In vitro characterization of the immunotoxic potential of several perfluorinated compounds (PFCs)

Toxicology and Applied Pharmacology 258 (2012) 248–255

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In vitro characterization of the immunotoxic potential of several perfluorinated compounds (PFCs) Emanuela Corsini a,⁎, Enrico Sangiovanni b, Anna Avogadro a, Valentina Galbiati a, Barbara Viviani a, Marina Marinovich a, Corrado L. Galli a, Mario Dell'Agli b, Dori R. Germolec c a b c

Laboratory of Toxicology, Department of Pharmacological Sciences, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy Laboratory of Pharmacognosy, Department of Pharmacological Sciences, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy National Toxicology Program, National Institute of Environmental Health Sciences, NIH, RTP, NC, USA

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Article history: Received 15 July 2011 Revised 20 October 2011 Accepted 9 November 2011 Available online 18 November 2011 Keywords: Perfluorinated compounds Immunosuppression PPAR-α receptor Cytokine Whole blood assay

a b s t r a c t We have previously shown that PFOA and PFOS directly suppress cytokine secretion in immune cells, with different mechanisms of action. In particular, we have demonstrated a role for PPAR-α in PFOA-induced immunotoxicity, and that PFOS has an inhibitory effect on LPS-induced I-κB degradation. These studies investigate the immunomodulatory effects of four other PFCs, namely PFBS, PFOSA, PFDA, and fluorotelomer using in vitro assays. The release of the pro-inflammatory cytokines IL-6 and TNF-α was evaluated in lipolysaccharide (LPS)-stimulated human peripheral blood leukocytes (hPBL) and in the human promyelocytic cell line THP-1, while the release of IL-10 and IFN-γ was evaluated in phytohemagglutinin (PHA)-stimulated hPBL. All PFCs suppressed LPS-induced TNF-α production in hPBL and THP-1 cells, while IL-6 production was suppressed by PFOSA, PFOS, PFDA and fluorotelomer. PFBS, PFOSA, PFOS, PFDA and fluorotelomer inhibited PHA-induced IL-10 release, while IFN-γ secretion was affected by PFOSA, PFOS, PFDA and fluorotelomer. Leukocytes obtained from female donors appear to be more sensitive to the in vitro immunotoxic effects of PFCs when their responses are compared to the results obtained using leukocytes from male donors. Mechanistic investigations demonstrated that inhibition of TNF-α release in THP-1 cells occurred at the transcriptional level. All PFCs, including PFOA and PFOS, decreased LPS-induced NF-κB activation. With the exception of PFOA, none of the PFCs tested was able to activate PPARα driven transcription in transiently transfected THP-1 cells, excluding a role for PPARα in the immunomodulation observed. PFBS and PFDA prevented LPS-induced I-κB degradation. Overall, these studies suggest that PFCs affect NF-κB activation, which directly suppresses cytokine secretion by immune cells. Our results indicate that PFOA is the least active of the PFCs examined followed by PFBS, PFDA, PFOS, PFOSA and fluorotelomer. © 2011 Elsevier Inc. All rights reserved.

Introduction Perfluorinated compounds (PFCs) are an emerging class of environmental contaminants commonly detected in blood samples of both wildlife and humans (Rayne and Forest, 2009; Suja et al., 2009), arising mainly from their use as surface treatment chemicals, polymerization aids, and surfactants. PFCs are of toxicological concern due to their persistence in the environment, and their potential

Abbreviations: PFCs, perfluorinated compounds; PFBS, perfluorobutane sulfonic acid; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonic acid; PFDA, perfluorodecanoic acid; Fluorotelomer, 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro-(8:2 Telomer); TNF-α, tumor necrosis factor-α; IL, interleukin; LBD, ligand binding domain. ⁎ Corresponding author. Fax: +39 02 50318284. E-mail address: [email protected] (E. Corsini). 0041-008X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2011.11.004

to bioaccumulate through the food chain (Fromme et al., 2009; Haug et al., 2011). PFCs comprise a heterogeneous class of chemicals consisting of an alkyl chain (4–14 carbons), which is partially or fully fluorinated, and have different functional groups attached. Among the PFCs are the perfluoroalkyl carboxylic acids (i.e. PFOA, PFDA), perfluoroalkyl sulfonic acids (i.e. PFOS, PFBS), perfluoroalkyl sulfonamides (i.e. PFOSA), and other polyfluorinated compounds, such as fluorotelomer alcohols (i.e. fluorotelomer). Although the production of two widely used PFCs, PFOS and PFOA, has been voluntarily phased-out by its primary manufacturer, both PFOS and PFOA are persistent environmental contaminants. These compounds are widely present in surface, ground, marine, and drinking waters at concentrations that vary from pg/l to μg/l. Some wastewaters contain PFCs at mg/l to low g/l levels (Rayne and Forest, 2009). Blood samples of occupationally exposed individuals and the general population in various countries were found to contain PFOS and PFOA at

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measurable levels. In the United States, the mean serum concentration in the general population was reported as 20.7 ng/ml for PFOS and as 3.7 ng/ml for PFOA (Calafat et al., 2006). PFOA serum concentrations reported in occupationally exposed humans were between 428 and 12,000 ng/ml (Costa et al., 2009; Steenland et al., 2010), and between 145 and 3490 ng/ml for PFOS (Olsen et al., 2007). The health effects of perfluoroalkyl-compounds in humans remain controversial, in spite of a number of experimental and epidemiological studies (Butenhoff et al., 2004; Emmett et al., 2006; Joensen et al., 2009; Steenland et al., 2010; Wang et al., 2011). Data from experimental animals demonstrated that the perfluorinated alkyl acids induce peroxisomal proliferation, hepatomegaly, altered steroidogenesis, developmental and reproductive toxicity, body weight loss associated with a wasting syndrome, and are carcinogenic in rodents (Biegel et al., 2001; Feng et al., 2009; Kennedy et al., 2004; Lau et al., 2004; Liu et al., 1996a, 1996b; Olsen et al., 1999; Pastoor et al., 1987). Several studies also indicated that PFOA suppressed antibody production, caused thymic and splenic atrophy, and altered T-cell populations (Yang et al., 2001, 2002a, 2002b). Recent studies have also shown that PFOS affects antibody production in the rodent immune system at levels found in the general human population (Peden-Adams et al., 2008). PFOS exposure suppressed immunity in mice resulting in a significant increase in emaciation and mortality in response to influenza A virus (Guruge et al., 2009). The pesticide sulfluramid, which is rapidly metabolized to PFOS, has been demonstrated in mice to target T-dependent, IgM antibody production at exposure levels 10-fold less than that observed with overt toxicity (Peden-Adams et al., 2007), further confirming the immune system as a sensitive target of PFC toxicity. The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily, and there are three primary subtypes: PPAR α, β, and γ. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis (Chinetti et al., 2000). Studies suggest that many of the biological effects of the PFCs are mediated through PPAR and because PFOA and PFOS both activate PPARα, the role of PPARα in PFOA and PFOS immunotoxicity has been investigated (DeWitt et al., 2011). However, the specific role of PPARs in PFC-immunotoxicity is still a matter of debate, and it is unclear whether or not there is a direct effect on immune cells. Some data suggest that PPARα mediates process connected with the immune system in an indirect fashion, by modulating lipid levels leading to hepatotoxicity and stress effects (Qazi et al., 2009). Yang et al. (2002a) compared the immunomodulating effects of PFOA in wild-type and PPARα null mice: the reductions in spleen weight and cellularity, in thymus weight and cellularity, and in mitogen-induced lymphocyte proliferation caused by PFOA in wildtype mice were not observed in PPARα null mice, indicating that PPARα plays a major role in the immunomodulation caused by PFOA. In contrast, as demonstrated by Qazi et al. (2009), the immunotoxicity of PFOS was only partially dependent upon PPARα activation: the reduction in thymus weight and in total number of thymocytes was only partially attenuated in PPARα-null animals. In vitro studies have demonstrated that PFOA is a more potent agonist of murine PPARα than PFOS (Takacs and Abbott, 2007), which may explain the differences in the immune responses in vivo. Effects independent of PPARα activation may have greater potential impact on humans exposed to PFCs as human hepatic PPARα expression is only one-tenth that of rodents (Kennedy et al., 2004). Furthermore, the affinity of PFOA and PFOS for human or murine PPARs is quite different. In vitro studies conducted on Cos-1 cells transfected with mouse or human PPAR α, β/δ, or γ reporter plasmids by Takacs (2007) demonstrated that PFOA (0.5–100 μM) significantly increased mouse and human PPARα and mouse PPARβ/δ activity. Whereas PFOS (1–250 μM) significantly increased activation of mouse PPARα and PPAR β/δ isoforms, no significant activation of mouse or human

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PPAR γ was observed with PFOA or PFOS. The mouse PPARα appears to be more sensitive to PFCs than the human PPARα, with PFOA having more activity than PFOS with both the mouse and human PPAR isoforms, further supporting the hypothesis of a different role for PPARα in PFOA and PFOS toxicity. Our understanding of the mechanisms underlying the immune effects of PFCs is still incomplete and insufficient to assess the relationship between structure–activity, PPAR activation and immune system toxicity. In the present study, the direct immunotoxicity of six PFCs, including PFOA and PFOS, was evaluated. Furthermore, the different PFCs were chosen based on different chain lengths (C4–C10) and functional groups (sulfonate, carboxyl and amino groups) to determine, if possible, the relationship between structure–activity and immune suppression. Materials and methods Chemicals. The chemical structures of the PFCs tested are reported in Fig. 1, and their chemical characteristics are shown in Table 1. Perfluorobutane sulfonic acid (PFBS; CAS# 375-73-5), perfluorooctane sulfonate (PFOS; CAS# 1763-23-1), perfluorooctanoic acid (PFOA; CAS# 335-67-1), perfluorodecanoic acid (PFDA; CAS# 335-76-2), 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro-(8:2 Telomer) (fluorotelomer, CAS# 678-39-7), and PFOSA (CAS# 754-91-6) were supplied through a contract with the National Toxicology Program (Battelle, Columbus, OH, USA). Chemical identity, purity, and stability were confirmed at Battelle by infrared spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Lipopolysaccharide from Escherichia coli serotype 0127:B8, antibodies against I-κB and β-actin, parthenolide, WY 14643 and all cell culture reagents were from Sigma (St Louis, MO, USA). Antibodies against phosphorylated-p65 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phytohemagglutinin (PHA) was from Invitrogen (Paisley, UK). Electrophoresis reagents were from Bio-Rad (Richmond, CA, USA). All reagents were purchased at the highest purity available. Cells. For all experiments using the human promyelocytic cell line THP-1 (Istituto Zooprofilattico di Brescia, Brescia, Italy), cells were diluted to 10 6 cells/ml in RPMI 1640 containing 2 mM L-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin, 50 μM 2mercaptoethanol, supplemented with 10% heated-inactivated fetal calf serum (media) and cultured in 37 °C in 5% CO2 incubator. For TNF-α release 0.5 × 10 6 cells were seeded in 24-well plates, while for Western blot analysis 4 × 10 6 cells were cultured in 15 ml polypropylene tubes. Cells were incubated with or without lipopolysaccharide (LPS) in the presence or absence of increasing concentrations of PFCs, or dimethyl sulphoxide (DMSO; 0.1% final concentration) as vehicle control, as described in the figure legends. Whole blood assay. Healthy young subjects (n = 8, 4 females and 4 males, average age 40 yrs), enrolled among colleagues of the researchers, were selected according to the guidelines of the Italian Health authorities and to the Declaration of Helsinki principles. Criteria for exclusion were abnormal laboratory values, medication known to affect the immune system, i.e. steroids and nonsteroidal anti-inflammatory drugs, or patients suffering from malignancies, inflammations, and infections. All subjects signed an informed consent and were informed about methods and aims of the study. Blood samples (5 ml) were taken by venous puncture with sodium citrate 0.5 M as anticoagulant. Sodium citrate was chosen instead of heparin or EDTA as anticoagulant, since functional assays were performed using the whole blood assay and heparin may be contaminated with endotoxin, while EDTA interferes with cell activation. Blood was diluted 1:10 with RPMI 1640 cell culture medium (Sigma, St Louis, USA) containing 2 mM L-glutamine, 0.1 mg/ml streptomycin, and 100 IU/ml penicillin. For the evaluation of cytokine production,

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Fig. 1. Chemical structure of the PFCs tested.

cultures were set up in 24-well plates containing 1 ml of 1:10 diluted whole blood, in medium alone or with increasing concentrations of PFCs or DMSO as vehicle control (0.1% final concentration) in the presence of 1 μg/ml LPS or 1.2 μg/ml PHA. For IL-6, and TNF-α release cells were incubated for 24 h, while for IL-10, and IFN-γ release cells were incubated for 72 h. Cells were incubated at 37 °C in a humidified 5% CO2 incubator. Lactate dehydrogenase. Cell viability was assessed by lactate dehydrogenase (LDH) leakage from damaged cells. LDH is a well-known indicator of cell membrane integrity and cell viability. LDH activity was determined in cell-free supernatants using a commercially available kit (Takara Bio Inc., Japan). Results are expressed as OD. Cytokine. Cytokine release was measured in cell-free supernatants obtained by centrifugation at 1200 rpm for 5 min and stored at −80 °C until measurement. Cytokine production was assessed by commercially available sandwich ELISAs (ImmunoTools GmbH, Friesoythe, Germany). ELISAs were performed according to the supplier's instructions. Results are expressed in pg/ml or as % of LPS treated cells. The limit of detection was 15.6 pg/ml for all cytokines tested. Table 1 Summary of the chemical characteristics of the PFCs tested. Name

CAS #

Molecular formula

Molecular weight

XLopPa

PFBS PFOSA PFOS PFOA Fluorotelomer PFDA

375-73-5 754-91-6 1763-23-1 335-67-1 678-39-7 335-76-2

C4HF9O3S C8H2F17NO2S C8HF17SO3 C8HF15O2 C10H5F17O C10HF19O2

300.1 499.1 500.1 414.1 464.1 514.1

2.3 4.8 5.0 4.9 5.7 6.3

a XLogP: partition coefficient or distribution coefficient calculated using version 3 of the algorithm to generate the XlogP value. Data obtained from PubChem.

Western blot analysis. The activation NF-κB was assessed by measuring cytosolic degradation of I-κB, and the phosphorylation of p65 (P-p65) by Western blot analysis. Briefly, cells were allowed to acclimatize for 1 h at 37 °C, and were then treated with PFCs (10 μg/ml) for 5 min, followed by LPS for 30 min. Cells were then collected, washed once with PBS, centrifuged and lysed in 100 μl of homogenization buffer (50 mM TRIS, 150 mM NaCl, 5 mM EDTA pH 7.5, 0.5% Triton X-100, 50 μM PMSF, 2 μg/ml aprotinin, 1 μg/ml pepstatin and 1 μg/ml leupeptin) and denatured for 10 min at 100 °C. The protein content of the cell lysate was measured using a commercial kit (Bio-Rad). 10 μg of extracted proteins were electrophoresed into a 12% SDS-polyacrylamide gel under reducing conditions. The proteins were then transferred to PVDF membranes (Amersham, Little Chalfont, UK). Western blots were visualized using primary antibodies for I-κB (1:2000), P-p65 (1:1500) and β-actin (1:2000) and developed using enhanced chemiluminescence (ECL, Amersham, Little Chalfont, UK). The image of the blot was acquired with the Molecular Imager Gel Doc XR (BioRad). The optical density of the bands was calculated and analyzed using the Image 1.47 program for digital image processing (Wayne Rasband, Research Service Branch, NIMH, NIH, Bethesda, MD, USA). Transient transfections. A luciferase reporter plasmid with three NF-κB sites from the E-selectin promoter as described previously (Brostjan et al., 1997) and kindly provided by N. Marx (Department of Internal Medicine II-Cardiology, University of Ulm, Ulm, Germany) was used. The expression vectors for PPARγ-LBD fused in frame with the Gal4 DNA binding domain (pGal4-PPARα-LBD) and the reporter vector containing five copies of the Gal4 upstream activating sequences (pGal4UAS-luciferase) driving the transcription of the luciferase reporter gene were kindly donated by Dr. Krister Bamberg (AstraZeneca, Mölndal, Sweden). THP-1 cells were transfected by the DEAE-dextran method (Sambrook and Russell, 2001). NF-κB

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driven transcription was performed by transfecting THP-1 cells with a mixture of DNA-dextran (750 μg/ml final concentration) and 70 ng NF-κB-luc reporter plasmid/well. PPARα transfection assays were performed by transfecting cells with the same amount of DEAEdextran with 70 ng of pGAL4UAS-luciferase and 35 ng of receptor vector (PPARα)/well. Cells were seeded in 96 well plates at a concentration of 1.5 × 10 5 cells/well and then incubated for 48 h in complete medium (FCS supplemented RPMI-1640). Cells were treated with increasing concentrations of PFOA and PFOS in the presence or absence of LPS (0.1 μg/ml) for 3 h in RPMI-1640 without FCS. At the end of the incubation, Britelite Plus reagent (Perkin Elmer, Milan, Italy) was added (100 μl/well). The luciferase assay was performed using a luminometer (Victor™ X3, Perkin Elmer, Milan, Italy). Results are expressed as luciferase activity and represent the mean ± SD values of four experiments performed in triplicate. Parthenolide was used as an inhibitor of NF-κB-driven transcription (88.4% inhibition at 20 μM, data not shown) whereas Wy 14643, a known PPARα agonist, (10 μM) was used as a positive control. Statistical analysis. All experiments were repeated at least three times, with representative results shown. Data are expressed as

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mean ± standard deviation (SD). Statistical analysis was performed using InStat software version 3.0a (GraphPad Software, La Jolla, CA, USA). Statistical differences were determined using ANOVA followed by Dunnett's multiple comparison test, the Tukey–Kramer multiple comparisons test or unpaired t test. Effects were considered significant if p ≤ 0.05. Results Effects of PFCs on cytokine release in human peripheral blood leukocytes The whole blood assay was used to assess the immunomodulatory effects of PFCs. Peripheral blood obtained from healthy volunteers was diluted 1:10 and treated with increasing concentrations of PFCs (0.1–10 μg/ml) in the presence of LPS (1 μg/ml) or PHA (1.2 μg/ml) for 24 and 72 h, respectively. As shown in Fig. 2, in leukocytes obtained from female and male donors, both sulfonate PFCs (Figs. 2A, C) and non-sulfonate PFCs (Figs. 2B, D) decreased LPSinduced release of TNF-α in a dose-related manner, with PFOA and PFBS being the less potent (p b 0.05, Tukey–Kramer multiple comparisons test). Leukocytes obtained from the female donor appear

Fig. 2. Effect of PFCs on LPS-induced release of TNF-α in peripheral blood leukocytes and in THP-1 cells. (A, B, C, D) Whole blood was diluted 1:10 in culture medium and treated with increasing concentrations of PFCs or DMSO (0.1% final concentration) vehicle control in the presence of 1 μg/ml LPS for 24 h. Each value represents the mean ± SD of triplicate wells from a single female or male donor. Data is representative of eight independent donors. (E, F) THP-1 cells were treated with increasing concentrations of PFCs or DMSO vehicle in the presence of 0.1 μg/ml LPS for 3 h as described in the Materials and methods. Each value represents the mean ± SD of three samples. * p b 0.05 vs LPS treated cells by Dunnett's multiple comparison test.

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to be more susceptible to PFBS-induced immunotoxicity when compared to the response obtained in male donors, suggesting a possible gender effect. Interestingly, as THP-1 is a promyelocytic leukemia cell line isolated from a 1 year old boy, the THP-1 cells responded similarly to leukocytes obtained from male donors. To further evaluate a potential gender effect in the immunomodulatory effects of PFCs, leukocytes obtained from female (n= 4) and male (n= 4) donors were treated with PFCs 10 μg/ml in the presence of LPS (1 μg/ml), and the effects on TNF-α release were compared. As shown in Fig. 3, leukocytes obtained from female donors appear to be more susceptible to PFCs-induced immunotoxicity, as assessed by the measurement of TNF-α production, when compared to the response observed in leukocytes obtained from male donors. Results are expressed as % of LPS treated cells, as the response to LPS alone varied from 838 to 2532 pg/ml in female donors, and from 2916 to 5910 pg/ml in male donors. Similarly, LPS-induced IL-6 release was suppressed by PFOSA, PFOS, PFDA, and fluorotelomer (Table 2), whereas PFOA and PFBS failed to modulate this response. To examine the effects on T-cell derived cytokines human peripheral blood cells obtained from a female donor were cultured with 10 μg/ml of PFCs (Table 2) in the presence of PHA (1.2 μg/ml). PFBS, PFOSA, PFOS, and fluorotelomer decreased T-cell derived PHA-induced IL-10 release, while IFN-γ release was affected by PFOSA, PFOS, PFDA and fluorotelomer.

Similar to what was observed following LPS stimulation, PFOA was the least active of the PFCs, with PFOSA, PFOS and fluorotelomer being the most potent inhibitors of T-cell cytokines. Data relative to the dose–response effects of PFCs on cytokine production in leukocytes obtained from male and female donors are supplied as additional data on the journal website. Due to donor variability and differential susceptibility to the PFCs, the gender effect was less evident for the other cytokines assessed. A larger study will be necessary to properly address the gender-specific immune effects of PFCs.

Effect of PFCs on LPS-induced TNF-α production in THP-1 cells Consistent with the findings in peripheral blood leukocytes, in the human promyelocytic cell line THP-1, PFCs were able to modulate LPS-induced TNF-α release (Figs. 2E and F) in a dose-dependent manner, with PFOA and PFBS being the less potent inhibitor of cytokine production (p b 0.05, Tukey–Kramer multiple comparisons test), and similar to the response observed in leukocytes obtained from male donors. The immunomodulatory effects observed in peripheral blood leukocytes and THP-1 cells were not due to cytotoxicity, as cell viability, as assessed by LDH leakage, was not affected following treatment with PFCs in either cell type (Table 3).

Fig. 3. Effect of PFCs on LPS-induced release of TNF-α in peripheral blood leukocytes obtained from female and male donors. Whole blood was diluted 1:10 in culture medium and treated with 10 μg/ml of the different PFCs or DMSO (0.1% final concentration) vehicle control in the presence of 1 μg/ml LPS for 24 h. Each dot represents a single female or male donor, the bar is the mean value, the dotted line at (100%) represents vehicle treated cells stimulated with LPS. * p b 0.05 by unpaired t test; ** p b 0.01 by unpaired t test.

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Table 2 Effects of PFCs on LPS-induced IL-6, and PHA-induced IL-10 and IFN-γ release from human peripheral blood leukocytes obtained from a female donor. PFCs

IL-6 (pg/ml)

IL-10 (pg/ml)

IFN-γ (pg/ml)

DMSO vehicle PFBS PFOSA PFOS PFOA Fluorotelomer PFDA

592 ± 60 488 ± 121 269 ± 43* 295 ± 42* 571 ± 103 202 ± 47* 311 ± 65*

1947 ± 313 1345 ± 326* 1057 ± 284* 1210 ± 160* 1514 ± 486 788 ± 32* 1290 ± 376

10094 ± 1035 7446 ± 1798 4245 ± 520* 5719 ± 1070* 8896 ± 1272 3340 ± 145* 7402 ± 485*

Whole blood was diluted 1:10 in culture medium and treated with PFCs (10 μg/ml) or DMSO (0.1% final concentration) as vehicle control in the presence of LPS (1 μg/ml) for 24 h or of PHA (1.2 μg/ml) for 72 h as described in the Materials and methods. Each value represents the mean ± SD of triplicate wells of a single donor, with * p b 0.05 vs PHA treated cells. Data is representative of three independent donors. In nonstimulated cells the release of cytokines was below the limit of detection.

Effects of PFCs on LPS-induced NF-κB driven transcription and PPARα activation in THP-1 cells Having established in these and previous studies that the TNF-α release data obtained from THP-1 cells was similar to that obtained with human peripheral blood leukocytes, we considered THP-1 cells an appropriate in vitro model to further study the molecular mechanisms underlying PFC-induced inhibition of TNF-α production (Corsini et al., 2011). As previous studies in our laboratory have shown them to be important targets for PFOA and PFOS (Corsini et al., 2011), the effects of PFCs on LPS-induced NF-κB activation and on PPARα activation, as possible mechanisms to explain the immunomodulatory effects observed, were investigated. The effects of PFCs on LPS-induced NF-κB activation were evaluated by examining I-κB degradation and phosphorylation of p65 by Western blot analysis, and NF-κB driven transcription by transient transfection with a luciferase reporter plasmid construct containing three NF-κB sites. As shown in Fig. 4A, PFOS, PFBS and PFDA prevented LPS-induced I-κB degradation, while PFOA, PFOSA and fluorotelomer had no effects. Despite their differing effects on LPS-induced I-κB degradation, all of the PFCs examined similarly inhibited LPS-induced phosphorylation of p65 at Ser276, which is required for optimal NFκB-mediated transcription (Fig. 4B) and NF-κB driven transcription (Fig. 5A). Taken together our data indicate that by interfering with LPS-induced NF-κB transactivation, PFCs prevent transcription and translation of TNF-α, resulting in a decrease in the release of this cytokine in monocytes. These findings are consistent with previously published results (Corsini et al., 2011), and confirm NF-κB as an important intracellular target of these compounds. As some PFCs have been shown to activate PPARα, we investigated the effects of selected PFCs in THP-1 cells transiently transfected with a luciferase reporter plasmid construct containing the LBD of Table 3 Effects of PFCs (10 μg/ml) on LDH leakage from human peripheral blood leukocytes (PBL) and THP-1 cells. PFCs

THP-1 t = 24 h

PBL t = 72 h

DMSO vehicle PFBS PFOSA PFOS PFOA Fluorotelomer PFDA

0.369 ± 0.008 0.374 ± 0.017 0.375 ± 0.007 0.363 ± 0.026 0.366 ± 0.005 0.375 ± 0.009 0.367 ± 0.013

0.879 ± 0.070 0.806 ± 0.098 0.873 ± 0.060 0.786 ± 0.054 0.761 ± 0.058 0.732 ± 0.089 0.846 ± 0.067

Whole blood diluted 1:10 in culture medium or THP-1 cells were treated with PFCs (10 μg/ml) or DMSO (0.1% final concentration) as vehicle control for 24 h (THP-1) and 72 h (PBL). LDH leakage was assessed as described in the Materials and methods. Results are expressed as OD values. Each value represents the mean ± SD of three samples.

Fig. 4. Effect of PFCs on LPS-induced NF-κB activation in THP-1 cells. THP-1 cells were treated with PFCs or DMSO vehicle in the presence of 0.1 μg/ml LPS for 30 min as described in the Materials and methods. NF-κB activation was assessed by I-κB degradation (A) and phosphorylation of p65 (B). Each value represents the mean ± SD of 3–4 independent experiments. * p b 0.05 vs LPS treated cells by Dunnett's multiple comparison test.

PPARα. As shown in Fig. 5B, only PFOA was able to significantly activate PPARα. All the other PFCs tested failed to activate the receptor, indicating that activation of PPARα is not a critical factor to initiate the immunomodulatory effects observed in THP-1 cells. As expected, the positive control Wy 14643 showed significant receptor activation (266 ± 40% vs 100% in vehicle treated cells, data not shown). Discussion Using human cells exposed to several PFCs in vitro, we have demonstrated that PFCs directly affected immune cell activation and reduced cytokine production (both pro- and anti-inflammatory). These results are consistent with previous studies that reported immunomodulation in experimental animals exposed to PFOA and PFOS, including altered inflammatory responses, cytokine production, reduced lymphoid organ weights and decreased antibody production (DeWitt et al., 2008, 2009). All of the PFCs studied, including PFOA and PFOS, decreased LPS-induced phosphorylation of p65 and NF-κB driven transcription. However, with the exception of PFOA, none of the PFCs tested was able to activate PPARα, as assessed in transiently transfected THP-1 cells, excluding a role for the this receptor in the immunomodulation observed. PFBS and PFDA prevented LPSinduced I-κB degradation, similar to what we observed in previous studies with PFOS (Corsini et al., 2011). Further studies are, however, necessary to characterize the mechanism of action of PFOSA and fluorotelomer. As plasma levels of PFOA and PFOS ranging between 3.7 and 12,000 ng/ml have been found in occupationally exposed populations, the concentrations (0.1–10 μg/ml) used in our in vitro studies would reflect highly exposed humans (Calafat et al., 2006; Costa et al., 2009; Olsen et al., 2007; Rayne and Forest, 2009;

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Fig. 5. Effect of PFCs on LPS-induced NF-κB promoter activity and PPARα activation. (A) THP-1 cells were treated with increasing concentrations of PFCs or DMSO vehicle in the presence of LPS 0.1 μg/ml for 3 h as described in the Materials and methods to assess NF-κB promoter activity. (B) THP-1 cells were treated with PFCs or DMSO as vehicle for 3 h to assess PPARα promoter activation as described in the Materials and methods. Each value represents the mean ± SD of 3–4 independent experiments. **p b 0.01 vs LPS treated cells by Dunnett's multiple comparison test.

Steenland et al., 2010). The lowest dose used in these studies (0.1 μg/ml) is 5–20 fold higher than background plasma concentrations of PFOA (5 ng/ml) and PFOS (20 ng/ml) in the general population. The exact role of PPARs in PFC-immunotoxicity is likely to be complex with some effects resulting from a PPAR-mediated mechanism, while others occur via a PPAR-independent mechanism. There is mounting experimental animal data demonstrating PPARα independence of some immune effects (DeWitt et al., 2009). We have demonstrated a role of PPARα in PFOA-induced inhibition of cytokine secretion in human cells exposed in vitro. While hypolipidemic drugs and other activators of PPARα have been shown to exhibit direct anti-inflammatory properties via inhibition of proinflammatory cytokine and chemokine secretion (Steffens and Mach, 2004), decreased PPAR activity has been associated with increased levels of inflammatory mediators in a number of cell types (Chinetti et al., 2000). In contrast, we have also shown that the inhibitory effect of PFCs on in vitro cytokine production by human leukocytes can occur independently of PPARα, and involves inhibition of NF-κB activation. NF-κB activation plays a key role in inflammation, immunity, cell proliferation, apoptosis and cytokine production in both T cells and monocytes/macrophages (Crabtree and Clipstone, 1994; Viatour et al., 2005). A detailed analysis of NF-κB in THP-1 cells showed that at concentrations that did not produce cellular cytotoxicity, all PFCs tested inhibited, at different levels, the signaling pathways that regulate NF-κB activation. All of the PFCs tested inhibited p65 phosphorylation and NF-κB driven transcription; and while it appears that PFOS, PFBS and PFDA act upstream by inhibiting I-κB degradation, PFOA, PFOSA and fluorotelomer do not interfere with LPS-induced I-κB degradation, suggesting a downstream effect. The phosphorylation of p65 is regulated by both cell- and stimulus-dependent activating kinases. Ser276 phosphorylation has a crucial role in the interaction with and the engagement of the cofactor CBP/p300, being therefore important in order to establish gene activation (Saccani et al., 2002; Vermeulen

et al., 2002, 2003). The phosphorylation of p65, which is required for optimal NF-κB dependent gene transcription (Schmitz et al., 2001), appears to be an important target of PFC-induced immunotoxicity. The PFCs used in this study were chosen based on their differing chain lengths (C4–C10) and functional groups (sulfonate, carboxyl and amino groups) to determine, if possible, the structure–activity relationship based on cytokine release as toxicological endpoints. Only a few studies have been performed to determine the structure– activity relationship of PFCs. Most of these studies have shown that the bioaccumulation potential and the toxicity of PFCs increase with increasing fluorinated carbon chain length and that PFCs with a sulfonate group tend to be more toxic than those with a carboxylated group (Hagenaars et al., 2011; Hu et al., 2002; Ji et al., 2008; Martin et al., 2003). Using effects on cytokine to assess potency, our results indicate that PFOA is the least active compound followed by PFBS, PFDA, PFOS, PFOSA and fluorotelomer. PFOS production has been phased out and PFBS is currently being produced as an alternative (3M, 2006). It has the same functional group as PFOS, but due to its shorter chain length, it has been shown to be less toxic and slightly less persistent in the environment (Lieder et al., 2009; Hagenaars et al., 2011). As compared to PFOS, PFBS is less potent in inhibiting both LPS and PHA-induced cytokine production, in agreement with the expected reduced bioavailability and indicative that PFCs with longer chain lengths tend to be more toxic than PFCs with shorter chain lengths. This is also true for PFOA (C8) and PFDA (C10). Comparison based on the functional groups of compounds with the same chain length indicates that PFCs with a sulfonate group are more potent than those with a carboxyl group (PFOS = PFOSA > PFOA). With regard to PFDA and fluorotelomer, both C10 with an acid and alcohol group respectively, the fluorotelomer was more active, inhibiting both LPS and PHA-induced cytokine production. Overall, we have demonstrated that in vitro exposure to PFCs directly inhibited cytokine production in cultured human leukocytes. In addition, we have shown differential effects in cells obtained from male and female donors, suggesting potential gender differences in sensitivity to the immunodulatory effects of PFCs. Furthermore, the phosphorylation of p65, and subsequent NF-κB driven transcription, appears to be an important target of PFC-induced immunotoxicity, which may account for the defective cytokine production observed. It is important to note that humans are exposed to multiple PFCs, and while the levels of some PFCs are decreasing due to changes in manufacturing processes, the levels of other PFCs have risen in recent years (Kato et al., 2011). Therefore, a possible additive role of PFCs and contributions by both receptor-mediated and PPAR-independent effects cannot be ruled out. In addition, as human PPARα expression is significantly less than that in rodents, the identification of effects independent of PPARα activation is important for evaluating human risk from exposure to PFCs. Supplementary materials related to this article can be found online at doi:10.1016/j.taap.2011.11.004. Conflict of interest Authors declare not having any financial or personal interest, nor having an association with any individuals or organizations that could have influenced inappropriately the submitted work. Acknowledgments We would like to thank Dr. Chad Blystone and Dr. Jamie DeWitt for their review and helpful suggestions. This research was supported in part by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health. This article may be the work product of an employee or group of employees of the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), however, the

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