Perfluorooctanesulfonate (PFOS) Conversion from N-Ethyl-N-(2-hydroxyethyl)-perfluorooctanesulfonamide (EtFOSE) in male Sprague Dawley rats after inhalation exposure.

Ethyl-N-(2-hydroxyethyl)-perfluorooctanesulfonamide (EtFOSE) was one of the key building blocks for many of the perfluorooctanesulfonyl-based chemistry and laboratory studies have shown that EtFOSE can metabolically degrade to perfluorooctanesulfonate (PFOS). Non-occupational contribution sources to PFOS are thought to occur in general population via diets, drinking water, air and dust. For workers, however, the exposure route was mostly airborne and the exposure source was predominantly to precursor compounds such as EtFOSE. We undertook this study to investigate how much EtFOSE was converted to PFOS in the serum for male rats after 6h of exposure to EtFOSE vapor (whole body) at ambient temperature, which simulated a work place exposure scenario. There were no abnormal clinical observations and all rats gained weight during study. Interim tail-vein blood samples, collected up to 21 days after exposure, were analyzed for Et-FOSE and PFOS concentrations by LC-MS/MS. Upon inhalation exposure, the biotransformation of EtFOSE to PFOS in serum in the male rats was rapid and very little EtFOSE was detected in the serum within 24h after EtFOSE exposure. The highest conversion to PFOS in serum after exposure to EtFOSE vapor appeared to occur between Day 8-14 post exposure. Considering the potential surface and fur adsorption of test compound in the whole-body exposure system, our data would support that at least 10% of the inhaled EtFOSE was biotransformed to PFOS in the serum based on the range of lower 95% CI (confidence interval) values. This information is valuable because it quantitatively translates EtFOSE exposure into serum PFOS concentration, which serves as a matrix for internal dosimetry (of PFOS exposure) that can be used as an anchor across species as well as between different exposure routes.

Four-week dietary supplementation with 10- and/or 15-fold basal choline caused decreased body weight in Sprague Dawley rats.

Choline is an essential nutrient utilized for phosphatidylcholine biosynthesis and lipoprotein packaging and secretion. Recently, choline supplementation has been used by athletes and the public for weight loss. However, the potential toxicological impact of choline dietary supplementation requires further investigation. This study examined the effects of choline dietary supplementation in Sprague Dawley rats for 4 weeks. Rats were fed diets containing basal choline levels (control) or 5-, 10-, or 15-fold (5×, 10×, or 15×) basal diet concentration. In groups fed choline-supplemented diets, there were no toxicologically relevant findings in clinical observations, food intake, clinical chemistry, liver weights, or liver histopathology. However, decreased mean body weights (8.5-10.2%) and body weight gains (24-31%) were noted for the 10× choline-supplemented (females only) and 15× choline-supplemented (both sexes) groups relative to the control groups from day 3 onward. These body weight effects were not related to a persistent reduction in average food intake. Serum cholesterol was increased in the 15× choline-supplemented male rats relative to the controls, an expected effect of choline supplementation; however, there were no changes in the serum cholesterol of female rats. Serum choline concentrations were increased in female rats relative to the male rats across all treatment groups. The maximum tolerated dose for male and female rats were the 15× and 10× choline supplements, respectively, based on decreased mean body weight and body weight gains. This study supported the conclusions of a clinical trial that showed a high choline diet can decrease body weight in humans.

A comparison of the pharmacokinetics of perfluorobutanesulfonate (PFBS) in rats, monkeys, and humans.

Materials derived from perfluorobutanesulfonyl fluoride (PBSF, C(4)F(9)SO(2)F) have been introduced as replacements for eight-carbon homolog products that were manufactured from perfluorooctanesulfonyl fluoride (POSF, C(8)F(17)SO(2)F). Perfluorobutanesulfonate (PFBS, C(4)F(9)SO(3)(-)) is a surfactant and potential degradation product of PBSF-derived materials. The purpose of this series of studies was to evaluate the pharmacokinetics of PFBS in rats, monkeys, and humans, thereby providing critical information for human health risk assessment. Studies included: (1) intravenous (i.v.) elimination studies in rats and monkeys; (2) oral uptake and elimination studies in rats; and (3) human serum PFBS elimination in a group of workers with occupational exposure to potassium PFBS (K(+)PFBS). PFBS concentrations were determined in serum (all species), liver (rats), urine (all species), and feces (rats). In rats, the mean terminal serum PFBS elimination half-lives, after i.v. administration of 30mg/kg PFBS, were: males 4.51+/-2.22h (standard error) and females 3.96+/-0.21h. In monkeys, the mean terminal serum PFBS elimination half-lives, after i.v. administration of 10mg/kg PFBS, were: males 95.2+/-27.1h and females 83.2+/-41.9h. Although terminal serum half-lives in male and female rats were similar, without statistical significance, clearance (CL) was significantly greater in female rats (469+/-40mL/h) than male rats (119+/-34mL/h) with the area under the curve (AUC) significantly larger in male rats (294+/-77microg.h/mL) than female rats (65+/-5microg.h/mL). These differences were not observed in male and female monkeys. Volume of distribution estimates suggested distribution was primarily extracellular in both rats and monkeys, regardless of sex, and urine appeared to be a major route of elimination. Among 6 human subjects (5 male, 1 female) followed up to 180 days, the geometric mean serum elimination half-life for PFBS was 25.8 days (95% confidence interval 16.6-40.2). Urine was observed to be a pathway of elimination in the human. Although species-specific differences exist, these findings demonstrate that PFBS is eliminated at a greater rate from human serum than the higher chain homologs of perfluorooctanesulfonate (PFOS) and perfluorohexanesulfonate (PFHxS). Thus, compared to PFOS and PFHxS, PFBS has a much lower potential for accumulation in human serum after repeated occupational, non-occupational (e.g., consumer), or environmental exposures.

Thyroid hormone status and pituitary function in adult rats given oral doses of perfluorooctanesulfonate (PFOS).

INTRODUCTION: Perfluorooctanesulfonate (PFOS) is widely distributed and persistent in humans and wildlife. Prior toxicological studies have reported decreased total and free thyroid 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 24h thereafter, measurements were made for serum FT4, TT4, triiodothyronine (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), 125 I-T4 was given to male and female SD rats by intravenous injection, followed in 2h by a single oral dose of 15 mg potassium PFOS/kg body weight. 125 I radioactivity was determined in urine and feces collected over a 24-h period and in serum and liver collected at 24h. (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 (3mg/kg-d), propyl thiouracil (PTU, 10 microg/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 6h, with values returning to control levels by 24h. TT4 was decreased by 55% over a 24-h period. TT3 and rT3 were decreased at 24h to a lesser extent than TT4. ME mRNA transcripts were increased at 2h and activity was increased at 24h. UGT1A mRNA transcripts were increased at 2 and 6h. (2) 125 I decreased in serum and liver relative to controls and consistent with a reduction in serum TT4. Concomitantly, 125 I 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 thyroid hormones 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.

An interlaboratory study of perfluorinated alkyl compound levels in human plasma.

We conducted an interlaboratory study which differed from the typical study of this type because of its emphasis on comparing intralaboratory variability in results. We sent specimens to six laboratories experienced in the analysis of perfluorinated alkyl compounds in blood matrices and that use stringent procedures to control and assure accuracy and precision. Each received an identical set of 60 plasma specimens that were analyzed in six completely independent batches. Split specimens were included so that within- and between-batch coefficients of variation could be calculated. All laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorohexanesulfonate (PFHxS) measured in the specimens in general showed a high level of agreement, although in some cases the agreement was only moderate. The average within- and between-batch coefficient of variation for PFOS was 9.1% and 9.3%; for PFOA was 14.5% and 14.5%; and for PFHxS was 14.5% and 17.0%. The recent availability of labeled internal standards, among other advances, has facilitated improvement in the accuracy and precision of the assays. Considering the degree of between-subject variation in levels among people in background-exposed populations, the results indicate that biomarker-based epidemiologic studies of associations with health could have reasonable precision.

Half-life of serum elimination of perfluorooctanesulfonate,perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers.

BACKGROUND: The presence of perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate (PFHS), and perfluorooctanoate (PFOA) has been reported in humans and wildlife. Pharmacokinetic differences have been observed in laboratory animals. OBJECTIVE: The purpose of this observational study was to estimate the elimination half-life of PFOS, PFHS, and PFOA from human serum. METHODS: Twenty-six (24 male, 2 female) retired fluorochemical production workers, with no additional occupational exposure, had periodic blood samples collected over 5 years, with serum stored in plastic vials at -80 degrees C. At the end of the study, we used HPLC-mass spectrometry to analyze the samples, with quantification based on the ion ratios for PFOS and PFHS and the internal standard (18)O(2)-PFOS. For PFOA, quantitation was based on the internal standard (13)C(2)-PFOA. RESULTS: THE ARITHMETIC MEAN INITIAL SERUM CONCENTRATIONS WERE AS FOLLOWS: PFOS, 799 ng/mL (range, 145-3,490); PFHS, 290 ng/mL (range, 16-1,295); and PFOA, 691 ng/mL (range, 72-5,100). For each of the 26 subjects, the elimination appeared linear on a semi-log plot of concentration versus time; therefore, we used a first-order model for estimation. The arithmetic and geometric mean half-lives of serum elimination, respectively, were 5.4 years [95% confidence interval (CI), 3.9-6.9] and 4.8 years (95% CI, 4.0-5.8) for PFOS; 8.5 years (95% CI, 6.4-10.6) and 7.3 years (95% CI, 5.8-9.2) for PFHS; and 3.8 years (95% CI, 3.1-4.4) and 3.5 years (95% CI, 3.0-4.1) for PFOA. CONCLUSIONS: Based on these data, humans appear to have a long half-life of serum elimination of PFOS, PFHS, and PFOA. Differences in species-specific pharmacokinetics may be due, in part, to a saturable renal resorption process.

Negative bias from analog methods used in the analysis of free thyroxine in rat serum containing perfluorooctanesulfonate (PFOS).

Decreases in serum total thyroxine (TT4) and free thyroxine (FT4) without a compensatory rise in thyroid stimulating hormone (thyrotropin or TSH) or histological changes of the thyroid have been observed in studies with perfluorooctanesulfonate (PFOS) treatments in rats. Prior observations do not fit the clinical profile of a hypothyroid state. PFOS is known to compete with fatty acids for albumin binding, and serum free fatty acids (FFA) are known to interfere with FT4 measurement using analog methods due to competition for protein binding. Therefore, we hypothesized that measured decreases in serum FT4 by analog methods in the presence of PFOS were due to carrier protein binding interference. We compared FT4 analog assay methods with a reference method using equilibrium dialysis (ED-RIA) for FT4 measurement in rat sera in vitro and in vivo. We also measured hepatic malic enzyme mRNA transcripts and activity as a marker for hepatic thyroid hormone response. PFOS did not reduce serum TT4 and FT4 in vitro at concentrations up to 200 microM. After three daily 5mg/kg oral doses of potassium PFOS to female rats, serum TSH and FT4 by ED-RIA were unchanged (although FT4 determined by two common analog methods was decreased), and malic enzyme was not suppressed. These data suggest that prior reports of reduced free thyroid hormone in the presence of PFOS were due to negative bias in analog methods and that short-term PFOS treatment does not suppress the physiological thyroid status in rats. A reference method such as ED-RIA should be used for determination of serum FT4 in the presence of PFOS.

Preliminary evidence of a decline in perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) concentrations in American Red Cross blood donors.

The purpose of this pilot study was to determine whether perfluorooctanesulfonate (PFOS,C(8)F(17)SO(3)(-)) and perfluorooctanoate (PFOA,C(7)F(15)CO(2)(-)) concentrations in American Red Cross blood donors from Minneapolis-St. Paul, Minnesota have declined after the 2000-2002 phase-out of perfluorooctanesulfonyl-fluoride (POSF, C(8)F(17)SO(2)F)-based materials by the primary global manufacturer, 3M Company. Forty donor plasma samples, categorized by age and sex, were collected in 2005, and PFOS and PFOA concentrations were compared to 100 (non-paired) donor serum samples collected in 2000 from the same general population that were analyzed at the time using ion-pair extraction methods with tetrahydroperfluorooctanesulfonate as an internal standard. Eleven of the 100 samples originally collected were reanalyzed with present study methods that involved (13)C- labeled PFOA spiked into the donor samples, original samples, control human plasma, and the calibration curve prior to extraction, and was used as a surrogate to monitor extraction efficiency. Quantification was performed by high performance liquid chromatography tandem mass spectrometry methods. Among the 100 serum samples analyzed for PFOS, the geometric mean was 33.1 ng ml(-1) (95% CI 29.8-36.7) in 2000 compared to 15.1 ng ml(-1) (95% CI 13.3-17.1) in 2005 (p<0.0001) for the 40 donor plasma samples. The geometric mean concentration for PFOA was 4.5 ng ml(-1) (95% CI 4.1-5.0) in 2000 compared to 2.2 ng ml(-1) (95% CI 1.9-2.6) in 2005 (p<0.0001). The decrease was consistent across donors' age and sex. To confirm these preliminary findings, additional sub-sets of year 2000 samples will be analyzed, and a much larger biomonitoring study of other locations is planned.

Organic Anion Transporting Polypeptides Contribute to the Disposition of Perfluoroalkyl Acids in Humans and Rats.

Perfluoroalkyl sulfonates (PFSAs) such as perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonate (PFOS) have very long serum elimination half-lives in humans, and preferentially distribute to serum and liver. The enterohepatic circulation of PFHxS and PFOS likely contributes to their extended elimination half-lives. We previously demonstrated that perfluorobutane sulfonate (PFBS), PFHxS, and PFOS are transported into hepatocytes both in a sodium-dependent and a sodium-independent manner. We identified Na+/taurocholate cotransporting polypeptide (NTCP) as the responsible sodium-dependent transporter. Furthermore, we demonstrated that the human apical sodium-dependent bile salt transporter (ASBT) contributes to the intestinal reabsorption of PFOS. However, so far no sodium-independent uptake transporters for PFSAs have been identified in human hepatocytes or enterocytes. In addition, perfluoroalkyl carboxylates (PFCAs) with 8 and 9 carbons were shown to preferentially distribute to the liver of rodents; however, no rat or human liver uptake transporters are known to transport these PFCAs. Therefore, we tested whether PFBS, PFHxS, PFOS, and PFCAs with 7-10 carbons are substrates of organic anion transporting polypeptides (OATPs). We used CHO and HEK293 cells to demonstrate that human OATP1B1, OATP1B3, and OATP2B1 can transport PFBS, PFHxS, PFOS, and the 2 PFCAs (C8 and C9). In addition, we show that rat OATP1A1, OATP1A5, OATP1B2, and OATP2B1 transport all 3 PFSAs. In conclusion, our results suggest that besides NTCP and ASBT, OATPs also are capable of contributing to the enterohepatic circulation and extended human serum elimination half-lives of the tested perfluoroalkyl acids.

Evaluation of Serum Lipid, Thyroid, and Hepatic Clinical Chemistries in Association With Serum Perfluorooctanesulfonate (PFOS) in Cynomolgus Monkeys After Oral Dosing With Potassium PFOS.

An oral dose study with perfluorooctanesulfonate (PFOS) was undertaken to identify potential associations between serum PFOS and changes in serum clinical chemistry parameters in purpose-bred young adult cynomolgus monkeys (Macaca fascicularis). In this study, control group (n = 6/sex) was sham-dosed with vehicle (0.5% Tween 20 and 5% ethanol in water), low-dose group (n = 6/sex) received 1 single K+PFOS dose (9 mg/kg), and high-dose group (n = 4-6/sex) received 3 separate K+ PFOS doses (11-17.2 mg/kg). Monkeys were given routine checkups and observed carefully for health problems on a daily basis. Scheduled blood samples were drawn from all monkeys prior to, during, and after K+PFOS administration for up to 1 year and they were analyzed for PFOS concentrations and clinical chemistry markers for coagulation, lipids, hepatic, renal, electrolytes, and thyroid-related hormones. No mortality occurred during the study. All the monkeys were healthy, gained weight, and were released back to the colony at the end of the study. The highest serum PFOS achieved was approximately 165 µg/ml. When compared with time-matched controls, administration of K+PFOS to monkeys did not result in any toxicologically meaningful or clinically relevant changes in serum clinical measurements for coagulation, lipids, hepatic, renal, electrolytes, and thyroid-related hormones. A slight reduction in serum cholesterol (primarily the high-density lipoprotein fraction), although not toxicologically significant, was observed. The corresponding lower-bound fifth percentile benchmark concentrations (BMCL1sd) were 74 and 76 µg/ml for male and female monkeys, respectively. Compared to the 2013-2014 geometric mean serum PFOS level of 4.99 ng/ml (0.00499 µg/ml) in US general population reported by CDC NHANES, this represents 4 orders of magnitude for margin of exposure.

Perfluorooctane Sulfonate-Induced Hepatic Steatosis in Male Sprague Dawley Rats Is Not Attenuated by Dietary Choline Supplementation.

Perfluorooctane sulfonate (PFOS) is an environmentally persistent chemical. Dietary 100 ppm PFOS fed to male mice and rats for 4 weeks caused hepatic steatosis through an unknown mechanism. Choline deficient diets can cause hepatic steatosis. A hepatic choline:PFOS ion complex was hypothesized to cause this effect in mice. This study tested whether dietary choline supplementation attenuates PFOS-induced hepatic steatosis in rats. Sprague Dawley rats (12/sex/group) were fed control, choline supplemented (CS), 100 ppm PFOS, or 100 ppm PFOS + CS diets for 3 weeks. Male rats fed both PFOS-containing diets had decreased serum cholesterol and triglycerides (TGs) on days 9, 16, and/or 23 and increased hepatic free fatty acids and TG (ie, steatosis). Female rats fed both PFOS diets had decreased serum cholesterol on days 9 and 16 and decreased hepatic free fatty acid and TG at termination (ie, no steatosis). Liver PFOS concentrations were similar for both sexes. Liver choline concentrations were increased in male rats fed PFOS (±CS), but the increase was lower in the PFOS + CS group. Female liver choline concentrations were not altered by any diet. These findings demonstrate a clear sex-related difference in PFOS-induced hepatic steatosis in the rat. Additional evaluated mechanisms (ie, nuclear receptor activation, mRNA upregulation, and choline kinase activity inhibition) did not appear to be involved in the hepatic steatosis. Dietary PFOS (100 ppm) induced hepatic steatosis in male, but not female, rats that was not attenuated by choline supplementation. The mechanism of lipid accumulation and the sex-related differences warrant further investigation.

Editor's Highlight: Perfluorooctane Sulfonate-Choline Ion Pair Formation: A Potential Mechanism Modulating Hepatic Steatosis and Oxidative Stress in Mice.

The mechanisms underlying perfluorooctane sulfonate (PFOS)-induced steatosis remain unclear. The hypothesis that PFOS causes steatosis and other hepatic effects by forming an ion pair with choline was examined. C57BL/6 mice were fed either a control diet or a marginal methionine/choline-deficient (mMCD) diet, with and without 0.003, 0.006, or 0.012% potassium PFOS. Dietary PFOS caused a dose-dependent decrease in body weight, and increases in the relative liver weight, hepatic triglyceride concentration and serum markers of liver toxicity and oxidative stress. Some of these effects were exacerbated in mice fed the mMCD diet supplemented with 0.012% PFOS compared with those fed the control diet supplemented with 0.012% PFOS. Surprisingly, serum PFOS concentrations were higher while liver PFOS concentrations were lower in mMCD-fed mice compared with corresponding control-fed mice. To determine if supplemental dietary choline could prevent PFOS-induced hepatic effects, C57BL/6 mice were fed a control diet, or a choline supplemental diet (1.2%) with or without 0.003% PFOS. Lipidomic analysis demonstrated that PFOS caused alterations in hepatic lipid metabolism in the PFOS-fed mice compared with controls, and supplemental dietary choline prevented these PFOS-induced changes. Interestingly, dietary choline supplementation also prevented PFOS-induced oxidative damage. These studies are the first to suggest that PFOS may cause hepatic steatosis and oxidative stress by effectively reducing the choline required for hepatic VLDL production and export by forming an ion pair with choline, and suggest that choline supplementation may prevent and/or treat PFOS-induced hepatic steatosis and oxidative stress.

Na+/Taurocholate Cotransporting Polypeptide and Apical Sodium-Dependent Bile Acid Transporter Are Involved in the Disposition of Perfluoroalkyl Sulfonates in Humans and Rats.

Among the perfluoroalkyl sulfonates (PFASs), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) have half-lives of several years in humans, mainly due to slow renal clearance and potential hepatic accumulation. Both compounds undergo enterohepatic circulation. To determine whether transporters involved in the enterohepatic circulation of bile acids are also involved in the disposition of PFASs, uptake of perfluorobutane sulfonate (PFBS), PFHxS, and PFOS was measured using freshly isolated human and rat hepatocytes in the absence or presence of sodium. The results demonstrated sodium-dependent uptake for all 3 PFASs. Given that the Na(+)/taurocholate cotransporting polypeptide (NTCP) and the apical sodium-dependent bile salt transporter (ASBT) are essential for the enterohepatic circulation of bile acids, transport of PFASs was investigated in stable CHO Flp-In cells for human NTCP or HEK293 cells transiently expressing rat NTCP, human ASBT, and rat ASBT. The results demonstrated that both human and rat NTCP can transport PFBS, PFHxS, and PFOS. Kinetics with human NTCP revealed Km values of 39.6, 112, and 130 µM for PFBS, PFHxS, and PFOS, respectively. For rat NTCP Km values were 76.2 and 294 µM for PFBS and PFHxS, respectively. Only PFOS was transported by human ASBT whereas rat ASBT did not transport any of the tested PFASs. Human OSTα/ß was also able to transport all 3 PFASs. In conclusion, these results suggest that the long half-live and the hepatic accumulation of PFOS in humans are at least, in part, due to transport by NTCP and ASBT.

Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid.

Previous studies suggested that perfluorooctanoate (PFOA) could activate the estrogen receptor (ER). The present study examined the hypothesis that PFOA can activate ER using an in vivo uterotrophic assay in CD-1 mice and an in vitro reporter assay. Pre-pubertal female CD-1 mice fed an estrogen-free diet from postnatal day (PND)14 through weaning on PND18 were administered 0, 0.005, 0.01, 0.02, 0.05, 0.1, or 1mg/kg PFOA or 17ß-estradiol (E2, 0.5mg/kg) from PND18-20. In contrast to E2, PFOA caused no changes in the relative uterine weight, the expression of ER target genes, or the morphology of the uterus/cervix and/or vagina on PND21. Treatment of a stable human cell line containing an ER-dependent luciferase reporter construct with a broad concentration range of PFOA caused no change in ER-dependent luciferase activity; whereas E2 caused a marked increase of ER-dependent luciferase activity. These data indicate that PFOA does not activate mouse or human ER.

A species difference in the peroxisome proliferator-activated receptor α-dependent response to the developmental effects of perfluorooctanoic acid.

This study examined the effect of prenatal perfluorooctanoic acid (PFOA) administration on pre- and postnatal development using peroxisome proliferator-activated receptor α (PPARα)-humanized mice to determine if species differences in receptor activity might influence the developmental effects induced by PFOA. Pregnant mice were treated daily with water or PFOA (3mg/kg) by po gavage from gestation day 1 (GD1) until GD17 and then either euthanized on GD18 or allowed to give birth and then euthanized on postnatal day 20 (PND20). No changes in average fetal weight, crown-to-rump length, or placental weight were observed on GD18. Expression of mRNA encoding the PPARα target genes acyl CoA oxidase (Acox1) and cytochrome P450 4a10 (Cyp4a10) in maternal and fetal liver was increased on GD18 in wild-type and PPARα-humanized mice but not in Pparα-null mice. On PND20, relative liver weight was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice. Hepatic expression of Acox1 and Cyp4a10 mRNA was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice on PND20. The percentage of mice surviving postnatally was lower in wild-type litters but not in litters from Pparα-null mice or PPARα-humanized mice. No changes in pup weight gain, onset of eye opening, or mammary gland development were found in any genotype. Results from these studies demonstrate that the developmental/postnatal effects resulting from prenatal PFOA exposure in mice are differentially mediated by mouse and human PPARα.

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.

In a prior 28-day dietary study in rats with 20 and 100 ppm K⁺ PFOS, activation of PPARα 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 PPARα 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 µg/mL (20 ppm and 100 ppm K⁺ PFOS, respectively), decreasing to 4 and 26 µg/mL by recovery Day 84. Thus, hepatic effects in male rats resulting from K⁺ PFOS-induced activation of PPARα 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.

Hepatocellular hypertrophy and cell proliferation in Sprague-Dawley rats from dietary exposure to potassium perfluorooctanesulfonate results from increased expression of xenosensor nuclear receptors PPARα and CAR/PXR.

The present study investigated the potential role for activation of PPARα and CAR/PXR by potassium PFOS (K⁺ PFOS) with respect to the etiology of hepatic hypertrophy and hepatocellular adenoma in rats. Male Sprague-Dawley rats were fed K⁺ PFOS (20 or 100 ppm) for either 1, 7, or 28 days. Wyeth 14,643 (Wy 14,643, 50 ppm) and phenobarbital (PB, 500 ppm) were the controls for PPARα and CAR/PXR activation, respectively. Measurements included: plasma ALT, AST, cholesterol, triglycerides, and glucose; liver protein and DNA content; liver activities of palmitoyl CoA oxidase (ACOX), Cyp4A, CYP2B, and CYP3A; induction of liver CYP4A1, CYP2E1, CYP2B1/2, and CYP3A1 proteins (SDS-PAGE and Western blots); liver and thyroid microscopic histopathology, apoptotic index, and cell proliferation index. Terminal body weight was decreased by K⁺ PFOS (100 ppm) and Wy 14,643. All test-compound treatments increased liver weight. Plasma lipids were decreased by both PFOS and Wy 14,643. After treatment for 1 day, K⁺ PFOS (100 ppm), PB, and Wy 14,643 increased mean hepatic DNA concentration and total hepatic DNA, and total DNA remained elevated after treatment for 7 days and 28 days (PB and Wy 14,643 only). Hepatic P450 concentration was elevated after 7 and 28 days by K⁺ PFOS and by PB. K⁺ PFOS and Wy 14,643 increased liver activities of ACOX and CYP4A as well as increased liver CYP4A1 protein. By 28 days of treatment, K⁺ PFOS and PB increased liver activities of CYP2B and CYP3A as well as increased liver CYP2B1/2 and CYP3A1 proteins, and Wy 14,643 increased CYP2B enzyme activity to a slight extent. All test compounds increased the liver cell proliferative index and decreased the liver apoptotic index. No histological changes of the thyroid were noted; however, PB and WY increased thyroid follicular cell proliferation index (seven-day treatment only), while K⁺ PFOS did not. The thyroid follicular cell apoptotic index did not differ between groups. The hepatomegaly and hepatocellular adenoma observed after dietary exposure of Sprague-Dawley rats to K⁺ PFOS likely are due to the increased expression of xenosensor nuclear receptors PPARα and CAR/PXR. Given the markedly lower or absent response of human hepatocytes to the proliferative stimulus from activation of PPARα and CAR/PXR, the hepatocellular proliferative response from activation of these receptors by PFOS observed in rats is not expected to be of human relevance.

Distribution of perfluorooctanesulfonate and perfluorooctanoate into human plasma lipoprotein fractions.

Some cross-sectional epidemiological studies have reported positive associations of serum concentrations of non-high density lipoprotein cholesterol with serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA). However, the strength of the reported associations is inconsistent for exposure-response across three orders of magnitude of serum PFOS and/or PFOA concentrations. These positive associations are unexpected based on toxicological/mechanistic studies, suggesting that the associations may have a biological, rather than a causal, basis. This study tested the hypothesis that PFOS and PFOA distribute into serum lipoprotein fractions such that increases in serum lipoproteins would result in corresponding increases in serum concentrations of PFOS and PFOA. Based on observed binding of PFOS and PFOA to isolated ß-lipoproteins in physiological saline (96% and 40% bound, respectively) in preliminary experiments using ultrafiltration and LC-MS/MS methods, binding to human donor plasma lipoprotein fractions was investigated by two density gradient methods. The majority of PFOS and PFOA recovered masses were found in lipoprotein-depleted plasma. Plasma density gradient fractionation data suggested that maximally 9% of PFOS distributes to lipoprotein-containing fractions, yet only 1% or less of PFOA is so distributed. These data do not support a strong role for plasma lipoprotein fractions in explaining the inconsistent dose-response associations reported in cross-sectional epidemiological studies.

Comparative pharmacokinetics of perfluorohexanesulfonate (PFHxS) in rats, mice, and monkeys.

Perfluorohexanesulfonate (PFHxS) has been found in biological samples from wildlife and humans. The human geometric mean serum PFHxS elimination half-life has been estimated to be 2665days. A series of studies was undertaken to establish pharmacokinetic parameters for PFHxS in rats, mice, and monkeys after single administration with pharmacokinetic parameters determined by WinNonlin(®) software. Rats and mice appeared to be more effective at eliminating PFHxS than monkeys. With the exception of female rats, which had serum PFHxS elimination half-life of approximately 2 days, the serum elimination half-lives in the rodent species and monkeys approximated 1month and 4months, respectively, when followed over extended time periods (10-24weeks). Collectively, these studies provide valuable insight for human health risk assessment regarding the potential for accumulation of PFHxS in humans.

Comparative pharmacokinetics of perfluorooctanesulfonate (PFOS) in rats, mice, and monkeys.

Perfluorooctanesulfonate (PFOS) has been found in biological samples in wildlife and humans. The geometric mean half-life of serum elimination of PFOS in humans has been estimated to be 4.8 years (95% CI, 4.0-5.8). A series of studies was undertaken to establish pharmacokinetic parameters for PFOS in rats, mice, and monkeys after single oral and/or IV administration of K(+)PFOS. Animals were followed for up to 23 weeks, and pharmacokinetic parameters were determined by WinNonlin® software. Rats and mice appeared to be more effective at eliminating PFOS than monkeys. The serum elimination half-lives in the rodent species were on the order of 1-2 months; whereas, in monkeys, the serum elimination half lives approximated 4 months. Collectively, these studies provide valuable insight for human health risk assessment regarding the potential for accumulation of body burden in humans on repeated exposure to PFOS and PFOS-generating materials.