Fetal Exposure of Rhesus Macaques to Bisphenol A Alters Cellular Development of the Conducting Airway by Changing Epithelial Secretory Product Expression

Background: Bisphenol A (BPA) exposure early in life results in organizational changes in reproductive organs, but the effect of BPA on conducting airway cellular maturation has not been studied. Late gestation is characterized by active differentiation of secretory cells in the lung epithelium. Objective: We evaluated the hypothesis that BPA exposure disrupts epithelial secretory cell development in the fetal conducting airway of the rhesus macaque. Methods: We exposed animals to BPA during either the second (early term) or the third (late term) trimester. There were four treatment groups: a) sham control early term, b) sham control late term, c) BPA early term (BPA-early), and d) BPA late term (BPA-late). Because cellular maturation occurs nonuniformly in the lung, we defined mRNA and protein expression by airway level using microdissection. Results: BPA exposure of the dam during late term significantly accelerated secretory cell maturation in the proximal airways of the fetus; both Clara cell secretory protein (CCSP) and MUC5AC/5B mRNA and protein expression increased. Conclusions: BPA exposure during late gestation accelerates secretory cell maturation in the proximal conducting airways. We identified a critical window of fetal susceptibility for BPA effects on lung epithelial cell maturation in the third trimester. This is of environmental health importance because increases in airway mucins are hallmarks of a number of childhood lung diseases that may be affected by BPA exposure.


METHODS
Animals: Animals were caged individually with a 0600-1800 hours light cycle with temperature maintained at 25-27°C. Animals were housed in stainless steel cages, fed a diet of Purina Monkey Chow, given water ad libitum via "lixit" device and provided seasonal produce, seeds and cereal as enrichment.
Lung Tissue Processing: The right cranial lobe was cannulated at the lobar bronchus and instilled with 1% paraformaldehyde at 25cm constant pressure for 1h. Tissue was fixed overnight (approximately 18 hrs) and the axial path airways microdissected and the generations noted.
Necropsies occurred on different days and care was taken to fix all lung tissue for the same length of time to allow comparison staining intensity. For high resolution histopathology, the right caudal lung lobe was cannulated and inflated at 25cm constant pressure with Karnovsky's fixative (0.9% glutaraldehyde/0.7% paraformaldehyde in cacodylate buffer, adjusted to pH 7.4, 330 mOsm) for 1h. Fixed lungs were sliced into cross sections and embedded in Araldite 502 resin. Blocks were sectioned at 1 micron and sections stained with methylene blue azure II (Van Winkle et al. 2004). The left cranial lung lobe was cannulated, inflated to capacity with RNALater and stored at -20°C until used for airway microdissection and qRT-PCR as described in ).
Immunohistochemistry and histochemistry: CCSP immunostaining-we used the avidin-biotin peroxidase method to detect primary antibody binding to the protein with a Vector ABC Kit following manufacturer's instructions. Nickel enhanced DAB (3,3'-diaminobenzidine; Sigma Chemical, St Louis MO) chromagen was used. A dilution series was used to determine optimal antibody concentration.
2 Morphometric histopathology: Whole slides were scanned at x40 on an Olympus VS110 virtual microscopy system scanner (Olympus, Tokyo, Japan). Scanned images were evaluated at x40 resolution for volume fraction and at x10 resolution for surface per volume. Standard morphometric approaches were used (Hsia 2010).The volume fraction of mucosubstancepositive cells was estimated using point (P) and intercept (I) counting of bronchial epithelium, utilizing vertical uniform random sine weighted probes and Visiopharm Integrator System software (Visiopharm, Hoersholm, Denmark). Volume fraction was calculated as: V v (epi) = P p = ΣP n /ΣP t where P p is the point fraction of P n , the number of test points hitting the positive epithelial cells, divided by P t , the total points hitting the reference space (epithelium). To Applied BioSystems Taqman® reagents, probes and primers. Purified RNA samples were stored 3 at -80°C until processed. cDNA (50ng) was made with the Taqman® cDNA RT-Transcription Kit (Applied Biosystems, Inc., Foster City, CA) and stored at -20°C. Reaction protocol: 25°C for 10 minutes, 48°C for 30 minutes, 95°C for 5 minutes then 4°C. All qRT-PCR samples were run in triplicate with RPL13A, a custom primer and probe assay, as the internal reference gene as this gene is recommended as a reference gene for rhesus monkey tissues. The sequences for