Available online at www.sciencedirect.com

Toxicology 243 (2008) 330–339

Thyroid hormone status and pituitary function in adult rats given oral doses of perfluorooctanesulfonate (PFOS)ଝ Shu-Ching Chang a, Julie R. Thibodeaux b,1, Mary L. Eastvold c, David J. Ehresman a, James A. Bjork d, John W. Froehlich e,2, Christopher Lau b, Ravinder J. Singh c, Kendall B. Wallace d, John L. Butenhoff a,∗ a Medical Department, 3M Company, St. Paul, MN 55144, United States b United States Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle Park, NC 27711, United States c Mayo Clinic and Foundation, Department of Laboratory Medicine and Pathology, Rochester, MN 55095, United States d University of Minnesota, Medical School, Department of Biochemistry and Molecular Biology, Duluth, MN 55812, United States e Pace Analytical Services, Inc., Minneapolis, MN 55414, United States Received 29 August 2007; received in revised form 18 October 2007; accepted 20 October 2007 Available online 26 October 2007

Abstract Introduction: Perfluorooctanesulfonate (PFOS) is widely distributed and persistent in humans and wildlife. Prior toxicological studies have reported decreased total and free hormones in serum without a major compensatory rise in thyrotropin (TSH) or altered thyroid gland histology. Although these animals (rats, mice and monkeys) might have maintained an euthyroid state, the basis for hypothyroxinemia remained unclear. We undertook this study to investigate the causes for the PFOS-induced reduction of serum total thyroxine (TT4) in rats. Hypotheses: We hypothesized that exposure to PFOS may increase free thyroxine (FT4) in the rat serum due to the ability of PFOS to compete with thyroxine for binding proteins. The increase in FT4 would increase the availability of the thyroid hormone to peripheral tissues for utilization, metabolic conversation, and excretion. We also hypothesized that PFOS does not directly interfere with the regulatory functions of the hypothalamic–pituitary–thyroid (HPT) axis in rats. Experiments: Three experimental designs were employed to test these hypotheses. (1) Female Sprague–Dawley (SD) rats were given a single oral dose of 15 mg potassium PFOS/kg body weight. At intervals of 2, 6, and 24 h thereafter, measurements were made for serum FT4, TT4, (TT3), reverse triiodothyronine (rT3), thryrotropin (TSH), and PFOS concentrations, as well as liver PFOS concentrations, UDP-glucuronosyltransferase 1A (UGT1A) family mRNA transcripts, and malic enzyme (ME) mRNA transcripts and activity. (2) To provide evidence for increased uptake and metabolism of thyroxine (T4), 125I-T4

ଝ Notice: The information in this document has been funded by 3M Company and the US Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. ∗ Corresponding author at: 3M Company, Medical Department, 3M Center 220-06-W-08, St. Paul, MN 55144, United States. Tel.: +1 651 733 1962; fax: +1 651 733 1773. E-mail addresses: [email protected] (S. Chang), [email protected] (J.R. Thibodeaux), [email protected] (M.L. Eastvold), [email protected] (D.J. Ehresman), [email protected] (J.A. Bjork), [email protected] (J.W. Froehlich), [email protected] (C. Lau), [email protected] (R.J. Singh), [email protected] (K.B. Wallace), [email protected] (J.L. Butenhoff). 1 Present address: University of North Carolina, School of Medicine, Chapel Hill, NC 27599, United States. 2 Present address: University of California, Department of Chemistry, Davis, CA 95616, United States.

0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2007.10.014 S. Chang et al. / Toxicology 243 (2008) 330–339 331 was given to male and female SD rats by intravenous injection, followed in 2 h by a single oral dose of 15 mg potassium PFOS/kg body weight. 125I radioactivity was determined in urine and feces collected over a 24-h period and in serum and liver collected at 24 h. (3) To assess the potentials effect of PFOS on the hypothalamic–pituitary–thyroid axis, over an 8-day period, groups of male SD rats were given PFOS (3 mg/kg-d), propyl thiouracil (PTU, 10 ␮g/mL in water), or PTU and PFOS in combination, with controls receiving 0.5% Tween® 20 vehicle. On days 1, 3, 7, and 8, TT4, TT3, and TSH were monitored. On day 8, pituitaries were removed and placed in static culture for assessment of thyrotropin releasing hormone (TRH)-mediated release of TSH. Results: (1) PFOS transiently increased FT4 and decreased TSH within 6 h, with values returning to control levels by 24 h. TT4 was decreased by 55% over a 24-h period. TT3 and rT3 were decreased at 24 h to a lesser extent than TT4. ME mRNA transcripts were increased at 2 h and activity was increased at 24 h. UGT1A mRNA transcripts were increased at 2 and 6 h. (2) 125I decreased in serum and liver relative to controls and consistent with a reduction in serum TT4. Concomitantly, 125I activity was increased in urine and feces collected from PFOS-treated rats. (3) During the 8 days of dosing with PFOS, TSH was not elevated in male rats, while TT4 and TT3 were decreased. Pituitary response to TRH-mediated TSH release was not diminished after 8-daily oral doses of PFOS. Conclusions: These findings suggest that oral dosing in rats with PFOS results in transiently increased tissue availability of the and turnover of T4 with a resulting reduction in serum TT4. PFOS does not induce a classical hypothyroid state under dosing conditions employed nor does it alter HPT activities. © 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Perfluorooctanesulfonate (PFOS); Thyroid hormones; Liver; 125I

1. Introduction The discrepancy seen is due to the fact that ana- log systems use labeled thyroxine (T4) analog to bind − Perfluorooctanesulfonate (PFOS, C8F17SO3 ) has with assay antibody in inverse proportion to the amount been found to be widely distributed in humans and of competing FT4; and the amount of assay antibody- wildlife (Butenhoff et al., 2006; Giesy and Kannan, bound labeled T4 analog is measured. However, a certain 2001; Hansen et al., 2001; Harada et al., 2004; Houde et amount of labeled T4 analog may be bound to endoge- al., 2006; Martin et al., 2004; Olsen et al., 2003, 2004). nous and assay serum carrier proteins with varying PFOS is resistant to environmental and metabolic degra- degrees of affinity, and the assay calibration takes this dation, and it appears to accumulate in the food chain into account. When a substance that effectively reduces (Johnson et al., 1984; Olsen et al., 2005; Seacat et al., serum carrier protein binding sites through competi- 2002). This finding has led to interests in environmental tive binding is introduced, such as PFOS or free fatty and health properties of PFOS at an international level acids, less labeled T4 analog is bound than is normally (OECD, 2002). accounted for in the assay calibration, forcing more Prior toxicological studies with PFOS have reported labeled T4 analog toward assay antibody binding sites, significant hypothyroxinemia that was characterized by resulting in an underreporting of FT4. The bias can be decreases in total and free thyroid hormones in serum minimized with a reference method, ED-RIA. ED-RIA without a major compensatory rise in thyroid stimulat- uses buffers that do not contain serum proteins so that ing hormone (TSH) (Lau et al., 2003; Luebker et al., binding interferences are greatly reduced. 2005; Seacat et al., 2002; Thibodeaux et al., 2003). As reported in our recent study (Chang et al., 2007), These observations did not fit the usual diagnosis of measurements of FT4 in serum containing PFOS by hypothyroidism and were more similar to the profile analog methods indeed are prone to negative bias due of non-thyroidal illness syndrome, or NTIS (Chopra, to displacement of bound T4 from carrier proteins in 1997; Larsen et al., 2003; Ravel, 1995). In these studies, serum by PFOS. When PFOS was added to rat sera in all of the thyroid hormones measured were performed vitro at concentrations ranging up to 200 ␮M, FT4 was using analog methods (with clinical laboratory auto- shown to be increased up to 260% over paired control analyzers); however, Seacat et al. (2002) and Luebker using ED-RIA but only 30% using a standard analog et al. (2005) also demonstrated that free thyroxine (FT4) method. Furthermore, total thyroxine (TT4) remained in PFOS-containing serum was unchanged compared to unchanged. controls when a reference method (equilibrium dialy- A negative bias in the analog method for measurement sis followed by radioimmunoassay, ED-RIA) for FT4 of serum FT4 after PFOS exposures was also demon- measurement was used. strated in vivo. Twenty-four hours following the last of 332 S. Chang et al. / Toxicology 243 (2008) 330–339 three consecutive daily oral doses of either vehicle or 2.2. Animals and husbandry 5 mg potassium PFOS/kg body weight to adult female Sprague–Dawley (SD) rats (Chang et al., 2007), we Male and female SD rats (8–10 weeks old, 200–250 g) observed that, compared to controls: (1) FT4 (by ED- were purchased from Charles River Laboratory (Portage, MI RIA) and TSH in serum were not altered by PFOS; or Raleigh, NC). All rats were group housed in standard solid (2) serum TT4 was decreased by almost 50%; and bottom cages. Purina Mouse/Rat Chow and tap water were pro- vided to all rats ad libitum throughout the study. Environmental (3) mRNA transcripts for hepatic malic enzyme (ME), controls for the animal room were set to maintain a temperature which responds to changes in thyroid hormones (Bogazzi of 72 ± 3 ◦F, humidity of 30–70%, a minimum of 10 exchanges et al., 1997; Hertz et al., 1991; Oppenheimer et al., 1977), of room air per hour and a 12 h light/dark cycle. Studies were were increased by approximately 20% (p < 0.05). How- performed in laboratories accredited by International Asso- ever, hepatic ME activity was unchanged. Based on these ciation for the Accreditation of Laboratory Animal Care. All preliminary data, the lack of change in FT4 (by ED-RIA), procedures involving rats were reviewed and approved by Insti- TSH, and ME activity suggested the PFOS-treated rats tutional Animal Care and Use Committees. Animals care and were in a euthyroid state. However, it remained unclear procedures followed the US Department of Health and Human as to why there was a reduction of serum TT4. We con- Services Guide for the Care and the Use of Laboratory Ani- sidered the plausibility that putative increased FT4 in mals Guidelines (Institute of Laboratory Animal Resources, serum by PFOS displacement might result in increased 1996). FT4 uptake, utilization, and metabolism by peripheral tissues, which in turn led to a net loss of TT4 (Menjo et 2.3. Study 1: effects of time and PFOS on serum thyroid al., 1999). hormones in vivo The goal of this study was to further investigate the short-term thyroid hormone status in rats treated To evaluate the acute effects of PFOS on serum thy- with PFOS. We hypothesized that: (1) PFOS com- roid hormone concentrations in vivo within 24 h of treatment, petes (directly or indirectly) with T4 for serum carrier groups of female SD rats (n = 5–15 per group) were given either a single dose of vehicle (0.5% Tween 20® in dis- protein binding sites, and the thyroid hormones dis- tilled water, three groups) or 15 mg potassium PFOS/kg body placement results in a transient elevation of FT4 after weight suspended in vehicle (three groups) at a dose volume an oral dose of PFOS; (2) this transient elevation of 1.0 mL/kg. The 15 mg/kg potassium PFOS dose was chosen in FT4 leads to increased turnover and elimination to achieve a target concentration of PFOS in serum of approxi- of T4; and (3) PFOS would not have an effect on mately 50–75 ␮g/mL, similar to the serum PFOS concentration the ability of the pituitary to release TSH or to reached after three daily 5 mg/kg-d doses in our previous study respond to hypothalamic thyrotropin releasing hormone (Chang et al., 2007). (TRH). Control and PFOS-treated rats were sacrificed by CO2 To test these hypotheses, serum thyroid hormones asphyxiation at 2, 6, and 24 h post-dosing with blood sam- (TT4, FT4, TSH, total triiodothyronine (TT3), and ples taken via abdominal aorta. After clotting, serum samples × reverse triiodothyronine (rT3)) were measured after were separated after centrifugation (2000 g for 15 min) and were flash-frozen using liquid nitrogen, pending analysis for PFOS exposures. Changes of these measurements PFOS concentration and thyroid hormones. Serum PFOS con- were related to biochemical markers ME and UDP- centrations were analyzed by LC/MS–MS in the 3M Medical glucuronosyltransferase 1A (UGT1A) in the liver. Department Bioanalytical Laboratory (St. Paul, MN, USA) as 125 Furthermore, urinary and fecal excretion of I from described in Chang et al. (2007). For this study, serum thy- 125I-labeled T4 after a single oral dose of PFOS in rats roid hormones were determined by Bayer ADVIA Centaur® and pituitary function with respect to TSH release in chemiluminometric analog assays (for TT4, TT3, and rT3 mea- response to TRH were investigated. surements) and by ED-RIA method (for FT4 measurements using Nichols Institute Diagnostics Free T4 by Equilibrium 2. Materials and methods Dialysis kits (Nichols Institute Diagnostics, San Clemente, CA)) (Mayo Medical Laboratories, Rochester, MN, USA). 2.1. Materials Serum TSH was measured at 6 and 24 h via rat TSH radioim- munoassay (National Hormone and Pituitary Program – ULCA All chemicals used in this study were reagent-grade and Harbor Medical Center, Torrance, CA, USA). In addition, were purchased from Sigma–Aldrich (St. Louis, MO), VWR liver tissues were collected at necropsy and flash-frozen in (West Chester, PA), Bachem (King of Prussia, PA), or Perkin- liquid nitrogen pending analysis of PFOS concentration (via Elmer (Boston, MA). Perfluorooctanesulfonate potassium salt LC/MS–MS) and assessments of hepatic biochemical markers (potassium PFOS, 86.9% pure) was supplied by 3M Specialty (ME and UGT1A mRNA transcript levels and ME activities) Material Division (St. Paul, MN). by methods described in Chang et al. (2007). S. Chang et al. / Toxicology 243 (2008) 330–339 333

2.4. Study 2: effects of PFOS on 125I elimination in vivo cut into blocks of ∼1mm3 and placed in 0.6 mL of Medium 199 (Medium 199 supplemented with 0.2% BSA, 13 mM Because our previous study observed that treatment with sodium bicarbonate, 10 mM HEPES, and 50 ␮M bacitracin, PFOS caused a reduction of serum TT4 in vivo but not in pH 7.4) in a 24-well flat bottom plate. The plate was kept on vitro, the objective of this study was to investigate if the ice until all pituitaries were in place. The plate was incubated PFOS-mediated reduction of serum TT4 in vivo might be in a shaking water bath at 37 ◦C for 30 min to allow for the the result of increased turnover of TT4. Male and female initial traumatic release of TSH. The medium was collected SD rats were injected with either 11 ␮Ci (male rats, n =4 by carefully aspirating with a pipette, and the pituitary tissues per group) or 9.3 ␮Ci (female rats, n = 5 per group) of 125I- were transferred to a new plate containing 0.6 mL of Medium T4 (specific activity = 1250 ␮Ci/␮g) followed by a subsequent 199 in each well. The plate was incubated at 37 ◦C, and the single oral dose of either vehicle (control) or 15 mg potas- medium was changed out every hour and frozen for future TSH sium PFOS/kg body weight. To determine the elimination of analysis. At 4 and 7 h, the pituitaries were incubated for 1 h in 125I, urine and feces were collected over a 24-h period after Medium 199 containing 50 nM of TRH. The addition of TRH PFOS administration. Serum and liver were harvested at the was to stimulate the secretion of TSH (Askew and Ramsden, end of 24 h. Serum, liver, urine, and feces were measured for 1984). At 11 h of incubation, the pituitaries were incubated for 125I radioactivities with a gamma counter (Perkin-Elmer® 1470 15 min in Medium 199 containing 60 mM KCl to release all Wizard Automatic Gamma Counter, Waltham, MA, USA). TSH. After 12 h of incubation, the tissue was removed from Serum TT4 concentration was analyzed by the Bayer ADVIA the plate and sonicated and frozen in 0.6 mL of Medium 199 Centaur® chemiluminometric method (Mayo Medical Labora- for determination of post-incubation hormone concentrations. tories, Rochester, MN, USA). The collected media were stored frozen at −20 ◦C pending TSH analysis via rat-specific RIA as described.

2.5. Study 3: pituitary function studies in vivo/ex vivo 2.6. Statistics (8-day repeat dose) The Student’s t-test was used to compare data resulting The synthesis and the release of thyroid hormones from from PFOS treatment to control values using either Microsoft thyroid gland is primarily stimulated by TSH, which in turn, is Office Excel or JMP® 5.1 – Windows – Release 5.1.2 (SAS secreted by pituitary. Pituitary release of TSH is controlled by Institute, Inc., Cary, NC, USA). All data are expressed as TRH, which is released from . Propylthiouracil means ± standard errors. (PTU) was used to inhibit thyroid hormone synthesis (in thy- roid) which leads to increased pituitary secretion of TSH 3. Results (Goldman and Cooper, 1993). The goal of this study is to evalu- ate the effects of PFOS only, PTU only, and combined treatment 3.1. Study 1: effects of time and PFOS on serum of PFOS and PTU, on TRH-mediated pituitary release of TSH. thyroid hormones in vivo The rationale for combined treatment of PFOS and PTU was to further ascertain whether the presence of PFOS could affect Effects of time and PFOS on serum thyroid hormone the release of TSH while PTU was applied during treatment measurements are shown in Table 1. Consistent with with PFOS. what we observed previously (Chang et al., 2007), serum Adult male SD rats (four groups of six rats) were given TT4 decreased significantly within 24 h with PFOS treat- either (1) vehicle, 0.5% Tween® 20 (oral gavage, 1 mL/kg); (2) 3 mg potassium PFOS/kg body weight per day suspended in ments. The decrease in serum TT4 was statistically 0.5% Tween 20® (oral gavage); (3) 10 ␮g/mL (10 ppm) PTU in significant at all time points. drinking water; or (4) 10 ppm PTU+3mgpotassium PFOS/kg Compared to control, there was no change in TT3 body weight per day (drinking water and oral gavage) for 7 and rT3 at 2 and 6 h. However, both TT3 and rT3 were consecutive days. reduced at 24 h. FT4 was increased significantly at 2 Both terminal serum samples (obtained from decapitation and 6 h (68 and 90%, respectively, over control) and it on day 8, 24 h after the last treatment) and interim serum returned to a level comparable to the control by 24 h. samples obtained by tail-bleeding on days 1, 3, and 7 were The transient increase in rat serum FT4 observed at 2 ® analyzed for TT4 and TT3 by Coat-a-Count RIA kits (Diag- and 6 h after PFOS treatment, as described above, was nostic Products Corporation, Los Angeles, CA, USA) and associated with a transient decrease of serum TSH at 6 h TSH by rat radioimmunoassay (Rat TSH Radioimmunoassay which returned to control levels by 24 h. OP No. NHEERL-H/RTD/EB/CL/2002-010-000, EPA, RTP, NC). The pituitary was removed on day 8 and evaluated Serum and liver PFOS concentrations are also listed for TSH release ex vivo in static tissue culture per method in Table 1. After PFOS administration, serum PFOS con- described by Goldman and Cooper (1993). Specifically, the centrations appeared to plateau at 6 h; this is similar to a anterior pituitary was hemisected along the midline and both previous study by Johnson et al. (1979) which reported halves were weighed. A hemisected fragment of pituitary was that in rats, Tmax equals to 6–8 h after receiving PFOS. 334 S. Chang et al. / Toxicology 243 (2008) 330–339 c * UGT1A transcript 36.22 b * ME activity 2.27 54.46 a ) 6 nd activities from adult female rats given * − 10 7.14 2.19 39.37 8.76 2.35 86.31 × ME transcript ( 11.30 11.94 2.97 g * * * g/g) ␮

Compared to controls, there were statistically sig- nificant increases in ME transcripts 2 h after PFOS treatment. In addition, ME activity was increased by 4, 12, and 43% over paired control at 2, 6, and 24 h, respec- tively, the increase becoming statistically significant at 24 h. Liver UGT1A mRNA transcripts from PFOS- treated rats were elevated over control values at 2 and 6 h post-treatment, and returned to control levels by 24 h.

3.2. Study 2: effects of PFOS on 125I elimination in vivo

Serum TT4 measurements, and 125I radioactivities for serum, liver, urine, and feces, are shown in Fig. 1 as the Fig. 2. Mean serum total thyroxine (TT4) concentrations in adult ® percent of control values. Compared to controls, serum male rats treated with (1) vehicle, 0.5% Tween 20 (oral gavage), (2) 3 mg/kg-d PFOS suspended in vehicle (oral gavage), (3) 10 ␮g/mL TT4 concentrations from PFOS-treated rats (both males (10 ppm) PTU in drinking water or (4) 10 ppm PTU + 3 mg/kg-d PFOS and females) were reduced by 55% at the end of 24-hour (drinking water and oral gavage) for 8 consecutive days (six rats per PFOS treatment. The decrease in serum TT4 correlated dose group). Interim serum samples were obtained on days 1, 3, 7, and with a decrease in serum 125I radioactivity as well. While 8 to measure serum TT4 by RIA (Diagnostic Products Corporation, liver 125I radioactivity was reduced by approximately Los Angeles, CA). Each point represents the mean serum TT4 deter- minations and error bars represent standard errors. Solid circle (᭹) 40 and 30% in males and females, respectively, urine represent serum TT4 values obtained from vehicle-treated rats and 125 and fecal I radioactivities were markedly increased in solid triangle () represent serum TT4 values obtained from PFOS- samples obtained from PFOS-treated rats, indicating an treated rats. Solid square () represent serum TT4 values obtained from increased turnover and a loss of thyroid hormones. PTU-treated rats and solid diamond () represent serum TT4 valued obtained from (PTU + PFOS)-treated rats. Serum TT4 values obtained from rats receiving PFOS or PTU or (PFOS + PTU) were statistically 3.3. Study 3: pituitary function studies in vivo/ex significantly lower than control rats at all time points (Student’s t, vivo (8-day repeat dose) p < 0.05).

Treatment of rats with PTU (with or without con- current PFOS treatment) significantly increased serum

Fig. 1. Serum TT4 concentrations (measured by Bayer ADVIA Centaur® chemiluminometric immunoassay) and 125I activities in serum, liver, urine, and feces (measured using Perkin-Elmer® Wizard 1470 Gamma Counter), represented as % over or under control, from rats receiving a 125I-T4 injection (11 and 9.3 ␮Ci for male and female rats, respectively) followed by a subsequent oral dose of either vehicle (0.5% Tween® 20) or 15 mg potassium PFOS/kg body weight. Open bars () represent male rats while solid bars () represent female rats. Asterisk (*) denotes significant difference from control (p < 0.05). 336 S. Chang et al. / Toxicology 243 (2008) 330–339

Fig. 3. Mean serum total triiodothyronine (TT3) concentrations in Fig. 4. Mean serum thyrotropin (TSH) concentrations in adult male adult male rats treated with (1) vehicle, 0.5% Tween® 20 (oral gav- rats treated with (1) vehicle, 0.5% Tween® 20 (oral gavage), (2) age), (2) 3 mg/kg-d PFOS suspended in vehicle (oral gavage), (3) 3 mg/kg-d PFOS suspended in vehicle (oral gavage), (3) 10 ␮g/mL 10 ␮g/mL (10 ppm) in drinking water or (4) 10 ppm PTU + 3 mg/kg-d (10 ppm) PTU in drinking water or (4) 10 ppm PTU + 3 mg/kg-d PFOS PFOS (drinking water and oral gavage) for 8 consecutive days (six rats (drinking water and oral gavage) for 8 consecutive days (six rats per per dose group). Interim serum samples were obtained on days 1, 3, dose group). Interim serum samples were obtained on days 1, 3, 7, and 7, and 8 to measure serum TT3 by RIA (Diagnostic Products Corpo- 8 to measure serum TSH by rat TSH RIA (EPA-NHEERL TSH rat ration, Los Angeles, CA). Each point represents the mean serum TT3 radioimmunoassay based on materials supplied by the National Hor- determinations and error bars represent standard errors. Solid circle mone and Pituitary Program, Torrance, CA). Each point represents (᭹) represent serum TT3 values obtained from vehicle-treated rats and the mean serum TSH determinations and error bars represent standard solid triangle () represent serum TT3 values obtained from PFOS- errors. Solid circle (᭹) represent serum TSH values obtained from treated rats. Solid square () represent serum TT3 values obtained from vehicle-treated rats and solid triangle () represent serum TSH values PTU-treated rats and solid diamond () represent serum TT3 valued obtained from PFOS-treated rats. Solid square () represent serum obtained from (PTU + PFOS)-treated rats. Serum TT3 values obtained TSH values obtained from PTU-treated rats and solid diamond () from rats receiving PFOS or PTU or (PFOS + PTU) were statistically represent serum TSH value obtained from (PTU + PFOS)-treated rats. significantly lower than control rats at all time points (Student’s t, Serum TSH values obtained from rats receiving PFOS were compara- p < 0.05). ble to control values. Serum TSH values from rats treated with PTU or (PFOS + PTU) were statistically significantly higher than control rats TSH levels and decreased TT4 (Fig. 2) and TT3 (Fig. 3) at all time points (Student’s t, p < 0.05). over the 8-day treatment period. TSH concentrations in sera from PFOS-only treated rats did not differ from therefore raised the question of whether the animals were the control group (Fig. 4), even though TT4 and TT3 rendered hypothyroid by PFOS. were significantly reduced (Figs. 2 and 3). Results from Production and release of metabolically active thyroid PTU + PFOS group did not differ significantly from hormones by the thyroid gland is regulated by pitu- PTU-treated group. In excised pituitaries from these itary secretion of TSH and hypothalamic secretion of rats, there was no effect of PFOS on the TRH-mediated TRH. This regulatory process for controlling circulating release of TSH in static tissue culture (Fig. 5). thyroid hormone concentrations constitutes what is com- monly referred to as the hypothalamic–pituitary–thyroid 4. Discussion axis (Menjo et al., 1999). In this system, serum-free (unbound) thyroxine and free triiodothyronine (FT4 and The series of experiments described herein was under- FT3, respectively) function as primary or secondary taken to develop a better understanding of the potential feedback signals, acting on the hypothalamus and/or effects of PFOS on serum thyroid hormones and physio- pituitary when there is an imbalance in their circulating logical thyroid status in rats. Reduced thyroid hormone concentrations. Although measurement of free thyroid levels in serum (hypothyroxinemia) have been reported hormones in serum can provide information on thyroid as a PFOS treatment-related effect in several toxicolog- status, the diagnosis of primary hypothyroidism is based ical studies (Butenhoff et al., 2002; Lau et al., 2003; mainly on a substantial elevation of TSH in response Luebker et al., 2005; Seacat et al., 2002; Thibodeaux to reduced free thyroid hormones (Larsen et al., 2003; et al., 2003); however, in these studies, reductions in Ravel, 1995; Sapin, 2001; Sapin and Schlienger, 2003). serum thyroid hormones (T3 and/or T4) occurred in In a previous study, Chang et al. (2007) demonstrated the absence of changes in thyroid gland histology or that measurement of FT4 by analog methods in serum clinically significant elevations of TSH. These findings containing PFOS is prone to negative bias in that PFOS S. Chang et al. / Toxicology 243 (2008) 330–339 337

a lowering of serum TT4, the large concentration of protein-bound thyroxine is able to act as a reservoir to maintain a constant concentration of FT4. In the experiments that we report here, a transient increase in serum FT4 and a corresponding transient decrease in serum TSH were observed over the first 6 h following a single oral dose of PFOS. By 24 h post-dose, the FT4 and TSH concentrations had returned to con- trol levels. However, TT4 in serum was reduced in a time-dependent manner. In addition to the increase in FT4, the increased liver UGT1A family mRNA transcripts observed at 2 and 6 h may have been representative of induction of increased Fig. 5. Thyrotropin (TSH) concentrations released from tissue cul- ture medium containing anterior pituitary excised from vehicle-treated glucuronidation and turnover of T4 (Barter and Klaassen, (open bars, ), PFOS-treated (left-striped bars, ), PTU-treated (right- 1992). It is unclear whether PFOS played a role in this striped bars, ), or (PFOS + PTU)-treated rats (crisscrossed bars, ). induction process or whether increases in tissue-active After plating (six rats per dose group), cells were allowed to reach equi- thyroid hormone contributed to this effect (Haberkorn et librium for 4 h and the TSH readings at 4 h were the baselines. TSH was al., 2003). The fact that TT3 was not as greatly reduced measured via RIA (EPA-NHEERL TSH rat radioimmunoassay based on materials supplied by the National Hormone and Pituitary Program, in our time-course experiment compared to TT4 (23 Torrance, CA). Each bar represents the mean serum TSH determina- and 55% reductions, respectively compared to control tions and error bars represent standard errors. For the PFOS-treatment values at 24 h post-dose) may represent a more promi- group, the net TSH released in response to the first and second TRH nent induction of T4-specific UGT isoforms, UGT1A1 stimulations was comparable to the control. While the net TSH released and/or UGT1A6, as opposed to the T3-specific UGT iso- from the PTU and (PFOS + PTU) groups were similar to the control during the first TRH challenge, they were significantly lower upon form, UGT2A2 (Vansell and Klaassen, 2002a,b). This the second TRH challenge. Asterisk (*) denotes significant difference would also pose a possible explanation for the lack of from control (p < 0.05). an elevation of TSH in our experiments, as it has been found that treatment of rats with inducers of T4-specific competes with FT4 for binding to serum proteins. There- UGT1A family isoforms in rat liver does not result in fore, to measure FT4 correctly with serum containing clinically significant increases in TSH and hypertro- PFOS, an equilibrium dialysis reference method (ED- phy/hyperplasia of the rat thyroid based on studies of RIA) should be used. Under this condition, PFOS was Klaassen and Hood (2001) and Vansell et al. (2004). found to increase the free fraction of T4 in concentration- The observation that liver ME mRNA transcripts dependent manner in rat serum in vitro while maintaining were increased 2 h following a dose of PFOS and that TT4 at a constant level. Twenty-four hours after PFOS liver ME activity was increased at 24 h also suggests a exposure, sera FT4 and TSH were not different between potential increased tissue response to thyroid hormone. PFOS-treated and control rats; however, mean TT4 con- However, PFOS is an agonist for the nuclear recep- centration in PFOS-treated rats was reduced to less than tor, PPAR␣ (Berthiaume and Wallace, 2002; Shipley half the control value. This led us to hypothesize that et al., 2004; Sohlenius et al., 1994; Takacs and Abbott, a PFOS-induced increase in FT4 via competitive dis- 2007; VandenHeuvel et al., 2006), and PPAR-␣ agonists, placement from serum binding sites may occur after including perfluorooctanoate (PFOA), a carboxylated oral dosing with PFOS and can lead to increased tis- analog of PFOS, have been shown to have thyromimetic sue uptake and turnover of T4 which would result in a effects in rat liver believed to be due to transcrip- lowering of serum TT4 while rebalancing the equilib- tional activation of thyroid-hormone-dependent genes rium between bound and free T4. When released by the such as liver ME, mitochondrial glycerol-3-phosphate thyroid gland into serum, thyroid hormones such as thy- dehydrogenase (␣G3PD), glucose-6-phosphate dehy- roxine are bound to serum carrier proteins for circulation. drogenase (G6PD), and S14 (Cai et al., 1996; Hertz et For T4, approximately 99.97% are bound to carrier pro- al., 1991,1993,1996). The activation of ME is believed to teins while a very small fraction circulates as unbound result from binding of PPAR␣/RXR (Retinoid X Recep- (free) fraction (approximately 0.03%). Since only free tor) heterodimer to a 5-flanking enhancer of the malic thyroid hormones are available to exert biological activ- enzyme promotor and is distinct from the action of thy- ity, FT4 can be obtained from (inactive) protein-bound roid hormone (Hertz et al., 1996; IJpenberg et al., 1997). T4 as needed. Under the conditions in which there is Therefore, the investigation of additional markers of liver 338 S. Chang et al. / Toxicology 243 (2008) 330–339 thyroid hormone response would confirm this interpre- lactin secretion in perfused euthyroid and hypothyroid rat pituitary tation. fragments. Horm. Res. 20, 269–276. Additional support for increased turnover of T4 Barter, R.A., Klaassen, C.D., 1992. UDP-glucuronosyltransferase 125 inducers reduce thyroid hormone levels in rats by an extrathyroidal comes from the I-labeled T4 elimination study. When mechanism. Toxicol. Appl. Pharmacol. 113, 36–42. 125 rats were pre-treated with I-radiolabeled T4 followed Berthiaume, J., Wallace, K., 2002. The impact of perfluoroactyl deriva- by a single oral dose of PFOS, a decrease in 125I activ- tives on peroxisome proliferation and mitochondrial biogenesis. ity in serum and liver and a corresponding increase The Toxicologist, 66. in 125I activity in urine and feces was observed in Bogazzi, F., Bartalena, L., Brogioni, S., Burelli, A., Grasso, L., Dell’Unto, E., Manetti, L., Martino, E., 1997. l-Thyroxine directly PFOS-treated rats compared to their controls. This cor- affects expression of thyroid hormone-sensitive genes: regulatory responded with decreased TT4 in PFOS-treated rats, effect of RXRbeta. Mol. Cell Endocrinol. 134, 23–31. and provided evidence to support the hypothesis that Butenhoff, J.L., York, R., Seacat, A.M., Luebker, D., 2002. the reduction in TT4 is due to increased turnover and Perfluorooctanesulfonate-induced perinatal mortality in rat pups elimination. is associated with a steep dose–response. The Toxicologist, 66. In order to investigate whether the lack of TSH- Butenhoff, J.L., Olsen, G.W., Pfahles-Hutchens, A., 2006. The appli- response was due to a compromised function of the cability of biomonitoring data for perfluorooctanesulfonate to the pituitary, the ability of the pituitary to respond to TRH environmental public health continuum. Environ. Health Perspect. stimulation was evaluated and the results were compared 114, 1776–1782. to the responses induced by a classical goitrogen, PTU. Cai, Y., Nelson, B.D., Li, R., Luciakova, K., dePierre, J.W., 1996. Thyromimetic action of the peroxisome proliferators clofibrate, Following the last dose, the pituitaries were removed and perfluorooctanoic acid, and acetylsalicylic acid includes changes cultured to monitor TSH concentrations over a 12-h incu- in mRNA levels for certain genes involved in mitochondrial bio- bation period during which TRH-stimulation of TSH was genesis. Arch. Biochem. Biophys. 325, 107–112. tested twice. Compared to control, serum TSH secretions Chang, S.C., Thibodeaux, J.R., Eastvold, M.L., Ehresman, D.J., Bjork, (during dosing) and TRH-mediated TSH releases (dur- J.A., Froehlich, J.W., Lau, C.S., Singh, R.J., Wallace, K.B., Buten- hoff, J.L., 2007. Negative bias from analog methods used in the ing static culture) were reduced by either PTU treatment analysis of free thyroxine in rat serum containing perfluorooctane- alone or PTU and PFOS combined treatment. On the sulfonate (PFOS). Toxicology 234, 21–33. other hand, there was no effect of PFOS on either serum Chopra, I.J., 1997. Clinical review 86: : is it TSH during dosing or TRH-mediated TSH release from a misnomer? J. Clin. Endocrinol. Metab. 82, 329–334. the pituitary during static culture. These observations Giesy, J.P., Kannan, K., 2001. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 35, 1339–1342. provide evidence that PFOS does not lower TT4 through Goldman, J.M., Cooper, R.L., 1993. Use of perfusion to evaluate hor- disruption of the hypothalamic–pituitary–thyroid axis monal release in vitro from rat pituitary and hypothalamic tissue, and does not interfere with TSH release. female reproductive toxicology. Methods in Toxicology, vol. 3B. In summary, findings from our study suggest that Academic Press, pp. 16–33. PFOS does not induce a hypothyroid state in rats under Haberkorn, V., Oziol, L., Goudonnet, H., 2003. 9-cis-Retinoic acid regulation of four UGT isoforms in hepatocytes from rats with the conditions of dosing. Neither does PFOS treat- various thyroid states. Pharm. Res. 20, 1568–1573. ment appear to alter the function of the hypothalamic– Hansen, K.J., Clemen, L.A., Ellefson, M.E., Johnson, H.O., 2001. pituitary–thyroid axis. Compound-specific, quantitative characterization of organic flu- orochemicals in biological matrices. Environ. Sci. Technol. 35, 766–770. Acknowledgements Harada, K., Saito, N., Inoue, K., Yoshinaga, T., Watanabe, T., Sasaki, S., Kamiyama, S., Koizumi, A., 2004. The influence of time, sex The authors wish to acknowledge the assistance of and geographic factors on levels of perfluorooctane sulfonate and Jill Hart (3M Medical Department, St. Paul, MN), Eliz- perfluorooctanoate in human serum over the last 25 years. J. Occup. abeth Foshay (3M Pharmaceuticals, St. Paul, MN), Mike Health 46, 141–147. Hinz (3M Corporate Health Physics, St. Paul, MN), Dr. Hertz, R., Aurbach, R., Hashimoto, T., Bar-Tana, J., 1991. Thy- romimetic effect of peroxisomal proliferators in rat liver. Biochem. A.F. Parlow (National Hormone and Pituitary Program J. 274 (Pt 3), 745–751. – UCLA Harbor Medical Center, Torrance, CA), and Dr. Hertz, R., Kalderon, B., Bar-Tana, J., 1993. Thyromimetic effect of Jerome Goldman (United States Environmental Protec- peroxisome proliferators. Biochimie 75, 257–261. tion Agency, Research Triangle Park, NC). Hertz, R., Nikodem, V., Ben-Ishai, A., Berman, I., Bar-Tana, J., 1996. Thyromimetic mode of action of peroxisome proliferators: activa- tion of ‘malic’ enzyme gene transcription. Biochem. J. 319 (Pt 1), References 241–248. Houde, M., Martin, J.W., Letcher, R.J., Solomon, K.R., Muir, D.C., Askew, R.D., Ramsden, D.B., 1984. Effect of repeated stimulation 2006. Biological monitoring of polyfluoroalkyl substances: a by thyrotropin-releasing hormone (TRH) on thyrotropin and pro- review. Environ. Sci. Technol. 40, 3463–3473. S. Chang et al. / Toxicology 243 (2008) 330–339 339

IJpenberg, A., Jeannin, E., Wahli, W., Desvergne, B., 1997. Polarity sulfonate, perfluorooctanoate, and other fluorochemicals in human and specific sequence requirements of peroxisome proliferator- blood. Environ. Health Perspect. 113, 539–545. activated receptor (PPAR)/retinoid X receptor heterodimer binding Oppenheimer, J.H., Silva, E., Schwartz, H.L., Surks, M.I., 1977. to DNA. A functional analysis of the malic enzyme gene PPAR Stimulation of hepatic mitochondrial alpha-glycerophosphate response element. J. Biol. Chem. 272, 20108–20117. dehydrogenase and malic enzyme by l-triiodothyronine. Char- Johnson, J.D., Gibson, S.J., Ober, R.E., 1979. Absorption of FC-143- acteristics of the response with specific nuclear thyroid hormone 14C in Rats After a Single Oral Dose. Riker Laboratories Inc., Saint binding sites fully saturated. J. Clin. Invest. 59, 517–527. Paul, MN. Ravel, R., 1995. Clinical Laboratory Medicine: Clinical Application Johnson, J.D., Gibson, S.J., Ober, R.E., 1984. Cholestyramine- of Laboratory Data. Mosby-Year Book Inc., Saint Louis. enhanced fecal elimination of carbon-14 in rats after administration Sapin, R., 2001. Serum thyroxine binding capacity-dependent bias in of ammonium [14C]perfluorooctanoate or potassium [14C] five free thyroxine immunoassays: assessment with serum dilu- perfluorooctanesulfonate. Fundam. Appl. Toxicol. 4, 972–976. tion experiments and impact on diagnostic performance. Clin. Klaassen, C.D., Hood, A.M., 2001. Effects of microsomal enzyme Biochem. 34, 367–371. inducers on thyroid follicular cell proliferation and thyroid hor- Sapin, R., Schlienger, J.L., 2003. Thyroxine (T4) and tri-iodothyronine mone metabolism. Toxicol. Pathol. 29, 34–40. (T3) determinations: techniques and value in the assessment of Larsen, P.R., Kronenberg, H.M., Melmed, S., Polonsky, K.S., 2003. thyroid function. Ann. Biol. Clin. (Paris) 61, 411–420. Williams Textbook of Endocrinology. Saunders: An Imprint of Seacat, A.M., Thomford, P.J., Hansen, K.J., Olsen, G.W., Case, M.T., Elsevier, Philadelphia, PA. Butenhoff, J.L., 2002. Subchronic toxicity studies on perfluorooc- Lau, C., Thibodeaux, J.R., Hanson, R.G., Rogers, J.M., Grey, tanesulfonate potassium salt in cynomolgus monkeys. Toxicol. Sci. B.E., Stanton, M.E., Butenhoff, J.L., Stevenson, L.A., 2003. 68, 249–264. Exposure to perfluorooctane sulfonate during pregnancy in Shipley, J.M., Hurst, C.H., Tanaka, S.S., DeRoos, F.L., Butenhoff, J.L., rat and mouse. II. Postnatal evaluation. Toxicol. Sci. 74, Seacat, A.M., Waxman, D.J., 2004. trans-activation of PPARalpha 382–392. and induction of PPARalpha target genes by perfluorooctane-based Luebker, D.J., York, R.G., Hansen, K.J., Moore, J.A., Butenhoff, J.L., chemicals. Toxicol. Sci. 80, 151–160. 2005. Neonatal mortality from in utero exposure to perfluorooc- Sohlenius, A.K., Andersson, K., Bergstrand, A., Spydevold, O., De tanesulfonate (PFOS) in Sprague–Dawley rats: dose–response, and Pierre, J.W., 1994. Effects of perfluorooctanoic acid – a potent biochemical and pharamacokinetic parameters. Toxicology 215, peroxisome proliferator in rat – on Morris hepatoma 7800C1 cells, 149–169. a rat cell line. Biochim. Biophys. Acta 1213, 63–74. Martin, J.W., Smithwick, M.M., Braune, B.M., Hoekstra, P.F., Muir, Takacs, M.L., Abbott, B.D., 2007. Activation of mouse and human D.C., Mabury, S.A., 2004. Identification of long-chain perfluo- peroxisome proliferator-activated receptors (alpha, beta/delta, rinated acids in biota from the Canadian Arctic. Environ. Sci. gamma) by perfluorooctanoic acid and perfluorooctane sulfonate. Technol. 38, 373–380. Toxicol. Sci. 95, 108–117. Menjo, M., Yamaguchi, S., Murata, Y., Hayashi, Y., Nagaya, T., Thibodeaux, J.R., Hanson, R.G., Rogers, J.M., Grey, B.E., Barbee, Ohmori, S., Refetoff, S., Seo, H., 1999. Responsiveness to thyroid B.D., Richards, J.H., Butenhoff, J.L., Stevenson, L.A., Lau, C., hormone is enhanced in rat hepatocytes cultured as spheroids com- 2003. Exposure to perfluorooctane sulfonate during pregnancy in pared with that in monolayers: altered responsiveness to thyroid rat and mouse. I. Maternal and prenatal evaluations. Toxicol. Sci. hormone possibly involves complex formed on thyroid hormone 74, 369–381. response elements. Thyroid 9, 959–967. Vanden Heuvel, J.P., Thompson, J.T., Frame, S.R., Gillies, P.J., 2006. OECD, 1999. Hazard Assessment of Perfluorooctane Sulfonate Differential activation of nuclear receptors by perfluorinated fatty (PFOS) and its Salts. Organisation for Economic Cooperation and acid analogs and natural fatty acids: a comparison of human, Development, Paris, France. mouse, and rat peroxisome proliferator-activated receptor-alpha, Olsen, G.W., Church, T.R., Miller, J.P., Burris, J.M., Hansen, K.J., -beta, and -gamma, liver X receptor-beta, and retinoid X receptor- Lundberg, J.K., Armitage, J.B., Herron, R.M., Medhdizadehkashi, alpha. Toxicol. Sci. 92, 476–489. Z., Nobiletti, J.B., O’Neill, E.M., Mandel, J.H., Zobel, L.R., 2003. Vansell, N.R., Klaassen, C.D., 2002a. Effect of microsomal enzyme Perfluorooctanesulfonate and other fluorochemicals in the serum of inducers on the biliary excretion of triiodothyronine (T(3)) and its American Red Cross adult blood donors. Environ. Health Perspect. metabolites. Toxicol. Sci. 65, 184–191. 111, 1892–1901. Vansell, N.R., Klaassen, C.D., 2002b. Increase in rat liver UDP- Olsen, G.W., Church, T.R., Larson, E.B., van Belle, G., Lundberg, glucuronosyltransferase mRNA by microsomal enzyme inducers J.K., Hansen, K.J., Burris, J.M., Mandel, J.H., Zobel, L.R., 2004. that enhance thyroid hormone glucuronidation. Drug Metab. Dis- Serum concentrations of perfluorooctanesulfonate and other flu- pos. 30, 240–246. orochemicals in an elderly population from Seattle, Washington. Vansell, N.R., Muppidi, J.R., Habeebu, S.M., Klaassen, C.D., 2004. Chemosphere 54, 1599–1611. Promotion of thyroid tumors in rats by -16alpha- Olsen, G.W., Huang, H.Y., Helzlsouer, K.J., Hansen, K.J., Butenhoff, carbonitrile (PCN) and polychlorinated biphenyl (PCB). Toxicol. J.L., Mandel, J.H., 2005. Historical comparison of perfluorooctane- Sci. 81, 50–59.