Effects of Gestational and Lactational Exposure to Organochlorine Compounds on Cellular, Humoral, and Innate Immunity in Swine

Houda Bilrha*, Raynald Roy*, Éric Wagner{dagger}, Marthe Belles-Isles*, Janice L. Bailey{ddagger} and Pierre Ayotte§,1

* Unité de recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL-CHUQ, Québec, QC, Canada G1V 4G2; {dagger} Division d’Hématologie-Oncologie, Hôpital Sainte-Justine, Montréal, QC, Canada H3T 1C5; {ddagger} Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, QC, Canada G1K 7P4; and § Unité de Recherche en Santé Publique, Centre de Recherche du CHUL-CHUQ, Université Laval, Québec, QC, Canada G1V 5B3

Received June 20, 2003; accepted September 5, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Few studies have characterized the immunotoxic potential of complex mixtures of organochlorines (OCs) that bear environmental relevance. We monitored immune parameters in male piglets exposed in utero and through lactation to an OC mixture which was designed to approximate that found in the traditional diet of Arctic aboriginal populations. Prepubertal sows were administered orally either corn oil (control group) or the OC mixture in increasing doses (low, medium, and high). The sows were inseminated with the semen from an untreated boar and OC treatment was continued throughout gestation and lactation (21 days). Blood was collected from the sows at delivery and monthly from piglets until 8 months of age for the determination of plasma OC concentrations and parameters of innate, cellular, and humoral immunity. Treatment with the OC mixture had no dose-dependent effect on the proportion of CD4+ and CD8+ T-cell subsets, and did not modulate the functional activity of the complement component C2. The proportion of CD4+CD8+ cells, CD8+DR+ cells, and the mitogenic lymphoproliferative response increased in OC-treated, 4-month-old piglets. At 6 months, the lymphoproliferative response to mitogen and the proportion CD4+CD8+ cells were still elevated in the OC-treated piglets, but the proportion of CD8+DR+ cells was decreased as compared to the controls. Animals in the high-dose group also exhibited a slight increase in polymorphonuclear leukocyte phagocytic activity at 8 months of age. Furthermore, the high dose decreased the antibody response to Mycoplasma hyopneumoniae. Our results indicate that developmental exposure to an environmentally relevant OC mixture alters the immune function in swine.

Key Words: organochlorine insecticides; polychlorinated biphenyls; immune system; swine; prenatal exposure.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Organochlorine compounds (OCs) constitute a group of environmental contaminants that are highly lipophilic and poorly metabolized, leading to their bioconcentration in the fatty tissues of organisms and biomagnification in aquatic food webs. This group of environmental contaminants includes pesticides (e.g., chlordane, aldrin, dieldrin, toxaphene, DDT) and industrial compounds (e.g., hexachlorobenzene (HCB), polychlorinated biphenyls (PCBs), polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs)). The immune system is one of the most sensitive target of OCs, especially substances that are structurally related to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which can bind to the aryl hydrocarbon receptor (Safe et al., 1990Go), forming a complex that triggers the expression of genes involved in cell proliferation and differentiation (Whitlock et al., 1991Go). Activation of this receptor by low doses of TCDD results in general immunosuppression and thymus hypoplasia (Esser, 1994Go; NRC, 1992Go). Exposure to TCDD during the development of the immune system results in more severe effects than if the chemical is administered during adult life. In some species, prenatal exposure may even be a prerequisite for immunosuppression (Birnbaum and Tuomisto, 2000Go; Gehrs and Smialowicz, 1999Go; Vos and Luster, 1989Go). Indeed, evidence in laboratory and wildlife animals suggests that the maturation of the immune system is especially vulnerable to the adverse effects of dioxinlike compounds, chlordane, DDT, hexachlorobenzene, kepone, and polycyclic aromatic hydrocarbons (Barnett et al., 1987Go; Bernhoft et al., 2000Go; Holladay et al., 1991Go; Ross et al., 1997Go). However, little is known regarding the effects of complex mixtures of several organochlorines, such as those found in the environment and food chains.

Populations that rely on species from the aquatic food chain for subsistence display high body burdens of several OCs and may be particularly at risk of immunotoxic effects. The Inuit population of Nunavik (Northern Quebec, Canada) displays a relatively high body burden of several OCs due to their high consumption of sea mammal fat as part of their traditional diet (Dewailly et al., 1993Go). We previously reported an association between prenatal exposure to several OCs (p,p'-DDE, hexachlorobenzene and dieldrin) and the risk of otitis media during the first year of life in Inuit infants from Nunavik (Dewailly et al., 2000Go). However, the possible confounding effects of smoking and omega-3 fatty acids may have influenced our results. In the Inuit population, women who smoke during pregnancy also tend to consume more traditional foods and are therefore more exposed to OCs than nonsmokers (Muckle et al., 2001aGo,bGo). Hence, adequate control of these confounders may be difficult to achieve in an epidemiologic design. Experimental studies in laboratory animals are needed to avoid these potential methodological problems and identify mechanisms of action that may be involved in mediating OC-induced immunotoxicity.

The objective of the present study was to evaluate the immunotoxicity of in utero and lactational exposure to a reconstituted mixture of organochlorine compounds, designed to resemble that found in sea mammal fat consumed by the Inuit, using the pig as an animal model. Sows were exposed for several months before insemination and throughout gestation and lactation to either corn oil or a mixture of OCs (low, medium, or high doses). The following parameters were determined in the piglets: (1) innate immunity by evaluating phagocytosis by polymorphonuclear cells (PMNs) and complement component C2 function; (2) cell-mediated immunity by evaluating the lymphoproliferative response to mitogenic stimulation and the expression of T-cell markers; and (3) humoral immunity by evaluating the specific antibody response to Mycoplasma hyopneumoniae vaccination.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal Treatment
Sixteen Landrace-Yorkshire-Duroc sows were randomly allocated to four treatment groups. The animals were housed in Université Laval swine facilities and received a standard diet once a day (10:00 A.M.), with fresh water being available ad libitum. The facility was held at an ambient temperature of approximately 20°C with a 10:14-h light:dark cycle. This research was conducted under the guidelines of the Université Laval committees for animal care, chemical safety, and ethics. Each group received a different dose of an organochlorine mixture from 4 months of age until weaning of their first litter. The composition of the organochlorine mixture is described in Table 1Go and was designed to approximate that found in the blubber of ringed seals from Northern Québec (Derek Muir, National Water Research Institute, Environment Canada, personal communication). The major component of the mixture is a PCB neat mix that was custom made by AccuStandard (New Haven, CT) and comprised the following components: 2,4,4'-trichlorobiphenyl (320 mg: 0.98% of total PCBs); 2,2',4,4'-tetrachlorobiphenyl (256 mg: 0.78% of total PCBs); 3,3',4,4'-tetrachlorobiphenyl (1.4 mg: 0.004% of total PCBs); 3,3',4,4', 5-pentachlorobiphenyl (6.7 mg: 0.02% of total PCBs); Aroclor 1254 (12.8 g: 39.28% of total PCBs); and Aroclor 1260 (19.2 g: 58.99% of total PCBs). 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT), technical toxaphene, {alpha}-hexachlorocyclohexane ({alpha}-HCH), aldrin, dieldrin, 1,2,4,5-tetrachlorobenzene, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDD), ß-HCH, hexachlorobenzene, mirex, {gamma}-HCH, and pentachlorobenzene were obtained from Aldrich Chemical Co. (Milwaukee, WI). Technical chlordane was supplied by Ceriliant (Austin, TX). The compounds were dissolved in corn oil to reach the appropriate concentration, and the resulting solution was placed in 2-ml gelatin capsules. The capsules were placed in bread slices and administered three times weekly at feeding time. The low-dose group received 1 µg, the medium-dose group 10 µg, and the high-dose group 100 µg PCBs/kg body weight/day. The doses of other OCs included in the mixture are listed in Table 1Go. The animals in the fourth group (the control group) were administered corn oil only. At puberty, the females were inseminated with semen from an untreated boar (Duroc). Eight to ten male piglets per dose group were nursed by their sow for 3 weeks and, after weaning, received a standard diet. The piglets were vaccinated against Mycoplasma hyopneumoniae at 5 and 7 weeks of age. Blood samples were collected monthly for organochlorine determination and for immunological measurements.


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TABLE 1 Mixture Composition and Organochlorine Doses for the Various Treatment Groups
 
Organochlorine Measurements
Fourteen PCB congeners (International Union for Pure and Applied Chemistry nos. 28, 52, 99, 101, 105, 118, 128, 138, 153, 156, 170, 180, 183, 187) and 18 chlorinated pesticides and metabolites (aldrin, {alpha}-chlordane, {gamma}-chlordane, p,p'-DDD, p,p'-DDE, p,p'-DDT, dieldrin, {alpha}-HCH, ß-HCH, {gamma}-HCH, heptachlor, heptachlor epoxide, hexachlorobenzene, mirex, cis-nonachlor, trans-nonachlor, oxychlordane, and pentachlorobenzene) were determined by high-resolution gas chromatography with electron capture detection (HRGC-ECD). Plasma samples (2 ml) were extracted with an ammonium sulfate:ethanol:hexane (1:1:3) solution, cleaned on Florisil columns, and analyzed on an HP-5890 series II gas chromatograph equipped with dual-capillary columns (Ultra-1 and Ultra-2; Hewlett Packard, Palo Alto, CA) and dual Ni-63 electron-capture detectors. Peaks were identified by their relative retention times obtained on the two columns, using a computer program developed in-house. Quantification was performed mainly on the Ultra-1 column. The limits of detection were 0.02 µg/l for PCB congeners and most pesticides and metabolites, except for p,p'-DDT (0.03 µg/l), ß-HCH (0.03 µg/l), dieldrin (0.08 µg/l), and heptachlor epoxide (0.08 µg/l). Plasma samples from the sows were also analyzed for five toxaphene congeners (Parlar nos. 26, 32, 50, 62, 69) by HRGC with mass spectrometry detection, but these compounds were not detected in any sample (limit of detection: 0.08 µg/l). Quality control procedures of the HRGC-ECD method as well as accuracy and precision data were reported in Rhainds et al. (1999)Go. OC analyses were performed at the toxicology laboratory of the Institut National de Santé Publique du Québec, which is accredited by the Canadian Association for Environmental Analytical Laboratories. Since OCs distribute mainly in body fat, concentrations in plasma samples were reported in µg/kg lipids. Total cholesterol, free cholesterol, and triglycerides were measured in plasma samples by standard enzymatic procedures, while phospholipids were determined according to the enzymatic method of Takayama et al. (1977)Go, using a commercial kit (Wako Pure Chemical Industries, Richmond, VA). The concentration of total plasma lipids was estimated according to the formula developed by Phillips et al. (1989)Go.

Immune System Parameters
Phagocytosis.
PMNs were recovered from the red cell pellet after Ficoll-Hypaque density gradient centrifugation of peripheral blood samples collected from piglets at 1, 5, and 8 months of age. The red cells were lysed with Immunolyse (Coulter, Hialeah, FL), and the PMNs were washed and resuspended in culture medium (RPMI-1640 medium). PMN concentration was adjusted to 1 x 106 cells/ml. Phagocytosis was assayed using the Vybrant Phagocytosis Assay Kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions and the technique of Wan et al. (1993)Go, with some modifications. A 100-µl volume of the E.coli (FITC-labeled) suspension was prewarmed at room temperature, briefly sonicated to disperse any aggregates, and added to 100 µl of cells. Following a 1-h incubation at 37°C, 5% CO2, the cells were washed, incubated for 1 min with 100 µl of Trypan Blue (250 µg/ml, pH 4.4), and placed on ice to stop the reaction. Tubes containing fluorescent particles with or without Trypan Blue were used as controls to indicate complete quenching or to eliminate background, respectively. The fluorescence intensity was measured by flow cytometry, and the results were expressed in percentage (%) of PMNs showing phagocytic activity.

Complement activity.
The functional activity of the second component (C2) of the classical pathway of complement activation was assessed in pigs at 1, 5, and 7 months after birth. C2 titration was performed as described by Ngan et al. (1977)Go, with minor modifications. Serial dilutions of pig plasma were added to optimally sensitized sheep erythrocytes, to which an optimal dilution of C2-deficient guinea pig serum was added. The dilution of pig plasma that induces one lytic event per cell (Mayer et al., 1961Go) represents the C2 functional titer. All plasma samples from a given pig were tested simultaneously using the same reagents. The results were expressed as the percentage of lytic activity displayed by an internal control (normal pig plasma) to compensate for minor day-to-day variations in the haemolytic assay.

Lymphoproliferative response.
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation, washed, and resuspended in culture medium (RPMI-1640 medium with 7.5% FCS, penicillin, streptomycin, and glutamine). The cells were distributed in triplicate (2 x 105 cells/well) in a 200-µl volume and incubated for 3 days at 37°C, 5% CO2, in the presence of phytohemagglutinin (PHA) (Sigma, Oakville, Ontario, Canada). A PHA concentration of 60 µg/ml was selected, which proved to yield optimal proliferation in preliminary studies. Twenty hours before the end of the incubation period, 1 µCi 3H-thymidine was added to each well. The cells were harvested on a filter (Tomtec apparatus, Wallac, Turku, Finland), and a melt-on scintillator sheet was applied (Meltilex A; Wallac, Turku, Finland). After drying, the radioactivity transferred on to the gel was measured in a micro-ß isotope counter (LKB Pharmacia, Piscataway, NJ). The lymphoproliferative response was evaluated at 1, 2, 4, and 6 months of age and expressed as total stimulation (in c.p.m.): 3H-thymidine uptake by the mitogen-stimulated lymphocytes minus the corresponding uptake by unstimulated lymphocytes.

Evaluation of T Lymphocyte Subsets
Monoclonal antibodies.
Lymphocyte subsets (CD4+, CD8+, CD4+CD8+) were evaluated at 1, 2, 4, 6, and 8 months of age. We also measured the swine leukocyte antigen SLA-DR on cytotoxic T cells since most of CD8+ cells bear this antigen in this species (Lunney et al., 1987Go; Thisthethwaite et al., 1983). Clones that produce monoclonal antibodies (mAb) specific for porcine CD4 (74-12-4, mouse IgG2b{kappa}) and CD8 (76-12-11, mouse IgG2a{kappa}) were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Anti-porcine CD4 and CD8 mAbs were purified by passage through a recombinant protein G-Agarose column (Gibco, Burlington, Ontario, Canada). Anti-CD4 and -CD8 mAbs were conjugated with fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively, using the FluoReporter Fluorescein-Ex protein labeling kit and the Protein-Protein cross-linking kit (Molecular Probes, Eugene, OR). Monoclonal antibodies specific for the SLA-DR (1053h2-18-1, mouse IgG2a{kappa}) were purchased from Research Diagnostics Inc. (Flanders, NJ). Anti-SLA-DR mAbs were used in indirect immunofluorescence with anti-mouse IgG (H+L)-FITC (Immunotech, Coulter, Hialeah, FL).

Flow cytometric analysis.
Flow cytometric analysis was performed by single and dual color fluorescence. Briefly, 1 x 106 PBMCs in 200 µl of phosphate buffered saline were incubated with an optimal concentration of fluorochrome-labeled monoclonal antibodies to identify the following lymphocyte subsets: (1) helper T cells: CD4-FITC; (2) cytotoxic T cells: CD8-PE; (3) double positive T lymphocytes: CD4-FITC and CD8-PE; and (4) cytotoxic T cells with SLA-DR expression: CD8-PE and SLA-DR-anti mouse IgG-FITC. The stained cells were washed, the red cells were lysed with a commercial lysing solution (Immunolyse, Beckman Coulter, Miami, FL), and the cells were then fixed with paraformaldehyde. After two washes, the cell pellets were suspended in 200 µl of Isoton II and analyzed on a Profile II flow cytometer (Coulter, Hialeah, FL). Cell labeling was measured on 5000 lymphocytes on a log fluorescence scale. The instrument performance was standardized daily with Immunocheck and Flow-set calibration beads (Beckman Coulter, Miami, FL).

Antibody Response to Mycoplasma hyopneumoniae
The piglets were vaccinated against Mycoplasma hyopneumoniae by 1-ml intramuscular injections of Mycoplasma hyopneumoniae bacterin at 5 and 7 weeks of age. An ELISA assay was performed by Biovet laboratory (St-Hyacinthe, Quebec, Canada) to determine anti-Mycoplasma hyopneumoniae IgG titers in plasma samples collected monthly from the piglets. The results were expressed as the percentage of animals showing a positive antigen-specific titer (i.e., >6 mg/l).

Statistical Analysis
All statistical analyses were performed using the SAS software (v 8.0, SAS Institute, Cary, NC). For continuous variables, the differences between mean values observed at several time points between experimental groups were tested using an analysis of variance (ANOVA) for repeated measurements (MIXED procedure). For the vaccine response (dichotomic variable), a logistic regression analysis for repeated measurements was used (GENMOD procedure) to test the differences between groups in the percentage of animals showing antibody titers above 6 mg/l at different time points. If the time x dose interaction term was statistically significant, multiple comparisons were made between the dose groups within each time stratum. In the absence of an interaction between time and dose, the interaction term was removed from the model and the differences between dose groups were tested globally for all time points. The level of statistical significance was set at 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Organochlorine Body Burden in Sows and Piglets
Concentrations of organochlorines in plasma samples collected from sows at delivery and from piglets 1 week after weaning (1 month of age) are presented in Tables 2Go and 3Go, respectively. With the exception of p,p'-DDE, OCs were not detected in plasma samples from animals in the control group. Most compounds were detected in the sows and piglets from the medium- and high-dose groups. Aldrin, chlordane isomers, heptachlor, and toxaphene congeners were not detected in any sample. The mean PCB plasma lipid concentration measured in sows at delivery increased nearly 10-fold between the low- and the medium-dose groups and by 8-fold between the medium- and the high-dose groups. Similar differences were observed for other OCs. For most compounds, the mean concentrations determined in piglets at 1 month of age were similar to those measured in maternal plasma samples.


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Table 2 Concentrations of Organochlorines in Plasma Samples (µg/kg of lipids) Collected at Delivery in Organochlorine-Treated or Control Sows
 

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TABLE 3 Concentrations of Organochlorines in Plasma Samples (µg/kg of lipids) of 1-Month-Old Piglets Born to Organochlorine-Treated or Control Sows
 
Immunological Parameters
Phagocytosis.
The phagocytic potential of PMNs collected from the piglets towards E. coli is presented in Figure 1Go. The analysis of variance for repeated measurements indicated a statistically significant interaction between time and dose. Gestational and lactational exposures to the OC mixture did not alter the phagocytic activity at 1 and 5 months of age. However, 8-month-old piglets from the high-dose group had a higher percentage of PMN-associated phagocytosis than the animals in all of the other groups.



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FIG. 1. Percentage of peripheral blood PMNs exhibiting phagocytic activity (uptake of FITC-labeled E.coli) in piglets at 1, 5, and 8 months of age. The piglets were exposed prenatally and through lactation to different doses of the OC mixture (see Table 1Go). Each bar represents the mean ± SD for eight to ten animals. ANOVAs revealed statistically significant differences between groups at 8 months of age. Within this time point, group means that were significantly different by multiple comparisons are indicated following different letters above the bars (p < 0.05); those marked by the same letters are not different.

 
Complement Component C2 Function.
Complement component C2 titers were similar at all time points and in all treatment groups (data not shown). An ANOVA for repeated measurements revealed no statistically significant interaction between time and dose and no effect of treatments.

Lymphoproliferative Response.
The capacity of T lymphocytes, isolated from peripheral blood at 1, 2, 4, and 6 months, to proliferate upon mitogenic stimulation (PHA, 60 µg/ml) is presented in Figure 2Go. The ANOVA for repeated measurements revealed a statistically significant interaction between time and dose. Additional ANOVAs for each time point separately indicated a lack of treatment-related effect at 1 and 2 months of age. However, at 4 and 6 months of age, a statistically significant, dose-dependent increase in PHA-induced lymphocyte proliferation was observed. The medium dose was the minimal effective dose in the proliferation assay.



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FIG. 2. Proliferative response (3H-thymidine incorporation in c.p.m.) to phytohemagglutinin of peripheral blood lymphocytes isolated from piglets at 1, 2, 4, and 6 months of age. The piglets were exposed prenatally and through lactation to different doses of the OC mixture (see Table 1Go). Each bar represents the mean ± SD for 8 to 10 animals. ANOVAs revealed statistically significant differences between groups at 4 and 6 months of age. Within these time points, group means that were significantly different following multiple comparisons are indicated by different letters above the bars (p < 0.05); those marked by the same letters are not different.

 
T Lymphocyte Subsets.
The proportions of CD4+, CD8+, CD4+CD8+, and CD8+/SLA-DR+ T lymphocytes in peripheral blood samples that were collected from the piglets at various time points are shown in Figure 3Go. ANOVAs for repeated measurements revealed statistically significant interactions between time and dose for all T-cell subsets evaluated in this study. ANOVAs performed within each time point revealed no dose–response relationship for CD4+ (Fig. 3AGo) and CD8+ (Fig. 3BGo) cells. The percentage of CD4+CD8+ T cells (Fig. 3CGo) increased in 4-month-old piglets from all OC-treated groups, but a clear dose–response relationship was not observed. In 6-month-old piglets, only the high-dose group displayed a mean percentage of CD4+CD8+ that was statistically greater than that in the control group. Finally, the OC treatment increased the expression of SLA-DR on cytotoxic T cells in 4-month-old piglets, but only the difference between the high-dose group and the control group was statistically significant. In contrast, at 6 months of age, all OC-treated groups displayed a lower percentage of CD8+/SLA-DR+ cells than that of the control group (Fig. 3DGo).



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FIG. 3. Percentage of CD4+ (A), CD8+ (B), CD4+/CD8+ (C), and CD8+/SLA-DR+ cells (D) in blood from piglets at 1, 2, 4, 6, and 8 months of age (flow cytometry analysis). The piglets were exposed prenatally and through lactation to different doses of the OC mixture (see Table 1Go). Each bar represents the mean ± SD for 8 to 10 animals. For percentages of CD4+/CD8+ and CD8+/SLA-DR+ cells, ANOVAs revealed statistically significant differences between groups at 4 and 6 months of age. Within these time points, group means that were significantly different following multiple comparisons are indicated by different letters above the bars (p < 0.05); those marked by the same letters are not different.

 
Antibody Response to Mycoplasma hyopneumoniae
Figure 4Go illustrates the antibody response to vaccination against Mycoplasma hyopneumoniae. A first dose of the vaccine was administrated at 5 weeks of age, followed by a booster dose 2 weeks later. Mean antibody titers (Fig. 4AGo) and proportions of piglets showing a positive response following vaccination (Fig. 4BGo) followed similar dose- and time-related trends. However, antibody titers were quite variable with standard deviations often exceeding mean values (data not shown). No difference was observed between the OC-treated groups and the control group for the first response to vaccination. At 2 months, the percentage of animals showing a positive response was above 65% in all groups. However, starting at 5 months of age, a gradual decrease occurred in the percentage of piglets with a positive IgG titer in the high-dose group. The results of a logistic regression analysis with repeated measurements indicated that, globally, between 2 and 8 months of age, the proportion of piglets showing a positive antibody response to the vaccine was lower in the high-dose group than in all other groups (Fig. 4BGo). Because of the large variability in antibody titers, the analysis of variance for these data did not reveal any statistical difference between groups.



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FIG. 4. Mean titer of IgG to Mycoplasama hyopneumoniae (A) and percentage of piglets showing a titer greater than 6 mg/l (B) following vaccination at 5 and 7 weeks of age. The piglets were exposed prenatally and through lactation to different doses of the OC mixture (see Table 1Go). Antibody measurements were performed monthly from 1 to 8 months of age (8 to 10 animals per dose group). Errors bars are not presented in panel A for the sake of clarity. *Proportions were lower in the high-dose group compared with all other groups (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our results show that, in the pig model, developmental exposure to a complex mixture of organochlorines, designed to approximate that found in sea mammal fat in the Canadian Arctic, modulates innate, cellular, and humoral immunity. We observed, in male piglets pre- and postnatally exposed to the OC mixture, alterations in phagocytosis by PMNs, proportions of T cells in peripheral blood, T-cell proliferative response to mitogen, and the antibody response to Mycoplasma hyopneumoniae vaccination.

This developmental study is unique because of the animal model used and the composition of the OC mixture fed to the animals. We could not find any study in the literature pertaining to immune system effects induced by a developmental exposure to a reconstituted complex OC mixture of relevance to the Arctic. Ross et al. (1997)Go administered orally to female rats during pregnancy and lactation one of the following treatments: (1) oil extracted from herring caught in the relatively uncontaminated Atlantic Ocean (control group); (2) oil extracted from herring caught in the contaminated Baltic Sea; or (3) oil extracted from Atlantic herring spiked with 2,3,7,8-TCDD (positive control group). In the positive control group, the authors reported immunosuppression characterized by decreased thymocyte and splenocyte proliferative responses to mitogens as well as decreased virus-associated natural killer (NK) cell activity and specific antibody responses. A similar pattern of effects was observed in rat pups exposed only to the Baltic Sea herring oil, but the effects were generally less pronounced (Ross et al., 1997Go). Interestingly, F1 rats from the control group treated with the Atlantic herring oil, the Baltic Sea herring oil group, or the positive control TCDD-exposed group that were infected with rat cytomegalovirus (RCMV) showed similar RCMV-specific IgG titers 12 days after infection. However, at 59 days postinfection, the rats in the Baltic sea herring oil group and the TCDD group had significantly lower titers than the animals in the control group. Hence, the long-term antibody response to virus infection, but not the primary response, was affected by treatment with the OC mixture, similar to the results obtained in our study following Mycoplasma hyopneumoniae vaccination.

Two studies with rodents investigated the effects on the immune system of a postnatal exposure to a mixture of contaminants present in the blubber (adipose tissue) of a heavily contaminated beluga whale from the St. Lawrence River (Fournier et al., 2000Go; Lapierre et al., 1999Go). In the first study (Lapierre et al., 1999Go), Fisher 344 rats were fed for 2 months a diet in which lipids were replaced by either blubber from the St. Lawrence beluga whale (high dose), blubber from a relatively clean Arctic beluga whale (control group), or a 50:50 mixture of blubbers from the St. Lawrence beluga whale and the Arctic beluga whale (medium dose). The authors did not report any difference between treatments for the various immune parameters evaluated: lymphoblastic transformation, NK cell activity, plaque forming cells, phagocytosis, oxidative burst, and immunophenotyping (Lapierre et al., 1999Go). In the second study (Fournier et al., 2000Go), groups of C57B1/6 mice were fed for a period of 90 days diets in which the fat consisted of either corn oil, beef oil, or blubber from a St. Lawrence beluga whale and an Arctic beluga whale in the following proportions (five groups): 100:0, 75:25, 50:50, 25:75, and 0:100. Mice in groups fed diets containing blubber from beluga whales were not different than the control mice for blastic transformation of splenocytes, NK cell activity, and weight of the spleen and the thymus. However, groups receiving diets with blubber from beluga whales showed decreased CD8+ T cells in the spleen, reduced phagocytosis by peritoneal macrophages, and reduced humoral response of splenic cells against sheep red blood cells (Fournier et al., 2000Go). In the latter study, the fact that similar immunosuppressive effects were observed in all groups of mice fed diets containing blubber from beluga whales, irrespective of their level of contamination, suggests a possible role of the lipid moiety, more specifically omega-3 fatty acids (n-3 PUFAs) that are present in high concentration in beluga whale fat and are well-known immunomodulators (Blok et al., 1991; Endres, 1996Go; Harbidge, 1998Go; Hardardottir and Kinsella, 1992Go). We avoided this problem by using a reconstituted OC mixture rather than lipids extracted from marine mammal blubber.

Increases in the proportion of peripheral blood CD4+CD8+ T cells, phagocytosis, and lymphoproliferative response were observed in our study, in contrast to the results described above. In addition to differences in mixture composition, the animals in our study were exposed during the period of immune system development, which can produce qualitative differences in immune responses compared to postnatal exposure (Holladay and Smialowicz, 2000Go). Elevation in some immune parameters can be linked to targeted immunosuppression in other parameters, as observed for heavy metals that can alter the balance between Th1- and Th2-lymphocytes (Fournié et al., 2002Go; Heo et al., 1996Go). Finally, the unique character of the immune system in swine may also explain differences in immune responses between our study and those performed with rodents, especially with regard to the lymphoproliferative response. Indeed, the swine immune system is not fully developed at birth (Becker et al., 1993Go), and spontaneous lymphocyte proliferation increases in neonatal pigs during the first weeks of life (Hoskinson et al., 1990Go).

The decrease in the secondary vaccinal response noted in the high-dose group in our study indicates that the OC mixture affected the production of antibodies by B cells and the memory response. This result increases the biological plausibility of associations noted previously in epidemiological studies between developmental OC exposure and decreased humoral immunity. Weisglas-Kuperus et al. (2000)Go reported that prenatal PCB exposure was associated with lower antibody levels to mumps and measles after primary vaccination in Dutch pre-school children. We previously reported an association between prenatal exposure to OCs and the risk of otitis media during the first year of life in Inuit infants (Dewailly et al., 2000Go). We also observed a lower concentration of IgM in newborns from a fish-eating population, living in the remote Lower North Shore region of the Gulf of St. Lawrence, compared to a reference population with lower prenatal OC exposure (Belles-Isles et al., 2002Go).

A trend toward delayed effects was noted for most of the changes in immune parameters induced by treatment to the OC mixture in the present study. Indeed, treatment-induced changes were only seen in animals 4 months of age and older. This suggests that developmental exposure to the OC mixture did not affect innate immunity but rather acquired immunity, perhaps through interference with the antigen presentation process and mechanisms involved in memory response. One might speculate that the decrease in antibody response to the vaccine could be related to the concomitant decrease in the expression of SLA-DR on CD8+ T cells, which was noted in piglets at 6 months of age. In the swine, a MHC class II molecule is expressed constitutively on all T cells (Lunney et al., 1987Go; Thisthethwaite et al., 1983). Furthermore, 90% of CD4+/CD8+ and CD4-/CD8+ T cells express the SLA-DR. In this species, CD4+/CD8+ T cells can potentially present antigens to other cells (Zuckermann et al., 1996Go). A decrease in expression of SLA-DR might affect antigen presentation and, in turn, stimulation of B cells to produce antibodies. A proper humoral immune response requires the interaction of three major immune cell types: antigen-presenting cells, helper T cells, and B cells. Alteration or dysfunction in any of these cells or cell interactions may result in aberrant antibody production (Luster et al., 1988Go). However, decreases in SLA-DR expression occurred to the same extent in all OC-treated groups, while the antibody response to the vaccine was decreased only in the high-dose group. This suggests that the expression of SLA-DR is not the only factor involved in the suppression of the antibody response to the vaccine. Additional studies are required to further elucidate the mechanisms involved in OC-induced suppression of humoral immunity.

Plasma concentrations of OCs in piglets were comparable with those observed in human populations exposed to the same compounds in Quebec (Canada). Indeed, Muckle et al., (2001b)Go reported in Inuit women giving birth in Nunavik a mean concentration of PCBs (sum of 14 congeners) in plasma lipids of 397 µg/kg, with values ranging from 72 to 1951 µg/kg. In our study, mean plasma lipid PCB concentrations in sows at delivery were 153, 1425, and 11,485 µg/kg for the low-, medium-, and high-dose groups, respectively. The mean plasma lipid concentration of p,p'-DDE in Inuit women was 386 µg/kg (range = 60 to 2260 µg/kg). The mean p,p'-DDE concentrations in plasma lipids from sows at delivery were 152, 1538, and 13,756 µg/kg. The mean concentrations of these major OCs in sows in the high-dose group are only five to six times higher than the maximal concentrations recently reported in Inuit women giving birth in Nunavik. Therefore, the body burdens of OCs achieved in the present study are relevant to the Inuit population of Nunavik. The concentrations of PCBs achieved in our study are also relevant to maternal PCB exposure in various environmentally exposed populations (Longnecker et al., 2003Go).

In summary, we observed in the pig model that gestational and lactational exposure to a mixture of OCs relevant to the Arctic alters the activity of several aspects of the immune system. Developmental exposure to OCs enhanced the phagocytic activity of PMNs and the proliferative response of T cells to mitogenic challenges. We also noted a suppression of SLA-DR expression by CD8+ T cells that may be related to the decreased humoral response observed in piglets from the high-dose group. Further studies are underway to investigate the mechanism by which developmental exposure to the OC mixture can interfere with normal humoral response, with a special focus on antigen-presenting cells.


    ACKNOWLEDGMENTS
 
Many thanks to Jean-Philippe Weber, Alain Leblanc and Évelyne Pelletier from the toxicology laboratory of the Institut National de Santé Publique du Québec for performing organochlorine analyses. This study was supported by grants from Indian and Northern Affairs Canada (Northern Contaminants Program) and Hydro-Québec/FRSQ Environment and Child Health Initiative.


    NOTES
 
1 To whom correspondence should be addressed at Unité de recherche en santé publique, Centre de Recherche du CHUL-CHUQ, 945 avenue Wolfe, Québec, QC, Canada G1V 5B3. Fax (418) 654-2148. E-mail: pierre. ayotte{at}inspq.qc.ca. Back


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 TOP
 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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