ToxSci Advance Access originally published online on May 4, 2006
Toxicological Sciences 2006 92(2):423-432; doi:10.1093/toxsci/kfl005
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Immunomodulatory Effects of Estradiol and Cadmium in Adult Female Rats
* INRS—Institut Armand-Frappier, Université du Québec, Pointe-Claire, Montréal, Quebec, Canada H9R 1G6; and Department of Oceanology, Université de Liège, B-4000 Liège, Belgium
1 To whom correspondence should be addressed at INRS—Institut Armand-Frappier, Université du Québec, 245 Hymus Boulevard, Pointe-Claire, Montréal, Quebec, Canada H9R 1G6. Fax: +1 514 630 8850. E-mail: daniel.cyr{at}iaf.inrs.ca.
Received February 10, 2006; accepted April 25, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A wide range of toxic effects has been associated with cadmium (Cd) exposure in mammals. However, the physiological factors that modulate these effects have received limited attention. We have previously demonstrated that neonatal exposure of rats to Cd during lactation results in sex-specific immunotoxic effects in both juvenile and adult rats. The objectives of this study were to determine the effects of 17ß-estradiol (E2) on the immunotoxicity of Cd in female rats. We compared the effects of 28 days of exposure to 0, 5, and 25 ppm cadmium chloride (CdCl2) through drinking water on ovariectomized Sprague-Dawley rats and on ovariectomized rats with E2 implant which mimicked the physiological level of E2 in female rat. Our results clarify the control of important immune functions by E2 at physiological level and demonstrate significant interactions between Cd and E2 effects on the cytotoxic activity of natural killer cells and phagocytosis of splenic cells as well as on the total number of thymocytes and of the four subpopulations of the thymocytes as defined by the expression of the cell-surface markers CD4 and CD8. Cd and E2 share several mechanisms of action that may account for these interactions. The estrogenic potential of Cd could also account for some of the observed effects. These interactions have to be taken into consideration in evaluating the risk of Cd immunotoxicity and the possible interactions with hormonal treatments.
Key Words: cadmium; estradiol; immunotoxic effects; ovariectomized rats.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cadmium (Cd) exposure has been associated with a wide range of toxic effects including nephrotoxicity, hepatotoxicity, alterations in bone formation, as well as effects on male reproductive physiology and immune system (Koller, 1998
; Nordberg, 1996; Waalkes et al., 1999). Cd concentrations in food normally range from 2 to 100 ppb and can reach levels that exceed 1 ppm in cigarette smoke. The daily intake of Cd is estimate to be approximately 10–50 µg but can reach levels of 200–1000 µg in highly contaminated areas (Nordberg, 1996). It has been reported that 90% of the patients with Itai-Itai disease, the most severe form of chronic Cd intoxication in humans, are postmenopausal women (Jarup et al., 1998). This suggests that estrogen depletion may play an important role in the development of this pathology. Cd exposure alters calcium homeostasis and bone decalcification caused by hormone depletion during menopause or following ovariectomy. Ovariectomy has also been reported to enhance Cd nephrotoxicity and hepatotoxicity following acute Cd exposure in Sprague-Dawley rats (Katsuta et al., 1993, 1994).
Cd can act as an endocrine disruptor by altering the homeostatic balance of a variety of hormones. Circulating concentrations of pituitary hormones, such as prolactin, adrenocorticotropin hormone, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone, were altered in male rats exposed to Cd (Lafuente et al., 2003). Acute exposure to Cd can also decrease progesterone production in female rats, and this effect is dependent on the stage of the estrus cycle at the time of exposure (Piasek and Laskey, 1994, 1999). More recently, it has been reported that Cd may have estrogenic properties. 17ß-Estradiol (E2) mediates its effects via two nuclear estrogen receptor (ER) isoforms, ER
and ERß (Hall et al., 2001). Cd has been reported to bind and activate ER in vitro and in vivo can mimic the effects of E2 on the uterus and mammary gland (Johnson et al., 2003; Stoica et al., 2000).
Hormones are known to influence the immune system, and a strong sexual dimorphism exists in the immune response. Female steroids such as E2 are important factors responsible for gender differences of immune system in mammals. E2 affects all aspects of the immune response: the production, differentiation, and maturation of immune cells as well as the nonspecific immune response, the humoral- and cell-mediated immune responses (Ansar Ahmed et al., 1985; Grossman, 1984, 1985, 1989; Medina et al., 2000). ERs are differentially expressed according to immune cell type, and the relative expression ratio of ER to ERß may be one of the factors determining the cell type–specific effects of E2 on immune cells (Phiel et al., 2005). The immunotoxic effects of Cd have been reported on the development of immune organs, on the differentiation of immune cells, and on specific and nonspecific immune responses (reviewed by Descotes, 1988, 1992; Koller, 1998). This indicates an impact on the immune system as a whole, which can lead to a significant decrease in host resistance. However, the variability of experimental results has revealed that the immunotoxicity of Cd not only depends on the speciation of the metal employed, the route, dose, and duration of exposure but also on the physiological status of the animal. This pointed to the need for environmentally relevant models to better assess the risk of Cd exposure upon the immune system and to address the impacts of physiological factors which may modulate immunotoxic effects. In a previous study, we investigated the effects of exposure of neonatal Sprague-Dawley rats to environmentally relevant doses of Cd via maternal milk. This neonatal exposure resulted in decreased body, kidney, and spleen weights of just-weaned females but not males (Pillet et al., 2005). These effects did not persist to adulthood. However, we have demonstrated sex-specific immunotoxic effects of neonatal exposure to these low levels of Cd, specifically on the cytotoxic activity of splenic natural killer (NK) cells of both juvenile and adult rats (Pillet et al., 2005).
Considering the estrogenic potential of Cd, the previously observed gender effects of neonatal exposures to low levels of Cd, and the role of estrogens in the endocrine regulation of the immune response, the present objective was to investigate the role of E2 on the immunotoxic effects of Cd in the rat.
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MATERIALS AND METHODS |
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Animals.
Ovariectomized (OV) rats and sham-operated (sham) intact female Sprague-Dawley rats (8 week old; Charles River Canada, St Constant, Quebec) were housed in a pathogen-free animal facility and cared for in compliance with the guidelines of the Canadian Council for Animal Care. Rats were weighed weekly and maintained under a constant photoperiod of 12:12 h light:dark. They received food and water ad libitum. All animal procedures used in this study were approved by the university animal care committee.
Experimental protocol and sample collection.
In order to determine the role of E2 on Cd immunotoxicity, seven experimental groups of animals were used. Half the ovariectomized rats were given a Silastic implant of E2 (OVE) while the remaining OV and sham rats received an empty implant. The day after surgery, OV and OVE rats were exposed to 0, 5, or 25 ppm CdCl2 in their drinking water for 28 days. Sham controls received water alone. The experimental groups were therefore, sham, OV, OV exposed to 5 ppm Cd, OV exposed to 25 ppm Cd, OVE, OVE exposed to 5 ppm Cd, OVE exposed to 25 ppm Cd.
At the end of the exposure period, the rats were euthanized by exsanguination followed by rupture of the diaphragm. Thymus, spleen, uterus, liver, and kidneys were removed aseptically and weighed. The kidneys were immediately frozen in liquid nitrogen and stored at – 80°C.
Cell suspensions from the spleen and thymus were obtained by passing the tissues through a stainless steel mesh in HBSS medium (pH 7.3; GIBCO, Mississauga, Ontario) supplemented with HEPES (25mM; GIBCO), penicillin (100 IU; Bio Media, Mississauga, Ontario), and streptomycin (100 µg/ml; Bio Media). Debris were removed by passing the cell suspension through nylon wool. Cells were washed twice in HBSS, and viability was determined by trypan blue exclusion. Splenocytes were then resuspended in RPMI medium (GIBCO) supplemented with heat-inactivated fetal bovine serum (10%; Bio Media), penicillin (100 IU), and streptomycin (100 µg/ml) (complete RPMI).
E2 administration.
E2 implants were prepared as previously described by Green et al. (1999) to produce physiological levels of E2 in female rats (Bridges, 1984; Bridges and Russell, 1981). Briefly, 16-mm segments of Silastic tubing (Dow Corning, Midland, MI) were filled with E2 (Sigma-Aldrich Chemicals, Oakville, Ontario). The ends of the implants were sealed with 3 mm of medical silicone type A (Dow Corning). Implants were washed for 2.5 days at 37°C in phosphate buffer (pH 7.4) supplemented with 1% bovine serum albumin (fraction V, ICN Biomedicals, Toronto, Ontario) to ensure that the newly made capsules had a constant E2 release rate. The implants were placed sc on the back of anesthetized rats (Isoflurane, Abbott Laboratories, Saint-Laurent, Quebec).
Cd analysis.
Kidneys were freeze dried for 36 h and digested with a mixed solution of hydrochloric and nitric acids (1:3, vol/vol) and slowly heated to 100°C until the digestion was complete. Atomic absorption spectrophotometry (ARL 3510) was used to determine Cd concentrations. A set of certified material samples (DORM-2, National Research Council, Institute for National Measurement Standards, Ontario, Canada) spiked with gradual concentrations of Cd was analyzed in parallel to ensure the accuracy of the method. Recoveries ranged from 98 to 102%.
Proliferative response of splenocytes.
An aliquot of splenocytes (5 x 105 cells) was cultured in triplicate in polystyrene flat-bottom, 96-well culture plates (Costar, Corning, NY) in complete RPMI supplemented with dextran (10 µg/ml; Sigma-Aldrich Chemicals) alone or with one of the three mitogens: concanavalin A (Con A) (5 µg/ml; Sigma-Aldrich Chemicals), phytohemagglutinin (PHA) (40 µg/ml; Sigma-Aldrich Chemicals), or lipopolysaccharide (LPS) (25 µg/ml; Sigma-Aldrich Chemicals). After 72 h at 37°C in a humidified chamber with 5% CO2, 0.5 µCi of 3H-TdR (1 mCi/ml, ICN Biomedicals, Costa Mesa, CA) was added to each well, and the cells were incubated for an additional 18 h. DNA from the cells was then collected on glass wool filters using a semiautomated cell harvester (Skatron Instruments, Sterling, VA) and the incorporated radioactivity measured using a liquid scintillation counter. Mean disintegrations per minute (DPM) for triplicate cultures were determined. The data were then expressed as a stimulation index, i.e., the ratio of the mean DPM of mitogen-stimulated cells to the mean DPM of unstimulated cells.
Cytotoxic activity of splenic NK cells.
The cytotoxic activity of splenic NK cells was assessed by measuring their ability to kill mice tumor cell line YAC-1 (American Type Culture Collection, Manassas, VA) according to the method of Brousseau et al. (1999). Briefly, YAC-1 cells were stained with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO; Sigma-Aldrich Chemicals) 12–18 h prior to use. Splenocytes and YAC-1 cells were combined at ratios of 20:1 and 80:1 in complete RPMI. A stained YAC-1 cell suspension was used to account for spontaneous mortality. Cultures were then centrifuged at 300 x g for 5 min at 20°C. After a 1-h incubation at 37°C, propidium iodide (PI; Sigma-Aldrich Chemicals) was added in order to obtain a final concentration of 50 µg/ml. The cells were then analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Using the CellQuest Pro software (Becton Dickinson), it was possible to discriminate between DiO-positive (DiO+) YAC-1 cells and DiO-negative (DiO–) splenocytes. Based on this distinction, the acquisition consisted of a minimum of 5000 DiO+ gated events. PI enters all cells but is actively excreted by living cells. PI-positive (PI+) cells thus represented dead cells. Therefore, double-positive (DP) PI+ and DiO+ cells were considered dead YAC-1 cells. The proportion of dead YAC-1 cells represented the cytotoxic activity of NK cells.
Ratio of active phagocytes in spleen.
The ratio of active phagocytes was determined by flow cytometry based on the protocol of Brousseau et al. (1999). Briefly, fluorescent latex beads (Molecular Probes Inc., Eugene, OR) were added to splenocytes at a ratio of 100 beads per cell. After a 1-h incubation at 37°C with agitation, cells were analyzed using a FACScan flow cytometer. The CellQuest Pro software allowed us to discriminate between cells that did not contain any beads, one bead, and cells that contained more than two beads. Results were expressed as the percentage of total splenocytes that engulfed more than two beads, which are considered as active phagocytes.
Phenotyping of thymocytes.
Thymocytes (2.5 x 105 cells) were incubated for 10 min on ice with human IgG (Cedarlane, Hornby, Ontario) at a final concentration of 50 µg/ml. Anti-rat PE-conjugated anti-CD4 and CY5-conjugated anti-CD8 (Cedarlane) were added at final concentrations of 0.5 µg/ml and 1 µg/ml, respectively. After a 30-min incubation on ice, thymocytes were centrifuged at 410 x g for 5 min at 4°C and washed three times in ice-cold HBSS. Cells were then analyzed using a FACScan flow cytometer and the CellQuest Pro software to discriminate between apoptotic and viable cells. The fluorescence of viable cells was measured by flow cytometry to determine cell populations using the CellQuest Pro software.
Statistical analysis.
Statistical analysis was performed using the STATISTICA (1998 edition, Statsoft, Tulsa, OK) and SigmaStat (version 2.0, Jandel Corporation, San Rafael, CA) software. Data were first tested for normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene test). When required, values were normalized by logarithmic transformation in order to allow for parametric tests. The effects of ovariectomy and E2 replacement as compared to sham controls were assessed by a one-way ANOVA. The effects of E2 and Cd exposure as well as the occurrence of interaction between these two factors were determined by a two-way ANOVA. When significant effects were pointed out, the Tukey a posteriori test was used to determine significant differences between groups. Significance was established at p < 0.05.
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RESULTS |
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Body and Organ Weights
Ovariectomy resulted in an 8% increase in body weight. Prior to receiving the E2 implant, ovariectomized rats were significantly heavier than sham controls (data not shown). However, 5 days after receiving the E2 implant, the differences in body weight between sham and OVE rats disappeared. The body weights of OV rats remained higher than OVE rats and sham controls (data not shown). This dimorphism in body weight was maintained until the end of the experiment. Cd exposure has no effect on the body weight.
Ovariectomy inhibited uterus growth and thymic regression. E2 implants restored normal uterine growth as well as thymic regression (Table 1). With the exception of kidneys, Cd exposure had no effect on organ weights of OV and OVE rats. Cd exposure increased renal weights independently of E2 administration (Table 1).
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Cd Accumulation in Kidneys
Renal Cd levels were used as an indicator of Cd exposure. Rats not exposed to CdCl2 had Cd levels that either were not detectable or were between the detection and quantification limits (0.10–0.5 µg/g dry weight, respectively), while kidneys from both OV and OVE females exposed to 25 ppm Cd contained three to four times more Cd than those of females exposed to 5 ppm Cd (Table 2). This illustrates the well-documented relationship between Cd exposure and its accumulation in the kidney (Cosma et al., 1991
; Hiratsuka et al., 1999; Nordberg, 1996; Thomas et al., 1985). We did not observe any effects of E2 on renal Cd accumulation.
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Proliferative Response of Splenocytes
Ovariectomy significantly increased the proliferation of splenocytes stimulated by LPS. E2 replacement in OVE rats restored the proliferative response of LPS-stimulated cells to the values observed in sham controls (Table 3). A similar trend was observed with PHA-stimulated splenocytes. These effects appeared significant when comparing OV and OVE rats to assess the combined effects of E2 and Cd exposure, while no effect was observed in Con A–stimulated cells. Cd exposure did not exert any effect on the proliferation response of splenocytes stimulated by Con A, LPS, or PHA in either OV or OVE rats (Table 3).
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Cytotoxic Activity of Splenic NK Cells
The cytotoxic activity of NK cells, as measured by the percentage of dead target cells (YAC-1), decreased as the ratio of splenocytes to YAC-1 cells decreased (data not shown), indicating a normal response pattern (Brousseau et al., 1999
). The cytotoxic activity patterns from both ratios were parallel. Accordingly, the 80:1 cell dilution was used to assess the effects of E2 and Cd. There were no differences in the cytotoxic activity of splenic NK cells between sham, OV, or OVE rats (Fig. 1A).
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A significant interaction was noted between the Cd and E2 effects (Fig. 1B). In the absence of E2, the cytotoxic activity of splenic NK cells was not affected by Cd exposure. In contrast, exposure to 25 ppm Cd significantly increased NK activity in OVE rats. Thus, a significant difference between the NK cell cytotoxic activity of OV and OVE females exposed to 25 ppm Cd was observed (Fig. 1B).
Phagocytic Activity of Splenocytes
Ovariectomy induced a slight but not significant reduction in the phagocytic activity of splenocytes as compared to sham controls. However, phagocytosis by cells from OVE females was significantly higher than those of the OV group (Fig. 2A).
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A significant interaction between the Cd and E2 effects was noted, which confirmed the difference between OV and OVE rats and revealed that exposure to 5 and 25 ppm Cd induced an increase in phagocytic activity of splenocytes in OV rats (Fig. 2B). In contrast, phagocytic activity of OVE rats exposed to Cd did not differ from nonexposed OVE rats (Fig. 2B). However, cells from OVE rats exposed to 25 ppm Cd had higher phagocytic activity than cells from OVE rats exposed to 5 ppm Cd (Fig. 2B).
Total Number and Phenotyping of Thymocytes
Ovariectomy prevented steroid hormone–induced thymic regression. As predicted from the literature, an increase in the total number of thymocytes in OV rats as compared to sham controls was observed (Fig. 3A). E2 administration restored the involution process of the thymus. However, while the weight of the thymus in OVE rats was similar to those of sham controls (Table 1), the number of cells was significantly lower (Fig. 3A).
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A significant interaction between the Cd and E2 effects was noted which confirmed the difference in cell number between OV and OVE rats. Although Cd exposure reduced the number of thymocytes in both OV and OVE females, this effect was significant only in OVE rats exposed to 25 ppm Cd (Fig. 3B).
The effects of ovariectomy and E2 administration on cell numbers affected all thymocyte subpopulations, as defined by the differential expression of the cell-surface markers CD4 and CD8 (Fig. 4, upper panel). These effects were significant for both the DP CD4+CD8+ and double-negative (DN) CD4–CD8– cells. The number of CD4+CD8– cells, precursors of peripheral T-helper lymphocytes (Th), in the thymus of OV rats was significantly higher than that in OVE rats but was not significantly different from sham controls (Fig. 4, upper panel).
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We also noted a significant interaction between the effects of Cd and E2 on the number of DP and Th precursors (Fig. 4, lower panel, B and C). The effects on DP cells were identical to those observed on total thymocytes, reflecting their relative abundance in this organ (Fig. 4, lower panel, B). Exposure to 5 ppm Cd significantly decreased the number of Th precursors in OV rats, while no effects were observed on OVE rats (Fig. 4, lower panel, C). Although not significant, we observed a similar pattern in the other thymocyte subpopulations, for which only the E2 effect was significant (Fig. 4, lower panel, A and D). There were no effects of Cd exposure on the relative proportions of thymocyte subpopulations. However, E2 treatment induced an enrichment of CD4+CD8– cells as compared to OV rats (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The endocrine regulation of immune functions is well established. The actions of E2 on the immune system are complex and differ according to its concentration and targeted functions. In the present study, there was an increase in LPS-induced proliferation of spleen lymphocytes in OV rats, while E2 decreased the proliferation of stimulated cells in sham controls and OVE rats. The higher proliferation of splenocytes stimulated by LPS in all the OV rats (regardless of the Cd exposure) as compared to all OVE rats confirmed this effect of E2. LPS stimulates B-cell proliferation, while Con A and PHA stimulate the proliferation of different T-cell subpopulations. In mice, E2 downregulates B-cell proliferation and lymphopoiesis (Hoffman-Goetz, 1999
; Medina et al., 2000). A reduction in B-cell lymphopoiesis in bone marrow and the resulting impact on the B-cell precursor migration to the spleen as described by Medina et al. (2000) may explain the lower spleen weights observed in our study in sham and OVE as compared to OV rats (data not show). We observed similar effects with splenocytes stimulated by PHA. Although previous studies had indicated that the modulation of lymphocyte proliferation by sexual hormones depended on the mitogen (and hence, the lymphocyte subpopulations), the status of the cell (mature, active, etc.), as well as the sex and the species of the animal, E2 essentially appeared to downregulate both T- and B-cell lymphopoiesis, as observed in this study (Bilbo and Nelson, 2001; Grossman, 1985; Hoffman-Goetz, 1999; McKenzie and Berczi, 1987). The absence of Cd effects on this process supports studies that have demonstrated the need for longer exposure periods and/or higher doses of Cd in order to induce immunotoxic effect on lymphocyte proliferation in rodents (Blakley, 1985; Malave and De Ruffino, 1984; Stacey et al., 1988; Thomas et al., 1985).
In the present study, ovariectomy did not affect the cytotoxic activity of splenic NK cells, as previously reported by Seaman et al. (1978). The implants used in our study were designed to produce physiological circulating levels of E2 (Bridges, 1984; Bridges and Russell, 1981). The similarity between the uterus weight of OVE and sham rats confirms the physiological level of E2 delivered by our implants as the uterus development is strongly regulated by E2 (Tsai et al., 1998). While supraphysiological levels of E2 have been reported to decrease NK activity in both OV and intact mice, the physiological level of hormone release by our implant did not affect NK activity (Nilsson and Carlsten, 1994; Seaman et al., 1978).
Our results indicated significant interactions between Cd and E2 effects on the cytotoxic activity of splenic NK cells. Cd had no effect in OV rats, while OVE females exposed to 25 ppm Cd had increased NK activity. Stacey et al. (1988) reported that there were no effects on NK activity in male rats exposed for 6 weeks to 5 ppm Cd in drinking water or to 2.5, 25, or 250 µg Cd/kg body weight by gavage five times per week. In contrast, Cifone et al. (1989a) observed a decrease in NK activity in female rats exposed to 200 or 400 ppm Cd in drinking water for the first month of exposure, but as the exposure continued, NK activities increased and surpassed those of controls. Our results indicated a lack of immunotoxic effects of Cd on the cytotoxic activity of splenic NK cells in the absence of E2. In the presence of E2, however, there was a significant increase in the susceptibility of NK cells to Cd stimulation.
Ovariectomy resulted in a reduction in the percentage of active phagocytes in the spleen, and OVE rats had a higher percentage of active phagocytes than OV rats. Saito et al. (1992) also observed a decreased in the percentage of phagocytes in the spleen of OV rats, and as in our experiment, this percentage increased when OV rats were administered E2. Ovariectomy had also been reported to decrease phagocytosis of rat macrophages (De Azevedo et al., 1997). In the sow, E2 induced an increase in phagocytosis of peripheral blood granulocytes (Magnusson and Einarsson, 1990). Our results on the effects of E2 on phagocytes in rats are in accordance with these studies.
There was a significant interaction between the effects of Cd and E2 on the proportion of active phagocytes in the spleen. Exposures to Cd increased the percentage of these phagocytes in OV but not in OVE rats, as compared to unexposed matched controls. We also noted that the percentage of active phagocytes in OVE rats exposed to 25 ppm Cd was significantly higher than that in OVE rats exposed to 5 ppm Cd. Interestingly, Thomas et al. (1985) reported an increase in the phagocytic activity of peritoneal macrophages of female mice exposed to 10, 50, or 250 ppm Cd in drinking water for 91 days. Our results suggest that both Cd and E2 can independently stimulate phagocytosis, but E2-induced stimulation tends to mask the toxic effects of Cd, which are more apparent in longer exposure to higher doses.
The thymus plays an important role in the development of the immune response. This organ gradually atrophies with age, mostly as a result of the increased concentrations in circulating sex steroids following the onset of puberty. Our results confirmed the impairment of thymic regression in OV rats and the reestablishment of this normal process in OVE rats, in accordance with previous studies on the role of E2 in the involution of the thymus (Safadi et al., 2000; Windmill et al., 1993). The lower number of thymocytes in OVE rats as compared to sham controls is in agreement with the studies of Leposavic et al. (2001), who also observed that OV rats exposed to E2 displayed fewer thymocytes than intact females, despite similar serum hormone concentrations. In the present study, all the thymocyte subpopulations, as defined by the expression of the cell-surface cluster domains CD4 and CD8, increased in OV rats and were lower in OVE rats which reflects the importance of E2 for thymocyte maturation and differentiation (Leposavic et al., 1996; Okasha et al., 2001; Rijhsinghani et al., 1996). The reduction of DN thymocytes in OVE rats suggests an impact on the early stages of thymocyte differentiation.
There was a significant interaction between the effects of Cd and E2 on the yield of total thymocytes as well as on the number of DP and CD4+CD8– cells. On one hand, exposure to 25 ppm Cd decreased the number of thymocytes in OVE but not in OV rats as compared to unexposed matched controls. The effect of Cd exposure on OVE females was observed on all the thymocyte subpopulations but was significant for DP cells, which represent more than 80% of thymocytes. On the other hand, exposure to 5 ppm Cd decreased the number of Th lymphocyte precursors in OV rats, although at this exposure dose, Cd had no effect on this cell subpopulation in OVE rats.
Our study demonstrated that there are interactions between E2 and immunotoxic effects of Cd. In a previous study we demonstrated that neonatal exposure to environmentally relevant levels of Cd via maternal milk produced gender-specific transitory and persistent alterations of immune functions. Cytotoxic activity of splenic NK cells was not affected when female rats were exposed to very low doses of Cd through maternal milk. In the present study, we addressed the role of E2 on the immunotoxicity of Cd. Clearly, these results indicate that the development and reproductive status as well as gender have to be taken into consideration for assessing the risk of Cd exposure.
E2 and Cd share different mechanisms of action, which could alter the different types of immune cells and their functions. Both Cd and E2 can act on common cellular pathways, leading to cell proliferation, activation, or apoptosis. For example, the levels of specific cytokines and the expression of their receptors strongly regulate proliferation, maturation, differentiation, functions, and apoptosis in immune cells. Interleukin-2 (IL-2) and its receptor (RIL-2) are involved in T lymphocyte differentiation and in activation of NK cells. The synthesis of IL-2 and RIL-2 can be affected by both E2 (Azenabor and Hoffman-Goetz, 2001; Karpuzoglu-Sahin et al., 2001) and Cd (Cifone et al., 1989b; Kayama et al., 1995; Liu et al., 1999; McMurray et al., 2001; Payette et al., 1995; Theocharis et al., 1991). Both Cd and E2 can also act on thymic epithelial cells (TECs), which are important regulators of thymocyte differentiation. TECs express elevated levels of the ERs, and E2 has been shown to affect the excretion of factors which control the differentiation of thymocytes (Sakabe et al., 1999; Savino et al., 1988; Stimson and Crilly, 1981). While the effects of Cd may be linked to its estrogenicity, some of its immunotoxic effects, as well as differences in sensitivity between rat strains, could account for differences in Cd effects on TECs (Morselt et al., 1988). While functional immunomarkers were assessed with normalized numbers of splenocytes, we cannot exclude potential cell type–specific effects of Cd on the viability of splenocyte subpopulations.
E2 mediates its effects primarily via ER and ERß. Cd has been shown to bind and activate ER, and low concentrations of Cd were able to induce estrogen-dependent gene expression (Stoica et al., 2000; Wilson et al., 2004). In vivo, Cd is able to mimic the effects of E2 on the uterus and mammary gland (Johnson et al., 2003). Interestingly, we did not observe any immunotoxic effects of Cd independent of E2, and the Cd effects appeared to enhance the E2 effects, reflecting the estrogenic potential of Cd except for the effects on NK cytotoxic activity, for which physiological levels of E2 had no effect.
In conclusion, Cd and E2 not only share certain similar cellular pathways but also can interact to alter immune functions. Furthermore, Cd can also modulate the hormonal balance and may exert estrogenic effects via interaction with ERs. This estrogenic potential of Cd may be responsible for the immunotoxic effects observed in the present study. The interdependence of Cd and E2 for eliciting effects on the immune system has not been previously reported and provides novel information for understanding the mechanisms by which Cd may modulate immune functions. These data also suggest that females may be at a greater risk than males for Cd-induced immunomodulation, and this information may be particularly useful for risk assessment.
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ACKNOWLEDGMENTS |
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J. Dufresne, M. Gregory, R. Biodon, L. Ménard, and M. Fortier are thanked for their technical assistance and helpful suggestions. This study was supported by Toxic Substances Research Initiative (Health Canada). S.P. was supported by the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture (Belgium) and M.D'E. was a recipient of an INRS—Institut Armand-Frappier studentship. M.F. is the recipient of a Canada Research Chair in Immunotoxicology.
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