ToxSci Advance Access originally published online on May 28, 2008
Toxicological Sciences 2008 105(1):173-181; doi:10.1093/toxsci/kfn099
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Published by Oxford University Press 2008.
Effects of Perfluorobutyrate Exposure during Pregnancy in the Mouse
* 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
Medical Department, 3M Corporation, St Paul, Minnesota 55144
The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709
1 To whom correspondence should be addressed 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.
Received February 14, 2008; accepted May 16, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Perfluorobutyrate (PFBA) is a perfluoroalkyl acid (PFAA) found in the environment. Previous studies have indicated developmental toxicity of PFAAs (perfluorooctane sulfonate [PFOS] and perfluorooctanoate [PFOA]); the current study examines that of PFBA. PFBA/NH4+ was given to timed-pregnant CD-1 mice by oral gavage daily from gestational day (GD) 1 to 17 at 35, 175, or 350 mg/kg (chosen to approximate the developmentally toxic doses of PFOA); controls received water. At GD 18, serum levels of PFBA were 3.8, 4.4, and 2.5 µg/ml, respectively, in the three treated groups. PFBA did not significantly affect maternal weight gain, number of implantations, fetal viability, fetus weight, or incidence of fetal malformations. Incidence of full-litter loss was significantly greater in the 350 mg/kg group, and maternal liver weights were significantly increased in the 175 and 350 mg/kg groups. In contrast to PFOA and PFOS, PFBA exposure during pregnancy did not adversely affect neonatal survival or postnatal growth. Liver enlargement was detected in the PFBA-exposed pups on postnatal day (PD) 1, but not by PD 10. Expression of selected hepatic genes in PFBA-exposed pups at PD 1 did not reveal any significant changes from controls. A significant delay in eye-opening in offspring was detected in all three PFBA groups, and slight delays in the onset of puberty were noted in the 175 and 350 mg/kg groups. These data suggest that exposure to PFBA during pregnancy in the mouse did not produce developmental toxicity comparable to that observed with PFOA, in part, due to rapid elimination of the chemical.
Key Words: perfluorobutyrate; mouse; pregnancy; developmental toxicity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The perfluoroalkyl acids (PFAAs) are a family of synthetic fluorocarbons consisting of a perfluorinated carbon tail and an acidic functional moiety, usually a carboxylate or sulfonate, which is essentially anionic under most environmental and physiological conditions. These compounds have strong surface tension reducing properties that have been exploited in numerous industrial and consumer applications (Renner, 2001
). However, the carbon-fluorine bond renders these compounds resistant to environmental and metabolic degradation. Certain PFAAs, in particular perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS), have exceptionally long serum elimination half-lives in humans (Olsen et al., 2007), and have been found to be widely distributed in human populations (Lau et al., 2007; Calafat et al., 2007) and in certain wildlife species (Houde et al., 2006). This has prompted intense investigation of potential adverse health impacts (Andersen et al., 2008; Butenhoff et al., 2004a; Kennedy et al., 2004; Lau et al., 2004, 2007; Scialli et al., 2007). Indeed, in rodent models, developmental toxicity has been reported for both PFOS and PFOA, featuring dose-related compromises of neonatal survival, growth and development (Abbott et al., 2007; Butenhoff et al., 2004b; Grasty et al., 2003, 2005; Lau et al., 2003, 2006; Luebker et al., 2005a, b; Rosen et al., 2007; Thibodeaux et al., 2003; White et al., 2007; Wolf et al., 2007).
One possible determinant of the differential expression of PFAA toxicity may involve their rate of elimination and body burden accumulation (primarily in liver, kidney and serum). Generally, these rates appear to be dependent on the carbon chain length, functional moieties and animal species (including humans). Available half-life estimates illustrate patterns of human > monkey > mouse 
rat; long carbon chain > short carbon chain; and sulfonate > carboxylate (see review, Lau et al., 2007). Mean half-lives for PFOS have been reported as 5.4 years for humans (Olsen et al., 2007), 150 days for monkeys and 100 days for rats; whereas those for perfluoro-butane sulfonate (PFBS) have been estimated as 1 month in humans and only 4 days in monkeys. Similarly, mean half-lives for PFOA have been reported as 3.8 years for humans, 20–30 days in monkeys (Butenhoff et al., 2004c), and 17–19 days for mice (Lau et al., 2005). Indeed, the considerably shorter half-life of PFBS compared with PFOS has been the basis for reformulation of some commercial products to include the shorter-perfluorinated-chain (C4 and C6) chemistry.
Perfluorobutyrate (PFBA) is another 4-carbon PFAA either synthesized by industry or formed as an atmospheric degradation product (D'Eon and Mabury, 2007; D'Eon et al., 2006; Martin et al., 2006; Skutlarek et al., 2006). Its recent detection in surface waters and wells in Minnesota has resulted in renewed investigation into potential health consequences of exposure via environmental media. Consistent with the trend of PFAA pharmacokinetic properties described above, much shorter half-lives of PFBA (than PFOA) were reported for humans (2–4 days), monkeys (40–90 h), rats (2–9 h), and mice (3–17 h) in a most recent study (Chang et al., 2008). These findings indicate a relatively short residence duration of PFBA in the body, particularly in comparison to PFOA, PFOS, and PFHxS. Therefore, lesser body burdens would be expected from repeated exposure to PFBA, and this may explain, in part, its milder observed toxicity, compared with that of PFOA in the rodent models (Chang et al., in press; Ikeda et al., 1985). However, no information is available concerning developmental toxicity of PFBA, and developmental toxicity in the mouse is a common feature with PFOS and PFOA (Lau et al., 2003, 2006; Thibodeaux et al., 2003), the current study was undertaken to ascertain whether PFBA might elicit adverse effects similar to PFOS and PFOA in the mouse model.
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MATERIALS AND METHODS |
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PFBA (ammonium salt; > 98% pure) was provided from 3M Company (St Paul, MN). For all studies, PFBA was diluted in deionized water and prepared fresh daily.
Animal treatment.
All animal studies were conducted in accordance with the Institutional Animal Care and Use Committee guidelines established by the U.S. Environmental Protection Agency's Office of Research and Development/National Health and Environmental Effects Research Laboratory. Procedures and facilities were consistent with the recommendations of the 1996 National Research Council's "Guide for the Care and Use of Laboratory Animals," the Animal Welfare Act, and Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Animal facilities were controlled for temperature (20–24°C) and relative humidity (40–60%) and kept under a 12-h light-dark cycle.
Timed-pregnant CD-1 mice were obtained from Charles River Laboratories (Raleigh, NC), where females were bred overnight. The sperm-positive females, considered to be at gestational day (GD) 0, were shipped to our facility on the same day. Upon arrival, mice were housed individually in polypropylene cages with Alpha-dri (Shepherd Specialty Papers, Kalamazoo, MI) bedding and provided pellet chow (LabDiet 5001, PMI Nutrition International LLC, Brentwood, MO) and tap water ad libitum.
The study was performed in three separate blocks, with approximately 40 mice per block (10 mice for each dose group). Upon arrival at the animal facility, mice were ranked by their weight and assigned to one of the four treatment groups: vehicle control (deionized water), 35, 175, or 350 mg PFBA/kg body weight. These doses were chosen to approximate the effective doses of 1, 5, and 10 mg/kg of PFOA used in a prior study (Lau et al., 2006). The elimination half-life for PFBA in female mice is about 3 h (Chang et al., in press), whereas the elimination half-life for PFOA is about 15–20 days (Lau et al., 2005). Therefore, in an attempt to simulate as closely as possible the PFOA doses used by Lau et al. (2006), an effort was made to match internal exposures based on the "area-under-the-curve" (AUC) estimates. A one-compartment pharmacokinetic model was used for both PFBA and PFOA. For comparison of AUC values for female mice given PFBA and PFOA during gestation, the AUC values for both compounds at a nominal dose of 5 mg/kg were estimated for GD 17 to 18, assuming continuous daily oral administration from GD 1. For PFBA, estimation was based on data from the single oral treatment study in nonpregnant female mice at 10 mg/kg reported by Chang et al. (in press) and used a volume of distribution (Vd) of 3.14 ml, uptake rate constant (K01) of 0.7/h, and clearance rate (CL) of 0.76 ml/h. For PFOA, estimates were based on a K01 for PFBA in female mice (0.7/h), a Vd of 6 ml (based on rat data scaled to the female mouse) and CL of 0.012 ml/h (based on an assumed elimination half-life of 15 days for the female mouse; Lau et al., 2005). The resulting AUCs (from the 17th to the 18th day of treatment) were estimated at 6956 h-µg/ml for PFOA and 197 h-µg/ml for PFBA. Therefore, to obtain equivalent estimated AUCs, the dose required for PFBA would be approximately 35 times that for PFOA.
From GD 1 to GD 17, mice were weighed daily and given PFBA or water by oral gavage at a volume of 10 ml/kg. At GD 18, mice from each dose group were subdivided. Some mice were sacrificed (24 h after last treatment) by CO2 asphyxiation for teratological evaluation. Trunk blood was collected after decapitation, and serum samples were prepared and analyzed for PFBA concentration. Maternal liver was dissected, weighed and stored frozen at –80°C until analysis for PFBA. The gravid uterus was removed, weighed, and examined; the numbers of the live and dead fetuses as well as resorptions were recorded. Live fetuses were weighed individually, and examined for external abnormalities. All fetuses were killed with an overdose of pentobarbital and fixed in Bodian's solution (2% formaldehyde, 5% acetic acid, 72% ethanol, 21% water) for visceral evaluation. Examination of the head, thoracic, and abdominal viscera was carried out using free-hand razor dissection. For mice without any fetus, their uteri were removed and stained with 2% ammonium sulfide for the detection of residual implantation site. Mice with negative staining were considered "nonpregnant"; those that stained positive but did not have any fetuses at term were categorized as having experienced "full-litter resorptions" (FLRs).
The remaining pregnant mice received an additional PFBA treatment on GD 18. On GD 19, these mice were monitored intermittently for parturition; condition of the newborns and numbers of live offspring were noted. The following day was designated as postnatal day (PD) 1. The number of live pups in each litter and their body weight were tabulated daily for the first 4 days after birth and at intervals of several days thereafter. The age at which the mouse neonates opened their eyes was tracked beginning on PD 10. All pups were weaned on PD 22 and separated by sex. The age at which the mouse offspring reached puberty was determined by monitoring vaginal opening in females and preputial separation in males beginning on PD 26, with their respective body weights noted. On PD 49, three pups for each sex were randomly selected from each litter and retained; these animals were weighed biweekly until 41 weeks of age.
After weaning, the nursing dams were sacrificed, their livers were weighed, and their uteri were removed, stained with 2% ammonium sulfide, and the residual implantation sites were counted. The postnatal survival rate was calculated based on the number of implantations for each pregnant mouse.
For study block 3, one male and one female pup from each litter were weighed and sacrificed at PD 1 and 10 by decapitation. Trunk blood was collected, and serum was prepared and stored frozen at –80°C for PFBA analysis. Livers were removed, weighed, and about 200 mg of fresh tissues were processed immediately in TRI reagent (Sigma Chemical, St Louis, MO) for RNA isolation according to the manufacturer's directions; the rest of the tissue was stored at –80°C for PFBA analysis.
Gene expression analysis.
Measurement of selected gene transcripts was carried out by quantitative PCR (qPCR) of reverse-transcribed cDNAs. These candidate genes were chosen for evaluation because their expression patterns have been shown to be markedly altered in the fetal liver of mice exposed to PFOA (Rosen et al., 2007). Two micrograms of total RNA was initially digested by 2 units of DNaseI (#M6101, Promega Corporation, Madison, WI) for 30 min at 37°C, followed by 10 min at 65°C in a buffer containing 40mM Tris (pH 8.0), 10mM MgSO4, and 1mM CaCl2. The RNA was then quantified by a Quant-iT RiboGreen RNA assay kit according to the manufacturer's protocol (#R11490, Invitrogen Corporation, Carlsbad, CA) and approximately 1.5 µg RNA was reverse-transcribed using a High Capacity cDNA Archive Kit according to the provided protocol (#4322171, Applied Biosystems, Foster City, CA). Amplification was performed on an Applied Biosystems model 7900HT Fast Real-Time PCR System in duplicate using 25 ng cDNA and TaqMan Universal PCR Master Mix (#4304437, Applied Biosystems) in a total volume of 12 µl. Gapdh was used as an endogenous reference gene because it was uniformly expressed and showed no significant change among all samples, with a cycling threshold standard deviation less than 0.22. The following TaqMan assays (Applied Biosystems) were included in this study: Gadph (#Mm999999_g1), Acox1 (#Mm00443579_m1), Me1 (#Mm00782380_s1), Gadd45b (#Mm00435123_m1), Peci (#Mm00478725_m1), Ehhadh (#Mm00470091_s1), Cyp3a41 (#Mm00776855_mH), C4a (#Mm00550309_m1), and Pdk4 (#Mm00443325_m1). Fold-change was calculated using the 2–
CT method (Livak and Schmittgen, 2001).
Determination of PFBA concentration.
Liver samples were thawed and homogenized (Polytron) in four volumes of water, based on individual weight, and re-frozen at –80°C. Serum and liver homogenates were shipped to 3M on dry-ice for analysis, according to the method described previously (Chang et al., in press), using 13C4-PFBA as the internal standard.
Data analysis.
Data are presented as means and standard errors. Statistical significance was determined for each outcome variable by ANOVA that included treatment dose, block and age (where appropriate) effect, using individual litter as the statistical unit. When a significant treatment effect or interaction (p < 0.05) was detected, each treatment group was then tested for difference from the control group using the Dunnett's test. Maternal weight gain was analyzed by ANOVA with repeated measure. Because of their heterogeneity of variance, data from the postweaning weight gain were log-transformed before ANOVA was conducted. Data for FLRs were analyzed by the nonparametric Fisher's exact test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Effects of PFBA on Maternal Weight Gain and Liver Weight
Control mice gained approximately 22 g during the course of pregnancy (Fig. 1). Overall, PFBA did not produce any significant change in maternal weight gains; the smaller mean gains of
20 g at the highest PFBA dose (350 mg/kg) likely reflected a greater incidence of full-litter loss (see below). In contrast, the maternal liver weights (both absolute and relative) at term were increased significantly by the PFBA exposure, in a dose-dependent manner (Fig. 2). The PFBA-induced liver enlargement was not limited to pregnant animals, as similar weight increases were noted in the nonpregnant mice treated for the same duration. Statistical significance was achieved in the 175 and 350 mg/kg dose groups. Interestingly, in a separate cohort of mice, upon cessation of chemical treatment at term, the liver weights in the PFBA-exposed dams returned to control level 3 weeks later (at postweaning age), even at the highest dose.
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Effects of PFBA on Pregnancy Outcomes
In utero exposure to PFBA produced a significant increase of FLRs at 350 mg/kg (33% vs. 7% in controls, Table 1). However, no changes were found in the number of implants, number of live fetuses, and fetal weight. Only a few anatomical abnormalities were observed in the mouse fetuses, but these birth defects were randomly distributed among the treatment groups, including controls, and therefore were not likely associated with PFBA exposure.
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Effects of PFBA on Postnatal Growth and Development
Prenatal exposure to PFBA did not affect survival of the newborns (Table 1). The chemical treatment also did not significantly affect the weight gains of the offspring from PD 1 to 21 (Fig. 3) or from PD 22 to 291 (Fig. 4). Neonatal liver weights were higher in the two high-dose groups of PFBA than that in controls at PD 1, but these changes were no longer detected by PD 10 (Fig. 5). Despite the lack of effect on growth, postnatal development of mouse pups exposed to PFBA in utero was slightly delayed (Table 2). Eye-opening in all three PFBA dose groups was about 1–1.5 days later than in the controls. Similarly, the pubertal landmark for female mice, vaginal opening, was delayed by 2–3 days in a dose-dependent manner, with the two high-dose groups reaching statistical significance. A significant delay of preputial separation, the pubertal landmark for male mice, was detected only at highest dose of PFBA.
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Effect of PFBA on the Gene Expression
To ascertain if PFBA-induced liver hypertrophy at PD 1 was associated with activation of hepatic genes, expression of selected candidate genes was evaluated by qPCR. These genes are associated with a number of biochemical pathways that reflect activation of nuclear receptors such as peroxisome proliferator–activated receptor-alpha (PPAR-
) and pregnane X-receptor (PXR). PFBA did not significantly alter the expression of these genes in the neonatal liver (Table 3).
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Serum and Liver Concentrations of PFBA
The maternal serum and liver concentrations of PFBA after 17 daily treatments during gestation are summarized in Table 4. At 24 h after the previous administration (approximately eight half-lives), only low levels of PFBA were found in the maternal circulation and liver; similar values were noted in the few nonpregnant female mice. Consistent with previous observations (Chang et al., in press
), accumulation of PFBA in the liver of pregnant mice is lower than that in the serum, averaging about 35–55% of the serum concentration. This is in stark contrast with PFOS and PFOA, where the liver accumulations are typically three to fourfolds larger than those in the serum (Thibodeaux et al., 2003; Wolf et al., in press). It is noteworthy that after eight half-lives (where approximately 0.4% of the original level is expected to remain), the relationship between administered doses and residual levels of PFBA was lost, in both serum and liver. For comparison, the serum and liver concentrations of mouse pups were evaluated at PD 1 and 10. Considerably lower PFBA levels were detected in the 1-day-old neonates than those seen in the dams at term; while the levels of the chemicals were barely at the detection limits, or below at PD 10. Interestingly, the liver-to-serum ratios of PFBA in the developing mice were similar to those in the dams and adults.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Results from the present study indicate that in utero exposure to PFBA in the mouse at doses up to 350 mg/kg/day did not compromise neonatal survival or postnatal growth, which are two hallmark features of developmental toxicity produced by the related eight-carbon analogs, PFOS and PFOA (Grasty et al., 2003
; Lau et al., 2003, 2006; Luebker et al., 2005a, b; Wolf et al., 2007). In addition, unlike the PFOA treatment, where latent elevations of body weight were noted in the adult offspring (Lau et al., 2006), no significant weight changes were seen in the PFBA-exposed offspring up to 42 weeks of postnatal age. The PFBA doses (175 and 350 mg/kg) were chosen to approximate the AUCs estimated to have resulted from doses of PFOA (5 and 10 mg/kg), at which severe developmental toxicity was previously indicated in CD-1 mice (Lau et al., 2006). However, it should be noted that the procedure used to estimate doses designed to produce roughly equivalent AUCs for pregnant mice given PFBA and PFOA was rather inexact, because it was based on the pharmacokinetic parameters of PFOA derived from rats and those of PFBA derived from the nonpregnant female mice (the only published information available to date). Additional study will be required to determine whether the AUCs for PFBA at 35, 175, and 350 mg/kg are indeed similar to those for PFOA at 1, 5, and 10 mg/kg of PFOA in pregnant mice. On the other hand, the biological effectiveness of PFBA administration is indicated by the increases in liver weight at 175 and 350 mg/kg doses, in both pregnant and nonpregnant mice.
Two possibilities may account for the absence of neonatal mortality and growth deficits after PFBA treatment (in contrast to PFOA). The first possibility is related to the pharmacokinetic properties of the chemicals. Despite our attempt to simulate the effective PFOA doses based on the AUC approximation, the half-life of PFBA is so short (approximately 3 h in nonpregnant female mice, Chang et al., in press) that after 24 h (before the subsequent daily treatment), > 99% of the PFBA would have been eliminated. Indeed, in the current study, after eight half-lives, only residual amounts of PFBA (2–4 µg/ml) were detected in the serum of the pregnant mice; while the differences among the treatment groups are no longer distinguishable, despite the 10-fold differences in the administered doses. Hence, each daily administration of PFBA amounted to repeated episodic exposure, and unlike PFOA, a steady-state level of PFBA was never reached (Lau et al., 2006), which might be required for the profound developmental effects of the C-8 chemical. The fact that our PFBA findings resemble those derived from PFOA exposure in the rat (Butenhoff et al., 2004b), where a similar short half-life of the chemical (2–4 h) was documented for the females (Lau et al., 2007), lends support to this hypothesis. The second possibility is associated with the modes of action for PFAAs. Previous studies have suggested that PFAAs (particularly those with 8–10 carbons) are moderately potent agonists of the PPAR- (Kudo et al., 2000, 2006; Maloney and Waxman, 1999; Shipley et al., 2004; Takacs and Abbott, 2007; Vanden Huevel et al., 2006). More recently, Abbott et al. (2007) reported that PPAR- was required for the PFOA-induced postnatal lethality and developmental deficits in the mouse. It was therefore not unreasonable to assume that PFBA might also activate PPAR- and the attendant biochemical functions. However, preliminary results from our laboratory indicated that PFBA was less potent than PFOA in activating this molecular signal (Wolf et al., 2008). Accordingly, PFBA may only be a weak PPAR- agonist, which in turn may contribute to its failure to elicit the same adverse effects as the PFAAs with longer carbon chains. This relationship of carbon chain length and biochemical potency is also evident for the sulfonates of PFAAs, where the potency of PFBS was shown to be approximately one-fiftieth of PFOS based on administered doses (Ehresman et al., 2007).
However, PFBA is not completely devoid of developmental influences, as subtle delays of maturation were observable in the mouse pups. These include slight delays in eye-opening (by about 1.5 days) in all three PFBA dose groups, and a significant delay in the onset of puberty in the 350 mg/kg group. With respect to reproductive outcome, whereas there was little to no discernible effects of PFBA on implantation and embryonic/fetal development, a significant increase in the incidence of FLRs was observed in the highest dose group (30% vs. 7% in controls). This profile of early pregnancy loss at high doses but otherwise unremarkable prenatal effects, coupled with slight postnatal developmental delays, is consistent with those reported previously with PFOA and PFOS exposure (Butenhoff et al., 2004b; Lau et al., 2003, 2006; Luebker et al., 2005a), and may represent a common feature of PFAA effects.
The significant elevations of liver weights in the 1-day-old pups were also noteworthy, although these changes were seen only in the 175 and 350 mg/kg dose groups, and were attenuated by PD 10. Interestingly, the dose-response profile and the magnitude of weight increases (30–41%) in the neonatal tissue are remarkably similar to those seen in the adult liver, regardless of pregnancy status (28–32%). These findings likely reflect a general hepatic response to PFBA in the mouse, and also indicate indirectly an effective transplacental transfer of the chemical from the maternal compartment to the fetuses, leading to similar liver enlargement. PFOA-induced hepatomegaly with attendant alterations of gene expression in the liver has been demonstrated previously in adult rat (Martin et al., 2007; Guruge et al., 2006) and mouse fetuses (Rosen et al., 2007). A majority of these robust changes are consistent with transactivation of PPAR- (Rosen et al., in press-a), although involvement of other nuclear receptors (such as the constitutive androstane receptor) is also likely (Rosen et al., in press-b). In that regard, it is reasonable to expect that liver enlargement produced by PFBA in the 1-day-old mice might be accompanied by corresponding changes in gene expression. In the present study, eight genes were selected for evaluation in the neonatal mouse liver by qPCR (Table 3). These candidate genes are associated with PPAR- transactivation and various biochemical responses to PFOA exposure. In contrast to the robust responses to PFOA detected in the fetal liver at term, with fold-changes ranging from 4 to 50, little deviations from controls were discerned in the PFBA-exposed neonatal liver. The pharmacokinetic and pharmacodynamic properties of PFBA and PFOA may account for these differences. First, because of the longer half-life of PFOA (compared with PFBA) in the mouse (Lau et al., 2005), steady-state levels of the chemical were likely reached in the dams after daily administration in the previous studies (Lau et al., 2006; Rosen et al., 2007); the robust gene expression in the fetal liver therefore might reflect the accumulation of PFOA in that tissue. Because of its short elimination half-life (Chang et al., in press), PFBA exposure was likely episodic in the pregnant mice, with chemical concentrations reaching a peak shortly after a bolus oral administration, followed by a rapid decline to very low levels before the next daily bolus dose. In addition, hepatic PFBA concentrations in adult rats and mice were significantly less than serum PFBA concentrations, thereby suggesting little uptake by the liver (Chang et al., in press). Similar findings were obtained from pregnant and neonatal mice (liver PFBA to serum PFBA ratios of 35–50%) in the current study (compared with those of PFOS in the rat of about 250–400%; Thibodeaux et al., 2003). Hence, the lack of discernable alterations of gene expression might be associated with the inability of the neonatal liver to accumulate PFBA. Secondly, a preliminary report (Wolf et al., 2008) indicated that the ability of PFBA to transactivate PPAR- in the mouse was considerably less than that of PFOA. Thus, the relatively small increase of liver weight elicited by PFBA (30% in the dams compared with the 2-fold elevation induced by PFOA; Lau et al., 2006), the transient nature of the effect (undetectable by PD 10 or sooner), and the relative lack of alterations in gene expression are all consistent with the weak agonistic potency of the chemical in activating PPAR- (the primary known mode of action for PFAAs), as well as its short resident time endogenously.
In contrast to PFOA exposure during pregnancy, PFBA treatment did not alter the maternal weight gain. The slightly lower weight in the 350 mg/kg group likely reflects the inclusion of those dams that had an early pregnancy loss (FLRs). As discussed previously, the hepatic effects of PFBA appear to be much less pronounced than those of PFOA. If liver weight increases were subtracted from maternal weight, some weight effect on body (minus liver) might have been present. However, whether the significant increase in the incidence of FLRs noted in the highest PFBA dose group indicate another facet of maternal effect remains to be elaborated.
In summary, results from the current study suggest that exposure to the C-4 chemical PFBA during pregnancy in the mouse did not recapitulate the profound adverse developmental effects previously reported with the C-8 chemicals PFOA and PFOS (neonatal morbidity and mortality, and postnatal growth deficits), although some subtler effects were still detectable. The milder responses of PFBA are likely accounted for by the rapid elimination of the chemical as well as its lower potency compared with the PFAAs with longer carbon chains.
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U.S. Environmental Protection Agency.
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NOTES |
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Disclaimer: This article has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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ACKNOWLEDGMENTS |
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We wish to thank Dr Mitch Rosen for his help in selecting the candidate genes for evaluation, and Ms Judith Schmid for her assistance in statistical analysis.
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