ToxSci Advance Access originally published online on February 14, 2008
Toxicological Sciences 2008 103(1):46-56; doi:10.1093/toxsci/kfn025
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Published by Oxford University Press 2008.
Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for the Involvement of Nuclear Receptors PPAR
and CAR
,1
* NHEERL/ORD, U.S. EPA, Research Triangle Park, North Carolina 27711
NHEERL Toxicogenomics Core, U.S. EPA, Research Triangle Park, North Carolina 27711
National Cancer Institute, Research Triangle Park, North Carolina 27711
1 To whom correspondence should be addressed at Environmental Carcinogenesis Division, National Health and Environmental Effects Research Lab, U.S. Environmental Protection Agency, 109 T.W. Alexander Dr., MD-B143-06, Research Triangle Park, NC 27711. Fax: (919) 541-0694. E-mail: corton.chris{at}epa.gov.
Received December 19, 2007; accepted February 4, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear receptor peroxisome proliferator–activated receptor alpha (PPAR
). To determine the role of PPAR in mediating PFOA transcriptional events, we compared the transcript profiles of the livers of wild-type or PPAR-null mice exposed to PFOA or the PPAR agonist WY-14,643 (WY). After 7 days of exposure, 85% or 99.7% of the genes altered by PFOA or WY exposure, respectively were dependent on PPAR. The PPAR–independent genes regulated by PFOA included those involved in lipid homeostasis and xenobiotic metabolism. Many of the lipid homeostasis genes including acyl-CoA oxidase (Acox1) were also regulated by WY in a PPAR–dependent manner. The increased expression of these genes in PPAR-null mice may be partly due to increases in PPAR
expression upon PFOA exposure. Many of the identified xenobiotic metabolism genes are known to be under control of the nuclear receptor CAR (constitutive activated/androstane receptor) and the transcription factor Nrf2 (nuclear factor erythroid 2–related factor 2). There was excellent correlation between the transcript profile of PPAR–independent PFOA genes and those of activators of CAR including phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) but not those regulated by the Nrf2 activator, dithiol-3-thione. These results indicate that PFOA alters most genes in wild-type mouse liver through PPAR, but that a subset of genes are regulated by CAR and possibly PPAR in the PPAR-null mouse. Key Words: peroxisome proliferator; perfluorinated alkyl acid; perfluorooctanoic acid; liver cancer.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Perfluoroalkyl acids (PFAA) including perfluorooctanoic acid (PFOA) are commonly used as surfactant processing aids in the production of fluoropolymers, coatings for clothing fabrics, upholstery and carpets, and paper products approved for food contact. PFAA are composed of a carbon backbone (typically ranging from C-4 to C-15), full substitution of hydrogen by fluorine, and a functional group (carboxylic acid in the case of PFOA). These chemicals are stable and persistent in the environment; wildlife and humans have measurable amounts in their tissues. PFAA are readily absorbed (Johnson and Ober, 1979
; Kemper, 2003), not known to be metabolized (Kuslikis et al., 1992; Ophaug and Singer, 1980; Vanden Heuvel et al., 1991), and poorly eliminated, with half-lives in humans estimated at 3.8 years for PFOA (Ehresman et al., 2005; Olsen et al., 2005). Production of PFOA was estimated in excess of 500 metric tons in the year 2000. Although the production of one PFAA, perfluorooctane sulfonate (PFOS) by its major manufacturer was phased out at the end of 2002, replacement PFAA chemicals (such as PFOA) are filling its void in the consumer and industrial markets.
PFOA is a member of a large class of structurally heterogeneous pharmaceutical and industrial chemicals called peroxisome proliferator chemicals (PPC) (Kennedy et al., 2004). The peroxisome proliferator–activated receptors (PPAR, β, and ), comprise a subset of the nuclear receptor superfamily and mediate many of the adaptive consequences of PPC exposure (Corton et al., 2000; Peraza et al., 2006). In the livers of mice and rats PPC exposure results in a predictable set of responses including increased expression of fatty acid β-oxidation genes, hepatocyte peroxisome proliferation, hepatomegaly, hepatocyte hypertrophy and hyperplasia, and increased incidence of liver tumors (Klaunig et al., 2003). For a number of PPC, these responses are abolished in PPAR-null mice (Anderson et al., 2004a, b; Lapinskas et al., 2005; Peters et al., 1997, 1998).
Like PPC, PFOA exposure in rats results in increases in markers of peroxisome proliferation, increased liver to body weights, hepatocyte proliferation and an increased incidence of liver tumors (summarized in Kennedy et al., 2004). PFOA and PFOS can activate PPAR and to a lesser extent, PPARβ and PPAR in trans-activation assays (Takacs and Abbott, 2007; Vanden Heuvel et al., 2006). Given the similarities between the responses altered by PFOA and other PPC, PFOA may mediate many of its effects linked to liver cancer through PPAR. However, increases in liver to body weights were observed in PFOA-exposed PPAR-null mice (Abbott et al., 2007; Yang et al., 2002), the biological basis for which is unknown.
In addition to activation of PPAR subtypes, PFAA may also mediate effects in the liver through other mechanisms. In a recent study, the transcript profiles of livers from rats treated with PFOA or PFOS were compared to a toxicogenomics reference database of over 600 compounds. As expected, PFOA and PFOS exhibited gene expression changes similar to other PPC. However, PFOA and to a lesser extent PFOS, increased the expression of marker genes for other nuclear receptors (Martin et al., 2007). These candidate nuclear receptors include constitutive activated/androstane receptor (CAR) and pregnane X receptor (PXR), which regulate the expression of xenobiotic metabolizing enzymes (XME) in response to exposure to drugs and environmental chemicals (Stanley et al., 2006; Timsit and Negishi, 2007). Similarities exist between the key events involved in liver tumor induction by PPC and compounds that activate CAR and PXR. Exposure to CAR or PXR activators can lead to increases in liver weight and hepatocyte hyperplasia that are abolished in mice nullizygous for the individual receptors (Chen et al., 2003; Huang et al., 2005; Staudinger et al., 2001; Yamamoto et al., 2004). Chronic exposure to the CAR activators phenobarbital (PB) or TCPOBOP (TC) leads to increases in liver cancer in a CAR-dependent manner (Huang et al., 2005; Yamamoto et al., 2004). Although PXR activators bind directly to the receptor, CAR can be activated through two distinct mechanisms. A number of compounds (e.g., TCPOBOP) bind directly to CAR leading to nuclear localization and transcriptional activation. In contrast, PB activates CAR through an ill-defined mechanism that may require phosphorylation events mediated by the adenosine monophosphate kinase (Blattler et al., 2007). By comparing the transcript profiles in wild-type and PPAR-null mice following exposure to either PFOA or WY-14,643, a selective PPAR activator, our group has recently suggested that PFOA may activate CAR regulated genes (Rosen et al., 2008).
In this study we describe a toxicogenomic dissection of the transcript profiles altered by PFOA exposure in the mouse liver. Using both original and historical data we show that most of the genes that were altered by PFOA were dependent on PPAR. However, there were many genes regulated by PFOA independently of PPAR involved in xenobiotic metabolism that are also the targets of the CAR activators, PB, and TCPOBOP. This toxicogenomic dissection indicates that PPAR and CAR control most of the genes regulated by PFOA in the mouse liver.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Animals, dosing, and tissue collection.
We used the tissues from a number of studies for the gene expression work. Study 1 was carried out at the U.S. Environmental Protection Agency (EPA), Research Triangle Park, NC. Male and female 129S1/SvlmJ wild-type (stock #002448) and PPAR
-null (Ppara-tm1Gonz/J, stock #003580) mice were obtained from Jackson Laboratories, (Bar Harbor, MA). Strain 129S1/SvImJ is recommended as the best approximate match to the 129S4/SvJae background of the PPAR-null strain. Breeding colonies of wild-type and PPAR-null mice were established in the EPA Reproductive Toxicology Facility in Research Triangle Park, NC. Genotypes of the mice were confirmed by PCR analysis (method provided by Jackson Laboratories) using genomic DNA prepared from tail biopsies taken from at least one pup from every litter. Mice were housed in ventilated Tecniplast cages (Tecniplast USA, Exton, PA) and provided pellet chow (LabDiet 5001, PMI Nutrition International, Brentwood, MO) and tap water ad libitum. Animal facilities were controlled for temperature (20–24°C) and relative humidity (40–60%), and kept under a 12-h light-dark cycle.
Four wild-type and PPAR-null male mice per group (6 months of age) were dosed by gavage for 7 consecutive days with PFOA (Fluka Chemical cat#77262, Steinheim, Switzerland) in distilled water or distilled water alone. Dose groups consisted of 1, 3, and 10 mg/kg/day PFOA. All dosing solutions were freshly prepared each day. At the end of the dosing period, animals were euthanized by CO2 asphyxiation and liver tissue was collected for preparation of total RNA and histopathology (described in Wolf et al., 2008). In the Wolf et al. study, all doses increased liver pathology including hepatocyte hypertrophy in wild-type mice and hepatocyte vacuolation in PPAR-null mice. In both strains there were increases in labeling indices at the 10 mg/kg dose.
Study 2 was carried out at Chemical Industry Institute of Toxicology (CIIT) Centers for Health Research (now Hamner Centers for Health Research) (Research Triangle Park, NC). Wild-type and PPAR-null mice 9–12 weeks of age on a mixed SV129/C57BL/6 background were used in these studies and have been described previously (Lee et al., 1995). The mice were originally obtained from Dr Frank Gonzalez, National Cancer Institute, National Institutes of Health (NIH) to establish a breeding colony at CIIT. Control and treated mice were provided with NIH-07 rodent chow (Zeigler Brothers, Gardeners, PA) and deionized, filtered water ad libitum. Lighting was on a 12-h light/dark cycle. Male mice were given by gavage one or three daily doses of the PPAR agonist WY-14,643 (ChemSyn Science, Lenexa, KS) at 50 mg/kg/day or the carrier methylcellulose (0.1%) and sacrificed either 12 h (one dose) or 24 h (three daily doses) after the last dose. A separate set of male mice were also fed a control diet or a diet containing WY (500 ppm) for 1 week. Liver tissue was collected one week after initiation of WY exposure and total RNA was prepared for gene profiling.
Study 3 was carried out at Baylor University (Houston, TX) in the laboratory of Dr David Moore. Female wild-type and CAR-null mice (8 weeks of age) (C57BL/6x129Sv) were treated with corn oil or the CAR activators PB (100 mg/kg/day for three days) or TCPOBOP (3 mg/kg on the first day and corn oil on days 2 and 3). Mice were sacrificed 72 h after initial exposure. Details of the experiment can be found on the Nuclear Receptor Signaling Atlas web site (www.nursa.org/10.1621/datasets.01003 verified 18 October 2007).
Study 4 was carried out at Johns Hopkins University (Baltimore, MD) in the lab of Dr Thomas Kensler. Ten-12-week-old wild-type and nuclear factor erythroid 2–related factor 2 (Nrf2)-null ICR mice were treated with 0.5 mmol/kg of dithiol-3-thione (D3T) by gavage in a suspension consisting of 1% Cremophor and 25% glycerol. Mice were sacrificed 6 or 24 h after treatment. Further details of the experiment are found in the original publication (Kwak et al., 2003). Portions of the livers from these studies were rapidly snap-frozen in liquid nitrogen and stored at –70°C until analysis. All animal studies were conducted under federal guidelines for the use and care of laboratory animals and were approved by Institutional Animal Care and Use Committees.
RNA isolation and analysis of gene expression.
For Study 1 liver, RNA was immediately isolated after tissue collection using a modified guanidium isothiocyanate method (TRIzol; Invitrogen) and was further purified using silica membrane spin columns (RNeasy; Qiagen, Valencia, CA). RNA integrity was assessed by the RNA 6000 LabChip kit using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Global gene expression changes were examined using the Affymetrix, Santa Clara, CA platform. Gene expression changes were assessed after exposure to 3 mg/kg/day PFOA or water controls in wild-type and PPAR-null mice. There were four animals per group; gene expression in each animal was assayed on a separate chip. Biotin-labeled Complementary RNA (cRNA) was produced from 15 µg total RNA using an Affymetrix "one-way" labeling kit (cat# 900493). Total cRNA was then quantified using a Nano-Drop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE) and evaluated for quality after fragmentation on a 2100 Bioanalyzer. Following overnight hybridization at 45°C to Affymetrix 430_2 GeneChips in an Affymetrix Model 640 GeneChip hybridization oven, the arrays were washed and stained using an Affymetrix 450 fluidics station as recommended by the manufacturer and scanned on an Affymetrix Model 3000 scanner. After scanning, raw data (Affymetrix .cel files) were obtained using Affymetrix GeneChip Operating Software (version 1.4). This software also provided summary reports by which array QA metrics were evaluated including average background, average signal, and 3'/5' expression ratios for spike-in controls, β-actin, and GAPDH. Gene expression was determined in Studies 2–4 as previously described (Anderson et al., 2004a, b; Kwak et al., 2003; www.nursa.org/10.1621/datasets.01003). Studies 1 and 3 used 430_2 chips and study 2 and 4 used the U74Av2 chips from Affymetrix.
Data (.cel files) was analyzed and statistically filtered using Rosetta Resolver version 6.0 software (Rosetta Inpharmatics, Kirkland, WA). Statistically significant genes were identified using one-way ANOVA with a false discovery rate (Benjamini–Hochberg test) of 
0.05 followed by a post hoc test (Scheffe) for significance. To directly compare the genes across GeneChip platforms, "best match" genes were obtained through the Affymetrix NetAffx web site (http://www.affymetrix.com/analysis/index.affx). There were a total of 8904 genes that were examined compared to the > 12,000 genes on the U74Av2 chip and > 45,000 genes on the 430_2 chip. Significant transcripts were evaluated for relevance to canonical pathway and biological function using Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com). Heat maps were generated using Eisen Lab Cluster and Treeview software (http://rana.lbl.gov/EisenSoftware.htm). Significant gene lists from ANOVA analysis were compared at the Gene Ontology level using MetaCore from GeneGo, Inc. (http://trials.genego.com/cgi/index.cgi#Information). The p values for MetaCore processes were calculated using a hypergeometric distribution in which the p value represents the probability of particular mapping arising by chance, given the numbers of genes in the set of all genes on processes, genes on a particular process and genes in the experiment. We considered significance at p 0.001 from the top 50 significant GO category list. A description of the PFOA microarray experiment is available through Gene Expression Omnibus at the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/geo/, as accession number GSE9786.
Real-time reverse transcription–PCR analysis.
The levels of expression of selected genes were quantified using real-time reverse transcription–PCR (RT-PCR) analysis from the 1, 3, and 10 mg/kg/day treatment groups in both mouse strains. Briefly, total RNA was reverse transcribed with murine leukemia virus reverse transcriptase and oligo(dT) primers. The forward and reverse primers for selected genes (Supplemental File 1) were designed using Primer Express software, version 2.0 (Applied Biosystems, Foster City, CA). The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real-time PCR analysis. The relative differences in expression between groups were expressed using cycle threshold (Ct) values as follows. The Ct values of the genes were first normalized with 18S ribosomal RNA of the same sample, and then the relative differences between control and treatment groups were calculated and expressed as relative increases, setting the control as 100%. Assuming that the Ct value is reflective of the initial starting copy and that there is 100% efficiency, a difference of one cycle is equivalent to a twofold difference in starting copy. Means and SE (n = 4) for RT-PCR data were calculated by Student's t test. The level of significance was set at p 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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PPAR
is Required for the Majority of Transcriptional Changes after PFOA ExposureTo uncover the gene regulatory networks that may contribute to liver tumor induction by PFOA, we examined transcript profiles in the livers of wild-type and PPAR
-null mice exposed to 3 mg/kg of PFOA each day for 7 days using Affymetrix full genome mouse chips. Significantly altered genes were identified using Rosetta Resolver as described in the "Materials and Methods." In wild-type mice, PFOA exposure altered 2.4% of the total number of genes queried (45,101), including 641 upregulated and 451 downregulated genes (Fig. 1A and Supplemental File 2). PFOA affected far fewer genes in PPAR-null mice, altering only 0.3% of the genes queried including 104 upregulated and 52 downregulated genes. Most of the genes regulated by PFOA in PPAR-null mice (117 genes) were also regulated similarly in wild-type mice. Analysis of the similarity of the gene changes in the two strains gave a Pearson's correlation of 0.529. PFOA also altered the expression of 39 genes solely in PPAR-null mice consisting of 28 upregulated genes and 11 downregulated genes. These results indicate that PPAR is required for the majority of transcriptional changes after PFOA exposure in the mouse liver.
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Comparison of the PFOA Transcript Profile with that of WY-14,643
To determine if the pattern of gene expression upon PFOA exposure is typical of a PPC, we compared the overall pattern of gene expression changes altered by PFOA with that of the prototypical activator of PPAR
, WY-14,643 (WY). Compared to PFOA, genes regulated by WY after 12 h, 3 or 7 days of exposure were more dependent on PPAR; a smaller number of genes were altered in the PPAR-null mice (18, 53 and 6 genes altered at 12 h, 3 days and 7 days, respectively) (Fig. 1B, and Supplemental File 3).
We directly determined the overlap in the genes regulated by PFOA and WY in wild-type versus PPAR-null mice as described in the "Materials and Methods." The genes altered by PFOA in wild-type and/or PPAR-null mice could be divided into 4 groups based on their expression behavior compared to WY (Fig. 2 and Supplemental File 4). The genes regulated by PFOA in wild-type mice only (449 genes) consisted of 398 genes that overlapped with those regulated by WY in a PPAR–dependent manner (group I) and 51 genes that were regulated by PFOA but not WY (group II). The group I genes shared common direction and magnitude of change by PFOA and WY. There were only four genes (Cyp2c39, Fads1, Keg1, Rpl8) in this group that exhibited opposite expression behavior between PFOA and WY. Many of the group I genes fell into functional categories typically regulated by PPC including lipid homeostasis, inflammation, cell proliferation and proteome maintenance (Anderson et al., 2004a, b; Currie et al., 2005; Wong and Gill, 2002) (Supplemental File 4). Of the 51 genes that were regulated by PFOA but not WY, most were downregulated and consisted of genes that exhibited relatively low magnitude expression changes. Included in this group were genes involved in amino acid metabolism (Adh5, Alas1, Dbt, Dhcr24, Hgd, Mlycd, Shmt1, Tat).
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In PPAR
-null mice, PFOA altered the expression of 81 genes including 62 that exhibited similar expression in both wild-type and PPAR-null mice (Fig. 2, group III). Interestingly, these genes were also regulated in WY-treated wild-type mice but not WY-treated PPAR-null mice. The 62 genes included many involved in lipid metabolism (described in greater detail below). Group IV genes consisted of 19 genes that were altered by PFOA only in PPAR-null mice and were dominated by xenobiotic metabolizing enzymes. The comparison between PFOA and WY gene expression patterns demonstrates that (1) PPAR is required for the majority of transcriptional changes after PFOA or WY exposure in the mouse liver, (2) PFOA regulates genes that are similarly regulated by a prototypical PPC in a PPAR–dependent manner, and (3) PFOA exhibits effects on gene expression that are not shared by WY including the regulation of a set of PPAR–independent genes.
Characterization of the PPAR–Independent Genes Regulated by PFOA
To determine the nature of the genes that were regulated by PFOA in a PPAR–independent manner, we first determined if PFOA and WY regulated the same or different sets of genes independently of PPAR. There was little overlap among the genes regulated independently of PPAR (Fig. 3). PFOA at 7 days or WY at 12 h, 3 days or 7 days regulate, for the most part, independent sets of genes. This is in contrast to the genes that were coordinately regulated by the compounds in wild-type mice.
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The PPAR
–independent genes regulated by PFOA were grouped into a number of functional categories (Fig. 4). More than 12% of the genes code for proteins involved in lipid homeostasis (Fig. 4A). These include those that are involved in fatty acid β-oxidation or degradation including 5730439E10Rik (similar to acyl-CoA dehydrogenase), Acaa1, Acadl, Acox1, Aldh3a2, Cyp4a10, Dci, Decr1, Ech1, Ehhadh, MGC25972 (similar to Cyp4a10), MGC29978 (3-ketoacyl-CoA thiolase B) and Peci. A number of genes involved in transport of fatty acids, cholesterol or bile acids were also identified including Abcd3, Apoc4, Dbi, Fabp1, Mttp, Pctp, and Slc25a20. For almost all of these genes, PFOA increased expression in both wild-type and PPAR-null mice; however, absolute expression in PPAR-null mice was generally less than wild-type mice.
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Another group of genes regulated by PFOA in both mouse strains were those involved in amino acid metabolism (Fig. 4B). These included genes with overlapping functions involved in methionine (Bhmt, Cbs, Cth, Inmtd, Mat1a), cysteine (Cdo1, Cth, Got1, Ldh1), glycine, serine, and threonine (Bhmt, Cbs, Cth, Dmgdh) and glutamate (Cps1, Gls2, Glud1, Glul) metabolism. In contrast to the lipid metabolism genes, most of these genes were downregulated. As with lipid homeostasis genes, the degree to which these genes were altered by PFOA was generally less in the PPAR
-null mice than wild-type mice.
Among the most striking changes induced by PFOA in PPAR-null mice were 49 probe sets (which collapsed to 38 genes) involved in xenobiotic metabolism, including those involved in Phase I (oxidation), Phase II (conjugation) and Phase III (transport) functions (Fig. 4C). Phase I genes included members of the aldo–keto reductase (Akr), alcohol dehydrogenase (Aldh), carboxylesterase (Ces), and cytochrome P450 (Cyp) families. Phase II genes included members of the glutathione S-transferase (Gst) and uridine diphosphate glucuronosyl transferase (Ugt) families as well as the sulfotransferase family. Phase III genes included members of the adenosine triphosphate–binding cassette transporter (Abc) and solute carrier (Slc) families. Most of these genes exhibited increased expression after PFOA exposure. In contrast to genes associated with lipid homeostasis and amino acid metabolism, the genes involved in xenobiotic metabolism in many cases exhibited greater expression in PPAR-null mice than wild-type mice. Fifteen of the genes were altered only in PPAR-null mice.
Additional categories of genes altered by PFOA included those involved in cell fate and angiogenesis (Ang1, Creg1, Igf1, Pdcd4, Tnfsf14), inflammatory responses (C3, C4, Ccl9, Cfh, Cxcl12, Cxcr3, H2-Bf, Igsf4a, Inhbe, Lect1, Ly6e, Mpeg1, Pigr, S100a8, Tnfsf14, Vpreb3), transcription factors (Creb3l3, Irf7, Nfia, Nfix, Nrbp2, Pou2f1, Rhox6, Tlx2, Zfp128, Zfp36l1, Zfp608), proteome maintenance and stress response (Ctsc, Ctse, Ela1, Hspa1a, Hspa1b, Itih2, Itih3, Mt3, Pcsk5, Prm1, Prss8, Psmb10, Tmprss4, Usp47) and structural proteins (Ankrd6, Col1a1, Fn1, Fndc4, Krt2-6g, Tube1).
Overlap in Genes Regulated by PFOA and Activators of CAR
One gene that was highly induced in PPAR-null mice by PFOA was Cyp2b10, a well-known target for the nuclear receptor CAR. Our group recently suggested that, in addition to activation of PPAR, exposure to PFOA might also lead to activation of murine CAR (Rosen et al., unpublished data). A number of PFOA-regulated genes (Ephx1, Gst family members and Por) are also regulated by Nrf2, a transcription factor activated by anti-oxidants and under conditions that induce oxidative stress (Kwak et al., 2003). To investigate the possibility that PFOA is regulating gene expression similar to activators of CAR or Nrf2, we compared the PFOA transcript profiles with those from the livers of mice exposed to CAR or Nrf2 activators. Wild-type and CAR-null mice were exposed to the CAR activators phenobarbital (PB) or (TCPOBOP), and wild-type and Nrf2-null mice were exposed to the Nrf2 activator D3T. The transcript profiles from these experiments were analyzed using the same procedures as those used for the PFOA experiment outlined in the "Materials and Methods." The comparison shows striking similarity in the direction and magnitude of the change between the PFOA PPAR-null genes and PB and TC but not D3T regulated genes (Fig. 5). The Pearson's correlation between the PFOA pattern in PPAR-null mice and PB or TC in wild-type mice was 0.86 and 0.84, respectively, greater than the comparison with PFOA in wild-type mice (0.55). Most of these genes were CAR-dependent as they were no longer altered by PB or TC in the same manner in the CAR-null mice. The Pearson's correlation with the PB and TC profiles in CAR-null mice dropped to essentially zero (-0.05 and 0.01 for PB and TC, respectively). There were a number of known target genes for PPAR (Acaa1, Acot1, Acox1, Decr1, Ech1, Fabp1, Peci) that were downregulated by TC but not PB in a CAR-dependent manner. There is evidence that PPAR and CAR antagonize each other, and this antagonism may stem from competition for limited amounts of shared coactivators (Columbano et al., 2001; Corton and Brown-Borg, 2005; Guo et al., 2007; Jia et al., 2005). In addition, exposure to PPAR activators results in translocation of CAR to the nucleus but suppression of Cyp2b10 and other CAR regulated genes (Guo et al., 2007). This antagonism may explain why there is a stronger CAR-like response to PFOA in the PPAR-null mice compared to wild-type mice. There was no correlation between the PFOA genes and those regulated by D3T in wild-type or Nrf2-null mice (Pearson's correlation 0.06). Overall, these results indicate that a subset of the PFOA genes in the PPAR-null mice are regulated by CAR but not Nrf2.
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Real-Time RT-PCR of Selected Genes
The lipid metabolism genes (Acox1, Cyp4a10, Dci), Cyp2a4/5 and Mt3 exhibited dose-dependent increases in wild-type mice that were attenuated in the PPAR
-null mice (Table 1; Supplementary Fig. 1). This was in contrast to the robust induction of genes involved in xenobiotic metabolism (Ark1b7, Cyp2b9/10, Gstmu1/3) or inflammatory and oxidative stress response (S100a8) in both wild-type and PPAR-null mice. The prototypical targets of PXR and Nrf2 (Cyp3a11, Nqo1) were not significantly altered. Myc, known to be involved in WY-induced hepatocyte proliferation was induced only in wild-type mice, and Jun and Gadd45alpha were not altered in either strain.
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We examined the expression of a number of transcription factors that may underlie the changes in the expression of lipid or xenobiotic metabolism genes. PPAR
exhibited approximately three to fivefold increases in expression after exposure, whereas PPARβ was not altered. Car (Nr1i3) expression was significantly increased in wild-type but not PPAR-null mice. Nrf2 but not its cytoplasmic negative regulator, Keap1 was modestly increased in both strains. Finally, PXR exhibited a dose-dependent increase in expression in wild-type mice and was induced at the highest dose in the PPAR-null mice. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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PFOA exposure in mice and rats leads to a number of changes typical of PPC including increases in fatty acid β-oxidation enzymes, increases in liver to body weights, hepatocyte hyperplasia and hypertrophy and hepatocellular adenomas (summarized in Kennedy et al., 2004
). Because these responses are associated with PPAR activation, liver effects of PFOA are assumed to be mediated by PPAR (Klaunig et al., 2003). However, a comparison of the effects of PFOA in wild-type and PPAR-null mice demonstrated that PFOA increased liver to body weights in both strains (Abbott et al., 2007; Yang et al., 2002), indicating that not all endpoints associated with liver cancer were PPAR dependent. In this study, we carried out a toxicogenomic dissection of the PFOA transcriptional response to determine the extent of the PPAR–independent changes and their functional significance for liver cancer.
We identified a set of genes that were regulated by PFOA independently of PPAR in the mouse liver, and these genes have a number of unique characteristics compared to those regulated by the prototypical PPAR agonist, WY. PFOA had a greater impact on PPAR–independent gene expression than WY. Fourteen % of all PFOA-regulated genes were PPAR independent, compared to 0.3–8% of WY genes (Fig. 1). The PPAR–independent genes regulated by the two chemicals were mostly nonoverlapping (Fig. 3). Thus, these profiling studies demonstrate that two PPC mediate most of their transcriptional effects through PPAR. Other PPC will likely have PPAR–independent transcriptional effects. For example, the PPC bezafibrate, a PPAR/β agonist would be expected to have non–PPAR targets even though this compound induces hallmarks of liver cancer, as well as liver cancer itself through a PPAR–dependent mode of action (Hays et al., 2005).
The genes regulated by PFOA in PPAR-null mice exhibit unique function and regulation. One group of genes (group III, Fig. 2) were regulated similarly by PFOA in both wild-type and PPAR-null mice but were regulated by WY only in wild-type mice. Many of the genes in this group exhibited greater absolute changes after PFOA exposure in wild-type compared to nullizygous mice and are involved in amino acid homeostasis and fatty acid β-oxidation, functions typically modulated by PPC through PPAR. One of the genes in this group included acyl-CoA oxidase (Acox1), considered to be a PPAR target gene predictive of peroxisome proliferation (Klaunig et al., 2003). One plausible mechanism by which these genes are regulated in the absence of PPAR is through PPARβ and PPAR, given that PPAR subtypes recognize and activate gene expression through a common DNA binding site (reviewed in Corton et al., 2000). In support of this, Acox1 expression increased after exposure to PPARβ or PPAR ligands in the livers of PPAR-null mice (DeLuca et al., 2000). Cell based trans-activation studies showed that PFOA activated PPARβ or PPAR, although to a lesser extent than PPAR (Maloney and Waxman, 1999; Takacs and Abbott, 2007; Vanden Heuvel et al., 2006). Additional PFOA-responsive genes that are involved in fatty acid β-oxidation and transport including Acaa1, Acaa1b, Aldh9a1, Cyp4a11, Ehhadh, Fabp1, and Txn have been shown to be regulated similarly by different PPAR subtypes (Supplemental Fig. 2). PPAR but not PPARβ expression itself is increased in both wild-type and PPAR-null mice after PFOA exposure. Taken together, the data indicates that PFOA may regulate the expression of a subset of genes through PPAR in PPAR-null mice.
Many of the PFOA PPAR–independent genes have functions in xenobiotic metabolism. Compared to the lipid and amino acid metabolism genes discussed above, the XME genes were either similarly regulated in both wild-type and PPAR-null mice or exhibited altered expression in PPAR-null mice only (groups III and IV, Fig. 2). In many cases the genes exhibited higher absolute expression changes in PPAR-null mice compared to wild-type mice. The XME genes are targets of chemical-activated transcription factors including CAR, PXR, Ah receptor and Nrf2. We examined the expression of CAR, PXR and Nrf2 by RT-PCR and found that these factors were increased in one or both mouse strains after PFOA exposure. On close inspection of the spectrum of XMEs that were modulated, CAR was the most likely candidate governing the changes in the XMEs. High levels of induction of the CAR target gene Cyp2b10 was observed for PFOA (our study) and for the CAR agonist TCPOBOP but not the PXR agonist pregnenolone 16-carbonitrile (Maglich et al., 2002). Even though CAR is an Ah receptor target gene, the induction of Cyp2b10 is relatively modest after exposure to the Ah receptor agonist β-naphthoflavone (Patel et al., 2007), and the prototypical Ah receptor target genes including Cyp1a1 were not altered in our array study. The most compelling evidence that CAR was involved in PFOA alterations in XME expression comes from a direct comparison between the profile of PFOA-regulated PPAR–independent genes and those regulated by CAR and Nrf2 activators (www.nursa.org/10.1621/datasets.01003; Kwak et al., 2003). We observed a high correlation (r2 > 0.8) between the PFOA profile in PPAR-null mice and the profiles from PB and TCPOBOP treated wild-type mice but not similarly treated CAR-null mice indicating that the correlation was dependent on CAR. In contrast there was no overlap between the PFOA profile and that of a Nrf2 activator. Our results are consistent with a recent study in which the PFOA profile from rat liver exhibited similarities to other compounds that induce XMEs through CAR (Martin et al., 2007). Recent studies from our group have also suggested that PFOA may influence molecular pathways such as those mediated by CAR when transcript profiles were generated on a different microarray platform (Rosen et al., 2008). Definitive proof that CAR is involved in the PFOA regulation of XMEs will come from studies in CAR-null mice.
Our working model for PFOA mode of action in the mouse liver is shown in Figure 6. The mode of action (MOA) is based in part on the proposed MOA for PPC as described by Klaunig et al. (2003) in which PPC, through activation of PPAR can regulate batteries of genes that contribute to key events required for liver cancer. Gene batteries involved in hepatocyte fate are thought to contribute to the increases in hepatocyte proliferation and decreases in apoptosis. Recent studies indicate a short inhibitory RNA cascade regulates c-Myc, controlling hepatocyte fate after exposure to PPC (Shah et al., 2007). Unique to other PPC, PFOA regulates genes involved in xenobiotic metabolism that may be dependent on CAR. In wild-type mice this regulation of XMEs by CAR is less pronounced compared to that in PPAR-null mice. The expression of the XMEs may be linked to the appearance of large electron-lucent nonmembrane bound spaces in PPAR-null but not wild-type mice after PFOA exposure; these spaces may be due to accumulation of PFOA (Wolf et al., 2008). Genes traditionally considered targets of PPAR involved in peroxisome proliferation and lipid metabolism were regulated in the PPAR-null mice to lower absolute levels indicating that a weaker but similar mechanism may underlie their expression changes including the activation of PPAR. The lack of robust regulation of these genes and the lack of observable cell proliferation at 3 mg/kg in PPAR-null mice (Wolf et al., 2008) indicates that oxidative stress and cell proliferation are unlikely to occur under these conditions. Although exposure to PB or TCPOBOP can result in liver cancer in mice, the lack of regulation of the large battery of CAR signature genes by PFOA in PPAR-null mice, especially those involved in cell proliferation makes it unlikely that PFOA at 1 or 3 mg/kg could cause liver cancer in PPAR-null mice through a CAR-dependent mechanism. Support for this comes from our RT-PCR studies in which we showed that there is little induction of the cell fate genes (Myc, Gadd45, Jun) at any dose level in PFOA treated PPAR-null mice.
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In summary, we performed a toxicogenomic dissection of the transcript profiles in the mouse liver after exposure to PFOA. We uncovered classes of genes that were regulated independently of PPAR
involved in lipid metabolism, possibly by PPAR and xenobiotic metabolism that mimics a CAR transcriptional signature. |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Intramural Programs of NMEERL, National Institute of Environmental Health Sciences, and the National Cancer Institute.
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NOTES |
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The information in this document has been funded by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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ACKNOWLEDGMENTS |
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We thank Drs Thomas Kensler and David Moore for making their Affymetrix data available for our comparison studies. We thank Drs Don Delker and Stephen Edwards for reviewing the manuscript and Ms Sharice Lloyd for performing preliminary studies.
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