Comparative Hepatic Effects of Perfluorooctanoic Acid and WY 14,643 in PPAR-α Knockout and Wild-type Mice

Abstract

Perfluorooctanoic acid (PFOA) is a chemical used in the production of fluoropolymers. Its persistence in the environment and presence in humans and wildlife has raised health concerns. Liver tumor induction by PFOA is thought to be mediated in rodents by PPAR-α. A recent US EPA scientific advisory board questioned the contribution of PPAR-α in PFOA-induced liver tumors. Liver response in CD-1, SV/129 wild-type (WT), and PPAR-α knockout (KO) SV/129 mice was evaluated after seven daily treatments of PFOA-NH₄ ⁺ (1, 3, or 10 mg/kg, p.o.) or the prototype PPARα-agonist Wyeth 14,643 (WY, 50 mg/kg). Livers were examined by light and electron microscopy. Proliferation was quantified after PCNA immunostaining. PFOA treatment induced a dose-dependent increase in hepatocyte hypertrophy and labeling index (LI) similar to WY in WT mice. Ultrastructural alterations of peroxisome proliferation were similar between WY-treated and 10 mg/kg PFOA-treated WT mice. KO mice had a dose-dependent increase in hepatocyte vacuolation but increased LI only at 10 mg PFOA/kg. WY-treated KO mice were not different from KO control. These data suggest that PPAR-α is required for WY- and PFOA-induced cellular alterations in WT mouse liver. Hepatic enlargement observed in KO mice may be due to an accumulation of cytoplasmic vacuoles that contain PFOA.

Introduction

Perfluorooctanoic acid (PFOA) is a fluorinated eight-carbon member of the perfluoroalkyl acid (PFAA) family. PFAAs are both hydrophobic and oleophobic and are used in the preparation of protectants on fabrics and surfactant for nonstick surfaces (Prevodurous et al. 2006). The potential health concerns for PFOA arise from its ubiquitous distribution and persistence in the environment, its presence in humans and wildlife, and its extraordinarily long biological half-life (Houde et al. 2006; Kannan et al. 2004; Olsen et al. 2007; Yamashita et al. 2005). Perfluoroalkyl acids, including PFOA, have been found in the blood and tissues of many species, including fish, marine birds, and marine mammals on several continents including Antarctica (Kannan et al. 2006; Lau et al. 2007; Olivero-Verbel et al. 2006; Sinclair et al. 2006; Tao et al. 2006; Van de Vijver et al. 2005; Van de Vijver et al. 2007; Verreault et al. 2005).

PFOA has been shown to induce a variety of effects in rodents, including liver toxicity and hepatocellular tumors in rats. Rats treated with up to 300 ppm PFOA in the feed for two years had enlarged hepatocytes, scattered hepatocellular necrosis and vacuolation, hepatic inflammation, and increased incidence of hepatic adenomas. PFOA has also been shown to increase liver weight and hepatic beta-oxidation (Biegel et al. 2001). PFOA showed tumor promoter activity by increasing the incidence of diethylnitrosamine (DEN)-initiated liver tumors after twelve months of treatment, but no liver tumors were found in PFOA-treated, noninitiated rats (Abdellatif et al. 1991).

Recently, the US Environmental Protection Agency (EPA) developed a draft risk assessment describing the potential for human health effects from exposure to PFOA (http://www.epa.gov/opptintr/pfoa/index.htm). This assessment was reviewed by the EPA’s Science Advisory Board (SAB, http://www.epa.gov/sab/panels/pfoa_rev_panel.htm). In their review of the assessment, the SAB identified several areas of concern regarding the data in support of the proposed mode of action (through trans-activation of peroxisome proliferator-activated receptor-alpha, PPAR-α) for the rodent liver tumor response from chronic exposure to PFOA. Although there was considerable evidence that rodent liver tumors develop subsequent to PPAR-α activation, there was concern that other events, unrelated to PPAR-α activation, had not been ruled out. Specifically, there was evidence that liver weights increased in PPAR-α knockout mice after treatment with PFOA (Yang et al. 2002). In addition, the short-term increase in hepatocyte proliferation that typically occurs after exposure to a PPAR-α agonist had not been documented. Finally, there is evidence that liver Kupffer cells can be activated in association with PPAR-α stimulation (Rose et al. 1997). The present study was designed to characterize the effects of PFOA and the prototypic PPAR-α agonist Wyeth 14,463 (WY) in mice without functional PPAR-α receptors in comparison to wild-type mice with functional receptors.

Materials and Methods

PPAR-α-null mice (129S4/SvJae-Pparatm1Gonz/J, stock #003580) and wild-type mice (129S1/SvlmJ, stock #002448), were originally purchased from the Jackson Laboratory (Bar Harbor, ME), and were maintained as an inbred colony on the 129/Sv background at the US EPA, Research Triangle Park, NC. Wild-type (SV/129 and CD-1) and PPAR-α knockout mice were given, by oral gavage daily for 7 days, 1, 3, or 10 mg PFOA (Fluka, St. Louis, MO) /kg body weight or 50 mg WY 14,463 (Sigma-Aldrich, St. Louis, MO) /kg. PFOA was prepared fresh daily in deionized water, and WY in 0.5% methylcellulose. Controls received either water or 0.5% methylcellulose at a volume of 10 mL/kg. All animals were housed four per cage in an AAALAC-International accredited US EPA animal facility, and all procedures involving the use of animals were approved by the Institutional Animal Care and Use Committee. At the end of the exposure period (twenty-four hours after the last treatment), mice were euthanized with CO₂ asphyxiation, followed by exanguination. At necropsy blood was collected for serum preparation, and livers were removed and weighed. A portion of the tissue was saved in 10% neutral buffered formalin or glutaraldehyde/paraformaldehyde solution for histological or ultrastructural examination. Another portion was removed for RNA preparation and transcriptional profiling (Rosen et al., 2008), and the remaining liver was flash-frozen on dry ice and stored at −80°C for analytical chemistry. The fixed livers were processed by routine methods for paraffin section and staining with hematoxylin and eosin (HE) for light microscopic examination or for epon embedment; they were then sectioned for uranyl acetate staining and ultrastructural examination by transmission electron microscopy. In addition to the HE-stained section, a companion paraffin section was processed for immunohistochemistry of proliferating cell nuclear antigen (PCNA) (Allen et al. 2006).

Liver sections were read without knowledge of treatment but with knowledge of genetic status. The livers from wild-type mice were scored based on severity of hepatocyte hypertrophy and hepatocyte vacuolation. The lesion scores were 0 = no lesion present, 1 = centrilobular hypertrophy, 2 = centrilobular and midzonal hypertrophy, 3 = panlobular hypertrophy, and 4 = panlobular hypertrophy with cytoplasmic vacuolation. The livers from PPAR-α knockout mice were scored based on severity of hepatocyte cytoplasmic vacuolation only. In knockout animals, there was no hepatocyte hypertrophy present unrelated to cytoplasmic expansion due to vacuole accumulation. The lesion scores were 0 = no vacuoles, 1 = few scattered cells had vacuolation, 2 = scattered clusters of vacuolated hepatocytes, 3 = most lobules had vacuolated hepatocytes, and 4 = majority of the hepatocytes were filled with large clear vacuoles.

To evaluate hepatocyte proliferation, tissue samples in paraffin blocks were sectioned and stained for PCNA by the Zymed-PCNA Staining Kit (Experimental Pathology Laboratories, Inc., Durham, NC). The percentage labeling indices (LI) were determined by counting the number of positive-stained PCNA in 900–1000 hepatocyte nuclei/animal. A Cytology/Histology Recognition Information System (CHRIS, Sverdrup Technology, Inc., Ft. Walton Beach, FL) was used to quantitate the LI from PCNA stained slides. Each image was examined and edited for accuracy (Allen et al. 2006; Medinsky et al. 1999). All liver slides were evaluated for LI without knowledge of treatment.

Analysis of PFOA in serum and liver was performed using a modification of a method originally developed by Hansen et al. (2001). Briefly, 25 μL of serum or homogenized liver was combined with 1 mL of 0.5 M tetrabutylammonium hydrogen sulfate (pH 10) and 2 mL of 0.25 M sodium carbonate and then vortexed for twenty minutes in a 15 mL polypropylene tube. Three hundred microliters of this mixture was then transferred to a fresh 15 mL polypropylene tube, and 25 μL of a 1 ng/μL solution of ¹³C2-PFOA (Perkin-Elmer, Wellesley, MA) was added as an internal standard. Five milliliters of methyl tert-butyl ether (MTBE) was then added, and the mixture was vortexed again for twenty minutes. The tube was centrifuged to separate the aqueous and organic phases, and 1 mL of the MTBE layer was extracted and transferred to a 5 mL polypropylene tube, where it was evaporated to dryness at 45°C under a gentle stream of dry nitrogen.

The residue was then redissolved in 400 μL of a 2 mM ammonium acetate/acetonitrile (1:1) solution and transferred to a polypropylene autosampler vial. Extracts were analyzed using an Agilent 1100 high-performance liquid chromatograph (Agilent Technology, Palo Alto, CA) coupled with an API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) (LC/MS/MS). Ten microliters of the extract was injected onto a Luna C18(2) 3×50 mm, 5 μm column (Phenomenex, Torrance, CA) using a mobile phase consisting of 30% 2 mM ammonium acetate solution and 70% acetonitrile at a flow rate of 200 μL/min. PFOA and ¹³C2-PFOA were monitored using parent and daughter ion transitions of 413 > 369 and 415 > 370, respectively. Peak integrations and areas were determined using Analyst software (Version 1.4.1, Applied Biosystems, Foster City, CA). For each analytical batch, matrix-matched calibration curves were prepared using mouse serum spiked with varying levels of PFOA (Fluka Chemical, Steinhiem, Switzerland), as described above.

For quality control, check standards were prepared by spiking large volumes of mouse serum at several arbitrary levels. These check standards were stored frozen and aliquots analyzed with each analytical set. In addition, control mouse serum samples were fortified at two or three levels in duplicate with known quantities of PFOA during the preparation of each analytical set. Duplicate-fortified and several check standards were run in each analytical batch to assess precision and accuracy. The limit of quantitation (LOQ) was set as the lowest calibration point on the standard curve. Analytical batches were considered to be acceptable if: matrix and reagent blanks had no significant PFOA peaks approaching the LOQ, the standard curve had a correlation coefficient > 0.98, and all standard curve points, fortified and check samples were within 70% to 130% of the theoretical and previously determined values, respectively.

The semiquantitative histology data, labeling indices, and PFOA concentrations were analyzed for statistical significance using JMP software (SAS, Cary, NC). The data were analyzed for pairwise difference to control using Student’s t test and for dose-response to relevant concurrent control using Dunnet’s multiple comparison test. Differences were significant when p < .05.

Results

Liver Weight and Histology

The PFOA-treated wild-type and CD-1 mice had a dose-dependent increase in relative and absolute liver weight, hepatocyte hypertrophy, and LI (Tables 1 and 2, Figure 1). Hepatocyte hypertrophy, increased liver weight, and increased LI were also present in the WY-treated CD-1 and wild-type mice (Tables 1 and 2, Figure 1). There was a dose-dependent increase in absolute and relative liver weight associated with vacuole accumulation in the knockout mouse after PFOA treatment (Table 3, Figure 2). The PFOA-treated PPAR-α knockout mouse response was different than the wild-type in that there was an increase in LI only at the high dose in knockout mice, whereas the wild-type mice had a nonstatistically significant increase in LI after treatment with 1 or 3 mg/kg PFOA.

Ultrastructure

Hepatocytes from wild-type control mice had prominent glycogen in the cytoplasm, and prominent golgi with associated rough endoplasmic reticulum (RER) throughout the cytoplasm. Mitochondria were numerous with 20–50/cell, peroxisomes were few with 0–3/cell and lipid vacuoles were rare (Figure 3A).

Hepatocyte cytoplasm of livers from wild-type WY-treated mice had a flocculent appearance and was moderately electron dense with a lack of glycogen (Figure 3B). Golgi was not a prominent feature, and RER was scarce. The cytoplasm contained numerous single membrane-bound structures with central moderately electron lucent areas; these structures were morphologically consistent with peroxisomes and were present at >50/cell. Mitochondria were also numerous, at >50/cell. The cytoplasm had scattered nonmembrane-bound, lipid-filled spaces which were, in some cells, focally moderate in number. The morphology of hepatocytes from PFOA-treated wild-type mice was the same in all dose groups, as described above for wild-type WY-treated animals with the addition of rarefaction of the cytoplasm as a prominent feature at the highest dose of PFOA (Figure 3C).

Hepatocytes from PPAR-α knockout control mice had prominent cytoplasmic glycogen and golgi with associated RER throughout the cytoplasm (Figure 4A). Mitochondria were numerous, with 20–50/cell. No peroxisomes were evident. The cytoplasm had numerous moderately electron-dense, nonmembrane-bound spaces morphologically consistent with lipids that ranged from the size of mitochondria to the size of nuclei and were present at 3–10/cell. The morphologic features of hepatocytes from WY-treated PPAR-α knockout mice were the same as described for PPAR-α knockout control (Figure 4B)

Hepatocytes from PFOA-treated PPAR-α knockout mice had decreased prominence of glycogen granules within the cytoplasm, less RER, and moderate rarefaction of the cytoplasm (Figure 4C). The most prominent feature was numerous large electron-lucent, nonmembrane-bound spaces throughout the cytoplasm that varied in size, with some as large as hepatocyte nuclei (Figure 4).

Serum and Tissue Analysis

Concentration of PFOA measured in the serum and liver was directly correlated with increasing dose to the animal (Table 4). The magnitude of accumulation of the compound was not different regardless of mouse strain or PPAR-α related genetics.

Discussion

Livers of wild-type mice had ultrastructural changes induced by PFOA that were essentially the same as those induced by WY. In contrast, PPAR-α knockout mice treated with PFOA had significant accumulation of cytoplasmic vacuoles in hepatocytes that was correlated with a dose-dependent increase in serum and liver concentrations of PFOA. These data suggest that functional PPAR-α is required for the cellular alterations induced by either WY or PFOA, and they further suggest that the electron-lucent material that accumulated in PFOA-treated PPAR-α knockout and wild-type mouse hepatocytes may contain or consist of PFOA. The alterations present after high-dose treatment with PFOA (10 mg/kg) in PPAR-α knockout mice are likely due to expansion of the hepatocyte cytoplasm from physical accumulation of material and an associated increase in cell number represented by an increased LI.

Kupffer cells did not appear to be hypertrophic or increased in number in treated animals and therefore not stimulated. A previous study in mice treated with PFOA at a dose that stimulated hepatocellular proliferation resulted in no increase in TNFα or IL-1β in the serum. This lack of increased TNFα and IL-1β suggested no association between Kupffer cell activity and PFOA-induced hepatocellular proliferation (Alsarra et al. 2006). The present study is consistent with this finding. Activation of the nuclear receptor RXR inhibits TNFα production by Kupffer cells and Kupffer cells do not express PPAR-α receptors, suggesting that it is unlikely that Kupffer cells are involved in PPAR-α agonist–induced hepatocellular proliferation and tumors (Alsarra et al. 2006).

PFOA and WY caused a dose-dependent increase in hepatocellular proliferation in wild-type mice, whereas increased proliferation in knockout mice was present only at the high dose of PFOA. Wild-type mice had a 2- and 3-fold increase in LI after treatment with 1 or 3 mg/kg PFOA, respectively, which was associated with increased liver weight and hepatocyte hypertrophy. In a previous study comparing PFOA to WY in rats, hepatic cell proliferation was increased after WY treatment, but PFOA did not increase the number of liver cells (Biegel et al. 2001). However, acute proliferation of hepatocyes may not have been detected in this study since the earliest post-treatment time point examined was 1 month, which does not rule out an early increase in cell proliferation that may have returned to control levels by 3–4 weeks (Biegel et al. 2001). In the present study, the mechanism by which hepatocyte proliferation was stimulated by the high dose of PFOA in knockout mice was not specifically identified, as neither apoptotic nor dead cells were present. It is possible that PFOA at high dose stimulated an alternative nuclear receptor–mediated pathway leading to a mitogenic response not requiring a functional PPAR-α receptor in the PPAR-α knockout mouse liver.

It is not clear which materials constitute the electron-lucent cytoplasmic vacuoles and clear areas in the PFOA-treated mouse livers. In a previous study, PFOA treatment resulted in an increased liver weight in PPAR-α knockout mice (Yang et al. 2002). The present study had the same response in knockout mice, which is attributed to the accumulation of cytoplasmic vacuoles. PPAR-α can be activated by binding to cellular fatty acids. PFOA, which has been shown to accumulate within the cytoplasm of liver cells, has structural similarity to fatty acids by virtue of hydrophobic and rigid perfluorinated carbon tails and strongly polar carboxyl groups (Kudo et al. 2007; Luebker et al. 2002; Maloney and Waxman 1999). In an investigation of PFOA binding to Fabp1, a fatty-acid–binding protein, PFOA was able to displace a fatty-acid molecule, suggesting that PFOA competes for binding with endogenous ligands that may include fatty acids and cholesterol (Luebker et al. 2002). PFOA may act as a ligand for PPAR-α, and PFOA bound to PPAR-α may be a mechanism by which PFOA is distributed within the hepatocyte in wild-type mice. PFOA’s physical chemical structure also makes it poorly miscible in water and lipid, thus promoting clumping to itself, a characteristic consistent with the large clear vacuoles present in the PPAR-α knockout mouse liver (Kissa 2001).

The present study illustrates that the presence of a functional PPAR-α is necessary for PFOA to induce effects in the liver similar to classic peroxisome proliferators. Mouse liver accumulates PFOA to a much greater degree than has been demonstrated in primates. Cynomolgus monkeys treated with 3 or 10 mg PFOA/kg body weight for six weeks had 77 ± 39 and 86 ± 33 μg/mL PFOA in the serum and 15.8 and 14.0 μg/g in the liver, respectively (Butenhoff et al. 2002). This result is in marked contrast to the present study, where the liver concentrations in mice were 3–4 times the mouse serum concentrations and were 10–20 times the primate liver concentrations after the same doses. The response to PFOA treatment in the primate was also different than the mouse in that although liver weight was elevated, there were no histologic alterations or hepatocellular proliferation (Butenhoff et al. 2002). Therefore, significant PFOA accumulation within the liver may be necessary to initiate the hepatic alterations associated with treatment.

In addition to toxicokinetic differences, there are significant functional similarities and differences between rodent and human PPAR-α. Both human and mouse PPAR-α agonism result in similar decreases in serum triglycerides and activation of genes that code for fatty acid oxidation enzymes (Cheung et al. 2004; Peters and Gonzalez 2005). In addition, the DNA and ligand-binding domains of PPAR-α have 100% and 94% homology, respectively, between the two species, suggesting the same peroxisome proliferator response element binding sites and similar ligand-binding affinity between the species (Yang et al. 2008). However, there has been some suggestion that there is a lower level of PPAR-α in human liver than mouse liver, implying that compared to the mouse, a greater concentration of ligand is required to fully activate the human PPAR-α (Peters and Gonzalez 2005). Human PPAR-α also does not activate genes that control cell proliferation. In contrast, mouse PPAR-α activates cell cycle control genes and stimulates hepatocyte cell proliferation (Cheung et al. 2004; Morimura et al. 2006; Peters and Gonzalez 2005; Yang et al. 2008). WY-treated mice expressing the human PPAR-α receptor overexpress p53, whereas wild-type mice have decreased p53 expression, suggesting that apoptosis is stimulated by the human PPAR-α but inhibited by the mouse PPAR-α (Morimura et al. 2006). The inherent differences in the PPAR-α response between mice and humans result in differences in the biological effects mediated by PPAR-α and the two species (Cheung et al. 2004; Peters and Gonzalez 2005; Yang et al. 2008). These data suggest that human liver would be considerably less susceptibile to respond to comparable doses of PFOA than the mouse.

The present study suggests that PFOA concentrates in the mouse liver and that the liver response in the presence of intact PPAR-α is morphologically different than in the PPAR-α knockout mouse. The increased liver weight associated with PFOA treatment of PPAR-α knockout mice is likely a result of accumulation of cytoplasmic vacuoles, which are associated with tissue accumulation of PFOA. A low likelihood for a hepatic tumor response in PFOA-exposed humans is suggested by the requirement for PPAR-α activation for the initiating key events in the mouse liver tumor pathway and by the fact that these events do not occur in mice with human PPAR-α.