Bisphenol A (BPA) is commonly used in the manufacturing of a wide range of consumer products, including polycarbonate plastics, epoxy resin that lines beverage and food cans, and some dental sealants. Consumption of food and beverages containing BPA represents the primary route of human BPA exposure, which is virtually ubiquitous. An increasing number of studies have evaluated the effects of BPA on immune responses in laboratory animals that have reported a variety of effects some of which have been contradictory. To address the divergent findings surrounding BPA exposure, a comprehensive chronic treatment study of BPA was conducted in Sprague-Dawley rats, termed the Consortium Linking Academic and Regulatory Insights on Toxicity of BPA (CLARITY-BPA). As a participant in the CLARITY-BPA project, our studies evaluated the effects of BPA on a broad range of immune function endpoints using spleen cells isolated from BPA or vehicle treated rats. This comprehensive assessment included measurements of lymphoproliferation in response to mitogenic stimuli, immunoglobulin production by B cells, and cellular activation of T cells, NK cells, monocytes, granulocytes, macrophages and dendritic cells. In total, 630 different measurements in BPA treated rats were performed of which 35 measurements were statistically different from vehicle controls. The most substantive alteration associated with BPA treatment was the augmentation of lymphoproliferation in response to pokeweed mitogen stimulations in 1 year old male rats, which was also observed in the reference estrogen ethinyl estradiol treated groups. With the exception of the aforementioned, the statistically significant changes associated with BPA treatment were mostly sporadic and not dose-dependent with only one out of five BPA dose groups showing a statistical difference. In addition, the observed BPA-associated alterations were mostly moderate in magnitude and showed no persistent trend over the one-year time period. Based on these findings, we conclude that the observed BPA-mediated changes observed in this study are unlikely to alter immune competence in adult rats.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12 h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized that TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes.

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.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical aryl hydrocarbon receptor (AhR) ligand, exhibits immune suppression in vivo and in vitro. Suppression of primary humoral immune responses in particular has been well characterized as one of the most sensitive functional immune endpoints in animals treated with TCDD. Previous studies have used purified TCDD to elucidate the mechanisms by which TCDD and dioxin-like compounds (DLC) impair IgM production by B cells, but did not represent the route by which animals and humans are likely to be exposed environmentally. In the studies reported here, mice were treated with TCDD adsorbed onto a well-defined synthetic silica phase of known purity and physical properties, followed by sensitization with sheep erythrocytes to initiate a humoral immune. We found that surfactant-templated mesoporous forms of amorphous silica provided an ideal combination of purity, dispersibility and textural properties for immobilizing TCDD. TCDD-adsorbed silica distributed to the spleen and liver after oral administration as assessed by induction of cyp1a1 gene expression. Most notably, TCDD delivered in the adsorbed state on amorphous silica and as a solute in corn oil (CO) produced similar suppression of the anti-sheep red blood cell immunoglobulin M antibody forming cell response (sRBC IgM AFC) response at equivalent doses of TCDD. These results suggest that TCDD immobilized on silicate particles found in soils distributes to the spleen and suppresses humoral immunity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic environmental contaminant found in soils and sediments. Because of its exceptionally low water solubility, this compound exists predominantly in the sorbed state in natural environments. Clay minerals, especially expandable smectite clays, are one of the major component geosorbents in soils and sediments that can function as an effective adsorbent for environmental dioxins, including TCDD. In this study, TCDD was intercalated in the smectite clay saponite by an incipient wetness method. The primary goal of this study was to intercalate TCDD in natural K-saponite clay and evaluate its immunotoxic effects in vivo. The relative bioavailability of TCDD was evaluated by comparing the metabolic activity of TCDD administered in the adsorbed state as an intercalate in saponite and freely dissolved in corn oil. This comparison revealed nearly identical TCDD-induced suppression of humoral immunity, a well-established and sensitive sequela, in a mammalian (mouse) model. This result suggests that TCDD adsorbed by clays is likely to be available for biouptake and biodistribution in mammals, consistent with previous observations of TCDD in livestock exposed to dioxin-contaminated ball clays that were used as feed additives. Adsorption of TCDD by clay minerals does not appear to mitigate risk associated with TCDD exposure substantially.

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Previous studies have demonstrated that most of the intraspecies variation in sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including suppression of antibody responses, in murine models is due to single nucleotide polymorphisms (SNPs) within the aryl hydrocarbon receptor (AhR) gene. The underlying reason for variation in sensitivity to TCDD-induced suppression of IgM responses among humans is not well understood, but is thought, in part, to be a result of different polymorphic forms of the AhR expressed by different individuals. In this study, the functional properties of six (P517S, R554K, V570I, V570I+P517S, R554K+V570I and P517S+R554K+V570I) human AhR variants were examined in the human B cell line, SKW 6.4. TCDD-induced Cyp1B1 and Cyp1A2 mRNA expression levels and Cyp1B1-regulated reporter gene activity, used for comparative purposes, were markedly lower in SKW cells containing the R554K SNP than in SKW-AHR+ (control AhR) cells. Furthermore, all AhR variants were able to mediate TCDD-induced suppression of the IgM response; however, a combined P517S+R554K+V570I variant partially reduced sensitivity to TCDD-mediated suppression of IgM secretion. Collectively, our findings show that the R554K human AhR SNP alone altered sensitivity of human B cells to TCDD-mediated induction of Cyp1B1 and Cyp1A2. By contrast, attenuation of TCDD-induced IgM suppression required a combination of all three SNPs P517S, R554K, and V570I.

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