Penta-BDE was purchased from Wellington Laboratories Inc. (Guelph, ON, Canada). RPMI 1640 media was obtained from Gibco BRL (Grand Island, NY, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD3e monoclonal antibody, Phycoerythrin (PE)-conjugated anti mouse CD8a monoclonal antibody, Cy-chrome-conjugated anti-mouse CD4 monoclonal antibody used in flow cytometry were purchased from Pharmingen Inc. (San Diego, CA, USA). MTS and PMS assay kits were from Promega (Madison, WI, USA). Mouse IgG1, IgM ELISA kit were purchased from BD Bioscience (San Diego, CA, USA). Extra materials and reagents were purchased from sigma chemical Co. (St. Louis, MO, USA).

Specific pathogen-free C57BL/6J mice were provided by Central Laboratory Animal Inc. (Korea). Animals aged 8 weeks were acclimatized for 1 week before treatment. Animals were cared in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The animal room was maintained at 23±2℃ and relative humidity between 55±10%. The light/dark cycle was maintained on 12-h intervals. Virgin female mice, aged 9 weeks, were mated with male in the proportion of 2:1. The day sperm plug was detected by vaginal smear was decided to be day 0 of gestation. The pregnant mice were randomly divided into four groups.

Penta-BDE and deca-BDE was dissolved in corn oil and orally administrated to mice at doses of 50, 100 and 200 mg/kg/day for penta-BDE and 0.5, 2.5, 12.5 g/kg/day for deca-BDE. Mice were treated from the day 0 of gestation to postnatal day 21.

On PND 21 and PND 63, dams and offsprings were sacrificed by CO₂ inhalation. Body weights of mice were measured at the time of dosing initiation and autopsy. Organ weight including spleen, thymus, liver and kidneys was weighed and cellularity of spleen and thymus was determined by counting with a hematocytometer after red blood cell lysis.

Hematology was performed by automatic hematological analyzer (ADVIA120, Bayer, Germany). Thymus and spleen were fixed in neutral aqueous, phosphate-buffered 4% solution of formaldehyde, wax-embedded according to a routine processing protocol, and 5 µm sections cut and stained with hematoxylin and eosin (H&E). The slides were examined under light microscope by toxicopathologist (Korean Society of Toxicologic Pathology).

Spleens and thymus were removed aseptically and kept on ice in complete RPMI 1640. The organs were gently pressed through cell strainer (100 µm, Nylon, BD falcon Co., MA, USA). Both cells were washed once in RPMI 1640. The cell suspension was transferred and centrifuged at 430 g for 10 min with 4℃. The supernatant was discarded and the pellet was suspended with RBC lysis buffer (0.15M NH₄Cl, 1M KHCO₃ 0.1 mM Na₂EDTA, pH=7.4) to remove RBCs. After storage for 10 min, on ice, the suspension cells were washed twice with completer RPMI 1640 and centrifuged at 430 g for 10 min with 4℃. Then cell pellet was resuspended in the appropriate culture media for the assay being performed. The cell concentration was adjusted to 2×10⁶ cells/ml in all studies.

For the evaluation of the proliferative potency of splenocytes, 50 µl of splenocytes (2×10⁶ cells/ml) were seeded in each of the 96-well flat-bottomed plates. Then, 50 µl of concanavalin A (5 µg/ml) or LPS (50 µg/ml) were added to the plates in duplicate. The plates were incubated for 72 h at 37℃ in 5% CO₂. Cell proliferation was assayed using CellTiter 96® aqueous nonradioactive cell proliferation assay kit according to the manufacturer's protocol.

After centrifugation of 700 µl of splenocytes or thymocytes (2×10⁶ cells/ml), splenocytes were stained with following antibodies; purified anti-mouse CD16/CD32 (blocking antibody), fluorescein isothisyanate (FITC)-conjugated anti-mouse CD3e monoclonal antibody (Pan T cell marker), phycpreythrin (PE)-conjugated anti-mouse CD8a monoclonal antibody (cytotoxic T cell marker), PE-Cy5-conjugated anti-mouse CD4 monoclonal antibody (helper T cell marker), FITC-conjugated anti-mouse CD11b monoclonal antibody (macrophage marker), PE-conjugated anti mouse CD19 monoclonal antibody (B cell marker), PE-Cy5-conjugated anti-mouse CD45R/B220 monoclonal antibody. After incubation, the cells were washed and fixed with 0.5% paraformaldehyde in PBS. Flow Cytometry analysis of splenocytes and thymocytes were performed with flow cytometer (Dual laser FACSCalibur™, Becton Dickinson, USA).

ELISA was used to measure total IgG1 and IgM antibody in mice sera, using ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, 96-well plates were coating with 50 µl of anti-mouse IgG1 diluted with coating buffer (carbonated buffer, pH 9.6) for 12 hours at 4℃. After washing with buffer (0.05% Tween-PBS), the plates were blocked with 1% bovine serum albumin (1% BSA-PBS) and diluted serum samples were added to wells of the plates in duplicate. After the incubation at 37℃ for one hour, the plates were washed 5 times with PBST, followed by addition of Biotin-anti-mouse IgG1 detection antibody and incubation at room temperature for one hour. Unbound detection antibody was washed from the plate after the incubation period with diluted wash buffer. Avidin-HRP was added, and incubated at room temperature for 15 min. Colorimetric detection was performed using ELISA plate reader (Molecular Device Co. USA).

All data were expressed as mean±standard deviation. Statistical analysis was performed using Sigma Stat Ver 2.0 (SPSS, USA). Data was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's method as a post hoc test. At p<0.05 was considered significant.

The results of body weight of dams orally administrated to penta-BDE have not shown the significant change over the control group (Table I). Body weights of PND21 exposed to deca-BDE were significantly decreased over control mice, but those of PND63 were recovered as control (Fig. 2, 3). Absolute and relative weight of spleens was decreased in dams exposed to penta-BDE, and especially significantly decreased in dams administrated to 100 mg/kg/day and 200 mg/kg/day. The thymi of penta-BDE exposed dams were weighed decreasingly (Table II, III).

In PND21, expose to penta-BDE 100 mg/kg/day and 200 mg/kg/day decreased significantly absolute and relative spleen and thymus weight. However no significant changes of spleen and thymus weight in PND63 occurred.

PBDE expose to dams had an effect on increase of the number of WBC and especially deca-BDE showed significantly increased the number of WBC, neutrophil and lymphocyte (Table IV). In contrast, there was slight decrease in the number of WBC, neutrophil and lymphocyte in offsprings (Table V). Histopathologically no significant changes in spleen and thymus were observed in mice treated with penta-BDE and deca-BDE, compared to control group.

When spleen cells from penta-BDE exposed to mice were stimulated with ConA as a T lymphocyte mitogen, the proliferation of these cells was dose-dependent (Fig. 4). Penta-BDE high dose dams and PND63 showed significant T cell proliferation (p<0.05). But T cell proliferation in deca-BDE treatment dams showed slight increase (Fig. 5).

Unlike T cell proliferation by ConA, there were no significantly changes of B cell proliferation by LPS between PBDEs treated dams, PND21 and PND63 (Fig. 6, 7).

Flow cytometric analysis of both splenocytes and thymocytes subpopulations was carried out to explore further the nature of the change in lymphoid organ weights in C57Bl/6J mice exposed to PBDEs from gestational day 6 to lactational day 21. Unlike dams and PND63, PND21 exposed to penta-BDE showed increase of total T cell population, CD3+/CD4+ T cell, and CD3+/CD8+ T cell and PND21 exposed to penta-BDE 100 mg/kg/day and 200 mg/kg/day were significant (Fig. 8, 9, 10). There were no significant differences between deca-BDE treatment groups.

B cell population was decreased in dams exposed to penta-BDE, but deca-BDE induced B cell population in spleen. PND21 showed B cell population to be decreased by PBDEs, but PND63 showed no significantly differences (Fig. 11). Deca-BDE showed significant decrease of CD11b+ macrophage population in PND21, but penta-BDE did not seem to be effective to dams and offsprings (Fig. 12).

Total IgG1 was detected increasingly in dams administrated with PBDEs by deca-BDE rather than penta-BDE. However, PND21 exposed to penta-BDE showed decrease of IgG1 production, and PND63 produced IgG1 similar to control (Fig. 13).

As the results of IgM level, all groups exposed to penta-BDE showed decrease of IgM production, but dams by deca-BDE produced more IgM than control (Fig. 14).