Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid

Toxicology 326 (2014) 62–73

Contents lists available at ScienceDirect

Toxicology journal homepage: www.elsevier.com/locate/toxicol

Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid Pei-Li Yao a , David J. Ehresman b , Jessica M. Caverly Rae c, Shu-Ching Chang b , Steven R. Frame c , John L. Butenhoff b , Gerald L. Kennedy d, Jeffrey M. Peters a, * a Department of Veterinary and Biomedical Sciences and The Center of Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802, USA b Medical Department, 3 M Company, St. Paul, MN 55144, USA c Haskell Global Centers for Health and Environmental Sciences, Newark, DE 19701, USA d Consultant to DuPont Company, Wilmington, DE 19805, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 August 2014 Received in revised form 9 October 2014 Accepted 17 October 2014 Available online 18 October 2014

Previous studies suggested that perfluorooctanoate (PFOA) could activate the estrogen receptor (ER). The present study examined the hypothesis that PFOA can activate ER using an in vivo uterotrophic assay in CD-1 mice and an in vitro reporter assay. Pre-pubertal female CD-1 mice fed an estrogen-free diet from postnatal day (PND)14 through weaning on PND18 were administered 0, 0.005, 0.01, 0.02, 0.05, 0.1, or 1 mg/kg PFOA or 17b-estradiol (E2, 0.5 mg/kg) from PND18–20. In contrast to E2, PFOA caused no changes in the relative uterine weight, the expression of ER target genes, or the morphology of the uterus/cervix and/or vagina on PND21. Treatment of a stable human cell line containing an ER-dependent luciferase reporter construct with a broad concentration range of PFOA caused no change in ER-dependent luciferase activity; whereas E2 caused a marked increase of ER-dependent luciferase activity. These data indicate that PFOA does not activate mouse or human ER. ã 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Perfluorooctanoate Uterus Estrogen receptor

1. Introduction Perfluoroalkyl acids (PFAAs) are compounds containing fluorine atoms on a carbon backbone with unique surfactant properties and are widely used for manufacturing and industrial applications (Andersen et al., 2008; Betts, 2007). Perfluorooctanoic acid (PFOA) is a PFAA that is reported to cause adverse effects in animal models. Humans can be exposed to PFOA because of its persistence in the environment (Post et al., 2012; Steenland et al., 2013) and its relatively long serum half-life (Olsen et al., 2007). PFOA is detectable in the blood of 99.9% of the general human population in the United States with an average serum PFOA concentration
4–5 ng/mL (
10–13 nM) (Kato et al., 2011). PFOA exposure has been associated with adverse effects in the liver and reproductive systems of animal models (Lau et al., 2007). The mechanism(s) that mediate(s) PFOA-induced adverse effects may exhibit species differences (Lau et al., 2007). For example, PFOA is a relatively weak

Abbreviations: ER, estrogen receptor; ERE, estrogen response element; PPAR, peroxisome proliferator-activated receptor; PFAA, perfluoroalkyl acid; PFOA, perfluorooctanoate; PR, progesterone receptor; TFF, trefoil factor. * Corresponding author. Tel.: +1 814 863 1387; fax: +1 814 863 1696. E-mail address: [email protected] (J.M. Peters). http://dx.doi.org/10.1016/j.tox.2014.10.008 0300-483X/ ã 2014 Elsevier Ireland Ltd. All rights reserved.

agonist for peroxisome proliferator-activated receptor (PPAR)-a, and can alter cell proliferation and lipid metabolism in rodent liver (Cheng and Klaassen, 2008; Takacs and Abbott, 2007; Wolf et al., 2008; Wolf et al., 2010). However, PPARa-mediated effects of PFOA in liver and other tissues are typically greater in rodent models as compared to human models (Albrecht et al., 2013; Bjork et al., 2011; Bjork and Wallace, 2009; Nakamura et al., 2009). These differences may be due to variation in binding affinity of PPARa agonists to human PPARa as compared to rodent PPARa. Developmental exposure of rodents to PFOA is reported to cause early pregnancy loss, reduced postnatal survival, defects in growth, and delays in developmental process (Lau et al., 2006; Wolf et al., 2010). Some of these developmental effects due to PFOA exposure are mediated by PPARa (Abbott et al., 2007), but evidence also exists that there is a species difference for these PPARa-dependent effects (Albrecht et al., 2013; Bjork et al., 2011; Bjork and Wallace, 2009; Nakamura et al., 2009) and that PFOA does not cause reduced postnatal survival by activation of human PPARa (Albrecht et al., 2013). Moreover, associations between PFOA exposure and alterations in development are not consistently observed in human studies (Fei et al., 2008; Olsen et al., 2009; Savitz et al., 2012a,b; Steenland et al., 2013). It has been suggested that PFOA may cause endocrine disruption through effects on the function of growth and sex hormones, including activation of the

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

estrogen receptor (ER) (Benninghoff et al., 2011; Du et al., 2013; Kjeldsen and Bonefeld-Jorgensen, 2013; White et al., 2011). However, the concentration of PFOA reported to elicit ERdependent activities is typically much higher than the average concentrations of PFOA observed in the general population or occupationally exposed humans (i.e.,
10–13 nM (Kato et al., 2011), 1.6 mM (Olsen et al., 2007), respectively). Further, other studies are inconsistent with the hypothesis that PFOA can activate the ER (Ishibashi et al., 2007, 2008). However, two recent cross-sectional epidemiological studies suggest an association between fetal exposure to PFOA and a delay of menarche, and/or a longer menstrual cycles in humans (Kristensen et al., 2013; Lyngso et al., 2014), suggesting that the female reproductive system may be sensitive to PFOA exposure. Since most studies showing effects modulated by activation of ER that could potentially influence the female reproductive tract use concentrations of PFOA that exceed those found in humans, it is of particular interest to note that a recent study showed that low dose PFOA caused changes in uterine histology in immature female CD-1 mice (Dixon et al., 2012). These investigators also provided evidence supporting the hypothesis that activation of ER mediated these PFOA-dependent effects in the reproductive tract (Dixon et al., 2012). However, although immature female CD-1 mice were exposed to three different doses of PFOA (0.01, 0.1 and 1.0 mg/kg), these effects were only observed at the lowest dose of PFOA (0.01 mg/kg) and no dosedependent effects were noted (Dixon et al., 2012). Thus, the present studies further examined the hypothesis that low dose PFOA exposure comparable to that found in humans causes ERdependent changes using an in vivo uterotrophic assay and an in vitro reporter assay. 2. Materials and methods 2.1. Animals and chemical treatment in vivo Timed-pregnant CD-1 mice (gestation day 14) were purchased from Charles River Laboratories (Raleigh, NC, USA). All mice used in this study were housed in a mouse facility at The Pennsylvania State University at a constant temperature (22.0  0.5  C) with 35–70% humidity and a 12:12 h (light:dark) photoperiod. The protocol for animal treatments was approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University, for examination in an Association for Assessment and Accreditation of Laboratory Animal Care Internationalapproved facility. Timed-pregnant CD-1 mice were initially fed standard lab chow diet and water ad libitum. From postnatal day (PND) 14, the dams were fed an estrogen-free, AIN-93-based pelleted diet (Dyets Inc., Bethlehem, PA, USA). Dams were allowed to give birth, and the female pups were weaned on PND18 and randomly divided into 8 groups. Two sets of timed pregnant CD1 mice were purchased. The pups from the first set of timed pregnant CD-1 mice were used for analysis of serum PFOA concentration, ER-dependent gene expression in the uterus, western blotting, and immunohistochemistry on PND21 (N = 10 pups per group). The pups from the second set of timed pregnant CD-1 mice were only used for histopathological analyses on PND21 (N = 5 pups per group). For both sets of mice, female pups were administered PFOA (0, 0.005, 0.01, 0.02, 0.05, 0.1, and 1.0 mg/kg/day dissolved in PBS; PBS served as the vehicle control for the 0 mg/kg/day group) or 17bestradiol (E2, 0.5 mg/kg/day) by oral gavage for 3 days from PND18– 20. PFOA was provided by DuPont Haskell Global Centers for Health and Environmental Sciences (Newark, DE, USA) and E2 was purchased from Sigma–Aldrich (St. Louis, MO, USA). On PND21, control and treated mice were euthanized by overexposure to carbon dioxide. Body weight and wet uterine weight were

63

measured, and the uterus/cervix and the vagina were collected from each pup. Tissues were fixed in 10% buffered formalin for histological analyses or immediately frozen in liquid nitrogen for protein and mRNA analysis. 2.2. Serum concentration of PFOA Serum was obtained from whole blood collected from PND21 control and treated CD-1 female pups (N = 10). Quantification of serum PFOA concentration was performed as previously described (Ehresman et al., 2007). The relationship between the dose of PFOA administered and the serum concentration of PFOA in PND21 pups was determined using linear regression (Prism 5.0, GraphPad Software Inc., La Jolla, CA, USA). 2.3. Histopathological analysis Formalin-fixed uterine/cervix and vaginal tissues (N = 5 per group) were embedded in paraffin blocks. Sections (5 mm) of both uterine horns and longitudinal sections of the uterine body (including the cervix), as well as sections of the vagina from each treatment group were stained with hematoxylin and eosin (H&E) by a standard staining procedure and assessed by a board-certified pathologist who had no knowledge of treatment. In the uteri, the severity of endometrial edema, epithelial hyperplasia of the mucosa, glandular epithelial hyperplasia, myometrial edema and myometrial hypertrophy/hyperplasia were scored. In the vaginas and cervices, the severity of squamous hyperplasia, squamous cornification, mucification and edema in the submucosa and stroma were scored. Histopathological severity scores ranged from 0 to 4, with 0 corresponding to no changes, 1 corresponding to minimal changes, 2 corresponding to mild changes, 3 corresponding to moderate changes and 4 corresponding to severe changes for each histopathological analysis. The average histopathological severity scores in the uterus/cervix and vaginas were calculated based on this scoring system. 2.4. Quantitative real-time polymerase chain reaction (qPCR) Expression of ER-dependent target genes in the uterus was measured by qPCR as previously described (Yao et al., 2014). Briefly, total RNA was isolated using RiboZol RNA extraction reagent (AMRESCO, Solon, OH, USA) using the manufacturer’s recommended procedures. cDNA was synthesized using 2 mg of total RNA as template mixed with M-MLV reverse transcriptase and random primers (Promega, Madison, WI, USA). DNA

Fig. 1. Average relative uterine weights of immature CD-1 mice. CD-1 female mice were treated with various doses of PFOA (0.005, 0.01, 0.02, 0.05, 0.1, or 1.0 mg/kg/ day) or 17b-estradiol (E2, 0.5 mg/kg/day) for 3 days from PND18–20 (N = 10 per group). On PND21, mice were euthanized and uteri weighed. The relative uterine weight to body weight (mg uterus/g body weight) was calculated. Values represent the mean  S.E.M. Values with different letters are significantly different at P  0.05.

64

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

Table 1 Serum PFOA concentration in CD-1 female mice dosed with PFOA from PND18– PND20. Daily PFOA dose (mg/kg/day)

Cumulative PFOA dose (mg/kg)

Mean serum PFOA (ng/mL)

Mean serum PFOA (nM)

Control 0.005 0.01 0.02 0.05 0.1 1.0

0 0.015 0.030 0.060 0.150 0.300 3.0

<1

NA 12.8  1.0 17.9  1.0 53.4  5.8 114.0  12.6 290.0  20.5 2830.0  233.5

5.3  0.4 7.4  0.4 22.1  2.4 47.2  5.2 120.1  8.5 1171.8  96.7

Serum values represent the mean  S.E.M. NA: not applicable.

amplification was carried out in 25 mL volumes containing SYBR Green PCR Supermix (Quanta Biosciences, Gaithersburg, MD, USA) using the iCycler iQ5 PCR thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) with 45 cycles of 95  C for 10 s, 60  C for 30 s and 72  C for 30 s. The GenBank accession number of the mouse ER target genes and the sequence of primers used to detect mRNA were: trefoil factor 1 (TFF1; NM_009362.2): forward, 50 -GGGATTCCCGTGGTGCTT-30 and reverse, 50 -TGGACCTTAGAAGGGACATTCTTC-30 ; TFF2 (NM_009363.3): forward, 50 -CTTGGTGTTTCCACCCACTT-30 and reverse, 50 -CACCAGGGCACTTCAAAGAT-30 ; TFF3 (NM_011575.2): forward, 50 -TGTCACATCGGAGCAGTGGTAAC-30 and reverse, 50 GCACCAGGGCACATTTGG-30 ; progesterone receptor (PR); NM_008829.2): forward, 50 - GACACTGGCTGTGGAATTTCC-30 and reverse, 50 -CCAGGATCTTGGGCAACTG-30 . The expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh; BC083149) was quantified as an internal control using the forward and reverse primers: 50 -GGTGGAGCCAAAAGGGTCAT-30 and 50 -GGTTCACACCCATCACAAACAT-30 . Each assay including a standard curve and a non-template control performed in triplicate. Five representative tissue samples per treatment group were randomly chosen for analysis. The relative mRNA level of each target gene was normalized to Gapdh because there was no difference in expression between groups detected for this gene product (data not shown). 2.5. Western blot analysis Total cellular protein was isolated from uterine tissues and quantitative western blot analysis was performed as previously described (Yao et al., 2014). Briefly, 30 mg of protein was separated by electrophoresis on 10% SDS-polyacrylamide gels, and transferred to a PVDF membrane (EMD Millipore Corporation, Billerica, MA, USA). Membranes were incubated with blocking buffer (5% dried milk in Tris buffered saline with Tween-20 (TBST)) at room temperature for 30 min and rinsed with TBST. Membranes were then incubated overnight with primary antibodies against: TFF1, TFF2, TFF3, PR, ERa, ERb or ACTIN (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4  C. After this overnight incubation, membranes were washed twice with TBST at room temperature for 10 min, and then incubated for 2 h with biotin-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature. After incubation with the biotinylated secondary antibodies, the membranes were washed again three times with TBST at room temperature and then with 125Istreptavidin. Membranes were then exposed to phosphorimager plates and the level of radioactivity quantified with filmless autoradiographic analysis (Packard Phosphorimager, PerkinElmer, Waltham, MA, USA). The relative expression level of specific proteins was normalized to that of ACTIN.

2.6. Immunohistochemistry The expression and the localization of TFF1, TFF2 and PR in the uterus and vagina were examined by immunohistochemistry. Sections (5 mm) of paraffin-embedded tissues were de-paraffinized and rehydrated, followed by antigen retrieval by heating in a 10 mM sodium citrate solution. Sections were incubated with 3% hydrogen peroxide to block the endogenous peroxidase activity and then incubated in the blocking buffer containing 10% horse serum. Primary antibodies used in this analysis were the same as those used for western blot analysis. Sections were incubated in primary antibody at 4  C overnight. Immunodetection was performed using a VectaStain ABC kit (Vector Labs, Burlingame, CA, USA) and DAB substrate (Vector Labs, Burlingame, CA, USA) using the manufacturer’s recommended procedures. Representative photomicrographs were obtained using a Nikon DS-Fi1 digital camera attached to Nikon Eclipse 50i microscope using NISElements F software (Nikon Inc., Melville, NY, USA). Five representative tissue samples per treatment group were randomly chosen for immunohistochemical analysis. A total of two sections per tissue sample, and ten fields per section were analyzed using ImageJ software to quantify relative protein expression (DABpositive regions) with the ImmunoRatio plugin (Version 1.49c). For PR, the localization was nuclear; and for TFF1/TFF2 the localization was cytosolic and nuclear. 2.7. ER-dependent reporter assay The human ovarian carcinoma cell line BG1-Luc4E2 (Rogers and Denison, 2000) was kindly provided by Dr. Michael Denison (University of California at Davis). The BG1-Luc4E2 cell line contains a stably integrated estrogen response element (ERE)luciferase construct, providing a useful model to detect estrogenic activity in response to estrogens and xenoestrogens. The BG1Luc4E2 cells were cultured in a-minimum essential media (a-MEM; Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA, USA) and 1% penicillin–streptomycin (Invitrogen, Grand Island, NY, USA) at 37  C with 5% carbon dioxide. Five days before PFOA treatment, (5  105) cells were seeded into six-well plates in phenol red-free MEM (Invitrogen, Grand Island, NY, USA) supplemented with 5% dextran-coated charcoal-treated FBS (Hyclone, Logan, UT, USA) to reduce estrogenic activity of culture media. Cells were then treated with the indicated final concentrations of PFOA in the absence or presence of E2 (5 nM) for 24 h and cell lysates were isolated. Cell lysates were prepared in 300 mL of lysis buffer and incubated with the substrate (Luciferase Assay System, Promega, Madison, WI, USA). The intensity of luminescence was measured on a GloMax-multi detection system (Promega, Madison, WI, USA). The concentrations of PFOA used in this reporter assay (1.2 nM–96 mM) included concentrations well below the average serum PFOA concentrations observed in the general population, the average range of serum PFOA concentrations in the general population, and the range of serum PFOA concentrations found in occupationally exposed humans and higher (Kato et al., 2011; Olsen et al., 2007). These concentrations of PFOA used included those measured in the serum of postnatally exposed pups from the in vivo study. For control and all treated groups, samples were examined in triplicate and the experiment was repeated three times. 2.8. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) and the Tukey’s test for post hoc comparisons (Prism 5.0 d, GraphPad Software, Inc., La Jolla, CA, USA).

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

65

Fig. 2. Histopathological changes in the uterus and vagina of immature CD-1 mice after treatment with either PFOA or E2. Uteri (left panels) and vaginas (right panels) were obtained from immature CD-1 female mice treated with or without various doses of PFOA or E2 for 3 days on PND18–20 (N = 5 per group). Histopathological changes were evaluated in paraffin-embedded tissues with H&E staining. Representative photomicrographs of sections of uterus from mice exposed to (A) 0 mg PFOA/kg body weight, (B) 0.01 mg PFOA/kg body weight, (C) 0.1 mg PFOA/kg body weight, (D) 1.0 mg PFOA/kg body weight, or (E) 0.5 mg E2/kg body weight. Note the similarities in mucosal epithelial (Ep) and glandular (G) epithelial height and myometrial (My) thickness, as well as the lack of endometrial (E) edema, in sections A–D. In contrast, note the overall increase in size of the section of uterus in section E, due to mucosal and glandular epithelial hyperplasia, myometrial hypertrophy, hyperplasia and edema, and endometrial edema. Representative photomicrographs of sections of vagina from mice exposed to (F) 0 mg PFOA/kg body weight, (G) 0.01 mg PFOA/kg body weight, (H) 0.1 mg PFOA/kg body weight, (I) 1.0 mg PFOA/kg body weight, or (J) 0.5 mg E2/kg body weight. Note the similarities in mucosal epithelial (Ep) height, submucosal (Su) edema and degree of mucosal epithelial mucification in the control and PFOA-treated groups. In contrast, note the mucosal epithelial squamous cornification (arrow in Section J), the very thickened mucosal epithelium, the edema in the stroma of the submucosa, and the lack of mucification in the section of vagina from the E2-treated group. Magnification sections A–D: 20; section E: 10; sections F–J: 40.

66

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

3. Results

3.2. Serum PFOA concentration

3.1. Uterine weight and body weight in pre-pubertal female CD-1 mice

After administration of PFOA for three days to pre-pubertal CD1 female mice from PND18–20, the average serum concentration of PFOA ranged from as low as 12.8 nM in mice dosed with 0.005 mg/ kg/day to 2.8 mM in mice dosed with 1.0 mg/kg/day (Table 1). Linear regression analysis revealed excellent correlation (r2 = 0.99) between the administered dose and the concentration of PFOA observed in serum.

Administration of PFOA at doses ranging from 0.005 to 1.0 mg/ kg/day to pre-pubertal CD-1 female mice from PND18–20 caused no changes in average body weight (data not shown) or relative wet uterine weight compared to controls on PND21 (Fig. 1). By contrast, E2 administration to pre-pubertal CD-1 female mice from PND18–20 caused no change in average body weight (data not shown) but a significant increase in relative uterine weight compared to controls on PND21 (
2.4-fold increase).

Fig. 3. Histopathological severity score in the uterus/cervix and vagina of immature CD-1 mice after treatment with either PFOA or E2. Uterus/cervix and vagina were obtained from control CD-1 female mice and CD-1 female mice treated with various doses of PFOA or E2 for 3 days from PND18–20 (N = 5 per group). The histopathological severity score for changes observed in the tissue was evaluated semi-quantitatively on a scale that ranged from 0 (change not present) to 4 (change present at highest level). (A) In the uterus/cervix, E2 caused mild to moderate edema of the endometrium, epithelial hyperplasia and myometrial hypertrophy/hyperplasia, while these changes were not evident in controls or PFOA-treated mice. (B) In the vagina, E2 administration resulted in mild to moderate squamous hyperplasia with epithelial cornification. These changes were not present in controls or in PFOAtreated mice. Controls and PFOA-treated mice often had mucification of vaginal epithelium. This change was not present in E2-treated mice. The ratio indicates the number of mice with the observation over the total number of mice evaluated. (C) The overall score for changes in the uterus/cervix or vagina was determined by adding individual scores together and the average overall score was calculated. No differences were observed between control and PFOA-treated groups. Scores of both control and PFOA-treated groups were less severe than the E2-treated group. Values represent the mean  S.E.M. Values with different letters are significantly different at P  0.05.

3.3. Uterine/cervical and vaginal histopathological analyses Sections of paraffin-embedded uterus/cervix and vaginas from either control, PFOA or E2 treated pre-pubertal CD-1 female mice were examined for histopathological changes. There were no histopathological observations in PFOA-treated mice that were discernible from those noted in control mice, nor were there increases in the incidence or severity (histopathological severity scores) of observations associated with the dose-dependent PFOA treatment. All observations noted were within the range of biological variability for mice of this strain and age. In contrast, changes observed in uterine/cervical or vaginal tissues from mice exposed to E2 were characteristic of exposure to E2 and these changes were easily discernible and clearly dissimilar from those observed in control and PFOA-treated mice. Edema was not noted in the endometrium of the uteri from mice in all of the control and PFOA-treated groups (Figs. 2 and 3), and was either not present or present at a minimal severity in the myometrium of control and PFOA-treated mice. Epithelial hyperplasia of the uterine endometrial mucosal and glandular epithelium ranged from minimal to mild in all of control and PFOA-treated groups. Myometrial hypertrophy/hyperplasia was either not present or present at a minimal level in mice from control or PFOA-treated groups (the highest incidence was 4 out of 5 in control mice). Squamous hyperplasia of the vaginal mucosal epithelium ranged from not present, to present at a mild degree in control mice (Figs. 2 and 3). Similarly, mucosal squamous hyperplasia in PFOA-treated mice was either not present or present at a minimal level. None of the control or PFOA-treated mice had squamous cornification of the vaginal mucosal epithelium. Vaginal mucosal epithelial mucification ranged from not present to present at a mild level in mice from control and PFOA-treated groups with no dose or treatment-related increase in incidence or severity of this observation discernible from control mice (Figs. 2 and 3). Edema of the submucosa of the vagina was either not present or present at a minimal level in control and PFOA-treated mice (Figs. 2 and 3). Sections of paraffin-embedded uterus/cervix and vagina from mice exposed to E2 showed typical histomorphological characteristics of E2 exposure, including moderate epithelial hyperplasia of the endometrial mucosal and glandular epithelium of the uterus; mild to moderate myometrial hypertrophy and hyperplasia in the uterus (Fig. 3A); marked squamous hyperplasia with moderate cornification of the vaginal mucosal epithelium (Fig. 3B); and minimal to mild edema of the submucosa of the vagina, the endometrium of the uterus and the myometrium of the uterus. Mucification of the vaginal mucosal epithelium was not observed. Collectively, these data indicate no consistent, dose-dependent differences in histopathologic severity scores in the uterus/cervix or vagina were observed between control and PFOA-treated groups; whereas the changes observed in pre-pubertal CD-1 female mice treated with E2 were markedly different as compared to both control and PFOA-treated groups (Fig. 3C).

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

67

3.4. ER-target gene expression

3.5. Estrogen response element activities in BG1-Luc4E2 cells

Administration of PFOA at doses ranging from 0.005 to 1.0 mg/ kg/day to pre-pubertal CD-1 female mice from PND18–20 caused no changes on PND21 in relative expression of mRNA encoding ER target genes in the uterus including TFF1, TFF2, TFF3 and PR as compared to controls (Fig. 4). By contrast, E2 administration to prepubertal CD-1 female mice from PND18–20 markedly increased relative expression of TFF1, TFF2, TFF3 and PR on PND21 as compared to controls (Fig. 4). The localization and expression of TFF1, TFF2 and PR uteri and vaginas were determined by immunohistochemistry. Administration of PFOA at doses ranging from 0.005 to 1.0 mg/kg/day to pre-pubertal CD-1 female mice from PND18–20 caused no consistent, dose-dependent changes on PND21 in the immunoreactivity for TFF1, TFF2 or PR in the uterus as compared to controls (Fig. 5). Similarly, administration of PFOA at doses ranging from 0.005 to 1.0 mg/kg/day to pre-pubertal CD-1 female mice from PND18–20 caused no consistent, dose-dependent changes on PND21 in the immunoreactivity for TFF1, TFF2 or PR in the vaginas as compared to controls (Fig. 6). By contrast, E2 administration to pre-pubertal CD-1 female mice from PND18– 20 markedly increased relative immunoreactivity for TFF1, TFF2 or PR on PND21 in both the uterus (Fig. 5) and vagina (Fig. 6) as compared to controls. Western blot analysis of uterine tissue was consistent with the immunohistochemical analysis. Whereas administration of PFOA at doses ranging from 0.005 to 1.0 mg/kg/day to pre-pubertal CD-1 female mice from PND18–20 caused no consistent, dose-dependent changes on PND21 in the expression of TFF1, TFF2, TFF3 or PR in the uterus as compared to controls (Fig. 7A), E2 administration to pre-pubertal CD-1 female mice from PND18–20 markedly increased relative expression of TFF1, TFF2 or PR on PND21 in the uterus compared to both PFOA-treated and controls (Fig. 7A). These collective observations indicate that relatively low dose exposure to PFOA to pre-pubertal CD-1 female mice from PND18–20 did not influence ER-target genes expression in the reproductive tract as compared to controls.

Since the predominant ER expressed in the uterus is ERa (Fig. 7B), comprehensive, dose-dependent analysis of whether PFOA could activate an ERa-dependent stable reporter cell line was performed. The stable human ovarian carcinoma cell line, BG1Luc4E2, has an ERE-luciferase construct stably integrated in its genome and expresses ERa, which when activated by estrogenic chemicals, drives expression of the ER-dependent luciferase activity in these cells (Rogers and Denison, 2000). Serum analysis on PND21 of PFOA concentration in pre-pubertal CD-1 female mice administered PFOA at doses ranging from 0.005 to 1.0 mg/kg/day from PND18–20 revealed concentrations from as low as 12.8 nM to as high as 2.8 mM (Table 1). Thus, for these studies, a broad range of PFOA concentration was used to test the estrogenic response in BG1-Luc4E2 cells following PFOA exposure and E2 exposure (as a positive control). The concentrations ranged from the doses below (1.2–2.4 nM), within (12 nM–2.4 mM) and above (12–24 mM) those observed in pre-pubertal CD-1 female mice administered PFOA at doses ranging from 0.005 to 1.0 mg/kg/day from PND18–20 on PND21 in vivo. Neither PFOA nor E2 caused morphological changes in BG1-Luc4E2 cells cultured in the presence of either compound compared to control cells (Fig. 8A). Luciferase activity was not increased by PFOA at any of the concentrations examined in BG1Luc4E2 cells, while luciferase activity was increased markedly (
11-fold) by E2 in BG1-Luc4E2 cells (Fig. 8B). Further, BG1-Luc4E2 cells co-cultured with both PFOA and E2 did not exhibit any dosedependent enhancement of E2-dependent luciferase activity as compared to controls (Fig. 8B). 4. Discussion One of the first reports that examined whether PFOA could activate ERa was the study by Ishibashi et al. who showed that PFOA did not activate either the human ERa or ERb at concentrations ranging from 0.01 to 10,000 mM using an in vitro yeast two-hybrid system (Ishibashi et al., 2007). Subsequent studies by others have provided evidence that in some cases support the hypothesis that PFOA could activate ERa in vivo/in vitro, while in other cases the

Fig. 4. The mRNA expression of ER-dependent genes in the uterus of immature CD-1 mice. The mRNA levels of (A) TFF1, (B) TFF2, (C) TFF3 and (D) PR in the uterus of control and treated CD-1 mice were determined by qPCR and normalized to Gapdh expression as compared to controls (N = 5 per group). E2 administration strongly induced ERresponsive gene expression, while PFOA caused no changes in expression of these genes. Values represent the mean  S.E.M. Values with different letters are significantly different at P  0.05.

68

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

Fig. 5. Immunohistological analysis of ER-dependent target proteins in the uterus of immature CD-1 mice. Uteri were obtained from control CD-1 female mice and mice treated with the indicated doses of PFOA or E2 for 3 days from PND18–20, and analyzed on PND21 (N = 5 per group). The expression and localization of TFF1, TFF2 and PR in the uterus of control (A, F, K), PFOA-treated (B–D, G–I, L–N) and E2-treated (E, J, O) mice shown in the representative photomicrographs were determined by quantifying the immunohistochemical localization of each respective protein (lower panel). Magnification = 40. Bar = 100 mm. Inset photomicrograph magnification = 100. Values with different letters are significantly different at P  0.05.

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

69

Fig. 6. Immunohistological analysis of ER-dependent target proteins in the vagina of immature CD-1 mice. Vaginas were obtained from control CD-1 female mice and mice treated with the indicated doses of PFOA or E2 for 3 days from PND18–20, and analyzed on PND21 (N = 5 per group). The expression and localization of TFF1, TFF2 and PR in the vagina of control (A, F, K), PFOA-treated (B–D, G–I, L–N) and E2-treated (E, J, O) mice shown in the representative photomicrographs were determined by quantifying the immunohistochemical localization of each respective protein (lower panel). Magnification = 40. Bar = 100 mm. Inset photomicrograph magnification = 100. Values with different letters are significantly different at P  0.05.

70

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

Fig. 7. Representative western blot analysis of ER-dependent target proteins in the uterus of immature CD-1 mice. Uteri were obtained from control CD-1 female mice and mice treated with the indicated doses of PFOA or E2 for 3 days from PND18–20 (N = 6 per group), and (A) analyzed for expression of TFF1, TFF2 and PR, or (B) ERa and ERb on PND21. Hybridization signals for each protein were normalized to expression of ACTIN. Normalized expression of TFF1, TFF2 and PR were analyzed by ANOVA. For expression of ERa and ERb, the relative ratio of the normalized ratios was calculated. Values represent the mean  S.E.M. Values with different letters are significantly different at P  0.05.

data obtained did not support this hypothesis. For example, PFOA can increase expression of an ERa target gene in fish and transactivate human ERa in an in vitro reporter assay (Benninghoff et al., 2011), whereas proliferation of a human breast cancer cell line is unaffected following exposure to PFOA at concentrations up to 50 mM while much lower concentrations of E2 markedly increased proliferation of these cells (Maras et al., 2006). Some of these differences observed between studies could be due to differences in the models examined, but also may be related to the concentrations of PFOA used for the studies that may not model the average concentrations of PFOA observed in both the general population and occupationally exposed humans (i.e.,
10–13 nM (Kato et al., 2011), 1.6 mM (Olsen et al., 2007), respectively), which were examined in the present studies. It was recently shown that low-dose exposure to PFOA during early postnatal development caused estrogenic-like activities in immature female CD-1 mice as shown by differences observed in the reproductive tract (Dixon et al., 2012). However, these effects were not observed in a dose-dependent fashion, and the authors hypothesized an inverted-U shaped dose response to explain these effects observed in a single dose group (0.01 mg PFOA/kg), but not the others (0.1 or 1.0 mg PFOA/kg) (Dixon et al., 2012). For this reason, the present study was designed to further examine the hypothesis that low-dose exposure of PFOA to immature female CD-1 mice would cause estrogenic effects in the reproductive tract using a protocol that essentially modeled the one used by Dixon et al., but with an extended dose range. In addition to the uterotrophic assay used by Dixon et al., the present study also examined expression of ER target genes and proteins in the uterus, performed internal dosimetry to quantify serum PFOA concentrations following neonatal exposure (without any influence from potential dietary estrogens), and examined the effect of PFOA and/ or E2 in a stable human cell line capable of detecting ERadependent activity.

In the present study, relative uterine weight on PND21 was unchanged in immature female CD-1 mice exposed to PFOA using doses ranging from 0.005 to 1.0 mg/kg between PND18 and PND20. Administration of E2 over the same postnatal timeframe increased relative uterine weight consistent with previous studies (Dixon et al., 2012). This is in contrast to the study by Dixon et al. where an increase in relative uterine weight was observed, but only at a dose of 0.01 mg PFOA/kg (Dixon et al., 2012). Sections of uterus/cervix and vagina from mice exposed to various doses of PFOA were not histomorphologically discernible from control mice with all observations noted being within the range of biological variability for mice of this strain and age. Control mice in the present study did not exhibit uterine endometrial or myometrial edema, but did have minimal to mild epithelial hyperplasia and myometrial hypertrophy/hyperplasia suggesting that these are normal findings in mice of this strain and age. Minimal to mild uterine epithelial hyperplasia was also observed in all PFOA-treated groups but was histomorphologically indistinguishable from control mice. Further, there was a complete absence of endometrial edema in the PFOA-treated groups in the present study. By contrast, in the study by Dixon et al. (Dixon et al., 2012), 37.5% of control mice had minimal endometrial edema but no control mice had epithelial hyperplasia or myometrial hypertrophy/hyperplasia. The reason for this difference cannot be determined from the present study. In the present study, administration of E2 caused an increase in the histopathological severity score of the uterus/cervix and the vagina. Control mice and PFOA-treated immature female CD1 mice had similar uterine and vaginal histopathological severity scores that were notably lower than the E2-treated group. In contrast, in the study by Dixon et al. (Dixon et al., 2012), higher uterine histopathological severity scores were observed in one PFOA-treated group (0.01 mg/kg), which they interpreted to be similar but less severe than those observed in the E2-exposed group. Dixon et al. also reported a dose-dependent, PFOA-induced change in mucification of vaginal mucosal epithelium, a finding that was not observed in the present study. The reason for this difference cannot be determined from the present study. To provide more sensitive analysis of estrogenic effects in the reproductive tract of immature female CD-1 mice treated with PFOA during early postnatal development, and analysis of ER target genes was performed. Administration of E2 markedly increased the expression of the ER target genes TFF1, TFF2, TFF3 and PR in the uterus, consistent with previous studies (Dunbier et al., 2010; Ing and Tornesi, 1997; Mhawech-Fauceglia et al., 2013; Prest et al., 2002). However, PFOA administration had no influence on the expression of mRNA encoding these uterine ER target genes as compared to controls. Similar results were observed in the uterus using immunohistochemistry and quantitative western blotting, where administration of E2 was the only treatment that caused an increase in expression of these ER target genes and proteins. Importantly, the range in serum concentration of PFOA observed in PFOA-treated immature female CD-1 mice encompassed the range in serum concentrations of PFOA observed in both the general population and occupationally exposed humans (i.e.,
10–13 nM (Kato et al., 2011), 1.6 mM (Olsen et al., 2007), respectively). Collectively, the qPCR, immunohistochemical analyses, and quantitative western blotting provide more compelling evidence that relatively low dose exposure of PFOA during early postnatal development in CD-1 female mice does not activate ER. Given the more extensive dose-dependent analysis in the present study, these data also indicate that an inverted-U shaped dose response as hypothesized by Dixon et al. are unlikely to explain the estrogenic effects observed in one group of immature female CD1 mice treated with 0.01 mg PFOA/kg during early postnatal development (Dixon et al., 2012).

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

Given the differences observed between these two studies, it is of interest to note that standard rodent diets can have significant estrogenic activities (Heindel and vom Saal, 2008; Thigpen et al., 1999a,b, 2013). This is important to note because the study by Dixon et al. used diets that contained estrogenic activity, whereas the present study used a diet that was estrogen-free. Due to the heterogeneity of dietary constituents used in producing standard rodent diets (Warden and Fisler, 2008), it remains possible that the effect observed in one group of immature female CD-1 mice exposed to one dose of PFOA during early postnatal development by Dixon et al. could be influenced by estrogenic activity of the diet used for these studies. This hypothesis deserves further evaluation. An ovarian cancer cell line, BG1-Luc4E2 cells, contain a stably integrated ERE-luciferase construct, allowing for the detection of estrogenic activity mediated by ERa in response to estrogen and environmental xenoestrogens (Rogers and Denison, 2000). Culturing BG1-Luc4E2 cells in medium with concentrations of PFOA that ranged from 1.2 nM to 96 mM did not increase ERa-dependent luciferase activity. Moreover, while culturing BG1-Luc4E2 cells in medium containing 5 nM E2 markedly increased ERa-dependent luciferase activity, co-culture of BG1-Luc4E2 cells with 5 nM E2 and PFOA with concentrations of PFOA that ranged from 2.4 nM to 2.4 mM did not further increase the ERa-dependent luciferase activity observed by E2 alone. These observations are in contrast to another study that found marginal estrogenic activity using a stable MVLN cell line containing an ERE-luciferase construct with concentrations greater than 10 mM (Kjeldsen and Bonefeld-

71

Jorgensen, 2013). The reason why stable BG1-Luc4E2 cells did not detect a change in ERa-dependent luciferase activity while MVLN cells did, cannot be determined from this study, but could be due to differences in the number of copies of the ERE-luciferase construct integrated into the genome of the two cell lines. However, it is important to point out that neither cell line detected changes in ERa-dependent luciferase activity using concentrations of PFOA that reflect both the general population and occupationally exposed humans (i.e.,
10–13 nM (Kato et al., 2011), 1.6 mM (Olsen et al., 2007), respectively. Additionally, two other studies reported that PFOA caused an increase in ERa-dependent reporter activity using transient transfection assays (Benninghoff et al., 2011; Du et al., 2013). It was reported that PFOA did not increase ERadependent luciferase activity when the cells were treated with PFOA alone, but when co-treated with 1 nM E2, PFOA did increase ERa-dependent luciferase activity (Du et al., 2013). Another study found that 0.1 and 1.0 mM PFOA increased ERa-dependent luciferase activity in HEK-293 T cells in the absence of E2 as compared to controls (Benninghoff et al., 2011). However, it is critical to point out that the models used for these analyses have inherent limitations. Du et al. used an expression vector encoding rat ERa, not human ERa. Thus, differences between rat and human ERa could influence these interpretations. More importantly, both studies relied on transient transfections, which does not require chromatin remodeling for transcriptional activity. By contrast, chromatin remodeling is required for ERa-dependent EREluciferase activity in BG1-Luc4E2 cells due to stable integration

Fig. 8. The effect of PFOA on ERa-dependent reporter activity in human ovarian carcinoma cells. BG1-Luc4E2 cells were cultured and treated with the indicated concentrations of PFOA in the absence or presence of E2 (5 nM). The concentrations of PFOA used were below and above the range of serum concentration observed in the in vivo study (Table 1). (A) Representative photomicrographs showing no significant changes in the morphology in either PFOA or E2 treated cells compared to control cells. Magnification = 20. (B) Cell lysates were collected and luciferase activities were determined. E2 caused a marked increase in luciferase activity in BG1-Luc4E2 cells, while PFOA had no effect on estrogen response element activities. The experiment was repeated three times with three independent samples per group. Values represent the mean  S.E.M. Values with different letters are significantly different at P  0.05.

72

P.-L. Yao et al. / Toxicology 326 (2014) 62–73

of the ERE-luciferase construct in these cells. Therefore, the transient transfection assays used for the studies by Du and Benninghoff potentially overestimated estrogenic activity of PFOA. A recent study also reported that PFOA can bind to human ERa with a similar binding pattern to E2 (Gao et al., 2013). However, the binding affinity was much weaker compared to E2, and only relatively high concentrations of PFOA (10–250 mM) were capable of causing a shift in ER structure from the resting state to the agonist-activated state (Gao et al., 2013). The combined results from the present study clearly show that PFOA does not exhibit dose-dependent estrogenic activities in immature female CD-1 mice dosed with PFOA during early postnatal development. Additionally, no inverse-U shaped dose response was observed in immature female CD-1 mice dosed with PFOA during early postnatal development for any endpoints. The conclusion that PFOA does not exhibit dose-dependent estrogenic activities in the reproductive tract of immature female mice dosed with PFOA during early postnatal development is based on several facts including the expanded dosing paradigm used for the present study, the extensive histopathological analyses, the quantitative analyses of ER target gene and protein expression, and the in vitro analyses using a stable ERa-dependent reporter cell line. The collective approaches and endpoints examined in the present study argue strongly that PFOA by itself does not activate either mouse or human ERa. Funding This work was supported by an unrestricted gift from the 3 M Company and the DuPont Company. Conflict of interest John L. Butenhoff, Shu-Ching Chang, and David J. Ehresman are employees of 3 M Company, a former manufacturer of PFOA. Jessica M. Caverly Rae and Steven R. Frame are employees of DuPont Company, a former manufacturer of PFOA. Gerald L. Kennedy, Jr. represents DuPont Company, a former manufacturer of PFOA. PeiLi Yao and Jeffrey M. Peters do not have competing interests. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements The authors gratefully acknowledge Dr. Michael Denison for providing the BG1-Luc4E2 cells used for these studies, and Lance Kramer for technical assistance with these studies. References Abbott, B.D., et al., 2007. Perfluorooctanoic acid induced developmental toxicity in the mouse is dependent on expression of peroxisome proliferator activated receptor-a. Toxicol. Sci. 98, 571–581. Albrecht, P.P., et al., 2013. A species difference in the peroxisome proliferatoractivated receptor a-dependent response to the developmental effects of perfluorooctanoic acid. Toxicol. Sci. 131, 568–582. Andersen, M.E., et al., 2008. Perfluoroalkyl acids and related chemistries– toxicokinetics and modes of action. Toxicol. Sci. 102, 3–14. Benninghoff, A.D., et al., 2011. Estrogen-like activity of perfluoroalkyl acids in vivo and interaction with human and rainbow trout estrogen receptors in vitro. Toxicol. Sci. 120, 42–58. Betts, K.S., 2007. Perfluoroalkyl acids: what is the evidence telling us? Environ. Health Perspect. 115, A250–256. Bjork, J.A., Wallace, K.B., 2009. Structure-activity relationships and human relevance for perfluoroalkyl acid-induced transcriptional activation of peroxisome proliferation in liver cell cultures. Toxicol. Sci. 111, 89–99.

Bjork, J.A., et al., 2011. Multiplicity of nuclear receptor activation by PFOA and PFOS in primary human and rodent hepatocytes. Toxicology 288, 8–17. Cheng, X., Klaassen, C.D., 2008. Critical role of PPAR-a in perfluorooctanoic acid- and perfluorodecanoic acid-induced downregulation of OATP uptake transporters in mouse livers. Toxicol. Sci. 106, 37–45. Dixon, D., et al., 2012. Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay. Reprod. Toxicol. 33, 506–512. Du, G., et al., 2013. Endocrine-related effects of perfluorooctanoic acid (PFOA) in zebrafish, H295R steroidogenesis and receptor reporter gene assays. Chemosphere 91, 1099–1106. Dunbier, A.K., et al., 2010. Relationship between plasma estradiol levels and estrogen-responsive gene expression in estrogen receptor-positive breast cancer in postmenopausal women. J. Clin. Oncol. 28, 1161–1167. Ehresman, D.J., et al., 2007. Comparison of human whole blood, plasma, and serum matrices for the determination of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and other fluorochemicals. Environ. Res. 103, 176–184. Fei, C., et al., 2008. Prenatal exposure to perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) and maternally reported developmental milestones in infancy. Environ. Health Perspect. 116, 1391–1395. Gao, Y., et al., 2013. Assessment of estrogenic activity of perfluoroalkyl acids based on ligand-induced conformation state of human estrogen receptor. Environ. Sci. Technol. 47, 634–641. Heindel, J.J., vom Saal, F.S., 2008. Meeting report: batch-to-batch variability in estrogenic activity in commercial animal diets – importance and approaches for laboratory animal research. Environ. Health Perspect. 116, 389–393. Ing, N.H., Tornesi, M.B., 1997. Estradiol up-regulates estrogen receptor and progesterone receptor gene expression in specific ovine uterine cells. Biol. Reprod. 56, 1205–1215. Ishibashi, H., et al., 2007. Estrogenic effects of fluorotelomer alcohols for human estrogen receptor isoforms a and b in vitro. Biol. Pharm. Bull. 30, 1358– 1359. Ishibashi, H., et al., 2008. Fluorotelomer alcohols induce hepatic vitellogenin through activation of the estrogen receptor in male medaka (Oryzias latipes). Chemosphere 71, 1853–1859. Kato, K., et al., 2011. Trends in exposure to polyfluoroalkyl chemicals in the U.S. population 1999–2008. Environ. Sci. Technol. 45, 8037–8045. Kjeldsen, L.S., Bonefeld-Jorgensen, E.C., 2013. Perfluorinated compounds affect the function of sex hormone receptors. Environ. Sci. Pollut. Res. Int. 20, 8031–8044. Kristensen, S.L., et al., 2013. Long-term effects of prenatal exposure to perfluoroalkyl substances on female reproduction. Hum. Reprod. 28, 3337–3348. Lau, C., et al., 2006. Effects of perfluorooctanoic acid exposure during pregnancy in the mouse. Toxicol. Sci. 90, 510–518. Lau, C., et al., 2007. Perfluoroalkyl acids: a review of monitoring and toxicological findings. Toxicol. Sci. 99, 366–394. Lyngso, J., et al., 2014. Menstrual cycle characteristics in fertile women from Greenland, Poland and Ukraine exposed to perfluorinated chemicals: a crosssectional study. Hum. Reprod. 29, 359–367. Maras, M., et al., 2006. Estrogen-like properties of fluorotelomer alcohols as revealed by MCF-7 breast cancer cell proliferation. Environ. Health Perspect. 114, 100–105. Mhawech-Fauceglia, P., et al., 2013. Trefoil factor family 3 (TFF3) expression and its interaction with estrogen receptor (ER) in endometrial adenocarcinoma. Gynecol. Oncol. 130, 174–180. Nakamura, T., et al., 2009. Microgram-order ammonium perfluorooctanoate may activate mouse peroxisome proliferator-activated receptor a, but not human PPARa. Toxicology 265, 27–33. Olsen, G.W., et al., 2007. Half-life of serum elimination of perfluorooctanesulfonate, perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ. Health Perspect. 115, 1298–1305. Olsen, G.W., et al., 2009. Perfluoroalkyl chemicals and human fetal development: an epidemiologic review with clinical and toxicological perspectives. Reprod. Toxicol. 27, 212–230. Post, G.B., et al., 2012. Perfluorooctanoic acid (PFOA), an emerging drinking water contaminant: a critical review of recent literature. Environ. Res. 116, 93–117. Prest, S.J., et al., 2002. The estrogen-regulated protein, TFF1, stimulates migration of human breast cancer cells. FASEB J. 16, 592–594. Rogers, J.M., Denison, M.S., 2000. Recombinant cell bioassays for endocrine disruptors: development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals. In Vitro Mol. Toxicol. 13, 67–82. Savitz, D.A., et al., 2012. Perfluorooctanoic acid exposure and pregnancy outcome in a highly exposed community. Epidemiology 23, 386–392. Savitz, D.A., et al., 2012. Relationship of perfluorooctanoic acid exposure to pregnancy outcome based on birth records in the mid-Ohio valley. Environ. Health Perspect. 120, 1201–1207. Steenland, K., et al., 2013. Epidemiologic evidence on the health effects of perfluorooctanoic acid (PFOA). Environ. Health Perspect. 118, 1100–1108. Takacs, M.L., Abbott, B.D., 2007. Activation of mouse and human peroxisome proliferator-activated receptors (a, b/d, g) by perfluorooctanoic acid and perfluorooctane sulfonate. Toxicol. Sci. 95, 108–117. Thigpen, J.E., et al., 1999. Phytoestrogen content of purified, open- and closedformula laboratory animal diets. Lab. Anim. Sci. 49, 530–536. Thigpen, J.E., et al., 1999. The phytoestrogen content of rodent diets. Environ. Health Perspect. 107, A182–183.

P.-L. Yao et al. / Toxicology 326 (2014) 62–73 Thigpen, J.E., et al., 2013. The estrogenic content of rodent diets bedding, cages, and water bottles and its effect on bisphenol A studies. J. Am. Assoc. Lab. Anim. Sci. 52, 130–141. Warden, C.H., Fisler, J.S., 2008. Comparisons of diets used in animal models of highfat feeding. Cell Metab. 7, 277. White, S.S., et al., 2011. Endocrine disrupting properties of perfluorooctanoic acid. J. Steroid Biochem. Mol. Biol. 127, 16–26.

73

Wolf, D.C., et al., 2008. Comparative hepatic effects of perfluorooctanoic acid and WY 14,643 in PPAR-a knockout and wild-type mice. Toxicol. Pathol. 36, 632–639. Wolf, C.J., et al., 2010. Developmental effects of perfluorononanoic acid in the mouse are dependent on peroxisome proliferator-activated receptor-a. PPAR Res. Yao, P.L., et al., 2014. Activation of peroxisome proliferator-activated receptor-b/d (PPAR-b/d) inhibits human breast cancer cell line tumorigenicity. Mol. Cancer Ther. 13, 1008–1017.