Evaluation of hepatic and thyroid responses in male Sprague Dawley rats for up to eighty-four days following seven days of dietary exposure to potassium perfluorooctanesulfonate

Toxicology 293 (2012) 30–40

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Evaluation of hepatic and thyroid responses in male Sprague Dawley rats for up to eighty-four days following seven days of dietary exposure to potassium perfluorooctanesulfonate Clifford R. Elcombe a , Barbara M. Elcombe a , John R. Foster b , Shu-Ching Chang c , David J. Ehresman c , Patricia E. Noker d , John L. Butenhoff c,∗ a

CXR Biosciences Ltd, Dundee DD1 5JJ, Scotland, United Kingdom AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, England, United Kingdom c Medical Department, 3M Company, St. Paul, MN 55144, USA d Southern Research Institute, Birmingham, AL 35205, USA b

a r t i c l e

i n f o

Article history: Received 19 July 2011 Received in revised form 27 December 2011 Accepted 28 December 2011 Available online 8 January 2012 Keywords: Potassium perfluorooctanesulfonate PPAR␣ CAR PXR PFOS Hepatomegaly Thyroid

a b s t r a c t In a prior 28-day dietary study in rats with 20 and 100 ppm K+ PFOS, activation of PPAR␣ and CAR/PXR were concluded to be etiological factors in K+ PFOS-induced hepatomegaly and hepatic tumorigenesis. The objective of this study was to evaluate persistence/resolution of K+ PFOS-induced, liver-related effects in male Sprague Dawley rats following a 7-day dietary exposure to K+ PFOS at 20 or 100 ppm. Groups of 10 rats per treatment were observed on recovery Day(s) 1, 28, 56, and 84 following treatment. Changes consistent with hepatic PPAR␣ and CAR/PXR activation noted on recovery Day 1 included: increased liver weight; decreased plasma cholesterol, alanine aminotransferase, and triglycerides; decreased liver DNA concentration and increased hepatocellular cytosolic CYP450 concentration; increased liver activity of acyl CoA oxidase, CYP4A, CYP2B, and CYP3A; increased liver proliferative index and decreased liver apoptotic index; decreased hepatocellular glycogen-induced vacuoles; increased centrilobular hepatocellular hypertrophy. Most effects resolved to control levels during recovery. Effects on plasma cholesterol, hepatocellular cytosolic CYP450 concentrations, liver apoptotic index, CYP3A, and centrilobular hepatocellular hypertrophy persisted through the end of the recovery period. Thyroid parameters (histology, apoptosis, and proliferation) were unaffected at all time points. Mean serum PFOS concentrations on recovery Day 1 were 39 and 140 ␮g/mL (20 ppm and 100 ppm K+ PFOS, respectively), decreasing to 4 and 26 ␮g/mL by recovery Day 84. Thus, hepatic effects in male rats resulting from K+ PFOS-induced activation of PPAR␣ and CAR/PXR resolved slowly or were still present after 84-days following a 7-day dietary treatment, consistent with the slow elimination rate of PFOS. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Perfluorooctanesulfonate (PFOS) is a perfluorinated surfactant that has been used commercially in applications requiring exceptional stability and high surface tension reducing properties. Degradation of “precursor” compounds metabolically (Xu et al., 2004) or in the environment (D’Eon et al., 2006; Rhoads et al., 2008)

∗ Corresponding author at: 3M Company, 3M Center 220-06-W-08, St. Paul, MN 55144, USA. Tel.: +1 651 733 1962; fax: +1 651 733 9066. E-mail addresses: [email protected] (C.R. Elcombe), [email protected] (B.M. Elcombe), [email protected] (J.R. Foster), [email protected] (S.-C. Chang), [email protected] (D.J. Ehresman), [email protected] (P.E. Noker), [email protected] (J.L. Butenhoff). 0300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2011.12.015

may also lead to formation of PFOS. Quantitation of PFOS in biomonitoring samples from humans (Hansen et al., 2001) and wildlife (Giesy and Kannan, 2001) demonstrated broad dissemination in the environment, and, since that time, numerous environmental sampling and biomonitoring studies have confirmed the broad dissemination of PFOS in the environment (Butenhoff et al., 2006; Houde et al., 2006; Lau et al., 2007). Recent trend studies suggest that body burdens of PFOS in the general population have been in decline since circa 2000–2002 (Calafat et al., 2007; Olsen et al., 2008; Sundström et al., 2011), when the major United States manufacturer ceased production (Renner, 2001). In repeat-dose toxicological studies in rodents (Bijland et al., 2011; Butenhoff et al., in this issue; Curran et al., 2008; Qazi et al., 2009; Seacat et al., 2003; Sohlenius et al., 1993) and non-human primates (Seacat et al., 2002), PFOS has been shown to produce responses that are consistent with activation of the xenosensor

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nuclear receptor NR1C1 (peroxisome proliferator-activated receptor ␣, or PPAR␣). These responses include hepatomegaly and hepatocellular hypertrophy, expansion of the smooth endoplasmic reticulum, changes in lipid metabolism, notably increased peroxisomal fatty acid ␤-oxidation, fatty acid ␻-hydroxylation of fatty acids, hypolipidemia, and, an increase in benign hepatocellular adenoma on chronic dietary dosing of Sprague Dawley rats (Butenhoff et al., 2012). Transactivation assays have confirmed PFOS as an agonist of PPAR␣ (Shipley et al., 2004; Takacs and Abbott, 2007; Vanden Heuvel et al., 2006; Wolf et al., 2008). Although PPAR␣ appears to be the primary target of PFOS, PFOS also has been shown to increase the expression of two other xenosensor nuclear receptors associated with hepatomegaly in rodents, NR1I3 (constitutive androstane receptor, or CAR) (Bjork et al., 2011; Elcombe et al., in this issue; Ren et al., 2009; Rosen et al., 2010) and NR1I2 (pregnane X receptor, or PXR) (Bijland et al., 2011; Bjork et al., 2011; Elcombe et al., 2012). We recently confirmed in a 28-day dietary study that the activation of PPAR␣ and CAR/PXR are etiological factors in K+ PFOSinduced hepatomegaly and hepatic tumorigenesis in rats (Elcombe et al., 2012). In that study, two dietary levels of K+ PFOS were used, 20 and 100 ppm (w/w), and separate groups of male rats were given dietary 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY) at 50 ppm (w/w) in diet and sodium phenobarbital (PB) at 500 ppm (w/w) in diet as positive controls for the activation of PPAR␣ and CAR/PXR, respectively. Treatment with K+ PFOS elicited responses characteristic of mixed activation of PPAR␣ and CAR/PXR, inducing the same changes as WY and PB, but to a lesser magnitude. The role of activation of xenosensor nuclear receptors such as PPAR␣, CAR, and PXR in producing hepatomegaly and liver tumors in rodents has been well-established (Klaunig et al., 2003; Lake, 2009). In the case of PPAR␣, the increase in liver weight results from increased peroxisomal mass and expansion of the smooth endoplasmic reticulum. CAR and PXR increase liver mass through induction of cytochromes with a consequent increase in cytochromal proteins. In addition, activation of PPAR␣, CAR, or PXR in rodents may stimulate replicative DNA synthesis, resulting in proliferation of hepatocytes, and may decrease apoptosis of hepatocytes, potentially leading to clonal expansion of preneoplastic foci and, ultimately, liver carcinogenesis. Although human liver may have some capacity to respond to the hepatocellular hypertrophic effects of activation of these xenosensor receptors, exposure of humans to agents known to activate these receptors has not been associated with increased risk of liver cancer. Furthermore, in recent years it has been demonstrated that, when the mouse forms of these receptors are replaced by the human forms, the human forms of the receptors appear incapable of supporting the hepatocellular hyperplastic response (Cheung et al., 2004; Gonzalez and Shah, 2008; Hirose et al., 2009; Ross et al., 2010; Shah et al., 2007). In addition to the increase in liver tumors observed in the chronic dietary study with K+ PFOS in Sprague Dawley rats, there was a statistically significant increase in thyroid follicular cell adenoma in male rats who received one year of 20 ppm dietary K+ PFOS and switched to control diet through termination of the study (stopdose group) when compared to male control rats or male rats that were fed with 20 ppm dietary K+ PFOS through study termination at 104 weeks (Butenhoff et al., 2012). The etiological basis of the increase in thyroid follicular tumors in the stop-dose group males has remained recondite and may possibly have been due to a chance occurrence. In the 28-day dietary study mentioned above (Elcombe et al., 2012), we further evaluated the apoptotic, proliferative, and histological responses in thyroid with K+ PFOS treatment; however, there were no changes when compared to the controls.

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Following the completion of the 28-day study (Elcombe et al., 2012), a seven-day dietary study at the same K+ PFOS dietary doses was conducted followed by an 84-day recovery observation period in order to allow evaluation of recovery from K+ PFOS-induced effects. The results of this recovery study are reported herein. 2. Materials and methods 2.1. Materials The potassium salt of perfluorooctanesulfonate (K+ PFOS, FC-95, Lot 217, 86.9% purity) was supplied by 3M Company (St. Paul, MN, USA) and was chemically stable during the course of the experiments. All other chemicals used in the study were reagent-grade with the highest obtainable purity. 2.2. Animals and husbandry Male Sprague Dawley rats (6–7 weeks old) were obtained from Charles River Laboratories (Margate, Kent, UK) and used in this study, because the aim of this study was to evaluate the mode of action for the chemically induced development of hepatocellular adenoma observed in a two-year dietary study with the same stock of rats. Upon arrival at the study facility, the rats were group-housed (2/cage) on sawdust in solid-bottomed polypropylene cages and they were acclimatized for at least 5 days before use. The rats were uniquely numbered by ear-punch and randomly assigned to groups. The room temperature was maintained within a range of 19–23 ◦ C with a relative humidity within a range of 40–70%. There was a nominal 14–15 air changes per hour and the light/dark cycle was 12/12 h. In-life procedures undertaken during the course of the studies were subject to the provisions of the United Kingdom Animals (Scientific Procedures) Act 1986. The study complied with all applicable sections of the Act and associated Codes of Practice for the Housing and Care of Animals used in Scientific Procedures and the Humane Killing of Animals under Schedule 1 to the Act, issued under section 21 of the Act. 2.3. Diets For this dietary study, K+ PFOS was incorporated into RMI powdered diet (Special Diet Services LTD, Stepfield, Witham, Essex, UK) at 20 ppm or 100 ppm concentration and the corresponding diet specifications were retained at the testing facility. The diets were prepared with an adjustment for purity. Rats in the control group received control RMI powdered diet ad libitum throughout the study while test group rats received diets containing K+ PFOS for 7 days followed by control RMI powdered diet during the recovery period when applicable. Drinking water was taken from the local supply and provided in bottles to all rats ad libitum. 2.4. Experimental design The study consisted of a control and two test groups, each with 40 male Sprague Dawley rats. Rats were given either control or test diets for 7 days followed by the control diet during the recovery period. On the first day following the 7-day dietary treatment, i.e., first day of recovery (recovery Day 1), and on recovery Days 28, 56, and 84, subgroups of 10 rats from each of the control and two K+ PFOS-treatment groups were sacrificed after overnight fasting. For all rats, clinical observations, body weights, and food consumption were monitored and recorded throughout the entire study. Specimens collected during necropsy were subjected to various biochemical endpoint analyses (serum and liver) as well as histological evaluations (liver and thyroid). In addition, the rates of cell proliferation and apoptosis were measured in liver and thyroid tissues. Levels of PFOS in the test diet, serum, liver, and liver cytosols were determined using LC–MS/MS techniques. 2.5. Experimental procedures 2.5.1. Test material administration The test materials were incorporated in the diet and were given to rats ad libitum for 7 days (see Section 2.3 for details). 2.5.2. Clinical observations, body weights, and food consumption All rats were observed daily throughout the study. Body weight was recorded for each rat at the start of the study and weekly thereafter (when applicable). All rats were weighed and body weights were recorded prior to necropsy. Food consumption was monitored and recorded throughout the study. 2.5.3. BrdU incorporation Cell proliferative responses in liver and thyroid were evaluated via bromodeoxyuridine (BrdU) immunohistochemistry. Five days prior to scheduled necropsies, rats were implanted subcutaneously with osmotic pumps (Alzet® 2ML1, Durect Corporation, Cupertino, CA, USA); each pump contained 2 mL of 15 mg/mL BrdU solution (in pH 7.4 PBS).

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2.5.4. Necropsy On recovery Days 1, 28, 56, and 84, after having been fasted overnight, groups 10 rats per treatment group were euthanized by CO2 asphyxiation and necropsied. Approximately 6–10 mL of blood was obtained via cardiac puncture and processed for serum (uncoated tubes) and plasma (lithium heparin-coated tubes) with centrifugation (2000 × g, 10 min, 8–10 ◦ C). Both serum and plasma samples were stored at approximately −70 ◦ C pending analyses. The liver was removed and weighed. For each rat, several 2 mm strips of liver tissue were excised from the center of the left lobe as well as the median lobe. One strip from each lobe was fixed in 10% neutral-buffered formaldehyde (NBF) for approximately 48 h for the evaluations of apoptotic index (by TUNEL assay) and cell proliferation (by BrdU immunohistochemistry). Approximately 1 cm of duodenum was obtained from the small intestine placed in NBF for approximately 48 h as a positive control for BrdU immunohistochemistry. For liver H&E histological examination of hematoxylin and eosin (H&E) stained tissue, one strip from each liver lobe (left and medium) was fixed in NBF for approximately 1 week. Approximately 0.25 g of liver was excised, weighed, flash-frozen in liquid nitrogen, and stored at approximately −70 ◦ C for DNA content determination. Two additional liver sections (weighing approximately 1 g each) were also flash-frozen in liquid nitrogen, stored at approximately −70 ◦ C pending test material concentration analyses. The remaining liver was weighed, scissor-minced in cold KCl solution (1.15%, w/v), and subjected to subcellular fractionation to obtain cytosol, heavy pellet, and microsomes. These liver fractions were stored at approximately −70 ◦ C pending analyses. The thyroid, including parathyroid together with trachea and esophagus, was removed from each rat and placed in NBF for approximately 48 h. These samples were used for cell proliferation assay by BrdU immunohistochemistry, analysis of apoptotic index by TUNEL, and H&E histology by light microscopy. 2.5.5. Biochemical measurements Clinical chemistry endpoints were determined in plasma samples collected using Roche Cobas Integra® 400 automated analyzer. Parameters measured included alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (CHOL), triglycerides (TRIG), and glucose (GLUC). Protein concentrations in liver heavy pellets and microsomes were determined using a modified Lowry protein assay (Lowry et al., 1951), with bovine serum albumin used as standards. The liver DNA content in whole tissue was measured spectrophotometrically using the diphenylamine reaction (Burton, 1956). Total cytochrome P450 (CYP450) concentration in liver microsomes was also determined by measuring the carbon monoxide difference spectrum of ferrocytochrome P450 (Omura and Sato, 1962). For liver heavy pellets, cyanide-insensitive acyl CoA oxidation (ACOX) was evaluated spectrophotometrically in order to determine peroxisome proliferation potential using palmitoyl CoA as a substrate (Bronfman et al., 1979). Because CYP4A, CYP2B, and CYP3A have been shown to correspond with increased PPAR␣, CAR, and PXR activities (Lake, 2009), several biochemical assays were performed in liver microsomes for their activities of: CYP4A activity (lauric acid 12-hydroxylation (12OH LAH)); CYP2B activity (pentoxyresorufin-O-depentylation (PROD)); and CYP3A activity (testosterone 6␤-hydroxylation (Test-6␤)). 2.5.6. Histopathology and morphometry Following fixation, all samples (liver, thyroid, and duodenum) were processed and embedded in paraffin. For liver and thyroid light microscopic evaluation, 5-␮m sections were cut and stained using H&E stain. The extent of in situ cell death (apoptosis) was measured using an indirect TUNEL labeling assay (Roche 11 684 817910) (Gavrieli et al., 1992). Because of the relatively low levels of apoptosis present in liver samples, the following morphometric method was applied to quantify the apoptotic rates: for each liver lobe present, the total number of apoptotic cells present was counted. The area occupied by the liver lobes was then calculated, and the number of hepatocytes nuclei counted/unit area using a 40× objective magnification at the microscope. A total of five areas were counted for each liver lobe, and the apoptotic index was then estimated as a percent of the total nuclei in the three liver lobes. The extent of cell proliferation was evaluated with BrdU incorporation via indirect BrdU labeling assay (Wijsman et al., 1992). For the estimation of proliferation index, the number of BrdU positive cells was counted and expressed as a percent of the total number of the hepatocyte nuclei in 10 fields, incorporating both a periportal area and a centrilobular area at 40× objective lens in a light microscope. 2.5.7. LC–MS/MS determination of PFOS concentrations LC–MS/MS methods were used to determine PFOS concentrations in serum, liver, and cytosol. Stable isotope-labeled 18 O2 -PFOS (Research Triangle Institute, Research Triangle Park, NC, USA) was used as the internal standard for the analysis of PFOS concentrations. The water used in the LC–MS/MS analysis was prepared by passing deionized water through a Milli-Q® water purification system followed by HPLC with a C18 column. All other chemicals used were reagent grade, purchased from either Sigma–Aldrich (St. Louis, MO, USA) or VWR (West Chester, PA, USA). Sample preparation and extractions for PFOS in serum and liver were completed following the general method described previously in Chang et al. (2007). Cytosol was processed in the manner as described by Chang et al. for the analysis of serum

samples for PFOS concentration. The LC–MS/MS conditions used for the analysis of PFOS were also described in detail in Chang et al. (2007). 2.5.8. Statistical and pharmacokinetic analyses Homogeneity of variance was assessed with Bartlett’s test (Sokal and Rohlf, 1969b). When Bartlett’s test was not significant (p > 0.001), ANOVA was performed (Snedecor and Cochron, 1967). In cases where ANOVA was significant (p ≤ 0.05), Dunnett’s t-test for multiple comparisons of treatment means with the control mean was performed with significance at p ≤ 0.05 (Dunnett, 1955, 1964). The foregoing tests were performed with JMP (version 5.1, SAS Institute, Inc., Cary, NC). If Bartlett’s test was significant for (p ≤ 0.001), the Kruskal–Wallis test (Sokal and Rohlf, 1969a) was performed on ranked data. When the Kruskal–Wallis test was significant (p ≤ 0.05), Dunn’s test (Dunn, 1964) for multiple comparisons of treated groups to the control was performed with significance at p ≤ 0.05. The Kruskal–Wallis and Dunn’s tests were performed with Minitab (version 16, Minitab Company, State College, PA) using the KrusMC.MAC macro. The two-sided Student’s t-test was performed in Microsoft® Excel® (Microsoft Office 2007) to test for significant differences in PFOS concentration parameters between the 20 ppm and 100 ppm dose groups with significance at p ≤ 0.05. Elimination rates of PFOS anion from serum and liver were analyzed using WINNONLIN® software, Version 1.1 (Scientific Consulting, Inc., Apex, NC).

3. Results 3.1. Clinical observations, body weights, and food consumption All rats survived to the scheduled necropsies, and there were no adverse clinical observations noted during the study. Terminal body weights are presented in Table 1. Administration of K+ PFOS at 20 ppm in the diet for 7 days followed by recovery (with control diet) led to decreased mean body weights relative to controls with statistical significance on recovery Days 21 and 28 for the 20 ppm group and on recovery Days 16, 21, and 28 for the 100 ppm group. Mean terminal body weights of rats receiving 20 ppm K+ PFOS or 100 ppm K+ PFOS at any other time point were lower than the respective controls but the differences were not statistically significantly different. Mean food consumption values, as grams of food per rat per week and grams of food consumed per kilogram body weight per day, and mean estimated daily intakes of test compounds per kilogram body weight are presented in Table 2. On a weekly basis or when adjusted for daily food intake by body weight, the food consumption by rats given K+ PFOS in diet was essentially similar to the amounts consumed by the control rats. Mean (±S.D.) daily test compound intakes were 1.93 ± 0.01 mg/kg/day and 9.65 ± 0.08 mg/kg/day, for the 20 ppm and 100 ppm K+ PFOS dietary dose groups, respectively. 3.2. Liver weights Data for mean absolute liver weight and mean liver weight as a percentage of terminal body weight are presented in Table 3. On recovery Day 1, mean absolute liver weight in the 100 ppm K+ PFOS dose group rats was statistically significantly increased by 15% relative to mean control liver weight, and mean liver weight relative to body weight was increased with statistical significance in both the 20 and 100 ppm dose groups by 12% and 21%, respectively, relative to controls. On recovery Day 28, the mean absolute liver weight in the 20 ppm dose group was reduced with statistical significance by 13% relative to controls; however, mean liver weight relative to body weight was similar in the 20 ppm dose group and control group, and the apparent reduction in absolute liver weight in the 20 ppm dose group may be a reflection of the statistically significant reduction observed in mean body weight in this group relative to the control group on recovery Day 28. The only other statistically significant difference in liver weight parameters between control and K+ PFOS-treated rats was noted on recovery Day 84, at which time mean liver weight relative to body weight in the K+ PFOS-treated rats were 13% greater than the control group

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Table 1 Mean (±standard deviation) body weights (g) in male Sprague Dawley rats at various time points following a seven-day dietary exposure to either control diet or diet containing K+ PFOS at 20 ppm or 100 ppm. Na

Control

20 ppm K+ PFOS

100 ppm K+ PFOS

Pre-dose

40/group

244.2 ± 26.4

234.7 ± 27.0

239.1 ± 29.8

Recovery day (RD) RD 1 RD 1 sacrifice groupc

40/group 10/group

294.9 ± 30.3 296.4 ± 30.1

284.0 ± 36.4 286.5 ± 34.1

279.6 ± 29.7 282.0 ± 32.5

RD 7 RD 16 RD 21 RD 28 RD 28 sacrifice group

30/group 30/group 30/group 30/group 10/group

339.5 371.9 412.2 428.2 423.3

± ± ± ± ±

36.9 41.5 46.8 50.9 46.0

322.4 350.2 384.8 397.0 382.2

± ± ± ± ±

38.8 42.0 46.8* 51.4* 30.8

318.1 346.1 382.1 396.1 378.2

± ± ± ± ±

33.9 39.4* 44.0* 47.4* 59.9

RD 35 RD 42 RD 49 RD 56 RD 56 sacrifice group

20/group 20/group 20/group 20/group 10/group

450.4 469.0 475.4 498.3 504.3

± ± ± ± ±

55.3 56.0 58.7 63.7 61.4

420.1 436.4 442.9 461.9 456.1

± ± ± ± ±

61.7 65.2 62.9 67.3 57.7

423.4 439.0 442.6 463.9 476.6

± ± ± ± ±

39.8 39.7 36.4 40.4 32.8

RD 63 RD 70 RD 77 RD 84 sacrifice group

10/group 10/group 10/group 10/group

515.2 528.1 539.5 557.8

± ± ± ±

70.2 72.9 75.8 78.7

489.4 501.7 514.6 524.3

± ± ± ±

83.8 85.2 89.3 89.5

470.0 477.9 488.2 509.8

± ± ± ±

48.7 49.1 48.7 54.1

Time b

a b c *

Sample size. Prior to test material administration. Represents a subset of all rats on study on the corresponding recovery day. Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.05).

Table 2 Mean (±S.D., N = 10/group) food consumption per week and day and mean estimated daily dose of K+ PFOS in mg/kg body weight in male Sprague Dawley rats following a seven-day dietary exposure to either control diet or to diet containing K+ PFOS at either 20 ppm or 100 ppm. Consumption parameter

Control

20 ppm K+ PFOS

100 ppm K+ PFOS

Mean weekly food consumption (g/rat/week) Mean daily food consumption (g/kg/day) Mean daily K+ PFOS dosea (mg/kg/day)

191.7 ± 12.7 94.2 ± 3.6 N.A.

191.7 ± 15.1 96.7 ± 4.4 1.93 ± 0.01

188.8 ± 23.1 96.5 ± 8.0 9.65 ± 0.08

a

Estimate based on dietary concentration of test compounds and daily food consumption in mg/kg body weight.

mean, without a noted difference in absolute liver weight. This latter observation may be the result of somewhat higher, but not statistically significantly different, mean body weight in the control group as compared to the treated groups, resulting in a statistically significant lower relative liver weight in the control group. 3.3. Biochemical measurements 3.3.1. Plasma clinical chemistries Plasma clinical chemistry results are presented in Table 4. Compared to controls, there were no rising ALT or AST concentrations in rats receiving test compounds, indicating a lack of overt hepatotoxicity. All mean ALT and AST concentrations from rats receiving

test compound were less than corresponding control values at all time points, though generally not with statistical significance. Mean plasma cholesterol was reduced by K+ PFOS at the end of recovery Day 1 (79% and 65% of control for 20 and 100 ppm K+ PFOS, respectively), at the end of recovery Day 28 (70% and 62% of control for 20 and 100 ppm K+ PFOS, respectively), and at the end of recovery Day 84 (81% of control for 100 ppm K+ PFOS only). Although mean plasma CHOL values in K+ PFOS-treated rats were somewhat lower than the control mean on recovery Day 56, they did not differ with statistical significance. Mean triglyceride concentrations in plasma were unaffected by dietary treatments with 20 ppm K+ PFOS; however, triglycerides were statistically significantly lower than time-matched controls in rats receiving 100 ppm

Table 3 Mean (±S.D., N = 10/group) liver weight (g) and liver-to-body weight ratios (%) in male Sprague Dawley rats at various time points following a seven-day dietary exposure to control diet or diet containing K+ PFOS at 20 ppm or 100 ppm. Liver weight measurement

Control

20 ppm K+ PFOS

100 ppm K+ PFOS

1

Absolute (g) Relative (%)

13.40 ± 1.38 4.53 ± 0.29

14.45 ± 1.71 5.06 ± 0.38**

15.42 ± 1.85* 5.48 ± 0.36***

28

Absolute (g) Relative (%)

18.04 ± 2.88 4.25 ± 0.34

15.76 ± 1.24 4.07 ± 0.34

17.46 ± 3.46 4.61 ± 0.44

56

Absolute (g) Relative (%)

20.19 ± 2.61 4.02 ± 0.36

18.23 ± 3.48 3.98 ± 0.38

19.19 ± 1.76 4.03 ± 0.27

84

Absolute (g) Relative (%)

20.23 ± 3.40 3.63 ± 0.39

21.56 ± 5.20 4.09 ± 0.51*

21.03 ± 2.74 4.12 ± 0.25*

Days following treatment

* ** ***

Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.05). Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.01). Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.001).

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Table 4 Mean (±S.D., N = 10/group) plasma clinical chemistry measures (alanine aminotransferase, aspartate aminotransferase, cholesterol, triglycerides, glucose) in male Sprague Dawley rats at various time points following a seven-day dietary exposure to control diet or diet containing K+ PFOS at 20 ppm or 100 ppm. Days following treatment

Clinical measure

Control

20 ppm K + PFOS

100 ppm K+ PFOS

1

ALT (U/L) AST (U/L) CHOL (mmol/L) TRIG (mmol/L) GLUC (mmol/L)

110.60 102.25 2.73 1.51 22.51

± ± ± ± ±

18.64 12.09 0.44 0.44 3.47

93.97 103.94 2.17 1.58 22.92

± ± ± ± ±

10.47 10.47 0.37** 0.53 3.58

90.04 109.97 1.77 1.02 22.26

± ± ± ± ±

16.66* 12.75 0.39*** 0.41* 2.93

28

ALT (U/L) AST (U/L) CHOL (mmol/L) TRIG (mmol/L) GLUC (mmol/L)

110.32 168.98 2.29 1.72 19.98

± ± ± ± ±

17.16 38.11 0.24 0.74 3.75

117.56 151.51 1.61 1.32 19.43

± ± ± ± ±

54.52 58.79 0.33*** 0.32 4.98

102.78 136.02 1.43 1.26 20.40

± ± ± ± ±

22.45 28.85 0.44*** 0.24 2.81

56

ALT (U/L) AST (U/L) CHOL (mmol/L) TRIG (mmol/L) GLUC (mmol/L)

90.54 124.75 2.19 1.70 17.87

± ± ± ± ±

15.55 24.75 0.35 0.63 2.67

89.07 128.30 1.96 2.02 19.34

± ± ± ± ±

22.47 26.87 0.26 0.70 2.82

103.85 120.31 2.00 1.26 23.18

± ± ± ± ±

29.44 22.49 0.37 0.34 3.29***

84

ALT (U/L) AST (U/L) CHOL (mmol/L) TRIG (mmol/L) GLUC (mmol/L)

84.15 184.34 2.17 1.66 18.43

± ± ± ± ±

16.68 44.30 0.36 0.50 2.24

84.82 186.13 1.98 2.35 17.70

± ± ± ± ±

18.03 40.44 0.40 0.99 2.20

99.36 177.97 1.75 1.58 19.86

± ± ± ± ±

36.44 66.32 0.38* 0.65 2.21

* ** ***

*

Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.05). Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.01). Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.001).

K+ PFOS on recovery Day 1 (68% of control mean), but not at subsequent time points. Compared to time-matched controls, mean plasma GLUC concentrations were not statistically significantly different in rats given dietary treatments of K+ PFOS during the any of the recovery phase except on recovery Day 56 on which rats from 100 ppm K+ PFOS had statistically significantly higher mean GLUC value than control (130% of control mean). 3.3.2. Liver biochemical assays To determine the proliferation status in liver, several biochemical parameters were evaluated and presented in Table 5. Compared to control, the mean hepatic concentration of DNA (mg DNA/g liver) was statistically significantly decreased on recovery Day 1 in the 100 ppm K+ PFOS groups (74% of control mean). Mean hepatic concentration of DNA in the 100 ppm K+ PFOS group remained decreased at the end of recovery Days 28 and 56 with statistical significance (72% and 77% relative to control, respectively) and was lower than the control mean (91% of control mean) on recovery Day 84 without being statistically significant.

Mean total DNA in liver (mg DNA/whole liver) was decreased with statistical significance relative to the control means on recovery Days 28 and 56 at both K+ PFOS dietary dose levels. Although the mean total DNA was not statistically significantly lower than the control mean on recovery Day 1, the mean values for K+ PFOStreated groups were somewhat less than the control value (97% and 85% for the 20 and 100 ppm dose groups, respectively). Liver microsomal CYP450 concentrations (nmol CYP450/mg protein) are also presented in Table 5. Mean CYP450 concentrations were increased at all recovery time points relative to control means in both the 20 and 100 ppm dose groups. On recovery Day 1, CYP450 concentrations were 123% and 189% in the 20 ppm and 100 ppm K+ PFOS dose groups, respectively, relative to control means, and were still elevated on recovery Day 84 (133% and 168% of the control mean). The results for liver tissue activities of ACOX (cyanideinsensitive palmitoyl CoA ␤-oxidation, a marker measuring peroxisomal fatty acid ␤-oxidation activity in liver heavy pellets) and 12-OH LAH (lauric acid 12-hydroxylation, a marker measuring CYP4A fatty acid ␻-hydroxylation activity in liver microsomes)

Table 5 Mean (±S.D., N = 10/group) liver DNA concentration, liver total DNA content, and liver microsomal P450 concentration in liver in male Sprague Dawley rats at various time points following a seven-day dietary exposure to control diet or diet containing K+ PFOS at 20 ppm or 100 ppm. Liver biochemical measure

Control

20 ppm K+ PFOS

100 ppm K+ PFOS

1

mg DNA/g liver mg DNA/whole liver nmol P450/mg protein

2.253 ± 0.163 30.21 ± 3.95 0.86 ± 0.12

2.043 ± 0.165 29.43 ± 3.33 1.07 ± 0.24*

1.669 ± 0.406*** 25.59 ± 6.37 1.63 ± 0.17***

28

mg DNA/g liver mg DNA/whole liver nmol P450/mg protein

2.179 ± 0.132 39.10 ± 5.30 0.65 ± 0.05

2.109 ± 0.339 31.71 ± 3.74** 0.87 ± 0.17**

1.562 ± 0.355*** 26.83 ± 6.27*** 1.33 ± 0.18***

56

mg DNA/g liver mg DNA/whole liver nmol P450/mg protein

2.162 ± 0.504 43.45 ± 10.91 0.68 ± 0.14

1.844 ± 0.259 33.14 ± 5.09* 0.95 ± 0.16**

1.655 ± 0.325* 32.10 ± 8.08* 1.12 ± 0.18***

84

mg DNA/g liver mg DNA/whole liver nmol P450/mg protein

1.915 ± 0.226 38.80 ± 7.55 0.60 ± 0.15

1.918 ± 0.408 40.44 ± 9.99 0.80 ± 0.12**

1.740 ± 0.153 36.91 ± 4.37 1.01 ± 0.11***

Days following treatment

* ** ***

Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.05). Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.01). Statistically significantly different from control (Dunnett’s t-test, p ≤ 0.001).

C.R. Elcombe et al. / Toxicology 293 (2012) 30–40

35

Fig. 1. Mean hepatic enzyme activities for (A) palmitoyl CoA oxidase, nmol NAD+ reduced/min/mg protein; (B) lauric acid 12-hydroxylation, nmol 12-hydroxy lauric acid formed/10 min/mg protein; (C) pentoxyresorufin-O-depentylation, pmol resorufin formed/min/mg protein; and (D) testosterone 6␤-hydroxylation, nmol hydroxytestosterone formed/min/mg protein in male Sprague Dawley rats (N = 10/treatment group) at various time points following a seven-day dietary exposure to either control diet or to diet containing K+ PFOS at either 20 ppm or 100 ppm. Error bars represent standard deviations. Open bars, strike bars, dotted bars, and solid bars denote 1, 28, 56, and 84 days following treatment, respectively. Statistical significance relative to control is indicated by asterisks with Dunnett’s t-test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) or by hash signs with Dunn’s test (### p ≤ 0.001).

are presented in Fig. 1, panels A and B, respectively. These markers were evaluated as a measure of PPAR␣ activation potential associated with the dietary treatment with K+ PFOS. Compared to time-matched controls, mean ACOX activity was statistically significantly increased in the 100 ppm K+ PFOS dose group through recovery Day 56 (127, 141, and 130% of control mean on recovery Days 1, 28, and 56, respectively). Mean ACOX activities were not increased relative to control means at any recovery period evaluation time point in rats given 20 ppm K+ PFOS. Compared to time-matched controls, mean 12-OH LAH activity was statistically significantly increased from 100 ppm K+ PFOS group during the study from recovery Day 1 through recovery Day 56 (206, 289, and 285% for recovery Days 1, 28, and 56, respectively). On recovery Day 84, 12-OH LAH activity in the 100 ppm dose group K+ PFOS, though 133% of the control mean, was not statistically significant. There were no increases in 12-OH LAH in rats given 20 ppm K+ PFOS. Results of measurements of PROD activity (pentoxyresorufinO-depentylation, a marker for CYP2B) are included in Fig. 1, panel C. PROD was used as a marker for the activation of CAR nuclear receptor. Administration of 20 ppm K+ PFOS in diet decreased mean PROD activity during all recovery periods of the study (49%, 50%, 44%, and 44% of control means on recovery Days 1, 8, 56, and 84, respectively). Administration of 100 ppm K+ PFOS in the diet initially increased mean PROD activity on Day 1 and Day 28 of recovery (362% and 162% of control mean, respectively), and resolved to a level that was similar to control mean on recovery Day 56 (96% of

control) and was decreased with statistical significance on recovery Day 84 (44% of control mean). Results of measurements for Test 6␤-OH (testosterone 6␤hydroxylation, a marker for CYP3A activity) are presented in Fig. 1, panel D. Test 6␤-OH was used as a marker for activation of the PXR nuclear receptor. Administration of 100 ppm K+ PFOS in diet increased mean Test 6␤-OH activity with statistical significance on recovery Days 1, 56, and 84 (232, 221, and 191% of control means on recovery Days 1, 56, and 84, respectively). 3.4. Histopathology of liver and thyroid tissues 3.4.1. Liver histopathology Evaluation of liver sections stained with H&E showed several treatment-related findings when compared to control rats (Table 6). First, a minimal to moderate (grades 1–3) periportal hepatocellular vacuolation considered to be due to intracytoplasmic glycogen accumulation was prominent in liver sections from all control group rats on the first day of the recovery period. The hepatocellular vacuolation was also present in liver sections from all K+ PFOS-treated rats at that time point, but with slightly decreased mean severity in the 100 ppm group. At all subsequent sacrifice time points, the incidence and mean severity of this finding were less in K+ PFOS-treated rats in an apparent dose-dependent manner. Second, a dose-related increased incidence and mean severity in minimal to moderate centrilobular hepatocellular hypertrophy was

36

C.R. Elcombe et al. / Toxicology 293 (2012) 30–40

Table 6 Summary of incidence and severity for hepatocellular hypertrophy and vacuolation in male Sprague Dawley rats sacrificed at various time points following a seven-day dietary exposure to control diet or diet containing K+ PFOS at 20 ppm or 100 ppm. Observation

1

28

Hepatocellular hypertrophy

56

84

1

28

Hepatocellular vacuolation

Control

20 ppm K+ PFOS

100 ppm K+ PFOS

Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity

0/10 0 0 0 0 0/10 0 0 0 0 0/10 0 0 0 0 0/10 0 0 0 0

8/10 8 0 0 1.0 5/10 4 1 0 1.2 4/10 4 0 0 1.0 5/10 5 0 0 1.0

10/10 2 8 0 1.8 10/10 0 9 1 2.1 10/10 0 9 1 2.1 10/10 3 7 0 1.7

Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity Total incidence Incidence by severity: Grade 1 Grade 2 Grade 3 Average severity

10/10 1 3 6 2.5 10/10 1 8 1 2.0 10/10 1 4 5 2.4 10/10 0 4 6 2.6

10/10 1 2 8 2.8 7/10 1 6 0 1.9 9/10 2 4 3 2.1 10/10 3 4 3 2.0

10/10 0 8 1 2.0 6/10 1 5 0 1.8 9/10 3 6 0 1.7 7/10 1 6 0 1.9

Days following treatment

56

84

present in rats given K+ PFOS at all recovery sacrifice time points. There was no evidence of an unequivocal diminution in this latter finding during the recovery period, indicating an incomplete reversibility. Results for determination of hepatocellular proliferation and hepatocellular apoptosis are presented in Fig. 2, panels A and B, respectively. The mean hepatocellular S-phase labeling index in K+ PFOS-treated rats, as determined by BrdU S-phase incorporation, was increased with statistical significance on recovery Day 1 (232% and 586% of control values, respectively, for 20 ppm and 100 ppm K+ PFOS) and was not increased during any subsequent recovery period. The mean hepatic apoptotic index was decreased in K+ PFOStreated rats at all recovery period observation time points (70, 42, 47, and 31% of control means, respectively, on recovery Days 1, 28, 56, and 84 days for 20 ppm K+ PFOS rats; and 42, 33, 19, and 29% of control means, respectively, on recovery Days 1, 28, 56, and 84 days for 100 ppm K+ PFOS rats). 3.4.2. Thyroid histopathology Microscopic examination of H&E stained thyroid gland sections did not reveal any findings considered to be related to K+ PFOS treatment. Data for cell proliferation in the thyroid are presented in Table 7. K+ PFOS treatment at either the 20 or 100 ppm dose had no effect on S-phase activity at any time point studied as compared to controls. The level of apoptosis in the thyroid follicles of all groups and at all time points was similar to controls. It was typical

to observe either no cells staining as apoptotic in the TUNEL assay, or, occasionally, just one cell per thyroid (data not shown). 3.5. LC–MS/MS determination of PFOS concentrations Results of PFOS concentrations in serum, liver, and liver cytosol over the recovery time period are presented in Table 8, along with the liver-to-serum and liver-to-cytosol concentration ratios. PFOS concentrations in serum, liver, and liver cytosol were statistically significantly different between the two K+ PFOS-treated groups and were in approximate proportion to dose and decreased with duration of recovery. At the end of 7-day 20 ppm and 100 ppm K+ PFOS dietary treatment (recovery Day 1), respectively, mean serum PFOS concentrations were 39.49 ± 7.76 ␮g/mL and 140.40 ± 14.05 ␮g/mL Table 7 Mean (±S.D., N = 10/group) thyroid follicular cell proliferation index (based on BrdU S-phase labeling) in male Sprague Dawley rats sacrificed at various time points following a seven-day dietary exposure to control diet or diet containing K+ PFOS at 20 ppm or 100 ppm. Days following treatment

Control

1 28 56 84

2.62 1.50 1.12 1.02

± ± ± ±

20 ppm K+ PFOS 0.21 0.33 0.33 0.31

2.70 1.70 1.30 1.21

± ± ± ±

0.56 0.27 0.26 0.24

100 ppm K+ PFOS 3.28 1.82 1.32 1.18

± ± ± ±

1.10 0.38 0.25 0.07

C.R. Elcombe et al. / Toxicology 293 (2012) 30–40

37

(Table 8). The respective liver PFOS mean concentrations were 123.92 ± 23.95 ␮g/g and 319.50 ± 46.64 ␮g/g (Table 8). At the end of 84 days recovery period, the respective serum PFOS concentrations were 4.38 ± 0.72 ␮g/mL and 25.79 ± 5.38 ␮g/mL while the respective liver PFOS concentrations were 24.99 ± 1.30 ␮g/g and 85.74 ± 16.33 ␮g/g. Liver cytosolic PFOS concentrations deceased over time in a manner similar to PFOS concentrations in serum and liver (Table 8). Liver PFOS concentrations were approximately 3–5 times higher than those in serum and approximately 7–10 times those in liver cytosol. In liver from control rats, PFOS was found at concentrations above the method lower limit of quantitation of 50 ppb in 4/10, 10/10, 9/10, and 10/10 rats on recovery Days 1, 28, 56, and 84, respectively (data not shown). The mean ± S.D. and range of these quantifiable values were 0.112 ± 0.039 ␮g/g, range = 0.0552–0.232 ␮g/g. No PFOS was quantifiable in serum or cytosol samples from control rats. Liver-to-serum PFOS concentration ratios in the 20 and 100 ppm were statistically significantly higher in the 20 ppm dose group than in the 100 ppm dose group on Days 1 and 84 of recovery. There were no differences between K+ PFOS-treated groups in liver-to-serum PFOS concentration ratios on Days 28 and 56 of recovery. However, there were no differences between the two treated groups in liver-to-cytosol PFOS concentration ratios at any recovery sacrifice time point. WINNONLIN® analysis of serum and liver PFOS concentration data during the recovery phase revealed that PFOS elimination halflives were on the order of one to two months. Specifically, the serum PFOS elimination half-lives during the 84-day recovery phase were 30.8 and 36.4 days for the 20 ppm and 100 ppm dose groups, respectively. Corresponding liver elimination half-lives were 68.1 and 48.3 days for the 20 and 100 ppm dose groups, respectively. 4. Discussion Fig. 2. Mean liver proliferative index (A), expressed as percent of cells positive for S-phase labeling with BrdU, and mean liver apoptotic index, expressed as percent of cells positive for apoptosis (B), in male Sprague Dawley rats (N = 10/treatment group) at various time points following a seven-day dietary exposure to either control diet or to diet containing K+ PFOS at either 20 ppm or 100 ppm. Open bars, strike bars, dotted bars, and solid bars denote 1, 28, 56, and 84 days following treatment, respectively. Error bars represent standard deviations. Statistical significance relative to control is indicated by asterisks with Dunnett’s t-test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) or by hash signs with Dunn’s test (# p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001).

In a 28-day dietary study reported in this issue (Elcombe et al., 2012), K+ PFOS elicited responses characteristic of mixed activation of PPAR␣ and CAR/PXR. The study reported herein evaluated resolution of K+ PFOS-induced, liver-related effects in male rats during an 84-day period following a 7-day dietary exposure to K+ PFOS at 20 or 100 ppm (w/w) in diet. This study focused on endpoints related to hepatocellular hypertrophy and included the activities of key marker enzymes associated with hepatic xenosensor nuclear receptors PPAR␣ (ACOX and CYP4A activities), CAR (CYP2B activity), and PXR (CYP3A activity) that exert ligand-specificity with

Table 8 Mean (±S.D.) PFOS concentrations in serum, liver, and liver cytosol, as well as liver-to-serum and liver-to-liver cytosol ratios in male Sprague Dawley rats at various time points following a seven-day dietary exposure to control diet or diet containing K+ PFOS at 20 ppm or 100 ppm. Days following treatment 1

28

56

84

Serum (␮g/mL)

20 ppm K+ PFOS 100 ppm K+ PFOS p-Valuea

39.49 ± 7.76 140.40 ± 14.05 ≤0.001

15.49 ± 1.60 53.82 ± 7.91 ≤0.001

8.03 ± 1.14 43.68 ± 5.51 ≤0.001

Liver (␮g/g)

20 ppm K+ PFOS 100 ppm K+ PFOS p-Value

123.92 ± 23.95 319.50 ± 46.64 ≤0.001

44.17 ± 4.36 169.30 ± 20.52 ≤0.001

32.99 ± 4.19 163.60 ± 36.99 ≤0.001

Cytosol (␮g/mL)

20 ppm K+ PFOS 100 ppm K + PFOS p-Value

12.85 ± 1.77 41.49 ± 8.56 ≤0.001

6.53 ± 1.71 24.05 ± 4.93 ≤0.001

4.24 ± 0.91 18.48 ± 4.18 ≤0.001

2.42 ± 0.75 8.75 ± 2.40 ≤0.001

Liver-to-serum ratio

20 ppm K+ PFOS 100 ppm K+ PFOS p-Value

3.2 ± 0.9 2.3 ± 0.5 ≤0.01

2.9 ± 0.5 3.2 ± 0.5 >0.05

4.1 ± 0.6 3.8 ± 0.8 >0.05

5.9 ± 1.2 3.4 ± 0.9 ≤0.001

Liver-to-cytosol ratio

20 ppm K+ PFOS 100 ppm K+ PFOS p-Value

9.8 ± 2.0 8.1 ± 2.4 >0.05

7.3 ± 2.3 7.2 ± 1.3 >0.05

8.0 ± 1.5 9.2 ± 2.5 >0.05

11.2 ± 3.5 10.3 ± 2.6 >0.05

a

Statistical significance level for the difference in values between the 20 ppm and 100 ppm dose groups based on two-sided Student’s t-test.

4.38 ± 0.72 25.79 ± 5.38 ≤0.001 24.99 ± 1.30 85.74 ± 16.33 ≤0.001

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C.R. Elcombe et al. / Toxicology 293 (2012) 30–40

PFOS (Ren et al., 2009; Rosen et al., 2009, 2010; Shipley et al., 2004; Takacs and Abbott, 2007; Vanden Heuvel et al., 2006; Wolf et al., 2008). These receptors play vital roles in the regulation of both intermediary metabolism and xenobiotic metabolism, and in the regulation of liver growth (Waxman, 1999). In rodent liver, the activation of xenosensor nuclear receptors, PPAR␣, CAR, and PXR may increase liver size by increasing replicative DNA synthesis (cell proliferation) and, sometimes in addition, by decreasing apoptosis (Klaunig et al., 2003; Lake, 2009). Seven days of dietary treatment with either 20 or 100 ppm K+ PFOS were sufficient to result in hepatic hypertrophy. Rats fed both K+ PFOS dietary concentrations and sacrificed on the first day of the recovery period following dietary treatment had increased relative liver weights as compared to controls. The latter finding was supported by microscopic observation of a dose-related increase in the incidence and severity of centrilobular hepatocellular hypertrophy. The decrease in hepatic DNA concentration and increase in hepatic P450 concentration also correlated well with hepatocellular hypertrophy observed on the first recovery day. In addition, the hepatic proliferative index was increased and the hepatic apoptotic index decreased in a dose-dependent manner, without a notable change in total DNA. Also at this time point, there was evidence in the 100 ppm group for induction of acyl CoA oxidase, lauric acid 12-hyroylase, pentoxyresorufin-O-depentylase, and testosterone 6 ␤-hydroxylase, indicative of activation by PFOS of the xenosensor nuclear receptors PPAR␣ and CAR/PXR. These changes were associated with mean serum PFOS concentrations of 39 and 140 ␮g/mL in the 20 and 100 ppm dose groups, respectively. Corresponding mean PFOS concentrations in liver and liver cytosol were 124 and 320 ␮g/g for liver and 13 and 41 ␮g/mL for liver cytosol. Although many of the hepatic responses observed on the first day of recovery attenuated over the course of the recovery period, minimal-to-mild centrilobular hepatocellular hypertrophy tended to persist. Mean absolute and relative liver weights had returned to control levels after 28 days of recovery. The apparent increase based on statistical significance in relative liver weight on recovery Day 84 likely was reflective of a heavier control body weight (558 g in control versus 524 g and 510 g in the 20 and 100 ppm dietary dose groups, respectively). This interpretation is supported by the lack of an increased relative liver weight on recovery Days 28 and 56. In addition, the relative liver weights for K+ PFOS-treated rats on recovery Day 84 were similar to those from controls and treated groups on recovery Day 56. Mean liver DNA concentration remained low in the 100 ppm group relative to controls through Day 56 of recovery. Mean liver P450 concentrations remained elevated relative to controls throughout the recovery period in both K+ PFOS-treated groups; although, they appeared to attenuate over time. Mean liver total DNA, similar to controls on recovery Day 1, was decreased at both treatment levels relative to controls on Days 28 and 56 of recovery, returning to control levels on recovery Day 84. The incidence and severity of centrilobular hepatocellular hypertrophy remained elevated and the incidence and severity glycogen-induced vacuolation remained decreased relative to controls in a dose-dependent manner through Day 84 of recovery. The enzyme activities responsive to PPAR␣ and CAR/PXR induction gradually attenuated during recovery. At the end of recovery Day 84, the activity of testosterone 6 ␤-hydroxylase remained elevated relative to controls, while activities of acyl CoA oxidase, lauric acid 12-hydroxylase, and pentoxyresorufin-O-depentylase approximated control levels of activity or were slightly lower than control levels. The persistent increase in P450 concentrations at the end of recovery corresponded to hepatocellular hypertrophy and may be the result of poor clearance of PFOS. The observed dose-dependent increase in liver proliferative index on recovery Day 1 was no longer present at other recovery time points. In contrast, the dose-dependent decreased liver

apoptotic index observed on recovery Day 1 persisted through the 84-day recovery period. Both of these effects are key events in the mode of action for rodent liver tumor formation for agents that activate PPAR␣ and CAR/PXR (Holsapple et al., 2006; Klaunig et al., 2003; Lake, 2009). The persistence of decreased hepatic apoptosis through 84 days of recovery in the present study suggests that damaged hepatocytes are not being removed and replaced and may increase the potential opportunity for clonal expansion of initiated/transformed hepatocytes on proliferative stimulus (Schattenberg et al., 2011). There were no significant elevations in liver enzymes (ALT or AST) during this study, suggesting absence of overt hepatic toxicity. A reduction of serum total cholesterol was evident at the beginning of the recovery period in K+ PFOS-treated rats of both dietary dose groups. This effect appeared to be stronger on Day 28 of recovery than on Day 1 and to return to levels similar to the controls on Days 56 and 84 of recovery; although, serum total cholesterol was reduced with significance in the 100 ppm group on recovery Day 84. The latter observation may represent a lingering depression of serum total cholesterol; however, it could also have been a chance observation, as a similar finding was not present on Day 56. Cholesterol lowering is consistent with previously reported observations (Bijland et al., 2011; Curran et al., 2008; Seacat et al., 2002, 2003) and likely is modulated, in part, by enhanced PPARmediated fatty acid oxidation. The attenuation of effects in recovery followed diminishing concentrations of PFOS in serum, liver, and liver cytosol. Similar to the previously reported serum elimination half-lives for PFOS (Benskin et al., 2009; Chang et al., in press), the serum PFOS elimination halflives during the 84-day recovery phase in the study reported herein were of the order of one month. Liver PFOS elimination half-lives were on the order of two months. Of most consequence from a potential risk assessment perspective is the persistent, dose-dependent decrease in liver apoptotic index. Decreased apoptosis factors into the mode of action for nongenotoxic carcinogens as it may lead to increased populations of mutated cells which, on proliferative stimulus, may fix and promote the mutations, thereby increasing cancer risk. In the course of this study, K+ PFOS did not appear to have any effect on the thyroid parameters evaluated (H&E histology, follicular epithelial cell proliferation, and follicular epithelial apoptosis). This is an interesting observation considering the fact that treatment with K+ PFOS resulted in activation of PPAR␣ and CAR/PXR in this study as well as in the 28-day study also reported in this issue (Elcombe et al., 2012). As discussed in detail in the just-cited article, other compounds that increase the expression of PPAR␣, CAR, and PXR have been shown to be capable of stimulating thyroid follicular epithelial proliferation in rats through a combination of increased phase II conjugation, increased expression of liver transport systems responsible for hepatocellular uptake of circulating thyroid hormones, as well as increased biliary efflux of conjugated and unconjugated hormones (Klaassen and Hood, 2001; Qatanani et al., 2005; Vansell and Klaassen, 2001, 2002a,b; Wieneke et al., 2009; Wong et al., 2005). In fact, in the 28-day study (Elcombe et al., 2012), both Wy 14,643 and phenobarbital, model activators of PPAR␣ and CAR/PXR, respectively, increased the thyroid follicular proliferation index as measured by BrdU incorporation; whereas, as in the present study, K+ PFOS did not. PFOS treatment in rats has been shown to reduce circulating thyroid hormone via displacement of thyroxine (T4) from serum binding sites and increased turnover in liver (Chang et al., 2008; Yu et al., 2009, 2011); however, in those studies, serum TSH and release of TSH from the pituitary were unaffected, suggesting maintenance of thyroid hormone homeostasis. It is possible that PFOS either does not induce or only weakly induces the specific enzymes and/or transporters responsible for the PB- and Wy 14,643-mediated increase in thyroid follicular

C.R. Elcombe et al. / Toxicology 293 (2012) 30–40

hyperplasia. It has been shown in rats that inducing compounds such as pregnenalone-16␣-carbonitrile (PCN, a specific PXR agonist) that increase serum TSH concentrations leading to stimulation of proliferation in the thyroid follicular epithelium do so by increasing the glucuronidation of triiodothyronine (T3); whereas, inducing compounds that only increase the glucuronidation of T4 do not produce an associated TSH increase (Klaassen and Hood, 2001). It has been demonstrated that the specific PPAR␣ agonist, nafenopin, like PFOS, is a weak competitor for T4 binding in serum which increases plasma free T4 as well as plasma and fecal clearance of T4 even in the presence of decreased 5 -deiodination (Kaiser et al., 1988); however, at the same time, plasma clearance of T3 decreased and TSH levels were maintained at euthyroid levels. Kaiser et al. suggested that increased T4 glucoronidation and biliary elimination explained the decrease in total T4. Therefore, it is possible that differences exist between PFOS and the inducers Wy 14,643 and phenobarbital relative to competition for serum T4 binding and/or induction in specific glucuronidation pathways for T4 and T3. In conclusion, the principal objective of this study was to evaluate the reversibility of the inductive effects of K+ PFOS treatment in rats over an 84-day period following seven days of dietary treatment at dose levels used in the 28-day study reported also in this issue (Elcombe et al., 2012). As in the 28-day study, after seven days of feeding K+ PFOS to male rats in the study reported herein, K+ PFOS exhibited the combined hepatic effects of PPAR␣ and CAR/PXR induction. Thus, the results obtained from this study offer further evidence supporting the role of K+ PFOS-mediated induction of PPAR␣ and CAR/PXR in the etiology of hepatocellular adenoma observed in the 104-week bioassay with K+ PFOS in Sprague Dawley rats (Butenhoff et al., 2012). Many of the K+ PFOSinduced effects present one day after the 7-day treatment period (recovery Day 1) resolved toward or returned to control levels during the 84-day recovery period. These included: hepatocellular proliferative index; activities associated with ACOX, CYP4A, and CYP2B; and liver weight. To an extent, these changes followed the elimination of PFOS anion from serum and liver over the 84-day period. Mean serum PFOS concentrations on recovery Day 1 were 39 and 140 ␮g/mL (20 ppm and 100 ppm K+ PFOS, respectively), decreasing to 4 and 26 ␮g/mL by recovery Day 84. Liver PFOS concentrations were generally 3–5 times those of the corresponding serum concentrations. However, several K+ PFOS-induced effects did not appear to subside significantly during the 84 days of recovery. These included: hepatocellular apoptotic index; centrilobular hepatocellular hypertrophy; liver microsomal total CYP450; and lowered cholesterol. Thus, hepatic effects in male rats resulting from K+ PFOS-induced activation of PPAR␣ and CAR/PXR resolved slowly or were still present after 84-days following a 7-day dietary treatment, consistent with the slow elimination rate of PFOS. Conflicts of interest John L. Butenhoff, Shu-Ching Chang, and David J. Ehresman are employed by 3M Company, a former manufacturer of PFOS and its salts as well as compounds that can degrade to PFOS. Cliff R. Elcombe and Barbara M. Elcombe (CXR Biosciences Ltd.), John R. Foster (AstraZeneca Pharmaceuticals), and Patricia E. Noker (Southern Research Institute) do not have competing interests other than employment in contract research work or data analyses for this study. Acknowledgments This work was funded by 3M Company. We wish to thank Dr. med. Vet. Ortwin Vogel (Toxicologic Pathology Consultancy) for his expert pathological evaluation of H&E stained tissues.

39

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