Timed-pregnant CD-1 mice were purchased from Charles River Laboratories (Raleigh, NC). Sperm-positive females [gestational day (GD) 0] were weighed upon arrival at the U.S. Environmental Protecion Agency (EPA). Animals were housed individually in polypropylene cages, and received food (LabDiet 5001; PMI Nutrition International LLC, Brentwood, MO) and tap water ad libitum
in polyethylene water bottles sealed with rubber stoppers and stainless-steel sipper tubes, as specified by White et al. (2009)
. Animal protocols were approved by the U.S. EPA’s Institutional Animal Care and Use Committee. Animals were treated humanely and with regard for alleviation of suffering.
Dosing solutions. PFOA (ammonium perfluorooctanoate; > 98% pure) was purchased from Fluka Chemical (Steinheim, Switzerland). PFOA was dissolved by agitation in deionized water at concentrations of 0.1 and 0.5 mg/mL (for 1 and 5 mg/kg doses, respectively) and prepared fresh daily, immediately before administration. PFOA-containing drinking water was prepared similarly, by serial dilution to a final concentration of 5 ng/mL (ppb). Drinking water was prepared fresh weekly, and cage bottles were refilled weekly after rinsing.
A study timeline is shown in Supplemental Material, (doi:10.1289/ehp.1002741). Timed pregnant P0
(parental generation) dams were randomly distributed among five treatment groups. Three groups were treated once daily by oral gavage on GDs 1–17 (designated “gestational”) with PFOA doses of 0 (control; n
= 10), 1 (n
= 12), or 5 mg/kg body weight (n
= 11). The remaining two groups received PFOA at 0 (n
= 7) or 1 mg/kg (n
= 10) as described above, but also received PFOA (5 ppb) in their drinking water (designated “chronic”) to approximate the 3.55 ppb PFOA present in the contaminated drinking-
water supply in Little Hocking, Ohio (Emmett et al. 2006
). These two groups received PFOA-containing drinking water throughout gestation (starting on GD7) and for the duration of the study, as did subsequent F1
offspring (except during F1
breeding and early gestation, to avoid exposing control males). Weekly water consumption was calculated per cage by weighing bottles when filled and again at the end of the week; the differential reflected consumption.
Figure 1 F1 female mammary gland development. Mammary whole-mounts illustrate morphology representative of treatment groups at PNDs 22, 42, and 63 (n = 6–7 females/treatment/age). Bars = 1,000 μm for PND22 and 2,000 μm for PND42 and PND63. (more ...)
dams were weighed daily throughout gestation. On PND1, F1
litters were weighed and sexed. F1
neonates were pooled and randomly redistributed to dams of their respective treatment groups, consistent with previous studies (Lau et al. 2006
; White et al. 2009
), equalizing litters to 12–13 neonates, with similar sex representation. Litters were monitored and weighed on PND10. On PND22, F1
offspring were weaned, and dams and 1–2 female offspring/litter were weighed and necropsied (n
= 5–7 litters/treatment
group). A subset of F1
females were maintained into adulthood and weighed and necropsied at PND42 and PND63 (n
= 6–8/treatment group).
Remaining adult F1 females were bred to age-matched control F1 males at 7–8 weeks of age, on the night of proestrus (determined by vaginal cytology). Breeding pairs remained together overnight only, and plug-positive females (GD0) were housed individually and monitored over gestation (n = 7–10 F1 dams/treatment group). On PND1, F2 neonates were weighed and sexed. F2 litters were equalized to 10 neonates for the lactational challenge experiment. F1 dams and 3 female offspring per F2 litter were sacrificed on either PND10 or PND22. The remaining F2 females were weaned and necropsied on either PND42 or PND63 (n = 4–8/treatment group).
The lactational challenge experiment was performed with F1 dams and their F2 litters on PND10, the peak of lactation. Dams were separated from offspring for 3 hr and then returned to their litters and allowed to nurse for 30 min. The time between reunion and initiation to nurse (arched back position over the litter) was recorded to the nearest second, as was the weight of the 10-pup litter before and after precisely 30 min of nursing, in order to estimate the volume of milk produced during the nursing period. Dams were euthanized and necropsied immediately after nursing.
Necropsy. All animals were terminated by decapitation; trunk blood was collected and serum was isolated and stored at –80°C in snap-top polypropylene tubes for PFOA analysis. Uteri were dissected from P0 and F1 dams, and implantation sites were visually identified by light macroscope (Leica WILD M420 macroscope; Leica, Wetzlar, Germany) to assess postimplantation loss per dam. Mammary glands were collected as described below.
Mammary gland preparation.
Mammary glands were removed from P0
dams on PND10 (F1
dams only) and PND22 (n
= 4–12/treatment group) because these times represent peak lactational output and weaning, respectively. In F1
offspring, a set of fourth and ﬁfth glands was removed from the skin and ﬂattened onto glass slides. Whole-mounts were ﬁxed in Carnoy’s solution, stained in carmine alum, and then dehydrated and cleared in xylene, as previously described (Fenton et al. 2002
). From dams only, a portion of the contralateral mammary gland was removed, placed in a histology cassette, ﬁxed in 10% neutral buffered formalin for 48 hr, and stored in 70% ethanol. These tissues were embedded in parafﬁn, and 5 μm sections were prepared and stained with hematoxylin and eosin (H&E). Whole-mounts and histological sections were visualized by light macroscope.
Mammary gland whole-mounts from F1 and F2 female offspring were scored on a 1–4 subjective, age-appropriate developmental scale (4 = excellent development/structure; 1 = poor development/structure). The number of primary ducts and large secondary ducts, lateral side branching, appearance of budding from the ductal tree, and longitudinal outgrowth of the epithelia were assessed. Because we did not address estrous cycle stage at the time of necropsy, we did not include stage-sensitive morphological traits in scoring criteria. Slides were separated by score during evaluation, compared within a score for consistency, and then recorded. Two individuals, blind to treatment, scored glands. Mean scores for the various ages and treatment groups were calculated and analyzed statistically for treatment and time-related differences.
H&E-stained lactating mammary gland sections from P0
dams were similarly scored on a 1–4 subjective scale. A value of 4 represented well-differentiated, functionally lactating tissue characterized by extensive epithelium, reduced adiposity, and presence of secretory alveoli, consistent with the peak of lactation (PND10, as previously described by Vorderstrasse et al. 2004
). A value of 1 represented little or diminishing presence of lobuloalveoli and extensive involution and regression of the tissue, with the presence of apoptotic bodies, increasing adiposity, and regressing alveoli, as anticipated at weaning (PND22). At both time points, dams were euthanized immediately after removal from litters to ensure comparable lactational morphology. Mammary glands representing the mean score or observation for each treatment group were photographed using the described macroscope and mounted camera (Photometrics CoolSNAP; Roper Scientiﬁc, Inc., Tucson, AZ).
Measurement of PFOA in serum.
Serum samples from the P0
dams at PND22 and from F1
offspring at PNDs 22, 42, and 63 were stored frozen in snap-top polypropylene vials until they were shipped on dry ice to the Centers for Disease Control and Prevention (CDC) laboratory. Serum PFOA measurements were performed by the CDC using the methodology described in detail by White et al. (2009)
Data analysis. Data were evaluated for dose effects using mixed-model analysis of variance in SAS (version 9.1; SAS Institute Inc., Cary, NC). For both generations, treatment-specific mean gestational weight gain was calculated for dams between GD1 and GD17, and treatment-specific mean body weights were determined for F1 and F2 offspring on PNDs 22, 42, and 63. In addition, we calculated F2 offspring body weight means at PNDs 1, 3, 5, 10, 14, and 17, based on whole-litter weights (divided by number of pups; litter used as the unit of measure before weaning). For all three generations, mean mammary gland lactational or developmental scores were calculated. Scores were analyzed using body weight at time of collection as a random effect, with litter as the unit of measure for neonatal scores. For both P0 and F1 dams, we calculated mean implant number, percentage of postimplantation (prenatal) loss, and percentage of postnatal survival. Differences between treatment groups were determined using Dunnett’s, Tukey’s, or Student’s t-tests, with significance set at p < 0.05 for all comparisons.