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CLARITY-BPA: Effects of Chronic Bisphenol A Exposure on the Immune System: Part 1 – Quantification of the Relative Number and Proportion of Leukocyte Populations in the Spleen and Thymus

Jinpeng Li, Anthony Bach, Robert B. Crawford, Ashwini S. Phadnis-Moghe, Weimin Chen, Shawna D’Ingillo, Natalia Kovalova, Jose E. Suarez-Martinez, Jiajun Zhou, Barbara L. F. Kaplan, and Norbert E. Kaminski.
Toxicology (2018) DOI: https://doi.org/10.1016/j.tox.2018.01.004 PMID: 29428349


Publication


Abstract

Bisphenol A (BPA) is extensively used in manufacturing of a broad range of consumer products worldwide. Due to its widespread use, human exposure to BPA is virtually ubiquitous. Broad human exposure coupled with a large scientific literature describing estrogenic activity of BPA in animals has raised public health concerns. To comprehensively evaluate the health effects of BPA exposure, a chronic toxicity study using a wide-range of BPA doses (2.5-25000 μg/kg bw/day) was conducted jointly by the NTP, thirteen NIEHS-supported grantees, and the FDA, which is called the Consortium Linking Academic and Regulatory Insights on Toxicity of BPA (CLARITY-BPA). As a participant in the CLARITY-BPA project, the objective of the current study was to evaluate the effects of chronic BPA exposure in Sprague-Dawley rats on the relative number and proportion of defined leukocyte populations in the spleen and the thymus. Toward this end, lymphoid tissues from a total of 641 rats were assayed after being continuously dosed with BPA or controls for up to one year. To comprehensively evaluate the effects of BPA on leukocyte compositions, extensive endpoints that cover major populations of leukocytes were assessed, including B cells, T cells, NK cells, granulocytes, monocytes, macrophages and dendritic cells. In total, of the 530 measurements in BPA-treated rats, 10 measurements were statistically different from vehicle controls and were mainly associated with either the macrophage or dendritic cell populations. Most, if not all, of these alterations were found to be transient with no persistent trend over the one-year time period. In addition, the observed BPA-associated alterations were mostly moderate in magnitude and not dose-dependent. Due to the aforementioned, it is unlikely that the observed BPA-mediated changes alone would adversely affect immune competence.

Figures


Figure 1. Percentage of CD3+ T cells in the thymus of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed a PND (Please leave postnatal day (PND) in the text) author 21. Thymocytes were isolated and surface stained with an anti-rat CD3 and quantified for the percentage of CD3+ T cells by flow cytometry. Results are presented as mean ± SE. n = 3–10 rats/treatment group/sex. *p < 0.05 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 2. Percentage of CD4+CD8+ double-positive T cells in the thymus of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at postnatal day (PND) 21. The percentage of CD4+CD8+ double-positive T cells in thymocytes was quantified by flow cytometry. Results are presented as mean ± SE. n = 3–10 rats/treatment group/sex. * p < 0.05 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 3. Spleen cellularity for rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at (A) postnatal day (PND) 21, (B) PND 90 or (C) 6 month. Splenocytes were isolated and counted on Beckman Coulter Counter with a size threshold of 4 μm after lysing red blood cells. The spleen cellularity was calculated as the number of splenocytes per milligram of spleen. Results are presented as mean ± SE. A) n = 5–10 rats/treatment group/sex. B) n = 6–10 rats/treatment group/sex. C) n = 4–10 rats/treatment group/sex, except that n = 0 in VH treated female group. * p < 0.05, ** p < 0.01 when compared to respective vehicle control (VH-Ov for female rats) by a two way ANOVA with Dunnett’s posttest. For more details about VH-Ov, please see the Statistical Analysis section in the Materials and Methods.

Figure 4. Summary of immune cell populations evaluated in the spleen and the corresponding cell markers utilized.

Both lymphoid and myeloid immune cell populations in spleens were quantified by flow cytometry. The cell markers used to identify each cell population are indicated. In all cases, the percentage of each cell population was calculated based on the total live leukocytes in spleens.

Figure 5. Percentage of CD3+ T cells in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at postnatal day (PND) 21. The percentage of CD3+ T cells in splenocytes was quantified by flow cytometry. Results are presented as mean ± SE. n = 5–10 rats/treatment group/sex. ** p < 0.01 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 6. Percentage of CD8+ T cells in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at 1 year of age. The percentage of CD8+ T cells in splenocytes was quantified by flow cytometry. Results are presented as mean ± SE.
n = 2–10 rats/treatment group/sex. * p < 0.05 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 7. Percentage of B cells in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at 6 months of age. The percentage of cell surface IgM+/IgG+ B cells in splenocytes was quantified by flow cytometry. Results are presented as mean ± SE. n = 4–10 rats/treatment group/sex, except that n = 0 in VH treated female group. * p < 0.05 was compared to respective vehicle control (VH-Ov for female rats) by a two way ANOVA with Dunnett’s posttest. For more details about VH-Ov, please see the Statistical Analysis section in the Materials and Methods.

Figure 8. Percentage of NKT cells in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at 1 year of age. The percentage of CD161a+ CD4/8+ NKT cells in splenocytes was quantified by flow cytometry. Results are presented as mean ± SE. n = 2–10 rats/treatment group/sex. * p < 0.05 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 9. Percentage of macrophages in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at 1 year of age. The percentage of CD11b+ CD11c− macrophages in splenocytes was quantified by flow cytometry. Results are presented as mean ± SE. n = 2–10 rats/treatment group/sex, n = 2 rats in 250 μg BPA/kg/day treated male group. * p < 0.05 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 10. Percentage of antigen presenting cells (APC) in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at 1 year of age. The percentage of MHCII+ CD11b+ CD11c− APCs in splenocytes was assessed by flow cytometry. Results are presented as mean ± SE. n = 2–10 rats/treatment group/sex. * p < 0.05 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 11. Percentage of classic dendritic cells (cDC) in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at postnatal day (PND) 90. The percentage of CD11b+ CD11c+ cDCs in splenocytes was quantified by flow cytometry. Results are presented as mean ± SE. n = 6–10 rats/treatment group/sex. * p < 0.05 when compared to respective vehicle control by a two way ANOVA with Dunnett’s posttest.

Figure 12. Percentage of mature cDC in the spleen of rats by treatment group and sex.

Rats were administered vehicle (VH, 0.3% aqueous carboxymethylcellulose), BPA or estrogen ethinyl estradiol (EE2) at the indicated dose level by oral gavage daily and sacrificed at 6 months of age. The percentage of CD11b+ CD11c+ MHCII+ mature cDCs in splenocytes was quantified by flow cytometry. Results are presented as mean ± SE. n = 4–10 rats/treatment group/sex, except that n = 0 in VH treated female group. ** p < 0.01, *** p < 0.001 when compared to respective vehicle control (VH-Ov for female rats) by a two way ANOVA with Dunnett’s posttest. For more details about VH-Ov, please see the Statistical Analysis section in the Materials and Methods.

Tables


Table 1. Effects of EE2 and BPA on thymic cellularity, the proportion of CD3+ T cells, CD4+CD8+ positive T cells, CD4+ T helper cells and CD8+ cytotoxic T cells in thymus.

Effects of EE2 and BPA on thymic cellularity, the proportion of CD3+ T cells, CD4+CD8+ double positive T cells, CD4+ T helper cells and CD8+ cytotoxic T cells in thymus.

Table 2. Effects of EE2 and BPA on spleen cellularity.

Table 3. Effects of EE2 and BPA on the percentage of lymphoid cell populations in splenocytes.

Table 4. Effects of EE2 and BPA on the percentage of spleen associated myeloid cell populations.

Table 5. Summary of experimental conditions, endpoints and the number measurements with observed BPA

Table 5. Summary of experimental conditions, endpoints and the number measurements with observed BPA effects in this study.

Supplemental Materials


Supplementary Data