- Email: [email protected]
Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro
Available online at www.sciencedirect.com
Veterinary Immunology and Immunopathology 121 (2008) 189–198 www.elsevier.com/locate/vetimm
Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro S. Ca´mara Pellisso´ a, M.J. Mun˜oz a, M. Carballo a, J.M. Sa´nchez-Vizcaı´no b,* a
CISA-INIA, Centro Investigacio´n Sanidad Animal (INIA), Valdeolmos, 28130 Madrid, Spain b Animal Health Department, UCM, Avda. Puerta de Hierro, 28040 Madrid, Spain
Received 26 April 2007; received in revised form 17 September 2007; accepted 26 September 2007
Abstract Heavy metals may affect the immune system of cetaceans. But no information exists on their effects on the bottlenose dolphin (Tursiops truncatus) immune system, although this species is a coastal top predator which can bioaccumulate high concentrations of them. This work studies the effects of Hg (1, 5 and 10 mg/L), Al (2,5, 25 and 50 mg/L), Cd (1, 10, 20 and 40 mg/L), Pb (1, 10, 20 and 50 mg/L) and Cr (1 and 10 mg/L), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. Cell viability, apoptosis, lymphocyte proliferation and phagocytosis were evaluated. Viability and lymphoproliferation were measured with Alamar Blue assay, and apoptosis and phagocytosis were evaluated with flow cytometry. Apoptosis was detected as mechanism of cell death after cadmium and mercury exposure. A significant reduction in the lymphoproliferative response was registered by exposure to 1 mg/L of mercury, 10 mg/L of cadmium and 50 mg/L of lead. Decreased phagocytosis was also observed at 5 mg/L of mercury, 50 mg/L of aluminium and 10 mg/L of cadmium. Chromium did not present any effects on any immune assay at the concentrations tested. The concentrations of heavy metals that were found to affect the functional activity of bottlenose dolphin leukocytes are within the environmental ranges reported in the tissues of bottlenose dolphins. These results support the hypothesis that exposure to these contaminants, particularly mercury and cadmium could lead to a reduction in host resistance to disease in these animals. # 2007 Elsevier B.V. All rights reserved. Keywords: Immunotoxicity; Bottlenose dolphin; Heavy metals; Viability; Apoptosis; Lymphoproliferation; Phagocytosis
1. Introduction The bottlenose dolphin (Tursiops truncatus) is a coastal predator at the top of the marine food chain, so it constitutes an excellent indicator of the health of the marine ecosystem (Wells et al., 2004). This dolphin species is considered a fish and squid eater (Culik, 2004). It tends to bioaccumulate high levels of Abbreviations: PI, propidium iodide; ppm, parts per million; SI, stimulation index. * Corresponding author. Tel.: +34 913944082; fax: +34 913943908. E-mail address: [email protected] (J.M. Sa´nchez-Vizcaı´no). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.09.009
persistent contaminants present in the marine environment, such as heavy metals (Law et al., 1991). Various studies have documented high levels of these chemicals in bottlenose dolphins in different parts of the world (Cardellicchio et al., 2000, 2002; Frodello et al., 2000; Parsons and Chan, 2001; Roditi-Elasar et al., 2003; Carballo et al., 2004). The available literature mainly reports concentrations in liver and kidney. Heavy metals are known to produce toxic effects on animals. The most sensitive target tissue affected by some toxins (or toxic components) is the immune system (Black et al., 1992; Raszyk et al., 1997). Extensive experimental investigations have shown that different heavy metals, such as mercury, cadmium, lead,
190
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
chromium or aluminium can cause immunomodulation in laboratory animals and also in humans (Koller, 1979; Descotes, 1988; Bernier et al., 1995; Lawrence and McCabe, 2002; Shrivastava et al., 2002; Synzynys et al., 2004). But to assess a possible immunosuppression induced by environmental xenobiotics in cetaceans, information on the functioning of the immune system of these species is needed. So, in the last few years, important efforts have been made to adapt methods for in vitro evaluation of the immune function in cetaceans (Romano et al., 1992; De Guise et al., 1995, 1996a, 1997; Noda et al., 2003; Beineke et al., 2004) and models using in vitro exposure of immune cells of marine mammals have been developed. These immunotoxicological assays have shown that different contaminants may cause immunosuppression in different marine mammals using parameters such as mitogeninduced proliferation, phagocytosis and natural killer cytotoxicity (De Guise et al., 1996b; Pillet et al., 2000; Nakata et al., 2002; Lalancette et al., 2003; Levin et al., 2004; Mori et al., 2006). Most of the studies in cetacean refer to the immunotoxicological potential of organochlorine compounds, but the effect of heavy metals on the immune function of cetaceans was only investigated in the beluga whale (Delphinapterus leucas) (De Guise et al., 1996b). But no studies have investigated the effects of heavy metals on the immune system of bottlenose dolphins. The aim of this study was to determine the action of five heavy metals (Al, Cd, Cr, Hg and Pb), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. These five heavy metals were chosen due to their previous detection in bottlenose dolphin tissues in many geographical areas, mainly in places of industrialized world (Das et al., 2003). Also their potential immunological impact in terrestrial and marine mammals has been considered for Hg, Cd and Pb, but still very little information is available about the immuno-modulating effect of Cr and Al. The immunotoxicity in this study was assessed using different parameters (cell viability, apoptosis, lymphocyte proliferation and phagocytosis).
(PbCl2, purity 99.9%) and chromium (CrCl2, purity 95%). All were purchased from Sigma–Aldrich Chemical Co. Concentrated stock solutions (10 g/L) were prepared by dilution in distilled water. All the toxics were then diluted in cell culture medium (RPMI1640) with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL sptreptomycin, 2 mM Lglutamine, 10 mM HEPES and 1% non-essential aminoacids (Sigma). The final incubation concentrations of metals were as follows: mercury, 1, 5 and 10 mg/L (ppm); aluminium, 2.5, 25 and 50 mg/L; cadmium, 1, 10, 20 and 40 mg/L; lead, 1, 10, 20 and 50 mg/L; and chromium, 1 and 10 mg/L. 2.2. Animals Blood samples were collected from nine captive bottlenose dolphins from four aquariums in Spain (Zoo Aquarium Madrid, Zoo Barcelona, L’Oceanogra´fic Valencia and Marineland). All individuals were healthy animals of both sexes and without haematological signs of disease. Blood samples were drawn from the ventral tail fluke into heparinized tubes and were kept at 48 C until the time of analysis, within 24 h after collection. Each assay was performed three to five times, depending upon blood availability. 2.3. Isolation of cells 2.3.1. Peripheral blood mononuclear cells (PBMCs) For assessment of lymphocyte viability, apoptosis and proliferation, the PBMCs were isolated following protocols designed by De Swart et al. (1993). Briefly, blood was diluted 1:1 with RPMI-1640 medium. The diluted blood was then carefully layered over a commercially available density gradient (Histopaque 1077, Sigma) and centrifuged at 400 g at room temperature for 30 min. The PBMC layer was carefully collected, and the recovered cells were washed twice in PBS at 200 g for 7 min at 4 8C. The cells were then resuspended in cell culture medium described previously, and their viability was determined by the trypan blue exclusion test. Cell viability prior to assays was >95%.
2. Materials and methods 2.1. Toxics tested Five heavy metals were selected for the study: mercury (HgCl2, purity 99.9%), aluminium (AlCl3, purity 99.9%), cadmium (CdCl2, purity 99.9%), lead
2.3.2. Peripheral blood leukocytes For evaluation of phagocytosis, leukocytes were isolated from peripheral blood adding a solution of NH4Cl for lysis of erythrocytes. The cells were then washed three times in PBS and suspended in supplemented RPMI. Viability of the cells was
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
determined by the trypan blue exclusion test. Cell viability prior to assays was always >95%. 2.4. Alamar Blue assay for cell viability Cell viability in leukocytes was determined by Alamar Blue assay, based on previous studies (Watters et al., 2004). Briefly, the PBMCs were seeded in triplicate in 96-well round-bottom plates at a density of 2 106 cells/mL and incubated in a total volume of 200 mL with and without metals at 37 8C in a humidified 5% CO2 incubator. After 48 h of incubation, 20 mL of Alamar blue were added to each well and cells were incubated an additional 24 h. Absorbance at 570 and 600 nm was measured using an automated microplate reader, and viability relative to untreated controls was calculated according to the manufacturer’s instructions by using this formula: ðeox 600 nm A570 nm eox 570 nm A600 nmÞ of treated sample 100 ðeox 600 nm A570 nm eox 570 nm A600 nmÞ of untreated control where eox600 nm = 117,216 (molar extinction coefficient of oxidized Alamar blue reagent at 600 nm), eox 570 nm = 80,586 (molar extinction coefficient of oxidized Alamar blue reagent at 570 nm), A570 nm = absorbance of sample at 570 nm and A600 = absorbance of sample at 600 nm. 2.5. Apoptosis assay To investigate the possible involvement of apoptosis in metals-induced cell death, flow cytometric analysis was performed. The Annexin-Vassay was used to detect phospatidylserine translocation on the membrane surface of cells undergoing apoptosis (Vermes et al., 1995). Cells were co-stained with Annexin V-FITC and propidium iodide (PI) (Roche). The PBMCs were cultured with or without different concentrations of metals. After 24 h, the cells were washed to remove excess chemicals and resuspended in 100 mL of Annexin V binding buffer containing 2 mL of Annexin V-FITC conjugate and 0.2 mg PI. After 15 min of incubation in the dark at room temperature, the cell solutions were diluted 1:4 with binding buffer. Apoptotic cells (Annexin-Vpositive and PI-negative) and necrotic cells (PIpositive) were detected and distinguished by flow cytometry using a FACScalibur flow cytometer (Becton Dickinson).
191
2.6. Alamar Blue assay for lymphocyte function This assay was performed to determine if metals suppressed the proliferative response of bottlenose dolphin lymphocytes. The PBMCs were adjusted to 2 106 cells/mL and were cultured with and without different concentrations of metals in 96-well flatbottom. The cells were either stimulated with concanavalin A (Con A: 10 mg/mL) or pokeweed mitogen (PWM: 2 mg/mL) for T cell or B and T cell mitogenesis, respectively. Each assay consisted of triplicate samples in a total volume of 200 mL of complete RPMI. Bovine PBMCs were used as an internal standard. Cells were incubated 72 h at 37 8C in the humidified 5% CO2 incubator. The function of PBMCs was determined using Alamar Blue assay according to previous reports (Ahmed et al., 1994; De Fries and Mitsuhashi, 1995). This method was adapted and optimized in bottlenose dolphin PBMCs in our laboratory. Briefly, 20 mL of Alamar blue dye was added to each well 24 h before measurement. Absorbances at 570 nm and 600 nm were then measured and the percent of reduction of Alamar blue was calculated according to the manufacturer’s instructions by using this formula: ðeox 600 nm A570 nm eox 570 nm A600 nmÞ ðered 570 nm A0 600 nm ered 600 nm A0 570 nmÞ 100 where ered570 nm = 155,677 (molar extinction coefficient of reduced Alamar blue at 570 nm), ered600 nm = 14,652 (molar extinction coefficient of reduced Alamar blue at 600 nm), A0 600 nm = absorbance of negative control wells which contained media plus Alamar blue but no cells at 600 nm and A0 570 = absorbance of negative control wells that contained media plus Alamar blue but no cells at 570 nm. Finally, results were expressed as relative stimulation index, comparing to control unexposed cells. 2.7. Phagocytosis assay Phagocytosis function of neutrophils and monocytes has been evaluated using flow cytometry. Cells were adjusted to 2 106 mL1 in complete medium and were incubated with or without metals at different concentrations for 3 h in polystyrene tubes at 37 8C in humidified atmosphere containing 5% CO2. Following the exposure period, phagocytic activity was measured using the protocol described by Stewart et al. (1986) with some modifications. Briefly, green fluorescent
192
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
latex beads of 1 mm diameter (Sigma–Aldrich Chemical Co.) were added to each cell suspension to obtain a concentration of 100 beads per cell, and then they were incubated at 4 8C (negative control) or 37 8C overnight under agitation. Phagocytosis was stopped by placing all the samples in ice. An aliquot of 500 mL of cell suspension was layered over a 3% gradient of bovine serum albumin (BSA) and centrifuged at 150 g for 8 min at 4 8C to remove non-phagocytosed microspheres. PI was added to cells just before the analysis by flow cytometry using a FACScalibur flow cytometer (Becton Dickinson). PI positive cells were categorized as dead cells and were excluded from the analysed phagocyting cells. Phagocytosis was tested in duplicate for each assay in five different experiments. For each sample, the fluorescence of 7000 events was read. Forward and side scatter were adjusted to eliminate cell debris. And granulocytes were gated for phagocytosis measurement. Results were analysed with CELLQUEST software to determine the percentage of leukocytes that had phagocytosed fluorescent beads. The phagocytic activity of cells exposed to metal was expressed as a percentage of the mean response observed with untreated cells. 2.8. Statistics The mean and standard deviation were determined for each assay. Statistical analysis consisted of a
parametric ANOVA test to compare groups when data were normally distributed. When data were not normally distributed, a non-parametric Kruskal–Wallis test was used. Significance was determined at p 0.05. 3. Results 3.1. Cell viability The sensitivity of bottlenose dolphin PBMCs to different concentrations of HgCl2, AlCl3, CdCl2, PbCl2 and CrCl2, after 72 h of exposure was shown in Fig. 1. Cellular viability determined by Alamar Blue assay showed no toxic effects for aluminium, lead and chromium during this time of incubation, but mercury (10 ppm) and cadmium (20 and 40 ppm) caused a significant decrease in cellular viability. 3.2. Apoptosis Two-color flow cytometry utilizing AnnexinV-FITC and PI was used to distinguish apoptotic lymphocytes from viable non-apoptotic and necrotic lymphocytes. Using this technique we can observe that only mercury and cadmium have an effect on apoptosis. As shown in Fig. 2, mercury can induce apoptosis at 1 and 5 ppm, but necrosis at these concentrations is not detected. At 10 ppm most of the cells present a PI and AnnexinVFITC dye. But these cells were not considered apoptotic because cell membranes permeable to PI might also
Fig. 1. Relative viability of bottlenose dolphin leukocytes exposed in vitro to metals determined by Alamar Blue assay (n = 4; mean S.D.). Values with asterisks are significantly different from unstimulated control ( p < 0.05).
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
193
Fig. 2. Changes induced in two cell populations of bottlenose dolphin leukocytes classified by FITC and PI fluorescence. Cells stained only with Annexin V-FITC (grey columns), corresponding to live cells undergoing apoptosis. Cells stained with FITC and PI (black columns), corresponding to dead/necrotic cells (n = 3; mean S.D.). Values with asterisks are significantly different from unstimulated control ( p < 0.05).
allow AnnexinV-FITC to penetrate into the cells to label phosphatidyl serine on the inner membrane side of nonapoptotic cells. In any case, they were considered as dead cells. Cadmium at 10 ppm significantly increases the number of cells in apoptosis without affecting the number of dead cells. At 20 and 40 ppm, however, we see an increase in both apoptotic cells and dead cells.
With the concentrations tested for aluminium, chrome and lead, we saw no increase in either category of cells. 3.3. Lymphocyte function The effect of heavy metals on the lymphocyte function of bottlenose dolphin PBMCs determined by Alamar Blue assay as shown in Fig. 3.
Fig. 3. Stimulation index of bottlenose dolphin lymphocytes cultured with HgCl2, AlCl3, CdCl2, PbCl2 or CrCl2. The cells were either stimulated with Con A (white columns) or with PWM (grey columns) (n = 4; mean S.D.). Values with asterisks are significantly different from unstimulated control ( p < 0.05).
194
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
Fig. 4. Phagocytic activities of bottlenose dolphin leukocytes following in vitro exposures to heavy metals. Results are expressed as a (%) of the control response (unexposed cells) (n = 5; mean S.D.). Values with asterisks are significantly different from control ( p < 0.05).
Mercury and cadmium are the metals which most affected the proliferation of lymphocytes. The lowest concentration of mercury that can reduce lymphoproliferation response was 1 ppm. At this concentration, mitogenic responses to Con A and PWM decreased 40% and 75%, respectively. Cadmium can reduce the proliferative response of bottlenose dolphin lymphocytes at 10, 20 and 40 ppm. This reduction is more marked in Con A-stimulated PBMCs. Lead, at 20 and 50 ppm, also affects proliferation but only by 10% with regard to the control group. No effect was observed with aluminium and chromium at the concentrations tested. 3.4. Phagocytosis Phagocytosis of fluorescent beads was evaluated in bottlenose dolphin leukocytes exposed to metals. Lead and chromium had no significant effect on the phagocytic function when compared to unexposed controls, as shown in Fig. 4. But mercury, cadmium and aluminium can modulate this function. At 5 and 10 ppm, mercury significantly reduced phagocytosis by 20% and 40%, respectively. Cadmium, at 10 and 20 ppm reduced phagocytosis by 25% and 45%, respectively. At 50 ppm, aluminium significantly reduced phagocytosis by 35%.
4. Discussion Heavy metals can produce alterations in the immune function (Bernier et al., 1995) and increase the incidence of infectious diseases in marine mammals (Siebert et al., 1999; Bennett et al., 2001). To evaluate this possibility we designed a study to determine whether five heavy metals, at concentrations founds in stranded bottlenose dolphin tissues (Parsons and Chan, 2001; Das et al., 2003; Law et al., 2003; Carballo et al., 2004), could have a toxic effect in bottlenose dolphin immune cells. In this study, increased toxicity was expressed as the decreased cell viability and the impairment of lymphocyte function and/or phagocytic activity upon exposure to mercury, cadmium, aluminium and lead, whereas chromium was relatively well tolerated by the immune cells of bottlenose dolphins. Mercury, the most immunotoxic metal in this study, induced the most severe suppression. A significant decrease in cell viability was detected at 10 ppm (50 mM). Our results have also shown that mercuryexposed cells of bottlenose dolphins die in a manner consistent with the induction of apoptosis, such as has been described in human lymphocytes (Guo et al., 1998; Shenker et al., 2000). At 1 ppm (5 mM) and 5 ppm (25 mM) we can detect the early stage of apoptosis, with no effect on the population of dead cells. And at
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
10 ppm, most of the mercury affected cells showed PI dye. It is likely that mercury at this concentration directly compromises the membranes of the cells and, consequently, their viability. However, both apoptosis and necrosis are mechanisms involved in the dead cell induced by mercury at high concentrations (Kim and Sharma, 2003). These data point out the necessity to develop more studies in bottlenose dolphin immune cells in order to determine if the cell death observed at 10 ppm of mercury is due to a direct induction of necrosis, secondary to apoptosis or is due to an induction of both mode of cytotoxicity. A reduction of lymphocyte function was also observed at low doses (1 ppm). This reduction is greater in PWM than in Con A stimulated lymphocytes. This may suggest that the activation of both T cells (stimulated by Con A and PWM) and B cells (stimulated by PWM) can be affected by mercury. Other studies in human cell lines showed that proliferation of both types of lymphocytes was reduced at similar concentrations (Kim and Sharma, 2003). Mercury also can affect phagocytosis response. Our results are similar to those observed in bovine neutrophils (De Guise et al., 2000). Seals phagocytes seemed less sensitive to mercury, showing a decreased activity at 100 mM (Pillet et al., 2000). Cadmium chloride is another highly toxic heavy metal known to modulate many immune functions in several species (Bernier et al., 1995). Our results showed that cadmium could also result in immunotoxic changes in bottlenose dolphin leukocytes, inducing apoptosis and affecting lymphocyte function and phagocytosis, like mercury but at higher concentrations. We can observe that cadmium at 10 ppm (90 mM) can induce apoptosis in bottlenose dolphin leukocytes and at 20 ppm (178 mM) and 40 ppm (356 mM) necrotic cells can also be detected. These data suggest that in vitro exposure to cadmium induces both pathways of cell death in bottlenose dolphin. This effect is consistent with previous studies in human lymphoid cell lines or PBMCs (Tsangaris and Tzortzatou-Stathopoulou, 1998; Hemdan et al., 2006) which indicate that cadmium has the ability to reduce cell vitality at low doses and is toxic at higher doses. Immunomodulatory activities of cadmium have also been investigated in several in vitro and in vivo model systems, but conflicting results have emerged. For example, in human cells, mitogenic activity was not significantly affected at 5 mM (Borella and Giardino, 1991). Other studies have documented that cadmium is not only toxic but may modulate immune responses (Krocova et al., 2000; Lawrence and McCabe, 2002). Our results showed a reduced activity of lymphocytes in
195
a dose-dependent manner from 10 ppm (90 mM) of cadmium. The lack of reduction of cell viability at this concentration indicates a direct effect on lymphocyte function. Similar studies in other species showed similar effects on proliferation, such as Con Astimulated beluga whale splenocytes or bovine lymphocytes that was reduced at concentrations of 10 mM (De Guise et al., 1996b, 2000), or mice splenocytes, at 20 ppm (Krocova et al., 2000). Other species seem to be more sensitive, like rat whose splenocytes were affected at 5 mM (Snyder and Valle, 1991). Phagocytic activity was reduced after cadmium exposure at 10 ppm. Other species such as harbour seal (Phoca vitulina) showed reduced phagocytosis in the same range (Pillet et al., 2000). Bovine leukocytes seem to be more sensitive because their phagocytic activity was decreased at 0.1–1 mM (De Guise et al., 2000). Lead is also a heavy metal that can alter the immune system, but in bottlenose dolphins only the lymphocytes activity is slightly affected. Previous investigations in different species showed conflicting results. Some studies described a lack of effect in viability or proliferation (Smith and Lawrence, 1988; Borella et al., 1990; De Guise et al., 1996b, 2000) whereas others have observed inhibitory or stimulatory effects of lead on immune cells. For example, on rat splenocytes or in human PBMCs, a significant inhibitory effect was described (Exon et al., 1985; Hemdan et al., 2005). But enhanced B and T cell proliferation (Lawrence, 1981; Warner and Lawrence, 1986) was observed in mice immune cells. Some authors have found that the effect on T cell proliferation depends on different subpopulations, so Pb can suppress Th1 response, but enhances Th2 clonal responses (McCabe and Lawrence, 1991; Heo et al., 1998; Chen et al., 1999; Shen et al., 2001). In bottlenose dolphins, there are no studies allowing to differentiate these responses, thus further investigations are needed. Our data showed that lead did not affect viability of bottlenose dolphin immune cells and we cannot observe any effect on apoptosis, similar to studies in other species (Shen et al., 2001). Neither was phagocytosis significantly affected by exposure to Pb in our study, similar to what has been described in other animals (De Guise et al., 2000). Aluminium is a metal present in the marine environment but few studies have evaluated its effect on the immune system. Only some authors (Gomez et al., 1986; Golub et al., 1993) have found changes, after oral exposure, in spleen or in cytokine production in laboratory animals. Our results showed that only phagocytosis is affected with a significant reduction, but at higher concentrations (50 ppm) than mercury and
196
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
cadmium. To our knowledge, similar studies of in vitro exposure to aluminium are not available. Chromium is a heavy metal found in the environment in different forms and is an essential nutrient required to promote the action of insulin for the utilization of sugars, proteins and fats. But some authors have observed various toxic reactions that affect the immune system, even though the mechanism of chromium-induced toxicity is not entirely understood (Borella et al., 1990; Chang et al., 1996; Lee et al., 2000; Shrivastava et al., 2002). But in bottlenose dolphin blood mononuclear cells we cannot detect any effect on any immune assay at the concentrations tested. In summary, an in vitro approach has been developed to test the effects of five heavy metals on the immune response of peripheral blood leukocytes from bottlenose dolphins. Using these assays we can confirm that mercury and cadmium are the most toxic metals for bottlenose dolphin leukocytes, followed by lead and aluminium, whereas chromium does not show any alteration. The immunotoxic concentrations reported in this study are within the ranges measured in some tissues of stranded bottlenose dolphins (Meador et al., 1999; Frodello et al., 2000; Parsons and Chan, 2001; Law et al., 2003; Carballo et al., 2004). The reduction of some functional activities of the bottlenose dolphin immune system may cause a significant weakness capable of altering host resistance to disease in freeranging cetaceans, as it has been suggested in other marine mammal species exposed to different pollutants (Siebert et al., 1999; Bennett et al., 2001). Our results provide a basis for explaining more complex processes which affect the health of those animals subjected to a marine environment in which the concentration of contaminants is such that it can provoke a loss of immunological resistance when faced with determined infectious agents present in the milieu. Acknowledgements The authors would like to thank Prof. A. Ferna´ndez from the University of Las Palmas de Gran Canaria, the staff of the aquariums at Zoo Aquarium Madrid, Zoo Barcelona, L’Oceanogra`fic and Marineland for providing blood samples from bottlenose dolphins; and Amalia, Juanjo, Carmen and G. Tejerizo for their collaboration. The present study was supported by the RTA2006-00168-00-00 GAN. References Ahmed, S.A., Gogal Jr., R.M., Walsh, J.E., 1994. A new rapid and simple non-radioactive assay to monitor and determine the
proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J. Immunol. Methods 170, 211–224. Beineke, A., Siebert, U., van Elk, N., Baumgartner, W., 2004. Development of a lymphocyte-transformation-assay for peripheral blood lymphocytes of the harbor porpoise and detection of cytokines using the reverse-transcription polymerase chain reaction. Vet. Immunol. Immunopathol. 98, 59–68. Bennett, P.M., Jepson, P.D., Law, R.J., Jones, B.R., Kuiken, T., Baker, J.R., Rogan, E., Kirkwood, J.K., 2001. Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environ. Pollut. 112, 33–40. Bernier, J., Brousseau, P., Krzystyniak, K., Tryphonas, H., Fournier, M., 1995. Immunotoxicity of heavy metals in relation to Great Lakes. Environ. Health Perspect. 9 (103 Suppl.), 23–34. Black, R.D., McVey, D.S., Oehme, F.W., 1992. Immunotoxicity in the bovine animal: a review. Vet. Hum. Toxicol. 34, 438–442. Borella, P., Giardino, A., 1991. Lead and cadmium at very low doses affect in vitro immune response of human lymphocytes. Environ. Res. 55, 165–177. Borella, P., Manni, S., Giardino, A., 1990. Cadmium, nickel, chromium and lead accumulate in human lymphocytes and interfere with PHA-induced proliferation. J. Trace Elem. Electrolytes Health Dis. 4, 87–95. Carballo, M., De la Pen˜a, E., Ferna´ndez, A., Aguayo, S., Espero´n, F., De la Torre, A., Mun˜oz, M.J., 2004. Accumulation of persistent organic chemicals and metals in stranded bottlenose dolphins (Tursiops truncatus) in Canary Islands (Spain). Toxicol. Appl. Pharmacol. 197, 235. Cardellicchio, N., Giandomenico, S., Ragone, P., Di Leo, A., 2000. Tissue distribution of metals in striped dolphins (Stenella coeruleoalba) from the Apulian coasts, southern Italy. Mar. Environ. Res. 49, 55–66. Cardellicchio, N., Decataldo, A., Di Leo, A., Giandomenico, S., 2002. Trace elements in organs and tissues of striped dolphins (Stenella coeruleoalba) from the Mediterranean Sea (Southern Italy). Chemosphere 49, 85–90. Chang, X., Mallard, B.A., Mowat, D.N., 1996. Effects of chromium on health status, blood neutrophil phagocytosis and in vitro lymphocyte blastogenesis of dairy cows. Vet. Immunol. Immunopathol. 52, 37–52. Chen, S., Golemboski, K.A., Sanders, F.S., Dietert, R.R., 1999. Persistent effect of in utero meso-2.3-dimercaptosuccinic acid (DMSA) on immune function and lead-induced immunotoxicity. Toxicology 132, 67–79. Culik, B.M., 2004. Review of small cetaceans. distribution, behaviour, migration and threats. UNEP/CMS p. 343. Das, K., Debacker, V., Pillet, S., Bouquegneau, J.M., 2003. Heavy metals in marine mammals. In: Vos, J.G., Bossart, G.D., Fournier, M., O’Shea, T.J. (Eds.), Toxicology of Marine Mammals. Taylor and Francis, London, pp. 135–167. De Fries, R., Mitsuhashi, M., 1995. Quantification of mitogen induced human lymphocyte proliferation: comparison of Alamar Blue assay to 3H-thymidine incorporation assay. J. Clin. Lab. Anal. 9, 89–95. De Guise, S., Flipo, D., Boehm, J.R., Martineau, D., Beland, P., Fournier, M., 1995. Immune functions in beluga whales (Delphinapterus leucas): evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry. Vet. Immunol. Immunopathol. 47, 351–362. De Guise, S., Bernier, J., Dufresne, M.M., Martineau, D., Beland, P., Fournier, M., 1996a. Immune functions in beluga whales (Delphinapterus leucas): evaluation of mitogen-induced blastic
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198 transformation of lymphocytes from peripheral blood, spleen and thymus. Vet. Immunol. Immunopathol. 50, 117–126. De Guise, S., Bernier, J., Martineau, D., Be´land, P., Fournier, M., 1996b. In vitro exposure of beluga whale lymphocytes to selected heavy metals. Environ. Toxicol. Chem. 15, 1357–1364. De Guise, S., Ross, P.S., Osterhaus, A.D., Martineau, D., Beland, P., Fournier, M., 1997. Immune functions in beluga whales (Delphinapterus leucas): evaluation of natural killer cell activity. Vet. Immunol. Immunopathol. 58, 345–354. De Guise, S., Bernier, J., Lapierre, P., Dufresne, M.M., Dubreuil, P., Fournier, M., 2000. Immune function of bovine leukocytes after in vitro exposure to selected heavy metals. Am. J. Vet. Res. 61, 339–344. De Swart, R.L., Kluten, R.M., Huizing, C.J., Vedder, L.J., Reijnders, P.J., Visser, I.K., UytdeHaag, F.G., Osterhaus, A.D., 1993. Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina). Vet. Immunol. Immunopathol. 37, 217–230. Descotes, J., 1988. Clinical immunotoxicology. J. Toxicol. Clin. Toxicol. 26, vii–viii. Exon, J.H., Talcott, P.A., Koller, L.D., 1985. Effect of lead, polychlorinated biphenyls, and cyclophosphamide on rat natural killer cells, interleukin 2, and antibody synthesis. Fundam. Appl. Toxicol. 5, 158–164. Frodello, J.P., Romeo, M., Viale, D., 2000. Distribution of mercury in the organs and tissues of five toothed-whale species of the Mediterranean. Environ. Pollut. 108, 447–452. Golub, M.S., Takeuchi, P.T., Gershwin, M.E., Yoshida, S.H., 1993. Influence of dietary aluminium on cytokine production by mitogen-stimulated spleen cells from Swiss Webster mice. Immunopharmacol. Immunotoxicol. 15, 605–619. Gomez, M., Domingo, J.L., Llobet, J.M., Tomas, J.M., Corbella, J., 1986. Short-term oral toxicity study of aluminium in rats. Arch. Farmacol. Toxicol. 12, 145–151. Guo, T.L., Miller, M.A., Shapiro, I.M., Shenker, B.J., 1998. Mercuric chloride induces apoptosis in human T lymphocytes: evidence of mitochondrial dysfunction. Toxicol. Appl. Pharmacol. 153, 250–257. Hemdan, N.Y., Emmrich, F., Adham, K., Wichmann, G., Lehmann, I., El-Massry, A., Ghoneim, H., Lehmann, J., Sack, U., 2005. Dosedependent modulation of the in vitro cytokine production of human immune competent cells by lead salts. Toxicol. Sci. 86, 75–83. Hemdan, N.Y., Emmrich, F., Sack, U., Wichmann, G., Lehmann, J., Adham, K., Lehmann, I., 2006. The in vitro immune modulation by cadmium depends on the way of cell activation. Toxicology 222, 37–45. Heo, Y., Lee, W.T., Lawrence, D.A., 1998. Differential effects of lead and cAMP on development and activities of Th1- and Th2lymphocytes. Toxicol. Sci. 43, 172–185. Kim, S.H., Sharma, R.P., 2003. Cytotoxicity of inorganic mercury in murine T and B lymphoma cell lines: involvement of reactive oxygen species, Ca(2+) homeostasis, and cytokine gene expression. Toxicol. In Vitro 17, 385–395. Koller, L.D., 1979. Effects of environmental contaminants on the immune system. Adv. Vet. Sci. Comp. Med. 23, 267–295. Krocova, Z., Macela, A., Kroca, M., Hernychova, L., 2000. The immunomodulatory effect(s) of lead and cadmium on the cells of immune system in vitro. Toxicol. In Vitro 14, 33–40. Lalancette, A., Morin, Y., Measures, L., Fournier, M., 2003. Contrasting changes of sensitivity by lymphocytes and neutrophils to mercury in developing grey seals. Dev. Comp. Immunol. 27, 735–747.
197
Law, R.J., Fileman, C.F., Hopkins, A.D., Baker, J.R., Harwood, J., Jackson, D.B., Kennedy, S., Martin, A.R., Morris, R.J., 1991. Concentrations of trace metals in the livers of marine mammals (seals, porpoises and dolphins) from waters around the British Isles. Mar. Pollut. Bull. 22, 183–191. Law, R.J., Morris, R.J., Allchin, C.R., Jones, B.R., Nicholson, M.D., 2003. Metals and organochlorines in small cetaceans stranded on the east coast of Australia. Mar. Pollut. Bull. 46, 1206–1211. Lawrence, D.A., 1981. In vivo and in vitro effects of lead on humoral and cell-mediated immunity. Infect. Immun. 31, 136–143. Lawrence, D.A., McCabe Jr., M.J., 2002. Immunomodulation by metals. Int. Immunopharmacol. 2, 293–302. Lee, D.N., Yen, H.T., Shen, T.F., Chen, B.J., 2000. Chromium-induced glucose uptake, superoxide anion production, and phagocytosis in cultured pulmonary alveolar macrophages of weanling pigs. Biol. Trace Elem. Res. 77, 53–64. Levin, M., Morsey, B., Mori, C., Guise, S., 2004. Specific noncoplanar PCB-mediated modulation of bottlenose dolphin and beluga whale phagocytosis upon in vitro exposure. J. Toxicol. Environ. Health A. 67, 1517–1535. McCabe Jr., M.J., Lawrence, D.A., 1991. Lead, a major environmental pollutant, is immunomodulatory by its differential effects on CD4+ T cells subsets. Toxicol. Appl. Pharmacol. 111, 13–23. Meador, J.P., Ernest, D., Hohn, A.A., Tilbury, K., Gorzelany, J., Worthy, G., Stein, J.E., 1999. Comparison of elements in bottlenose dolphins stranded on the beaches of Texas and Florida in the Gulf of Mexico over a one-year period. Arch. Environ. Contam. Toxicol. 36, 87–98. Mori, C., Morsey, B., Levin, M., Nambiar, P.R., De Guise, S., 2006. Immunomodulatory effects of in vitro exposure to organochlorines on T-cell proliferation in marine mammals and mice. J. Toxicol. Environ. Health A. 69, 283–302. Nakata, H., Sakakibara, A., Kanoh, M., Kudo, S., Watanabe, H., Nagai, N., Miyazaki, N., Asano, Y., Tanabe, S., 2002. Evaluation of mitogen-induced responses in marine mammal and human lymphocytes by in-vitro exposure of butyltins and non-ortho coplanar PCBs. Environ. Pollut. 120, 245–253. Noda, K., Aoki, M., Akiyoshi, H., Asaki, H., Shimada, T., Ohashi, F., 2003. Evaluation of the polymorphonuclear cell functions of bottlenose dolphins. J. Vet. Med. Sci. 65, 727–729. Parsons, E.C., Chan, H.M., 2001. Organochlorine and trace element contamination in bottlenose dolphins (Tursiops truncatus) from the South China Sea. Mar. Pollut. Bull. 42, 780–786. Pillet, S., Lesage, V., Hammill, M., Cyr, D., Bouqueneau, J.M., Fournier, M., 2000. Effects of mercury and zinc on phagocytic activity of peripheral blood leukocytes of two species of seals from the St Lawrence Estuary. Mar. Pollut. Bull. 40, 921–927. Raszyk, J., Toman, M., Gajduskova, V., Nezveda, K., Ulrich, R., Jarosova, A., Docekalova, H., Salava, J., Palac, J., 1997. Effects of environmental pollutants on the porcine and bovine immune systems. Vet. Med. (Praha) 42, 313–317. Roditi-Elasar, M., Kerem, D., Hornung, H., Kress, N., Shoham-Frider, E., Goffman, O., Spanier, E., 2003. Heavy metal levels in bottlenose and striped dolphins off the Mediterranean coast of Israel. Mar. Pollut. Bull. 46, 503–512. Romano, T.A., Ridgway, S.H., Quaranta, V., 1992. MHC class II molecules and immunoglobulins on peripheral blood lymphocytes of the bottlenosed dolphin Tursiops truncatus. J. Exp. Zool. 263, 96–104. Shen, X., Lee, K., Konig, R., 2001. Effects of heavy metal ions on resting and antigen-activated CD4(+) T cells. Toxicology 169, 67–80.
198
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
Shenker, B.J., Guo, T.L., Shapiro, I.M., 2000. Mercury-induced apoptosis in human lymphoid cells: evidence that the apoptotic pathway is mercurial species dependent. Environ. Res. 84, 89–99. Shrivastava, R., Upreti, R.K., Seth, P.K., Chaturvedi, U.C., 2002. Effects of chromium on the immune system. FEMS Immunol. Med. Microbiol. 34, 1–7. Siebert, U., Joiris, C., Holsbeek, L., Benke, H., Failing, K., Frese, K., Petzinger, E., 1999. Potential relation between mercury concentrations and necropsy findings in cetaceans from German waters of North and Baltic Seas. Mar. Pollut. Bull. 38, 285–295. Smith, K.L., Lawrence, D.A., 1988. Immunomodulation of in vitro antigen presentation by cations. Toxicol. Appl. Pharmacol. 96, 476–484. Snyder, C.A., Valle, C.D., 1991. Lymphocyte proliferation assays as potential biomarkers for toxicant exposures. J. Toxicol. Environ. Health. 34, 127–139. Stewart, C.C., Lehnert, B.E., Steinkamp, J.A., 1986. In vitro and in vivo measurement of phagocytosis by flow cytometry. Methods Enzymol. 132, 183–192.
Synzynys, B.I., Sharetskii, A.N., Kharlamova, O.V., 2004. Immunotoxicity of aluminum chloride. Gig. Sanit. 70–72 (in Russian, with English abstract). Tsangaris, G.T., Tzortzatou-Stathopoulou, F., 1998. Cadmium induces apoptosis differentially on immune system cell lines. Toxicology 128, 143–150. Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C., 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51. Warner, G.L., Lawrence, D.A., 1986. Stimulation of murine lymphocyte responses by cations. Cell. Immunol. 101, 425–439. Watters, J.W., Kraja, A., Meucci, M.A., Province, M.A., McLeod, H.L., 2004. Genome-wide discovery of loci influencing chemotherapy cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 101, 11809–11814. Wells, R.S., Rhinehart, H.L., Hansen, L.J., Sweeney, J.C., Townsend, F.I., Stone, R., Casper, D.R., Scott, M.D., Hohn, A.A., Rowles, T.K., 2004. Bottlenose dolphins as marine ecosystem sentinels: developing a health monitoring system. EcoHealth 1, 246–254.
Veterinary Immunology and Immunopathology 121 (2008) 189–198 www.elsevier.com/locate/vetimm
Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro S. Ca´mara Pellisso´ a, M.J. Mun˜oz a, M. Carballo a, J.M. Sa´nchez-Vizcaı´no b,* a
CISA-INIA, Centro Investigacio´n Sanidad Animal (INIA), Valdeolmos, 28130 Madrid, Spain b Animal Health Department, UCM, Avda. Puerta de Hierro, 28040 Madrid, Spain
Received 26 April 2007; received in revised form 17 September 2007; accepted 26 September 2007
Abstract Heavy metals may affect the immune system of cetaceans. But no information exists on their effects on the bottlenose dolphin (Tursiops truncatus) immune system, although this species is a coastal top predator which can bioaccumulate high concentrations of them. This work studies the effects of Hg (1, 5 and 10 mg/L), Al (2,5, 25 and 50 mg/L), Cd (1, 10, 20 and 40 mg/L), Pb (1, 10, 20 and 50 mg/L) and Cr (1 and 10 mg/L), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. Cell viability, apoptosis, lymphocyte proliferation and phagocytosis were evaluated. Viability and lymphoproliferation were measured with Alamar Blue assay, and apoptosis and phagocytosis were evaluated with flow cytometry. Apoptosis was detected as mechanism of cell death after cadmium and mercury exposure. A significant reduction in the lymphoproliferative response was registered by exposure to 1 mg/L of mercury, 10 mg/L of cadmium and 50 mg/L of lead. Decreased phagocytosis was also observed at 5 mg/L of mercury, 50 mg/L of aluminium and 10 mg/L of cadmium. Chromium did not present any effects on any immune assay at the concentrations tested. The concentrations of heavy metals that were found to affect the functional activity of bottlenose dolphin leukocytes are within the environmental ranges reported in the tissues of bottlenose dolphins. These results support the hypothesis that exposure to these contaminants, particularly mercury and cadmium could lead to a reduction in host resistance to disease in these animals. # 2007 Elsevier B.V. All rights reserved. Keywords: Immunotoxicity; Bottlenose dolphin; Heavy metals; Viability; Apoptosis; Lymphoproliferation; Phagocytosis
1. Introduction The bottlenose dolphin (Tursiops truncatus) is a coastal predator at the top of the marine food chain, so it constitutes an excellent indicator of the health of the marine ecosystem (Wells et al., 2004). This dolphin species is considered a fish and squid eater (Culik, 2004). It tends to bioaccumulate high levels of Abbreviations: PI, propidium iodide; ppm, parts per million; SI, stimulation index. * Corresponding author. Tel.: +34 913944082; fax: +34 913943908. E-mail address: [email protected] (J.M. Sa´nchez-Vizcaı´no). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.09.009
persistent contaminants present in the marine environment, such as heavy metals (Law et al., 1991). Various studies have documented high levels of these chemicals in bottlenose dolphins in different parts of the world (Cardellicchio et al., 2000, 2002; Frodello et al., 2000; Parsons and Chan, 2001; Roditi-Elasar et al., 2003; Carballo et al., 2004). The available literature mainly reports concentrations in liver and kidney. Heavy metals are known to produce toxic effects on animals. The most sensitive target tissue affected by some toxins (or toxic components) is the immune system (Black et al., 1992; Raszyk et al., 1997). Extensive experimental investigations have shown that different heavy metals, such as mercury, cadmium, lead,
190
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
chromium or aluminium can cause immunomodulation in laboratory animals and also in humans (Koller, 1979; Descotes, 1988; Bernier et al., 1995; Lawrence and McCabe, 2002; Shrivastava et al., 2002; Synzynys et al., 2004). But to assess a possible immunosuppression induced by environmental xenobiotics in cetaceans, information on the functioning of the immune system of these species is needed. So, in the last few years, important efforts have been made to adapt methods for in vitro evaluation of the immune function in cetaceans (Romano et al., 1992; De Guise et al., 1995, 1996a, 1997; Noda et al., 2003; Beineke et al., 2004) and models using in vitro exposure of immune cells of marine mammals have been developed. These immunotoxicological assays have shown that different contaminants may cause immunosuppression in different marine mammals using parameters such as mitogeninduced proliferation, phagocytosis and natural killer cytotoxicity (De Guise et al., 1996b; Pillet et al., 2000; Nakata et al., 2002; Lalancette et al., 2003; Levin et al., 2004; Mori et al., 2006). Most of the studies in cetacean refer to the immunotoxicological potential of organochlorine compounds, but the effect of heavy metals on the immune function of cetaceans was only investigated in the beluga whale (Delphinapterus leucas) (De Guise et al., 1996b). But no studies have investigated the effects of heavy metals on the immune system of bottlenose dolphins. The aim of this study was to determine the action of five heavy metals (Al, Cd, Cr, Hg and Pb), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. These five heavy metals were chosen due to their previous detection in bottlenose dolphin tissues in many geographical areas, mainly in places of industrialized world (Das et al., 2003). Also their potential immunological impact in terrestrial and marine mammals has been considered for Hg, Cd and Pb, but still very little information is available about the immuno-modulating effect of Cr and Al. The immunotoxicity in this study was assessed using different parameters (cell viability, apoptosis, lymphocyte proliferation and phagocytosis).
(PbCl2, purity 99.9%) and chromium (CrCl2, purity 95%). All were purchased from Sigma–Aldrich Chemical Co. Concentrated stock solutions (10 g/L) were prepared by dilution in distilled water. All the toxics were then diluted in cell culture medium (RPMI1640) with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL sptreptomycin, 2 mM Lglutamine, 10 mM HEPES and 1% non-essential aminoacids (Sigma). The final incubation concentrations of metals were as follows: mercury, 1, 5 and 10 mg/L (ppm); aluminium, 2.5, 25 and 50 mg/L; cadmium, 1, 10, 20 and 40 mg/L; lead, 1, 10, 20 and 50 mg/L; and chromium, 1 and 10 mg/L. 2.2. Animals Blood samples were collected from nine captive bottlenose dolphins from four aquariums in Spain (Zoo Aquarium Madrid, Zoo Barcelona, L’Oceanogra´fic Valencia and Marineland). All individuals were healthy animals of both sexes and without haematological signs of disease. Blood samples were drawn from the ventral tail fluke into heparinized tubes and were kept at 48 C until the time of analysis, within 24 h after collection. Each assay was performed three to five times, depending upon blood availability. 2.3. Isolation of cells 2.3.1. Peripheral blood mononuclear cells (PBMCs) For assessment of lymphocyte viability, apoptosis and proliferation, the PBMCs were isolated following protocols designed by De Swart et al. (1993). Briefly, blood was diluted 1:1 with RPMI-1640 medium. The diluted blood was then carefully layered over a commercially available density gradient (Histopaque 1077, Sigma) and centrifuged at 400 g at room temperature for 30 min. The PBMC layer was carefully collected, and the recovered cells were washed twice in PBS at 200 g for 7 min at 4 8C. The cells were then resuspended in cell culture medium described previously, and their viability was determined by the trypan blue exclusion test. Cell viability prior to assays was >95%.
2. Materials and methods 2.1. Toxics tested Five heavy metals were selected for the study: mercury (HgCl2, purity 99.9%), aluminium (AlCl3, purity 99.9%), cadmium (CdCl2, purity 99.9%), lead
2.3.2. Peripheral blood leukocytes For evaluation of phagocytosis, leukocytes were isolated from peripheral blood adding a solution of NH4Cl for lysis of erythrocytes. The cells were then washed three times in PBS and suspended in supplemented RPMI. Viability of the cells was
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
determined by the trypan blue exclusion test. Cell viability prior to assays was always >95%. 2.4. Alamar Blue assay for cell viability Cell viability in leukocytes was determined by Alamar Blue assay, based on previous studies (Watters et al., 2004). Briefly, the PBMCs were seeded in triplicate in 96-well round-bottom plates at a density of 2 106 cells/mL and incubated in a total volume of 200 mL with and without metals at 37 8C in a humidified 5% CO2 incubator. After 48 h of incubation, 20 mL of Alamar blue were added to each well and cells were incubated an additional 24 h. Absorbance at 570 and 600 nm was measured using an automated microplate reader, and viability relative to untreated controls was calculated according to the manufacturer’s instructions by using this formula: ðeox 600 nm A570 nm eox 570 nm A600 nmÞ of treated sample 100 ðeox 600 nm A570 nm eox 570 nm A600 nmÞ of untreated control where eox600 nm = 117,216 (molar extinction coefficient of oxidized Alamar blue reagent at 600 nm), eox 570 nm = 80,586 (molar extinction coefficient of oxidized Alamar blue reagent at 570 nm), A570 nm = absorbance of sample at 570 nm and A600 = absorbance of sample at 600 nm. 2.5. Apoptosis assay To investigate the possible involvement of apoptosis in metals-induced cell death, flow cytometric analysis was performed. The Annexin-Vassay was used to detect phospatidylserine translocation on the membrane surface of cells undergoing apoptosis (Vermes et al., 1995). Cells were co-stained with Annexin V-FITC and propidium iodide (PI) (Roche). The PBMCs were cultured with or without different concentrations of metals. After 24 h, the cells were washed to remove excess chemicals and resuspended in 100 mL of Annexin V binding buffer containing 2 mL of Annexin V-FITC conjugate and 0.2 mg PI. After 15 min of incubation in the dark at room temperature, the cell solutions were diluted 1:4 with binding buffer. Apoptotic cells (Annexin-Vpositive and PI-negative) and necrotic cells (PIpositive) were detected and distinguished by flow cytometry using a FACScalibur flow cytometer (Becton Dickinson).
191
2.6. Alamar Blue assay for lymphocyte function This assay was performed to determine if metals suppressed the proliferative response of bottlenose dolphin lymphocytes. The PBMCs were adjusted to 2 106 cells/mL and were cultured with and without different concentrations of metals in 96-well flatbottom. The cells were either stimulated with concanavalin A (Con A: 10 mg/mL) or pokeweed mitogen (PWM: 2 mg/mL) for T cell or B and T cell mitogenesis, respectively. Each assay consisted of triplicate samples in a total volume of 200 mL of complete RPMI. Bovine PBMCs were used as an internal standard. Cells were incubated 72 h at 37 8C in the humidified 5% CO2 incubator. The function of PBMCs was determined using Alamar Blue assay according to previous reports (Ahmed et al., 1994; De Fries and Mitsuhashi, 1995). This method was adapted and optimized in bottlenose dolphin PBMCs in our laboratory. Briefly, 20 mL of Alamar blue dye was added to each well 24 h before measurement. Absorbances at 570 nm and 600 nm were then measured and the percent of reduction of Alamar blue was calculated according to the manufacturer’s instructions by using this formula: ðeox 600 nm A570 nm eox 570 nm A600 nmÞ ðered 570 nm A0 600 nm ered 600 nm A0 570 nmÞ 100 where ered570 nm = 155,677 (molar extinction coefficient of reduced Alamar blue at 570 nm), ered600 nm = 14,652 (molar extinction coefficient of reduced Alamar blue at 600 nm), A0 600 nm = absorbance of negative control wells which contained media plus Alamar blue but no cells at 600 nm and A0 570 = absorbance of negative control wells that contained media plus Alamar blue but no cells at 570 nm. Finally, results were expressed as relative stimulation index, comparing to control unexposed cells. 2.7. Phagocytosis assay Phagocytosis function of neutrophils and monocytes has been evaluated using flow cytometry. Cells were adjusted to 2 106 mL1 in complete medium and were incubated with or without metals at different concentrations for 3 h in polystyrene tubes at 37 8C in humidified atmosphere containing 5% CO2. Following the exposure period, phagocytic activity was measured using the protocol described by Stewart et al. (1986) with some modifications. Briefly, green fluorescent
192
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
latex beads of 1 mm diameter (Sigma–Aldrich Chemical Co.) were added to each cell suspension to obtain a concentration of 100 beads per cell, and then they were incubated at 4 8C (negative control) or 37 8C overnight under agitation. Phagocytosis was stopped by placing all the samples in ice. An aliquot of 500 mL of cell suspension was layered over a 3% gradient of bovine serum albumin (BSA) and centrifuged at 150 g for 8 min at 4 8C to remove non-phagocytosed microspheres. PI was added to cells just before the analysis by flow cytometry using a FACScalibur flow cytometer (Becton Dickinson). PI positive cells were categorized as dead cells and were excluded from the analysed phagocyting cells. Phagocytosis was tested in duplicate for each assay in five different experiments. For each sample, the fluorescence of 7000 events was read. Forward and side scatter were adjusted to eliminate cell debris. And granulocytes were gated for phagocytosis measurement. Results were analysed with CELLQUEST software to determine the percentage of leukocytes that had phagocytosed fluorescent beads. The phagocytic activity of cells exposed to metal was expressed as a percentage of the mean response observed with untreated cells. 2.8. Statistics The mean and standard deviation were determined for each assay. Statistical analysis consisted of a
parametric ANOVA test to compare groups when data were normally distributed. When data were not normally distributed, a non-parametric Kruskal–Wallis test was used. Significance was determined at p 0.05. 3. Results 3.1. Cell viability The sensitivity of bottlenose dolphin PBMCs to different concentrations of HgCl2, AlCl3, CdCl2, PbCl2 and CrCl2, after 72 h of exposure was shown in Fig. 1. Cellular viability determined by Alamar Blue assay showed no toxic effects for aluminium, lead and chromium during this time of incubation, but mercury (10 ppm) and cadmium (20 and 40 ppm) caused a significant decrease in cellular viability. 3.2. Apoptosis Two-color flow cytometry utilizing AnnexinV-FITC and PI was used to distinguish apoptotic lymphocytes from viable non-apoptotic and necrotic lymphocytes. Using this technique we can observe that only mercury and cadmium have an effect on apoptosis. As shown in Fig. 2, mercury can induce apoptosis at 1 and 5 ppm, but necrosis at these concentrations is not detected. At 10 ppm most of the cells present a PI and AnnexinVFITC dye. But these cells were not considered apoptotic because cell membranes permeable to PI might also
Fig. 1. Relative viability of bottlenose dolphin leukocytes exposed in vitro to metals determined by Alamar Blue assay (n = 4; mean S.D.). Values with asterisks are significantly different from unstimulated control ( p < 0.05).
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
193
Fig. 2. Changes induced in two cell populations of bottlenose dolphin leukocytes classified by FITC and PI fluorescence. Cells stained only with Annexin V-FITC (grey columns), corresponding to live cells undergoing apoptosis. Cells stained with FITC and PI (black columns), corresponding to dead/necrotic cells (n = 3; mean S.D.). Values with asterisks are significantly different from unstimulated control ( p < 0.05).
allow AnnexinV-FITC to penetrate into the cells to label phosphatidyl serine on the inner membrane side of nonapoptotic cells. In any case, they were considered as dead cells. Cadmium at 10 ppm significantly increases the number of cells in apoptosis without affecting the number of dead cells. At 20 and 40 ppm, however, we see an increase in both apoptotic cells and dead cells.
With the concentrations tested for aluminium, chrome and lead, we saw no increase in either category of cells. 3.3. Lymphocyte function The effect of heavy metals on the lymphocyte function of bottlenose dolphin PBMCs determined by Alamar Blue assay as shown in Fig. 3.
Fig. 3. Stimulation index of bottlenose dolphin lymphocytes cultured with HgCl2, AlCl3, CdCl2, PbCl2 or CrCl2. The cells were either stimulated with Con A (white columns) or with PWM (grey columns) (n = 4; mean S.D.). Values with asterisks are significantly different from unstimulated control ( p < 0.05).
194
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
Fig. 4. Phagocytic activities of bottlenose dolphin leukocytes following in vitro exposures to heavy metals. Results are expressed as a (%) of the control response (unexposed cells) (n = 5; mean S.D.). Values with asterisks are significantly different from control ( p < 0.05).
Mercury and cadmium are the metals which most affected the proliferation of lymphocytes. The lowest concentration of mercury that can reduce lymphoproliferation response was 1 ppm. At this concentration, mitogenic responses to Con A and PWM decreased 40% and 75%, respectively. Cadmium can reduce the proliferative response of bottlenose dolphin lymphocytes at 10, 20 and 40 ppm. This reduction is more marked in Con A-stimulated PBMCs. Lead, at 20 and 50 ppm, also affects proliferation but only by 10% with regard to the control group. No effect was observed with aluminium and chromium at the concentrations tested. 3.4. Phagocytosis Phagocytosis of fluorescent beads was evaluated in bottlenose dolphin leukocytes exposed to metals. Lead and chromium had no significant effect on the phagocytic function when compared to unexposed controls, as shown in Fig. 4. But mercury, cadmium and aluminium can modulate this function. At 5 and 10 ppm, mercury significantly reduced phagocytosis by 20% and 40%, respectively. Cadmium, at 10 and 20 ppm reduced phagocytosis by 25% and 45%, respectively. At 50 ppm, aluminium significantly reduced phagocytosis by 35%.
4. Discussion Heavy metals can produce alterations in the immune function (Bernier et al., 1995) and increase the incidence of infectious diseases in marine mammals (Siebert et al., 1999; Bennett et al., 2001). To evaluate this possibility we designed a study to determine whether five heavy metals, at concentrations founds in stranded bottlenose dolphin tissues (Parsons and Chan, 2001; Das et al., 2003; Law et al., 2003; Carballo et al., 2004), could have a toxic effect in bottlenose dolphin immune cells. In this study, increased toxicity was expressed as the decreased cell viability and the impairment of lymphocyte function and/or phagocytic activity upon exposure to mercury, cadmium, aluminium and lead, whereas chromium was relatively well tolerated by the immune cells of bottlenose dolphins. Mercury, the most immunotoxic metal in this study, induced the most severe suppression. A significant decrease in cell viability was detected at 10 ppm (50 mM). Our results have also shown that mercuryexposed cells of bottlenose dolphins die in a manner consistent with the induction of apoptosis, such as has been described in human lymphocytes (Guo et al., 1998; Shenker et al., 2000). At 1 ppm (5 mM) and 5 ppm (25 mM) we can detect the early stage of apoptosis, with no effect on the population of dead cells. And at
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
10 ppm, most of the mercury affected cells showed PI dye. It is likely that mercury at this concentration directly compromises the membranes of the cells and, consequently, their viability. However, both apoptosis and necrosis are mechanisms involved in the dead cell induced by mercury at high concentrations (Kim and Sharma, 2003). These data point out the necessity to develop more studies in bottlenose dolphin immune cells in order to determine if the cell death observed at 10 ppm of mercury is due to a direct induction of necrosis, secondary to apoptosis or is due to an induction of both mode of cytotoxicity. A reduction of lymphocyte function was also observed at low doses (1 ppm). This reduction is greater in PWM than in Con A stimulated lymphocytes. This may suggest that the activation of both T cells (stimulated by Con A and PWM) and B cells (stimulated by PWM) can be affected by mercury. Other studies in human cell lines showed that proliferation of both types of lymphocytes was reduced at similar concentrations (Kim and Sharma, 2003). Mercury also can affect phagocytosis response. Our results are similar to those observed in bovine neutrophils (De Guise et al., 2000). Seals phagocytes seemed less sensitive to mercury, showing a decreased activity at 100 mM (Pillet et al., 2000). Cadmium chloride is another highly toxic heavy metal known to modulate many immune functions in several species (Bernier et al., 1995). Our results showed that cadmium could also result in immunotoxic changes in bottlenose dolphin leukocytes, inducing apoptosis and affecting lymphocyte function and phagocytosis, like mercury but at higher concentrations. We can observe that cadmium at 10 ppm (90 mM) can induce apoptosis in bottlenose dolphin leukocytes and at 20 ppm (178 mM) and 40 ppm (356 mM) necrotic cells can also be detected. These data suggest that in vitro exposure to cadmium induces both pathways of cell death in bottlenose dolphin. This effect is consistent with previous studies in human lymphoid cell lines or PBMCs (Tsangaris and Tzortzatou-Stathopoulou, 1998; Hemdan et al., 2006) which indicate that cadmium has the ability to reduce cell vitality at low doses and is toxic at higher doses. Immunomodulatory activities of cadmium have also been investigated in several in vitro and in vivo model systems, but conflicting results have emerged. For example, in human cells, mitogenic activity was not significantly affected at 5 mM (Borella and Giardino, 1991). Other studies have documented that cadmium is not only toxic but may modulate immune responses (Krocova et al., 2000; Lawrence and McCabe, 2002). Our results showed a reduced activity of lymphocytes in
195
a dose-dependent manner from 10 ppm (90 mM) of cadmium. The lack of reduction of cell viability at this concentration indicates a direct effect on lymphocyte function. Similar studies in other species showed similar effects on proliferation, such as Con Astimulated beluga whale splenocytes or bovine lymphocytes that was reduced at concentrations of 10 mM (De Guise et al., 1996b, 2000), or mice splenocytes, at 20 ppm (Krocova et al., 2000). Other species seem to be more sensitive, like rat whose splenocytes were affected at 5 mM (Snyder and Valle, 1991). Phagocytic activity was reduced after cadmium exposure at 10 ppm. Other species such as harbour seal (Phoca vitulina) showed reduced phagocytosis in the same range (Pillet et al., 2000). Bovine leukocytes seem to be more sensitive because their phagocytic activity was decreased at 0.1–1 mM (De Guise et al., 2000). Lead is also a heavy metal that can alter the immune system, but in bottlenose dolphins only the lymphocytes activity is slightly affected. Previous investigations in different species showed conflicting results. Some studies described a lack of effect in viability or proliferation (Smith and Lawrence, 1988; Borella et al., 1990; De Guise et al., 1996b, 2000) whereas others have observed inhibitory or stimulatory effects of lead on immune cells. For example, on rat splenocytes or in human PBMCs, a significant inhibitory effect was described (Exon et al., 1985; Hemdan et al., 2005). But enhanced B and T cell proliferation (Lawrence, 1981; Warner and Lawrence, 1986) was observed in mice immune cells. Some authors have found that the effect on T cell proliferation depends on different subpopulations, so Pb can suppress Th1 response, but enhances Th2 clonal responses (McCabe and Lawrence, 1991; Heo et al., 1998; Chen et al., 1999; Shen et al., 2001). In bottlenose dolphins, there are no studies allowing to differentiate these responses, thus further investigations are needed. Our data showed that lead did not affect viability of bottlenose dolphin immune cells and we cannot observe any effect on apoptosis, similar to studies in other species (Shen et al., 2001). Neither was phagocytosis significantly affected by exposure to Pb in our study, similar to what has been described in other animals (De Guise et al., 2000). Aluminium is a metal present in the marine environment but few studies have evaluated its effect on the immune system. Only some authors (Gomez et al., 1986; Golub et al., 1993) have found changes, after oral exposure, in spleen or in cytokine production in laboratory animals. Our results showed that only phagocytosis is affected with a significant reduction, but at higher concentrations (50 ppm) than mercury and
196
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
cadmium. To our knowledge, similar studies of in vitro exposure to aluminium are not available. Chromium is a heavy metal found in the environment in different forms and is an essential nutrient required to promote the action of insulin for the utilization of sugars, proteins and fats. But some authors have observed various toxic reactions that affect the immune system, even though the mechanism of chromium-induced toxicity is not entirely understood (Borella et al., 1990; Chang et al., 1996; Lee et al., 2000; Shrivastava et al., 2002). But in bottlenose dolphin blood mononuclear cells we cannot detect any effect on any immune assay at the concentrations tested. In summary, an in vitro approach has been developed to test the effects of five heavy metals on the immune response of peripheral blood leukocytes from bottlenose dolphins. Using these assays we can confirm that mercury and cadmium are the most toxic metals for bottlenose dolphin leukocytes, followed by lead and aluminium, whereas chromium does not show any alteration. The immunotoxic concentrations reported in this study are within the ranges measured in some tissues of stranded bottlenose dolphins (Meador et al., 1999; Frodello et al., 2000; Parsons and Chan, 2001; Law et al., 2003; Carballo et al., 2004). The reduction of some functional activities of the bottlenose dolphin immune system may cause a significant weakness capable of altering host resistance to disease in freeranging cetaceans, as it has been suggested in other marine mammal species exposed to different pollutants (Siebert et al., 1999; Bennett et al., 2001). Our results provide a basis for explaining more complex processes which affect the health of those animals subjected to a marine environment in which the concentration of contaminants is such that it can provoke a loss of immunological resistance when faced with determined infectious agents present in the milieu. Acknowledgements The authors would like to thank Prof. A. Ferna´ndez from the University of Las Palmas de Gran Canaria, the staff of the aquariums at Zoo Aquarium Madrid, Zoo Barcelona, L’Oceanogra`fic and Marineland for providing blood samples from bottlenose dolphins; and Amalia, Juanjo, Carmen and G. Tejerizo for their collaboration. The present study was supported by the RTA2006-00168-00-00 GAN. References Ahmed, S.A., Gogal Jr., R.M., Walsh, J.E., 1994. A new rapid and simple non-radioactive assay to monitor and determine the
proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J. Immunol. Methods 170, 211–224. Beineke, A., Siebert, U., van Elk, N., Baumgartner, W., 2004. Development of a lymphocyte-transformation-assay for peripheral blood lymphocytes of the harbor porpoise and detection of cytokines using the reverse-transcription polymerase chain reaction. Vet. Immunol. Immunopathol. 98, 59–68. Bennett, P.M., Jepson, P.D., Law, R.J., Jones, B.R., Kuiken, T., Baker, J.R., Rogan, E., Kirkwood, J.K., 2001. Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environ. Pollut. 112, 33–40. Bernier, J., Brousseau, P., Krzystyniak, K., Tryphonas, H., Fournier, M., 1995. Immunotoxicity of heavy metals in relation to Great Lakes. Environ. Health Perspect. 9 (103 Suppl.), 23–34. Black, R.D., McVey, D.S., Oehme, F.W., 1992. Immunotoxicity in the bovine animal: a review. Vet. Hum. Toxicol. 34, 438–442. Borella, P., Giardino, A., 1991. Lead and cadmium at very low doses affect in vitro immune response of human lymphocytes. Environ. Res. 55, 165–177. Borella, P., Manni, S., Giardino, A., 1990. Cadmium, nickel, chromium and lead accumulate in human lymphocytes and interfere with PHA-induced proliferation. J. Trace Elem. Electrolytes Health Dis. 4, 87–95. Carballo, M., De la Pen˜a, E., Ferna´ndez, A., Aguayo, S., Espero´n, F., De la Torre, A., Mun˜oz, M.J., 2004. Accumulation of persistent organic chemicals and metals in stranded bottlenose dolphins (Tursiops truncatus) in Canary Islands (Spain). Toxicol. Appl. Pharmacol. 197, 235. Cardellicchio, N., Giandomenico, S., Ragone, P., Di Leo, A., 2000. Tissue distribution of metals in striped dolphins (Stenella coeruleoalba) from the Apulian coasts, southern Italy. Mar. Environ. Res. 49, 55–66. Cardellicchio, N., Decataldo, A., Di Leo, A., Giandomenico, S., 2002. Trace elements in organs and tissues of striped dolphins (Stenella coeruleoalba) from the Mediterranean Sea (Southern Italy). Chemosphere 49, 85–90. Chang, X., Mallard, B.A., Mowat, D.N., 1996. Effects of chromium on health status, blood neutrophil phagocytosis and in vitro lymphocyte blastogenesis of dairy cows. Vet. Immunol. Immunopathol. 52, 37–52. Chen, S., Golemboski, K.A., Sanders, F.S., Dietert, R.R., 1999. Persistent effect of in utero meso-2.3-dimercaptosuccinic acid (DMSA) on immune function and lead-induced immunotoxicity. Toxicology 132, 67–79. Culik, B.M., 2004. Review of small cetaceans. distribution, behaviour, migration and threats. UNEP/CMS p. 343. Das, K., Debacker, V., Pillet, S., Bouquegneau, J.M., 2003. Heavy metals in marine mammals. In: Vos, J.G., Bossart, G.D., Fournier, M., O’Shea, T.J. (Eds.), Toxicology of Marine Mammals. Taylor and Francis, London, pp. 135–167. De Fries, R., Mitsuhashi, M., 1995. Quantification of mitogen induced human lymphocyte proliferation: comparison of Alamar Blue assay to 3H-thymidine incorporation assay. J. Clin. Lab. Anal. 9, 89–95. De Guise, S., Flipo, D., Boehm, J.R., Martineau, D., Beland, P., Fournier, M., 1995. Immune functions in beluga whales (Delphinapterus leucas): evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry. Vet. Immunol. Immunopathol. 47, 351–362. De Guise, S., Bernier, J., Dufresne, M.M., Martineau, D., Beland, P., Fournier, M., 1996a. Immune functions in beluga whales (Delphinapterus leucas): evaluation of mitogen-induced blastic
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198 transformation of lymphocytes from peripheral blood, spleen and thymus. Vet. Immunol. Immunopathol. 50, 117–126. De Guise, S., Bernier, J., Martineau, D., Be´land, P., Fournier, M., 1996b. In vitro exposure of beluga whale lymphocytes to selected heavy metals. Environ. Toxicol. Chem. 15, 1357–1364. De Guise, S., Ross, P.S., Osterhaus, A.D., Martineau, D., Beland, P., Fournier, M., 1997. Immune functions in beluga whales (Delphinapterus leucas): evaluation of natural killer cell activity. Vet. Immunol. Immunopathol. 58, 345–354. De Guise, S., Bernier, J., Lapierre, P., Dufresne, M.M., Dubreuil, P., Fournier, M., 2000. Immune function of bovine leukocytes after in vitro exposure to selected heavy metals. Am. J. Vet. Res. 61, 339–344. De Swart, R.L., Kluten, R.M., Huizing, C.J., Vedder, L.J., Reijnders, P.J., Visser, I.K., UytdeHaag, F.G., Osterhaus, A.D., 1993. Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina). Vet. Immunol. Immunopathol. 37, 217–230. Descotes, J., 1988. Clinical immunotoxicology. J. Toxicol. Clin. Toxicol. 26, vii–viii. Exon, J.H., Talcott, P.A., Koller, L.D., 1985. Effect of lead, polychlorinated biphenyls, and cyclophosphamide on rat natural killer cells, interleukin 2, and antibody synthesis. Fundam. Appl. Toxicol. 5, 158–164. Frodello, J.P., Romeo, M., Viale, D., 2000. Distribution of mercury in the organs and tissues of five toothed-whale species of the Mediterranean. Environ. Pollut. 108, 447–452. Golub, M.S., Takeuchi, P.T., Gershwin, M.E., Yoshida, S.H., 1993. Influence of dietary aluminium on cytokine production by mitogen-stimulated spleen cells from Swiss Webster mice. Immunopharmacol. Immunotoxicol. 15, 605–619. Gomez, M., Domingo, J.L., Llobet, J.M., Tomas, J.M., Corbella, J., 1986. Short-term oral toxicity study of aluminium in rats. Arch. Farmacol. Toxicol. 12, 145–151. Guo, T.L., Miller, M.A., Shapiro, I.M., Shenker, B.J., 1998. Mercuric chloride induces apoptosis in human T lymphocytes: evidence of mitochondrial dysfunction. Toxicol. Appl. Pharmacol. 153, 250–257. Hemdan, N.Y., Emmrich, F., Adham, K., Wichmann, G., Lehmann, I., El-Massry, A., Ghoneim, H., Lehmann, J., Sack, U., 2005. Dosedependent modulation of the in vitro cytokine production of human immune competent cells by lead salts. Toxicol. Sci. 86, 75–83. Hemdan, N.Y., Emmrich, F., Sack, U., Wichmann, G., Lehmann, J., Adham, K., Lehmann, I., 2006. The in vitro immune modulation by cadmium depends on the way of cell activation. Toxicology 222, 37–45. Heo, Y., Lee, W.T., Lawrence, D.A., 1998. Differential effects of lead and cAMP on development and activities of Th1- and Th2lymphocytes. Toxicol. Sci. 43, 172–185. Kim, S.H., Sharma, R.P., 2003. Cytotoxicity of inorganic mercury in murine T and B lymphoma cell lines: involvement of reactive oxygen species, Ca(2+) homeostasis, and cytokine gene expression. Toxicol. In Vitro 17, 385–395. Koller, L.D., 1979. Effects of environmental contaminants on the immune system. Adv. Vet. Sci. Comp. Med. 23, 267–295. Krocova, Z., Macela, A., Kroca, M., Hernychova, L., 2000. The immunomodulatory effect(s) of lead and cadmium on the cells of immune system in vitro. Toxicol. In Vitro 14, 33–40. Lalancette, A., Morin, Y., Measures, L., Fournier, M., 2003. Contrasting changes of sensitivity by lymphocytes and neutrophils to mercury in developing grey seals. Dev. Comp. Immunol. 27, 735–747.
197
Law, R.J., Fileman, C.F., Hopkins, A.D., Baker, J.R., Harwood, J., Jackson, D.B., Kennedy, S., Martin, A.R., Morris, R.J., 1991. Concentrations of trace metals in the livers of marine mammals (seals, porpoises and dolphins) from waters around the British Isles. Mar. Pollut. Bull. 22, 183–191. Law, R.J., Morris, R.J., Allchin, C.R., Jones, B.R., Nicholson, M.D., 2003. Metals and organochlorines in small cetaceans stranded on the east coast of Australia. Mar. Pollut. Bull. 46, 1206–1211. Lawrence, D.A., 1981. In vivo and in vitro effects of lead on humoral and cell-mediated immunity. Infect. Immun. 31, 136–143. Lawrence, D.A., McCabe Jr., M.J., 2002. Immunomodulation by metals. Int. Immunopharmacol. 2, 293–302. Lee, D.N., Yen, H.T., Shen, T.F., Chen, B.J., 2000. Chromium-induced glucose uptake, superoxide anion production, and phagocytosis in cultured pulmonary alveolar macrophages of weanling pigs. Biol. Trace Elem. Res. 77, 53–64. Levin, M., Morsey, B., Mori, C., Guise, S., 2004. Specific noncoplanar PCB-mediated modulation of bottlenose dolphin and beluga whale phagocytosis upon in vitro exposure. J. Toxicol. Environ. Health A. 67, 1517–1535. McCabe Jr., M.J., Lawrence, D.A., 1991. Lead, a major environmental pollutant, is immunomodulatory by its differential effects on CD4+ T cells subsets. Toxicol. Appl. Pharmacol. 111, 13–23. Meador, J.P., Ernest, D., Hohn, A.A., Tilbury, K., Gorzelany, J., Worthy, G., Stein, J.E., 1999. Comparison of elements in bottlenose dolphins stranded on the beaches of Texas and Florida in the Gulf of Mexico over a one-year period. Arch. Environ. Contam. Toxicol. 36, 87–98. Mori, C., Morsey, B., Levin, M., Nambiar, P.R., De Guise, S., 2006. Immunomodulatory effects of in vitro exposure to organochlorines on T-cell proliferation in marine mammals and mice. J. Toxicol. Environ. Health A. 69, 283–302. Nakata, H., Sakakibara, A., Kanoh, M., Kudo, S., Watanabe, H., Nagai, N., Miyazaki, N., Asano, Y., Tanabe, S., 2002. Evaluation of mitogen-induced responses in marine mammal and human lymphocytes by in-vitro exposure of butyltins and non-ortho coplanar PCBs. Environ. Pollut. 120, 245–253. Noda, K., Aoki, M., Akiyoshi, H., Asaki, H., Shimada, T., Ohashi, F., 2003. Evaluation of the polymorphonuclear cell functions of bottlenose dolphins. J. Vet. Med. Sci. 65, 727–729. Parsons, E.C., Chan, H.M., 2001. Organochlorine and trace element contamination in bottlenose dolphins (Tursiops truncatus) from the South China Sea. Mar. Pollut. Bull. 42, 780–786. Pillet, S., Lesage, V., Hammill, M., Cyr, D., Bouqueneau, J.M., Fournier, M., 2000. Effects of mercury and zinc on phagocytic activity of peripheral blood leukocytes of two species of seals from the St Lawrence Estuary. Mar. Pollut. Bull. 40, 921–927. Raszyk, J., Toman, M., Gajduskova, V., Nezveda, K., Ulrich, R., Jarosova, A., Docekalova, H., Salava, J., Palac, J., 1997. Effects of environmental pollutants on the porcine and bovine immune systems. Vet. Med. (Praha) 42, 313–317. Roditi-Elasar, M., Kerem, D., Hornung, H., Kress, N., Shoham-Frider, E., Goffman, O., Spanier, E., 2003. Heavy metal levels in bottlenose and striped dolphins off the Mediterranean coast of Israel. Mar. Pollut. Bull. 46, 503–512. Romano, T.A., Ridgway, S.H., Quaranta, V., 1992. MHC class II molecules and immunoglobulins on peripheral blood lymphocytes of the bottlenosed dolphin Tursiops truncatus. J. Exp. Zool. 263, 96–104. Shen, X., Lee, K., Konig, R., 2001. Effects of heavy metal ions on resting and antigen-activated CD4(+) T cells. Toxicology 169, 67–80.
198
S. Ca´mara Pellisso´ et al. / Veterinary Immunology and Immunopathology 121 (2008) 189–198
Shenker, B.J., Guo, T.L., Shapiro, I.M., 2000. Mercury-induced apoptosis in human lymphoid cells: evidence that the apoptotic pathway is mercurial species dependent. Environ. Res. 84, 89–99. Shrivastava, R., Upreti, R.K., Seth, P.K., Chaturvedi, U.C., 2002. Effects of chromium on the immune system. FEMS Immunol. Med. Microbiol. 34, 1–7. Siebert, U., Joiris, C., Holsbeek, L., Benke, H., Failing, K., Frese, K., Petzinger, E., 1999. Potential relation between mercury concentrations and necropsy findings in cetaceans from German waters of North and Baltic Seas. Mar. Pollut. Bull. 38, 285–295. Smith, K.L., Lawrence, D.A., 1988. Immunomodulation of in vitro antigen presentation by cations. Toxicol. Appl. Pharmacol. 96, 476–484. Snyder, C.A., Valle, C.D., 1991. Lymphocyte proliferation assays as potential biomarkers for toxicant exposures. J. Toxicol. Environ. Health. 34, 127–139. Stewart, C.C., Lehnert, B.E., Steinkamp, J.A., 1986. In vitro and in vivo measurement of phagocytosis by flow cytometry. Methods Enzymol. 132, 183–192.
Synzynys, B.I., Sharetskii, A.N., Kharlamova, O.V., 2004. Immunotoxicity of aluminum chloride. Gig. Sanit. 70–72 (in Russian, with English abstract). Tsangaris, G.T., Tzortzatou-Stathopoulou, F., 1998. Cadmium induces apoptosis differentially on immune system cell lines. Toxicology 128, 143–150. Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C., 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51. Warner, G.L., Lawrence, D.A., 1986. Stimulation of murine lymphocyte responses by cations. Cell. Immunol. 101, 425–439. Watters, J.W., Kraja, A., Meucci, M.A., Province, M.A., McLeod, H.L., 2004. Genome-wide discovery of loci influencing chemotherapy cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 101, 11809–11814. Wells, R.S., Rhinehart, H.L., Hansen, L.J., Sweeney, J.C., Townsend, F.I., Stone, R., Casper, D.R., Scott, M.D., Hohn, A.A., Rowles, T.K., 2004. Bottlenose dolphins as marine ecosystem sentinels: developing a health monitoring system. EcoHealth 1, 246–254.