* Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711;
Department of Psychology, University of Delaware, Newark, Delaware 19716;
3M, Medical Department, St. Paul, Minnesota 55133
Received February 24, 2003; accepted April 16, 2003
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ABSTRACT |
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Key Words: perfluorooctane sulfonate; postnatal; toxicity; rodent.
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INTRODUCTION |
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The potential toxicity of organic fluorinated chemicals in general, and PFOS in particular, has not been well characterized, and even less is known about mechanisms of their toxic action. In adult monkeys and rodents, hepatic toxicity and altered thyroid hormone economy are notable findings (Luebker et al., 2002; Seacat et al., 2002
; 2003
; Sohlenius et al., 1993
; Thibodeaux et al., 2003
), and interference with mitochondrial bioenergetics and cellcell communication through gap junctions have been implicated as potential mechanisms of toxicity (Berthiaume and Wallace, 2002
; Hu et al., 2002
; Starkov and Wallace, 2002
). The preceding article (Thibodeaux et al., 2003
) describes the maternal and prenatal toxicity of PFOS in rats and mice. In this study, evaluation of the developmental toxicity of PFOS is extended to the postnatal period. Survival of the newborn rodents, their growth and development, as well as their thyroid status have been examined, and alterations induced by in utero exposure to PFOS have been correlated with maternal and neonatal body burden in rats.
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MATERIALS AND METHODS |
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Animal Treatment
Pregnant Sprague-Dawley rats and CD-1 mice, bred within a 4-h period in the afternoon and overnight, respectively, were obtained from Charles River Laboratories (Raleigh, NC). Those animals with spermatozoa in a vaginal smear and/or with a copulatory plug were considered to be at gestation day (GD) 0. Animals were housed individually in polypropylene cages with heat-treated pine shavings for bedding and were provided pellet chow (LabDiet 5001, PMI Nutrition International, Brentwood, MO) and tap water ad libitum. Animal facilities were controlled for temperature (2024°C) and relative humidity (4060%), and operated under a 12-h light-dark cycle.
Rats.
Perfluorooctane sulfonate was freshly prepared in 0.5% Tween-20 vehicle and administered to pregnant dams daily by gavage at doses of 1, 2, 3, 5, or 10 mg/kg from GD 2 until GD 21. Controls received vehicle alone at an equivalent volume (1 ml/kg). On GD 22, rats were monitored at hourly intervals. Time of parturition for each animal, number of live offspring, and conditions of the newborns were noted. The following day was designated as postnatal day (PD) 1. The number of live pups in each litter was tabulated daily, and their body weight was determined at intervals of several days. For this study, the litter size was not adjusted, despite the loss of neonates, unless only three or fewer pups survived within a litter (typically seen in the high-dosage groups). In such cases, the surviving pups were distributed randomly to nursing dams within the same dosage group with a litter size of less than 10. The age at which the neonates opened their eyes was tracked beginning on PD 12. All pups were weaned on PD 21 and separated by gender. The age at which the rat offspring reached puberty was determined by tracking vaginal opening in females beginning on PD 30 and preputial separation in males beginning on PD 40. For the female rats, when vaginal opening was completed, estrous cycles were monitored by daily evaluation of vaginal cytology, according to the method described by Cooper and Goldman (1999).
In a follow-up study, newborns from the 5 mg/kg PFOS dosage group were cross-fostered with controls immediately after parturition. In this experiment, 10 control and 10 PFOS-exposed litters were subdivided evenly into four groups: (1) control pups remaining with their dams; (2) PFOS-exposed pups remaining with their dams; (3) PFOS-exposed pups transferred to control nursing dams; and (4) control pups transferred to PFOS-treated dams. Survival of the neonates was monitored for 3 days after birth.
In another study, pregnant rats (1728 per dosage group) were treated with PFOS (0, 1, 2, 3, or 5 mg/kg), as previously described. Four pups from each litter were sacrificed by decapitation within 24 h after birth, and trunk blood and liver were collected, pooled within each litter, and stored frozen for PFOS and thyroid hormone analyses. The remaining neonates were randomized and redistributed to the nursing dams within their respective dosage groups, with litter size kept at 1012 pups to maintain a uniform nutritive status. Redistribution of pups was repeated at intervals of several days. All pups were weaned on PD 21. On PDs 2, 5, 9, 15, 21, 28, and 35, pups of both genders were randomly chosen from several litters (one sample per litter for preweaning age point), weighed, and sacrificed by decapitation. Trunk blood was collected and serum prepared for PFOS and thyroid hormone analyses. Liver weights were recorded. Brains were removed quickly and dissected for hippocampus and prefrontal cortex on a cold plate (Thermoelectrics, Wilmington, DE); these brain tissues were then frozen immediately on dry ice and stored at 80°C for subsequent determination of choline acetyltransferase activity. At the early age points, samples were pooled from several pups within each litter: On PD 2, four pups were pooled for each sample; on PD 5, three pups were pooled; on PDs 9 and 15, two pups were pooled. In addition, at weaning, one male and one female pup were chosen randomly from eight individual litters of controls and eight litters of the 3 mg/kg dosage group for the T-maze delayed alternation test. The behavioral experiment was performed in two separate blocks, each with eight controls and eight PFOS-exposed pups.
Mice.
PFOS was prepared as for rats and administered by gavage at doses of 1, 5, 10, 15, and 20 mg/kg from GD 1 until GD 17. Controls received an equivalent volume of 0.5% Tween-20 vehicle (10 ml/kg). The day of birth was designated PD 0. Condition of the newborn mice was examined as described for the rat. Litter size and weight gain of the pups were monitored; all pups were weaned on PD 21. Age at eye opening was tracked beginning on PD 12. In a separate study, pregnant mice (2122 per dosage group) were given PFOS as described above. Pups of both genders were randomly selected from several litters, weighed, and sacrificed within 24 h of birth and on PDs 3, 7, 14, 21, 28, and 35. Liver samples were collected and weighed. Trunk blood was collected and serum prepared for thyroid hormone analysis. At the early age points, samples were pooled from several pups within each litter: On PD 3, four neonates were pooled for each sample; on PD 7, two pups were pooled. Litters were culled to 1012 pups at intervals of several days.
Radioimmunoassays
T4 and T3.
Serum samples were thawed, and levels of total thyroxine (T4), free T4, and triiodothyronine (T3) were measured in duplicate with the respective radioimmunoassay (RIA) kits (Diagnostics Products Corporation, Los Angeles, CA), according to the method described in detail in the preceding article (Thibodeaux et al., 2003). Internal standards from rat sera were used to monitor interassay differences.
Thyroid-stimulating hormone (TSH).
Serum samples were thawed, and the level of TSH was quantified by RIA. The assay was performed using the following materials supplied by the National Hormone and Pituitary Program (Torrance, CA): iodination preparation NIDDK-rTSH-I-9, reference preparation NIDDK-rTSH-RP-3, and antiserum NIDDK-antirat TSH-RIA-6. Iodination material was radiolabeled with 125I (Perkin Elmer/New England Nuclear, Boston, MA) by a modification of the chloramine-T method of Greenwood et al. (1963). Labeled TSH was separated from unreacted iodide by gel filtration chromatography, as described previously (Goldman et al., 1986
). The assay was conducted according to procedures described in the preceding article (Thibodeaux et al., 2003
).
Choline Acetyltransferase (ChAT) Assay
Assays were conducted essentially as described by Lau et al. (1987). Frozen rat brain tissues were thawed and homogenized (Polytron, Brinkman Instruments, Westbury, NY) in 79 volumes of ice-cold 10-mM phosphate buffer (pH 7.4). An aliquot of 30 µl of the homogenate was mixed with 30 µl of the incubation buffer containing final concentrations of 60 mM sodium phosphate (pH 7.9), 200 mM NaCl, 20 mM choline chloride, 17 mM MgCl2, 1 mM EDTA, 0.2% Triton X-100, 0.12 mM physostigmine, 0.6 mg/ml bovine serum albumin, and 0.4 mM [14C]acetyl-coenzyme A (NEN Life Science, Boston, MA). Blanks for each treatment group were prepared from pooled homogenate and kept on ice. Samples were preincubated on ice for 15 min, then transferred to a 370C water bath for an additional 30 min of incubation. The reaction was stopped by placing the samples on ice, and labeled acetylcholine was then extracted and counted by scintillation spectrometry. Enzyme activity was expressed as nmol of acetylcholine formed per g of tissue per 30 min.
T-Maze
The apparatus and procedure have been described in detail by Freeman and Stanton (1991). In brief, on PD 21, rat pups were housed in individual cages and deprived of food and water overnight. Beginning in the morning of PD 22, animals were acclimated to maze running with two goal-box training sessions and a session of 12 forced runs. These sessions occurred 4 h apart (e.g., at 8 A.M., noon, and 4 P.M.). Goal-box sessions consisted of placing the animal in a goal arm (one arm for the first session and the other for the second session) containing 0.05 ml of light cream reward. This process was repeated six times for each session. The forced-run session consisted of 12 forced-run trials (six to each maze arm in random order) in which the trap door to only one arm of the T-maze was raised, and the animal was rewarded when it broke the photoelectric beam at the end of that goal arm. At the end of each training session, animals received supplementary cream in their home cages in order to maintain them at 85% of their predeprivation body weight and to equalize the amount of light cream given to each subject in a session, depending on the number of rewarded trials. In the morning of PD 23, subjects were trained on delayed alternation over two sessions that were separated by a 6-h interval. The first session consisted of two 12-trial blocks and the second of three 12-trial blocks. On each training trial, an animal was placed in the maze and given a pair of runs. On the first run, the animal was allowed to enter only one of the two goal arms of the T-maze. This forced run was followed immediately by the second run in which the animal was allowed to choose either arm of the maze but was rewarded only for choosing the arm opposite from that entered on the previous run. Performance was scored as percentage of correct determinations to enter the choice arm, with 50% reflecting random success. The studies were conducted in four separate blocks, each representing two male and two female pups (from two individual litters) for controls and two males and two females for the PFOS (3 mg/kg) group. Statistical analysis did not indicate a gender effect in the T-maze performance; therefore, results from male and female rats were pooled.
Determination of PFOS Concentrations
Serum samples were diluted, and liver samples were homogenized in five volumes of reagent-grade water. An aliquot of each dilution was spiked with appropriate internal standards. Acetonitrile (5 ml) was added as an extraction solvent, which also served to precipitate the proteins. The samples were shaken at 300 rpm for 20 min and centrifuged at 850 x g for 10 min. The supernatant was transferred to a clean tube, diluted with 40 ml of water, and passed through a preconditioned C18 SPE cartridge. PFOS was eluted from the SPE cartridge with 2 ml methanol and analyzed by high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS), according to the method described by Hansen et al. (2001).
Data Analysis
Data are presented as means and standard errors, and evaluated by ANOVA. For the neonatal survival, cross-foster, and developmental landmark studies, the individual litter was used as the statistical unit. For other postnatal studies, because neonates were randomized at birth (within each treatment group) and litters were culled during the course of the study, the individual pup was used as the statistical unit. When a significant treatment effect or interaction was detected, Duncans multiple-range test or Dunnetts t-test was performed post hoc for comparisons between treated groups and controls. Statistically significant differences were determined at p 0.05.
The U.S. Environmental Protection Agency (EPA) now uses the benchmark dose (BMD) approach (Barnes et al., 1995; Crump, 1984
) for noncancer risk assessment (EPA, 1995
). This approach is designed to provide a more quantitative alternative to dose-response assessment than the no-observed-adverse-effect-level (NOAEL) process by constructing mathematical models to fit all data points in the dose-response study and to take data variance into consideration. In this study, BMD5 and BMDL5 values were calculated for maternal and developmental toxicity after PFOS exposure. BMD5 refers to the central estimate of the administered dose predicted to cause a 5% increase in response above background, and BMDL5 is defined as the corresponding lower limit of the 95% confidence interval on the BMD (Allen et al., 1994
). Benchmark Dose Software (EPA, 2000
) was used to calculate the BMD5 values. Selection of a specific curve-fitting model for the BMD determination was based on the Akaikes Information Criterion (AIC) value. The AIC is equal to 2L + 2p, where L is the log-likelihood at the maximum likelihood estimates for the parameters, and p is the number of model parameters estimated. The model that demonstrates "goodness of fit" with the lowest AIC value is presumed to be the most appropriate.
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RESULTS |
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DISCUSSION |
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The pathophysiological mechanisms underlying PFOS-induced neonatal mortality are largely unknown at present. However, in a preliminary study, Grasty and coworkers (2002) reported a similar pattern of neonatal death when PFOS was given to the pregnant rats during only the last few days of gestation, although a higher daily dosage of the fluorochemical was used. In this regard, it will be interesting to compare the body burdens of PFOS in newborn rats after various exposure paradigms (e.g., Butenhoff et al., 2002
; Grasty et al., 2002
; current study) and between rat and mouse newborns to ascertain whether postnatal toxicity of the fluorochemical can be correlated to a critical body burden. These results together suggest that the organ systems developing late in gestation may be particularly vulnerable to PFOS. This hypothesis is also consistent with the relatively unremarkable teratological findings. Considering that PFOS-induced organ toxicity is incompatible with postnatal survival, maturation of the lung and pulmonary function is a plausible target of PFOS toxicity (Lau and Kavlock, 1994
). Indeed, the profile of neonatal mortality induced by PFOS is reminiscent of the developmental toxicity of nitrofen, an herbicide that perturbed fetal lung maturation, ultimately leading to compromised cardiopulmonary function and death in newborn rats (Lau et al., 1986
, 1988
; Stone and Manson, 1981
). The previous teratological finding of enlarged right atrium in the PFOS-exposed fetus (Thibodeaux et al., 2003
), possibly suggesting pulmonary hypertension, is also consistent with this hypothesis.
PFOS-exposed rat pups surviving beyond the first few days exhibited growth retardation. Deficits of body weight gain were seen in the 2 mg/kg and higher dosage groups, accompanied by a trend toward developmental delays, as illustrated by eye opening. This pattern of growth deficit and developmental delay also extended to neonatal mice exposed to PFOS prenatally. These results suggest that PFOS may interfere with cellular or functional maturation of target organs, possibly via alterations of thyroid hormones. The importance of thyroid hormones in regulating growth and development is well established (Legrand, 1986; Mussa et al., 2001
; Porterfield, 1993
; Stein et al., 1991
). In previous studies, PFOS has been shown to depress circulating thyroid hormones in the adult monkey, rat, and mouse (Seacat et al., 2002
, Thibodeaux et al., 2003
). Levels of T4 and T3 in pregnant rodents were markedly reduced by PFOS. Prior to fetal thyroid gland development, the only source of thyroid hormones for the conceptus is the maternal circulation. Thus, the abrupt falls of T4 and T3 in PFOS-treated pregnant rodents would be expected to deprive the developing organism of these key endogenous signals. However, the actual availability of thyroid hormones (particularly T3) in the embryonic/fetal target cells has not been determined; the impact of the maternal hypothyroid state could have been counteracted by upregulation of placental deiodinase activity (Versloot et al., 1998
). On the other hand, prenatal exposure to PFOS was associated with hypothyroxinemia in neonatal rats, because both total and free T4 levels were significantly and persistently reduced. The lack of similar effects on neonatal serum T3 (unlike the responses to PFOS in the adult rats) or TSH (consistent with adult findings) is not well understood, though not necessarily unique. Previous studies with polychlorinated biphenyls and polybrominated diphenyl ethers have shown a similar profile of thyroid hormone imbalance (Goldey et al., 1995
; Morse et al., 1996
; Rosiak et al., 1997
; Zahalka et al., 2001
; Zhou et al., 2002
). One possibility for the maintenance of serum T3 in the PFOS-exposed rat pups may involve upregulation of the liver type I deiodinase that converts T4 to T3.
Development of the brain in general and the cholinergic neurotransmitter system in particular are critically dependent on thyroid hormones (Gould and Butcher, 1989; Kalaria and Prince, 1986
; Rami et al., 1989
; Sawin et al., 1998
; Virgili et al., 1991
). The neurotransmitter biosynthetic enzyme choline acetyltransferase is exquisitely sensitive to thyroid status (Rami et al., 1989
; Sawin et al., 1998
). Propylthiouracil (PTU)-induced neonatal hypothyroidism has been shown to suppress ChAT activity and impair learning and memory (Christy Carter, 2003, personal communication; Sawin et al., 1998
). Circulating T4 and T3 were profoundly depressed in the PTU-treated rat pups, whereas PFOS suppressed only serum T4, and to a lesser extent than that produced by PTU. Yet small but significant reductions of ChAT activity were seen in the prefrontal cortex of PFOS-treated pups, whereas ChAT activity in another brain region richly endowed with cholinergic neurons, the hippocampus, was unaffected. Importantly, the PFOS-induced hypothyroxinemia and marginal reductions of cortical ChAT activity were not accompanied by notable deficits in learning and memory acquisition, perhaps due to the mildness of the thyroid hormone imbalance. Indeed, only subtle changes in behavioral performance produced by congenital hypothyroidism have been previously reported (MacNabb et al., 2000
).
In subchronic studies with adult rat and monkey, liver enlargement and hepatic toxicity were associated with PFOS exposure (Seacat et al., 2002, 2003
). In our studies where rodents were exposed to PFOS only during pregnancy, maternal liver weights in rats were unaltered but those in mice were significantly elevated, compared to controls (Thibodeaux et al., 2003
). Whereas significant increases of relative liver weight were detected in both rat and mouse offspring, these changes were more notable and consistent in the mouse. These findings suggest that the developing liver is another potential target for PFOS action.
Although the results were limited to the first few days of life, several points can be made concerning the body burden of PFOS in the neonatal rats:
In summary, in utero exposure of PFOS to laboratory rodents severely compromised postnatal survival. Morbidity and mortality were dose dependent, with the rat being more sensitive than the mouse. Persistent growth deficits and developmental delays were seen in the surviving animals. Hypothyroxinemia was observed in PFOS-exposed rat pups, with an attendant small reduction of ChAT activity in the prefrontal cortex, but significant deficits in learning behaviors were not detected. Thus, postnatal evaluations of rodent offspring exposed to PFOS in utero revealed adverse developmental outcomes at a dosage lower than that associated with teratological findings; additional investigation will be required to elucidate the pathophysiological mechanisms underlying the PFOS toxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be sent at: Mail Drop 67, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: lau.christopher{at}epa.gov.
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