Evaluation of Prenatal Exposure to Bisphenol Analogues on Development and Long-Term Health of the Mammary Gland in Female Mice
Tucker DK, Hayes Bouknight S, Brar SS, Kissling G, Fenton S
Environmental Health Perspectives(2018) DOI: https://doi.org/10.1289/EHP3189 PMID:30102602
Background: Continued efforts to phase out bisphenol A (BPA) from consumer products have been met with the challenges of finding safer alternatives.
OBJECTIVES: This study aimed to determine whether early life exposure to BPA, and its related analogues, bisphenol AF (BPAF) and bisphenol S (BPS), could affect female pubertal mammary gland development and long-term mammary health in mice.
METHODS: Timed pregnant CD-1 mice were exposed to vehicle, BPA (0.5, 5, 50 mg/kg), BPAF (0.05, 0.5, 5 mg/kg), or BPS (0.05, 0.5, 5 mg/kg) via oral gavage between gestation days 10-17. Mammary glands were collected from resulting female offspring at PND 20, 28, 35 and 56, and at 3, 8, and 14 months for whole mount, histopathological evaluation, and qPCR; serum steroid concentrations were also measured at these time points.
RESULTS: In the bisphenol-exposed mice, accelerated mammary gland development was evident during early puberty and persisted into adulthood. By late adulthood, mammary glands from bisphenol-exposed female offspring exhibited adverse morphology compared with controls; most prominent were undifferentiated duct ends, significantly more lobuloalveolar hyperplasia and perivascular inflammation, and various tumors, including adenocarcinomas. Effects were especially prominent in the BPAF 5 mg/kg and BPS 0.5 mg/kg groups. Serum steroid concentrations and mammary mRNA levels of Esr1, Pgr, Ar, and Gper1 were similar to controls.
CONCLUSIONS: These data demonstrate that prenatal exposure of mice to BPAF or BPS induced precocious development of the mammary gland, and that siblings were significantly more susceptible to spontaneous pre-neoplastic epithelial lesions and inflammation, with an incidence greater than that observed in vehicle- and BPA-exposed animals.
Figure 1.Study Design Summary
Time-pregnant mice were gavage dosed from gestation day (GD) 10.5 until 17.5, twice daily (BID), with bisphenol A (BPA) 50 (n = 11), 5 (n = 12), or 0.5 (n = 13) mg/kg body weight (bw); bisphenol AF (BPAF) 5 (n = 11), 0.5 (n = 11), or 0.05 (n = 10) mg/kg bw; bisphenol S (BPS) 5 (n = 12), 0.5 (n = 11), or 0.05 (n=12) mg/kg bw; or vehicle control (pure sesame oil, n=12). Procedures were performed on various postnatal days (PND), including 3, 8 and 14 months (mo.).
Figure 2. Pubertal assessment in CD-1 prenatally-exposed female offspring
Pubertal assessment in CD-1 female offspring prenatally exposed to bisphenol A (BPA), bisphenol AF (BPAF), or bisphenol S (BPS). Data are reported as mean age with standard error bars (days old) at A) Vaginal opening (VO) and B) Occurrence of first estrus for the various doses of bisphenols. No statistical differences were observed when bisphenol-exposed animals were compared with vehicle controls using mixed effects ANOVA, with a Dunnett’s post-hoc test. Number of animals per group is noted in Table 1.
Figure 3. Quantitative mammary gland measures in female offspring at PND 20
Sholl analysis (number of intersections, branching density) and quantitative mammary gland measurements in bisphenol A (BPA), bisphenol AF (BPAF), or bisphenol S (BPS) prenatally-exposed female offspring at postnatal day (PND) 20. Mean ± SEM. *p < 0.05 and **p< 0.01 is statistically significant compared with vehicle control using Mann Whitney test. Jonckheere’s test was used to determine statistical dose-response trends within treatment group (denoted with a bar). Number of animals per group is noted in Table 1.
Figure 4. Representative mammary gland whole mounts on PND 20 and 35
Bisphenol AF (BPAF)-exposed females on postnatal day (PND) 20 are in the top panel and bisphenol S (BPS)-exposed females on PND 35 are in the bottom panel. Each image represents the mean scores shown in Table 1 for these ages/dose groups. A and E) Vehicle Control, B) BPAF 0.05 mg/kg, C) BPAF 0.5 mg/kg, D) BPAF 5 mg/kg, F) BPS 0.05 mg/kg, G) BPS 0.5 mg/kg and H) BPS 5 mg/kg. Scale bar=1 mm (A-H). Arrowheads denote terminal end buds increased in mid and high dose BPAF groups, LN=lymph nodes, and arrows indicate the area of the fat pad in which the 4th and 5th glands typically meet. BPS-treated animals demonstrated precocious epithelial growth, and 4th and 5th gland ends met sooner than controls. Number of animals per group is noted in Table 1.
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Figure 5. Occurrence of terminal end buds (TEBs) in mammary glands of 3-month old females
Representative images shown are A) Control and B) 0.5 mg/kg bisphenol AF (BPAF)-exposed mammary whole mounts. Inset: Arrows represent putative TEBs retained at 3 mo. in BPAF-exposed mammary gland. Scale bar=1 mm (A-B, inset). C) Occurrence of retained TEBs was determined as a percentage of total animals in each dose group (apparent/none) with TEBs. Vehicle (n=8), BPA 0.5 (n=11), BPA 5 (n=11), BPA 50 (n=9), BPAF 0.05 (n=9), BPAF 0.5 (n=8), BPAF 5 (n=7), BPS 0.05 (n=10), BPS 0.5 (n=8), and BPS 5 (n=8).
Figure 6. Histological presentation of mammary lesions after early life bisphenol analogue exposure
Representative images illustrate some non-neoplastic lesions: A) Squamous metaplasia, 20x, bar = 10 µm, B) Intraductular hyperplasia 20x, bar = 10 µm, and D) Perivascular inflammation, 10x, bar = 20 µm. Representative image of a neoplastic lesion in C) Papillary adenocarcinoma, 20x. Bar = 10 µm.
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Figure 7. Steroid serum levels from exposed females during pubertal development
Serum estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA) concentrations at postnatal days (PND) A, E, I, M) PND 20; B, F, J, N) PND 28; C, G, K, O) PND 35; and D, H, L, P) PND 56, respectively. Mean ± SEM. *p<0.05 is statistically significant compared with vehicle control by Dunnett’s test. Number of animals per group is noted in Table 1.
Figure 8. Expression of steroid receptor mRNA in the mammary glands at 8 and 14 months
Expression of steroid receptor mRNA in the mammary glands of bisphenol A (BPA), bisphenol AF (BPAF), or bisphenol S (BPS)-exposed females at A) 8 and B) 14 months. Mean ± SEM. * p < 0.05 and ** p < 0.01 is statistically significant compared with vehicle control. n= 3-4 litters represented, except for 8 mo. old BPS 0.05 mg/kg group (n=1).
- Figure 8 (2 MB)
- Figure 8 Excel Raw Data - 8 mos (1 MB)
- Figure 8 Excel Raw Data - 14 mos (1 MB)
- Figure 8 Graphpad Raw Data (45 KB)
Table 1. Pubertal mammary gland development scores
Pubertal mammary gland development scores of female offspring exposed in utero to bisphenol A (BPA), Bisphenol AF (BPAF), and bisphenol S (BPS).
Table 2. Mammary gland whole mount evaluation
Mammary gland whole mount evaluation results in 3-month old mice
Table 3. Mammary gland lesion incidences by age and treatment group
Mammary gland lesion incidences by age and treatment group following prenatal exposure to bisphenol A (BPA), bisphenol AF (BPAF), and bisphenol S (BPS).
Table 4. Evaluation of combined incidences
Evaluation of combined incidences of mammary gland inflammation, neoplasia, non-neoplastic lesions, or lymph node effects at 14 months of age following prenatal exposure to bisphenol A (BPA), bisphenol AF (BPAF), or bisphenol S (BPS).
Supplemental Figure S1. Pubertal and adult body weight gain in female offspring
Pubertal and adult body weight gain in female offspring of CD-1 dams treated from gestation day (GD) 10 - GD 17 with A) bisphenol A (BPA), B) bisphenol AF (BPAF), or C) bisphenol S (BPS). * p < 0.05 is statistically significant on PND 35 in BPAF 0.5 mg/kg/bw and BPS 5 mg/kg/bw groups compared to vehicle control using ANOVA and a Dunnett’s t-test. Litters: Vehicle (n = 9), BPA 0.5 (n = 12), BPA 5 (n = 12), BPA 50 (n = 9), BPAF 0.05 (n = 9), BPAF 0.5 (n = 11), BPAF 5 (n = 11), BPS 0.05 (n = 12), BPS 0.5 (n = 11) and BPS (n = 10).
Supplemental Figure S2. Female serum hormone levels at 3, 8, and 14 months
Effects of prenatal bisphenol A (BPA), bisphenol AF (BPAF), or bisphenol S (BPS) exposure on female serum hormone levels at 3, 8, and 14 months. A, D, G, and J = 3 months; B, E, H, and K = 8 months; and C, F, I, and L = 14 months. Mean ± SEM. Litter n=2-6 per treatment group. *p<0.05 and **p < 0.01 is statistically significant compared to vehicle control by ANOVA and Dunnett’s test.
Supplemental Table S1. Record of animals with mammary gland diagnoses by age
Record of vehicle and treated animals > 3 months of age with mammary gland diagnoses by age
Supplemental Table S2. Estrous cycle phase and duration at PND63-83
Number of days (d) spent in estrus, diestrus and proestrus following in utero exposure to bisphenol A (BPA), bisphenol AF (BPAF), or bisphenol S (BPS) during postnatal days (PND) 63-83