Effects of whole life exposure to Bisphenol A or 17α-ethinyl estradiol in uterus of nulligravida CD1 mice

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) with known estrogenic activity. Exposure to BPA in adult mice was shown previously to increase uterine pathology with associated alterations in the immune response and fibrosis. Reported here are uterine histopathology findings from CD1 mice exposed to BPA or 17α-ethinyl estradiol at multiple doses from conception through postnatal day 90. Along with uterine pathology, impacts of exposure on collagen accumulation and F4/80 positive macrophage numbers, as an indicator of immune response in the endometrium and myometrium, are presented. These companion data are from offspring (F1) of the dams analyzed for effects of adult exposures published in the Reproductive Toxicology manuscript titled “Strain-Specific Induction of Endometrial Periglandular Fibrosis in Mice Exposed during Adulthood to the Endocrine Disrupting Chemical Bisphenol A” (doi: 10.1016/j.reprotox.2015.08.001).


a b s t r a c t
Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) with known estrogenic activity. Exposure to BPA in adult mice was shown previously to increase uterine pathology with associated alterations in the immune response and fibrosis. Reported here are uterine histopathology findings from CD1 mice exposed to BPA or 17α-ethinyl estradiol at multiple doses from conception through postnatal day 90. Along with uterine pathology, impacts of exposure on collagen accumulation and F4/80 positive macrophage numbers, as an indicator of immune response in the endometrium and myometrium, are presented. These companion data are from offspring (F1) of the dams analyzed for effects of adult exposures published in the Reproductive Toxicology manuscript titled "Strain-Specific Induction of Endometrial Periglandular Fibrosis in Mice Exposed during Adulthood to the Endocrine Disrupting Chemical Bisphenol A" (doi: 10  The dose response analysis affords comparative assessment of uterus estrogen-sensitivity and identification of differences between strains and life-stages

Data
The results in Table 1 are pathology analysis presenting the incidence of distended glands, uterine cysts, and the density of gland nests in the endometrium for control and each BPA and EE exposure group at PND 21, 49, and 90. Significant age-related increases in distended glands were observed in most exposure groups. As expected for the CD1 strain, the density of gland nests was very low in all groups at PND90. No significant exposure related differences in gland nest densities Examples of photomicrographs for H&E and picrosirius red-stained uterine sections from control, 30 ppm BPA, and 0.01 ppm EE exposure groups are presented in Fig. 1. The percentage of F4/80-positive cells was used as a marker of immune response and was quantified for CD1 mice at PND90 in control and each BPA or EE exposure group (Fig. 2)

Animal husbandry and necropsy
All animal procedures were performed in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee and followed recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Mice were acclimated to a polycarbonate-free caging system that limited contamination from exogenous estrogenic compounds. The composition of the control and test diets used were described in detail previously [1][2][3]. Dosing was experimentally designed to span a range of exposures below the acceptable oral limit of BPA for humans (50 mg/kg body weight/day) and approach the no observed adverse effect level (NOAEL) for BPA of 50000 mg/kg/day [4][5][6]. Diets containing BPA at known concentrations (0.03, 0.3, 3, 30, and 300 ppm) resulted in doses of 4, 40, 400, 4000, and 40000 mg/kg/day, and concentrations of EE (0.0001, 0.001, and 0.01 ppm) resulted in doses of 0.02, 0.2, and 1 mg/kg/day. Control and experimental diet was fed to sires, dams and analyzed offspring ad libitum throughout their lifespan. Dams and sires were exposed to BPA or EE for two weeks prior to mating. To best replicate the human exposure throughout the differing life stages, offspring exposure occurred through maternal consumption of test diet during the fetal and lactation periods, with uninterrupted dietary exposure continuing through sacrifice at PND90. The female offspring (F1) were separated from dams at PND 21 and housed by exposure group 5 per cage. The nulligravida females analyzed here were sacrificed in estrus based on a cytological analysis of vaginal lavage, with staging confirmed based on uterus morphology. Full details for the study can be found in Kendig et al. [2].

Histology and immunohistochemistry
Tissues were embedded, sectioned, and stained with H&E as previously described [1,2,7]. Stained sections were examined on a Nikon Eclipse 55i microscope using a DS-Fi1 CCD camera controlled with Digital Sight Software (Nikon; Melville, NY). Uterine tissue from CD1 offspring at PND21, 49, and 90 was examined for distended glands, cysts, and gland nests as described [1].

Statistical analysis
The observer was blinded to exposure group. The litter was used as the statistical unit; one animal was randomly selected from each litter as a representative of that litter. Pathology was assessed with statistical differences analyzed using Fisher's exact test or χ 2 test for trend when assessing across age.
Gland nest density was analyzed using a one-way ANOVA and Dunnett's multiple comparison test. The percentage of F4/80-positive cells was analyzed using a two-way ANOVA and Bonferroni post-hoc test, in which exposure and location in uterus (endometrium or myometrium) were used as the main effects. Outliers were removed based on Grubb's test. Significance between differences in values was defined as p o0.05. All data was analyzed using GraphPad Prism s v5/v6 software (GraphPad; La Jolla, California).