The present studies demonstrate that exposure to PFOA throughout gestation (GD 1–17) or during the latter half of gestation (GD 10–17) at doses 10- to 30-fold lower than previously investigated are sufficient to produce abnormal mammary gland development in CD-1 mice. In both studies, low doses of PFOA elevated relative liver weights and stunted mammary gland development. Full-gestational exposure to ≥ 0.3 mg/kg PFOA resulted in reduced mammary gland developmental scores compared with controls at perinatal (PNDs 14 and 21), peripubertal (PND 42), and adult (PND 84) time points. Moreover, exposure to 30-fold lower doses of PFOA (0.01 mg/kg) during GD 10–17 suppressed mammary gland development as well. These data suggest that prenatal exposure to PFOA may alter mammary gland development in CD-1 mice at doses lower than investigated here. Additionally, effects on mammary tissue were observed at doses of PFOA lower than those required to exert an effect on the liver and the mammary effects persisted longer. These findings implied that in CD-1 mice, the mammary gland was more sensitive to prenatal PFOA exposure than was the liver.
In a previous study by Yang et al. (2009)
, peripubertal exposures to PFOA in Balb/C and C57Bl/6 mice resulted in mammary gland growth effects at doses ≥ 5 mg/kg but not at ≤ 1 mg/kg, even though elevated liver weights were observed at 1 mg/kg. Thus, the mammary gland effects presented from our studies appear to be the result of an increased sensitivity in the CD-1 mouse strain. We postulate that intraspecies differences in effects are more likely due to the timing of exposure, as there are strain differences in timing of puberty (Nelson et al., 1990
); yet, mammary gland morphology remains fairly consistent during stages of development (i.e., puberty and pregnancy). Further research is needed to determine if the sensitivity is attributed to timing of exposure or the mouse strain utilized or if there are other novel mechanisms underlying this apparent sensitivity in CD-1 mice.
The developmental scoring method utilized in these studies, which incorporated both qualitative and quantitative endpoints, accurately predicted the long-term mammary gland developmental delays seen in PND 84 full-gestation–treated animals. Following full-gestational PFOA exposure, several TEBs remained in PFOA-treated mammary glands at PND 84. Other studies have shown that the extended presence of TEBs, in general, can lead to long-term adverse effects on the gland, including a higher risk for mammary tumor formation following exposure to carcinogens (Russo and Russo, 1978
) and altered lactation (Rayner et al., 2005
). Although it is unclear whether there are lasting adult effects on the mammary gland due to late-gestation exposure, evaluation of mammary tissues indicated that prenatal exposure to doses as low as 0.01 mg PFOA/kg can also lead to developmental delays. Thus, we did not identify an NOAEL for PFOA-induced mammary gland developmental effects in CD-1 mice.
In the full-gestational study, relative liver weights were elevated in the 0.3 mg/kg group, which is lower than the previously reported liver LOAEL of 1.0 mg/kg in this mouse strain (1.0 mg/kg was the lowest dose utilized; Lau et al., 2006
; Wolf et al., 2008
). Another recent study found that prenatal exposure to 0.3 mg/kg of PFOA elevated absolute liver weights independent of BW differences (Onishchenko et al., 2011
). Importantly, in the late-gestation study, we used exposure periods that were half the length previously used; yet, this study produced comparable effects on liver weights at the same doses reported previously (Lau et al., 2006
). This warrants strength to the supposition that the LOAEL for PFOA-induced liver hepatomegaly in CD-1 mice is lower than 1.0 mg/kg and that the NOAEL is approximately 0.1 mg/kg.
PFOA did not appear to affect brain weights in our studies. However, the presence of PFOA in the tissue warrants further investigation, as it may impart other effects to the brain. A recent study (Johansson et al., 2008
) showed that low-level developmental exposure to PFOA produced behavioral effects in mice that extended into adulthood. Onishchenko et al. (2011)
reported that prenatal exposure to 0.3 mg/kg affected activity levels in mice independent of brain weight changes. The presence of PFOA in the neonatal brain, coupled with its absence after four weeks of age, suggests that PFOA passes through the fetal mouse blood-brain barrier but is not able to pass through the fully functional barrier that is normally formed by the time of birth (Bauer et al., 1993
Theoretically, animals dosed for longer periods are expected to exhibit higher serum concentrations than those exposed for shorter periods. However, as seen in , there are similarities in the serum PFOA concentration of 1.0 mg/kg females from the full-gestation study compared with those of the late-gestation study at PND 7. Other studies have found that there are comparable serum concentrations in adult CD-1 mice following differing lengths of exposure (Lau et al., 2006
). It is postulated that these similarities in PFOA serum concentrations are attributable to differences in clearance rates relative to exposure length. In a study by Lou et al. (2009)
, with repeated low doses of PFOA, net urinary elimination rates of PFOA were found to be high, potentially due to saturation of renal resorption. However, in the same study, urinary elimination rates were found to be low when resorption saturation was not reached. Thus, it is speculated that the longer exposure parameters for the full-gestation study offspring resulted in higher urinary elimination rates and consequently higher blood clearance rates, which may have reduced the serum PFOA concentration to comparable levels of the offspring exposed during late gestation. Additionally, it is important to consider the functional capacity of the developing urinary system in pups at this age relative to their ability to eliminate toxicants.
A temporal comparison revealed that the mean serum PFOA concentration and calculated blood burden of the 1.0 mg/kg group female offspring were greatest at PND 14 in the full-gestation study, whereas in the 1.0 mg/kg female offspring of the late-gestation study, the mean serum concentration was greatest at PND 1 and the calculated PFOA blood burden was greatest from PNDs 7 to 14 (, Supplementary tables 4
). A similar trend to that observed in the late-gestation females was also seen in mice given a single prenatal dose of PFOA with analytically measured, not calculated, body burdens (Fenton et al., 2009
After parturition, offspring of PFOA-treated lactating dams experience extended exposure via milk consumption (Fenton et al., 2009
). In addition to the PFOA that is transported into milk from the blood supply, the grooming habits of rodents further contribute to milk-borne exposure; PFOA present in urine of PNDs 1–10 offspring is consumed by dams and subsequently recirculated back into the maternal system (Rodriguez et al., 2009
). Due to the relationship between maternal grooming habits and pup exposure, pup urinary excretion rates can also influence the availability of PFOA for recycling by the dam. Therefore, higher urinary excretion rates in pups may indeed account for greater serum PFOA concentrations and blood burdens at the second week of age in those exposed throughout gestation compared with those exposed from GD 10 to 17.
Importantly, serum PFOA concentrations found in the 0.01 mg/kg group are lower than those measured in young children living in areas highly contaminated with PFOA, such as the Ohio River Valley in West Virginia (Emmett et al., 2006
). Emmett et al. (2006)
reported that the 2004 mean serum PFOA concentrations were near 600 ng/ml in children aged 2–5 years from the Ohio River Valley area. Approximately 2 years later, and after some exposure intervention, Frisbee et al. (2009)
reported mean serum concentration of 77.6 and 59.9 ng/ml in children from the C8 Health Project of age < 12 and 12–19 years, respectively. The late-gestation study conducted here observed mice only until weaning, which would be equivalent to 2–3 years of age in humans. Due to the elimination rates of PFOA in female mice (t1/2
~16 days, Lou et al., 2009
), it can be assumed that serum PFOA levels of pups in the 0.1 mg/kg treatment group would have decreased after PND 21 to levels approaching, if not lower, than those reported in children by Frisbee et al. (2009)
. Importantly, from the full-gestation study mammary glands, we observed that the developmental effects were not transient and in fact were apparent at adulthood, although PFOA exposure had ceased 12 weeks earlier. These findings are of great concern considering that children are likely to be exposed to PFOA prenatally, as well as throughout life. Therefore, it is important to determine the human relevance of the observed endpoints in relation to internal dosimetry to establish a benchmark dose for the PFOA mammary gland effects.
It is also critical to establish a mode of action (MOA) for the developmental mammary gland growth effects following PFOA exposure to determine whether this MOA is biologically relevant to humans. The liver toxicity and general developmental effects of PFOA are believed to be mediated by activation of peroxisome proliferator–activated receptor-alpha (PPAR-α) (Abbott et al., 2007
; Rosen et al., 2009
; Wolf et al., 2008
); yet, there are data to suggest that PFOA-induced mammary gland effects are mediated by other pathways (Zhao et al., 2010
). Previous studies using PPAR-α knockout (KO) mice reported normal lactation after exposure to PFOA (Zhao et al., 2010
). Changes in serum progesterone reported in the study of Zhao et al. (2010)
suggest that stimulatory, and potentially inhibitory, mammary gland effects may be mediated through endocrine disruption. PFOA may indirectly affect branching morphogenesis through modulation of progesterone synthesis (Zhao et al., 2010
), and other endocrine-disrupting effects of PFOA have been reported (White et al., 2011).
In the future, we plan to further compare mammary gland developmental effects in PPAR-α wild-type and KO mice after prenatal PFOA exposure to determine whether PPAR-α activation is involved in this outcome.
In summary, an NOAEL was not achieved in either study for PFOA-induced mammary gland effects in CD-1 mice, as altered mammary gland development was observed in offspring of dams treated with the lowest PFOA dose utilized in each study. As these are the lowest doses of PFOA tested in CD-1 mice thus far, additional studies are necessary to determine an NOAEL, as well as to establish the human relevance of PFOA-induced mammary gland effects.