PFBA (ammonium salt; > 98% pure) was provided from 3M Company

(St Paul, MN). For all studies, PFBA was diluted in deionized water and

prepared fresh daily.

Animal treatment. All animal studies were conducted in accordance with

the Institutional Animal Care and Use Committee guidelines established by the

U.S. Environmental Protection Agency’s Office of Research and Development/

National Health and Environmental Effects Research Laboratory. Procedures and

facilities were consistent with the recommendations of the 1996 National

Research Council’s ‘‘Guide for the Care and Use of Laboratory Animals,’’ the

Animal Welfare Act, and Public Health Service Policy on the Humane Care and

Use of Laboratory Animals. Animal facilities were controlled for temperature

(20–24C) and relative humidity (40–60%) and kept under a 12-h light-dark cycle.

Timed-pregnant CD-1 mice were obtained from Charles River Laboratories

(Raleigh, NC), where females were bred overnight. The sperm-positive

females, considered to be at gestational day (GD) 0, were shipped to our

facility on the same day. Upon arrival, mice were housed individually in

polypropylene cages with Alpha-dri (Shepherd Specialty Papers, Kalamazoo,

MI) bedding and provided pellet chow (LabDiet 5001, PMI Nutrition

International LLC, Brentwood, MO) and tap water ad libitum.

The study was performed in three separate blocks, with approximately 40

mice per block (10 mice for each dose group). Upon arrival at the animal

facility, mice were ranked by their weight and assigned to one of the four

treatment groups: vehicle control (deionized water), 35, 175, or 350 mg PFBA/

kg body weight. These doses were chosen to approximate the effective doses of

1, 5, and 10 mg/kg of PFOA used in a prior study (Lau et al., 2006). The

elimination half-life for PFBA in female mice is about 3 h (Chang et al., in

press), whereas the elimination half-life for PFOA is about 15–20 days (Lau

et al., 2005). Therefore, in an attempt to simulate as closely as possible the PFOA

doses used by Lau et al. (2006), an effort was made to match internal exposures

based on the ‘‘area-under-the-curve’’ (AUC) estimates. A one-compartment

pharmacokinetic model was used for both PFBA and PFOA. For comparison of

AUC values for female mice given PFBA and PFOA during gestation, the AUC

values for both compounds at a nominal dose of 5 mg/kg were estimated for

GD 17 to 18, assuming continuous daily oral administration from GD 1. For

PFBA, estimation was based on data from the single oral treatment study in

nonpregnant female mice at 10 mg/kg reported by Chang et al. (in press) and

used a volume of distribution (Vd) of 3.14 ml, uptake rate constant (K01) of

0.7/h, and clearance rate (CL) of 0.76 ml/h. For PFOA, estimates were based on

a K01 for PFBA in female mice (0.7/h), a Vd of 6 ml (based on rat data scaled to

the female mouse) and CL of 0.012 ml/h (based on an assumed elimination

half-life of 15 days for the female mouse; Lau et al., 2005). The resulting AUCs

(from the 17th to the 18th day of treatment) were estimated at 6956 h-lg/ml for

PFOA and 197 h-lg/ml for PFBA. Therefore, to obtain equivalent estimated

AUCs, the dose required for PFBA would be approximately 35 times that for

PFOA.

From GD 1 to GD 17, mice were weighed daily and given PFBA or water

by oral gavage at a volume of 10 ml/kg. At GD 18, mice from each dose group

were subdivided. Some mice were sacrificed (24 h after last treatment) by CO2

asphyxiation for teratological evaluation. Trunk blood was collected after

decapitation, and serum samples were prepared and analyzed for PFBA

concentration. Maternal liver was dissected, weighed and stored frozen at

80C until analysis for PFBA. The gravid uterus was removed, weighed, and

examined; the numbers of the live and dead fetuses as well as resorptions were

recorded. Live fetuses were weighed individually, and examined for external

abnormalities. All fetuses were killed with an overdose of pentobarbital and

fixed in Bodian’s solution (2% formaldehyde, 5% acetic acid, 72% ethanol,

21% water) for visceral evaluation. Examination of the head, thoracic, and

abdominal viscera was carried out using free-hand razor dissection. For mice

without any fetus, their uteri were removed and stained with 2% ammonium

sulfide for the detection of residual implantation site. Mice with negative

staining were considered ‘‘nonpregnant’’; those that stained positive but did not

have any fetuses at term were categorized as having experienced ‘‘full-litter

resorptions’’ (FLRs).

The remaining pregnant mice received an additional PFBA treatment on GD

18. On GD 19, these mice were monitored intermittently for parturition;

condition of the newborns and numbers of live offspring were noted. The

following day was designated as postnatal day (PD) 1. The number of live pups in

each litter and their body weight were tabulated daily for the first 4 days after birth

and at intervals of several days thereafter. The age at which the mouse neonates

opened their eyes was tracked beginning on PD 10. All pups were weaned on PD

22 and separated by sex. The age at which the mouse offspring reached puberty

was determined by monitoring vaginal opening in females and preputial

separation in males beginning on PD 26, with their respective body weights

noted. On PD 49, three pups for each sex were randomly selected from each litter

and retained; these animals were weighed biweekly until 41 weeks of age.

After weaning, the nursing dams were sacrificed, their livers were weighed,

and their uteri were removed, stained with 2% ammonium sulfide, and the

residual implantation sites were counted. The postnatal survival rate was

calculated based on the number of implantations for each pregnant mouse.

For study block 3, one male and one female pup from each litter were

weighed and sacrificed at PD 1 and 10 by decapitation. Trunk blood was

collected, and serum was prepared and stored frozen at 80C for PFBA

analysis. Livers were removed, weighed, and about 200 mg of fresh tissues

were processed immediately in TRI reagent (Sigma Chemical, St Louis, MO)

for RNA isolation according to the manufacturer’s directions; the rest of the

tissue was stored at 80C for PFBA analysis.

Gene expression analysis. Measurement of selected gene transcripts was

carried out by quantitative PCR (qPCR) of reverse-transcribed cDNAs. These

candidate genes were chosen for evaluation because their expression patterns

have been shown to be markedly altered in the fetal liver of mice exposed to

PFOA (Rosen et al., 2007). Two micrograms of total RNA was initially

digested by 2 units of DNaseI (#M6101, Promega Corporation, Madison, WI)

for 30 min at 37C, followed by 10 min at 65C in a buffer containing 40mM

Tris (pH 8.0), 10mM MgSO4, and 1mM CaCl2. The RNA was then quantified

by a Quant-iT RiboGreen RNA assay kit according to the manufacturer’s

protocol (#R11490, Invitrogen Corporation, Carlsbad, CA) and approximately

1.5 lg RNA was reverse-transcribed using a High Capacity cDNA Archive Kit

according to the provided protocol (#4322171, Applied Biosystems, Foster

City, CA). Amplification was performed on an Applied Biosystems model

7900HT Fast Real-Time PCR System in duplicate using 25 ng cDNA and

TaqMan Universal PCR Master Mix (#4304437, Applied Biosystems) in a total

volume of 12 ll. Gapdh was used as an endogenous reference gene because

it was uniformly expressed and showed no significant change among all

samples, with a cycling threshold standard deviation less than 0.22. The

following TaqMan assays (Applied Biosystems) were included in this study:

Gadph (#Mm999999_g1), Acox1 (#Mm00443579_m1),Me1 (#Mm00782380_s1),

Gadd45b (#Mm00435123_m1), Peci (#Mm00478725_m1), Ehhadh

(#Mm00470091_s1), Cyp3a41 (#Mm00776855_mH), C4a (#Mm00550309_m1),

and Pdk4 (#Mm00443325_m1). Fold-change was calculated using the 244CT

method (Livak and Schmittgen, 2001).

Determination of PFBA concentration. Liver samples were thawed and

homogenized (Polytron) in four volumes of water, based on individual weight,

and re-frozen at 80C. Serum and liver homogenates were shipped to 3M on

dry-ice for analysis, according to the method described previously (Chang

et al., in press), using 13C4-PFBA as the internal standard.

Data analysis. Data are presented as means and standard errors. Statistical

significance was determined for each outcome variable by ANOVA that

included treatment dose, block and age (where appropriate) effect, using

individual litter as the statistical unit. When a significant treatment effect or

interaction (p < 0.05) was detected, each treatment group was then tested for

difference from the control group using the Dunnett’s test. Maternal weight

gain was analyzed by ANOVA with repeated measure. Because of their

heterogeneity of variance, data from the postweaning weight gain were logtransformed

before ANOVA was conducted. Data for FLRs were analyzed by

the nonparametric Fisher’s exact test.

F1 Male CD-1 Mice