The effects of maternal exposure to bisphenol A on allergic lung inflammation into adulthood

Abstract

Bisphenol A (BPA) is a high-production volume chemical classified as an environmental estrogen and used primarily in the plastics industry. BPA's increased usage correlates with rising BPA levels in people and a corresponding increase in the incidence of asthma. Due to limited studies, the contribution of maternal BPA exposure to allergic asthma pathogenesis is unclear. Using two established mouse models of allergic asthma, we examined whether developmental exposure to BPA alters hallmarks of allergic lung inflammation in adult offspring. Pregnant C57BL/6 dams were gavaged with 0, 0.5, 5, 50, or 500 μg BPA/kg/day from gestational day 6 until postnatal day 21. To induce allergic inflammation, adult offspring were mucosally sensitized with inhaled ovalbumin containing low-dose lipopolysaccharide or ip sensitized using ovalbumin with alum followed by ovalbumin aerosol challenge. In the mucosal sensitization model, female offspring that were maternally exposed to ≥ 50 μg BPA/kg/day displayed enhanced airway lymphocytic and lung inflammation, compared with offspring of control dams. Peritoneally sensitized, female offspring exposed to ≤ 50 μg BPA/kg/day presented dampened lung eosinophilia, compared with vehicle controls. Male offspring did not exhibit these differences in either sensitization model. Our data demonstrate that maternal exposure to BPA has subtle and qualitatively different effects on allergic inflammation, which are critically dependent upon route of allergen sensitization and sex. However, these subtle, yet persistent changes due to developmental exposure to BPA did not lead to significant differences in overall airway responsiveness, suggesting that early life exposure to BPA does not exacerbate allergic inflammation into adulthood.

FIG. 1.

Airway cell influx in the mucosal sensitization model. Adult female (N = 4–6) or male (N = 5–8) offspring of dams treated daily with BPA (50 or 500 µg/kg) or the vehicle control were sensitized (i.t.) with OVA alone or OVA + LPS prior to aerosol challenge. Differential cell counts of H&E stained BAL cells (200 cells counted/slide) were acquired using coded slides. Graphs depict the mean percentage (± SEM) of eosinophils (A, D), neutrophils (B, E), and lymphocytes (C, F) in developmentally exposed adult female (A–C) and male (D–F) offspring. Please note that in order to make comparison of sex differences easier, the same y-axis scaling for each cell type is used to present data from female and male offspring. An asterisk (*) indicates p ≤ 0.05, for comparisons of means between the same sensitization/challenge group, but different BPA exposure groups. Data are representative of findings from two independently performed experiments.

FIG. 2.

Developmental exposure to BPA alters overall lung inflammation. Adult female (N = 5–6) or male offspring (N = 3–6) of BPA exposed or vehicle control dams were sensitized (i.t.) with OVA alone or OVA + LPS prior to aerosol challenge. Scoring was performed by three independent scientists, using coded slides. Representative images are from H&E-stained lung tissue at 40× magnification (A, C) and depict the cumulative histopathological assessment. Graphs (B, D) present the mean inflammation scores for each group (error bars = SEM). An asterisk (*) indicates p ≤ 0.05 when mean values from the same sensitization/challenge group, but different BPA exposure groups, were compared.

FIG. 3.

Effects of developmental exposure to BPA on T-lymphocyte subpopulations in the lung. Lung tissues were examined from adult female (N = 5–7) or male offspring (N = 5–8) from dams exposed to BPA or vehicle control. Immune cells were obtained from intact lung, stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry, as described in the Materials and Methods section. (A) Representative dot plots show the gating strategy for lung lymphocyte populations. After gating on CD45⁺ cells (all leukocytes), fluorescence minus one (FMO) controls were used to identify CD4⁺ and CD8⁺ cells, or CD4⁺ cells that are CD25⁺FoxP3⁺ (T_regs). The number in each region of the dot plot indicates the percentage of cells in that gate. (B–G) The graphs depict the average number (± SEM) of CD4⁺ (B, E), CD8⁺ (C, F), and CD4⁺CD25⁺FoxP3⁺ (D, G) lymphocytes after mucosal allergic sensitization and challenge. Asterisks (*) indicate p ≤ 0.05 when mean values from the same sensitization/challenge group, but different BPA exposure groups, were compared. The same y-axis scaling for each cell type is used to simplify comparisons across sex.

FIG. 4.

Exposure to lower maternal doses of BPA does not affect airway cell populations in a mucosal sensitization model. Airway cells from adult female (A–C) or male (D–F) offspring (6–8 mice/group) maternally exposed to 0, 0.5, or 5 µg BPA/kg/day were assessed. The total number of cells collected per mouse was determined, and differential cell counts were performed to enumerate leukocytes in BAL cells (200 BAL cells counted/mouse). The graphs depict the average number (± SEM) of eosinophils (A, D), neutrophils (B, E), and lymphocytes (C, F). The same y-axis scaling is used for each cell type to simplify comparison of data from female and male offspring; however, the scaling is different from the data presented in Figure 1. An * indicates p ≤ 0.05 between same sensitization/challenge group, but different maternal BPA dose.

FIG. 5.

Lung lymphocyte populations in offspring maternally exposed to < 50 μg BPA/kg/day. Adult female (A–C), or male offspring (D–F) from dams exposed daily to BPA or vehicle control were compared after mucosal sensitization modeling. Immune cells from the lung were obtained, processed, and analyzed as in Figure 3. Data are expressed as mean number (± SEM) of each lymphocyte subset from 6–8 mice/group. Asterisks (*) indicate p ≤ 0.05 between same sensitization/challenge group, but different maternal BPA doses. The same y-axis scaling for each cell type is used to simplify sex comparisons.

FIG. 6.

Maternal BPA exposure alters several endpoints in offspring using a peritoneal sensitization model. Dams were treated with BPA doses ranging from 0.5 to 500 µg/kg/day from day of pregnancy 6 through postpartum day 21. Adult female offspring were sensitized by i.p. injection of OVA in alum and challenged with OVA aerosol. Airway cells, lung-derived immune cells, and blood were obtained from mice in each group. Total cellular recovery was determined and differential cell counts of H&E stained airway cells (200 cells/slide) were assessed by morphology to determine the percentage and number of eosinophils (A), neutrophils (B), and lymphocytes (C). Lung-derived (interstitial) immune cells were collected by collagenase digestion and stained for flow cytometry. Graphs show the average number of lung eosinophil (D) and CD4⁺ lymphocyte (E) populations in each group. OVA-specific IgE was accessed by stacking ELISA (F). All data are expressed as mean ± SEM for 4–5 mice/group; *p ≤ 0.05 between same sensitization/challenge group, but different BPA exposure dose.

FIG. 7.

Maternal exposure to BPA did not adversely affect airway hyperresponsiveness measurements of lung resistance (A) or elastance (B) in adult female offspring. Pressure measurements, postbaseline, were obtained in response to airway challenge with saline and increasing concentrations of aerosolized methacholine using a jet nebulizer. Resistance and elastance measurements to describe overall airway function were derived from the pressure measurements. Data are expressed as mean value for each group + SEM from 4–5 mice per group. *p ≤ 0.05 between mice of the same sensitization/challenge group, but different maternal BPA exposure dose.