Toxicological Sciences

ToxSci Advance Access originally published online on October 17, 2006
Toxicological Sciences 2007 95(1):108-117; doi:10.1093/toxsci/kfl135

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Published by Oxford University Press 2006.

Activation of Mouse and Human Peroxisome Proliferator–Activated Receptors (, ß/, ) by Perfluorooctanoic Acid and Perfluorooctane Sulfonate

Margy L. Takacs and Barbara D. Abbott¹

Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, North Carolina 27711

¹ To whom correspondence should be addressed at NHEERL Building, US Environmental Protection Agency, 2525 East Highway 54, Durham, NC 27713. Fax: (919) 541-4017. Email abbott.barbara{at}epa.gov.

Received September 21, 2006; accepted October 11, 2006

	ABSTRACT

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This study evaluates the potential for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) to activate peroxisome proliferator–activated receptors (PPARs), using a transient transfection cell assay. Cos-1 cells were cultured in Dulbecco's Minimal Essential Medium (DMEM) with fetal bovine serum in 96-well plates and transfected with mouse or human PPAR, ß/, or reporter plasmids. Transfected cells were exposed to PFOA (0.5–100µM), PFOS (1–250µM), positive controls (i.e., known agonists and antagonists), and negative controls (i.e., DMEM, 0.1% water, and 0.1% dimethyl sulfoxide). Following treatment for 24 h, activity was measured using the Luciferase reporter assay. In this assay, PFOA had more transactivity than PFOS with both the mouse and human PPAR isoforms. PFOA significantly increased mouse and human PPAR and mouse PPARß/ activity relative to vehicle. PFOS significantly increased activation of mouse PPAR and PPARß/ isoforms. No significant activation of mouse or human PPAR was observed with PFOA or PFOS. The PPAR antagonist, MK-886, significantly suppressed PFOA and PFOS activity of mouse and human PPAR. The PPAR antagonist, GW9662, significantly suppressed PFOA activity on the human isoform. In conclusion, this study characterized the dose response and differential activation of mouse and human PPAR, ß/, by PFOA and PFOS. While this model allows opportunities to compare potential activation by perfluoroalkyl acids, it only evaluates the interaction and activation of the PPAR reporter constructs and is not necessarily predictive of a toxicological response in vivo.

Key Words: PPAR; PFOA; PFOS; transient transfection assay; Cos-1 cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 
Perfluoroalkyl acids (PFAAs) are organic fluorochemicals in which carbon-hydrogen bonds are replaced by carbon-fluorine bonds, with the exception of a few terminal carboxyl groups. Fully fluorinated hydrocarbons are stable at high temperatures, nonflammable, and not readily degraded or metabolized. The chemical composition of PFAAs renders this class of compounds stable and persistent in humans, wildlife, and the environment (Giesy and Kannan, 2001, 2002; Grasty et al., 2003; Hansen et al., 2001; Kannan et al., 2001a,b; Lau et al., 2004; Taves, 1968). Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are prominent representatives of the PFAAs and have a broad industrial and consumer application for use in surfactants, coating on fabrics, upholstery and carpets, paper products used for food preservation, fire-fighting foams, and pesticides (Olsen et al., 2005; Renner, 2001). The estimated half-lives in humans are 3.8 years for PFOA and 5.4 years for PFOS. Both compounds are readily absorbed and poorly eliminated in humans and wildlife, and have been shown to activate peroxisome proliferator–activated receptors (PPARs) (Ehresman et al., 2005; Grasty et al., 2003; Lau et al., 2006; Maloney and Waxman, 1999; Shipley et al., 2004). The activation of PPARs by PFOA and PFOS is known to alter enzyme activities and effect changes in gene expression (PFOA: Kennedy et al., 2004; PFOS: Intrasuksri et al., 1998; Shipley et al., 2004).

PFOA and PFOS in rats and mice showed developmental toxicity and other adverse effects in vivo. These effects included reduction of fetal weight, cleft palate, anasarca, delayed ossification of bones, and cardiac abnormalities, as well as, decreased neonatal survival following in utero exposure, reduction in mean post natal body weight, and a significant delay in sexual maturation (Butenhoff et al., 2004; Case et al., 2001; Christian et al., 1999; Gortner, 1980; Grasty et al., 2003; Kennedy et al., 2004; Peraza et al., 2006; Starkov and Wallace, 2002; Thibodeaux et al., 2003; Wetzel, 1983; Yang et al., 2002). The wide distribution of PFAAs in the environment, their persistence in humans and wildlife, and the accumulating evidence of developmental toxicity have drawn considerable attention from the public and regulatory agencies (Ehresman et al., 2005; Maloney and Waxman, 1999; Shipley et al., 2004; U.S. Environmental Protection Agency, 2003).

PPARs are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. These transcription factors alter target gene expression in response to endogenous and exogenous ligands and are associated with lipid metabolism, energy homeostasis, and cell differentiation (Escher and Wahli, 2000; Hihi et al., 2002; Shipley et al., 2004). PPARs are similar to the classical steroid, thyroid, and retinoid hormone receptors in regards to ligand modulation (Escher and Wahli, 2000; Keller and Wahli, 1997; Mangelsdorf et al., 1995; Peraza et al., 2006). The three PPAR isoforms, PPAR, ß/, and , are encoded by separate genes, are expressed in various tissues, and have specific roles during development and in the adult. PPAR isoforms are found in all mammals, amphibians, and teleosts examined to date (Escher and Wahli, 2000; Keller et al., 2000; Peraza et al., 2006; Peters et al., 2005; Wahli, 2002). In mammals, PPAR plays a role in lipid homeostasis, inflammation, and peroxisome proliferation and is well expressed in the liver, heart, kidney, skeletal muscle, intestine, pancreas, lung, placenta, and adipose tissue (Auboeuf et al., 1997; Berry et al., 2003; Escher and Wahli, 2000; Hihi et al., 2002; Mukherjee et al., 1997; Sugden and Holness, 2004). PPARß/ has a role in reverse cholesterol transport, wound healing, and cell proliferation and apoptosis and has been shown to be ubiquitously expressed in both humans and rodents with an important role during development (Berry et al., 2003; Braissant and Wahli, 1998; Braissant et al., 1996; Hihi et al., 2002; Kim et al., 2006; Lim et al., 1999; Peraza et al., 2006; Rodie et al., 2005). PPAR plays a role in lipid storage, adipocyte differentiation, and control of inflammation and is abundantly expressed in adipose tissue, placenta, skeletal muscle, liver, heart, and bone marrow stromal cells (Asami-Miyagishi et al., 2004; Auboeuf et al., 1997; Berry et al., 2003; Escher and Wahli, 2000; Hihi et al., 2002; Mukherjee et al., 1997; Rodie et al., 2005; Tarrade et al., 2001; Vidal-Puig et al., 1997). PPAR isoforms play a role in implantation of the embryo, maintaining pregnancy, growth and development of the fetus, and initiation of labor at term and have specific expression patterns during development in the uterus, embryo, placenta, and extra-embryonic membranes (i.e., amnion and yolk sac) (Asami-Miyagishi et al., 2004; Berry et al., 2003; Braissant and Wahli, 1998; Ding et al., 2003a,b; Dunn-Albanese et al., 2004; Komar and Curry, 2002; Peraza et al., 2006; Tarrade et al., 2001).

Several studies have shown significant activation of the PPAR isoforms by PFAAs in Cos-1 and 3T3-L1 cells using various binding and reporter constructs in cell-based assays (Bility et al., 2004; Maloney and Waxman, 1999; Shipley et al., 2004; Vanden Heuvel et al., 2006). The purpose of our study was to evaluate the potential for PFAAs to activate specific mouse and human PPAR, ß/, isoforms and to determine the effect of specific PPAR antagonists on that activation.

	MATERIAL AND METHODS

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Chemicals.
PFOS (potassium salt; > 98% pure) and PFOA (ammonium salt; > 98% pure) were purchased from Fluka Chemical (Steinheim, Switzerland). WY-14643 (4-chloro-6-(2,3-xylidine)-pyrimidinylthio acetic acid), MK-886 sodium (15-deoxy-D^12,14-prostaglandin J2), L-165,041, Troglitazone, GW9662, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO). WY-14643 and L165,041 were dissolved in DMSO to give a 25mM stock solution. Troglitazone and GW9662 were prepared as 10mM stock solutions and MK-886 was prepared as a 20mM stock solution. Dilutions were prepared in serum-free Dulbecco's Minimal Essential Medium (DMEM) and in triplicate. PFOA and PFOS were dissolved in deionized-distilled water (Picopure Hydro Services and Supplies, Inc., Durham, NC) to give a 25mM stock solution. PFOA was dissolved in water at room temperature and PFOS was dissolved in boiling water. Dilutions of PFOA and PFOS were prepared in serum-free DMEM and in triplicate to give 12 replicates for each dose concentration (i.e., n = 3 independent dilutions with four replicates per dilution).

Plasmids.
Mouse and human PPAR, PPARß/, and PPAR plasmids that were used in this study are the vectors developed and described in Bility et al. (2004) and were kindly provided by Dr Jeffery M. Peters and Dr John P. Vanden Heuvel (Penn State University, PA). The ligand-binding domain of mouse or human PPAR, PPARß/, or PPAR was fused to the DNA-binding domain of the yeast transcription factor Gal4 under the control of the SV40 promoter. This plasmid also encoded the UAS-firefly luciferase reporter under the control of the Gal4 DNA response element. The plasmids were transformed into competent DH5 bacterial cells (Gibco, Grand Island, NY) and isolated using Wizard Plus Miniprep kits (Promega, Madison, WI). Plasmid DNA was verified by AseI and DraIII restriction enzyme digestion fragment size (New England BioLabs, Inc., Ispwich, MA) via gel electrophoresis and using known agonists for each isoform to confirm activation in transfected cells.

Cell culture and transactivation assay.
Cos-1 cells (ATCC, Manassas, VA) were cultured in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco) and 0.2 mg/ml streptomycin and 200 U/ml penicillin (Gibco) at 37°C/5% CO₂. Cells were plated at a density of 10⁴ cells per well of a 96-well plate containing 100 µl of culture medium per well. The medium was replaced with 10 µl serum-free DMEM 24 h after plating, at which time cotransfection with 1 µg/µl plasmid DNA and 0.25 ng/µl of pSEAP2-control vector (Biosciences Clontech, Palo Alto, CA) was carried out using FuGENE 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN) following the manufacturer's recommended procedures for 96-well plates. After 3 h incubation at 37°C/5% CO₂, 100 µl of DMEM + FBS was added to each well and cells were maintained overnight in the culture medium. Following overnight culture, the culture medium was aspirated and replaced with serum-free DMEM containing PFOA or PFOS at concentrations of 0.5–100µM for PFOA and 1–250µM for PFOS to evaluate PPAR activation. Solutions of PFOA, PFOS, positive, and negative controls were prepared fresh on the days of treatment. The PPAR agonist, WY-14643 (10µM for mouse and human), and antagonist, MK-886 (20µM for mouse and human), were used as positive controls for the activation and antagonism of PPAR, respectively. The PPARß/ agonist, L165,041 (40µM for mouse and human), was used as a positive control for PPARß/ activation. Since a PPARß/ antagonist was not available, there is no positive control for the antagonism of mouse or human PPARß/. Troglitazone (30µM for mouse and 3µM for human) and GW9662 (3µM for mouse and 1µM for human) were used as positive controls for PPAR as agonist and antagonist, respectively. Dose response curves for each positive control (agonists and antagonists) were determined to identify optimum concentrations to activate or inhibit mouse and human PPAR, PPARß/, and PPAR activity.

Twenty-four hours after the treatment of the transfected Cos-1 cells with either the positive control compounds, PFOA, or PFOS, with or without the appropriate antagonist, the cells were washed once in cold phosphate-buffered saline (pH 7.4) and lysed with reporter lysis buffer (Promega) for 30 min; luciferase activity was measured using the Luciferase reporter assay kit (Promega) and a LUMIstar Galaxy Luminometer (BMG Labtechnologies, Durham, NC) according to the manufacturer's recommended procedures.

Transfection and cell viability control assays.
Transfection efficiency was evaluated using a SEAP concentration (secreted form of human placental alkaline phosphatase via the pSEAP2-control vector) assay. A SEAP expression control vector was transfected into cells and 24 h later, cell culture medium was collected from each well (100 µl) prior to cell washing and lysis. The concentration of SEAP was determined using the Chemiluminescent SEAP assay kit (BD Biosciences Clontech). Luciferase activity values were normalized for transfection efficiency by multiplying the measured firefly luciferase activity values by the corrected SEAP activity values obtained from the same well. Cytotoxicity assays were performed using a CellTiter-Blue cell viability assay kit (Promega) according to the fluorescence detection method. Analyses showed no cytotoxicity at the PFOA or PFOS concentrations tested.

Statistical methods.
Statistical analyses were performed using Prism 4.0, GraphPad Software (San Diego, CA). Differences between treatments were determined using analysis of variance (ANOVA) followed by Dunnett's post hoc test and Bonferroni posttest, with p < 0.05 deemed significant. The fold inhibition by antagonist of normalized luciferase activity was calculated relative to agonist-treated cells, and represents the mean values ± SE based on n = 3 independent dilutions, with each value being an average of duplicate determinations.

	RESULTS

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Positive and Negative Controls
Each of the mouse and human reporter plasmids was tested in Cos-1 cells for activation by known specific agonists and reduction of that activity by antagonists. Positive and negative controls for each mouse and human PPAR isoform are shown in Figure 1A–1F. Dose response curves for each positive control (agonists and antagonists) were determined to identify optimum concentrations to activate or inhibit mouse and human PPAR, PPARß/, and PPAR activity. While the data for these dose-response assays are not shown, the maximum fold induction of each agonist was as follows: mouse PPAR, 23-fold at 30µM WY-14643; human PPAR, three-fold at 50µM WY-14643; mouse PPARß/, 30-fold at 50µM L165,041; human PPARß/, 15-fold at 40µM L165,041; mouse PPAR, five-fold at 50µM Troglitazone; and human PPAR, four-fold at 30µM Troglitazone. The maximum percent inhibition of agonist activity by antagonist was as follows: mouse PPAR, 90% at 90µM MK-866; human PPAR, 65% at 100µM MK-866; mouse PPAR, 47% at 50µM GW9662; and human PPAR 45% at 80µM GW9662. Rather than use the maximal levels of activation or inhibition, positive control concentrations for assays of PFAA activation were selected from the mid-range of the dose response curves in order to be within scale of the PFAAs to be tested.

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 1 (A–F) Activation of positive and negative controls for mouse and human PPAR, ß/, isoforms. Stock solutions of positive controls (agonist, antagonist) were dissolved in DMSO and dose concentrations were prepared in serum-free DMEM. The figures represent an average of three independent dilutions for each mouse and human construct (i.e., n = 3 independent dilutions with four replicates per dilution). Cell lysates were analyzed for luciferase activity after 24 h of treatment. Significant difference between activity in cultures exposed to agonist + antagonist is represented by a letter and asterisks represent the significant difference between the agonist activity and negative controls (DMSO, water). Abbreviations used: NC, negative control (0.1% DMSO or 0.1% water in solid bar and 0.1% DMSO or 0.1% water + antagonist in striped bar); PC, positive control (agonist in solid bar and agonist + antagonist in striped bar). DMSO and water, 0.1% v/v.

 
The positive and negative controls shown in Figure 1 are the concentrations that were run concurrently with each PFOA and PFOS experiment, and represent the average of three independent assays of the agonists and antagonists for each mouse and human construct. The mouse and human PPAR positive and negative controls are shown in Figure 1A and 1B, respectively. The mouse PPAR agonist, 10µM WY-14643, significantly activated the reporter construct (15-fold induction) and when challenged with the PPAR antagonist, MK-886 at 20µM, resulted in a significant 17% inhibition (ANOVA, p < 0.001). The human PPAR construct was not significantly activated by 10µM WY-14643 (one-fold induction) and there was no significant suppression of this activity in the presence of 20µM MK-886. For the mouse PPARß/ (Fig. 1C), the agonist, 40µM L165,041, resulted in a significant increase in activity relative to the negative controls (28-fold induction) (ANOVA, p < 0.001). Likewise, the human PPARß/ vector (Fig. 1D), was significantly activated by the agonist, 40µM L165,041 (13-fold induction) (ANOVA, p < 0.001). The PPAR agonist, 30µM Troglitazone, significantly stimulated the activity of the mouse PPAR construct (three-fold induction) (Fig. 1E), and a significant 80% inhibition occurred when challenged with the antagonist, 3µM GW9662 (ANOVA, p < 0.001). The activity of the human PPAR construct (Fig. 1F) was also significantly stimulated by the agonist, 3µM Troglitazone (two-fold induction) (ANOVA, p < 0.05), and coexposure to the antagonist, 1µM GW9662, resulted in a 23% inhibition; however, this was not a statistically significant effect. The negative controls, 0.1% DMSO, and 0.1% water did not show a significant activation or suppression of activity in any of the mouse or human PPAR isoforms, either alone or when challenged with the respective antagonists.

Activation of PPAR
Activation of mouse and human PPAR isoforms by PFOA and PFOS are shown in Figure 2 (results of 12 independent experiments). The PPAR inhibitor, MK-886, was used to challenge the activity at each concentration of PFOA and PFOS. PFOA activated mouse and human PPAR in a dose-dependent manner (Fig. 2A and 2B, respectively). The activity of PFOA in the mouse construct (Fig. 2A) increased with dose at 10, 20, 30, and 40µM showing a significant increase in activity compared to the negative water control (e.g., the highest significant activation was 2.5-fold at 40µM) (ANOVA, p < 0.05). At 3 and 10µM, there was a significant difference in activation between PFOA and PFOA + MK-886 (ANOVA, p < 0.05), with 29% inhibition of activity at 3µM PFOA and 44% inhibition at 10µM PFOA. The activity of PFOA in the human PPAR construct (Fig. 2B) also increased with dose and at 30 and 40µM showed a significant increase in activity compared to the negative water control (e.g., the highest activation was 1.88-fold at 40µM) (ANOVA, p < 0.05). At 10µM PFOA there was a significant difference between activation by PFOA and PFOA + MK-886, with 58% inhibition (ANOVA, p < 0.05). The maximum activation achieved was observed at 40µM PFOA in both the mouse (2.3 relative light units [RLU]) and human (1.76 RLU) PPAR constructs.

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 2 (A–D) The activation of mouse and human PPAR expression plasmids by PFOA (A, B, respectively) or PFOS (C, D, respectively) after transfection into Cos-1 cells. Dilutions of PFOA and PFOS were prepared in serum-free DMEM and in triplicate (i.e., n = 3 independent dilutions with four replicates per dilution). The negative water control was included to show activation of nontreated cells. Cell lysates were analyzed for luciferase activity 24 h after treatment. Fold inhibition of the normalized luciferase activity was calculated relative to the agonist-treated cells. Significant difference between the agonist and antagonist is represented by a letter and asterisks represent the significant difference between the agonist activity and negative water control (ANOVA). Abbreviation used: NC, negative control (0.1% water in black bar and 0.1% water + antagonist in white bar). Concentrations examined: PFOA, 0.5, 1, 3, 5, 10, 15, 20, 30, 40µM; PFOS, 1, 10, 20, 30, 60, 90, 120, 150, 250µM; MK-886, 20µM (in mouse PPAR and human PPAR). Water, 0.1% v/v.

 
PFOS also activated the mouse PPAR construct (Fig. 2C) with a significant increase in activity at 120µM PFOS, compared to the negative water control (e.g., highest significant activation was 1.5-fold at 120µM) (ANOVA, p < 0.01). In addition, at 60 and 120µM PFOS concentrations coexposure with MK-886 resulted in a significant 23 and 41% inhibition of activity compared with PFOS alone, respectively (ANOVA, p < 0.05). In the human PPAR construct, PFOS did not significantly increase activity (Fig. 2D) compared to the negative water control. However, there was a significant difference in activity at 120µM between PFOS and PFOS + MK-886, with 33% inhibition (ANOVA, p < 0.05). There was also a significant difference in activity at 250µM between PFOS and PFOS + MK-886, however, at this concentration PFOS + antagonist had increased activity compared to PFOS alone (ANOVA, p < 0.05). The maximum activation was observed at 120µM PFOS in both the PPAR mouse (1.50 RLU) and human (1.40 RLU) constructs.

Activation of PPARß/
The activation of mouse and human PPARß/ by PFOA and PFOS is shown in Figure 3 (results of 12 independent experiments). There was an increase in activity across the dose range of PFOA for the mouse PPARß/ construct (Fig. 3A), and at 40–80µM the increased activity was significant compared to the negative water control (e.g., the highest significant activation was 7.5-fold at 80µM) (ANOVA, p < 0.05). PFOA did not stimulate activity of the human PPARß/ construct (Fig. 3B) at any dose compared to the negative water control. PFOS activated mouse PPARß/ at 20 and 30µM with a significant increase in activity compared to the negative water control (e.g., the highest significant activation was 1.7-fold at 20µM) (ANOVA, p < 0.05) (Fig. 3C). The human PPARß/ construct did not show a significant activation by PFOS at any dose when compared to the negative water control (Fig. 3D).

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 3 (A–D) The activation of mouse and human PPARß/ by PFOA (A, B, respectively) or PFOS (C, D, respectively) after transfection into Cos-1 cells. Dilutions of PFOA and PFOS were prepared in serum-free DMEM and in triplicate (i.e., n = 3 independent dilutions with four replicates per dilution). The negative water control was included to show activation of nontreated cells. Cell lysates were analyzed for luciferase activity 24 h after treatment. Asterisks represent the significant difference between the agonist activity and negative water control (ANOVA). Abbreviation used: NC, negative control (0.1% water in black bar). Concentrations used: PFOA, 10, 15, 20, 30, 40, 50, 60, 70, 80µM; PFOS, 10, 15, 20, 30, 40, 50, 60, 70, 80µM. Water, 0.1% v/v.

 
Activation of PPAR
The activation of mouse and human PPAR by PFOA and PFOS is shown in Figure 4 as the results of 12 independent experiments. PFOA did not significantly increase the activity of the PPAR construct at any dose compared to the negative water control for either mouse (Fig. 4A) or human (Fig. 4B) construct. In addition, for the mouse PPAR construct (Fig. 4A), at each concentration PFOA + GW9662 resulted in an increased activity level over PFOA alone with 30 and 100µM concentrations showing a significant increase in activity over PFOA alone (ANOVA, p < 0.05). For the human construct PFOA + GW9662 had similar activity compared to PFOA alone (no significant difference), except at the 50µM concentration where PFOA + GW9662 produced a significant 58% inhibition compared to PFOA alone (Fig. 4B). PFOS did not significantly activate the PPAR mouse or human construct at any dose compared to the negative water control (Fig. 4C and 4D, respectively). Likewise, there is no significant difference in PFOS activity when challenged with a 3µM concentration of inhibitor, GW9662, for either mouse or human construct.

View larger version (45K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 4 (A–D) The activation of mouse and human PPAR by PFOA (A, B, respectively) or PFOS (C, D, respectively) after transfection into Cos-1 cells. Dilutions of PFOA and PFOS were prepared in serum-free DMEM and in triplicate (i.e., n = 3 independent dilutions with four replicates per dilution). The negative water control was included to show activation of nontreated cells. Cell lysates were analyzed for luciferase activity 24 h after treatment. Fold inhibition of the normalized luciferase activity was calculated relative to the agonist-treated cells. Significant difference between the agonist and antagonist is represented by a letter and asterisks represent the significant difference between the agonist activity and negative water control (ANOVA). Abbreviation used: NC, negative control (0.1% water in black bar and 0.1% water + antagonist in white bar). Concentrations used: PFOA, 1, 5, 10, 20, 30, 40, 50, 75, 100µM; PFOS, 1, 10, 20, 30, 60, 90, 120, 150, 250µM; GW9662, 3µM (mouse PPAR), 1µM (human PPAR). Water, 0.1% v/v.

 
Table 1 provides a summary of PFOA and PFOS activation in the mouse and human PPAR constructs showing the actual RLU and providing estimates of the lowest observed effect level (LOEL) values. PFOA and to a lesser extent PFOS were able to significantly activate mouse and human PPAR and this activity was inhibited by the antagonist. The mouse, but not the human, PPARß/ construct was also significantly activated by PFOA and PFOS. Neither mouse nor human PPAR constructs were activated by PFOA or PFOS.

View this table: [in this window] [in a new window]   TABLE 1 Summary for the LOEC for PFOA and PFOS Transactivation of Mouse and Human PPAR, ß/, and

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 
PPARs, members of the nuclear hormone receptor superfamily, can be activated by various xenobiotics, drugs, and natural fatty acids. These transcription factors regulate genes involved in lipid metabolism, energy homeostasis, and cell differentiation (Braissant et al., 1996; Vanden Heuvel et al., 2006). High profile PFAAs such as PFOA and PFOS have been shown to induce peroxisome proliferation, alter lipid metabolism, and exhibit developmental toxicity in laboratory animals but little is known about the mode of action in the developing embryo, fetus, or neonate. The potential toxicity of PFAAs during embryonic and neonatal development is a matter of concern and the difficulties inherent in extrapolating from laboratory animal models to the developing human make it a priority to have testing models to evaluate the ability for PFAA compounds to activate PPAR isoforms across species. The objective of this study was to evaluate the activity of PFOA and PFOS using mouse and human PPAR, ß/, -isoforms. The agonists and antagonists selected for this study were chosen for their selective activation or antagonism of mouse and human PPAR, ß/, isoforms (Berger et al., 1999; Bility et al., 2004; Burgess et al., 2005; Kehrer et al., 2001; Lehmann et al., 1995; Maloney and Waxman, 1999; Peraza et al., 2006; Shipley et al., 2004; Teruel et al., 1999). There are several known agonists and antagonists for the mouse and human PPAR isoforms; compounds that were suitable for use in culture were selected for the transfected Cos-1 cell assays. The agonist for mouse and human PPAR, WY-14643 exhibited a 15- and one-fold increase in activity over the negative controls, respectively (i.e., DMSO and water). The agonist, L165,041, for mouse and human PPARß/ exhibited a 28- and 13-fold increase in activity over the negative controls and the agonist for PPAR, Troglitazone, showed a three- and two-fold increase in activity for mouse and human PPAR over the negative controls. Our studies confirm that WY-14643, L165,041, and Troglitazone are effective agonists for mouse and human PPAR, PPARß/, PPAR isoforms, respectively, and were particularly active with the mouse PPAR constructs. The antagonist for PPAR, MK-886, resulted in 20% inhibition of agonist activity in both mouse and human constructs in Cos-1 cells exposed to both agonist and antagonist simultaneously. The PPAR antagonist, MK-886, is a noncompetitive inhibitor (Kehrer et al., 2001) and this may have contributed to the inconsistent suppression of WY-14643 activity in the mouse and human PPAR constructs. Another PPAR antagonist, GW6471 (Sigma), was attempted for better inhibition of WY-14643 activity in both the mouse and human constructs; however, GW6471 came out of solution at 50µM during incubation in DMEM at 37°C/5% CO₂, and slowed cell growth substantially at that concentration (data not shown). There was no specific antagonist available for PPARß/. The PPAR antagonist, GW9662, induced 20 and 80% inhibition of agonist activity in the human and mouse PPAR constructs, respectively.

As summarized in Table 1, PFOA showed significant increases in activity in both mouse and human PPAR constructs across the dose response while PFOS induced only the mouse PPAR construct, however, PFOA and PFOS activities were both reduced by the PPAR antagonist. PFOA and PFOS also increased the activity of the mouse PPARß/, but not the human PPARß/ construct. Neither PFOA nor PFOS activated the mouse or the human PPAR construct. However, there were instances of unexpected and significant increases in activity with the PPAR construct after simultaneous exposure to agonist and antagonists. It is not clear what the increased activity of PFOA or PFOS + GW9662 versus PFOA or PFOS alone indicates in the cells transfected with PPAR, and future studies will be needed to examine this interaction.

Our study is in agreement with the current literature examining PFOA and PFOS activity and extends the knowledge of PPAR, ß/, activation across species. The present study confirms that PFOA can activate mouse and human PPAR and mouse PPARß/. In vitro models in various cell types and using a variety of expression vector approaches have shown that PFOA activates PPAR in the range of 10–100µM, that the human PPAR isoform is less sensitive to PFOA than the rodent counterpart, and that mouse PPARß/ is activated by PFOA but not the human PPARß/ counterpart (Intrasuksri et al., 1998; Maloney and Waxman, 1999; Shipley et al., 2004; Vanden Heuvel et al., 2006). Interestingly, the lack of activity for PFOA on PPAR reported in the present study is consistent with what has been reported in another lab which also used Cos-1 cells (Maloney and Waxman, 1999) but inconsistent with that reported using 3T3-L1 cells (Vanden Heuvel et al., 2006). Vanden Heuvel et al. reported activation of mouse and human PPAR in 3T3-L1 cells at 100 and 200µM PFOA, respectively. In Cos-1 cells, we did not detect activity with mouse or human PPAR with 100µM PFOA. This suggests that there may be a cell-type dependent effect on activation of the PPAR vector.

PFOS has been shown to activate mouse and human PPAR in 3T3-L1 cells (Vanden Heuvel et al., 2006) and in Cos-1 cells (Shipley et al., 2004), and these findings are in agreement with our results in Cos-1 cells. PFOS was also reported to activate mouse and human PPAR, but not PPARß, in 3T3-L1 cells (Vanden Heuvel et al., 2006). However, in Cos-1 cells, we did not detect activation of mouse or human PPAR by PFOS. We did observe PFOS activation of the mouse PPARß/ construct in Cos-1 cells. Thus, the only studies to date examining PFOS activity on PPARß and PPAR vectors (Vanden Heuvel's study and ours) used different cell types and report different outcomes. This is similar to the different outcomes for PPARß/ responses to PFOA discussed previously and may reflect an influence of cell type on outcome.

The concentrations of PFOA and PFOS used in our studies were within the ranges found in maternal serum of rats and mice (0.69–199 µg/ml) (PFOS: Lau et al., 2003; Thibodeaux et al., 2003; PFOA: Lau et al., 2006) following doses that caused developmental toxicity or mortality in offspring (1µM 0.5 ppm). In vivo studies investigating the effects of PFOA during pregnancy in the rat and mouse detected full-litter resorptions and neonatal mortality as well as development toxicity, including retarded postnatal growth and significant delays in reaching sexual maturity (Lau et al., 2004, 2006). Exposure to PFOS during pregnancy in rats resulted in cleft palate, anasarca, and heart defects, compromised postnatal survival of neonatal rats and mice, and delayed growth and development (Lau et al., 2003; Thibodeaux et al., 2003). Our in vitro studies clearly demonstrate that PFOA can significantly activate mouse and human PPAR and mouse PPARß/ isoforms and to a lesser extent, PFOS can activate mouse PPAR and ß/ isoforms. During embryonic development, these isoforms are often coexpressed in developing tissues and organs and the relative levels vary between cell types (Braissant et al., 1996). Inappropriate activation (i.e., increase or decrease) of one or more PPAR isoforms during critical stages of development could cause adverse developmental effects or lethality.

The PPAR transactivation model may be useful for screening other perfluorinated compounds. It is important to note several basic concepts when using this model. Activity in this model only evaluates the potential for a compound to interact with the PPAR ligand–binding domains and activate the reporter and is not necessarily predictive of the chemical's ability to produce a toxicological response in vivo. It is also possible that in an in vivo setting, PFOA and PFOS could produce biological responses that are independent of the PPAR receptors. There is also the potential in an in vitro model for endogenous ligands to be produced by the cells which could contribute to the activation of the reporters. Lastly, the use of antagonists is important because while a good correlation between compound interaction and luciferase activity is observed in transfection cell assays, this is not necessarily reflective of specific activity that would be eliminated by a competitive antagonist for the ligand-binding domain. The present work provides unique information on PPAR, ß/, and isoform activation by using isoform-specific antagonists as a positive control to confirm the specificity of the receptor response.

In conclusion, this transactivation model has shown (1) PFOA is more capable than PFOS in activating PPAR and that the mouse PPAR is more responsive than human, (2) PFOA and PFOS activated mouse, but not human, PPARß/, and (3) neither PFOA nor PFOS activated mouse or human PPAR reporters. It is our goal to use this model to evaluate other selected PFAAs for agonist and/or antagonist activity and to use this information to assist in selection of compounds for further testing in vivo.

	NOTES

 
Disclaimer: This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency. Mention of trade names of commercial products does not constitute endorsement/recommendation for use.

	ACKNOWLEDGMENTS

 
The authors appreciate the generous gift of the mouse and human PPAR, PPARß/, and PPAR plasmids, which were designed by Dr John P. Vanden Heuvel and provided for our use by Dr Jeffrey M. Peters, from their laboratories at the Department of Veterinary and Biomedical Science and The Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University. Our gratitude is also extended to Kathy Bobseine and Mary Cardon, who provided invaluable advice and instruction, allowing the model to be established and optimized in our laboratory.

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