Evidence for the involvement of xenobiotic-responsive nuclear receptors in transcriptional effects upon perfluoroalkyl acid exposure in diverse species☆
Introduction
A wide range of species including wildlife and humans have measurable amounts of perfluoroalkyl acids (PFAA) in their tissues [1]. PFAA are composed of a carbon backbone (typically ranging from 4 to 15 carbons), full substitution of hydrogen by fluorine, and a functional group (carboxylic acid in the case of perfluorooctanoic acid (PFOA) and a sulfonic acid group for perfluorooctane sulfonate (PFOS)). PFAA have a number of uses including surfactant-processing aids in the production of fluoropolymers, coatings for clothing, fabrics, upholstery and carpets, and paper products approved for food contact. PFAA are readily absorbed [2], [3], are not known to be metabolized [4], [5], [6], and are poorly eliminated, with half-lives in humans estimated at 3.8 years for PFOA [7], [8]. Production of PFOA was estimated in excess of 500 metric tons in the year 2000. Although the production of PFOS by its major manufacturer was phased out at the end of 2002, likely contributing to the decline in PFOS body burdens [9], replacement PFAA chemicals (such as PFOA) have filled its void in the consumer and industrial markets.
Many PFAAs are members of a large class of structurally heterogeneous pharmaceutical and industrial chemicals called peroxisome proliferator chemicals (PPC) [10], [11]. The peroxisome proliferator-activated receptors (PPARα, β/δ, and γ), comprise a subset of the nuclear receptor superfamily and mediate many of the adaptive consequences of PPC exposure through direct or indirect activation [12], [13]. PPC exposure results in a predictable set of responses in the rodent liver. These responses include increased expression of fatty acid β-oxidation genes, hepatocyte peroxisome proliferation, hepatomegaly, hepatocyte hypertrophy and hyperplasia, and increased incidence of liver tumors [10]. For a number of PPC including WY-14,643, bezafibrate, and di-2-ethylhexyl phthalate, these responses are abolished in PPARα-null mice [14], [15], [16], [17], [18], [19] demonstrating the importance of PPARα activation as a key event in the PPARα mode of action [10].
PFOA exposure in rats increases liver tumor incidence. Carcinogenesis is thought to arise through activation of PPARα and induction of responses associated with a typical PPC (summarized in ref. [11]). PFOA and PFOS can activate PPARα and to a lesser extent, PPARβ and PPARγ in trans-activation assays [20], [21], [22]. Most of the gene expression changes (∼85%) in the mouse liver after PFOA exposure were shown to be PPARα-dependent as transcriptional levels were altered in wild-type mice but not PPARα-null mice [23], [24]. Wild-type but not PPARα-null mice exposed to PFOA exhibited a number of pathological and histological features associated with PPC-like effects [25]. Another PFAA, perfluorodecanoic acid (PFDA) was shown to induce a typical marker gene of PPC exposure (Cyp4a14) in wild-type but not PPARα-null mice [26] and PFDA down-regulated a number of bile acid transporters in wild-type but not PPARα-null mice [27]. Taken together, many of the hepatic responses associated with exposure to PFOA and other PFAAs are likely due to activation of PPARα.
In addition to activation of PPARα, PFAA may also mediate effects in the liver through other nuclear receptors. Increases in liver to body weights were observed in PFOA-exposed PPARα-null mice [28], [29], the biological basis for which is unknown. 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 under control of other nuclear receptors [30]. 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 [31], [32]. Activators of CAR and PXR such as phenobarbital (PB) and pregnenolone 16 alpha-carbonitrile (PCN), respectively regulate an overlapping set of XMEs and growth regulatory genes in the rat liver [33]. Like PPC, 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 [34], [35], [36], [37]. Chronic exposure to the CAR activators PB or 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) leads to increases in liver cancer in wild-type but not CAR-null mice [36], [37]. By comparing the transcript profiles in wild-type and PPARα-null mice after PFOA exposure to those of wild-type and CAR-null mice exposed to CAR activators, our group showed that the PPARα-independent genes regulated by PFOA largely overlapped with those regulated by CAR [23], [24]. PFDA was shown to regulate the CAR target gene and protein Cyp2b10 in wild-type but not CAR-null mice [26]. Taken together, there is growing evidence that PFAAs including PFOA activates multiple nuclear receptors in mouse liver including PPARα and CAR.
There is interest in understanding the potential toxicological effects of increasing body burdens of PFAAs on wild species. A number of studies have examined the effects of PFAA exposure on liver gene expression in birds and fish. Chickens have been used as a surrogate species to study the effects of PFAAs in wild birds. The chicken genome does not encode PXR or CAR genes but rather the chicken X receptor (CXR) gene, which has about equal sequence and functional similarity to PXR and CAR. CXR acts as the main xenobiotic sensing nuclear receptor in the chicken and possibly other birds [38]. Microarray studies using the Affymetrix chicken gene chip showed that chickens exposed to PFOA or PFOS exhibited alterations of some XMEs in liver [39]. A microarray study on the gene expression in the livers of wild common cormorants in which the levels of environmental contaminants including PFAAs, dioxins, and PCBs were measured showed correlations between PFOS and a number of genes but few XMEs [40]. Despite the evidence that chickens and other bird species possess a PPARα gene and can respond modestly to the effects of a classical PPARα activator ciprofibrate [41], there was no evidence in these microarray studies that exposure to PFAAs led to activation of PPARα.
Some fish species possess a PXR but not a CAR gene [42], [43]. Compared to mammals, there is little information about what genes PXR is regulating in fish. However, there were correlations between activation of PXR and the PXR-regulated genes MDR1 and CYP3A in fish [43]. Two microarray studies have been recently published revealing the gene expression changes in the livers of common carp or rare minnow after PFAA exposure. Both studies observed alterations in a number of XMEs after exposure [44], [45]. Studies in which different species of fish were exposed to activators of PPARα revealed species differences in PPARα-like responses. Catfish, bluegill, rainbow trout and zebrafish were responsive; medaka were marginally responsive; and common carp and sea bass were non-responsive [46], [47], [48], [49], [50], [51]. In fathead minnows, there were minor increases in palmitoyl-CoA oxidase activity (an indicator of PPARα activation) in livers after exposure to PFOA; these increases were observed at one low concentration but not higher concentrations [52]. The microarray studies in rare minnow and common carp did not show evidence for activation of PPARα by PFAAs [44], [45]. Overall, studies in the livers of birds and fish provide some marginal evidence that CAR/PXR-regulated responses are playing a role in gene expression changes after exposure to PFAAs.
Understanding the mechanism of chemical toxicity in the rat is hampered by the lack of genetic models abundantly available in mice. However, toxicogenomic analyses in which global gene expression profiles of chemicals with different modes of action are directly compared can be invaluable to make predictions as to the molecular basis for toxicity. Given that the microarray profiles in the rat liver after exposure to PFOA and PFOS indicated the involvement of CAR and PXR [30], we compared the profiles produced by PFOA and PFOS from available studies to known activators of CAR, PXR, and PPARα in rat liver. Additionally, we reanalyzed and reviewed microarray data from birds and fish after PFAA exposure to determine if transcriptional responses could be attributable to activation of xenobiotic-responsive nuclear receptors. Finally, we make correlations between expression of CAR and regulated genes in the livers of different human populations and levels of PFAAs in their blood.
Section snippets
Materials and methods
A summary of the animal studies is shown in Table 1. The doses selected in these studies (with the exception of Study 2) would be expected to elicit close to a maximal transcriptional response. We used the output files (.cel) from microarray studies, which utilized DNA chips from Affymetrix. The raw data files analyzed in this project were either downloaded from a public database, such as Gene Expression Omnibus (GEO), or communicated through original authors. All of the Affymetrix (Santa
Transcriptional changes in the livers of rats exposed to PFAAs
We compared expression profiles generated on the Affymetrix chip platform after exposure to PFOA and PFOS to a number of xenobiotics and drugs, which activate well-characterized pathways of XME expression. The list of studies used to make the comparisons is shown in Table 1. We examined liver gene expression from two studies: PFOA or PFOS in male rats [30] and PFOS in female rats [54]. To address whether CAR or PXR is activated after exposure to PFAAs, we compared the expression profiles in the
Conclusions
A number of conclusions can be drawn from this study and those studies cited. (1) PFAAs vary in their ability to activate PPARα in vivo based on microarray and conventional RT-PCR data in rats and mice. Our microarray analysis indicates that PFOA is a stronger activator of PPARα than PFOS in the rat liver. Most of the genes regulated by PFOA require PPARα in the mouse liver [23], [24]. (2) There is some evidence that CAR and PXR are activated after PFAA exposure in the rodent liver. Our results
Conflict of interest
None.
Acknowledgements
We thank Drs. Mitch Rosen and Bill Ward for reviewing the manuscript and Drs. David Dix, Heidrun Ellinger-Ziegelbauer, and Tom Sutter for contributions of their Affymetrix data files to this study. This research was partly supported by the Japanese Ministry of Environment under the Global Environment Conservation Research Fund (2004-2008) to KSG. PKSL was supported by the Hong Kong Research Grants Council (CityU 1401/05M).
References (80)
- et al.
Role of PPARalpha in mediating the effects of phthalates and metabolites in the liver
Toxicology
(2005) - et al.
The transcriptional response to a peroxisome proliferator-activated receptor alpha agonist includes increased expression of proteome maintenance genes
J Biol Chem
(2004) - et al.
Involvement of the peroxisome proliferator-activated receptor alpha in the immunomodulation caused by peroxisome proliferators in mice
Biochem Pharmacol
(2002) - et al.
CAR and PXR: the xenobiotic-sensing receptors
Steroids
(2007) - et al.
Differential expression of chicken hepatic genes responsive to PFOA and PFOS
Toxicology
(2007) - et al.
Expression of PXR CYP3A and MDR1 genes in liver of zebrafish
Comp Biochem Physiol C Toxicol Pharmacol
(2005) - et al.
Toxicity evaluation of perfluorooctane sulfonate (PFOS) in the liver of common carp (Cyprinus carpio)
Aquat Toxicol
(2008) - et al.
Toxicogenomic analysis of the hepatic effects of perfluorooctanoic acid on rare minnows (Gobiocypris rarus)
Toxicol Appl Pharmacol
(2008) - et al.
Induction of lauric acid hydroxylase activity in catfish and bluegill by peroxisome proliferating agents
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol
(1998) - et al.
Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp (Cyprinus carpio)
Aquat Toxicol
(2003)
Effect of clofibrate, a peroxisome proliferator, in sea bass (Dicentrarchus labrax), a marine fish
Environ Res
Evaluation of a rodent peroxisome proliferator in two species of freshwater fish: rainbow trout (Onchorynchus mykiss) and Japanese medaka (Oryzias latipes)
Ecotoxicol Environ Saf
Induction of peroxisome proliferation in rainbow trout exposed to ciprofibrate
Toxicol Appl Pharmacol
Identification of genes responsive to PFOS using gene expression profiling
Environ Toxicol Pharmacol
The Nrf2-Keap1 defence pathway: role in protection against drug-induced toxicity
Toxicology
Comparison of the expression profiles induced by genotoxic and nongenotoxic carcinogens in rat liver
Mutat Res
Perfluorooctane sulfonate-induced changes in fetal rat liver gene expression
Toxicology
Transcriptional activation of cytochrome P450 CYP2C45 by drugs is mediated by the chicken xenobiotic receptor (CXR) interacting with a phenobarbital response enhancer unit
J Biol Chem
Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues
Mol Cell Endocrinol
The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes
Toxicol Appl Pharmacol
Regulation of apoptosis by peroxisome proliferators
Toxicol Lett
Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes
Toxicol Appl Pharmacol
PPARalpha: mechanism of species differences and hepatocarcinogenesis of peroxisome proliferators
Toxicology
Are PFCAs bioaccumulative? A critical review and comparison with regulatory criteria and persistent lipophilic compounds
Environ Sci Technol
Lack of evidence for perfluorodecanoyl- or perfluorooctanoyl-Coenzyme A formation in male and female rats
Biochem Toxicol
Metabolic handling of perfluorooctanoic acid in rats
Proc Soc Exp Biol Med
Tissue distribution, metabolism, and elimination of perfluorooctanoic acid in male and female rats
Biochem Toxicol
Evaluation of the half-life (T1/2) of elimination of perfluorooctanoate (PFOA) from human serum
Toxicologist
Historical comparison of perfluorooctanesulfonate, perfluorooctanoate, and other fluorochemicals in human blood
Environ Health Perspect
Decline in perfluorooctanesulfonate and other polyfluoroalkyl chemicals in American Red Cross adult blood donors 2000–2006
Environ Sci Technol
PPARalpha agonist-induced rodent tumors: modes of action and human relevance
Crit Rev Toxicol
The toxicology of perfluorooctanoate
Crit Rev Toxicol
Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators
Annu Rev Pharmacol Toxicol
The toxicology of ligands for peroxisome proliferator-activated receptors (PPAR)
Toxicol Sci
Role of PPAR alpha in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14 643
Carcinogenesis
Role of peroxisome proliferator-activated receptor alpha in altered cell cycle regulation in mouse liver
Carcinogenesis
Overlapping transcriptional programs regulated by the nuclear receptors peroxisome proliferator-activated receptor alpha, retinoid X receptor, and liver X receptor in mouse liver
Mol Pharmacol
Role of peroxisome proliferator-activated receptor-alpha (PPARalpha) in bezafibrate-induced hepatocarcinogenesis and cholestasis
Carcinogenesis
Cited by (78)
Role of per- and polyfluoroalkyl substances in the cardiorenal system: Unraveling crosstalk from the network of pollutants and phenotypes
2025, Journal of Environmental Sciences (China)Carcinogenesis: Mechanisms and Evaluation
2021, Haschek and Rousseaux's Handbook of Toxicologic Pathology: Volume 1: Principles and Practice of Toxicologic Pathology
- ☆
Disclaimer: 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.