Effects of perfluorooctanoic acid on mouse mammary gland development and differentiation resulting from cross-foster and restricted gestational exposures

Abstract

The adverse consequences of developmental exposures to perfluorooctanoic acid (PFOA) are established in mice, and include impaired development of the mammary gland (MG). However, the relationships between timing or route of exposure, and consequences in the MG have not been characterized. To address the effects of these variables on the onset and persistence of MG effects in female offspring, timed pregnant CD-1 dams received PFOA by oral gavage over various gestational durations. Cross-fostering studies identified the 5mg/kg dose, under either lactational- or intrauterine-only exposures, to delay MG development as early as postnatal day (PND) 1, persisting beyond PND 63. Intrauterine exposure during the final days of pregnancy caused adverse MG developmental effects similar to that of extended gestational exposures. These studies confirm a window of MG sensitivity in late fetal and early neonatal life, and demonstrate developmental PFOA exposure results in early and persistent MG effects, suggesting permanent consequences.

Fig. 1

Schematic of study design and implementation for (A) the late-life effects cross-foster study, (B) the early-life effects cross-foster study, and (C) the restricted-exposure study.

Fig. 2

MG development of female offspring in the late-life effects cross-foster study. (A) Whole mount preparations of mammary tissue from female offspring are shown at PND 22 (25×; a lymph node appears as a large darkly staining object), PND 42 (50×), and PND 63 (50×). Glands pictured are representative of mean respective scores (given in Table 1; N = 10–13 adult females per treatment group at PND 22, N = 9–18 per group at PND 42, N = 9–17 per group at PND 63). *Significant treatment effect by ANOVA, compared to control; p < 0.05. (B) On the left, whole mounts from representative adult female offspring at 18 months of age (16×). Large arrows indicate unusual, darkly staining foci; one small arrow in 5U indicates peripheral, localized increases in epithelial density observed in some PFOA-exposed animals at 18 months. On the right, histopathologic images from contralateral glands in the same animal show ductal areas that might account for darkly staining foci observed on whole mounts (400×; N = 5–12 females per group at 18 months). The arrow in 5U identifies an area of inflammation. In 5U + L the large arrow identifies an area of increased stromal density; the small arrow points to a potentially hyperplastic region of ductal epithelium. These ductal pathologies were observed in all treatment groups; some ductal inflammation was also seen in controls.

Fig. 3

MG development of female offspring in the early-life effects cross-foster study. Whole mount preparations of mammary tissue from female offspring at PND 1, 3, 5 (64×), and PND 10 (50×). The arrow in 5U + L on PND 1 identifies ductal epithelium. Glands pictured are representative of mean respective scores (Table 1; N = 4 litters per treatment group at each time point; three pup glands scored per litter). *Significant treatment effect by ANOVA, compared to control; p < 0.05.

Fig. 4

Serum PFOA concentrations in dams and female offspring from the early-life effects cross-foster study (offspring from the late-life effects study also shown on right for comparison; reported in [14]). Data are shown as mean ± S.E.M. bars; numerical values are shown for non-zero controls, and for control dams nursing treated pups at PND 1. (A) Among the dams, direct treatment with PFOA consistently yielded higher serum level than nursing treated pups (5U) alone. (B) Among offspring, lactationally exposed females (5L) exhibited serum concentrations that increased until PND 10, when they converged upon concentrations observed in the intrauterine (5U) and combined exposure (5U + L) groups. At PND 1, treated pups (5U, 5U + L) exhibited higher serum PFOA concentrations than treated dams (nursed 5L, 5U + L pups). By PND 10 all exposed offspring groups were becoming similar to one another in serum concentrations, and were becoming increasingly similar in serum concentrations to their paired dams. In the late-life effects study (right), by PND 63 serum from female offspring in all treatment groups were near background levels, at less than 1000 ng/ml. Statistical comparisons are provided in the text.

Fig. 5

MG differentiation of lactating dams in the early-life effects cross-foster study. Whole mount preparations of mammary tissue from lactating dams are shown on PND 1, the first day of lactation (40×). Glands pictured are representative of lactating dams in respective groups at LD 1 (body weights given in Table 2; N = 4 dams per treatment group at each time point).

Fig. 6

MG development of female offspring in the restricted-exposure study. (A) Whole mount preparations of mammary tissue are shown at PND 32 (16×). Glands pictured show morphology representative of respective treatment groups at given time points (Table 1; N = 10–20 females per treatment group at PND 32). *Significant treatment effect by ANOVA, compared to control; p < 0.05. (B) On the left, whole mount preparations of mammary tissue at 18 months of age are shown (16×). Large arrows identify unusual, darkly staining foci; small arrows identify peripheral, localized increases in epithelial density observed in some PFOA-exposed animals at 18 months. On the right, histopathologic images from contralateral glands in the same animal show ductal areas that might account for darkly staining foci observed on whole mounts (400×; N = 4–11 females per treatment group at 18 months). The large arrow in GD 15–17 identifies an area of increased stromal density; the small arrow points to a focus of inflammation. In GD 10–17 the arrow identifies a large, potentially hyperplastic region of ductal epithelium. These ductal pathologies were observed in all treatment groups; some ductal inflammation was also seen in controls.