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Toxicol Sci. 2010 May; 115(1): 214–224.
Published online 2010 Jan 29. doi: 10.1093/toxsci/kfq030
PMCID: PMC2855353
PMID: 20118188

Perfluorooctanoic Acid Effects on Steroid Hormone and Growth Factor Levels Mediate Stimulation of Peripubertal Mammary Gland Development in C57Bl/6 Mice

Yong Zhao,*† Ying S. Tan,*† Sandra Z. Haslam,*† and Chengfeng Yang*†‡,1
Author information Article notes Copyright and License information PMC Disclaimer

Associated Data

Supplementary Materials

Abstract

Perfluorooctanoic acid (PFOA) is a synthetic, widely used perfluorinated carboxylic acid and a persistent environmental pollutant. It is an agonist of peroxisome proliferator–activated receptor α (PPARα). Studies have shown that PFOA causes hepatocellular hypertrophy, tumorigenesis, and developmental toxicity in rodents, and some of its toxicity depends on the expression of PPARα. Our recent study revealed a stimulatory effect of peripubertal PFOA treatment (5 mg/kg) on mammary gland development in C57Bl/6 mice. The present study was designed to examine the underlying mechanism(s). It was found that mammary gland stimulation by PFOA was similarly observed in PPARα knockout and wild-type C57Bl/6 mice. The presence of ovaries was required for PFOA treatment (5 mg/kg) to stimulate mammary gland development with significant increases in the levels of enzymes involved in steroid hormone synthesis in both PFOA-treated wild-type and PPARα knockout mouse ovaries. PFOA treatment significantly increased serum progesterone (P) levels in ovary-intact mice and also enhanced mouse mammary gland responses to exogenous estradiol (E), P, and E + P. In addition, PFOA treatment resulted in elevated mammary gland levels of epidermal growth factor receptor (EGFR), estrogen receptor α, amphiregulin (Areg, a ligand of EGFR), hepatocyte growth factor, cyclin D1, and proliferating cell nuclear antigen (PCNA) in both wild-type and PPARα knockout mouse mammary glands. These results indicate that PFOA stimulates mammary gland development in C57Bl/6 mice by promoting steroid hormone production in ovaries and increasing the levels of a number of growth factors in mammary glands, which is independent of the expression of PPARα.

Keywords: perfluorooctanoic acid, mammary gland development, puberty, progesterone, growth factors, peroxisome proliferator–activated receptor α

Perfluorooctanoic acid (PFOA), a synthetic perfluorinated carboxylic acid, has been widely used in the production of many fluoropolymers for making numerous industrial and consumer products because of its capacity to resist extreme temperatures and stresses. Biomonitoring studies showed that PFOA has been detected in the sera of humans and wild life (Fromme et al., 2009; Harada and Koizumi, 2009; Lau et al., 2007). Classic toxicological studies suggested that the acute toxicity of PFOA is low to moderate in mice, rats, and rabbits (Kennedy et al., 2004; Kudo and Kawashima, 2003). PFOA treatment of adult animals mainly causes liver hypertrophy and a common tumor triad consisting of hepatocellular carcinomas, pancreatic acinar cell tumors, and Leydig cell tumors (Biegel et al., 2001; Kennedy et al., 2004). Recent studies revealed general development toxicity and the specific effects on mammary gland development upon PFOA treatment during various critical developmental stages (Abbott, 2009; Lau et al., 2004; White et al., 2007, 2009; Wolf et al., 2007; Yang et al., 2009). We found that PFOA treatment (5 mg/kg body weight [BW]) for 4 weeks during the peripubertal period (3–7 weeks of age) resulted in increased numbers of terminal end buds (TEBs) and stimulated terminal ducts (TDs) in C57Bl/6 mouse mammary glands (Yang et al., 2009). However, the mechanism by which peripubertal PFOA treatment stimulates mammary gland development in C57Bl/6 mouse strain is not known.

In general, pubertal mammary gland development is mainly controlled by steroid hormones, growth hormones (GHs), and growth factors (Ciarloni et al., 2007; Imagawa et al., 2002; Kleinberg and Ruan, 2008; Sternlicht, 2006). Estradiol (E) and progesterone (P) produced by the ovaries promote mammary gland development. Among other important factors that are involved in stimulating pubertal mammary gland development are GH, insulin-like growth factor I (IGF-I), amphiregulin (Areg, a member of epidermal growth factor [EGF] family; Ciarloni et al., 2007; Imagawa et al., 2002; Kleinberg and Ruan, 2008; Sternlicht, 2006), and hepatocyte growth factor (HGF; Accornero et al., 2009; Niranjan et al., 1995; Soriano et al., 1998). In the pubertal mouse, pituitary GH acts through its receptor on mammary stromal cells to increase the expression of IGF-I, which, in turn, stimulates TEB formation and epithelial branching in a paracrine manner (Ciarloni et al., 2007; Imagawa et al., 2002; Kleinberg and Ruan, 2008; Sternlicht, 2006). Also, GH increases estrogen receptor α (ERα) levels in the rat mammary gland (Feldman et al., 1999). Areg, induced by E, is an essential mediator of ERα function in pubertal mammary gland development acting in a paracrine manner by binding to the EGF receptor (EGFR) to increase epithelial proliferation, TEBs formation, and ductal elongation (Ciarloni et al., 2007). HGF, synthesized in the stroma, is important for normal mammary ductal development by stimulating the proliferation, motility, and morphogenesis of nearby epithelium (Accornero et al., 2009; Niranjan et al., 1995; Soriano et al., 1998). Although it was reported that PFOA treatment increases the serum E levels in adult male CD rats (Biegel et al., 2001), it is not known whether peripubertal PFOA treatment has an effect on the levels of steroid hormones and growth factors in female mice.

The ligand-activated nuclear receptor peroxisome proliferator–activated receptor α (PPARα) plays important roles in regulating inflammatory responses, cell proliferation, and differentiation (Abbott, 2009; Mandard et al., 2004; Rizzo and Fiorucci, 2006). PFOA is an agonist of PPARα, and studies have shown that PPARα plays critical roles in various toxicities of PFOA in rodents. For example, it was found using PPARα knockout 129S1/SvlmJ mice that the general developmental toxicity caused by PFOA depends on the expression of PPARα (Abbott et al., 2007). Whether the effects of PFOA treatment on mouse mammary gland development depend on the expression of PPARα is not known.

In the present study, the potential mechanisms by which peripubertal PFOA treatment stimulates the mammary gland development in C57Bl/6 mice were investigated. It was found that mammary gland stimulation by PFOA was similarly observed in PPARα knockout and wild-type mice. The stimulatory effect of PFOA did not occur in ovariectomized (OVX) mice. PFOA treatment increased the levels of serum P in ovary-intact mice, which was likely due to the elevated levels of steroid hormone synthesis enzymes in the ovary. Moreover, PFOA treatment also upregulated the protein levels of EGFR, ERα, Areg (a ligand of EGFR), HGF, cyclin D1, and proliferating cell nuclear antigen (PCNA) in both wild-type and PPARα knockout mouse mammary glands. These results indicate that PFOA stimulates mammary gland development in C57Bl/6 mice by promoting steroid hormone production in ovaries and increasing the levels of a number of growth factors in mammary glands, and this stimulatory effect dose not depend on the expression of PPARα.

MATERIALS AND METHODS

Animals.

Three-week-old female C57Bl/6 mice were purchased from Charles River Laboratories (Portage, MI). Mice were weighed upon arrival and randomly distributed into different treatment groups. These mice were used for all experiments with wild-type mice. The PPARα knockout C57BL/6 breeding mice were obtained from Taconic Farms, Inc. (Hudson, NY), the sole source for mice with this gene deletion in C57Bl/6 background. All mice received food (8640 Harlan Teklad 22/5 Rodent Diet) and tap water ad libitum and were housed in microisolator cages. Animal facilities were maintained on a 12:12-h light-dark cycle, at 20–24°C and 40–50% relative humidity. All animal protocols were reviewed and approved by the Michigan State University Institutional Animal Care and Use Committee.

Study protocols.

PFOA, as its ammonium salt (>98% pure), was obtained from Fluka Chemical (Steinheim, Switzerland). 17β-Estradiol (E) and P were purchased from Sigma Chemical Co. (St Louis, MO). The stock solutions of E (1 mg/ml) and P (5 mg/ml) were made in 100% ethanol or in 0.85% saline with gum Arabic, respectively. The stock solutions were diluted in 0.85% saline to the final concentrations for injection. PFOA dosing solution was prepared fresh daily in deionized water and given to animals as described previously (Yang et al., 2009). Briefly, C57Bl/6 wild-type or PPARα knockout female mice received either vehicle control (deionized H2O) or PFOA at 5 mg/kg BW by oral gavage, once daily, 5 days per week for 4 weeks starting at 21 days of age (5–10 mice per group). Because the half-life of PFOA is 16–19 days in mice, the mice were not treated on the weekend. BW and appearance of vaginal opening were monitored daily. The animals were sacrificed after 4 weeks of treatment. The estrous cycle status was determined by vaginal smear at termination.

To study whether PFOA itself has a direct hormonal effect (E or P) on mammary gland development, C57Bl/6 mice were OVX at 3 weeks of age, followed by 1-week recovery, and dosed for 4 weeks with vehicle control or PFOA 5 mg/kg BW as described previously (10 mice per group), starting at 28 days of age. BW was monitored daily. The mice were terminated 24 h after the last dose of PFOA. Success of OVX was confirmed by uterine weight: the average uterine weight of OVX mice (5.05 ± 1.04 mg) was significantly lower than that of ovary-intact mice (45.96 ± 4.58 mg).

To study whether PFOA treatment affected mammary gland responses to hormones, E, P, or E + P, ovary-intact C57Bl/6 mice were dosed with vehicle control or PFOA 5 mg/kg BW for 4 weeks starting at 21 days of age. BW and appearance of vaginal opening were monitored daily. The mice were OVX 24 h after the last dose of PFOA. After 1-week recovery, the mice were injected sc daily for 5 days with vehicle control (0.85% saline), E (1 μg/0.2 ml per mouse), P (1 mg/0.2 ml per mouse), or E + P (1 μg + 1 mg/0.2 ml per mouse). The mice were terminated 24 h after the last hormone injection.

Necropsy and mammary gland whole-mount analysis.

Mammary glands, ovaries, and livers were harvested at the time of termination. Tissues were fixed in 10% neutral formalin, paraffin embedded, and then 5-μm sections were prepared and stained with hematoxylin and eosin (H&E). One pair of abdominal and inguinal mammary glands from each animal was prepared as whole mount (Yang et al., 2009). Whole-mount and histological sections were visualized by light microscopy. H&E sections of ovaries and mammary glands were reviewed, blind to treatment, for treatment-related differences and pathological changes.

Whole-mount preparations of the inguinal glands were scored for growth and developmental status. Photomicrographs were made of whole mounts using a Nikon SMZ-2T microscope with QImaging MicroPublisher 5.0 RTV camera (QImaging, Surrey, British Columbia, Canada) at ×10 magnification for quantification of (1) longitudinal growth, as determined by ductal length extending from the lymph node to the most distal terminal branches; (2) numbers of TEBs; and (3) stimulated/enlarged TDs. TEBs are defined as enlarged (>100 μm in diameter), multilayered ductal tips surrounded by adipocytes and located at the periphery of the gland. Stimulated TDs are defined as TEB-like structures (75–100 μm in diameter) occurring within the body of the gland and are distinguished from unstimulated TDs, which averaged 50 μm in diameter. Two independent measures were performed. Mean values for each treatment group were calculated and analyzed statistically for treatment-related differences (Yang et al., 2009).

Measurement of serum steroid hormones and their binding proteins.

Serum total E levels were determined using Delfia Estradiol time-resolved fluoroimmunoassay (Cat no.: 1244-056; PerkinElmer, Turku, Finland) following the manufacture's instructions. Briefly, the serum total E was analyzed in duplicates in a 96-well plate, with each well containing 25 μl of blank, E standards, positive control (Lyphochek Immunoassay Plus Control; Bio-Rad Laboratories, Irvine, CA), or unknown mice serum samples. The levels of serum P were measured by ELISA (Cat no.: 11-PROHU-E01; Alpco Diagnostics, Salem, NH) following the manufacture's instructions. The serum P was determined in a 96-well plate, with each well containing 25 μl of P standards, blank, positive control, or unknown mice serum samples. Serum albumin was assayed by an ELISA kit from Alpco Diagnostics (Cat no.: 41-ALBMS-E01) in a 96-well plate, with each well containing 100 μl of albumin standards, blank, or diluted mice serum samples. Sex hormone–binding globulin (SHBG) was analyzed by Western blot using a specific anti-SHBG antibody (Ab) from Santa Cruz Biotechnology, Inc. (Cat no.: sc-32890; Santa Cruz, CA).

Quantitative PCR array and quantitative reverse transcription–PCR analysis.

Total RNA was extracted from livers by TRIzol Reagent (Invitrogen Corp., Carlsbad, CA), purified by RT2 qPCR-Grade RNA Isolation Kit (SABiosciences, Frederick, MD). Total RNA was quantified by Nanodrop 3300 (ThermoScientific, Wilmington, DE). The quality of RNA was controlled by the A260:A280 ratio higher than 2.0 and confirmed by electrophoreses showing a fraction of each total RNA sample with sharp 18S and 28S ribosomal RNA (rRNA) bands. Three micrograms of total RNA was used for making the first-strand complementary DNA (cDNA; in 20-μl volume) using RT2 First Strand Kit (SABiosciences) following the manufacture's instructions. The generated first-strand cDNAs (20 μl) was diluted to 150 μl with double-stilled water (ddH2O). Then, 1 μl was used for one PCR reaction (in a 96-well plate) for mouse liver drug metabolic enzyme quantitative PCR (qPCR) array (Cat no.: PAMM-002; SABiosciences) and qPCR analysis for the selected genes related to steroid hormone synthesis/metabolism and function. Each PCR reaction (25 μl) includes 12.5 μl of 2× SABiosciences RT2 qPCR Master Mix (SYBR Green), 1 μl of diluted first-stand cDNA synthesis reaction, and 11.5 μl of ddH2O. Total 84 target genes, 5 house keeping genes, and 7 controls were in one 96-well plate for the PCR arrays. The primers for qPCR analysis of the following genes were purchased from SABiosciences: hydroxysteroid (17-β) dehydrogenase 1 (HSD17β1; Cat no.: PPM32668A); HSD17β2 (Cat no.: PPM37004E); HSD17β4 (Cat no.: PPM24933A); sulfotransferase family 1A, member 1 (SULT1A1; Cat no.: 04355A); SULT1E1 (Cat no.: PPM04009B); Uridine diphosphate glucuronosyltransferase 2 family, polypeptide A1 (Ugt2A1; Cat no.: PPM03892A); and Ugt2B1 (Cat no.: 26924A). The qPCR was performed with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA). The qPCR was performed with the following program—step 1: 95°C, 10 min; step 2: 40 cycles of 95°C, 15 s; 60°C, 1 min; step 3: dissociation curve 95°C, 1 min; 65°C, 2 min (optics off); 65–95°C at 2°C per minute (optics on). The data were analyzed and normalized using the online software (http://www.sabiosciences.com/pcr/arrayanalysis.php) from SAbiosciences. Three animal liver samples were analyzed from either control group or PFOA-treated group.

Western blot analysis.

Freshly frozen mammary glands were homogenized in Radioimmunoprecipitation buffer containing the protease inhibitor cocktail, PMSF, and sodium orthovanadate from Santa Cruz Biotechnology, Inc. (Cat no.: sc-24948). Ovarian proteins were isolated from the phenol solution after RNA extraction by TRIzol Reagent following the manufacture's instruction. Protein concentration was determined by Bio-Rad DC Protein Assay Kit. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. All the following primary Abs were purchased from Santa Cruz Biotechnology, Inc.: goat polyclonal anti-aromatase (CYP19A1) Ab (Cat no.: sc-14245), rabbit polyclonal anti-HSD17β1 Ab (Cat no.: sc-32872), goat polyclonal anti-HSD3β1 Ab (Cat no.: sc-30820), mouse monoclonal anti-Areg Ab (Cat no.: sc-74501), rabbit polyclonal anti-ERα Ab (Cat no.: sc-542), goat polyclonal anti-IGF-I Ab (Cat no.: sc-1422), goat polyclonal anti-HGF Ab (Cat no.: sc-1358), and mouse monoclonal anti-GAPDH Ab (Cat: sc-32233). Rabbit polyclonal anti-cyclin D1 Ab (Cat no.: AHF0102) was purchased from Invitrogen Corp. and mouse monoclonal anti-PCNA Ab from Calbiochem (Cat no.: NA-03; Gibbstown, NJ). Thirty micrograms of total protein per sample was loaded onto 12% SDS–polyacrylamide gel electrophoresis gels for Western blot analysis. The gels were transferred to Polyvinylidene Fluoride membrane in cool room (4°C) at 30 V overnight. Then, the membranes were blocked with 5% milk for 1 h at room temperature, followed by three washes with 0.1% Tween 20 in 1× PBS (PBST). The membranes were incubated with primary Abs diluted with 1:1 of PBS:Odyssey block buffer (Abs final concentration 0.2–0.5 μg/ml) for 2 h at room temperature. After three washes with PBST, the blots were incubated with the Alexa Fluor–labeled secondary goat anti-mouse, goat anti-rabbit, or donkey anti-goat Ab for 1 h at room temperature. Then, the blots were scanned using the Odyssey Infrared Imaging System (Li-Cor, Inc., Lincoln, NE) after three washes. Three animals from control- or PFOA-treated group were analyzed. The resulting bands were analyzed using Odyssey Infrared Imaging System software.

Double immunofluorescent staining of Areg and ERα in mouse mammary gland sections.

Mammary gland sections (5 μm) were prepared and subjected to antigen retrieval and immunostaining as previously described (Aupperlee et al., 2005). Briefly, sections were first blocked with normal rabbit serum in PBS, followed by incubation with goat anti-mouse Areg Ab (1:40 in PBS–0.5% Triton X-100;. Cat no.: AF989; R&D Systems, Inc., Minneapolis, MN) at 4°C overnight. After brief wash, sections were incubated with an Alexa 488–labeled rabbit anti-goat secondary Ab (1:100 in PBS; Molecular Probes, Eugene, OR) at room temperature for 30 min and then blocked with goat anti-mouse immunoglobulin G fragment antigene binding portion (1:100 in 1% phosphate buffer saline albumin) for 1 h, followed by incubation with normal goat serum in PBS for 30 min. The sections were then incubated with a mouse anti-ERα monoclonal Ab (1:10 in PBS–0.5% Triton X-100; Cat no.: NCL-ER-6F11; Novocastra Laboratories Ltd, UK) at 4°C overnight, followed by incubation with an Alexa 546–labeled goat anti-mouse secondary Ab (1:200 in PBS; Molecular Probes) at room temperature for 30 min and counterstained with 4′,6-diamidino-2-phenylindole. The stained sections were visualized with a Nikon Eclipse TE2000-U fluorescence microscope (Nikon, Inc., Melville, NY), and the captured fluorescent images were analyzed using MetaMorph software. A minimum of 1000 cells were counted for each section, and a minimum of two to three tissue sections per animal were analyzed. Three animals from control- and PFOA-treated groups were analyzed. The number of Areg- and/or ERα-positive cells is expressed as the percentage of total luminal epithelial cells counted.

Statistical analysis.

Values are presented as mean ± SD for all studies including gene expression array analyses. Differences between control and treatment groups were determined using Student's t-tests, and differences were considered significant at p < 0.05.

RESULTS

PFOA Stimulatory Effect on C57Bl/6 Mouse Mammary Gland Requires the Presence of Ovaries

OVX C57Bl/6 mice were treated with vehicle control (deionized H2O) or PFOA (5 mg/kg BW) daily for 4 weeks and the mammary gland whole mount was analyzed. The mammary glands of OVX vehicle control- or PFOA-treated mice failed to develop compared with ovary-intact mice. There was no outgrowth of ducts or presence of TEBs in control- or PFOA-treated OVX mice (Fig. 1). This was in sharp contrast to the significant stimulatory effect of PFOA on mammary gland development previously observed in the ovary-intact mice (Yang et al., 2009). These results indicate that PFOA does not have a direct hormonal stimulatory effect on C57Bl/6 mouse mammary gland development.

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PFOA has no stimulatory effect on mammary gland development in OVX mice. C57Bl/6 mice were OVX and treated with vehicle control or PFOA (5 mg/kg) for 4 weeks as described in the “Materials and Methods” section. Mice were sacrificed 24 h after the last treatment and mammary gland whole mount was prepared as described in the “Materials and Methods” section. Representative photomicrographs of mammary gland whole-mount images from (A) vehicle control–treated mice and (B) PFOA-treated mice are shown.

Effect of PFOA Treatment on Mouse Serum Levels of Steroid Hormones and Their Binding Proteins

E and P are two major steroid hormones produced mainly by the ovaries that play crucial roles in mammary gland development. As shown in Figure 2A, serum total E levels between vehicle control– and PFOA-treated mice in proestrus or estrus cycle stages are not significantly different. However, PFOA treatment significantly increased serum P levels about threefold in mice that were in proestrus or estrus stages (Fig. 2B). Serum steroid hormone–binding proteins (SHBPs) are capable of binding E and other steroid hormones, thus affecting hormone bioavailability. Serum SHBG and albumin are the most important SHBPs. Because we did not detect a significant difference in serum E levels between vehicle control– and PFOA-treated mice, we measured serum SHBG and albumin to determine whether PFOA treatment had an effect on the levels of SHBPs. Western blot analysis showed no significant differences in serum SHBG levels between vehicle control– and PFOA-treated mice (data not shown). Serum albumin levels were 37.1 ± 2.5 mg/ml (n = 5) and 35.8 ± 3.8 mg/ml (n = 5) in vehicle control– and PFOA-treated mice, respectively, and were not significantly different.

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Effect of PFOA treatment on serum levels of E and P. C57Bl/6 mice were sacrificed 24 h after the last vehicle control or PFOA (5 mg/kg) treatment, and serum was collected for the measurement of (A) E and (B) P levels as described in the “Materials and Methods” section. Data are presented as mean ± SD. “*” or “#” indicates p < 0.05, compared with vehicle control–treated animals in estrus and proestrus, respectively.

PFOA Treatment Increases Mouse Mammary Gland Responses to E, P, or E + P Treatment

The significant increase in serum levels of P in PFOA-treated mice led us to investigate whether PFOA treatment altered mammary gland responses to E, P, or E + P. To address this question, 3-week-old mice were treated with vehicle control or PFOA for 4 weeks and then the mice were OVX. After 1-week recovery, the mice were treated with vehicle control or hormones as described in the “Materials and Methods” section. As shown in Figure 3, there were more stimulated TDs in the mammary glands of mice treated with PFOA and 5-day E or P, compared with that of mice treated with vehicle control and 5-day E or P. Moreover, the mammary glands of mice treated with PFOA and 5-day P or E + P also had more side branches. These data suggest that PFOA treatment increases mouse mammary gland responses to these hormones.

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Effect of PFOA exposure on mammary gland responses to exogenous E, P, or E + P treatment after OVX. C57Bl/6 mice were first treated with vehicle control or PFOA (5 mg/kg) for 4 weeks, and then OVX; injected sc with vehicle control (0.85% NaCl solution), E (1 μg per mouse), P (1 mg per mouse), or E + P (1 μg E + 1 mg P per mouse) for 5 days; and sacrificed 24 h after the last treatment. Mammary gland whole mount was prepared as described in the “Materials and Methods” section. Representative photomicrographs of mammary gland whole-mount images from (A) vehicle control–treated mice and (B) PFOA-treated mice are shown. Note, more enlarged TDs (5dE) or more side branches (5dP, 5dE+P) are indicated by arrows in mammary glands of PFOA-treated mice after hormone treatments.

The Stimulatory Effect of PFOA Treatment on Mammary Gland Development Is Independent of the Expression of PPARα

PFOA is an agonist of PPARα, and a recent study showed that the general developmental toxicity of PFOA in mice depends on the expression of PPARα (Abbott et al., 2007). However, whether the effect of PFOA treatment on mouse mammary gland development depends on PPARα expression is not known. To address this question, PPARα knockout mice from C57Bl/6 background were given the same PFOA treatment as described previously for wild-type mice (Yang et al., 2009). The stimulatory effect of PFOA on mammary gland development was observed in PPARα knockout mice (Fig. 4) similar to that observed in wild-type mice (Yang et al., 2009). Increased numbers of TEBs and stimulated TDs were detected in mammary glands of PFOA-treated PPARα knockout mice. These results demonstrate that the stimulatory effect of peripubertal PFOA treatment on mammary gland development in C57Bl/6 mice is independent of the expression of PPARα.

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PFOA stimulates mammary gland development in PPARα knockout C57Bl/6 mice. Three-week-old PPARα knockout C57Bl/6 mice were treated with vehicle control or PFOA (5 mg/kg) for 4 weeks as described in the “Materials and Methods” section. Mice were sacrificed 24 h after the last vehicle control or PFOA treatment and mammary gland whole mount was prepared as described in the “Materials and Methods” section. Representative photomicrographs of mammary gland whole-mount images from (A) vehicle control—treated or (B) PFOA-treated PPARα knockout mice are shown. White arrows indicate stimulated TEBs and black arrows stimulated duct ends.

Effect of PFOA Treatment on Expression Levels of Liver Drug Metabolic Enzymes in Wild-Type and PPARα Knockout Mice

Previous studies identified liver as one of the major targets of PFOA treatment (Kennedy et al., 2004; Kudo and Kawashima, 2003). Because liver drug metabolic function is another important factor that may affect the serum levels of steroid hormones and thus mammary gland development, we investigated the effect of PFOA treatment on the expression levels of liver drug metabolic enzymes in wild-type and PPARα knockout C57BL/6 mice using a liver drug metabolic enzyme qPCR array. The qPCR array contains 84 critical genes involved in the metabolism of drugs, toxic chemicals, hormones, and micronutrients; the array results are presented in Supplementary table 1. We also analyzed by quantitative reverse transcription–PCR the effects of PFOA treatment on the expression levels of an additional six liver drug metabolic enzymes (HSD17β4, SULT1A1, SULT1E1, Ugt1A1, Ugt2A1, and Ugt2B1) that are critically involved in steroid hormone metabolism but were not included in the qPCR array. Among the total 90 liver drug metabolic enzyme genes analyzed by qPCR array and qPCR, the expression levels of 34 genes in wild-type and 19 genes in PPARα knockout mice were significantly up- or downregulated in the PFOA-treated mice (Table 1, Supplementary table 1). The majority of upregulated genes were detoxification enzymes. The liver metabolic enzymes relevant to steroid hormone metabolism and whose expression levels were significantly changed by PFOA exposure are summarized in Table 1. Interestingly, the expression levels of the majority of enzymes that promote steroid hormone metabolism and excretion were significantly downregulated in wild-type mice, but they were not changed in PPARα knockout mice (Table 1). The expression level of an enzyme (HSD17β4) that converts E to estrone was also significantly upregulated by PFOA treatment in wild-type mice but not in PPARα knockout mice. Since PFOA treatment caused similar effects on mammary glands in both wild-type and PPARα knockout mice, these results suggest that the effect of PFOA treatment on the expression of liver drug metabolic enzymes may not significantly contribute to the stimulation of mouse mammary grand development by PFOA.

TABLE 1

Effect of PFOA Treatment on the Expression Levels of Liver Drug Metabolic Enzymes Related to Steroid Hormone Metabolism/Excretion in Wild-Type and PPARα Knock Mice

Description of liver drug metabolic enzymesPFOA-treated wild-type mice (fold of change)aPFOA-treated PPARα knockout mice (fold of change)
Cytochrome P450, family 1, subfamily a, polypeptide 1−2.5 ± 0.45*No change
Cytochrome P450, family 1, subfamily a, polypeptide 2−3.4 ± 1.14*No change
Catechol-o-methyltransferase−2.1 ± 0.32*No change
HSD17β2−3.4 ± 0.62*No change
HSD17β43.9 ± 1.05*No change
SULT1A1−7.1 ± 2.14*No change
SULT1E1−14.4 ± 1.24*No change
Ugt2A1−2.3 ± 0.78*No change
Ugt2B1−2.0 ± 0.31No change

Note. Three-week-old wild-type and PPARα knockout C57Bl/6 mice were treated with vehicle control or PFOA (5 mg/kg) for 4 weeks and sacrificed 24 h after the last treatment. Total RNA was extracted from frozen liver samples and used for the synthesis of first-strand cDNAs that were used for liver drug metabolic enzyme qPCR array analysis as described in the “Materials and Methods” section.

aFold change is expressed as mean ± SD (n = 3).
*p < 0.05, compared with respective vehicle control–treated mice.

PFOA Treatment Increases Steroid Hormone Synthetic Enzymes in the Ovaries of Wild-Type and PPARα Knockout C57Bl/6 Mice

The differential effects of PFOA treatment on liver steroid hormone metabolic enzyme gene expressions in wild-type and PPARα knockout C57Bl/6 mice and the increase in serum levels of P in PFOA-treated mice led us to examine the effect of PFOA treatment on the levels of critical enzymes involved in steroid hormone production in ovaries. Three enzymes (aromatase, HSD3β1, and HSD17β1) were chosen for analysis based on their important roles in the production of E and P. Aromatase is a key enzyme for the production of E, converting testosterone to E and androstenedione to estrone (Payne and Hales, 2004). HSD3β1 is a critical enzyme for conversion of pregnenolone to P and androstenediol to testosterone, which is the precursor for the production of E (Payne and Hales, 2004). HSD17β1 is another important enzyme in the production of E, mainly by converting estrone to E (Payne and Hales, 2004). Western blot analysis showed that PFOA treatment of wild-type mice increased the protein levels of HSD3β1 and HSD17β1 by 1.6 ± 0.5-fold (n = 3) and 2.8 ± 1.2-fold (n = 3, p < 0.05), respectively, but had no effect on the levels of aromatase (Fig. 5). In PFOA-treated PPARα knockout mice, the protein level of HSD17β1 was increased 2.4 ± 0.64-fold (n = 3, p < 0.05), but the levels of HSD3β1 and aromatase were not changed (Fig. 5). These results suggest that PFOA treatment may increase serum steroid hormone levels by increasing the levels of synthetic enzymes in ovaries.

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Effect of PFOA treatment on protein levels of steroid hormone synthetic enzymes in mouse ovaries. Wild-type and PPARα knockout mice were sacrificed 24 h after the last vehicle control or PFOA (5 mg/kg) treatment. Protein samples prepared from frozen ovaries were used for Western blot analysis as described in the “Materials and Methods” section. The protein levels of HSD3β1, HSD17β1, and aromatase in three individual vehicle control– or PFOA-treated ovaries were analyzed by Western blot. GAPDH served as a protein loading control. Representative Western blot from one animal in each group is shown.

PFOA Treatment Increases the Protein Levels of Areg, HGFα, ERα, EGFR, Cyclin D1, and PCNA in Wild-Type and PPARα Knockout Mouse Mammary Glands

In addition to steroid hormones, local growth factors also play important roles in mammary gland development (Imagawa et al., 2002). We examined the effect of PFOA treatment on the expression levels of Areg, IGF-I, and HGF, growth factors critically involved in mammary gland development. Western blot analysis showed that PFOA treatment increased the protein levels of Areg, IGF-I, and HGFα in wild-type mammary glands by 3.5 ± 2.1-fold (n = 3), 4.1 ± 1.7-fold (n = 3, p < 0.05), and 1.4 ± 0.3-fold (n = 3), respectively (Fig. 6). The protein levels of Areg and HGFα in PPARα knockout mouse mammary glands were also increased 2.7 ± 0.6-fold (n = 3, p < 0.05) and 1.5 ± 0.2-fold (n = 3, p < 0.05), respectively, by PFOA treatment (Fig. 6). Because the expression of Areg could be induced by E, and Areg is an essential mediator of ERα function in mammary gland development (Ciarloni et al., 2007), and Areg is an EGFR ligand, we examined the effect of PFOA on the expression levels of ERα and EGFR in mammary glands. PFOA treatment caused 2.4 ± 1.0-fold (n = 3) and 2.8 ± 1.1-fold (n = 3, p < 0.05) increases of ERα and EGFR protein levels, respectively, in the wild-type mammary glands (Fig. 6). Similarly, PFOA treatment also increased the protein levels of ERα and EGFR by 1.8 ± 0.2-fold (n = 3) and 2.0 ± 0.4-fold (n = 3, p < 0.05), respectively, in PPARα knockout mammary glands (Fig. 6). We also analyzed mammary gland protein levels of cyclin D1 and PCNA, two of the most commonly used markers reflecting active cell proliferation. PFOA treatment caused 3.3 ± 1.1-fold (n = 3, p < 0.05) and 3.1 ± 0.4-fold (n = 3, p < 0.05) increases of cyclin D1 and PCNA protein levels, respectively, in wild-type mouse mammary glands (Fig. 6). Similarly, PFOA treatment increased the protein levels of cyclin D1 and PCNA by 2.4 ± 0.8-fold (n = 3, p < 0.05) and 2.0 ± 0.5-fold (n = 3, p < 0.05), respectively, in PPARα knockout mouse mammary glands (Fig. 6). Together, these results indicate that PFOA treatment raises the levels of steroid hormones, growth factors, and receptors that, in turn, promote mouse mammary gland cell proliferation.

An external file that holds a picture, illustration, etc. Object name is toxscikfq030f06_ht.jpg

Effect of PFOA treatment on protein levels of Areg, HGF, IGF-I, ERα, EGFR, cyclin D1, and PCNA in mouse mammary glands. Wild-type and PPARα knockout mice were sacrificed 24 h after the last vehicle control or PFOA (5 mg/kg) treatment. Protein samples were prepared from frozen mammary glands and used for Western blot analysis as described in the “Materials and Methods” section. The protein levels of Areg, HGF, IGF-I, ERα, EGFR, cyclin D1, and PCNA in three individual vehicle control– or PFOA-treated mouse mammary glands were analyzed by Western blot. GAPDH served as a protein loading control. Representative Western blot from one animal in each group is shown.

The upregulation of Areg and ERα protein levels in PFOA-treated wild-type and PPARα knockout mouse mammary glands was further confirmed by double immunofluorescent staining of ERα and Areg (Figs. 7A and 7B). There was a significant increase in the number of Areg-positive luminal epithelial cells in PFOA-treated mammary glands compared with those in vehicle control–treated mice (Fig. 7C). Consistent with recent findings that Areg expression is induced by E in mouse mammary glands, the Areg staining colocalized with ERα staining (Figs. 7A and 7B). Moreover, PFOA-treated mammary glands had significantly more Areg and ERα double-positive staining cells than vehicle control–treated mouse mammary glands (Fig. 7C).

An external file that holds a picture, illustration, etc. Object name is toxscikfq030f07_3c.jpg

Effect of PFOA treatment on the expression of Areg and ERα in mouse mammary luminal epithelial cells (LECs). Wild-type and PPARα knockout mice were sacrificed 24 h after the last vehicle control or PFOA (5 mg/kg) treatment, and mammary glands were processed for dual immunofluorescent staining of Areg and ERα as described in the “Materials and Methods” section. Representative images of double immunofluorescence staining of Areg (green) and ERα (red) in mammary gland sections from vehicle control– or PFOA-treated (A) wild-type or (B) PPARα knockout mice. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Quantitation of Areg- and/or ERα-positive mammary LECs in vehicle control– or PFOA-treated (C) wild-type or (D) PPARα knockout mice. Data (mean ± SD, n = 3) are presented as percentage of total LECs. a,bp < 0.05, compared with vehicle control–treated mice. Blue bar, ERα-positive cells; red bar, Areg-positive cells; and yellow bar, Areg and ERα double-positive cells.

DISCUSSION

The peripubertal period of breast development is considered an important window of susceptibility to environmental exposures that may predispose humans to increased breast cancer risk later in life (Fenton, 2006). Our recent study found that peripubertal treatment with PFOA, one of the most widely present persistent organic environmental pollutants, caused a stimulatory effect on mammary gland development. After 4 weeks of treatment, we observed increased numbers of TEBs and stimulated TDs in C57Bl/6 mouse mammary glands (Yang et al., 2009). A time-course study looking at the effects of PFOA after 1, 2, or 3 weeks of treatment also showed stimulatory effects detectable as early as after 1 week of treatment but that were maximal after 4 weeks of treatment (Yang and Haslam, unpublished observation). Studies have shown that TEB structures are sensitive to chemical carcinogen treatment and their presence at the time of carcinogen exposure is positively associated with mammary tumor multiplicity in rodent models (Fenton, 2006). The increased numbers of TEBs in the PFOA-treated developing glands could enhance the sensitivity of the gland to the actions of chemical carcinogens. Thus, it is critical to understand the underlying mechanism by which PFOA stimulates mammary gland development.

The best characterized mechanism by which environmental pollutants accelerate mammary gland development is through the estrogen-like effect of endocrine-disrupting chemicals (EDC) that can mimic the effect of endogenous estrogen (Fenton, 2006; Russo and Russo, 1978). Although the stimulatory effects observed in PFOA-treated C57Bl/6 mouse mammary gland and uterus are similar to estrogen-induced effects in these tissues (Yang et al., 2009), it was not known whether PFOA does so through a direct estrogenic effect. The results from our study in OVX C57Bl/6 mice demonstrate that PFOA does not have a direct stimulatory effect. This finding also extends the previous observations from two in vitro studies showing that PFOA does not possess direct estrogenic activity (Ishibashi et al., 2007; Maras et al., 2006).

PFOA is an agonist of PPARα, a ligand-activated nuclear receptor that can regulate gene expression and many important biological functions (Abbott, 2009; Mandard et al., 2004; Rizzo and Fiorucci, 2006). Although it was found that the general developmental toxicity of PFOA resulting from gestational/prenatal exposure in CD-1 mice depends on the expression of PPARα (Abbott et al., 2007), whether the effects of PFOA treatment on peripubertal mouse mammary gland development require the presence of PPARα is not known. We found that peripubertal PFOA treatment caused similar stimulatory effects on mammary gland development in PPARα knockout and wild-type C57Bl/6 mice. This finding establishes that the stimulatory effect of PFOA on mammary gland development in C57Bl/6 mice is independent of the expression of PPARα. In this regard, a previous study found that PPARα knockout mice have normal mammary gland development and function (Lee et al., 1995).

Because the stimulatory effects of PFOA treatment on the mammary gland and uterus are similar to estrogen-induced effects in these organs, we reasoned that PFOA may exhibit an indirect estrogenic effect by increasing the levels of E and/or other steroid hormones. We did not detect a significant increase of serum E levels in PFOA-treated C57Bl/6 mice. Instead, we found about a threefold increase of serum P levels in PFOA-treated mice. Further analysis revealed elevated protein levels of two important enzymes (HSD3β1 and HSD17β1) in the ovary of wild-type mice and HSD17β1 in PPARα knockout mice that are capable of increasing the synthesis of E and P. Previous studies showed that PFOA treatment increased serum E levels by about twofold in male CD rats (Biegel et al., 2001). A possible explanation for the lack of a detectable effect of PFOA on serum E levels herein could be that the E levels in female mice are variable and significantly affected by the stage of estrous cycles. Indeed, it was found that the E levels in female mice fluctuate greatly even within the same estrous cycle stage (Silberstein et al., 2006).

The finding that the serum P levels are significantly higher in PFOA-treated mice is of great interest. Although it is well known that P plays an important role in pregnancy-induced mammary gland development, much less is known about the role of P in peripubertal mammary gland development. We have recently found that E and P have comparable stimulatory effects on mammary gland development in OVX C57Bl/6 pubertal mice as evidenced by similar increases in the numbers of TEBs and stimulated TDs (Aupperlee and Haslam, unpublished observation). In this study, we found that PFOA could increase serum P levels and enhance mammary gland responses to E, P, or E + P treatment by increasing the number of stimulated TDs and side branches in the PFOA-treated OVX wild-type mice. These findings indicate that PFOA not only affects steroid hormone production but also alters the mammary gland response of OVX mice to exogenous steroid hormone treatment. It will be of interest to further investigate the role of P in the stimulatory effect of PFOA on mammary gland development using P receptor knockout mice.

In addition to E and P produced by the ovary, growth factors such as Areg, HGF, and IGF-I produced locally in the mammary gland have also been shown to play important roles in pubertal mammary gland development (Ciarloni et al., 2007; Imagawa et al., 2002; Kleinberg et al., 2000). The present study showed that the protein levels of Areg, HGF, Erα, and EGFR in mammary glands of both PFOA-treated wild-type and PPARα knockout C57Bl/6 mice were elevated. IGF-I protein levels were only increased in PFOA-treated wild-type mice but not in PPARα knockout mice, which suggests that IGF-I may not play a significant role in the stimulatory effect of PFOA on mouse mammary gland development or that there are redundant roles in these factors that promote pubertal mammary gland development. It has been found that E induces Areg and EGFR gene expression (Deroo et al., 2009; Martínez-Lacaci et al., 1995), and our recent study found that E or P treatment significantly increases the expression of Areg protein levels in the mammary gland of peripubertal 3-week-old C57Bl/6 mice (Aupperlee and Haslam, unpublished observation). It is likely that upregulation of Areg levels in mouse mammary gland by PFOA may result from elevated levels of steroid hormones in PFOA-treated mice. We also analyzed the protein levels of EGF and fibroblast growth factor (FGF), two other mammary gland growth factors. In the case of FGF, there was no significant difference between control- and PFOA-treated mice; EGF protein was not detectable by Western blot (Yang and Haslam, unpublished observation). We observed significant increases of ERα and Areg in mammary epithelium. HGF, an estrogen-regulated mammary gland growth factor produced in the stroma (Zhang et al., 2002), was also increased by PFOA treatment. Thus, it is likely that PFOA affects both mammary epithelium and stroma. Further studies are needed to investigate the specific mechanisms by which PFOA treatment increases the levels of ERα and EGFR. Given the important roles of growth factors in mammary gland development, the upregulation of Areg, HGF, ERα, and EGFR may be critically involved in the stimulatory effect of PFOA on mouse mammary gland development.

Because of its widespread presence in the environment, in wildlife, and in humans, and its persistence and accumulation properties, PFOA has raised significant health concerns. In recent years, general population accidental exposures to high levels of PFOA have been reported (Emmett et al., 2006; Hölzer et al., 2008), raising the need for detailed analysis of the toxicological effects of PFOA and the underlying mechanisms. The findings in the present report are significant in three aspects. First, our finding that PFOA does not have a direct hormonal stimulatory effect on mammary gland development extends the previous observations that PFOA does not possess a direct estrogenic effect on cultured cells (Ishibashi et al., 2007; Maras et al., 2006). Second, our observation that PFOA treatment significantly increases the levels of serum P provides a rationale for determining if serum P levels are increased in pubertal girls and women exposed to PFOA. If so, serum P levels may serve as a potential biomarker for monitoring PFOA exposure in women and PFOA may also be considered as an EDC. Third, our finding that the stimulatory effect of PFOA on mammary gland development is independent of the expression of PPARα is of particular interest in relation to previous studies in mice that have shown that PPARα is required for the development of PFOA-induced hepatocellular tumors. The liver tumorigenesis findings have been considered to be not relevant to humans because PFOA is not capable of causing hepatocelular tumors in animal livers expressing a humanized PPARα (Yang et al., 2008). Therefore, these findings are very important for studying the potential effects of PFOA on human breast development and they extend the previous PFOA toxicological studies. Environmental exposures that increase the number of TEBs or prolong their presence in developing mammary gland could enhance the sensitivity of the gland to the action of carcinogens, thus increasing breast cancer risk later in life. If it is determined that PFOA treatment results in increased susceptibility to mammary tumorigenesis, it is highly possible that this effect will be independent of the expression of PPARα. This raises a concern of a potential cancer-promoting effect of PFOA in the human breast. Further studies are warranted to determine if PFOA treatment enhances mammary tumorigenesis in animal models.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

Breast Cancer and the Environment Research Centers grant (U01 ES/CA 012800) from the National Institute of Environmental Health Science and the National Cancer Institute, National Institutes of Health, U.S. Department of Health and Human Services.

Supplementary Material

[Supplementary Data]

Acknowledgments

The authors thank Jeffery Leipprandt, Jessica Bennett, and Lisa Ann Zustiak for their excellent technical assistance in animal model studies. The contents of the article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Environmental Health Science or National Cancer Institute, National Institutes of Health. The authors declare that there are no conflicts of interest.

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