Environ. Sci. Technol. 2009, 43, 8416–8422
Prenatal and Postnatal Impact of Perfluorooctane Sulfonate (PFOS) on Rat Development: A Cross-Foster Study on Chemical Burden and Thyroid Hormone System
PND 0. Only transcript level of transthyretin, TH binding protein, in group TT significantly increased to 150% of the control on PND 21. The results showed that prenatal PFOS exposure and postnatal PFOS exposure induced hypothyroxinemia in rat pups to a similar extent, which suggested that in utero PFOS exposure and postnatal PFOS accumulation, especially though maternal milk, are matters of great concern.
WEN-GUANG YU,† WEI LIU,† Y I - H E J I N , * ,† X I A O - H U I L I U , † F A - Q I W A N G , †,‡ L I L I U , ‡ A N D SHOJI F. NAKAYAMA§ School of Environmental and Biological Science and Technology, Dalian University of Technology, Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, Linggong Road 2, Dalian 116024, China, Division of Hygienic Toxicology, School of Public Health, China Medical University, North 2 Road 92, Shenyang, Liaoning 110001, China, and National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive, MS 190, Cincinnati, Ohio 45268
Perfluorooctane sulfonate (C8F17SO3-, PFOS), manufactured for over 50 years, is an environmentally and biologically stable compound used widely in industrial and household applications. Toxicological studies suggest that PFOS is correlated with multiple toxicities such as hepatotoxicity, carcinogenicity, immunotoxicity as well as reproductive and developmental effects (1, 2). The major manufacturer, 3M, instituted a voluntary phase-out of production of perfluoroalkyl sulfonates (PFOS and related chemicals) in 2000. PFOS, however, has been found worldwide in wildlife and humans due to its persistent and bioaccumulative tendencies (3). Those studies have raised growing public concern on the health and environmental effects of PFOS and related compounds. The fact that PFOS was detected in human blood, plasma, and serum all over the world is indicative of human risk to PFOS exposure. Of particular concern is the presence of PFOS in human cord blood (4-6) and breast milk (7-9), which suggests that neonates can be exposed to PFOS both prenatally and postnatally. The effects of PFOS on the developmental outcomes have been extensively studied in rodents and, to some extent, in humans. In rodents, administration of PFOS during pregnancy resulted in fetal weight deficit, neonatal survival reduction, postnatal growth retardation, developmental delay, structure anomalies, and neurobehavioral defects (10-16). In humans, negative association between PFOS concentration in cord serum and birth weight, ponderal index and head circumference size was reported (17). Children born to mothers with higher plasma PFOS levels have been found to be more likely to start sitting without support at a later age than those born to mothers with lower exposure levels in Denmark (18). The adverse effects of PFOS on growth and neurological development are potentially related to thyroid hormone (TH) deficiency. Thyroid hormones, mainly thyroxine (T4) and 3,3′,5-triiodothyronine (T3), play important roles in regulating metabolism, growth and development, especially for the development of central nervous system and brain function (19). In humans, hypothyroxinemia (low in T4) during fetal or postnatal period, even when serum T3 concentration is normal, induces permanent functional abnormalities in children (20). Severe TH deficiency during fetal and neonatal periods results in the syndrome of cretinism characterized by mental retardation, deafness, and ataxia (21). Under normal physiological conditions in mammals, thyrotropin-releasing hormone secreted from the hypothalamus stimulates the anterior pituitary to release thyrotropin (TSH), which then simulates the thyroid to synthesize the prohormone, T4. T4 is converted to biologically active T3 by outer-ring deiodination or inactive reverse T3 (rT3) by inner-ring deiodination. Serum T4 and T3 are transported to target tissues by the carrier proteins such as transthyretin (TTR), thyroxine-binding globulin and albumin. The two important pathways of TH metabolism are deiodination catalyzed by deiodinases and conjugation with glucuronic
Received June 1, 2009. Revised manuscript received August 18, 2009. Accepted August 24, 2009.
Perfluorooctane sulfonate (PFOS), an environmentally persistent organic pollutant, has been reported to be transferred to the developing organisms via both placenta and breast milk. A cross-foster model was used to determine whether prenatal or postnatal exposure to PFOS alone can disturb the TH homeostasis in rat pups, and if so, which kind of exposure is a major cause of TH level alteration. Pregnant rats were fed standard laboratory rodent diet containing 0 (control) or 3.2 mg PFOS/kg throughout gestation and lactation period. On the day of birth, litters born to treated and control dams were crossfostered, resulting in the following groups: unexposed control (CC), pups exposed only prenatally (TC), only postnatally (CT) or both prenatally and postnatally (TT). Serum and liver PFOS concentrations, serum total thyroxine (T4), total triiodothyronine (T3), reverse T3 (rT3) levels, and hepatic expression of genes involvedinTHtransport,metabolism,andreceptorswereevaluated in pups at the age of postnatal days (PNDs) 0, 7, 14, 21, or 35. PFOS body burden level in pups in group CT increased, while those in group TC dropped as they aged. Neither total T3 nor rT3 in pups was affected by PFOS exposure. Gestational exposure to PFOS alone (TC) significantly (p < 0.05) decreased T4 level in pups on PNDs 21 and 35, 20.3 and 19.4% lower than the control on the same PND, respectively. Postnatal exposure to PFOS alone (CT) also induced T4 depression on PNDs 21 and 35, 28.6 and 35.9% lower than controls, respectively. No significant difference in T4 level (p > 0.05) was observed between TC and CT on these two time points. None of the selected TH related transcripts was affected by PFOS in pups on * Corresponding author phone: +86-411-84708084; fax: +86-41184708084; e-mail:
[email protected]. † Dalian University of Technology. ‡ China Medical University. § U.S. Environmental Protection Agency. 8416
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Introduction
10.1021/es901602d CCC: $40.75
2009 American Chemical Society
Published on Web 09/29/2009
acid catalyzed by uridine diphosphoglucuronosyl transferases (UGTs). Thyroid hormones, mainly T3, bind to the TH receptors (TRR and TRβ) in target tissues to produce a biological response. Meanwhile, serum TH negatively regulates the synthesis of thyrotropin-releasing hormone and TSH secretion. Multiple physiological steps within these processes can be disturbed by xenobiotic exposure resulting in thyroid imbalance (22). Several studies have demonstrated that treatment with PFOS can decrease serum T4 and T3 without a compensatory elevation of TSH in rat dams and neonates (11, 12, 15). Specifically, depressed serum total and free T4 levels were detected in pups born to and nursing on PFOS-dosed dams (12). Administration of PFOS to rats for 6 weeks prior to mating, during mating, through gestation and lactation resulted in significant decrease in total T4 on postnatal day 5 (15). Because postnatal PFOS burden in pups in these studies reflects both prenatal and postnatal exposure, previous analyses did not completely separate out associations for prenatal and postnatal exposures. Those findings raised the questions that whether prenatal or postnatal exposure to PFOS alone can disturb the TH homeostasis in rat pups, and if so, which kind of exposure is a major cause of TH level alteration. Accordingly, the present study employed a cross-foster model to address these questions. If the TH was affected by PFOS, whether the alterations in TH levels were associated with the changes in hepatic TH related gene expression were also determined. Serum and liver PFOS concentrations, serum TH (T4, T3, and rT3) levels, and hepatic expression of genes involved in TH transport, metabolism and receptors were evaluated in rat pups at the age of various postnatal days under different conditions of PFOS exposure.
Materials and Methods Chemicals. The potassium salt of PFOS (CAS number 279539-3; hereafter referred to as PFOS), 98% purity, was purchased from Fluka (Buchs, Switzerland). Tetrabutylammonium hydrogen sulfate (high-performance liquid chromatography [HPLC] grade) was the product of Acros Organics (Geel, Belgium). Methanol (HPLC grade) and methyl tertbutyl ether (HPLC grade) were obtained from Fisher (Fairlawn, NJ) and Tedia (Fairfield, OH), respectively. Diet Preparation. PFOS was solublized in 0.5% Tween20 to prepare solution at the concentration of 0.32 mg/mL. Ten mL of PFOS solution was added to 1 kg of diet powder (standard laboratory rodent diet) purchased from Experimental Animal Care Center of Dalian Medical University (Dalian, China). The PFOS-adulterated chow was mixed thoroughly to form a homogeneous mixture at the concentration of 3.2 mg PFOS/(kg feed). Control feed was prepared without PFOS adulteration. In the preliminary experiment for establishment of the cross-foster animal model, neither maternal food consumption or body weight during the gestation and lactation period nor the postnatal survival rate of pups was affected by PFOS at the dose of 3.2 mg /(kg feed) (Supporting Information (SI) Table S1). Thus, the findings of thyroid hormone toxicity evaluation following 3.2 mg/(kg feed) of PFOS administration through gestation and lactation would not be confounded by significant maternal toxicity as well as postnatal mortality. Animals Husbandry and the Cross-foster Study Design. Adult Wistar rats (180-200 g in body weight) were housed in standard plastic cages (3-4 animals per cage) and maintained at controlled room temperature 23 ( 2 °C, relative humidity 45% ( 10% and a 12:12 h light:dark cycle. After acclamation for one week, females were bred overnight. The day on which sperms were detected in vaginal smears was considered as gestation day 0 (GD 0). The pregnant rats were housed individually and provided with rodent chow con-
taining PFOS at concentrations of 0 (control, n ) 20) or 3.2 mg/kg feed (n ) 20) throughout gestation and lactation. Dams were allowed to deliver naturally. The day of birth was considered as postnatal day (PND) 0. Two control dams were dead from dystocia resulting in 38 litters in total. Two control litters and two PFOS-treated litters were used for sample collection on PND 0. Other litters were cross-fostered within 12 h of birth to yield the following four groups: (1) Litters from control dams fostered by other control dams (CC, unexposed control, n ) 8), (2) Litters from treated dams fostered by control dams (TC, prenatal exposure, n ) 8), (3) Litters from control dams fostered by treated dams (CT, postnatal exposure, n ) 8), and (4) Litters from treated dams fostered by other treated dams (TT, prenatal + postnatal exposure, n ) 10). The litter size was adjusted to 10 pups (five males and five females where possible). No pups were coupled with their original birth mother. The pups were weaned on PND 21 and housed in unisexual groups on a litter basis. After PND 21, the weaned pups were provided the same feed as that of their rearing mother, which means that pups in groups CT and TT received PFOS directly from PFOS-adulterated feed. Samples Collection. Pups were weighed and sacrificed on PNDs 0, 7, 14, 21, or 35. The blood and liver were collected. The blood from the two control (or PFOS-treated) litters was pooled into one sample, and the livers in each litter were pooled by gender on PND 0. On PND 7, two males and two females per nursing dam were sacrificed and the blood (or the liver) was pooled together. On PNDs 14, 21, or 35, one male and one female (if possible) per nursing dam were sacrificed. The blood was clotted for 2 h and centrifuged at 1800g for 20 min. The serum was removed for determination of TH and PFOS concentrations. The livers were rinsed in physiological saline solution, blotted, weighed, and immediately frozen in liquid nitrogen and stored at -80 °C until further processing. Determining PFOS Concentrations in Serum and Liver. PFOS in the serum and liver samples were extracted using a previously published method (23). Briefly, liver samples were homogenized in Milli-Q water for 1.5 min followed by sonication for approximately 30 min. Serum samples or liver homogenate aliquots were mixed with 1 mL of 0.5 M tetrabutylammonium hydrogen sulfate (pH 10.0) and 2 mL of 0.25 M sodium carbonate buffer in 15 mL polypropylene tubes. After the addition of 5 mL of methyl tert-butyl ether (MTBE), the mixture was vortexed for 20 min followed by centrifugation at 3000 rpm for 15 min. The MTBE layer was transferred into a new polypropylene tube. The residual mixture was rinsed with MTBE and separated again. Two rinses were combined, evaporated under nitrogen, and then resuspended in 1 mL of methanol. Serum samples were then passed through a nylon filter (0.45 µm pore size; Whatman). Liver samples were further cleaned up using a Presep-C Agri solid phase extraction column (Wako, Osaka, Japan). Concentrations of PFOS in extracted solutions were quantified by a Shimadzu 2010A liquid chromatography-mass spectrometer (Shimadzu, Kyoto, Japan). Detail analytical conditions were provided in SI Table S2. The limits of quantification (LOQ) for serum and liver samples were determined as 0.5 µg/L and 0.0025 µg/g, respectively. Serum Thyroid Hormone Analysis. Serum total T4, T3, and rT3 were analyzed using commercially available radioimmunoassay (RIA) kits with 125I as a tracer (North Institute of Biological Technology, Beijing, China). The LOQs for T4, T3, and rT3 RIA kits were determined to be 20, 0.2, and 0.04 ng/mL, respectively. The intra- and interassay coefficients of variation for all hormones were less than 10 and 15%, respectively. Quantitative Reverse Transcriptase-Polymerase Chain Reaction RNA Isolation and Reverse Transcription. Total RNA was isolated from the livers derived from pups on PNDs VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Genes Studied, With Abbreviations and Summary of Biological Functions genbank accession no.
simple name
gene name
summary of biological function(s)
NM_021653
type 1 deiodinase
DIO1
activates the prohormone thyroxine (T4) to bioactive 3,3′,5-triiodothyronine (T3) by outer ring deiodination.
NM_057105
uridine diphosphoglucuronosyl transferase 1A1
UGT1A1
UGT1A1 and UGT1A6 are two isozymes of uridine diphosphoglucuronosyl transferases (UGTs), the enzyme system playing a role in thyroid hormone glucuronic acid conjugation and biliary excretion.
NM_012683
uridine diphosphoglucuronosyl transferase 1A6
UGT1A6
NM_012681
transthyretin
TTR
binds T4 and T3 and plays a role in thyroid hormone transport in serum.
NM_001017960
thyroid hormone receptor R
TRR
binds the promoter of the Na+/H+ exchanger NHE1 and mediates thyroid hormone induced transcriptional activation.
NM_012672
thyroid hormone receptor β
TRβ
nuclear hormone receptor for T3; mediates the biological activities of thyroid hormone
0 and 21 using the RNAiso reagent (Takara, Japan). The ultraviolet absorbance of 260 nm (A260) was used to estimate total RNA concentration. The quality of the RNA was electrophoretically verified by ethidium bromide staining as well as by A260/A280 ranging between 1.9 and 2.1. Complementary DNA (cDNA) was synthesized using a PrimeScript RT reagent kit (Takara). Reverse transcription (RT) reaction was carried out in a Tpersonal thermocycler (Biometra, Goettingen, Germany) under the following conditions: 15 min at 37 °C, 5 s at 85 °C. RT control (no reverse transcriptase was added to the reaction mixture) was set to monitor the presence of contaminating genomic DNA in the polymerase chain reaction step. Real-Time Polymerase Chain Reaction. The hepatic genes studied, including genes closely related to TH transport (TTR), metabolism (DIO1, UGT1A1 and UGT1A6) and receptors (TRR and TRβ) are listed in Table 1. Primers sequences and efficiencies for reference and target genes are shown in SI Table S3. Quantitative polymerase chain reaction (PCR) was performed on a Rotor-Gene 3000 instrument (Corbett Research, Sydney, Australia) using SYBR Premix Ex Taq reagent kit (Takara). A negative control (without cDNA template) and RT control were set for each gene per PCR run. The PCR reaction parameters used were 10 s at 95 °C and 40 cycles at 62 °C for 20 s, followed by the dissociation stage. The average CT (cycle threshold) of the duplicate measurements was calculated. The relative gene expression in PFOS-exposed groups in comparison with the control was quantified by the 2-∆∆CT method (24). Data Analysis. Data analyses were performed using SPSS software version 11.5 (SPSS, Chicago, IL). All values are expressed as mean ( standard error. The normality of the data was analyzed by Shapiro-Wilk test. Statistical significance of differences in TH levels and selected genes expression between PFOS-treated groups (CT, TC, and TT) and controls (CC) was evaluated by analysis of variance (ANOVA) followed by Bonferroni post hoc test for multiple comparisons. Prior to ANOVA, data were analyzed for homogeneity of variances by Levene’s test. Statistical differences in selected end points between litters (at birth) born to control dams and litters born to PFOS-treated dams were evaluated by independentsample t test. The level of significance was set as 0.05. 8418
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Results and Discussion Body and Liver Weights in Rat Pups. No clinical abnormality was observed by the daily physical examinations. The offspring in PFOS-treated groups did not differ significantly from controls with respect to body weight on PND 0, 14, 21, and 35 (SI Table S4), which suggests that exposure to PFOS throughout gestation and/or lactation affected neither the ability of dams to feed and nurse their pups nor the quality and quantity of the milk. The relative liver weight significantly increased in pups (on PNDs 21 and 35) exposed to PFOS prenatally and postnatally (SI Table S4). PFOS Concentrations in Serum and Liver. Serum and liver PFOS concentrations measured in litter samples collected on PNDs 0, 7, 14, 21, and 35 are summarized in Table 2. The occurrence of PFOS transfer from dams to pups, both via placenta and milk, was observed. Litters in group CT (postnatal exposure only) increased in serum PFOS concentration in a time dependent manner after birth, with the highest concentration of 7.04 µg/mL in females on PND 35. In contrast, litters in group TC (in utero exposure only) decreased in serum PFOS as they aged. Mean serum PFOS concentrations in males and females declined 94.1% and 82.9% on PND 35, respectively, compared to the concentration measured in litters born to treated dams at birth. On various postnatal ages, liver PFOS concentrations were higher than the respective serum PFOS concentrations. The mean PFOS liver/serum ratios were comparable to that reported by Chang et al. (25), in which higher levels of PFOS were also detected in the liver than in serum (by a factor of 2-4) in pups before weaning. The mean ratio on PND 0 was 1.18, probably reflecting the minimal enterohepatic cycling in utero (26). Mean serum PFOS concentration in males in TC on PND 35 was approximately 40% of that in females and t test further revealed a significant difference (p < 0.05) in serum (but not liver) PFOS level between males and females, resulting in the relative high mean PFOS liver/serum ratio 8.17 in males. Males typically have larger plasma volume, and higher average organ blood flow than female after weaning, which may in part account for the apparent lower serum concentration in males. Epidemiological surveys of chemical burden on humans support our findings that PFOS is transferred to the devel-
TABLE 2. Concentrations of PFOS in Serum and Liver in Rat Pups on Postnatal Days (PNDs) 0, 7, 14, 21, and 35a PFOS b
PFOS[liver]/PFOS[serum]c
PNDs
groups
0
LC LT
