Elucidating the Molecular Basis of Adverse Health Effects from

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Elucidating the Molecular Basis of Adverse Health Effects from Exposure to Anthropogenic Polyfluorinated Compounds Using Toxicoproteomic Approaches Nicole Hansmeier,*,† Tzu-Chiao Chao,‡ Julie B. Herbstman,§ Lynn R. Goldman,∥ Frank R. Witter,⊥ and Rolf U. Halden*,# †

Department of Biology, University of Osnabrück, Barbarastrasse 11, Osnabrück 49076, Germany Department of Biology, University of Regina, 3737 Wascana Parkway, Regina, Saskatchewan S4S0A2, Canada § Department of Environmental Health Sciences, Columbia University, 722 West 168th Street, New York, New York 10032, United States ∥ School of Public Health and Health Services, The George Washington University, 950 New Hampshire Avenue, Washington, DC 20052, United States ⊥ Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, Maryland 21287, United States # Center for Environmental Security, The Biodesign Institute, Arizona State University, 781 East Terrace Mall, Tempe, Arizona 85287, United States ‡

ABSTRACT: Linear, short-chain polyfluorinated and perfluorinated alkyl compounds, often referred to as PFCs, have been in worldwide use as surfactants and polymer precursors for decades, and environmental dispersal of these highly persistent compounds represents a public health threat. Whereas ubiquitous low-level exposure to these compounds has been demonstrated in human populations from around the world, the exact mechanisms of toxicity and their toxic potency remain subject to investigation and scientific dispute. As with other environmental exposures, a major hurdle for gaining a better understanding of their human health impacts is the limited utility of cell culture and animal models serving as convenient, yet imperfect proxies to human physiology and disease. The present communication provides a brief overview of the current understanding of potential health effects of PFC exposure and examines how new toxicoproteomic methodologies can provide insight into the molecular mechanism of PFC exposure. Furthermore, we showcase an exemplary data set to illustrate how toxicoproteomic, population-wide studies might overcome limitations of animal models to more fully understand the metabolism and effects of PFCs and other environmental stressors where it matters most, in human populations experiencing real-world, chronic, low-level exposures. KEYWORDS: proteomics, PFOA, PFOS, infant health, exposure, PPAR, HNF4A properties exerting the unwanted toxicity.9 A number of molecular targets have been proposed based on animal and human cell line studies.9,11 However, large knowledge gaps still exist concerning the mechanisms of action of this emergent class of pollutants.9,11 The objective of this communication is to provide a brief overview of the current understanding of potential health effects of PFC exposures and discuss how new toxicoproteomic methodologies can provide critical information on the molecular mechanisms of this emergent group of pollutants. We further provide an example and outlook on how this

1. INTRODUCTION Polyfluoroalkyl compounds (PFCs), such as perfluoroalkyl sulfonates (PFASs) and perfluorocarboxylates (PFCAs), have been in worldwide use for decades,1 but their potential to cause adverse effects to public health has been recognized only recently.2 Their long persistence in the natural and built environment,3 bioaccumulation potential,4 and prevalence in wildlife and human populations5 have raised serious environmental and human health concerns, resulting in calls for implementation of tighter regulations.6 Today, human exposure to PFCs is ubiquitous5 and begins prenatally,7 with mounting evidence linking it to a range of adverse outcomes including hepatic, immunotoxic, reproductive, neurobehavioral, developmental, hormonal, and other effects.8−10 It is imperative to understand on a mechanistic basis the health risks posed by PFCs and the underlying structural © 2014 American Chemical Society

Special Issue: Environmental Impact on Health Received: September 18, 2014 Published: October 28, 2014 51

dx.doi.org/10.1021/pr500990w | J. Proteome Res. 2015, 14, 51−58

a

2D-DIGE

2D-DIGE/ transcriptomics iTRAQ

Huang et al. 201361

Haagenaars et al. 201360

iTRAQ

2D-DIGE

Tan et al. 201266

Roland et al. 201362

52

European eel European eel

zebrafish larvae European bullhead mice

mice

zebrafish

human

rare minnow human

organism

PBMC (in vitro)b PBMC (in vivo)b

liver

gill tissue

larvae

testis

liver hepatic cell line HepG2 hepatic cell line L-02 liver

organ/cell line

no growth effects

liver hypertrophy N/A

develop. abnormalities N/A

reduced sperm count

N/A

N/A

N/A no cytotoxicity

phenotype

+

+

+

+

+

+

+

+

+ +

+

+

+

+

+

+

+ +

lipid/steroid metabolism and transport

+

+

+

+

+

+

+

energy metabolism diverse

immune system; cytoskeleton

blood coagulation; cytoskeleton; signal transduction

vesicle transport; cytoskeleton

vesicle transport; cytoskeleton

development/cell division; translation

cell cycle; Ca2+ homeostasis; macromolecule catabolism vacuolar trafficking; transcription/translation; macromolecule catabolism immune response; cytoskeleton; macromolecule catabolism; translation; apoptosis cell cycle; Ca2+ homeostasis; transcription/translation; amino acid metabolism; endocrine system; ion transport endocrine system; translation; immune response

outcome

Listed are only studies with statistical analyses. bPBMC (peripheral blood mononuclear cells); energy metabolism (processes of generating energy from nutrients).

2D-DIGE

2D-DIGE

Dorts et al. 201159

Roland et al. 201463

2D-PAGE

PFOS Shi et al. 200965

Zhang et al. 201468

2D-PAGE 2D-PAGE

approach

PFOA Wei et al. 200867 Scharmach et al. 201264

study

stress response

Table 1. Selected Toxicoproteomic Studies Conducted to Unravel the Mechanism of PFC Toxicitya

Journal of Proteome Research Perspective


Journal of Proteome Research

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approach can be extended to a population-wide assessment of toxicity effects from environmental exposures in general and PFCs in particular.

In epidemiological studies, PFOA and PFOS exposures during fetal development were found to inversely affect birth weight in humans.49 Furthermore, studies on children revealed a PFC-induced reduction of humoral immune response,50 disturbed thyroid function and hypothyroidism,51 delayed onset of puberty,52 as well as metabolic syndromes in overweight children.53 Moreover, a long-term investigation on a pregnancy cohort recruited 1988−1989 in Denmark reported that in utero exposure to PFOA may be linked to semen quality impairment and infertility 19−21 years later.54 PFCs also appear to negatively impact adult health with possible links to kidney and testicular cancer, colitis, altered hormone and thyroid functions, elevated cholesterol and uric acid levels, and increased liver proteins in serum.14 For further details of PFC toxicology on human and animals the reader is directed to recent reviews by Stahl et al.55 and Lau et al.56 In summary, there is overwhelming evidence that PFCs negatively impact human health, with exposure of infants and children posing risks of abnormal development and health issues later in life. The toxicity of PFC exposures has been characterized predominantly in rodents in the past decade,56 and little progress has been made to pinpoint the underlying, specific molecular mechanisms. In animal studies proposing a mode of action, relevance of the findings for human physiology is uncertain. For example, the peroxisome proliferator-activated receptor (PPAR) alpha pathway was found to be strongly affected by PFOS and PFOA exposure in rats and is associated with hepatotoxicity, immunotoxicity, endocrine disruption, developmental toxicity, and carcinogenicity,56 yet in humans there is only a weak correlation between PPAR alphaexpression and PFOS/PFOA exposure.57 While acute effects on human livers were not reported, the elevated levels of cholesterol and serum liver enzyme as well as potential disruption of steroid functions imply some subtle effects that may very well follow a different pathway than those identified in rodents. It is therefore imperative to study more deeply the underlying biochemical mechanisms of PFC toxicity in humans to explain the observed links between exposure and health outcomes. This information is crucial for a mechanism-based evaluation of PFC-associated health risks and for an elucidation of commonalities and differences in the modes of toxicity among the various PFC classes.

2. NATURE OF PFCs, FUNCTION, AND USES Structurally, PFCs are a diverse class of fluorocarbons composed of a fluorinated carbon backbone of varying length, commonly in the C3−C14 range, terminated by a charged moiety such as a carboxylate, phosphonate, or sulfonate group. The fluorinated carbon backbone provides extremely low surface tension and accounts for the water and lipid repellency of this group of anthropogenic compounds. The latter property combined with an extreme stability, nonreactivity, and high effectiveness at low concentrations distinguishes PFCs from other chemical surfactants.3,12 With such useful properties, PFCs have found their way into a large variety of applications in the engineering, chemical, electronics, and medical industries.13,14 They are also used in numerous common consumer products including paint, cookware, food packaging, polish, fireretardants, floor wax, cosmetics, pesticides, cleaners, and stainresistant textile and fabric coatings.5,15 According to a report of the Organization for Economic Co-operation and Development (OECD), over 600 PFCs were in use worldwide in 2007.16 With two new PFC compounds being introduced annually,16 between 600 and 750 PFCs are estimated to be manufactured and used worldwide in 2014. Concerns about PFCs and their use were raised first after observing bioaccumulation and biomagnification of these synthetic compounds in terrestrial food chains.4,17,18 Diverse PFCs were found worldwide in the environment and in wildlife, including in animals inhabiting remote areas, such as polar bears in the arctic.19,20 The extent of PFC occurrence in the environment has been subject to multiple excellent reviews.8,9,14,21,22 3. EXPOSURE AND HEALTH RISKS OF PFCs The ubiquity of PFCs makes human exposure inevitable. Established exposure routes include ingestion, absorption, and inhalation from sources as diverse as drinking water, food, consumer products, dust, aerosols, and chemical manufacturing plants.23−30 Blood samples collected from human populations worldwide reflect the omnipresence of PFCs typically at levels in the ng/mL range.31 For instance, a geometric mean serum concentration of 5.2 ng/mL for perfluorooctanoic acid (PFOA) and 30.4 ng/mL for perfluorooctanesulfonate (PFOS) was reported for the U.S. population in 2000.5 Serum levels in workers producing or using PFOA were much higher than those found in the general population.9 Maximum PFOA concentrations reported for serum of a worker in the 1990s were above 100 000 ng/mL (100 ppm). Awareness of those exposure levels triggered governmental regulatory agencies to call for a full review of PFC toxicity. The largest body of evidence for PFC toxicity was gathered from animal models, showing PFC exposure to elicit liver enlargements,32,33 hepatocellular hypertrophy,34 hepatocellular adenomas,35 testicular and pancreatic tumors,9,36 immunotoxicity,37 neurotoxicity,38 reproductive39or developmental deficits40,41 as well as thyroid hormone alterations and endocrine disruption.42 In particular, the developing fetus appears to be sensitive to PFC-induced disruptions; both PFOS and PFOA are able to cross the placental barrier, resulting in birth weight reduction, pregnancy loss, and developmental delays in rodents.43−48

4. TOXICOPROTEOMICS Proteomics may aid in the elucidation of mechanisms underlying adverse human health effects in PFC-exposed populations, and toxicoproteomic analyses are particularly attractive and promising.58 The latter method utilizes massspectrometry-based identification and quantification of proteins from cells or tissues to uncover changes in proteome composition resulting from toxic exposures. Derived molecular data can then be linked to epidemiological information, thereby providing molecular evidence for health sequelae observed following established harmful exposures. To date, only a limited number of toxicoproteomic studies on PFC effects have been conducted, mostly involving a variety of animal models and human cell lines.59−68 Predominantly, two-dimensional gel electrophoresis (2D-GE) and to a lesser extent liquid chromatography tandem mass spectrometry (LC− MS/MS)-based shotgun approaches were used (see Table 1). Almost all studies relied on exposure of test animals or cell lines to defined amounts of PFOS or PFOA before either the whole 53



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proteome of the organism (such as zebrafish larvae)65 or specific organs such as gills,59 testes,68 or liver60,66,67 were profiled and compared with unexposed controls. One study took a hybrid approach by studying both laboratory-exposed fish and eels captured from Belgian rivers.63 These comparative analyses individually identified between 18 and 93 proteins affected by exposure to PFOS and PFOA.65,68 Overall, a lack of overlap in the affected proteins (or homologues) among these studies59−68 can be noted, a finding that may be explained in part by the use of differing animal models, experimental conditions, and analytical methodologies. In addition, four studies on fish proteomes59,62,63,67 reported the identification of only a few proteins due to a lack of genome information on the respective animal model used.59,62,63,67 Yet a surprising number of commonalities can be found between these studies when the functions and pathways of the identified proteins are considered rather than the identity of the protein itself. For example, all animal and human studies found some level of stress and inflammation response from the research subjects after PFC exposure (Table 1). In particular, proteins indicative for oxidative stress were found to be differentially expressed, such as glutathione S-transferases and peroxiredoxins. Such changes also have been found in various non-PFC-related exposure studies,69−73 and therefore likely represent nonspecific responses. More specific responses were found in toxicoproteomic studies of fish, murine liver, and testes as well as of human hepatocytes, in which proteins involved in the metabolism of lipids and steroids were differentially expressed. This supports previous findings from animal and human studies identifying the liver as a principal organ and locus of PFOA/PFOS toxicity. Specifically, the proteome studies verified the effect of PFOS on the regulation of PPAR alpha in mice.66 Moreover, these proteomic studies provided a potential explanation for the apparently conflicting reports on the effects of PFCs on the human liver. A comparative proteome survey on the human liver cell line HepG2 revealed the nuclear receptor HNF4a (hepatocyte nuclear factor 4 alpha) and proteins under its control to represent main targets of PFOA as opposed to the PPAR-alpha pathway in mice.64 This alternative mechanism may explain differences in health outcomes observed between exposed humans and mice while also highlighting the challenge of translating with confidence observations made in rodents to health effects in human populations. Because HNF4a is known to play an active role in liver development and embryogenesis,74 in utero exposure to PFOA and other PFCs may be especially harmful to humans and demands further attention. Consistent with this result, related steroidogenic enzymes also were found to be altered in their expression in PFC-exposed animals and cell lines.61,64−66,68 This includes a number of apolipoproteins, which play crucial roles in the metabolism and transport of lipids.75 Of special interest is the fact that their deregulation could be directly responsible for altered cholesterol levels. Another example is a protein of the cytochrome P450 family and key enzyme in the initial steps of steroidogenesis, CYP11A1, which was identified in a proteome study of testes from PFC-exposed mice.68 Dysfunctions of this class of enzymes are associated with infertility and altered levels of testosterone and estrogen,76 providing a potential mechanistic explanation for the observed reproductive toxicity of PFCs. These proteome data are also in excellent agreement with a qPCR-based study77 in which the

apolipoprotein A1 pathway was implicated in elevated cholesterol values as a consequence of PFC exposure. Because the metabolism of steroids, lipids, and energy is intricately linked, it is not easy to untangle the network of pathways adversely impacted by PFCs. However, the various studies conducted thus far indicate that one mode of action of PFC toxicity is via alteration of the expression of key transcription factors, such as PPAR-alpha (in mice) or HNF4a (in human), which then cause wide-reaching effects throughout the regulatory network. In summary, even the limited amount of studies in nonstandardized systems available today demonstrates the usefulness of toxicoproteomics for furthering our understanding of PFC toxicity on a molecular basis. Available data provide a mechanism-based foundation, which can assist in interpreting epidemiological, outcome-based investigations. Proteome data available to date confirm the role of PFCs, such as PFOS and PFOA, as modulators of hepatic metabolism even at environmentally relevant, low, or moderate levels, that is, in the parts per billion range. The resulting changes in lipid metabolism, while subtle, have far-reaching consequences on energy metabolism, cholesterol, and hormonal levels.

5. EVALUATION OF EXPOSURE MARKERS IN HUMAN POPULATIONS BY EXAMPLE OF AN INFANT COHORT While the results gleaned from whole animal and in vitro systems of human cells can be helpful, their applicability to human physiology is known to be limited. Combining toxicoproteomics with human epidemiological studies therefore represents a powerful but largely untapped resource for monitoring molecular effects and for predicting human health effects of long-term, environmental exposures. A number of criteria have to be met to successfully expand the proteomic methodology from animals and in vitro human cell assays to human populations in a practical fashion: (1) Specimens need to be sampled using non- or minimally invasive methods, with prime targets representing body fluids such as blood and urine. (2) The protein composition of these samples has to reflect the physiology of relevant organs, tissues, and pathways and ideally should inform on potential adverse health effects even in presymptomatic populations. (3) Proteins within these samples have to be of sufficient quality and quantity to be identified and quantified using proteomic approaches. These challenges will come to the fore when studying adverse effects from exposures in vulnerable subpopulations such as children and infants. In a previous study, using cord blood samples from a well-characterized Baltimore cohort of mothers and babies (the Tracking Health Related Environmental Exposures or THREE study),7,78,79 we were able to extract proteomic data (>1000 proteins) from as little as 100 μL of serum using LC−MS/MS.80 Thus, cord blood may be an interesting and noninvasive biopsy material for assessing exposure and health of infants. Toxicoproteomic studies directed explicitly at the effect of PFOS and PFOA exposure thus far have focused on target organs such as liver and testes,66,68 and it is uncertain whether resultant changes in these organs also would be reflected in blood or other body fluids of exposed individuals, including infants. One important question to answer therefore is whether proteins identifiable in readily accessible biospecimens, such as 54



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Table 2. Differentially Expressed Proteins Identified in Cord Blood of Babies Exposed to Differing Levels of PFOS/PFOAa protein, accession no. apolipoprotein B (ApoB), P04114 alpha-1B-glycoprotein (A1BG), P04217 alpha-2-antiplasmin (SerpinF), P08697 alpha-2-HS-glycoprotein (FetuinA), P02765 alpha-1-acid glycoprotein 1 (A1AG1), P02763 transferrin (TRFE), P02787 kininogen-1 (KNG), P01042

Protscore (unused)b

seq cov (%)

no. of peptides (95% cov.)

iTRAQ mean ratiosc

94.72 15.67 9.31 24.05

33 39 25 55

47 8 5 17

11.6

51

6

7.9 ± 6.2

immune system process; transport

101.68 17.5

82 37

111 9

3.6 ± 2.1 2.7 ± 1.2

transport; blood coagulation; ion homeostasis proteolysis; immune response; inflammation; apoptosis; blood coagulation transport; steroid metabolic process; immune response

0.3 2.2 0.4 3

± ± ± ±

0.2 0.5 0.1 1.1

vitamin-D-binding protein (VTBP), P02774 hemopexin (HPX), P02790 hemoglobin (HBA), P69905

29.02

61

16

2.1 ± 1

17.35 16.99

47 70

15 17

0.3 ± 0.1 21 ± 4.2

hemoglobin (HBB), P68871

15.9

93

22

11 ± 3.5

function/process lipid and steroid transport immune system process; transport inflammation; proteolysis; blood coagulation immune system process; development; endocrine function

transport; ion homeostasis; anti-inflammatory factor transport; blood circulation; hydrogen peroxide catabolic process; positive regulation of cell death transport; blood circulation; hydrogen peroxide catabolic process; positive regulation of cell death

a We created a pool of six highly exposed samples with serum PFOA concentrations of 2.8 to 4.2 ng/mL (mean: 3.6 ng/mL) and PFOS 4.4−18.1 ng/mL (mean: 11.4 ng/mL) and compared them with six serum samples containing a mean PFOA concentration of 0.6 ng/mL (range: 0.4 to 0.8 ng/mL) and PFOS of 1.9 ng/mL (range: 0.4−2.7 ng/mL) (N. Hansmeier, T.-C. Chao, L. R. Goldman, F. R. Witter, R. U. Halden, unpublished). b Proteins were analyzed as before80 and identified with ProteinPilot Software v4.0 (Applied Biosystems/MDS SCIEX)82 searched against the UniProt human proteome database version March 20th, 2009 (Protscore ≥1.3, at least two unique peptides >95% confidence, 95%), p value ≤0.05 (two-tailed ANOVA), greater than a 1.2-fold expression change.

ultimately may serve to demonstrate PFC-induced proteome alterations on an individual basis in human subpopulations experiencing real-world environmental exposures. A key question is whether proteomic studies of blood can capture modes of toxicity located in the liver and potentially in other organs of humans and newborns. The vast dynamic range of protein concentrations81 remains a methodological challenge, yet with analytical capabilities improving, proteomics has the potential to provide ever deeper insights into the toxic mechanisms of harmful environmental exposures.

cord blood, are useful for determining the mechanism of PFC toxicity in infants. To answer this question, we conducted a feasibility study that leveraged a cord blood repository available from the Baltimore THREE cohort for which PFOA and PFOS levels had been determined previously.7,78 The THREE study is a crosssectional cohort study designed to investigate prenatal exposure to environmental chemicals and resultant adverse impacts on newborns detectable at birth. This study cohort consists of 300 cord blood serum samples collected from singleton birth between November 26, 2004 and March 16, 2005. Within this cohort, every individual exhibited detectable levels of PFOS and PFOA within a range between 0.2−34.8 ng/mL and 0.3−7.1 ng/mL, respectively.7 Because we were interested in the early detection of exposures, we specifically selected samples from asymptomatic children (i.e., those with normal birth metrics for birth weight, ponderal index, and head circumference) and performed a semiquantitative proteome comparison of pooled cord serum samples of newborns highly exposed to PFOS/ PFOA (meanPFOA: 3.6 ng/mL, meanPFOS: 11.4 ng/mL) compared with a control group (meanPFOA: 0.6 ng/mL, meanPFOS: 1.9 ng/mL). With a heavy-isotope labeling approach using isobaric tags for relative and absolute quantitation (iTRAQ), we were able to identify 11 proteins differing significantly in abundance (see Table 2). The plurality of the identified proteins was involved in immune response and inflammation (kininogen, alpha-1B-glycoprotein, alpha-1-acidglycoprotein 1, vitamin-D-binding protein) and lipid/steroid metabolism (apolipoprotein B). These preliminary results have not yet been confirmed with orthogonal methods and come from a small group of study subjects; however, they are in general agreement with tissue-specific studies, suggesting that PFOS/PFOA-induced changes in expression of major proteins, such as apolipoprotein B, may be detectable in blood samples. This and similar studies to be conducted in the future

6. CONCLUSIONS AND OUTLOOK There is mounting evidence that ongoing exposures to PFCs exert a negative impact on human health. Pinpointing the mode of toxicity is difficult when relying entirely on studies of whole animals and human cell lines. Initial proteomic investigations highlight the feasibility of MS-based surveys to gain important insights into the molecular mechanisms underlying PFCassociated health outcomes. First evidence available from the analysis of pooled cord blood samples suggests the possibility of detecting proteomic changes in newborns triggered by environmental exposures to PFCs. These studies could be strengthened in the future by extending proteome analyses to individuals as opposed to pooled samples of exposed subpopulation and to include other tissues, such as the placental trophoblast. It is conceivable that PFC exposure may influence pregnancy outcomes by compromising the protective functions of the placenta. Altered expression of proteins involved in transport functions of the placenta may represent both a plausible mechanism of toxicity and a convenient marker of placental injury. As more proteomic data become available for human populations environmentally exposed to harmful pollutants, this information will aid in filling existing gaps in our understanding of toxic mechanisms and in 55



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(8) Houde, M.; De Silva, A. O.; Muir, D. C.; Letcher, R. J. Monitoring of perfluorinated compounds in aquatic biota: an updated review. Environ. Sci. Technol. 2011, 45 (19), 7962−7973. (9) Lau, C.; Anitole, K.; Hodes, C.; Lai, D.; Pfahles-Hutchens, A.; Seed, J. Perfluoroalkyl acids: a review of monitoring and toxicological findings. Toxicol. Sci. 2007, 99 (2), 366−394. (10) Adams, D. E. C.; U, H. R. Fluorinated Chemicals and the Impacts of Anthropogenic Use; Oxford University Press: New York, 2010; Vol. 1048, pp 539−560. (11) Houde, M.; Martin, J. W.; Letcher, R. J.; Solomon, K. R.; Muir, D. C. Biological monitoring of polyfluoroalkyl substances: A review. Environ. Sci. Technol. 2006, 40 (11), 3463−3473. (12) Rayne, S.; Forest, K. Perfluoroalkyl sulfonic and carboxylic acids: a critical review of physicochemical properties, levels and patterns in waters and wastewaters, and treatment methods. J. Environ. Sci. Health, Part A: Toxic/Hazard. Subst. Environ. Eng. 2009, 44 (12), 1145−1199. (13) Lewandowski, G.; Meissner, E.; Milchert, E. Special applications of fluorinated organic compounds. J. Hazard Mater. 2006, 136 (3), 385−391. (14) Post, G. B.; Cohn, P. D.; Cooper, K. R. Perfluorooctanoic acid (PFOA), an emerging drinking water contaminant: a critical review of recent literature. Environ. Res. 2012, 116, 93−117. (15) Buck, R. C.; Franklin, J.; Berger, U.; Conder, J. M.; Cousins, I. T.; de Voogt, P.; Jensen, A. A.; Kannan, K.; Mabury, S. A.; van Leeuwen, S. P. Perfluoroalkyl and polyfluoroalkyl substances in the environment: terminology, classification, and origins. Integr. Environ. Assess. Manage. 2011, 7 (4), 513−541. (16) OECD. Lists of PFOS, PFAS, PFCA, Related compounds and chemicals that may degrade to PFCA. ENV/JM/MONO 2007, 15. (17) Loi, E. I.; Yeung, L. W.; Taniyasu, S.; Lam, P. K.; Kannan, K.; Yamashita, N. Trophic magnification of poly- and perfluorinated compounds in a subtropical food web. Environ. Sci. Technol. 2011, 45 (13), 5506−5513. (18) Muller, C. E.; De Silva, A. O.; Small, J.; Williamson, M.; Wang, X.; Morris, A.; Katz, S.; Gamberg, M.; Muir, D. C. Biomagnification of perfluorinated compounds in a remote terrestrial food chain: LichenCaribou-wolf. Environ. Sci. Technol. 2011, 45 (20), 8665−8673. (19) Giesy, J. P.; Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 2001, 35 (7), 1339−1342. (20) Greaves, A. K.; Letcher, R. J.; Sonne, C.; Dietz, R.; Born, E. W. Tissue-specific concentrations and patterns of perfluoroalkyl carboxylates and sulfonates in East Greenland polar bears. Environ. Sci. Technol. 2012, 46 (21), 11575−11583. (21) Lindstrom, A. B.; Strynar, M. J.; Libelo, E. L. Polyfluorinated compounds: past, present, and future. Environ. Sci. Technol. 2011, 45 (19), 7954−7961. (22) Ahrens, L. Polyfluoroalkyl compounds in the aquatic environment: a review of their occurrence and fate. J. Environ. Monit. 2011, 13 (1), 20−31. (23) Freberg, B. I.; Haug, L. S.; Olsen, R.; Daae, H. L.; Hersson, M.; Thomsen, C.; Thorud, S.; Becher, G.; Molander, P.; Ellingsen, D. G. Occupational exposure to airborne perfluorinated compounds during professional ski waxing. Environ. Sci. Technol. 2010, 44 (19), 7723− 7728. (24) Xu, Z.; Fiedler, S.; Pfister, G.; Henkelmann, B.; Mosch, C.; Volkel, W.; Fromme, H.; Schramm, K. W. Human exposure to fluorotelomer alcohols, perfluorooctane sulfonate and perfluorooctanoate via house dust in Bavaria, Germany. Sci. Total Environ. 2013, 443, 485−490. (25) D’Hollander, W.; de Voogt, P.; De Coen, W.; Bervoets, L. Perfluorinated substances in human food and other sources of human exposure. Rev. Environ. Contam. Toxicol. 2010, 208, 179−215. (26) Eschauzier, C.; Hoppe, M.; Schlummer, M.; de Voogt, P. Presence and sources of anthropogenic perfluoroalkyl acids in highconsumption tap-water based beverages. Chemosphere 2013, 90 (1), 36−41. (27) Zhang, T.; Sun, H. W.; Wu, Q.; Zhang, X. Z.; Yun, S. H.; Kannan, K. Perfluorochemicals in meat, eggs and indoor dust in

developing diagnostic tools for assessing and managing toxic exposures before they can manifest in adverse human health effects.

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AUTHOR INFORMATION

Corresponding Authors

*N.H.: Phone: ++ 49 (0)541 969 2855. Fax: ++ 49 (0)541 969 3942. E-mail: [email protected]. *R.U.H.: Tel: +1 (480) 727-0893. Fax: +1 (480) 965-6603. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported in part by the Johns Hopkins Center for a Livable Future and by Award Numbers R01ES015445 and 1R01ES020889 of the National Institute of Environmental Health Sciences (NIEHS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS or the National Institutes of Health (NIH).

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ABBREVIATIONS HNF4A, hepatocyte nuclear factor 4 alpha; iTRAQ, isobaric tags for relative and absolute quantitation; LC−MS/MS, liquid chromatography tandem mass spectrometry; OECD, Organization for Economic Co-operation and Development; PFUA, perfluoroundecanoic acid; POSF, perfluorooctane sulfonyl fluoride; PFPA, perfluorinated phosphonic acid; PAP, polyfluoroalkyl phosphate ester; PFNA, perfluorononanoic acid; PFC, polyfluoroalkyl compounds; PFOS, perfluorooctanesulfonate; PFOA, perfluorooctanoic acid; PPAR, peroxisome proliferator-activated receptor; qPCR, quantitative polymerase chain reaction; THREE, Tracking Health Related Environmental Exposures Study; 2D-GE, two-dimensional gel electrophoresis

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REFERENCES

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