, in HaCat cells, that endosulfan activates the Raf, MEK y ERK1/2 pathway [10]. It has also been previously reported that PMA stimulation of fish lymphocytes also uses ERK1/2 signaling [31]. Our current supposition is that early exposure to activate this pathway may lead to a lack of response to subsequent signals. The present study shows that endosulfan induces, in splenocytes, a sustained phosphorylation of ERK1/2 for at least 180 min, which is a significant increase compared to the control group (Table 1). These data confirm a report from Bulayeva and Watson (2004) in which the GH3/B6/F1O cell line (selected for the high expression of the membrane receptor for ER-alpha [mER-a]) exhibited rapid activation of the ERK1/2 pathway when exposed to sub picomolar and nanomolar concentrations of insecticide [32]. In this study, the authors found this activation to rapidly decrease after 10 min in a time dependent manner. Additionally, Narita et al. (2007) have reported the activation of the ERK1/2 pathway by ER-a in the HMC-1 cell line of human mastocyte origin [27]. In addition to the known response of the ER-a to endosulfan, the increase in the expression of pERK1/2 reported in this study could also be the result of the excessive production of ROS, which is able to inactivate the MAP-kinase phosphatases (MKPs) and leads to the sustained activation of MAPK/ERK1/2 [33]. In a previous study, our laboratory has found that exposure to endosulfan induces high expression of ROS in tilapia derived macrophages [3]. Exposure to endosulfan could in turn have an anti-apoptotic effect, as shown in Fig. 2, where a marked decrease in the percentage of apoptosis of endosulfan pre-exposed splenocytes treated with the apoptosis inducing drug etoposide can be seen. Our data may be related with previous work which showed that

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endosulfan has profound effects on cell signaling systems: the insecticide was shown to decrease the activity of effector caspase 3 and caspase 7 [13] and to increase the levels of the anti-apoptotic protein Bcl-2 [34]. Thus the inhibition of etoposide-induced apoptosis observed in our system after endosulfan exposure may be explained by these previous findings. It is interesting to note that reduction in apoptosis was also found in endosulfan-exposed cells not treated with etoposide. This is in concordance with data published by Kannan et al. (2000) where Jurkat cells exposed to 20 mM of endosulfan only presented 2.2% of apoptosis [35]. The anti-apoptotic effect that we have seen may explain the induction of senescence in cells exposed to endosulfan. According to Sasaki et al. (2001), senescent cells may be resistant to apoptosis [36]. Fig. 3 show that endosulfan promotes senescence in splenocytes. These data coincide with Nowak and Kingsford (2003) who demonstrated that 69% of hepatocytes from Tandanus tandanus, exposed in vivo to 1.0 mg L
1 of endosulfan, present lipofuscin granules (a pigment marker of cell senescence) [17]. Moreover, Caglar et al. (2003) suggested that senescence in mouse liver cells treated with endosulfan is due to oxidative stress [37]. This hypothesis and the data presented here confirm previous observation on increased ROS production by this insecticide in tilapia [3] and another report from Brandl et al. (2011) on the induction of senescence in mesenchymal stem cells under oxidative stress [38]. In conclusion, these results provide experimental evidence of endosulfan-mediated unspecific activation of immune cells in an in vitro model which confirms the endosulfan-mediated immunoexitability seen in vivo. With respect to possible mechanisms responsible for these observations, our data indicate that even at very low levels, exposure to organochlorinated pesticides such as endosulfan resulted in increased expression of pERK1/2, splenic cells activation, and a decrease in apoptosis culminating with high levels of cell senescence. The data presented here indicate that exposure of immune cells to endosulfan deregulates their function and might facilitate the development of diseases. Thus, Nile tilapia is useful as an immune-toxicological model. The study of populations of fish inhabiting highly polluted environments needs to be promoted to provide further information on the etiology of pollutant-mediated diseases.

Acknowledgments This study was supported by P3E 2010 (number 108126) program from the Universidad de Guadalajara and by PROMEP-SEP funding for CA-UDG-482. The first author received a grant from CONACyT-Mexico (number 48460). We gratefully acknowledge BSc. Alfredo Molina Sahagun, of CESAJ, A.C. and Aquamol S.C. de R.L., for providing the fish, and Dr. Eduardo Juarez-Carrillo for the fish bioassay laboratory. We thank MSc. Jesse Haramati for excellent revising and reading of the manuscript.

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