C57BL/6 mice (6–8 weeks; NCI, Bethesda, MD) were housed in prewashed polysulfone microisolator cages under pathogen-free conditions. Mice received soy-free, AIN76-semi-PD1RR chow (Test Diet, Richmond, IN) to avoid phytoestrogenic effects that might mimic possible estrogenic effects of BPA. Glass water bottles and reverse osmosis-purified water were used. Paired mice were checked daily for vaginal plugs, indicating pregnancy. Pregnant females were housed singly and initially treated by oral gavage with 50 or 500 μg BPA/kg/day (d) (Sigma-Aldrich, St Louis, MO) or peanut oil vehicle control, beginning on gestational day 6 and continuing daily through postnatal day (PND) 21. An estrogen control was not used as BPA may act via other receptors in addition to ERs; thus it is not clear that BPA and natural estrogens will have the same effects (Chung et al., 2011
). The results of these initial experiments and new information in the literature prompted the subsequent examination of maternal exposure to lower BPA doses (0.5 and 5 μg BPA/kg/day). Dosing to the dam was initiated on the 6th day of pregnancy to eliminate any possible effects of BPA on implantation, which occurs in the mouse between the 4th and 5th day of gestation, and all pups were weaned on PND 21, at which time all maternal transfer of BPA ceased. Offspring were not directly given any BPA. The selected dose range spans the current tolerable daily intake of 50 μg BPA/kg/day (USEPA, 2010
). Maternal treatment with these doses of BPA did not affect offspring bodyweight, which was monitored over the life span of the offspring, pregnancy duration, litter size, or the sex ratio of litters (Roy et al., 2012
Using a subset of pregnant dams, we determined the amount of circulating BPA detected following this oral dose of BPA. BPA was measured at the Laboratory of Analytical Chemistry, New York State Department of Health, using high performance liquid chromatography coupled with electrospray triple-quadrupole mass spectrometry (Padmanabhan et al., 2008
). Four hours after gavage of pregnant mice, free and total BPA were measured (7 mice per treatment group). The mean circulating levels of total BPA in pregnant mice given 50 µg/kg/day were 2.941ng/ml, whereas mean circulating BPA levels in vehicle control–treated dams was 0.434ng/ml (p
= 0.0173). Interestingly, in the BPA-treated group, none of the samples tested were below the limit of quantitation (LOQ), whereas in the samples from the group dosed with peanut oil vehicle, 4 out of 7 samples were below the LOQ.
On PND 21, offspring were weaned and housed in same-sex groups under the BPA-controlled conditions described above until maturity (6–10 weeks). Over the course of this research, several separate cohorts of maternally exposed mice were generated over a period of many months. Vehicle-treated dams were included in all pregnancy cohorts, so that BPA exposed offspring and vehicle-exposed offspring were age matched and derived from a single shipment of nulliparous mice (i.e., each batch of mice that we used as dams). Our experimental strategy utilized the available offspring in the various exposure groups as they were available. For all presented data, the offspring of vehicle- and BPA-treated dams were impregnated and treated in the same time frame. For experiments, age and sex-matched, maternally exposed, mature offspring were derived from separate vehicle- or BPA-treated dams. For example, a group with an N = 5 contained five mice, each from a different exposed dam of the same vehicle or BPA dose. Animal work was done in accordance with protocols approved by the University of Rochester Animal Care and Use Committee.
Mucosal sensitization model
. Mice were anesthetized with avertin ((2,2,2-tribromoethanol); Aldrich, Milwaukee, WI) and intratracheally (i.t.
) sensitized on days 0, 1, and 2 with 100 μg of endotoxin-depleted ovalbumin (OVA), with, or without, 100ng of Escherichia coli
lipopolysaccharide (LPS) (O55:B5; Sigma-Aldrich) in PBS (50 μl/mouse). OVA (Grade V, Sigma-Aldrich) was endotoxin-depleted using Endotoxin Detoxi-Gel (Pierce, Rockford, IL). This protocol results in TH
2-type allergic airway inflammation following aerosol OVA challenge (Eisenbarth et al., 2002
). Mice were challenged twice per day on days 14, 15, and 16 with aerosolized 1% OVA in PBS (1h), using a jet nebulizer (DeVilbiss, Somerset, PA) and constant airflow rate. Forty-eight hours after the final aerosol challenge, mice were sacrificed by avertin overdose. We chose the 48-h time point based on prior studies and to better compare outcomes with mice in the peritoneal sensitization groups (Hogaboam et al., 2008
; Lawrence et al., 2008
Peritoneal sensitization model
. Mice were ip sensitized on days 0 and 4 with PBS or 100 μg OVA in PBS with 100 μl Imject Alum (Pierce; 200 μl/mouse). Although this is an artificial route of allergen encounter, this widely used model relies on the robust TH
2-promoting properties of injected alum. Mice were challenged once (1h), on day 11, with aerosolized 1% OVA in PBS, using a challenge chamber, jet nebulizer, and constant airflow rate. Forty-eight hours postaerosol challenge, mice were sacrificed using avertin overdose. In previous studies, inflammatory cells, cytokines, and OVA-specific IgE were detected 48h after single aerosol challenge (Hogaboam et al., 2008
; Lawrence et al., 2008
). Based on this work, we elected to use the time point for the current study.
Histological analysis. Lungs were fixed in situ with 10% neutral-buffered formalin, embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin (H&E). Coded samples were scored for tissue inflammation by at least two different scientists using the following scale: 0, no airway involvement, few/no inflammatory cells in tissue; 1, limited inflammation, small number of airways with inflammatory cell clusters, most airways have no inflammation; 2, modest inflammation, several airways with small inflammatory cell clusters, no areas of intense infiltrate; 3, moderate-to-intense inflammation, many airways with inflammatory cells and occasional areas of intense infiltrate; 4, significant airway inflammation with multiple intense foci, massive perivascular/periairway inflammation, most/all airways involved. The combined scores of five lobes resulted in an overall number. The maximum possible inflammation score for each whole lung was 20.
. Lungs were lavaged as previously described (Warren et al., 2000
). The supernatant from the first 1ml wash was frozen at −80°C for cytokine analysis, whereas recovered cells from all washes were pooled. A TC-10 (Bio-Rad, Hercules, CA) was used to calculate the total number of cells recovered. Bronchoalveolar lavage (BAL) cells (5×104
) were spun onto glass microscope slides using a cytospin (Shandon Scientific, Waltham, MA), fixed, and stained with H&E.
Flow cytometric analysis of lung-derived immune cells
. The pulmonary vasculature was perfused (PBS) via the right ventricle, and immune cells were obtained from collagenase-digested whole lung as described previously (Neff-LaFord et al., 2007
). Leukocytes were filtered, washed, and counted, and nonspecific binding was blocked using anti-CD16/32 and rat IgG. Subsequently, cells were stained with fluorochrome-conjugated antibodies against cell surface CD4, CD8, CD11b, CD11c, CD25, CD45.2, MHCII, Siglec-F, or the intracellular protein, FoxP3 (eBioscience, San Diego, CA; BD Pharmingen, San Jose, CA). For the latter, cells were permeabilized with saponin postfixation and incubated with an anti-FoxP3 antibody. A live/dead stain (Invitrogen, Carlsbad, CA) was used to selectively gate on live cells during analyses. Events were acquired using an LSR II cytometer (BD Biosciences, Franklin Lakes, NJ) with subsequent analysis using FlowJo 8.8.7 (Tree Star, Ashland, OR).
OVA-specific antibody measurements
. Circulating OVA-specific IgE levels were analyzed by stacking ELISA (Hogaboam et al., 2008
). Serial dilutions of OVA-specific IgE (GeneTex, Irvine, CA) and plasma from unexposed/unsensitized/unchallenged C57BL/6 mice were used as positive and negative controls, respectively. Plasma from exposed mice was serially diluted, and biotinylated anti-mouse IgE subclass antibody (eBioscience) was used, in combination with avidin-peroxidase and ABTS substrate (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, Sigma-Aldrich) to detect OVA-specific IgE in the samples.
Measurement of airway hyperresponsiveness. Airway responsiveness to methacholine was assessed using the FlexiVent, invasive, airway plethysmography system (Scireq, Chandler, AZ) and analyzed using Lab Chart Version 6 software (ADInstruments, Inc., Colorado Springs, CO). Mice were paralyzed with succinylcholine, anesthetized with avertin, tracheostomized, and intubated with a cannula connected to a ventilator. The system was calibrated for airflow and pressure. Pressure measurements, postbaseline, were obtained in response to airway challenge with saline and increasing concentrations of aerosolized methacholine in saline (0.1, 0.3, 1, 3, 10, 30, and 100mg/ml) using a jet nebulizer. Resistance and elastance measurements to describe overall airway responsiveness were derived from the pressure measurements. Resistance is a measurement describing a reduction in airflow due to factors such as bronchoconstriction, whereas elastance describes the “stretching/recoil” potential of the airway.
Statistical analysis. Given that the dams, not offspring, were directly exposed, all offspring from a litter are considered as a single statistical unit. Statistical analyses were performed on all measured endpoints using JMP software (Version 9, SAS, Cary, NC). Mean differences between independent variables were compared among treatment groups using two-way ANOVA, followed by a post hoc test (Tukey’s-Kramer HSD [Honestly Significant Difference]). Also, nonparametric analyses of data were performed using a Kruskal-Wallis test. Differences between two groups at a single point in time were evaluated using a Student’s t-test or Tukey’s-Kramer HSD test. Differences were considered significant when p values were less than 0.05. All statistical analyses were conducted within animals of the same sex.