Arch Toxicol (2009) 83:805–815 DOI 10.1007/s00204-009-0424-0
ORGAN TOXICITY AND MECHANISMS
Chronic eVects of perXuorooctanesulfonate exposure on immunotoxicity in adult male C57BL/6 mice Guang-Hui Dong · Ying-Hua Zhang · Li Zheng · Wei Liu · Yi-He Jin · Qin-Cheng He
Received: 20 January 2009 / Accepted: 19 March 2009 / Published online: 3 April 2009 © Springer-Verlag 2009
Abstract A paucity of data exists to corroborate the few studies that report immune suppression after exposure to perXuorooctanesulfonate (PFOS). In this study, adult male C57BL/6 mice were exposed to PFOS daily via gavage for 60 days [0, 0.5, 5, 25, 50, or 125 mg/kg total administered dose (TAD)]. The results showed that liver mass was signiWcantly increased at ¸5 mg PFOS/kg TAD and in a dose-dependent manner. Lymphocyte proliferation and natural killer cell activity were altered in male mice. Plaque forming cell (PFC) response was suppressed beginning at 5 mg/kg TAD. Based on the liver mass and PFC response, the no observed adverse eVect level and lowest observed adverse eVect level for male mice exposed PFOS for 60 days was 0.5 and 5 mg/kg TAD, respectively. Measured PFOS serum concentrations at these dose levels were 0.674 § 0.166 and 7.132 § 1.039 mg/l, respectively. These results indicate that PFOS exposure can aVect the immunity function in mice at levels approximately 50-fold for highly exposed human populations.
G.-H. Dong (&) · Y.-H. Zhang · Y.-H. Jin · Q.-C. He School of Public Health, China Medical University, 92 North 2nd Road, Heping Districts, Shenyang 110001, People’s Republic of China e-mail:
[email protected] L. Zheng Department of Immunology, College of Basic Medical Science, China Medical University, Shenyang 110001, People’s Republic of China W. Liu · Y.-H. Jin (&) Department of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, People’s Republic of China e-mail:
[email protected]
Keywords PerXuorooctanesulfonate · Immunotoxicity · PFC response
Introduction PerXuorooctanesulfonate [PFOS; CF3(CF2)7SO3¡], which is produced synthetically or from the metabolism of other perXuorinated chemicals (PFCs) (ButenhoV et al. 2006), has extreme thermal, biological, and chemical stability as well as hydrophobic and lipophobic characteristics. These properties make it widely to be used as industrial surfactants and emulsiWers and in numerous consumer products. PFOS has aroused scientists’great concern in recent years due to its widespread occurrence in the environment, in the wildlife and in humans (Calafat et al. 2007; Giesy and Kannan 2001; Hart et al. 2008; Jin et al. 2007; Monroy et al. 2008). Furthermore, it has shifted among biological populations via biological concentration and magniWcation, leading to excessive accumulation among the higher trophic level of food chain (such as predator and human beings) (de Vos et al. 2008; Houde et al. 2006; Tittlemier et al. 2007; Vestergren et al. 2008). One of our recent studies indicate that the serum PFOS level in un-occupationally exposed individuals from Shenyang of China increased signiWcantly from 0.03 g/l in 1987 to 22.4 g/l in 2002, and from 1999 to 2002, serum PFOS concentration also increased 13-fold (Jin et al. 2007). PerXuorooctanesulfonate is the predominately PFCs found in both human and wildlife blood samples and accumulates primarily in the blood and liver (Olsen et al. 2003; Houde et al. 2006). Levels of PFOS in human serum concentrations vary depending on the population evaluated, a review of environmental and human biomonitoring studies indicates that PFOS has been detected in human sera at
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mean levels ranging from approximated 10 to 75 g/l serum (Lau et al. 2007). A number of mammalian toxicology studies were conducted to assess the health eVects of exposure to PFOS, recent studies indicated that PFOS can cause hepatotoxicity, tumors of the liver, and of the thyroid and mammary glands, developmental toxicity, reproductive toxicity, neurotoxicity, and endocrine-disrupting (3M Company 2002; Abbott 2008; Ankley et al. 2005; Chang et al. 2008; Martin et al. 2007). Another study conducted by 3M Company revealed an increase in bladder cancer mortality among workers exposed to PFOS (Alexander et al. 2003). Immunotoxicity has also been reported (Keil et al. 2008; Lefebvre et al. 2008; Peden-Adams et al. 2008; Zheng et al. 2008). Mice was administered by gavage (5–40 mg PFOS/ kg body weight/day) for 7 days displayed splenic and thymic atrophy accompanied by reduced spleen and thymus cellularity and inhibition of lymphocyte proliferation and natural killer (NK) cell activity (Zheng et al. 2008). Rat exposed to dietary PFOS (2–100 mg PFOS/kg diet) for 28 days produced a signiWcant reduction in serum total immunoglobulin (Ig) G1 levels (Lefebvre et al. 2008). Also, another study showed that PFOS at levels reported in humans was immunosuppressive to adult mice when administered by gavage for 28 days (Peden-Adams et al. 2008), although these low doses were not associated with overt signs of toxicity. However, to our knowledge, few studies have assessed the chronic eVects of PFOS exposure on immune system. In the present study, the eVects of PFOS on the immune system were assessed in adult C57BL/6 male mice after oral exposure by gavage for 60 days. In this study, we evaluated the immune function in experiments designed to corroborate the reported altered immune function observed in mice and to establish no observed adverse eVect level (NOAEL) and lowest observed adverse eVect level (LOAEL) values from dose–response studies of immune function.
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1640 medium (with L-glutamine and sodium bicarbonate), phosphate-buVered saline (PBS; with or without Ca2+ and Mg2+), and penicillin/streptomycin were purchased from Cellgro (Mediatech, Herndon, VA, USA). The fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), concanavalin A (ConA) (type IV-S), lipopolysaccharide (LPS; E. coli 0111:B4) and sodium dodecyl sulfate (SDS) were purchased from Sigma (St Louis, MS, USA). Nitroblue tetrazolium chloride (NBT), nicotinamide adenine dinucleotide (NAD+) and phenazine methosulfate (PMS) were from Amresco (Solon, OH, USA). Lactate dehydrogenase (LDH) substrate includes the following compositions: NBT, NAD, PMS, and sodium lactate. The Xuorescent antibodies, mouse LgG2a (isotype control), Xuorescein isothiocyanate (FITC) conjugated rat-antimouse CD3 (monoclonal), phycoerythrin (PE) conjugated rat anti-mouse CD4 (monoclonal), peridinin chlorophyll protein (Percp) conjugated rat anti-mouse CD8 (monoclonal), and rat anti-mouse CD45R/B220 antibodies conjugated to FITC were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Luma Plate, UniWlters, and Microscint 20™ were procured from Packard (Meriden, CT, USA). Triton X, tissue culture plates and disposables were purchased from Sigma (St Louis, MS, USA). YAC-1 cells were purchased from ATCC (Manassas, VA, USA). High performance liquid chromatography (Agilent1100)/ mass spectrometry (Agilent MSD SL) was purchased from Agilent (USA). Tetrabutylammonium hydrogen sulfate (HPLC grade) was purchased from Acros Organics (Geel, Belgium). Sodium carbonate (>99.5%) was obtained from Kanto Chemical (Tokyo). Methyl tertiary-butyl ether (MTBE; HPLC grade), methanol (HPLC grade), acetonitrile (HPLC grade), and ammonium acetate (>97%) were obtained from Wako Pure Chemical Industries (Osaka). Animals and treatment
Materials and methods Chemicals, antibodies, and supplies PerXuorooctane sulfonic acid potassium salt (PFOS; FW 538.22; purity >98%) was purchased from Fluka Chemical (Steinheim, Switzerland, Lot & Filling code: 367360/1 21101, EC No. 2205287). Potassium PFOS suspensions were prepared in de-ionized water with 2% Tween® 80 at concentrations. Sheep red blood cells (SRBCs) in Alsever’s solution were obtained from Laboratory Animal Research Center of China Medical University (Shenyang China). Guinea-pig serum was used as a source of complement for the development of hemolytic plaques [plaque forming cell (PFC) assay]. Roswell Park Memorial Institute (RPMI)-
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All experiments were approved by the Institutional Animal Ethics Committee and conformed to NIH guidelines on the ethical use of animals. EVorts were made to minimize the number of animals used and their suVering, and approved by the Animal Care and Use Committee in China Medical University. All experiments were performed on male C57BL/6 mice (8–10 weeks old; weighting 18–22 g) purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Science, China (SLAC, Shanghai, China). Sixty adult C57BL/6 male mice were randomly divided by weight into six groups of ten per group. Once distributed into groups the mice acclimated to the new cage conditions and the new treatment room (12-h light/dark cycle (light 06:00– 18:00 hours, dark 18:00–06:00 hours), 22.4 § 1.3°C, 60– 65% relative humidity) for 10 days before dosing was
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initiated. Exposures consisted of oral administration of PFOS delivered in de-ionized water with 2% Tween 80. Control mice received de-ionized water with 2% Tween 80 only. C57BL/6 mice were dosed once daily via oral gavage for 60 days (0, 8.33, 83.33, 416.67, 833.33, or 2083.33 g PFOS/kg body weight/day) to yield a targeted total administered dose (TAD) over the 60 days of 0, 0.5, 5, 25, 50, or 125 mg PFOS/kg body weight. Food intake and body weight of all animals was measured daily for 60 days. Each dose was adjusted per body weight of each mouse by adjusting the gavage volume between 100 and 130 l. Bedding, food, and water were changed twice a week and mice were observed daily. We chose the dose of 0.5 mg/kg TAD because PedenAdams et al. (2008) reported that this dose reduced IgM production and also in order to compare doses or concentrations reported in other studies (Keil et al. 2008; Lefebvre et al. 2008; Peden-Adams et al. 2008). Body and organ mass and organ cellularity Body mass was measured from 1 day prior to exposures to the termination of the experiment. On the 61st day (24 h after last treatment), mice were bled by retro-orbital puncture under light diethyl ether anesthesia and subsequently sacriWced by cervical dislocation. Blood samples were collected and held at room temperature for 30 min, centrifuged at 4°C to separate serum, and serum was frozen at ¡80°C until analyzed for corticosterone by radioimmunoassay (RIA) or PFOS concentration. Spleen, thymus, liver, and kidneys were collected and weighed. All balances were calibrated, using standard weights, prior to use. Organ mass was normalized for body weight and reported as a somatic index [(organ weight/body weight) £ 100]. Additionally, spleen and thymus were aseptically processed into single-cell suspensions by gentle grinding with the use of sterile, frosted microscope slides for functional immune endpoints and T cell immunophenotype determinations. A Coulter Counter (model ZF; Hialeah, FL, USA) was used to obtain cell counts from theses single-cell suspensions. Alterations in cell viability following treatment were assessed after red blood cell lysis. Serum PFOS analysis Details of the analytical procedure to measure PFOS have been outlined previously (Hansen et al. 2001; Zheng et al. 2008). BrieXy, 0.5 ml of serum, 1 ml of 0.5 M tetrabutylammonium hydrogen sulfate solution and 2 ml of sodium carbonate buVer (0.25 M, pH 10) were added to 15 ml polypropylene tube and thoroughly mixed. Following addition of 5 ml of MTBE to the solution, the organic and aqueous layers were separated by centrifugation, and the organic layer was removed. The aqueous mixture was rinsed with MTBE and separated twice. The solvent was evaporated at
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room temperature under a nitrogen gas Xow, and the sample was then reconstituted in 0.5 ml of methanol. The sample was then passed through a nylon Wlter (Autovial R5 PUNYL; 0.45-m pore size; Whatman Japan, Tokyo) to remove any suspended materials and insoluble particles. Each extracted solution was analyzed by liquid chromatography–mass spectrometry (LC/MS) as previously described (Jin et al. 2007). BrieXy, each methanol extract (injection volume, 10 l) was chromatographed using HPLC at a Xow rate 0.2 ml/min. Gradient conditions were used in the mobile phase. Initial mobile phase conditions were 35:65 CH3CN/10 mM CH3COONH4 buVer, followed by a 5-min ramp to 45:55, hold until 20 min. The chromatographic column was kept at 40°C. Mass spectra were taken using an LC/MS system equipped with an orthogonal spray interface, employing electrospray ionization in the negative mode. The electrospray probe and ion source were operated at following conditions: capillary voltage 4 kV, fragmentor voltages 200 V, nebulizer pressure 50 psi, desolvation temperature 350°C, and desolvation gas Xow rate 101/min. Molecular ion 499 was selected as the primary ion for PFOS (C8F17SO3). The limit of detection of the method used for PFOS measurement was 0.01 mg/l. Corticosterone Serum samples were thawed and levels of corticosterone were measured in duplicate using an RIA kit (ICN Biomedical Inc., Costa Mesa, CA, USA) as described previously (Francis et al. 2000). Splenic and thymic CD4/CD8 subpopulations Spleen or thymus cells were labeled with Xuorescent (PE or Percp) rat IgG2 monoclonal antibodies speciWc for mouse CD4 or CD8 (rat anti-mouse). The antibody dilution used for FACS analysis was 1:5 (v/v) for FITC conjugated rat-anti-mouse CD3, 1:2 (v/v) for PE conjugated rat antimouse CD4 and 1:2 (v/v) for Percp conjugated rat antimouse CD8, respectively, following the manufacturer’s instructions. In this procedure, single-cell suspensions of splenocytes and thymocytes were washed and resuspended in PBS (pH 7.4; containing 0.1% sodium azide and 1% bovine serum albumin). Monoclonal antibodies were incubated with cells for 30 min at 6°C in the dark. Red blood cells were lysed (0.83% NH4CI, 0.1% NaHCO3, and 0.006% EDTA in distilled water; pH 7.0) and removed by several washings with PBS. Lastly, the cells were Wxed with 1% paraformaldehyde and stored at 6°C in the dark. Flow cytometric analysis was performed using a Becton Dickinson Xow cytometer (FACSCalibur; San Jose, CA, USA). Nonstained cells and isotypic antibody controls were used to establish gates for the CD4/CD8 subpopulations in
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thymic and splenic cells. Data are represented as absolute number of cells, determined by multiplying the percent gated cells by the total number of nucleated cells obtained by the Coulter Counter. Lymphocyte proliferation assay The splenocytes were washed three times in RPMI-1640 supplemented with 10% FBS and then resuspended in RPMI 1640 medium. The concentration of splenocytes was adjusted to 5 £ 106 nucleated cells/ml. The proliferation ability of the lymphocytes was determined by MTT stain assay. Hundred microliters of aliquots of the resulting cell suspensions were dispensed into 96-well plates (5 £ 105 cells/well) containing triplicate wells of either 10 g/ml mitogen ConA, 10 g/ml LPS or supplemented RPMI-1640 (unstimulated wells), the Wnal culture volume was 200 l in each well. The plates were incubated for 48 h at 37°C in a humidiWed 5% CO2–air mixture. After addition of 10 l MTT per well, the plate was incubated for 4 h and then 100 l of 20% SDS (Sigma) were pipetted into each well to dissolve formazan crystals. After incubation at 37°C in 5% CO2 atmosphere overnight, the microplate was read on a Bio-Rad microplate reader (Model 550) using test wavelength of 570 nm. The ratio of the optical density (OD) of stimulated to the OD of unstimulated cultures was used as the stimulation index. The proliferation index was calculated by the equation: proliferation index = A value of ConA or LPS-stimulated cells/A value of nonstimulated cells. Natural killer cell activity Splenocytes from each mouse were prepared as described above. For the determination of NK cell activity, LDH release assay was carried according to previous report (Wu et al. 1996; Zheng et al. 2008) with some modiWcations. The splenocytes were washed and suspended in complete RPMI-1640 medium, then were counted and diluted to 1.0 £ 106 nucleated cells/ml. The amount of the LDH released from the lysed target cells was determined to measure NK activity. The cell line Yac-1 was used as the target cell. Yac-1 was washed with complete RPMI-1640 medium, counted and Wnally diluted to the concentration of 1.0 £ 105/ml with the medium. The same volume of Yac-1 cells and splenocytes were added to the wells of 96 roundbottom microwell plates (the cell ratio of eVector-to-target is 10:1). Three wells were used for every mouse. To ensure cell–cell contact, the plate was centrifuged at a low speed for 2 min. After 4-h incubation at 37°C in a humid atmosphere with 5% CO2, the plate was centrifuged at 1,000 rpm/min for 5 min. The supernatant from each well (100 l) was transferred into the corresponding wells of a 96 Xat-bottom microwell plate. Then 100 l of lactic acid
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dehydrogenase substrate mixture was added to each well. After 3 min, 50 l of cold medium was added to each well to stop reactions. Finally, a microtiter plate reader (BioRad, Modal 550) was used for evaluation of changes in the absorbance at a wavelength of 490 nm. The release of LDH from Yac-1 cells was expressed as absorbance. The percentage of NK cell activity was calculated by the formula: NK cell activity = [(E ¡ S)/(M ¡ S)] £ 100%. Where E represents the experimental release of LDH activity from target cells incubated in the presence of lymphocytes, M represents the maximum release of the LDH activity determined by lysing the target cells with 1% of NP-40, and S is the spontaneous release of the LDH activity from target cells incubated in the absence of lymphocytes. Antibody plaque forming cell assay The number of plaque forming cell (PFC) was determined using the Jerne plaque assay (Jerne and Nordin 1963). BrieXy, 4 days prior to euthanasia, mice were administered 0.1 ml of a 25% SRBC suspension in PBS via intraperitoneal injection. All SRBCs for the experiments were drawn from a single donor animal. Spleen cells collected from individual animals (0.1 ml; 1 £ 106 cells/0.1 ml), 0.4 ml of 0.5% “low melting point” agarose (GIBCO, Grand Island, NY, USA) in RPMI-1640 medium, and 50 l of a suspension of 5% SRBC were added to test tubes at 37°C and poured onto microscope slides containing a bottom layer of 0.5% agarose in water. The slides were then incubated for 2 h at 37°C and 5% CO2. Guinea pig serum diluted 1:4 in RPMI-1640 was added to the slides and after another 40 min (37°C and 5% CO2) incubation the number of plaques was counted and values were expressed as PFC per 106 cells. For the preparation of guinea pig serum as the complement source, animals were anesthetized before cardiac puncture and blood samples were collected. Statistics All experiments were repeated two times (two trials). The sample sizes for all treatment in the experimental trials were 5 and 5 for trials 1 and 2, respectively. Experimental trials were tested for trial by treatment interactions and when statistically possible data from trials were combined. Data were tested for normality (Shapiro–Wilks W-test) and homogeneity (Bartlett’s test for unequal variances) and, if needed, appropriate transformations were made. Transformations when required are outlined in the Wgure legends. A one-way analysis of variance was used to determine diVerences among doses for each endpoint using SAS software (Version 9.13; SAS Institute Inc., Cary, NC, USA) in which the standard error used a pooled estimate of error variance. When signiWcant diVerences were detected by the
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dose-dependent increase in the concentrations of PFOS in serum from exposed mice. Changes in serum corticosterone among the treatment groups were shown in Fig. 3. The corticosterone level (mean § SEM) in the sera of control animals at the time of sacriWce was 426 § 29.47 ng/ml. Treatment with the dose of 0.5, 5, and 25 mg PFOS/kg TAD did not aVect serum corticosterone levels. In contrast, the corticosterone levels were signiWcantly increased in animals treatment with the TAD dose of 50 mg (576.94 § 35.17) or 125 mg (634.83 § 44.25) PFOS/kg, respectively.
F test (P < 0.05), Dunnett’s t test was used to compare treatment groups to the control group.
Results Animal body weight, food intake, and organ mass There were no signiWcantly diVerences in body weight among the treatment groups at the beginning of the experiment. In the 60-day study, body weight (mean § SEM) in control animals on day 0 (pretreatment) was 22.48 § 0.41 g and increased to 25.58 § 0.54 g by day 60. In contrast, Wnal body weight were signiWcantly lower relative to controls in mice exposure to 25, 50, and 125 mg/kg TAD of PFOS (P < 0.05; Fig. 1). Also, in groups treatment with the dose of 50 and 125 mg/kg TAD of PFOS, changes in daily food intake (mean § SEM) showed signiWcant deterioration from their own pre-exposed baseline (P < 0.05). However, food intake in animals that were treated with the dose of 0.5 and 5 mg/kg TAD PFOS was similar to that in controls (Fig. 2). At the last day of the treatment, body, spleen, thymus, and kidney mass were signiWcantly decreased compared to the control following exposure to 25, 50, and 125 mg/kg TAD of PFOS (Table 1). Furthermore, liver mass was signiWcantly increased at dose as low as 5 mg PFOS/kg TAD.
Splenic and thymic cellularity Treatment with 25, 50, or 125 mg PFOS/kg TAD resulted in downtrend of splenic and thymic cellularity following 60 days of treatment (Fig. 4). Especially for the group of mice exposed to 125 mg PFOS/kg TAD, the splenic and thymic cellularity was found to be signiWcantly decreased by 55 and 70% following the 60-day exposure compared with control mice [(4.22 § 0.32) £ 107 vs. (9.40 § 0.45) £ 107 for splenic; (3.42 § 0.36) £ 107 vs. (11.49 § 0.56) £ 107 for thymic], respectively. Lymphocyte immunophenotypes CD4/CD8 marker analysis was performed on splenic and thymic lymphocytes to determine population changes in functional cell types or during stages of maturation, respectively. FACS analysis of splenic and thymic T lymphocytes demonstrated that the numbers of all T cell CD4/CD8
Serum PFOS and corticosterone Concentrations of PFOS in serum of mice exposed to either of Wve doses of PFOS were shown in Table 1. There was a 27
25
23
Bo dy Wei g ht (g )
Fig. 1 Body weight (mean) change trend of adult male C57BL/6 mice treated with PFOS orally for 60 days. Body weight was measured daily after treatment of animals; n = 10 in each group. TAD total administered dose over the course of 60 day
21
Control 19
0.5 mg/kg TAD 5 mg/kg TAD 25 mg/kg TAD
17
50 mg/kg TAD 125 mg/kg TAD
15
0
3
6
9
12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60
days
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Arch Toxicol (2009) 83:805–815 5.0 4.5 4.0 3.5
Food Intake (g)
Fig. 2 Change in food intake (mean) after treatment with 2% Tween 80 (control) or with 0.5, 5, 25, 50, or 125 mg TAD PFOS/ kg of body weight for 60 days. Food intake was measured daily after treatment of animals; n = 10 in each group. TAD total administered dose over the course of 60 days
3.0 2.5 2.0
Control 1.5
0.5 mg/kg TAD 5 mg/kg TAD
1.0
25 mg/kg TAD 50 mg/kg TAD
0.5
125 mg/kg TAD 0.0
0
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
51
54
57
60
days
subpopulations were signiWcantly decreased beginning at 25 mg PFOS/kg TAD (Tables 2, 3). Also, there was a signiWcantly trend toward decreased B cells with increasing PFOS exposure beginning at 25 mg PFOS/kg TAD. NK cell function, lymphocyte proliferation, and plaque forming cell assessments At the end of treatment, the eVects of PFOS on the NK activity of mice were studied by LDH release assay. The average values (mean § SEM) of NK cell activity in control animals was 31.14 § 1.93. Treatment with the dose of 5 mg PFOS/kg TAD signiWcantly increased the NK cell activity by 38% (45.43 § 4.74%). In contrast, treatment
with the dose of 50 mg/kg TAD (20.28 § 2.51%) and 125 mg/kg TAD (15.67 § 1.52%) PFOS resulted in a marked decrease in the levels of NK cell activity (Fig. 5). To determine the eVects of PFOS on lymphocyte proliferation, mitogen-induced proliferation responses (B cell and T cell) of C57BL/6 mice splenocyte was measured by MTT method. As shown in Fig. 6, the average absorbances (mean § SEM) of B lymphocytes from 50 mg/kg TAD (1.64 § 0.08) and 125 mg/kg TAD PFOS groups (1.35 § 0.10) were lower than the control (2.34 § 0.20). The eVect of PFOS on the number of PFC production per 106 spleen cells is shown in Fig. 7. There was a signiWcant trend toward decreased PFC production with increasing PFOS exposure. The suppression of this response was
Table 1 Body mass and organ mass in adult male C57BL/6 mice treated with PFOS orally for 60 days PFOS (mg/kg TAD)
n
PFOS concentration in serum (mg/l)
Control
10
0.048 § 0.014
0.5
10
0.674 § 0.166*
5
10
25
10
50
10
125
10
Body weight changea
Spleen massb
Thymus massb
Kidney massb
3.10 § 0.13
0.49 § 0.02
0.29 § 0.02
1.54 § 0.03
5.17 § 0.12
2.58 § 0.15
0.48 § 0.01
0.27 § 0.02
1.62 § 0.05
5.21 § 0.17
7.132 § 1.039*
2.81 § 0.18
0.45 § 0.01
0.25 § 0.01
1.51 § 0.06
5.78 § 0.13*
21.638 § 4.410*
1.09 § 0.16*
0.32 § 0.01*
0.18 § 0.01*
1.39 § 0.05
6.67 § 0.11*
65.426 § 11.726*
¡2.46 § 0.30*
0.30 § 0.02*
0.14 § 0.01*
1.27 § 0.06*
8.17 § 0.21*
120.670 § 21.759*
¡4.77 § 0.11*
0.23 § 0.01*
0.12 § 0.01*
1.29 § 0.04*
11.47 § 0.12*
Data are reported as mean § SEM. Body mass and organ mass data did not require transformation for statistical analysis TAD total administered dose over the course of 60 days * SigniWcantly diVerent from respective control (P · 0.05) a Body weight change denotes change in body weight from start to Wnish: [Final body weight (g) ¡ start body weight (g)] b Calculated as: [organ weight (g)/body weight (g)] £ 100
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Liver massb
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811
800
Corticosterone (ng/mL)
700
* *
600 500 400 300 200 100 0 Control
0.5 mg/kg TAD
5 mg/kg TAD
25 mg/kg TAD
50 mg/kg TAD
125 mg/kg TAD
PFOS
Fig. 3 Changes in serum corticosterone (mean § SEM) levels in adult male C57BL/6 mice following oral exposure to PFOS for 60 days. Serum corticosterone levels were measured at the end of 60 days of treatment at the time of sacriWce; n = 10 in each group. *SigniWcantly diVerent from control (P · 0.05). TAD total administered dose over the course of 60 days 1.40E+08 Spleen Thymus
Tota l Cellula rity
1.20E+08 1.00E+08 8.00E+07
* *
6.00E+07
*
* *
4.00E+07
*
2.00E+07 0.00E+00
Control
0.5 mg/kg 5 mg/kg TAD 25 mg/kg TAD TAD
50 mg/kg TAD
125 mg/kg TAD
PFOS
Fig. 4 Splenic and thymic cellularity in adult male C57BL/6 mice following an oral exposure to PFOS for 60 days. Data are presented as mean § SEM. *SigniWcantly diVerent from respective control (P · 0.05). The data were log transformed as required for statistical analysis; n = 10 in each group. TAD total administered dose over the course of 60 days
dose-responsive beginning at exposures of 5 mg PFOS/kg TAD. Therefore, we identiWed a NOAEL of 0.5 mg PFOS/kg TAD and a LOAEL of 5 mg/kg TAD.
Discussion Within the past decade, considerable attention has been paid to PFOS and related PFCs because of their presence in humans, biota, and environmental media. However, the toxicokinetics and mode(s)/mechanism(s) of action for toxicologic eVects have not yet been thoroughly described. Although immunosuppression has been identiWed as an end point of concern by the U.S. Environmental Protection Agency (2006), a paucity of data exists to corroborate the
few studies that report immune suppression after exposure to PFOS. In this study, we evaluated lymphoid organ weights and immune responses in adult C57BL/6 mice following exposure to the PFOS. Exposure to PFOS (25–125 mg/kg TAD) via gavage for 60 days statistically reduced body weight gain and food consumption, decreased spleen mass and thymus mass, and increased liver mass, which was consistent with similar observations in other studies evaluating PFOS, perXuorooctanoic acid (PFOA), and perXuorinated insecticide N-ethyl perXuorooctane sulfonamide (N-EtPFOSA) (DeWitt et al. 2008; Keil et al. 2008; Lefebvre et al. 2008; Peden-Adams et al. 2007; Yang et al. 2000, 2001, 2002; Zheng et al. 2008). In addition, 5 mg PFOS/kg TAD, corresponding to mean daily PFOS exposure values of 0.083 mg/kg body weight/day, also increased liver mass when given via gavage for 60 days in this study. This is contrast to the results of Peden-Adams et al. (2008), who reported that organ (spleen, thymus, and liver) and body mass were not altered in both male and female mice exposed to 5 mg PFOS/kg TAD over 28 days. However, increased liver/body weight (liver mass) was evident only in female rats exposed 2 mg/kg dietary PFOS, corresponding to means daily PFOS exposure values of 0.15 mg PFOS/kg body weight/day, or total mean PFOS exposure values 4.2 mg/kg body weight over 28 days (Lefebvre et al. 2008). Our data therefore suggest that in rodents, decreased immune organ mass may be a sensitive indicator of immunotoxicity at concentrations ¸0.083 mg PFOS/kg per day (5 mg PFOS/kg TAD) for 60 days, a dose that result in a serum concentration of 7.132 mg/l. This concentration is approximately 4- to 50-fold greater than the concentration of PFOS reported in sera of occupationally exposed humans (Organization for Economic Co-operation and Development 2002). In the current study, there was a nonlinear relationship between NK cell function and oral PFOS exposure in male mice. At the levels of 0.5 and 5 mg PFOS/kg TAD, PFOS did cause increased NK cell activity, however, the ability of spleen NK cell was signiWcantly decreased in a dose-dependent manner when exposed to PFOS at level of 50 and 125 mg/kg TAD. Consistent with our previous study, exposure to 20 and 40 mg PFOS/kg per day for 7 days were suYcient to produce decreased NK cell activity (Zheng et al. 2008). Also, Keil et al. (2008) studied the eVect of gestational exposure to PFOS on immune function in mice and found that NK cell activity was suppressed only in 8week-old male oVspring mice at the 1 and 5 mg/kg per day PFOS treatments for 17 days (42.5 and 32.1% decrease, respectively). However, in female mice, NK cell function was not altered following exposure to PFOS (Peden-Adams et al. 2008) and N-EtPFOSA (Peden-Adams et al. 2007). Peden-Adams et al. (2007, 2008) have shown that male
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Table 2 Splenic CD4/CD8 subpopulations in adult male C57BL/6 mice treated with PFOS orally for 60 days PFOS (mg/kg TAD)
CD4+ (cells £ 107)
DP (cells £ 105)
DN (cells £ 107)
CD8+ (cells £ 107)
B220+ (cells £ 107)
Control
2.11 § 0.12
10.17 § 0.80
5.99 § 0.28
1.20 § 0.07
5.39 § 0.35
0.5
2.30 § 0.03
9.63 § 0.30
6.20 § 0.20
1.35 § 0.03
5.45 § 0.32
5
1.89 § 0.09
8.55 § 0.43
5.58 § 0.15
1.14 § 0.05
5.67 § 0.45
25
1.54 § 0.07*
7.03 § 0.31*
4.66 § 0.14*
1.06 § 0.04
4.89 § 0.31
50
1.02 § 0.06*
5.17 § 0.28*
4.06 § 0.24*
0.79 § 0.05*
4.14 § 0.30*
125
0.56 § 0.04*
3.89 § 0.31*
3.21 § 0.25*
0.41 § 0.03*
3.62 § 0.27*
Data are reported as the mean absolute number of cells § SEM. Absolute values were determined by multiplying the percent gated cells by the total number nucleated cells counted in the spleen. DP = CD4+/CD8+, DN = CD4¡/CD8¡; n = 10 in each group TAD total administered dose over the course of 60 days * SigniWcantly diVerent from control (P · 0.05)
Table 3 Thymic CD4/CD8 subpopulations in adult male C57BL/6 mice treated with PFOS orally for 60 days CD4+ (cells £ 107)
DP (cells £ 107)
DN (cells £ 106)
CD8+ (cells £ 106)
Control
1.54 § 0.18
8.21 § 1.20
6.64 § 0.92
3.36 § 0.21
0.5
1.43 § 0.24
7.76 § 1.15
6.81 § 0.74
3.71 § 0.26
5
1.21 § 0.11
6.31 § 0.64
5.77 § 0.85
3.09 § 0.18
25
1.10 § 0.07
5.01 § 0.83*
5.12 § 0.41
2.58 § 0.23*
50
0.91 § 0.10*
4.32 § 0.41*
4.39 § 0.53
2.14 § 0.15*
125
0.67 § 0.06*
3.18 § 0.46*
3.65 § 0.37*
1.87 § 0.14*
PFOS (mg/kg TAD)
Data are reported as the mean absolute number of cells § SEM. Absolute values were determined by multiplying the percent gated cells by the total number nucleated cells counted in the thymus. DP = CD4+/CD8+, DN = CD4¡/CD8¡; n = 10 in each group TAD total administered dose over the course of 60 days * SigniWcantly diVerent from control (P · 0.05)
60
*
50
40
NK Activity (% )
Fig. 5 Splenic NK cell activity was determined by LDH release assay in adult male C57BL/6 mice following oral exposure to PFOS for 60 days. When signiWcant diVerences were detected by the F test (P < 0.05), Dunnett’s t test was used to compare treatment groups to the control group. Data are presented as mean § SEM. *SigniWcantly diVerent from control (P · 0.05; n = 10 in each group). TAD total administered dose over the course of 60 days
30
* *
20
10
0 Cont rol
0.5 mg/kg TAD
5 mg/kg TAD
25 mg/kg TAD
PFOS
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50 mg/kg TAD 125 mg/kg TAD
Arch Toxicol (2009) 83:805–815
813
3.0 ConA LPS
P roliferation Index
2.5 2.0 *
* *
1.5 1.0 0.5 0.0 Control
0.5 mg/kg TAD
5 mg/kg TAD
25 mg/kg TAD
50 mg/kg TAD
125 mg/kg TAD
PFOS
Fig. 6 Splenic lymphocyte proliferation was measured using the MTT method in adult male C57BL/6 mice following oral exposure to PFOS for 60 days. The splenic were isolated from each group of mice. The cells (5 £ 106/well) were cultured without or with 10 g/ml ConA or LPS for 48 h, and the cell proliferation was measured by MTT assay. A value of 1 for the proliferative index represents cell proliferation obtained in ConA-nontreated or LPS-nontreated cells. Unstimulated counts were not signiWcantly diVerent between groups, therefore, data are represented as the proliferation index. Data are presented as mean § SEM. *SigniWcantly diVerent from control (P · 0.05; n = 10 in each group). TAD total administered dose over the course of 60 days
700
PFC/1 0 6 spleen cells
600 500
*
400 *
300
*
200
*
100 0 Control
0.5 mg/kg TAD
5 mg/kg TAD
25 mg/kg TAD
50 mg/kg TAD
125 mg/kg TAD
PFOS
Fig. 7 Sheep red blood cell (SRBC)-speciWc IgM plaque forming cell (PFC) response was measured using the Cunningham modiWcation of the Jerne plaque assay in adult male C57BL/6 mice following oral exposure to PFOS for 60 days. Data are presented as mean § SEM. *SigniWcantly diVerent from control (P · 0.05). PFC data were log transformed for statistical analysis; n = 10 in each group. TAD total administered dose over the course of 60 days
mice appear to be more sensitive to the eVects of PFOS than female mice. Suppression eVects of PFOS (50 and 125 mg/kg TAD) were observed on the proliferation of mice splenic leukocytes in response to the T cell mitogen ConA and B cell mitogen LPS, suggestive of the involvement of PFOS in proliferation-suppressing activities and subsequent modula-
tion of T cells and B cells. Yang et al. (2002) found that a dietary concentration of 0.02% PFOA for 10 days (reported as 20 mg/kg per day) could decrease T cell and B cell mitogen-induced lymphoproliferation. Contrary to these studies, Peden-Adams et al. (2008) have reported that PFOS did not suppress splenic lymphocytes proliferation, but showed an increasing trend in T cell and B cell proliferation in the 28day exposure although this was not statistically signiWcant. Also, there were no signiWcant eVects of PFOS on splenocyte proliferation in rats for either the mitogens (Lefebvre et al. 2008). These discrepancy may be more likely related to the dose administered (50 and 125 mg/kg TAD vs. 5 mg/ kg TAD), and the immune responses of mice were also more sensitive to PFOS than rats. A review of the expression of peroxisome proliferator activated receptor (PPAR) studies indicates that the inhibition of PFOA occurred via a PPAR (Abbott 2008). PPAR is the primary PPAR isoform expressed in lymhocytes and is more highly expressed in B cells than in T cells (Cunard et al. 2002). PFOS is a known agonist for PPAR (Shipley et al. 2004). Several studies have suggested that PPAR agonists including PFOS, PFOA, and WY14,643 have profound eVects on murine immune responses including decreased spleen weight and splenocyte number and suppression of antibody response (Cunard et al. 2002; DeWitt et al. 2008; Keil et al. 2008; Lefebvre et al. 2008; Yang et al. 2000, 2002; Zheng et al. 2008). However, Relative to PFOA, PFOS was shown to be a less potent PPAR activator (Abbott 2008). Moreover, as PPAR is diVerentially expressed in species and eVects mediated by PPAR vary by species, knowledge of the role of PPAR in the PFOS-induced proliferation-suppressing is important to understanding human and wildlife health risks due to this compound (Peden-Adams et al. 2008). Also, since all mice that were included in this study received immunization with SRBCs in addition to PFOS, it should be taken into account that the immunization diVerentially contributes to the phenotype seen in the Wgures above. The data from our study indicated that reductions in PFC responses to SRBC, a measure of T-dependent IgM responses, occurred at lower doses and PFOS serum concentrations than those required to reduce spleen and thymus organ mass. In this study, spleen and thymus mass reductions occurred at doses of ¸25 mg PFOS/kg TAD, corresponding to mean PFOS serum concentration of 21.638 mg/l. PFC production was reduced at a dose of 5 mg/kg TAD for 60 days (0.083 mg/kg body weight/day PFOS) and a PFOS serum concentration of 7.132 mg/l which we identiWed as a LOAEL. The NOAEL for PFC response in mice exposed to gavage PFOS for 60 days was 0.5 mg/kg TAD (0.0083 mg/kg body weight/day). PedenAdams et al. (2008) reported that, in male B6C3F1 mice, the PFC production decreased at serum PFOS levels
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814
91.5 g/l (1.66 g PFOS/kg body weight/day for 28 days = 0.05 mg PFOS/kg TAD) that were 14-fold lower than the average blood concentrations of occupationally exposed humans and in the upper range of concentrations reported for the general population, making this the lowest reported LOAEL for PFOS which approximately 100-fold lower than the PFOS exposure levels in present study (0.05 mg/kg TAD over 28 days vs. 5 mg/kg TAD over 60 days). Furthermore, another published data assessing PFC response for SRBC-speciWc IgM production indicated that LOAEL in the male pups of mice gestational exposed to PFOS for 17 days was 5 mg/kg per day or total mean PFOS exposure values of 85 mg/kg body weight over 17 days (Keil et al. 2008). These discrepancies may be more likely related to the term of PFOS exposure. The mechanism disrupting antibody secretion is not known. Yang et al. (2001) demonstrated a 75% decrease in splenic CD19+ B cells following exposure to PFOA. In this study, numbers of B220+ B cells were not signiWcantly decreased following exposure to PFOS at doses of 5 and 25 mg/kg TAD, suggesting the deWcit in SRBC-speciWc IgM production was not only due to a decreased total number of splenic B cells. As one manifestation of severe environmental or physiologic stress, increased serum corticosterone concentration is known to suppress immunoglobulin production and to impair immune function. In this study, PFOS exposure (50 or 125 mg PFOS/kg TAD) signiWcantly increased the corticosterone concentration in serum, indicating that PFOS has a stimulatory eVect on the stress axis. So, these eVects of PFOS on immune function may be partly and probably mediated through hypothalamic–pituitary–adrenal axis. However, this study also showed, even in the group treatment with the dose of 5 or 25 mg PFOS/kg TAD, neither the food intake, body weight, and numbers of B220+ B-cells nor the serum corticosterone concentration were marked diVerence comparing with the control group, but the PFC response were still signiWcantly suppressed comparison with to normal C57BL/6 mice, which conWrms PFC as a relevant and sensitive immune parameter. In summary, the LOAEL for signiWcant changes in immune parameters in mice exposed to gavage PFOS for 60 days was 5 mg PFOS/kg TAD gavage for reduced PFC production in male mice, and this eVect was not based on alterations observed in the CD4/CD8 lymphocytic subpopulation data. As the Xow cytometric data demonstrates splenic and thymic CD4+/CD8¡ and CD4¡/CD8+ subpopulations were signiWcantly altered in male mice beginning at the PFOS exposure levels of 25 mg/kg TAD. The LOAEL for signiWcantly increased liver mass (percent liver/body weight values) was 5 mg PFOS/kg TAD gavage in mice. Taken together, the results suggest that the eVects of PFOS on immune responses in the mice may, in part, be secondary to hepatic changes. A summary of human biomonitor-
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ing studies indicates that PFOS residues in adult blood or serum are generally present at levels lower than 150 g/l (Lau et al. 2007), considerably lower than the micrograms per gram levels achieved in the serum of PFOS-treated mice. For selected endpoints, PFOS was immunosuppressive to mice at serum PFOS levels within range of those measured in humans (Peden-Adams et al. 2008). The mechanistic basis for the eVects of PFOS in rodent models remains to be determined, and may provide insight into the extent to which human immune responses are inXuenced by PFOS exposure. Acknowledgments This research was supported by Grants from the National Natural Science Foundation of China (20707041 and 20837004) and Grants from the Research Found for the Doctor Program of Higher Education (20070159015).
References 3M Company (2002) 104-Week dietary chronic toxicity and carcinogenicity study with perXuorooctane sulfonic acid potassium salt (PFOS; T-6295) in rats. Final Report 3M T-6295 (Covance study no. 6329-183), vols I–IX. 3M, St Paul, 4068 p Abbott BD (2008) Review of the expression of peroxisome proliferators activated receptors alpha (PPARalpha), beta (PPARbeta), and gamma (PPARgamma) in rodent and human development. Reprod Toxicol. doi:10.1016/j.reprotox.2008.10.001 Alexander BH, Olsen GW, Burris JM, Mandel JH, Mandel JS (2003) Mortality of employees of a perXuorooctanesulphonyl Xuoride manufacturing facility. Occup Environ Med 60:722–729 Ankley GT, Kuehl DW, Kahl MD, Jensen KM, Linnum A, Leino RL, Villeneuvet DA (2005) Reproductive and developmental toxicity and bioconcentration of perXuorooctanesulfonate in a partial lifecycle test with the fathead minnow (Pimephales promelas). Environ Toxicol Chem 24:2316–2324 ButenhoV JL, Olsen GW, Pfahles-Hutchens A (2006) The applicability of biomonitoring data for perXuorooctanesulfonate to the environmental public health continuum. Environ Health Perspect 114:1776–1782 Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Tully JS, Needham LL (2007) Serum concentrations of 11 polyXuoroalkyl compounds in the U.S. population: data from the national health and nutrition examination survey (NHANES). Environ Sci Technol 41:2237–2242 Chang SC, Thibodeaux JR, Eastvold ML, Ehresman DJ, Bjork JA, Froehlich JW, Lau C, Singh RJ, Wallace KB, ButenhoV JL (2008) Thyroid hormone status and pituitary function in adult rats given oral doses of perXuorooctanesulfonate (PFOS). Toxicology 243:330–339 Cunard R, DiCampli D, Archer DC, Stevenson JL, Ricote M, Galss CK, Kelly CJ (2002) WY14,643, an PPAR alpha ligand, has profound eVects on immune responses in vivo. J Immunol 169:6806–6812 de Vos MG, Huijbregts MA, van den Heuvel-Greve MJ, Vethaak AD, Van de Vijver KI, Leonards PE, van Leeuwen SP, de Vooqt P, Hendriks AJ (2008) Accumulation of perXuorooctane sulfonate (PFOS) in the food chain of the Western Scheldt estuary: comparing Weld measurements with kinetic modeling. Chemosphere 70:1766–1773 DeWitt JC, Copeland CB, Strynar MJ, Luebke RW (2008) PerXuorooctanoic acid-induced immunomodulation in adult C57BL/6J or C57BL/6N female mice. Environ Health Perspect 116:644–650
Arch Toxicol (2009) 83:805–815 Francis J, MohanKumar SMJ, MohanKumar PS (2000) Correlations of norepinephrine release in the paraventricular nucleus with plasma corticosterone and leptin after systemic lipopolysaccharide: blockade by soluble IL-1 receptor. Brain Res 867:180–187 Giesy JP, Kannan K (2001) Global distribution of perXuorooctane sulfonate in wildlife. Environ Sci Technol 35:1339–1342 Hansen KJ, Clemen LA, Ellefson ME, Johnson HO (2001) CompoundspeciWc, quantitative characterization of organic Xuorochemicals in biological matrices. Environ Sci Technol 35:766–770 Hart K, Kannan K, Isobe T, Takahashi S, Yamada TK, Miyazaki N, Tanabe S (2008) Time trends and transplacental transfer of perXuorinated compounds in melon-headed whales stranded along the Japanese coast in 1982, 2001/2002, and 2006. Environ Sci Technol 42:7132–7137 Houde M, Bujas TA, Small J, Wells RS, Fair PA, Bossart GD, Solomon KR, Muir DC (2006) BiomagniWcation of perXuoroalkyl compounds in the bottlenose dolphin (Tursiops truncatus) food web. Environ Sci Technol 40:4138–4144 Jerne NK, Nordin AA (1963) Plaque formation in agar by single antibody-producing cells. Science 140:405 Jin Y, Saito N, Harada KH, Inoue K, Koizumi A (2007) Historical trends in human serum levels of perXuorooctanoate and perXuorooctane sulfonate in Shenyang, China. Tohoku J Exp Med 212:63–70 Keil DE, Mehlmann T, Butterworth L, Peden-Adams MM (2008) Gestational exposure to perXuorooctane sulfonate suppresses immune function in B6C3F1 mice. Toxicol Sci 103:77–85 Lau C, Anitole K, Hodes C, Lai D, Pfahles-Hutchens A, Seed J (2007) PerXuoroalkyl acids: a review of monitoring and toxicological Wndings. Toxicol Sci 99:366–394 Lefebvre DE, Curran I, Armstrong C, Coady L, Parenteau M, Liston V, Barker M, Aziz S, Rutherford K, Bellon-Gagnon P, Shenton J, Mehta R, Bondy G (2008) Immunomodulatory eVects of dietary potassium eprXuorooctane sulfonate (PFOS) exposure in adult Sprague-Dawley Rats. J Toxicol Environ Health A 71:1516–1525 Martin MT, Brennan RJ, Hu W, Avanoqlu E, Lau C, Ren H, Wood CR, Corton JC, Kavlock RJ, Dix DJ (2007) Toxicogenomic study of triazole fungicides and perXuoroalkyl acids in rat livers predicts toxicity and categorizes chemicals based on mechanisms of toxicity. Toxicol Sci 97:595–613 Monroy R, Morrison K, Teo K, Atkinson S, Kubwabo C, Stewart B, Foster WG (2008) Serum levels of perXuoroalkyl compounds in human maternal and umbilical cord blood samples. Environ Res 108:56–62 Olsen GW, Church TR, Miller JP, Burris JM, Hansen KJ, Lundberg JK, Armitage JB, Herron RM, Medhdizadehkashi Z, Nobiletti JB, O’Neill EM, Mandel JH, Zobel LR (2003) PerXuorooctanesulfonate and other Xuorochemicals in the serum of American Red Cross adult blood donors. Environ Health Perspect 111:1892–1901
815 Organization for Economic Co-operation and Development (2002) Hazard assessment of perXuorooctane sulfonate (PFOS) and its salts. Available at: http://www.oecd.org/dataoecd/23/18/2382880.pdf Peden-Adams MM, EuDaly JG, Dabra S, EuDaly A, Heesemann L, Smythe J, Keil D (2007) Suppression of humoral immunity following exposure to the perXuorinated inseticide sulXuramid. J Toxicol Environ Health A 70:1130–1141 Peden-Adams MM, Keller JM, EuDaly JG, Berger J, Gilkeson GS, Keil DE (2008) Suppression of humoral immunity in mice following exposure to perXuorooctane sulfonate. Toxicol Sci 104:144–154 Shipley JM, Hurst CH, Tanaka SS, DeRoos FL, ButenhoV JL, Seacat AM, Waxman DJ (2004) Trans-activation of PPARalpha and induction of PPAR alpha target genes by perXuorooctane-based chemicals. Toxicol Sci 80:151–160 Tittlemier SA, Pepper K, Seymour C, Moisey J, Bronson R, Cao XL, Dabeka RW (2007) Dietary exposure of Canadians to perXuorinated carboxylates and perXuorooctane sulfonate via consumption of meat, Wsh, fast foods, and food items prepared in their packaging. J Agric Food Chem 55:3203–3210 U.S. EPA (U.S. Environmental Protection Agency) (2006) SAB review of EPA’s draft risk assessment of potential human health eVects associated with PFOA and its salts. Available at: http:// yosemite.epa.gov/sab/sabpeople.nsf/WebCommittees/BOARD Vestergren R, Cousins IT, Trudel D, Wormuth M, Scheringer M (2008) Estimating the contribution of precursor compounds in consumer exposure to PFOS and PFOA. Chemosphere 73:1617– 1624 Wu R, Coleman N, Stanley M (1996) DiVerent susceptibility of cervical keratinocytes containing human papillomavirus to cellmediated cytotoxicity. Chin Med J 109:854–858 Yang Q, Xie Y, DePierre JW (2000) EVects of peroxisome proliferators on the thymus and spleen of mice. Clin Exp Immunol 122:219–226 Yang Q, Xie Y, Eriksson AM, Nelson BD, DePierre JW (2001) Further evidence for the involvement of inhibition of cell proliferation and development in thymic and splenic atrophy induced by the peroxisome proliferator perXuorooctanoic acid in mice. Biochem Pharmacol 62:1133–1140 Yang Q, Abedi-Valugerdi M, Xie Y, Zhao X-Y, Möller G, Nelson BD, Depierre JW (2002) Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perXuorooctanoic acid. Int Immunopharmacol 2:389–397 Zheng L, Dong GH, Jin YH, He QC (2008) Immunotoxic changes associated with a 7-day oral exposure to perXuorooctanesulfonate (PFOS) in adult male C57BL/6 mice. Arch Toxicol. doi:10.1007/ s00204-008-0361-3
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