Document KR2mrQwx4DJ3zX00ygEmqbEmX

Taft/ Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1 8 0 0 /Cincinnati, OH 45202-3957/Tel: 513.381.2838/Fax: 513.381.0205/ www.taftlaw.com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix / Beijing Robert a . Bilott 513.357.9638 bllott@taftlaw.com June 12, 2009 TSCA Confidential Business Information Center (7407M) EPA East - Room 6428, Attn: Section 8(e) & FYI U.S. Environmental Protection Agency 1200 Pennsylvania Avenue, NW Washington, DC 20460-0001 Re: Submission To TSCA 8(e)/FYI Database Re: PFQA/PFOS 3B < ff tt c " To TSCA 8(e)/FYI Database: We are hereby providing the following information for inclusion in the TSCA 8(e)/ FYI databases with respect to PFOA/PFOS: 1. Fenton, S.E., et al., "Analysis of PFOA in Dosed CD-1 Mice Part 2: Reprod.Disposition of PFOA in Tissues Mice and Their Pups," TanodxicFollu. id(2s0F0r9o),m Pregnant and Lactating doi:10.1016/j.reprotox.2009.02.012; Reprod. Toxicol.2. von Ehrenstein, O.S., et al., "Polyfluoroalkyl Chemicals in the Serum and Milk of Breastfeeding Women," (2009), doi:10.1016/j.reprotox.2009.03.001; and 3. Hines, E.P., et al., "Phenotypic Dichotomy Following Developmental Exposure to Perfluorooctanoic Acid (PFOA) in Female CD-1 Mice: Low LDiofes,e"s30In4dMucoeleEcluelvaart&edCSeellruulamr ELenpdtoincrainnodloIgnsyu9li7n-,1a0n5d(2O0v0e9r)w. eight in Mid- 89090000309 890 900 RAB:mdm Enclosure 11434740.1 CO N IN N tN O CB iH 3 / 0M 8M A TM 03 P-3 Accepted Manuscript Title: Analysis o f PFOA in Dosed CD-I M ice Part 2: Disposition o f PFOA in tissues and fluids from pregnant and lactating mice and their pups , RcprwhKtm L-; 'JriicofcKty Authors: Suzanne E. Fenton, Jessica L. Reiner, Shoji F. Nakayama, Amy D. Delinsky, Jason P. Stanko, Erin R Hines, Sally S. W hite, Andrew B. Lindstrom, Mark J. Strynar, Syrago-Styliani E. Petropoulou PD: DOI: Reference: S0890-6238(09)00040-9 doi: 10.1016/j.reprotox.2009.02.012 RTX 6230 To appear in: Received date: Revised date: Accepted date: Reproductive Toxicology 4-2-2009 20-2-2009 25-2-2009 Please cite this article as: Fenton SE, Reiner JL, Nakayama SF, Delinsky AD, Stanko JP, Hines EP, White SS, Lindstrom AB, Strynar MJ, Petropoulou S-SE, Analysis o f PFOA in Dosed CD-I Mice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups, Reproductive Toxicology (2008), doi: 10.1016/j.reprotox.2009.02.012 This is a PDF file o f an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. i Analysis of PFOA in Dosed CD-I Mice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups. Suzanne E. Fenton*', Jessica L. Reinerb, Shoji F. Nakayamab, Amy D. Delinsky*, Jason P. Stanko*, Erin P. Hines*, Sally S. White*'*1, Andrew B. Lindstrom*, Marie J. Stiynar', and Syrago-Styliani E. Petropouk>ub* *Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. EPA, MD-67, Research Triangle Park, N C 27711, USA *Oakridge Institutefo r Science and Education (ORJSE)Research Participant, Human Exposure and Atmospheric Sciences Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, NC 27711, USA cHuman Exposure and Atmospheric Sciences Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, NC 27711, USA dCurriculum in Toxicology, University o fNorth Carolina, Chapel Hill, NC 27599, USA *Current address: Division o fLaboratory Sciences, National Centerfo r Environmental Health, Centersfo r Disease Control and Prevention. Atlanta, GA 30341, USA *Corresponding author and address: Suzanne E. Fenton, Ph.D. U.S. Environmental Protection Agency Mail Drop 67 Research Triangle Park, NC 27711 USA Tel: 919-541-5220 Fax:919-541-4017 E-mail: fenton.suzanne@CDa.gov 2/20/2009 Running title: PFOA disposition in lactation Abbreviations ANOVA analysis o f variance BW body weight GD gestational day LOD limit of detection LOQ limit of quantitation MS mass spectrometer PFAA perfluoroalkyl acid PFOA perfluorooctanoic acid PFOS perfluorooctane sulfonate PND postnatal day SEM standard error of the mean UPLC ultra performance liquid chromatography 2/20/2009 3 Abstract Previous studies in mice with multiple gestational exposures to perfluorooctanoic acid (PFOA) demonstrate numerous dose dependent growth and developmental effects which appeared to worsen if offspring exposed in utero nursed from PFOA-exposed dams. To evaluate the disposition of PFOA in the pregnant and lactating dam and her offspring, time-pregnant CD-I mice received a single 0 ,0 .1 ,1 , or 5 mg PFOA/kg BW dose (N=25/dose group) by gavage on gestation day 17. Maternal and pup fluids and tissues were collected over time. Pups exhibited significantly higher serum PFOA concentrations than their respective dams, and their body burden increased after birth until at least 8 days old, regardless of dose. The distribution of milk:serum PFOA varied by dose and time, but was typically in excess of 0.20. These data suggest that milk is a substantial PFOA exposure route in mice and should be considered in risk assessment modeling designs for this compound. Key words: PFOA; serum; amniotic fluid; urine; milk; mammary gland; dosimetry; disposition 2/20/2009 Page 3 of 32 P-7 y 4 1 1. Introduction 2 Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acid (PFAA) 3 family of man-made, fhiorinated organic compounds used in a number of consumer 4 goods and industrial surfactants due to their grease and water-repellant properties. The 5 use of PFAAs in many common applications, such as stain repellants for clothing, 6 carpeting, and upholstery, and the stability of the carbon-fluorine bond have made them 7 ubiquitous in the environment. The predominant route o f exposure in North American 8 and European consumers is likely oral intake, including drinking water, while inhalation 9 and dermal absorption comprise routes o f lesser exposure [1-5], 10 PFAAs are persistent, readily absorbed, not known to be metabolized, and are 11 poorly eliminated, with half-lives in humans ranging from roughly 4-8 years [2-4], In 12 fact, the arithmetic and geometric mean half-lives Ofserum elimination, respectively, 13 were 5.4 years [95% confidence interval (Cl), 3.9-6.9] and 4.8 years (95% Cl, 4.0-5.8) 14 for PFOS; 8.5 years (95% Cl, 6.4-10.6) and 7.3 years (95% Cl, 5.8-9 2) for PFHS; and 15 3.8 years (95% Cl, 3.1 -4.4) and 3.5 years (95% Cl, 3.0-4.1) for PFOA [4J. 16 These characteristics led to increased concern for the potential health risks of 17 PFAAs and a program to reduce product and emission content of PFOA and related 18 chemicals was recently initiated [1J. PFAAs are continually detected worldwide in both 19 human and wildlife samples [3,6-9]. A recent analysis of American Red Cross blood 20 donors indicated a reduction o f 60% in blood perfluorooctane sulfonate (PFOS) and 25% 21 in blood PFOA levels between the years 2000 and 2006[ 10]. However, while the 22 production of and potential for human and wildlife exposures to certain PFAAs has been 23 reduced in the US in recent years, it is not clear that perfhiorinated compounds produced 2/20/2009 Page 4 of 32 p.8 5 1 in other countries will not continue to replace them in the US marketplace or in the 2 contribution to worldwide exposure. 3 Much of the recent health effects research on PFOA in mice, commonly 4 associated with gestational exposures o f 0.01-5 mg PFOA/kg BW, has focused on 5 developmental toxicities such as decreased maternal weight gain, reduced neonatal 6 survival and body weight, as well as later life effects such as pubertal delays, mammary 7 gland abnormalities, and excessive weight gain [2,11 -16]. Early postnatal adverse health 8 observations prompted studies examining the effect of PFOA on maternal lactation and 9 health effects o f the nursing offspring. White et al. [14] described reduced epithelial 10 differentiation on postnatal day (PND) 10 in mammary glands o f CD-I mouse dams 11 exposed to 5 mg PFOA/kg BW from GDI-17, as well as delays in epithelial involution 12 and alterations in milk protein gene expression on PND20. In addition, female offspring 13 of exposed dams displayed stunted mammary epithelial branching and growth on PND 10 14 and PND20. In a cross-foster study utilizing CD-I mice, Wolf et al. [16] reported that 15 although in utero exposure to 5 mg PFOA/kg BW from GDI -17 in the absence of 16 lactational exposure was sufficient to induce postnatal body weight deficits and 17 developmental delays, pup survival from birth to weaning was affected only in those both 18 in utero and lactationally exposed. Furthermore, recent studies [15] have shown that 19 unexposed neonates lactationally exposed to PFOA quickly developed mammary gland 20 growth deficits and that control dams nursing in niero-exposed pups (dams exposed via 21 pup grooming) demonstrated slowed differentiation of their own mammary glands that 22 was evident in whole mount preparations of the tissue by the 5thday of lactation. These 2/20/2009 Page 5 of 32 P-9 V 6 1 results support a role for impaired lactational development and possibly a significant 2 lactational transfer of PFOA in the observation of early growth effects. 3 The concern for potential prenatal and neonatal exposures in humans has been 4 raised further by the detection of PFAAs in human breast milk and cord blood and the 5 development-related outcomes associated with these observations. So et al. [17] indicated 6 a range of 47-210 ng/L (0.047-0.21 ng/ml) PFOA in 19 samples o f breast milk from 7 Chinese women. PFOA was detected in only one of 12 human milk samples collected 8 from 1996-2004 in Sweden at a concentration o f492 pg/ml (0.492 ng/ml; [18], and a 9 mean of 43.8 pg/ml (0.044 ng/ml) was reported for 45 U.S. breast milk samples collected 10 in 2004 [19], Two studies recently determined a negative association between PFOA and 11 growth indices in children with median cord serum levels of 1.6 ng/ml PFOA in the U.S. 12 [20] and 5.6 ng/ml PFOA in Denmark [21]. While only one sample was found to contain 13 PFOA in the Karrman et al. [18] study, these researchers reported a significant milk to 14 serum correlation (r2= 0.7-0.8, p<0.05) for other PFAAs detected. Furthermore, Tao et 15 al. [19] suggested that there may be preferential partitioning of PFOA to milk compared 16 to other PFAAs and also indicated that women who were nursing for the first time 17 exhibited 49% higher concentrations of PFOA in breast milk than women who had 18 nursed previously, although inter-individual variation, daily milk output and milk protein 19 concentration were not taken into consideration. The only study that has evaluated the 20 distribution coefficient o f PFCs between blood and milk in animal models was a 21 pharmacokinetic study of placental and lactational transport of PFOA in rats[22]. 22 Although female rats are known to have a serum PFOA half-life of only a few hours [23], 23 unlike mice which have a Vi-life of about 15 days [ 13], the study [22] indicated 2/20/2009 Page 6 of 32 7 1 concentrations in rat milk approximately ]0 times less than that of maternal plasma and 2 that the milk concentrations were generally of the same magnitude as the concentrations 3 in pup plasma. 4 The increasing amount of research confirming the developmental toxicity of 5 PFAAs in animal studies, coupled with their detection in human cord blood and milk, 6 supports the need for examining the disposition of PFAAs during pregnancy/lactation in 7 an appropriate animal model in order to fully establish the association between 8 prenatal/neonatal exposure and offspring effects. While other studies have examined the 9 pharmacokinetics of PFAAs in limited contexts, little data currently exist on the 10 disposition o f PFOA in pregnant and lactating mice or their offspring. We recently 11 developed an analytical method for the analysis of PFOA in mouse serum, urine, milk, 12 mammary tissue, amniotic fluid, and pups [24], Utilizing these methods, we report here 13 data on the distribution o f PFOA in various matrices o f pregnant and lactating CD-1 14 mice, as well as the serum concentration and total body load of their offspring, following 15 a single exposure o f PFOA on GD 17. These data will allow us to reduce the 16 uncertainties in risk assessment for this particular PFAA. 17 18 19 20 21 22 23 24 2/20/2009 Page 7 of 32 * |ieSTii8|g?3^**'Sflfi^S',V.i ? [ i t f i g j p. 11 8 1 2. M aterials and methods 2 2. / Chemicals 3 PFOA (ammonium salt; >98% pure), used in animal exposures, was purchased 4 from Fluka Chemical (Steinheim, Switzerland). PFOA was completely dissolved by 5 agitation in deionized tap water, in which PFOA was below the level of detection (LOD 6 0.5 ng/L for water), and prepared fresh just prior to use. 7 8 2.2 Animals 9 All animal studies were conducted as approved by the National Health and 10 Environmental Effects Research Laboratory Institutional Animal Care and Use 11 Committee. Confirmed timed pregnant female CD-I mice (=100) were purchased from 12 Charles River Laboratories (Raleigh, NC). Pregnant mice were received at the U.S. 13 EPA's Laboratory Animal Care facility on gestation day (GD) 14 (day of sperm-positive 14 designated as GDO). Upon arrival, mice (approximately 12 weeks old) were weighed and 15 randomly distributed among PFOA treatment groups. They were housed individually in 16 polypropylene cages with Alpha-dri (Shepherd Specialty Papers, Kalamazoo, MI) 17 bedding and nesting materials. They were provided pelleted chow (LabDiet 5001, PM1 18 Nutrition International LLC, Brentwood, MO) and tap water ad libitum (both contained 19 PFOA at concentrations below the LOD). Animal facilities were controlled for 20 temperature (20-24C) and relative humidity (40-60%), and kept under a 14:10-h light21 dark cycle. Mice (=25/dose group) received either wateT vehicle or a single dose (0.1, 22 1.0 or 5.0 mg/kg) of PFOA (in water; 10 pl/g) by oral gavage on GD 17. 23 2/20/2009 Page 8 of 32 p. 12 9 1 2.3 Animal Assessments and Sample Collection 2 Live dam body weights were recorded on GDI7, GDI 8 (prior to parturition), 3 PND1 (day after parturition), and PNDs 2,4, 8,11, and 18. On GD18,24 hr after the 4 PFOA exposure, five dams in each dose group were sacrificed and trunk blood, urine, 5 amniotic fluid (fluid immediately surrounding each fetus), and the 4th and 5th mammary 6 gland were collected. Liver weight, total number o f fetuses (live, dead, or resorbed), and 7 fetal weights were determined. One entire fetus from each litter was euthanized by 8 decapitation and quick frozen on dry ice in a 15 ml screwcap vial. Remaining fetuses 9 were quickly euthanized and discarded. The dam mammary gland, urine, and amniotic 10 fluid were kept on ice, and then frozen until assayed. The trunk blood was allowed to 11 clot; serum was collected after centrifugation and frozen until assayed. All samples were 12 kept frozen at -80 C. 13 A similar routine was followed on PND1 (48 hr after exposure, n=5 dams/dose 14 group). Weights of the dam, pups, dam liver, and the number of live pups in each litter 15 were recorded. A single pup from each litter was weighed, euthanized, and quick frozen 16 in a collection vial (including all blood). Blood from all remaining pups in each litter was 17 pooled into a single vial, allowed to clot, and separated to serum by centrifugation. Dam 18 and pup serum, dam urine and mammary tissue were frozen until assayed. All remaining 19 litters, in all dose groups, were equalized to 10 pups each on PND1. Biological samples, 20 including a single pup and pup serum, as described for PND1, were also collected on 21 PNDs 4, 8 and 18 (n=5 dams/dose group), at the same time of day. 22 Milk collection was attempted, following administration of oxytocin (1U/ml, i.p., 23 20 min prior to milking) on both GD 18 and PND 1, but was unsuccessful. Milk was 2/20/2009 Page 9 of 32 p. 13 10 1 collected on PNDs 2,8 ,1 1 , and 18 following a 2 hr separation o f the pups from the dam 2 and an oxytocin stimulus. The milk was vacuum aspirated using low, pulsatile pressure, 3 into a pre-weighed microcentrifuge tube. Collected milk was weighed and frozen until 4 analyzed. Biological samples including urine, dam and pup serum, amniotic fluid, 5 mammary tissue, whole pup, and milk were analyzed for PFOA using the methods 6 described briefly below and in our companion paper [24], 7 8 2.4 PFOA sample analyses 9 Briefly, the analysis of PFOA was performed using a Waters AcquityTM Ultra 10 Performance liquid chromatography system interfaced with a Waters Quattro Premier XE 11 triple quadrupole mass spectrometer (UPLC-MS/MS) (Waters, Milford, MA). Either 25 12 or 50 pL of serum and amniotic fluid (50 pL used for controls), 20 pL aliquots of urine 13 and milk, and 300 pL o f pup or mammary tissue homogenates were utilized as starting 14 material for these analyses. Samples were extracted, purified, and concentrated or diluted 15 exactly as described by Reiner et al. [24). 10-40 pL of the prepared sample, depending on 16 the concentration of the original exposure, was injected and run on the UPLC-MS/MS 17 [24], Refer to Reiner et al. [24] for method performance and quality control steps that 18 were performed to insure the precision and accuracy of the methods used. The limit of 19 quantitation (LOQ) for these experiments were 5 ng/ml (serum), 1 ng/mi (amniotic fluid, 20 urine, milk), and 1 ng/g (whole pups, mammary tissue). 21 22 23 2/20/2009 Page 10 ot 32 * . ' ' r *o s5 c . V r. y f. -fc -g V r* = 4 g # g s a n ? e.-iixtx..;? ii^ = t jS w S v r f T 5 * * ? < te w IS s 4 a = sS :; p. 14 11 1 2.5 Urinary crealinine measures 2 Creatinine concentrations were measured as a basis to evaluate PFOA in mouse 3 urine. The QuantiChrom creatinine assay (BioAssay Systems, Hayward, CA) exhibited 4 an LOD of 0.10 ng/ml and was linear up to 300 ng/ml. Thirty pi of each urine sample was 5 prepared and evaluated at 510 nm singly or in duplicates (five duplicates per set of 20 6 samples) according to the manufacturer's instructions. The inter-assay coefficient of 7 variation (CV) ranged from 4.0-6.8% and the intra-assay CV ranged from 0.3-16.1 %, 8 with an average of 4.9%. The assay standard accuracy ranged from 0.2-8.4%. Urinary 9 PFOA is reported as corrected for creatinine concentrations (ng PFOA/g creatinine). 10 11 2.6 Computations and Statistics 12 Reported PFOA concentrations have bear adjusted for dilution or concentration 13 factors, as well as creatinine levels (ng/g; urine), or the weight of the tissue evaluated 14 (ng/g; mammary tissue and whole pups). Serum, amniotic fluid, milk and urinary 15 concentrations are reported as ng/ml. Averages, proportions, and statistical comparisons 16 were calculated with SAS 9.1 (SAS Institute; Cary, NC). Statistical significance was 17 determined using a Proc GLM ANOVA, with a Dunnett's post-hoc comparison, and 18 significance was set at p<0.05. 19 20 21 22 2/20/2009 Page 11 of 32 SJV7! |;J 5 p. 15 12 1 3. Results 2 J. 1 Biological Outcomes 3 This is the first study to report single dose disposition of PFOA in pregnant and 4 lactating mice and their offspring. The doses chosen were based on previous reports in 5 CD-I mice [3,14,16] demonstrating developmental health outcomes following multiple 6 gestational PFOA exposures. A single PFOA exposure cmG D I7 did not affect the 7 number of live fetuses (on GDI 8), implantation sites, or live-bom pups (on PND1), or 8 dam body weights (data not shown). Unlike studies using multiple gestational PFOA 9 exposures [13,25], there was no change in pup body weight, dam liver weight, and dam 10 liver:BW ratios, within the PFOA dose Tange administered in this study (Figure 1). The 11 rise in dam liver:BW ratio between GDI 8 and PND1, which persisted until weaning, was 12 due to the dramatic decrease in body weight at parturition, as this single late gestation 13 PFOA exposure failed to change mean liver weight in exposed dams, compared to control 14 values, at any time evaluated. 15 16 3.2 PFOA Concentrations Prior to Birth 17 The mean concentration of PFOA in the amniotic fluid and serum of the exposed 18 dams 24 hr after exposure is shown (Figure 2; amniotic fluid controls average 3.8 ng/ml). 19 The average concentration of PFOA detected in dam serum was about twice the amniotic 20 fluid concentration at each dose evaluated (amniotic fluid was 68.8, 51.8, and 40% of 21 dam serum levels at 0.1,1, and 5 mg PFOA/kg BW, respectively). A comparison of the 22 amount of PFOA in an entire GDI 8 fetus (body burden/pup+standard error of the mean 23 [SEM]; Figure 5) to the GD18 PFOA concentration in amniotic fluid (ng/ml; assuming 1 2/20/2009 Page 12 of 32 p. 16 13 1 ml total volume) reveals 2.3-, 3.1-, and 2.7-fold increased PFOA in the pup vs. the fluid 2 in which it was contained in utero for 0.1,1, and 5 mg/kg dose groups, respectively. 3 4 3.3 PFOA Concentrations in the Dams 5 The serum and urine PFOA concentrations were evaluated in dams that were 6 nursing litters of approximately 10 pups (PND1 equalized; minimal pup loss over time). 7 As expected, dam sera contained the highest PFOA concentrations o f any matrix 8 evaluated, regardless o f dose (Figure 3; all serum controls <LOQ). The rise in circulating 9 serum PFOA with increasing dam exposures was proportional to the change in dose 10 delivered, regardless of stage of lactation (i.e., mean 9.9-fold and 5.1-fold increases 11 between 0.1 -1.0 mg/kg and 1.0-5.0 mg/kg exposures, respectively). 12 A one-time PFOA exposure of 0.1 mg/kg produced an average dam serum PFOA 13 concentration (Figure 3A) of 144-226 ng/ml at 24 and 48 hr after exposure, respectively, 14 which was reduced to an average of 44 ng/ml near the peak of lactation (PND8), and had 15 risen to a mean of 123 ng/ml by PNDJ 8, a time when the pups' primary caloric intake 16 came from rodent chow and not milk. The U-shaped serum concentration curve observed 17 in the 0.1 mg PFOA/kg dose group was also present in the 1 and 5 mg/kg exposure 18 groups. 19 As shown in Figure 3 (A-C; control urine and mammary gland PFOA <LOQ), 20 although the concentrations of PFOA cannot be compared directly between serum, urine, 21 and mammary tissue, due to the difference in units, it was evident that much less PFOA 22 was being excreted in dam urine than was present in dam serum, and that mammary 23 tissue contained a considerable amount of PFOA. While a U-shaped response in dam 2/20/2009 Page 13 of 32 14 1 excretion of PFOA (urine) was not as pronounced as that of serum, mammary tissue 2 demonstrated a strong U-shaped response, with the lowest concentrations measured near 3 the peak of lactation, and a significant rise in concentration apparent again at PND18 4 (p<0.05). 5 When aspirated milk PFOA values were evaluated (Figure 3D; 1 control >LOQ), 6 a U-shaped curve over time was again evident. As depicted in Table 1, the percentage of 7 PFOA in milk (compared to serum) was substantial. Comparing the milk concentrations 8 to the closest matched dam serum concentrations (by time), the amount o f PFOA in the 9 milk consistently ranged from 1/10 to 1/2 that of dam serum PFOA across dose and time. 10 It appeared that the day of lactation on which milk PFOA was measured had an important 11 influence on this relationship. The milk:serum PFOA ratio was greatest in early and late 12 lactation (PND2 and PND18), ranging from 15-56% (means of 33% early and 26% late), 13 while near the peak o f lactation (PND8 and 11), the PFOA milkiserum ratio ranged from 14 11-27% (mean 17.7%). It was not possible to accurately measure the volume of milk 15 obtained at aspiration, but precise weights were compared. On PNDs 2,8, 11, and 18, the 16 average weight of milk obtained via aspiration of control mice was 0.072,0.1906, 17 0.2547, and 0.0457 g, respectively, demonstrating over a 3.5-fold increase in weight from 18 PND2 to 11 and a 5.6-fold drop from PND11 to 18. 19 20 3.4 PFOA Concentrations in the Pups 21 Pup serum PFOA concentration was evaluated on PNDs 1,4, 8, and 18. In 22 comparing the average PFOA concentrations in PND1 pups vs. their respective dams 23 (Figure 4A; whole control pups and control serum < LOQ), it appeared that circulating 2/20/2009 Page 14 of 32 p. 18 15 1 pup serum PFOA concentrations were significantly higher than those measured in dams, 2 regardless of dose (p<0.05). Although pups possessed a substantially higher serum PFOA 3 concentration than dams, the difference in pup and dam blood volumes at those stages of 4 pup development are considerable. Regardless of those differences, heightened 5 circulating PFOA in pup sera reflected increased exposures, proportional to dose 6 throughout lactation (i.e., mean 10.4-fold and 4.3-fold increases between 0.1-1.0 mg/kg 7 and 1.0-5.0 mg/kg exposures, respectively). 8 Unlike their dams, pups did not demonstrate U-shaped serum PFOA 9 concentration curves (Figure 4B). Pup serum PFOA concentrations continued to exceed 10 the average dam serum PFOA concentrations over time, until PND18 when the pup and 11 dam concentrations approached 1:1. When the PFOA concentration (ng/g) was evaluated 12 in whole pups (pup and blood; Figure 5 left panels), a decline in PFOA concentration was 13 detected over time, across all doses. However, when the rapidly increasing body weight 14 of the pups was taken into consideration to calculate the total amount of PFOA in the 15 neonate (as shown in Figure 1), a completely different trend was noted (Figure 5 right 16 panels). Regardless of exposure dose, PFOA body burden (adjusted for weight) rose 17 through the peak of lactation and had begun to decline by PND18, demonstrating an 18 inverse U-shaped curve. When the administered PFOA dose and measured body burden 19 in whole pups (body weight taken into effect) were compared the administered 20 PFOA measured PFOA ratio was no longer proportional throughout lactation, and unlike 21 the ratios reported for dam and pup serum PFOA. Mean body burden ratios of 13.2-fold 22 (range 11.1-17.8) and 4.3-fold (range 3.2-5.1) increases between 0.1-1.0 mg/kg and 1.023 5.0 mg/kg exposures, respectively, were determined. 24 2/20/2009 Page 15 of 32 p. 19 16 1 4. Discussion 2 These data confrnn that on a concentration-based comparison, gestationally 3 PFOA-exposed pups exhibited a significantly larger serum PFOA load than their dam. 4 That substantia] serum PFOA load in pups was evident 24 hr after a single exposure, and 5 was apparently due to blood-borne (transplacental) transfer. Another important discovery 6 is the U-shaped PFOA concentration over time, regardless of dose, in the dam mammary 7 tissue, milk, and serum. This unique PFOA response was not detected in pups or pup 8 serum, and was evident to a lesser extent in the dams' urinary excretion curves. However, 9 when PFOA body burden in whole pups was the unit of measure, an inverse U-shaped 10 curve was apparent, and the PFOA burden of pups is proposed to increase due to milk11 borne PFOA intake. 12 The decline in concentration seen in the milk, mammary and serum U-shaped 13 curves is hypothesized to be due to hydro-dilution associated with increased blood and 14 milk volumes. Several physiological conditions are changed during lactation that have 15 been well documented in rats and directly relate to mice as their lactation period is of the 16 same length. A decrease in total plasma proteins due to increased blood volume, cardiac 17 output, and blood flow to certain tissues, such as the mammary gland has been reported 18 in rats [26, 27]. Elevated blood volume is due to increased plasma volume [27). Milk 19 yield (g/hr) in rats was reported to reach its peak by PND 10 [27] and the rat mammary 20 gland reaches its maximum size (as % body weight) by PND15 [26], with a steep rise in 21 size from PND5-15. Rat mammary gland blood flow and volume of milk produced are 22 directly related, when measured on PND15 [26], Total serum proteins are lower in 23 lactating rats that those measured in non-lactating rats [27], and in humans, serum 2/20/2009 Page 16 of 32 17 ] albumin concentration decreases during pregnancy and early lactation [28], Further, at 14 2 d postpartum, the cardiac output of lactating rabbits was 30% higher than that in non3 lactating animals, and the mammary gland was the only organ shown to increase in 4 weight, relative to body weight [29]. 5 Although a complete set of data that could address the exact reason for the U6 shaped curves during lactation was not collected in this study, the aspirated milk weights 7 did reveal a dramatic increase in milk volume (assumed due to weight change) from 8 PND2 up to the peak of lactation (PND11). This dramatic change in volume (weight) 9 may explain the decrease in milk PFOA concentration seen between PND2 and PND11. 10 PFOA also appears to concentrate in serum and milk near the end of lactation (PND 18, 11 for example) when pups are eating more chow and suckle less often. Mammary gland 12 blood flow has been reported to decrease by half in a 24 hr period, when suckling rat 13 offspring are removed from the dam [26], and this fall in mammary blood flow is directly 14 associated with decreased cardiac output and % blood flow used by the mammary gland. 15 In this study a precipitous drop in weight of milk collected between the peak o f lactation 16 and PND 18 was noted, indicating a rapid decrease in milk volume. Therefore, the U17 shape of the dam PFOA curves are proposed to be driven by physiological dilution and 18 concentration of the PFOA load over the period of lactation, reaching the greatest dilution 19 at or near the peak of lactation when the milk volume produced by the dam and 20 consumed by the pups is the greatest. Increased consumption of milk up to PND11 likely 21 directly contributed to the accumulation of body burden in the pup over this life stage. 22 A significant contribution of milk-borne PFOA transfer in CD-I mice was 23 detected in these studies. Previous reports in rats [22] and humans [18] have estimated 2/20/2009 Page 17 of 32 ^sUxrLVt*--?;..**$*Vh&?.v4Ife*ae<i*rfo p. 21 18 1 that the dam PFOA milkiserum distribution ratio was 0.1 and 0.01, respectively. In the 2 present study, the distribution ratio ranged from slightly more than 0.5 to 0.1 in mice, 3 depending on dose, with the lowest doses tested demonstrating the highest ratios over 4 time. If the milk PFOA concentrations had been measured near the peak of lactation only 5 (days 8-11), the 0.1 milkrsera distribution estimate previously reported for rats in mid6 lactation [22] may have also been presumed true in mice. However, at two periods during 7 lactation (early and late) spikes of increased milkrserum ratios appeared, regardless of 8 dose, with a substantial peak in milk PFOA concentrations on PND2. Although volumes 9 of milk large enough to perform analytical measures prior to PND2 were not able to be 10 obtained, we suspect, based on the significant PFOA concentrations in the PND 1 11 mammary gland, that substantial milk PFOA concentrations would have been evident on 12 PND1, as well, primarily due to being condensed in small milk volumes. 13 In previous reports by Lau [13], Wolf [16], White [14] and co-workers, decreased 14 body weight gain and neonatal mortality were evident on several days just after birth in 15 CD-1mouse litters gestationally exposed to 3 mg/kg PFOA and higher. In fact, in a 16 cross-foster study [16] demonstrating decreased body weight gain at 5 mg/kg from in 17 utero exposure only, significant decreases in body weight gain were detected in the 3 18 mg/kg dose group only when in utero exposed mice were also allowed to nurse from a 19 PFOA exposed dam. Even at 5 mg/kg, there was no evidence of decreased pup body 20 weight or neonatal mortality in the current study, following a single gestational PFOA 21 exposure. Our PFOA measurements in whole pups indicate that the PFOA body burden 22 accumulates in early life, and begins a decline as pups mature, open their eyes, and begin 23 to eat chow and drink water. Our data and those demonstrating deleterious health 2/20/2009 Page 18 of 32 p. 22 19 1 outcomes suggest that the milk of gestationally PFOA-exposed mice was a major source 2 of continued exposure to this compound for the developing pups. 3 As expected, large differences in dam and pup serum PFOA concentrations from 4 those previously reported [14,16] were noticed, and those differences bring to light the 5 issue of single vs. multiple dose kinetics. As noted for PFOS, single dose kinetics may 6 differ substantially from those involving repeated doses [30], Concentration dependent 7 changes in clearance can result in discrepancies between single and repeated dose 8 kinetics. 9 A limited number of epidemiological studies have revealed associations between 10 health outcomes (birth weight, head circumference) and cord blood or maternal serum 11 PFOA concentrations in humans [20,21], while other studies failed to detect associations 12 with later developmental milestones in infants [31]. Several studies have now measured 13 PFAAs in human milk [17-19, 32, 33], however only one study has been able to 14 approximate the milkrserum relationship of PFOA transfer [18]. The reported 0.01 15 (1/100,b) relationship was determined from a single voluntarily contributed sample at 3 16 weeks postpartum. According to the mouse milk:serum PFOA distribution over time that 17 we report herein, the values reported in one human [18] and rats [22] may not be 18 representative of the PFOA distribution to milk throughout lactation in those species. 19 In conclusion, these studies confirmed and further defined considerable PFOA 20 exposures to mouse offspring following a single gestational exposure. They also 21 demonstrated the accumulation of chemical over time in whole pups, which likely results 22 from milk-borne PFOA, an exposure that had previously been incompletely assessed in 23 other species. A single 0.1 mg/kg PFOA exposure to a pregnant mouse induced 2/20/2009 Page 19 of 32 ** 5^*S S r.T * x i= r ^ in u -s ; ...jg ? a*ff-- -s. a a *5 *3 t S b r*r- p. 23 20 1 circulating serum PFOA concentrations of 44-216 ng/ml in dams and 117-326 ng/ml in 2 pups; values similar to or lower than serum PFOA concentrations of children that were 3 accidentally exposed via DuPont production plant emission (34], Because of evidence 4 [15, 35] demonstrating neonatal and latent health effects following developmental 5 exposures to PFOA in mice, associated with higher circulating PFOA levels than those 6 reported here, continued studies evaluating exposure-effect relationships are warranted in 7 children. 8 9 10 11 12 13 Acknowledgements 14 The authors would like to thank Drs. Barbara Abbott (US EPA, Reproductive Toxicology 15 Division) and Chester Rodriguez (National Center for Computational Toxicology, US 16 EPA) for their constructive criticisms of this manuscript. We acknowledge the excellent 17 care of these animals by New Year Tech, Inc. (Restin, VA). The research in this article 18 has been reviewed by the National Health and Environmental Effects Research 19 Laboratory, US Environmental Protection Agency (EPA), and approved for publication. 20 Findings in this report are those of the authors and approval does not signify this report 21 reflects EPA policy. The use of trade names or commercial products does not constitute 22 endorsement or recommendation for use. 23 24 2/20/2009 Page 20 of 32 p. 24 21 1 References 2 [1] U.S. Environmental Protection Agency, Announcement of the 2010/15 PFOA 3 Stewardship Program by Administrator Stephen L. Johnson. (2006) Available at 4 http://www.epa.gov/opptintr/pfoa/pubs/pfoastewardship.htm. Accessed 2/3/2009. 5 6 [2] M.E. Andersen, J.L. Butenhoff, S.C. Chang, D.G. Farrar, G.L. Kennedy, Jr., C. 7 Lau, G.W. Olsen, J. Seed and K.B. Wallace, Perfluoroalkyl acids and related cbemistries8 -toxicokinetics and modes of action, Toxicol Sci, 102 (2008), 3-14. 9 10 (3] C. Lau, K. Anitole, C. Hodes, D. Lai, A. Pfahles-Hutchens and J. Seed, 11 Perfluoroalkyl acids: a review of monitoring and toxicological findings, Toxicol Sci, 99 12 (2007), 366-394. 13 14 [4] G.W. Olsen, J.M. Burris, D.J. Ehresman, J.W. Froehlich, A.M. Seacat, J.L. 15 Butenhoff and L.R. Zobel, Half-life of serum elimination of perfluorooctanesulfonate, 16 perfluorohexanesulfonate, and perfhiorooctanoate in retired fluorochemical production 17 workers, Environ Health Perspect, 115 (2007), 1298-1305. 18 19 [5] D. Trudel, L. Horowitz, M. Wormuth, M. Scheringer, l.T. Cousins and K. 20 Hungerbuhler, Estimating consumer exposure to PFOS and PFOA, Risk Anal, 28 (2008), 21 251-269. 22 23 [6] K.S. Guruge, P.M. Manage, N. Yamanaka, S. Miyazaki, S. 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Olsen, D.C. Mair, T.R. Church, M.E. Ellefson, W.K. Reagen, T.M. Boyd, 42 R.M. Herron, Z. Medhdizadehkashi, J.B. Nobileni, J.A. Rios, J.L. Butenhoff and L.R. 43 Zobel, Decline in perfluorooctanesulfonate and other polyfluoroalkyl chemicals in 44 American Red Cross adult blood donors, 2000-2006, Environ Sci Technol, 42 (2008), 45 4989-4995. 2/20/2009 Page 21 of 32 * ` I d rift m 3* p. 25 ->'v-::"*vl- 22 1 2 [11] J.L. Butenhoff, D.W. Gaylor, J.A. Moore, G.W. Olsen, J. Rodricks, J.H. Mandel 3 and L.R. Zobel, Characterization of risk for general population exposure to 4 perfluorooctanoate, Regul Toxicol Pharmacol, 39 (2004), 363-380. 5 6 [12] C. Lau, J.L. Butenhoff and J.M. Rogers, The developmental toxicity of 7 perfluoroalkyl acids and their derivatives, Toxicol Appl Pharmacol, 198 (2004), 231-241. 8 9 [13] C. Lau, J.R. Thibodeaux, R.G. Hanson, M.G. Narotsky, J.M. Rogers, A.B. 10 Lindstrom and M.J. Strynar, Effects of perfluorooctanoic acid exposure during pregnancy 11 in the mouse, Toxicol Sci, 90 (2006), 510-518. 12 13 [14] S.S. White, A.M. Calafat, Z. Kuklenyik, L. Villanueva, R.D. Zehr, L. Helfant, 14 M J . Stiynar, A.B. Lindstrom, J.R. Thibodeaux, C. Wood and S.E. Fenton, Gestational 15 PFOA exposure of mice is associated with altered mammary gland development in dams 16 and female offspring, Toxicol Sci, 96 (2007), 133-144. 17 18 [15] S.S. White, K. Kato, L.T. Jia, B.J. Basden, A.M. Calafat, E.P. Hines, J.P. Stanko, 19 C.J. Wolf, B.D. Abbott and S.E. Fenton, Effects o f perfluorooctanoic acid on mouse 20 mammary gland development and differentiation resulting from cross-foster and 21 restricted gestational exposures, Reprod Toxicol (2008). 22 23 [16] C.J. Wolf, S.E. Fenton, J.E. Schmid, A.M. Calafat, Z. Kuklenyik, X.A. Bryant, J. 24 Thibodeaux, K.P. Das, S.S. White, C.S. Lau and B.D. Abbott, Developmental toxicity of 25 perfluorooctanoic acid in the CD-I mouse after cross-foster and restricted gestational 26 exposures, Toxicol Sci, 95 (2007), 462-473. 27 28 [17] M.K. So, N. Yamashita, S. Taniyasu, Q. Jiang, J.P. Giesy, K. Chen and P.K. Lam, 29 Health risks in infants associated with exposure to perfluorinated compounds in human 30 breast milk from Zhoushan, China, Environ Sci Technol, 40 (2006), 2924-2929. 31 32 [18] A. Karrman, I. Ericson, B. van Bavel, P.O. Damerud, M. Aune, A. Glynn, S. 33 Lignell and G. Lindstrom, Exposure of perfluorinated chemicals through lactation: levels 34 of matched human milk and serum and a temporal trend, 1996-2004, in Sweden, Environ 35 Health Perspect, 115 (2007), 226-230. 36 37 [19] L. Tao, K. Kannan, C.M. Wong, K.F. Arcaro and J.L. Butenhoff, Perfluorinated 38 compounds in human milk from Massachusetts, U.S.A, Environ Sci Technol, 42 (2008), 39 3096-3101. 40 41 [20] BJ . Apelberg, F.R. Witter, J.B. Herbstman, A.M. Calafat, R.U. Halden, L.L. 42 Needham and L.R. Goldman, Cord serum concentrations of perfluorooctane sulfonate 43 (PFOS) and perfluorooctanoate (PFOA) in relation to weight and size at birth, Environ 44 Health Perspect, 115 (2007), 1670-1676. 45 2/20/2009 Page 22 of 32 ' 5 . n -. J i ` 3 r= s> a a ^ tr r . L a to M ig tE S PEPET Rp p. 26 23 1 [21] C. Fei, J.K. McLaughlin, R.E. Tarone and J. Olsen, Fetal growth indicators and 2 perfluorinated chemicals: a study in the Danish National Birth Cohort, Am J Epidemiol, 3 168 (2008), 66-72. 4 5 [22] P.M. Hinderliter, E. Mylchreest, S.A. Gannon, J.L. Butenhoff and G.L. Kennedy, 6 Jr., Perfluorooctanoate: Placental and lactational transport pharmacokinetics in rats, 7 Toxicology, 211 (2005), 139-148. 8 9 [23] J.P. Vanden Heuvel, B.I. Kuslikis, M.J. Van Rafelghem and R.E. Peterson, Tissue 10 distribution, metabolism, and elimination of perfluorooctanoic acid in male and female 11 rats, J Biochem Toxicol, 6 (1991), 83-92. 12 13 [24] J.L. Reiner, S.F. Nakayama, A.D. Delinsky, J.P. Stanko, S.E. Fenton, A.B. 14 Lindstrom and M.J. Strynar, Analysis of PFOA in dosed CD1 mice: Part 1. Methods 15 development for the analysis of tissues and fluids from pregnant and lactating mice and 16 their pups, Reprod Toxicol (2008). 17 18 [25] B.D. Abbott, C.J. Wolf, J.E. Schmid, K.P. Das, R.D. Zehr, L. Helfant, S. 19 Nakayama, A.B. Lindstrom, M.J. Strynar and C. Lau, Perfluorooctanoic acid induced 20 developmental toxicity in the mouse is dependent on expression of peroxisome 21 proliferator activated receptor-alpha, Toxicol Sci, 98 (2007), 571-581. 22 23 [26] A. Hanwell and J.L. Linzell, The effects of engorgement with milk and of 24 suckling on mammary blood flow in the rat, J Physiol, 233 (1973), 111-125. 25 26 [27] K. Suzuki, H. Hirose, R. Hokao, N. Takemura and S. Motoyoshi, Changes of 27 plasma osmotic pressure during lactation in rats, J Vet Med Sci, 55 (1993), 561-564. 28 29 [28] M. Dean, B. Stock, R J. Patterson and G. Levy, Serum protein binding of drugs 30 during and after pregnancy in humans, Clin Pharmacol Ther, 28 (1980), 253-261. 31 32 [29] C.S. Jones and D.S. Parker, Mammary blood flow and cardiac output during 33 initiated involution of the mammary gland in the rabbit, Comp Biochem Physiol A Comp 34 Physiol, 91 (1988), 21-25. 35 36 [30] L.A. Harris and H.A. Barton, Comparing single and repeated dosimetry data for 37 perfluorooctane sulfonate in rats, Toxicol Lett, 181 (2008), 148-156. 38 39 [31] C. Fei, J.K. McLaughlin, R.E. Tarone and J. Olsen, Perfluorinated chemicals and 40 fetal growth: a study within the Danish National Birth Cohort, Environ Health Perspecl, 41 115(2007), 1677-1682. 42 43 [32] W. Volkel, O. Genzel-Boroviczeny, H. Demmelmair, C. Gebauer, B. Koletzko, 44 D. Twardella, U. Raab and H. Fromme, Perfluorooctane sulphonate (PFOS) and 45 perfluorooctanoic acid (PFOA) in human breast milk: results of a pilot study, Int J Hyg 46 Environ Health, 211 (2008), 440-446. 2/20/2009 Page 23 of 32 p. 27 V-v-I-"r-'-'i 24 1 2 [33] O.S. von Ehrenstein, Fenton, Suzanne E., Kato, Kayoko, Kuklenyik, Zsuzsanna, 3 Calafat, Antonia M., Hines, Erin P ., Polyfluoroalkyl Chemicals in toe Serum and Milk of 4 Breastfeeding Women Reprod Toxicol, In Press (2009). 5 6 [34] E.A. Emmett, F.S. Shofer, H. Zhang, D. Freeman, C. Desai and L.M. Shaw, 7 Community exposure to perfhiorooctanoate: relationships between serum concentrations 8 and exposure sources, J Occup Environ Med, 48 (2006), 759-770. 9 10 [35] E.P. Hines, White, Sally S., Stanko, Jason P., Gibbs-Floumoy, Eugene A., Lau, 11 Christopher, Fenton, Suzanne E ., Phenotypic Dichotomy Following Developmental 12 Exposure to Perfluorooctanoic Acid (PFOA) in Female CD-I Mice: Low Doses Induce 13 Elevated Serum Leptin and Insulin, and Overweight in Mid-life, Molec Cell Endocrinol, 14 In Press (2009). 15 16 17 2/20/2009 Page 24 of 32 p. 28 25 1 Figure legends: 2 Figure 1. Dam tissue weights and average pup weights following a single gavage PFOA 3 exposure mi GDI7. PFOA was without effect on several biological end points (p>0.05), 4 such as dam body weight measured on several postnatal days (PND) and on gestation day 5 (GD)18 (not shown). (A) Dam liver weight, (B) livenbody weight ratio, and (C) pup 6 body weight over time or numbers of live pups or fetuses (not shown) were also 7 unchanged by a single PFOA exposure Data are shown as Mean + SEM or as a mean 8 ratio. 9 Figure 2. Comparison of gestation day (GD)18 dam serum and amniotic fluid PFOA 10 concentrations. PFOA concentrations were significantly higher in dam serum than 11 amniotic fluid at all doses evaluated (p<0.05). Data are shown as Mean + SEM. 12 Figure 3. PFOA concentrations in exposed dams. PFOA concentrations were measured 13 in dam serum (A; ng/ml), urine (B; ng/g creatinine), and mammary tissue (C; ng/g tissue 14 weight) on postnatal days (PND) 1,4, 8 and 18. PFOA concentrations were measured in 15 aspirated milk samples collected on PNDs 2, 8, 11, and 18 (D; ng/ml). Although panels 16 A-C and B-D cannot be directly compared (due to different units), the U-shaped 17 concentration curve present in dam serum (regardless of dose) was also detected in 18 mammary tissue and aspirated milk. Data are shown as Mean + SEM .fDenotes a single 19 reliable measurement at this time due to insufficient volumes in other dams at this dose 20 and time. 21 Figure 4. Neonatal transfer of PFOA to pups. (A) A significantly higher PFOA 22 concentration in pup vs. dam serum on PND1 was noted (p<0.05; v:v). (B) Pooled pup 23 serum PFOA concentrations did not demonstrate a U-shaped curve, but gradually 2/20/2009 Page 25 of 32 S i ^=1 i^-3 '=-J-3 ***-m m i m ^ S i i 26 1 declined over lime, presumably due to dilution of dose by increased growth-related blood 2 volume. Data are shown as Mean + SEM. 3 Figure 5. Whole pup PFOA concentrations. PFOA concentrations were measured in a 4 representative whole pup (pup and blood; ng/g; left panels) from each litter. Although 5 there is a consistent downward trend in PFOA concentration over time, the rapidly 6 increasing blood volume and body weight changes must be taken into consideration when 7 interpreting these data. Body weight-adjusted values (right panels; [ng/g PFOA 8 measures* g body weight = body burden]) demonstrate an accumulation of exposure until 9 late in the lactational period. Data are shown as Mean + SEM. 10 11 12 2/20/2009 Figure 1. 3 .5 (A) Dam Liver Weight W eight (g) Control 0.1 1 PFOA Exposure (mg/kg) (B) Dam Liver: BW Ratio 5 Liver: BW Ratio (%) Control 0.1 1 PFOA Exposure (mg/kg) 5 Control 0.1 1 PFOA Exposure (mg/kg) GD18 QPND1 Q PN D4 0 PND8 PND18 5 Page 27 of 32 _** - *-^^* * .<*** __ .^ , , ---- #-- * p. 31 ^fssga^jy Figure 2. 9000 8000 E 7000 w 6000 5000 < uO. 4000 a. 3000 2000 1000 0 0.1 5 PFOA Exposure (mg/kg) Page 28 of 32 p. 32 S ii, irm M m PFOA (ng/ml) Figure 3 25 2Dt 15 10 5 0 GDIS PHOI PND4 PNDS PND1S (A) Dam Serum PFOA G D I! PND1 PH M PNOS PNOIS (B) Dam Urine PFOA GDIS PND1 PMD4 n m PHD1 PFOA (ng/g creatinine) (C) Dam Mammary Gland PFOA PFOA (ng/g) PFOA (ng/ml) (D) Dam Milk PFOA Time PMD2 PNDS PND11 PN01B Page 29 of 32 p. 33 Figure 4. (A) Dam vs. Pup Serum PFOA 18000 T E 16000 . PND 1 Dam 0c 14000 . PND 1 Pup < 12000 . Ou. 10000 . . 8000 . E3w 6000 . 0 4000 . cn 2000 . 0. dt 0.1 PFOA Exposure (mg/kg) (B) Pup Serum PFOA Over Time 1 4 8 18 Postnatal Day (PND) 1 4 8 18 Page 30 of 32 1 O PFOA (ng/g) I o 00 PFOA (ng/g) . .'.it > tc2o 13 tn GDI8 1 4 8 18 asm s 3 * 1 mg/kg GD18 A Body Burden PFOA (ng) Body Burden PFOA (ng) s 1 1 |g Body Burden PFOA (ng) 33 * fi ii&Vji 1 Wife i" Jhj*"*>* &V IS J lj&iff)eIf'AP.'i;-,:.. Sii!, Spj; tjf'iv-;' fe/ mt p. 34 Page 31 of 32 Tables ay* 'a if"' ' l.'!v:, m,w, * . . il|<<n|n .i;-:.i;ii:^i!|s'!S!itid5^Wii! Table 1. Milk-borne PFOA* as a percentage of dam serum concentrations over lactation. Single GD17PF0A exposure PND 1 serum PFOA comparison PND 4 serum PFOA comparison PND 2 milk PND 8 serum PFOA comparison PND 6 milk PND 8 serum PFOA comparison PND 11 milk PND 18 serum PFOA comparison PND 18 milk 0.1 mg PFOA/kg 15% 31% 27% 11% 36% 1.0 mg PFOA/kg 37% 56% 21% 21% 24% 5.0 mg PFOA/kg 25% 36% 13% 13% 18% r PFOA= perfluorooctanoic acid, QDgestational day, PND*postnatal day. The milk.serum PFOA ratio reported above was calculated as: [concentration of milk PFOA/concentratlon of serum PFOA]*100" % milk:serum for each dam within a dose group. These values were averaged and reported above. s 32 of 32 o COOl . GjModel ^ ^ 23 1 1 -8 R eproductive Toxicology xxx (2009) xxx-xxx Contents lists available at ScienceOirect Reproductive Toxicology jo u rn a l h o m ep a g e : w w w .e ls e v ie r.c o m /lo c a te /fe p ro to x p. 36 , <ii Polyfluoroalkyl chemicals in the serum and milk of breastfeeding women * Ondine S. von Ehrenstein3 *, Suzanne E. Fentonb, Kayoko Katoc, Zsuzsanna Kuklenyikc, o Antonia M. CaJafatc, Erin P. Hinesb>1 4 ` UCLA School o f Public Health. University o f California. Los Angeles. CA. United States 5 k US Environmental Protection Agency. ORD. NHEEKL. Reproductive Toxicology Division. KTP. NC. U nited Slates t ' Centers fo r Disease Control 6r Prevention. Division o f Laboratory Science. Notional Centerfo r Environmental Health Atlanta. CA. United States 7 __________ ____ _______________ _______________________________________________________ A RTICLE INFO ----------------------------- --- to Article history: i < Received 2 8 January 2009 12 Received in revised fo rm 27 February 2009 >3 Accepted 2 M arch 2009 i4 Available o n lin e xxx is _ _ ------------------------------------------------------is Keywords: t r P o lyflu o ro a lkyl chem icals is P e rfluo ro a lkyl acids is P e rfttiorooctanoic acid 2t> P erfluoroocrane su lfon ic acid 21 Serum 22 Breast m ilk 23 Lacrarion ABSTRACT Polyfluoroalkyl chemicals (PFCs) comprise a group of man-made organic compounds, some of which are persistent contaminants with developmental toxicity shown in laboratory animals. There is a paucity of human perinatal exposure data. The US EPAconducted a pilot study (Methods Advancement in Milk Analysis) including 34 breastfeeding women in North Carolina. M ilk and serum samples were collected at 2 -7 weeks and 3 -4 months postpartum: 9 PFCswere assessed in m ilk and 7 in serum. Periluorooctane sulfonic acid (PFOS). perfluocooctanoic acid (PFQA). periluorononanoic acid (PFNA).and perfluorohexane sulfonic acid (PFHxS) were found in nearly 100%of the serum samples. PFOSand PFOAwere found at the highest concentrations. PFCswere below the lim it of detection in most milk samples. Serum concentra tions of PFOS. PFOAand PFHxS were lower (p <0.01 ) at the second visit compared to the first visit. Living in North Carolina 10 years or longer was related to elevated PFOS. PFOA and PFNA (p < 0.03). These pilot data support the need to further explore perinatal PFCexposuresand potentially related health effects, as planned in the upcoming National Children'sStudy which provided the framework for this investigation. 2009 Published by Elsevier Inc. 24 1. In tro d u c tio n 2s Polyfi uoroalkyl chem icals ( PFCs) com prise a large group o f ma n2s made fluo rinated organic compounds used in num erous consumer 2 7 products and industrial applications such as food packaging m ate28 ria l, non-stick cookware, protective coatings fo r textiles, carpets. 29 and paper, surface car coatings o r treatm ents, as w elt as in sur30 (actants fo r com m ercial and industrial applications [1 ]. PFCs. and si more specifically perfluoroalkyl acids (PFAAs), have been detected 32 in w ild life , fish used for hum an consum ption, and sera o f humans 3 3 in many d ifferen t geographical areas w orldw ide [2-19J. N ation34 ally representative US sera biom onitoring data in subjects 12 years AhbrrvnmoRs. Cl, confidence in te rva l; IQR. in te rq u a rtile range; LOO, lim it o f detection; LOQ, lim it o f q u a n tifica tio n ; Pfaas, p erflu o ro a lkyl acids; PFOSA. perfhtorooctane sulfonam ide; Et-PK)SA-AcO H,2-(N-M hyf-peTntorooctane solfonam ido) acetic a cid ; Me-PFOSA-AcOH, 2-fN -m ethyl-perfluorooctane sulfonam ido) acetic acid; PFHxS, perfluorohexane su lfon ic a cid; PFOS. perf)uorooctane sulfonic acid; PFOA. p erfluorooctanoic acid; PFNA. perfluorononanok acid: PFC. polyflu o roa lkyl chem icals; W TC. W orld Trade O n te i. Q 2 * C orresponding auth or a t; UCLA School o f Public H ealth. PO Boot 1772. Los Angr les.CA 90095-1772. U nited Slates. Tel.: I 310 206 5324; fax: * I 310 794 1805 E-mail address: ovchren9ucta.edu (0.5. von Ehrenstein). 1C urrent address: US E nvironm ental Protection Agency, N ational Center fo r Expo sure Analysts, Environm ental M edia Assessment C roup. M a il code 8243-01. Research Triangle Park, NC 27711, U nited States. 0890-6238/S - see fro n t matte? O 2009 Published by Elsevier Inc. d o t:)0.1016^reprotox.2009.03.001 and older demonstrated widespread exposure to perfluorooctane sulfonic acid (PFOS). perfluorooctanoic acid (PFOA), and p erflu orononartoic acid (PFNA) during the last decade (20.211. Exposures of lactating wom en and young children to PFCs have not been frequently studied, although a num ber o f anim al and recent hum an studies have suggested transfer to breast m ilk and across the placental barrier (22-26J. Developmental and reproductive health effects in anim als, including reduced b irth w eight and gestational length, developm ental delays and structural defects especially in relation to PFOA and PFOS exposure have increasingly raised concerns, although the developm ental toxicity in laboratory anim als was shown at doses 1 0 0 -5 0 0 times o f those seen in hum an sera [2 .2 7 -2 9 1.Some exposure assessments in cord blood suggested that PFAAs can also cross the placental barrier in humans (3 0 .3 1 1. Apelberg et al. |2 3 | recently reported average cord blood concen trations o f 4 .9 ng/m l (PFOS) and 1.6ng/m l (PFOA) (n -2 9 9 ). w h ile Spliethoff et al.. reported the detection of PFAAs in new bom blood spots confirm ing the transfer o f PFAAs in u tero [32]. In tw o recent epidem iological studies. PFAA cord blood concen(rations w ere related to anthropom etric indicators o f fetal gro w th at b irth, and m aternal pregnancy serum PFAA concentrations w ere associated w ith child b irth w eight [22.24J. Based on the Danish National Birth Cohort, inverse associations w ere reported betw een gestational PFOA exposure and b irth w eight w hile no effects w ere reported for markers of fetal growth at birth, or postnatal developm ental milestones (24.33). m 37 so 0 >2 <3 u , 7 *9 51 53 u w i? u 59 M -ifSv asr-!JL"' p. 37 ,Q |l|Lgdclrcpcs&l 1-8 - '*r a- *; y-v;; \ r: it#:.: ; 2 OS von Ehrenstein et o L f Reproductive Toxicology xxx (2009) xxx-xxx 6 6? 63 64 65 66 67 66 69 70 71 72 73 74 75 76 77 79 79 <0 61 62 63 6 85 86 87 86 6 90 91 92 93 94 95 06 97 96 99 100 *0< ICC 103 10 105 106 107 108 109 HO m 112 i3 114 115 116 117 118 119 120 121 122 123 124 126 176 127 Data on hum an m ilk PFC concentrations are still sparse. The available d ata based on small sample sizes from China (34 ], Sweden (3 5 ]. G erm any and Hungary 13 6 ], suggested detectable levels ofpre d om inantly PFOAand PFOS.The concentrations ofPFOS(131 p g/m l) and PFOA (4 3 .8 p g /m l) in 45 m ilk samples collected in 2004 from w om en aged 2 2 -4 3 years residing in Massachusetts have been reported recen tly [2 5 ). Studies investigating the p artition o f PFCs in to m ilk are largely lacking. One e a rlie r study in Sweden (n = 12) suggested tra n s fe r o f only about 1% o f PFC concentration in serum in to m ilk (3 5 ). Tem poral concentration changes in serum o r m ilk o f lactating w om en are unknown, as no study has assessed concen tratio n s in th e same wom an at tw o tim e points during lactation. To evaluate in fan t and m aternal exposure to PFCs and to a range o f o th e r environm ental com ponents, as w ell as to compare concentrations across biological fluids (3 7 ). the US Environm en ta l Protection Agency (US EPA) conducted a p ilo t study entitled M ethods Advancem ent fo r M ilk Analysis (M A M A ). This p ilo t study was carried o u t to develop reliable collection and analysis methods fo r th e N ational C hildren's Study, including 100.000 children from pre-conception to age 21 |3 8 ). W e previously reported the M AMA findings regarding phthalates |3 7 ) and the biological components o f human m ilk |3 9 ). 2 . M a te ria ls a n d m ethods 2.1. Study design and population The design o f the EPA MAMA study and bask m ethods has been described in d e ta il p revio u sly (3 9 f h i b rie f. 34 healthy. English-speaking breastfeeding wom en betw een 18 and 58 years o f age w ere re cruite d via newspaper advertisem ents, u n i v e rs ity em ail p u b lica tio n s, and flie rs d istrib u te d toe tin k ia n s specializing in wom en's h e a lth o r p e d ia trics b y an EPAcontractor( W estat Inc.. Chape) H ill NC).The question n aire assessm ent and th e co lle ctio n o f m ilk and serum specimens w ere conducted a t the EPA's H um an Studies F a cility d in k (Chapel H ill NC) between December 2004 and July 2005. W om en w ere breastfeeding th e ir firs t, second o r th ird c h ild and w ere n o t required to e xclusively breastfeed fo r p a rticip a tio n in th is study. The wom en donated m ilk a m i serum sam ples at 2 -7 w e e k s (ts t v is it: n - 18 m ilk : *3 4 serum }, and a t 3 -4 m o nth s (2nd v is it: n -2 0 m ilk ; n -3 0 serum ) postpartum . The p a rtic ip a tio n o f hum an subjects h i the M AM A study was approved by the in s titu tio n a l Review Boards o f th e U niversity o f N orth C arolina's School o f M edicine (IRB num ber 03-EPA-20?) and th e Centers fo r D iseaseControl and P revention (IRB num ber 3961). The w om en p a rticip a te d in verbal and w ritte n inform ed consent p rio r to adm m istra tio n o f a com prehensive questionnaire w hich d id not include questions regarding th e o ffsp rin g o f study participants (39). 2.2. Questionnaire A q uestionnaire regarding m aternal residence, occupation, and d ie ta ry and life s ty le factors was adm inistered to participants a t the firs t d in k v is it. Ques tio n n a ire ite m s w ere selected to address p o te n tia l routes o f exposure to m u ltip le enviro n m en ta l chem kals (phthalates. phenols. PCBs. dioxins, PFCs. persistent o rg a n k p o llu ta n ts , m etals, and brom inated flam e retardants). The current analy s is included th e fo llo w in g questions th a t w ere th o u gh t to p o te n tia lly relate to PFC exposure routes: "H ow lo n g have you live d in N o rth C arolina?" (4 0 -4 2 ) and "Does yo u r home have an enclosed garage attached?". The la tte r question was selected as som e a pp licatio n s used in and around cars contain PFCs, e.g., external and in te rn a l surface car coatings o r treatm ents. 2 3 . Sample collection and preparation The w om en w ere asked to fast fo r 1.5 h before sam ple collection. The MAMA sam ple co lle ctio n procedures fo r serum and m ilk w ere published previously (391 Sam pling d e ta ils, in clu d in g tim e o f day (betw een 9 AM and 2 PM) and th e am ount o f bodily flu id colle cte d , were recorded m the co lle ctio n log. M ilk (90 m l o r -3 oz) was expressed in th e EPA c lin k using a com m ercially available e le c trk breast pum p ( M edela. M cH enry, II). MHk was pum ped in fo PFC-free bottles and divided in to 3 m l a liq u ots in PFC-free polypropylene-tubes. W om en's blood samples (about 2 0 m l). w ete collected in to non-heparinized glass vacutainer tubes (Becton D kkinson, Franklin Lakes. NJ) by an EPA nurse via venipuncture. A fter 1 h at room tem pera tu re to a llow fo r c lo ttin g , blood samples were spun at 3 000rpm fo r 15 m in at room tem perature and serum was collected.A ll samples w ere stored a t -2 0 Cand shipped o n d ry ice to th e CDCs D ivision o f Laboratory Sciences, N ational Center fo r Environ m ental H ealth (A tla nta . GA) fo r analysis. A t th e CDC. a ll samples were stored at o r below -2 0 'C u n til analyzed. Table 1 U m ks o fq ua n tifica tion ( L0Q) in m ilk and lim its o fdetection (IO D ) in serum (n g/m l). P olyftuoroalkyl cbem kals M i LOQ Serum LOD 2-(N -ethy1-perfluorooctane sulfonam ido) acetic acid 2-(N 'incthyFperfluon>octanr sulfonam ido) acetic acid Perfluorobutane sulfonate Perfluorodecanoate Prrfluorohexane sulfonate (PFHxS) Perfluorononanoate (PFNA) Perfluorooctane sulfonam ide (PFOSA) Perfluorooctane sulfonate (PFOS) Perfluotooctanoate (PFOA) 4 Denotes not measured in serum . 0.60 0.60 0J0 0.60 0J0 OJO 0.15 060 OJO 0.20 OJO 0.10 0.10 0.05 0.05 0.10 2.4. Analysis o f m ilk and serum /o r PFCs In serum and m ilk , we determ ined the concentrations o f PFOS. PFQA. PFNA. PFHxS. perfluoroocune sulfonam ide (PFOSA). 2-(N -raethyFperfluorooctane sulfonam kto) acetic a dd (Me-PFO$A-AcOHl 2-(N -ethyi-perfluorooctane sulfonam ido) acetic a dd {Et-PFOSA-AcOH); perfluorobutane sulfonic a d d and perftuorodecanok a dd w ere o n ly measured in m ilk . The a nalytical m ethod involved autom ated solid-phase extra ctio n (SPE) coupled to reversed-phase high perform ance liq u id chrom atography (HPLC)-tandem mass spectrom etry (M S/M S). Samples were run in singlets and were re-analyzed o n ly i f the w ater and/or m a trix blanks w ere above 3 x lim it o f detection (LODX The a n a lytka l procedures invo lvin g th e use o f stan dards. q u a lity co ntro l, and blanks, as w e ll as autom ated sam ple extra ctio n w ere conducted as published previously [4 3 -4 6 j. The samples from both v is its w ere ana lyzed together in M atch 2006 (serum ) and November 2006 (m ilk ). For m ilk , sam ple preparation was conducted using autom ated o ff-lin e SPE|43J. O ne-m l o f m ilk , to w hich we added 3 m l o f 0.1 M fo rm k acid and 50 p i o f intern a l standard so lu tio n , was vortex-m ixed and sonkated. and placed on a Zym ark Rapid Trace Station (2ym ark Corp,, H opkinton. M A). PFCs from the m ilk w ere extracted on an O asis-KIB SPE colum n (W aters C orporation. M ilfo rd . M A). The SPE eluate was evaporated at 55 ~C to - lOOpJ under a stream o f d ry nitrogen (UHP grade) in a Zym ark Turbovap evaporator, and reconstituted w ith 300 p i ofO .IX fo rm k acid. The reconstituted m ilk extract (-4 0 0 p i) was transferred to a polypropylene autosam p le r via) fo r the o n -lin e SPE-HPLC-MS/MS analysis, perform ed using a Surveyor HPLC system {Therm oFirm igan.San Jose. CA, U SAl including one s ix-p o rt sw itching valve (Rheodyne MX7960. Rohnert Park. CA. USA) and one a dd itio na l Surveyor LC pum p, coupled w ith a Therm oFiim igan TSQ Q uantum U ltra criple-quadrupok mass spec trom eter equipped w ith a heated electrospray ion iza tion (HES1)interface.The HPLC pum p operated a t a 300 p l/m in flo w rate w ith 20 mM am m onium acetate (pH 4 ) in w ater (m obile phase A ) and a ceto nitrile (m obile phase B). The extract was injected in to the liq u id chrom atograph system fo r concentration o f the PFCs by o n -lin e SPE on a Betasil C8 precoluron (3 m m 4 10 m m . 5 p m : Therm oHypersiF Keystone. Bellefonre. PA, USA), chrom atographk separation on a Betasil CB a n a lytka l HPLC colum n (2.1 m m x 50 nun, 5 p m ; Therm oHypersiFKeystone). and detection and quantifica tio n by negative-ion HESFMS/MS. For serum , we used a m odification o f the on-line SPE coupled to HPLC-MS/MS approach described before |47). B riefly, w e added 250 p i o f 0.1 M fo rm k acid and 25 p i o f in te rn a l standard solution to 100p i o f serum, and the spiked serum was vortex-m ixed and sonkated. The samples w ere placed on a Sym biosis o n -lin e SPE system (Spark H olland. Plainsboro. NJ) fo r the precoocentration o f the analytes on a Polaris 0 8 cartridge (7 p m . 10 m m * 1m m : Spark H olland L The analytes w ete transferred onto a Betasil C8 HPLC colum n (3 m m x 50m m . 5 p m ; Therm oHypersiF Keystone. Bellefonte. PA), separated by HPLC (m obile phase A: 2 0 m M am m onium acetate in w ater. pH 4; m obile phase B: m ethanol), and detected by negative-ion Tm bolonspray-M S/M S on an API 4000 mass spectrom eter (A pplied Biosysrems. Fos te r CHy.CA). Reportable breast m ilk PFCconcentrations can fa ll below the lO D due to concentration factors that are part 0! the extraction protocol. Thus lim it o f q u a n tifi cation ( LOQ)(3xLOD) is used fo r m ilk samples and L0D is used fo r an other biological m edia, w here sample concentration is not required. The LOD in serum and the LOQ In m ilk are show n in Table 1. 2.5. Biological marker analysis Selected biologies in m ilk and serum were analyzed fo r each wom an according to U bC orp's standard operating procedures fo r these assays as previously reported in d erail 139]. The assessed endpoints w ere in m ilk : Secrerory im m unoglobulin A, pro la ctin . tissue necrosis factor- (TN F-a). interle u kin -6 (IL -6 ). triglycerides, glucose, and estra d io l: and in serum: prola ctin , tmmunogfobms, TN F-a, IL-6. triglycerides, glucose, and estradioL In this investigation, the m ilk and serum concentration o f the biological m arkers were used to explore possible relationships w ith th e detectable PFCs. ase-- : 12 190 11 1 1 134 135 136 137 136 >39 140 *41 142 1*3 144 146 146 47 149 149 50 151 52 153 154 155 56 tS7 158 159 160 161 16S 163 164 165 466 167 166 *69 70 71 7? 173 174 76 176 177 7 6 179 <60 161 16? 83 64 p. 38 a i von Ehrenstein a l /Reproductive Toxicology xxx (2009)xxx-xxx 3 1*10*2 Q 5 Percentage (n um b e r) o f scrum and m ilk samples w ith PFCs' LOD at v is it 1 (serum n - 3 4 ; m ilk n - IS ) and v is it 2 (serum n -3 0 : m ilk n -2 0 ) P erB uoroalkyLidds Serum LOD %( it) M iHc>LOQ X(n) PFOS V is it 1 V is it 2 0 (3 4 ) 1 0 0 (3 0 ) 0 0 PFOA V is it! V is it 2 0 (3 4 ) 1 0 0 (3 0 ) 0 0 PFHxS V is it 1 V is it 2 1 0 0 (3 4 ) 1 0 0 (3 0 ) 0 0 PFNA V is it 1 V is it 2 9 7(3 3 ) 1 0 0 (3 4 ) 0 0 PFOSA V is it ! V isir 2 4 4 (1 5 ) 7 3 (2 2 ) 0 1 5 (3 ) Me-PFOSA-AcOH V is iti V is it'2 53 (IS ) 5 0(15) 5 0 (1 ) 0 Et-PFOSA-AcOH V isit 1 V is it 2 0 5 0 (1 ) 00 P erftuoiobutane sulfonate V isit 1 V isit 2 b b 0 0 P e rfh io ro d e c a n o a te V is iti V is it 2 b b 0 0 * PFOS: p erfh io fo o cta n e sulfonate; Et-PFOSA-AcOH: 2 -(N -e ftiyl-p erflu o ro ocu n e su lfon a m ido ) acetic a cid ; Me-PFOSA-AcOH: 2 -fN -m eth yt-p rrfluo ro o cta ne suMonam ido) acetic acid; PFHxS: perfluorohexane su lfon ic acid; PFOS: perftuorooctanyl sulfonate; PFOA: perflu o ro octan o k a d d ; PFNA: perihaorononanok acid. k Denotes n o t m easured in serum . res 2.6. Statistical analysis res We calculated the percenlage o f detection fo r each analyte in serum and m ilk. re7 and deternuned the m edian, range, mean, standard e rro r, and selected percentiles, res For values below the LOO.values equal to LOD/sqr2 were used [48.49|. Further artalrfis yses. in c lu d in g re lationships betw een v is its and across m edia, w ere conducted fo r those analytes fo r w hich the frequency o f detection (>LOD) was >60X at both visits. Forthose wom en w ho donated 2 serum samples, the m edian difference between the concentrations fo r the same PFC a t v is it 1 and v is it 2 was calculated and assessed w ith th e W iktm o n signed-rank te st (non-param etric). Spearman correlation coeffi cients and related p values w ere calculated fo r correlations between the PFCs at v is it 1 and v is it 2 . and between the PFCs and the biological m arkers in m ilk and serum. Relations between a p rio ri selected variables assessed by questionnaire and the PFCs w ere evaluated using W ilctxton scores (rank sums) te s t The c u t-o ff points fo r cat egorizing selected variables w ere decided a p rio ri based on assum ptions according to data previously reported [4 0 -4 2 |. and to achieve approxim ately equal d istrib u tio n o f num bers o f subjects across categories. Tw o-sided p values are reported. A ll analyses were conducted w ith SAS version 9 (SAS In stitu te . Cary. NCJ. 3. Results The m edian age o f the wom en in this study was 31.3 years (in terq u artile range (IQ R): 27.1-34.2 years), and the children's m edian ages w ere 5.5 weeks (IQR: 4 -6 w eeks) at visit 1 and 13 w eeks (1 3 -1 4 w eeks) at visit 2. Three o f the analytes. PFHxS, PFOS and PFOA, w ere detected in 1 0 0 * o f wom en's serum samples at both visits, PFNA was detectable in 97% a t visit 1 and in 100% o f wom en's samples at visit 2 (Table 2). In contrast, in m ilk samples o f ju s t 4 w om en, only 3 o f the analytes w ere >L0Q: Et-PFOSAAcOH (I.O n g /m l) and Me-PFOSA-AcOH (0.7 ng/m i) w ere detected in 1 w om an at visit 1. and PFOSA was detected in 3 wom en at the 2nd visit (0 .3 . 0.5. and 0.6 ng/m i). The rem ainder o f the m ilk samples from both collections w ere measured and found to have concentrations < LOQ. The distribution o fPFCserum concentrations is shown in Table 3. Highest concentrations w ere found for PFOS w ith median values of 20.0n g /m l at th e first visit and 16.9 ng/m l at the second visit. PFOS concentrations w ere alm ost six-fold higher than the concentration o fthe analyte w ith the next highest value. PFOA, w ith median values o f 3 3 and 2.9 ng/m l at the first and second visit, respectively. M edian serum concentrations w ere significantly (p < 0 .0 1 ) low er for PFOS. PFOA and PFHxS assessed at visit 2 compared to the concentration assessed at visit 1. based on samples o f 30 wom en w ho donated serum samples at both visits w ith the differences shown in Table 4. Accordingly, the concentrations o f the detected serum PFCs are reported for each visit (Table 3). Serum concentra tions o f the same PFC w ere significantly correlated between the tw o visits (Table 4 ). Due to the lim ited num ber o f breast m ilk sam ples w ith detectable PFC concentrations, we could not calculate the 190 191 19? 199 19* 1 <99 197 190 *9 209 1 20? 203 204 205 206 207 209 209 210 211 712 213 71* 7i5 ?t6 7*7 7*9 270 27t 27? 273 224 275 276 277 278 279 230 Table 3 D istrib u tio n (m ean, standard e rror, m edian, selected percentiles,IQ R ) o f PFCs* in serum samples a t v is it 1 ( u - 34} and v is it 2 (n -3 0 ) in ng/m l. M ean(SEM ) 10th percentile 2 5 th percentile M edian 75th percericiie 9 0th percentile 95th percentile IQR PFOS V isit t 2 1 0 (1 .9 ) n .7 13.2 20.0 30.1 37.6 45.7 16.9 V isit 2 1 8 .8 (15 ) 9.70 1 4 j0 109 22.6 30.2 35.5 8.60 PFOA V isit f 3 .9 9 (0 3 5 ) 1.50 230 3.50 4.60 6.0 8.70 2.40 V isit 2 3.0 (0 .2 1) 1.45 2.40 2 0 0 3.70 4.65 5.0 1.30 PFHxS VisU 1 V is it 2 1.94 (0.27) 1 .5 0 (0 3 2 ) 0.70 0.50 1JD 0.70 1.55 2.40 u s 1.70 3.40 2-90 3.80 4.60 1.40 1.00 PFNA V isit 1 1.22 (0 J 2 ) 0.40 070 1.10 1.60 2.00 2.70 OSO V isit 2 133 (0.09) 0.75 ux> 1.20 IS O IS O 2.40 OSO PFOSA V isit 1 0.07(0.01 ) <LOD <L0D <LOD 0.10 a io 0.10 0.07 V isit 2 0 .0 9 (0 1 )1 ) <LOD <L0D a io 0.10 0.15 0.20 0.07 Me-PFOSA-AcOH V is iti 0 .23(0.02) V isit 2 0.24 (0.02) "LO D <LOD <LOD <LOD 0.20 an 030 030 030 0.40 0.40 0.50 0.16 0.16 a PFOS: PerfU iotooctane sulfonate: Et-PFOSA AcOH: 2~(N-eihyt-perih>orooctane sulfonam ido) acetic acid; Me-PFOSA-AcOH: 2HN-m erhy] perRuorooctane sulfonam ido) acetic acid; PFHxS: perfluorohexane sulfonic acid; PFOS: periluorooctanyl sulfonate; PFOA: p eriluorooctanok acid; PFNA: perfluorononanoic acid. Values measured <tOD were im puted by LOD/sqr2. p. 39 4 O S. von Etirem rein ot. / Xrprotfunrve Toxicology xxx (2009) xxx-xxx Table 4 D ifference and co rre la tio n in PfC serum concentrations (n g /m l) betw een v is it one and v is it tw o. T ables C orrelations between concentration o f PfC and in te rie u kin -6 in serum a t v is it 1 (n -3 4 ) and 2 ( r r - 30). M edian d iffe re nce (IQ ft) p v a tu * C orrelation co e fficie n ts* PFOS PFOA PFHxS PFNA - 2 JO ( - 7 J . 1.0) -0 .S S (-1 .4 0 .0 .0 ) -0 .4 0 (-0 .8 0 . -0 .1 0 ) 0.11 (-0 .2 0 .0 .5 0 ) <0.01 <0.001 <0.001 0.10 0l82 0.82 087 0.71 * W ilcoxon sign e d -ta nk te st (non-param etric) ( n - 30). p v a tu e <0001 <0001 <0001 <0.001 PFOS V is iti V is it 2 PFOA V is it t V is it 2 C orrelation co efficie n t, o ' -0 .2 1 039 -0 .1 5 0.07 p value 0.20 003 040 0.70 Spearman co rre la tio n co efficie n t <r and related p value (n - 30). PFHxS V is it] -0 .1 1 0.50 V is it 2 231 p artitio n coefficient from serum to m ilk, b u t can conclude th a t m ilk 232 concentrations w ere notably low er than serum concentrations. PFNA V is iti 233 Based on self-reported data, w om en had lived in N orth Carolina V is it 2 038 -0 .0 0 3 -0 0 8 084 18 0.70 234 fo r (m ean. SEM ) 14.6 (1 3 2 ) years. Interestingly, w om en w ho had * Spearman correlation coefficient n and related p value. Bolded values signify 235 reported liv in g in N o rth Carolina fo r 10 years o r m ore compared significant correlations. 236 to those w ho had reported living in N orth Carolina less than 10 237 years, had h ig h er serum concentrations o f PFNA, PFOA, and PF0S 236 (p < 0.03) (Fig. 1). Furtherm ore, living in a house w ith an enclosed Serum concentrations o f IL-6 w ere positively correlated w ith 239 garage attached as com pared to living in a house w ith no enclosed PFOS (p = 0 .0 3 ) and PFHxS (p = 0 .0 4 ) at the second visit (Table 5). 240 garage attached, suggested a relation to higher concentrations o f None o f the other selected biological markers in serum or in m ilk 241 PFH xS(ng/m l: m edian, IQR, visit 1: 2.2 (1 .4 ) vs. 1.1 (0 .6 ).p < 0.001: showed a significant correlation w ith the PFCserum concentrations 242 v is it 2 :1 .5 (1 .4 ) vs. 0.9 (0 .7 ) p -0 .0 3 ) and o f PFOS (visit 1 :2 5 .4 (1 6 3 ) at e ith e r collection tim e point. There was no significant relationship 243 vs. 14.4 ( 9 3 ) .p -0 .0 1 ; visit 2: 21.2 (11.5) vs. 1 4 .5 (7 .8 )p -0 .1 ). between m aternal age or parity and PFC serum concentrations in our study (data not shown). Due to the small numbers and lack of racial diversity in this p ilo t study based on convenience sampling (only 3 wom en reported themselves as Black/African-Am erican, one as Asian and one as Hispanic), w e could not analyze PFC con centrations by ethnic group. 2*4 245 246 247 246 2 260 2i 252 253 264 4. Discussion P F N A V isit 1 PFNA V isit 2 P FO A V isit 1 P FO A V isit 2 P F C b y V is it (a )a n d (b ) Serum concentrations o f PFNA. PFOA. and PFO $(ns/jnl)com |>arm g liv in g in N orth C arolina > 10 to <10 years at v is it 1 and v is it 2. Data are show n as box and whisker representations; open circles denote mean values w ith the medians drnoted as a stra ig h t lin e , p <0.03 in W ilcoxon Scores (rank sum s) tests fo r groups l0 ye a rsvs.< t0 ye a rsa t v is it I and v is it 2 fo r each PfC . Num bers o f subjects in each group: >10years: n - 16. v is it 1; n * 15. v is it 2; O O years: n - 18. v is it 1 .n 15. v is it 2. In this p ilot study o f healthy lactating North Carolina wom en. 6 of the 7 PFCs analyzed in serum w ere detectable at 2 -7 weeks and 3 -4 m onths postpartum . PFOS. PFOA. PFNA. and PFHxS w ere found in nearly 100% o f the serum samples. PFOS. followed by PFOA and PFHxS w ere the compounds detected at the highest concentrations. Only a small proportion o f m ilk samples had detectable values o f 3 o f the 9 PFCs analyzed in m ilk. Interestingly, serum levels w ere low er for PFOS. PFOA, and PFHxS at the second visit compared to the first visit, and prolonged tim e lived in North Carolina, as w ell as living in a home w ith enclosed garage attached, suggested a relation to elevated serum concentrations o f certain PFCs in our sample: however, these analyses were unadjusted and based on a small nonrandom sample in this pilot study and thus should be considered exploratory. W e can conclude th at postnatal exposure to PFCs via breast m ilk is likely to be low during the tim e period captured in our investigation. Data on PFC serum concentrations oflactating wom en are sparse and based on sm all sample sizes. Available data relevant fo r preand postnatal exposures to PFCs are sum marized in Table 6. Only one earlier study assessed both serum and m ilk levels, in 12 lac tating w om en in Sweden, and reported sim ilar serum values to ours for PFOS(median: 18.7 n g/m t)and PFOA(3.8 ng/m l) w h ile con centrations o f PFHxS w ere higher (4 .0 ng/m l) in the Swedish study (35J. Based on data from the Danish National Birth Cohort, prena tal m aternal serum concentrations appeared to be higher for PFOS and PFOA in Denmark than seen postpartum in our study. Interest ingly, in the Danish study, concentrations were low er in the second than in the first trim ester, possibly due to dilution of the PFCs w ith blood volume expansion due to pregnancy, but values in cord blood (n - 5 0 ) confirm ed fetal exposures 124,33) (Table 6). The serum PFC concentrations seen in our study compare w ell w ith US serum data from NHANES 2 0 0 3-2 0 0 4 . assessed in representative samples of 265 266 257 256 259 260 261 262 263 264 265 266 267 266 269 270 271 272 273 274 275 276 277 778 27 ?0 ?Si 262 263 264 765 266 967 p. 40 i -m=?' a* /. :r -'- O S von Ehremtein et a t / Reproductive Toxicologyxxx (2009) xxx-xxx 5 Table 6 Published data on average PFC c Location,year o f sa m p lii M assachusetts. USA. 2054 Leip zig/M un ich Germ any, 2006 G ybr. H ungary 1996/97 Zhousan. C hina. 2004 Sweden. rxJivktu at m atched sera and m ilk (2 0 0 4 ); pooled com posite m ilk sam ples (1 99 6 -2 00 4 ), B altim ore. M D.USA. 2004-2005 ; m aterna) s i and cord blood. M a trix. studypO R uU pn.and sam ple size ' M ilk:con ve n ie n ce sam ple, ag 22-43:years, n -4 5 , nursing tl firs t tim e : n -3 4 , nursed >1: r M ilk : convenience sam pling zt hosp ita l sam ples, n - 1$ (M unich) M iDcbank, r r - 38 (Leipzig) M others o f,p re te rm infan ts, n -1 3 (H ungary) a t 3 -7 weeks p o s tp a rtu m PFCconcentration as re p o rt ! PROS(m eaivSD X 131(103)pg/m ) PFOA: 43.8 (33.1)pg/m l PFHxS: 14.5 ( 13.7)p g/m l PFNA: 7 3 6 (4 .7 0 )p g /m l PFHpA. PFDA, PFUnOA. PFDoDA. PFBS:aB<LOD PFOSfmedian. range) M unich: 113 (2 8-2 3 9 )ng /L Leipzig: 123 (3 3 -3 0 9 ) ng/1. H ungary. 330 (9 6 -6 3 9 ) ng/L PFOA. a ll: <LOD (<LO D -460) ng/L M ilk : convenience sam pling at h ospital volunteers, n - 19 M ilk and serum : convenience sam ple p rim iparous w om en, n - 12 Pooled annual com posite m ilk samples (n -2 5 -9 0 ) R an g e s*(n g /L ) PFOS; 4 5 -3 6 0 , PPCJA; 47-210; PFHxS: 4 -1 0 0 ; PFNA: 6 3 -6 2 ; PFOA: 3 3 -1 5 ; PFUnOA: 7.6-56 MiOc (m ean. SD) ng/m k p ro s: 0301 (0.117); PFHxS: 0.085(0.047): PFOSA: 0.013 (0.009); PFNA: NA; PFOA, PFOA. PFUnOA: ND Date o f m ilk co lle ctio n : 3 weeks p o s tp a rtu m Cord blood, h osp ita l based, singleton d elive rie s (n -2 9 3 ) Serum (m ean, SD) a g /m l: PFOS: 20.7 (103fe PFHxS: 4.7 (2 3 ); PFOSA: 0 3 4 (0.16); PFNA: 0 8 0 (0 5 5 ); PFOA: 3.8 (LO ); PFOA: 053 (0 4 1 ); PFUnOA: 0 4 0 (0 3 5 ) Com posite mSk ng/m k PFOS0 30 9 (1996)-0.123 (2 00 4 k PFHxS: 0337 (1996>-0316(2004); PFOSA: <0307 (1 996)-<0007 (2004); PFNA: 0.028 ( 199G )-0320 (2004); PFOA: <0309(1996>-<0209 (2 0 0 4 ) PFOA (m edian, range): 1 5(0 .3-7 .1 ) PFOS (m edian, range) ng/m k 5.0 ( <LOD (-0 2 > -3 4 3 ) Denm ark. 1996-2004 Japan, 2 003 M aternal plasm a: 1st trim e ste r I n - 1399). 2nd trim e ste r (n -2 0 0 ) Cord M ood, n - 30 M aternal plasm a: 3rd trim e ste r (n - IS ) cord M ood (n - IS ) M aternal, 1 st trim e ste r: PFOS (n g/m l.m ean. SD): 3 5 3 (13X>X PFDA: 5j6 (2 5 ) M aternal. 2nd trim e ste r: PFOS: 2 99 (1 1 .0 ); PFOA: 4 .5 (1 9 ) Cord blood: PFOS: 11.0(4.7); PFOA: 3 .7 (3 .4 ) M a te rna l 3 rd trim e ste r serum range: PFOS (4 9-1 7 .6 ng/rnIX PFOA (<LOD to 2 3 ng/m IX PFOSA (<LO D to<LO D ) Cord M ood: PFOS (1 5 -5 3 ng/mIX PFOA (<LOD to < U X PFOSA (<LOD to <L0D) * PFC LODs fo r serum , blood and m ilk varied in the d iffe re n t studies as reported in the o rig in a l references. No averages reported by authors. Percentage q uantified > LOD* PFOS: 96X PFOA: 89X PFHxS: S IX PFNA: 64X PFHpA. PFDA. PFtlnDA. PFDoDA. PFBS: <8X PFOS:100% PFOA: 16X PFOS.PFOA. PFHxS. PFNA. PFDA. PFUnOA: 100% M ilk : PFOS. PFHxS: 100X (r - 12); PFOSA: 67X (n-8X P FN A : 16X (n -2 ):P F 0 A :B X (ii-l) Serum : PROS. PfHxS. PF0A.PFNA. PFDA. FFUnfM : 100X (n-12X PFOSA: 7 5 X (n -9 ) PFOS: 9 9 * PFOA: tOOX: Er-PFOSA-AcOH. Me-PFOSA-AcOH. PFBuS.PFHpA. PFUA-PFDoA: I-4 0 X M aternai. 1st trim ester: PFOS: 100X. PFOA: WOX (except i t - 1) M a te rn a l 3rd trim ester. PFOS: 100X, PFOA: 20X. PFOSA: OX Cord M ood: PfOS: 100X.PFOA. OX. PFOSA: OX Reference (2 5 ) (3 6 ] |3 4 | |3 5 ) [2 2 3 3 J [24) (3 0 | fem ales aged 12 and above, showing m edian concentrations for PFOS and PFOA o i 18.2 ng/m l (1QR: 12.4-27.3 n g /m l) and 3 .6 ng/m l (IQR: 2 .5 -5 .2 n g/m l), respectively (20.211. Based on the NHANES data, n atio n -w id e serum concentrations dropped for PFOS. PFOA and for PFHxS betw een 1999/2000 and 2 0 0 3/2 0 0 4 w hile those for PFNA increased in the same tim e period |2 0 (. O ur average levels are som ew hat low er than reported for fem ales in the US in 1989 (5) but s im ila r to other findings in samples collected between 1999 and 2 0 0 5 (6,10,20.21.50|. In a recent US investigation, median cord blood levels forPFOS and PFOA o f 5 and 1.6 ng/m l, respectively, w ere reported [23 J.This is about a third to a fourth ( PFOS) and 5 0 * ( PFOA) of the concentrations w e found in m aternal serum samples, and also about h alfthe concentration reported for cord blood from Denm ark (241. Table 6. A few investigations o f PFCs in human m ilk have been con ducted in Sweden. China. Denm ark and recently in the US (Table 6 ) (2 5 ,3 4 -3 6 ]. Only one study assessed both serum and m ilk concen trations and detected PFOS and PFHxS in all 12 m ilk samples at mean concentrations o f 0.201 and 0.085 ng/m l respectively, sug gesting partitioning o f on average IX from serum to m ilk (35J. In the Chinese study, values o f PFOS and PFOA in m ilk samples (n = 19) w ere in the range o f0 .0 4 5 -0 .3 6 and 0.047-0.21 ng/m l. respectively 134). M ilk concentrations are sum marized in Table 6, supporting our findings of low er values in m ilk than in serum, as w e ll as 300 301 30? 303 304 305 306 307 308 30 3i0 3ii p. 41 r; 6 O S on Buensletn et at/Reproductive Toxicology m (2009)xxx-xxx 912 913 914 915 316 31? 318 319 320 321 322 329 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 338 3*0 341 342 343 344 345 348 347 348 34 350 3S1 352 363 354 355 306 7 356 359 360 361 36? 363 964 366 966 967 968 969 370 371 372 373 374 37S 376 377 suggesting regional differences in exposure levels [25,34-361. PFAAs are strongly bound to the protein fraction o f hum an blood [1 0 ,5 1 -5 3 ). T he protein concentration in hum an blood contains m ain ly album in and few er beta-lipoproteins and is about 3 -5 tim es h ig her than th e protein fraction in hum an m ilk (casein and lactalb u m in ). It has been shown th a t strongly protein-bound drugs are less likely to transfer to hum an m ilk than sm all non-ionic lipophilic com pounds [5 4 [. This may explain w hy PFAA concentrations are m uch low er in hum an m ilk than in m aternal serum, although trans fe r o f PFAAs to m ilk has been observed in anim al studies, albeit at m uch higher serum concentrations o f PFAAs |2 6 ). In our study, concentrations o f PFOS, PFOA and PFHxS in serum w e re low er a t the second visit com pared to concentrations at the firs t visit. Since PFC concentrations measured in hum an sera have h alf-lives ranging betw een 3.4 years fo r PFOA, 4.6 years for PFOS, and 7.1 years for PFHxS [55 ). these data suggest th a t processes related to depuration into breast m ilk m ight be occurring that w e could not assess (possibly because w e measured m ilk con centrations too late in lactation), o r th a t there m ight be m aternal m etabolic changes during lactation that m ay relate to this change (i.e .. changes in blood volum e, body w eight, o r hepatic activities). Because PFCs are tig h tly bound to serum proteins, serum protein levels during lactation could have affected the concentrations o f PFCs in serum . U nfortunately, w e did not measure serum album in to test this hypothesis. A lternatively, if PFCs p artition m ore into liv e r than serum in the course o f lactation, serum concentrations o f PFCs could be affected as w e ll. Tire possible transfer o f PFCs to m ilk may also vary at d ifferent tim es during lactation. The nature o f the relationship betw een the suggested decline o f PFC values in serum to concentrations in m ilk are yet u nd ear and insufficient d ata exist to d ate to explain the relationship at this point. Few ear lie r reports suggested declines in breast m ilk during lactation for lipo ph ilic com pounds including dioxins. PCBs. and PBDEs [56.57). N o other study to our knowledge, has investigated PFC concentra tio n changes in serum o r m ilk over tim e during lactation assessed in the same w om en at tw o tim e points. However, it should be noted th a t our findings are based on a relatively sm all num ber o f a volun tee r non-random sam ple o f wom en and need replication in a larger study for fu rth e r confirm ation. Tao et al. conducted a regression analysis of PFOS and PFOA concentrations in breast m ilk collected a t various tim e points from 25 d ifferen t w om en w ith in the first 6 months postpartum ; they concluded that values increased over tim e o f lactation |2 5 ). However, since these findings w ere based on m ilk samples o f d ifferent subjects rath er than com paring changes over tim e in the same w om en, the differences m ay be due to intraindividual variatio n . Our investigation suggested th a t living in North Carolina for a prolonged tim e period o f 10 years and m ore was related to higher serum concentrations o f PFNA, PFOA, and PFOS in our pilot study. However, fu rth e r evaluation o f this explorative finding is required. Point sources m ay lead to elevated exposures, as indicated by serum concentrations o f PFOA in persons living near a US facility using and producing this com pound, th a t w ere notably higher than among the general US population [58 ). A system atic surface w ater survey conducted in North Carolina showed large variation in concentra tio n on a sm all scale indicating a series o f source inputs around the Cape Fear Drainage Basin th at m ay potentially result in pockets w ith increased exposures |4 0 ). Comparing serum PFC concentrations among donors at the 6 Am erican Red Cross Blood Bank locations across the US showed highest concentrations for PFOS and second highest fo r PFOA in Charlotte. North Carolina, in samples collected in 2 0 0 0-2 0 0 1 |4 1 ), w ith a substantial decline observed in samples collected in 2 0 0 6 at the same locations |4 2 |. Recently, elevated plasma concentrations especially o f PFOA. PFNA, and PFHxS have been reported for personnel involved in the W orld Trade Cen te r (W TC) disaster (i.e ., from fire-fig h tin g foams used to combat the W TC fire o r directly from the W TCs degradation) |5 9 ) further 371 supporting the notion o f source related local variations o f human 37 exposures to certain PFCs. W om en w ho reported living in a home 380 w ith an enclosed garage attached also had increased concentrations 981 o f PFHxS and PFOS in our sample. This may be due to certain m ate 382 rials used in and around cars containing PFCs. such as post-m arket 383 applications o f external and internal surface car coatings or treat 38 m ents. However, due to the sm all sample size in this pilot study, we 986 could not analyze the impacts o f other variables, especially socio 386 economic factors; these findings are thus explorative and should be 987 interpreted cautiously. 368 The pro-inflam m atory cytokine IL-6 was positively correlated to 38 PFOS and PFHxS, respectively, at the second collection tim e point 390 possibly indicating that certain PFAAs may be related to inflam m a 391 tory processes. In line w ith these findings are recent results from experim ental studies in mice, reporting suppression o f im m une 302 383 responses follow ing exposure to PFOS in utero [60 ]. W e did not see 994 correlations w ith other a priori selected biological m arkers assessed 395 in m ilk or serum , i.e., im m unoglobulin, estradiol, prolactin orTN F- 396 a . Rodentstudies using PFOA in concentrations orders o fm agnitude 397 higher than M AM A serum concentrations have shown a suppres 396 sion o f genetic markers o f inflam m ation after an acute exposure to 399 PFOA [61). Because our findings are explorative, future studies may 400 w ant to address the role o f chronic exposure to low dose PFCs in 401 the inflam m atory process. 40? In this pilot study the num ber o f wom en was relatively sm all and 403 confines the investigation o f associations, possible exposure path 404 ways. and tim e trends after birth. In addition, the sample was not 405 selected random ly, thus selection bias cannot be excluded. How 406 ever. the participation o f wom en was unlikely to be related to PFC <07 exposures o r to certain PFC exposure sources since they w ere most 406 likely not aware o f th eir PFC exposures. A further lim itatio n o f our 409 study is that w e could not collect m ilk samples sooner after b irth in 4t0 view o f ethical constraints in asking for the colostrum m ilk. Studies 411 in m ice measuring PFOA concentration over the course o f lactation 412 have shown that the peak in m ilk PFOA concentration occurs soon 419 after b irth (Fenton et aL. in this issue), a tim e that was not follow ed Q3 414 in the M AM A collection scheme. O verall, the findings reported are 41& explorative and need further evaluation. 416 In conclusion, although infant exposure via breast m ilk is likely 4 ? to be low, the cum ulative daily infant intake of PFCs via breast m ilk 418 per kg body w eight could be appreciable fo r some populations or 4*9 groups (Table 6 ). Since toxicological and pharm acokinetic data for 4?|) PFC exposed infants are lacking, it is largely unknown if potential 421 health effects in infants or during childhood may be related to cur rent exposure levels of PFCs. In urero exposure should continue to 422 <23 be a concern as the M AMA serum PFAA concentrations are sim ilar 424 ro values reported in tw o separate studies that have shown inverse 426 associations between m aternal serum or cord blood PFAA concen 426 trations and infant b irth w eight [22,24]. Thus, the findings o f this 427 pilot study underscore the im portance o f biom onitoring m aternal 426 and infant exposure to PFC as w ell as the need for further study 4? o f the potential human health effects o f PFCs. In the upcoming US 490 National Children's Study [38] PFC exposures in pregnant and lactating w om en and th e ir children in North Carolina and across the 431 492 US w ill be furth er studied. *33 C onflict o f in terest The authors declare that there are no conflicts of interest. 494 435 Acknow ledgm ents The research in this article has been reviewed by the National Health and Environm ental Effects Research Laboratory. US Environ- 06 7 498 p. 42 Z::F: ?> y-; O S von Ebnnstetn et at./Reproductive Toxicology mat (2009) mot-mat 7 m en tal Protection Agency ( EPA), and the Centers for Disease Control mo and Prevention (CDC) and approved for publication. Approval does 44i not signify this report reflects EPA or CDC policy. The findings in 44? this report are those o f the authors and do not reflect the views o f 44? th e CDC The use o f trade names or com m ercial products does not 444 constitute endorsem ent or recom m endation for use. 44? This w o rk was supported in part by the Intram ural Research Pro- 44? gram at th e Eunice KennedyShriverN ational In stitu te o f Child H ealth 447 and Hum an Developm ent, National Institutes o f H ealth. Bethesda, 44? M D . 44? Partial extram ural funding was provided through the rec- 4so om m endation o f th e National Children's Study Intra-Agency 4si Coordinating C om m ittee. 45? 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Concentrations of phthalate m etabolites in m ilk , urine, salvia and serum o f lactaring N orth Carolina wom en. Environ H ealth Perspect. d oi: 10.l289/ehp.116l0 available via h ttp ://d x .d o i.o rg /1Online 22 August 2008). (38) Landrigan PJ, Trasande L Thorpe L, et al. The N ational C hildren's Study: a 21-year prospective study o f 100.000 Am erican children. Pediatrics 2006;118:2173-86. |39) H in ts EP. Rayner JU Barbee R. et a l Assays for endogenous com ponents o f hum an m Hk: com parison o f fresh and frozen samples and corresponding ana lyte s in serum .J Hum Loci 2007:23:144-56. 140) Nakayama S. Strynar MJ. Hetfam L er a l Perftuorinated com pounds in The cape fe a r drainage basin in N orth C arolina. Environ Sci Technol 2007:41(15): 5 2 7 1 -6 . (41) Olsen GW. Church TR, M ille r JP.et al. Petfluorooctanesulfbate ad other ftuo ro chem icals in the serum o f Am erican Red Cross Aduh Blood Donors. Environ H ealth Perspect 20(3:111:1892-901. [4 2 ] Olsen GW. M air DC. Church TR. et a l. Decline in perfluoroctanesuffonate and other polyfluoroalsfcy chem icals in Am erican Red Cross adirlt Mood donors. 2000-2006. Environ Set Technol 2008;42:4989-95. (43} Kuklenyik Z.RerchJATuByJS.ee aLAutom ated solid-phase e xtraction and mea surem ent o fperftuorinated organic acids and amides in hum an serum and m ilk. Environ Sci Technol 2004;38:3698-704. [44) CDC T hird national re po rt on hum an exposure to environm ental chem icals, h ttp r/fw w w .cdcgov/exposurereport/: 2005. |4 5 ) Taylor JK. Q uality assurance o f chem ical measurements. Chelsea. M I: le w is Publishers; 1987. [4 6 ) Van LeetiwenSPf, Karrm an A van Bavei 8. De Boer J. Lindstrom C. Struggle fo r q u a lity in determ ination o f perftuorinated contam inants in environm ental and hum an samples. Environ Sci Technol 2006:40:7854-60. 1471 K uklenyik Z. Needham LL C alafat AM. Measurement o f 16 p erftuorinated organic acids and amides in hum an serum using o n-line solid-phase e xtraction. Anal Chero 2005;77:6085-91. [4 8 ] H orrum g RW. Reed LD. E stim ation o f average concenuaion in the presence o t nondetectable values. App) Occup Environ Hyg 2008:46-51. (49) CDC (Centers fo r Disease C ontrol and Prevention) General docum en ta tio n on laboratory data. General inform ation about the NHANES 2003-2004 laboratory m ethodology and public dara files. Available: 516 517 618 519 5?0 52 522 523 524 525 525 527 528 629 630 531 632 533 534 535 536 $37 S39 S3 5*0 541 . 52 543 544 64 649 547 548 549 sso 551 662 553 56 555 556 567 556 55 560 561 562 563 564 565 566 567 568 $69 570 $7T $72 $73 574 575 57$ 577 578 579 580 581 SA? 583 584 585 566 587 588 509 590 58 592 533 584 59$ 696 587 588 59 600 SO* p. 43 S?8888885I2 v `.\:r 7 ' " . ',: -i& Cy-V V . 8 O.S. von Ehrenstan er a t / R eprotfttaivr Toxicology xxx (2009) xxx-xxx w h ttp ://w w w .c d c -gov/nchs/dala/nhanes/nhanes_03jt>4/lab_c_generaid<x;.pdf; 1561 Hooper K, She J. Sharp M ,c t a l D epuration o f polybrom inated d ip h e nyl ethers eoa 2006. (PBDEs) and polychlorinated biphenyls (PCBs) in breast mHk from C alifornia 604 [5 0 j Olsen GW. M air DC, Reagen WK. e t aL P relim inary evidence o f a decline in per- firs t-tim e m others (p rim ip ara e ). Environ H ealth Perspect 2007:115:1271-5. $06 flu oro o cta nesulfonare ( PFOS)and perfiuorooctanoate(PFQ A)concentrations in 157] LaKindJS, B e rlin CM,P arkC N .etal. M ethodology fo r characterizing d istrib u tio n s 606 A m erican Red Cross blood donors. Chemosphere 2007;68:105-11. o f increm entalbody burdens o f23,T*-TC D D and DDE from breast m ilk m N orth 607 [S I | Jones JD. H u W . De Coen W . et al. B inding o f p erflu o n na te d fa tty acids to serum Am erican nursing Infants. JToxicol Environ H ealth A 2000;59:605-39; 606 proteins. E vnviron Toxicol Chem 2003;22:2639-49. (58} Em m ett EA. Shofer PS. Zhang H . e t a l C om m unity exposure to perfluorooc- 609 [5 2 } H an X, Snow 7A . Kemper R A ,etal. B inding o fp e rflu o ro o cta n o ic acid to ra t and tanoate: relationships between serum concentrations and exposure sources, j 610 hum an plasm a proteins. Chem Res Toxicol 2003;16:775-81. Oceup Environ Med 2006:48:759-70. on [S 3| Bhattachavya AA. G rune T. Curvy S. C rystallographic analysis reveals com m on |5 9 ] Tao U Kannan K, Aldous KM, e t a l B iom onitoring o f perfluorochem tcals in 6i? m odes o f b in d in g o f m edium and king-chain fa tty acids to hum an serum a lbu- plasm a o f New York State personnel responding to the W orld Trade Center 6>3 m in .J M o l B iot2003;303:721-3 2. disaster. E nviron Sci Technot 2008;42:3472-8. 614 [5 4 | Aschenbrenner DS. VeaWe SJ. D rug therapy in nursing. U ppim cott. W illia m s & 160) K eil DE. M ehlm ann T. B u tte rw o rth L. et a l Gestational exposure to perRuo* 616 W ilk in s ; 2008. rooctane sulfonate suppresses im m une function in B6C3R m ice. Toxicol Sci ie 155} Olsen GW , B u rris JM, Ehresman DJ. et al. H a lf-life o f serum e lim in a tio n o f 2 0 0 8 :1 0 3 :7 7 -8 5 . 617 perO uorooctanesulfonate. perfluorohexanesulfonate. andperfluoiooctanoate |6 l] Guruge KS. Yeung LW. Yamanaka N. et a l Gene expression p rofiles in ra t live r it in re tire d ftuo ro ch em ka l production w orkers. Environ H ealth Perspect treated w ith perfluorooctanoic acid (PFOA). Toxicol Sci 2006:89(1):9 3 -107. 2 0 0 7 ;1 1 5 :1 2 9 8 -3 0 5 . p. 44 ELSEVIER M olecular and Cellular Endocrinology 304 (2009)97-105 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology jo u rn a l h o m ep a g e : w w w .e ls e v ie r.c o m /lo c a te /m c e Phenotypic dichotomy following developmental exposure to perfluorooctanoic acid (PFOA) in female CD-I mice: Low doses induce elevated serum leptin and insulin, and overweight in mid-life* Erin P. Hines3'*, Sally S. W hite6,Jason P. Stanko\ Eugene A. Gibbs-Floumoyc, Christopher Laua, Suzanne E Fenton3 * Reproductive Toxicology D ivision, Office o f Research and Devebpment, Notional Health and Environmental Effects Research Laboratory, US. Environmental Protection Agency, Research Triangle Park, NC27711, United States b C urriculum in Toxicology, UNC Chape) HfH, Chapel H ill NC 27599. United Stores c Biological and Biomedical Sciences Progran0nitiative fo r Maximizing Student Diversity. UNC Chapel HRK Chapel H ill NC27S99, United States ARTI CLE I NFO A rticle history: Received 27 January 2009 Accepted 24 February 2009 Keywords: PFOA O verw eight Leptin D evelopm ental exposure O besity O variectom y ABSTRACT The synthetic surfactant, perfluorooctanoic acid (PFOA) is a proven developmental toxicant in mice, caus ing pregnancy loss, increased neonatal mortality, delayed eye opening, and abnormal mammary gland growth in animals exposed during fetal life. PFOA is found in the sera and tissues of wildlife and humans throughout the world, but is especially high in the sera o f children compared to adults. These studies in CD-I mice aim to determine the latent health effects of PFOA following: ( l ) an in utero exposure, (2) an in utero exposure followed by ovariectomy (ovx). or (3} exposure as an adult. Mice were exposed to 0 ,0 .0 1 ,0 .1 .0 3 .1 ,3 . or 5 mgPFOA/kg BW for 17 days of pregnancy or as young adults. Body weight was reduced in the highest doses on postnatal day (PND) 1 and at weaning. However, the lowest exposures (0 .0 1 -0 3 mg/kg) significantly increased body weight, and serum insulin and leptin (03)1-0.1 mg/kg) in m id-life after developmental exposure. PFOAexposure combined w ith ovx caused no additional increase in m id-life body weight. At 18 months of age, the effects of in utero PFOA exposure on body weight were no longer detected. White adipose tissue and spleen weights were decreased at high doses of PFOA in intact developmentally exposed mice, and spleen weight was reduced in PFOA-exposed ovx mice. Brown adipose tissue weight was significantly increased in both ovx and intact mice at high PFOA doses. Liver weight was unaffected in late life by these exposure paradigms. Finally, there was no effect ofadult expo sure to PFOAon body weight. These studies demonstrate an important window of exposure for low-dose effects of PFOAon body weight gain, as well as leptin and insulin concentrations in mid-life, at a lowest observed effect level of 0.01 mg PFOA/kg BW. The mode of action of these effects and its relevance to human health remain to be explored. Published by Elsevier Ireland Ltd. Abbreviations: ANOVA. analysis o f variance; BM I. body mass index; BW. body w eight; C8. eight-carbon; CV. coefficient o f va ria tio n ; DES. d ie tylstilb e stro l; E j. estradiol; GO,g estational day; t )n . H a lf-life ; IACUC. In s titu tio n a l A n im a l Care and Use Com m ittee; LH. lu te in izin g horm one; LOD. lim it o f d ete ction : LOQ,lim it o fq ua n tita tio n : NHANES. N ational H ealth and N u tritio n Exam ination Survey: NMR. nuclear m agnetic resonance; NOAEL. no observable adverse effect leve l: ovx. ovariectom ized; PFAA. p erfluoroalkyt acid; PFOA, perfluorooctanoic acid; PFOS. perfhiorooctane sulfonate; PND. postnatal day; PPAR, peroxisom e proliferator-activared receptors; SMR, standardized m o rta lity ra tio . * Disclaimer: The inform a tio n in th is docum ent has been funded by the US. Environm ental Protection Agency. It has been subjected to review by the N ational H ealth and E nvironm ental Effects Research Laboratory and approved fo r publication. Approval does not sign ify th a t the contents reflect the view s o f the Agency, nor does m ention of trade names or com m ercial products constitute endorsem ent o r recom m endation fo r use. C orresponding author. C urrent address: US. E nvironm ental Protection Agency. N ational Center fo r Exposure Analysis. Environm ental Media Assessment Group. Research T riangle Park. NC 27711. U nited States. T e l: 1 919541 4204;fax: 1 919 S41 2985. E-moil address: htnes.erineepa.gov (E.P. Hines). 0303-7207/5 - see fro n t m atter. Published by Elsevier Ireland Ltd. doi;10.1016/j.m ce.2009.02.021 98 EJ>. Hines e t o L / Molecular am t Cellular Endocrinology 304 (2009) 97-105 1. Introduction Perfluorooctanoic acid (PFOA). one o f the eight carbon (C 8) perfluoroalkyl acids (PFAAs). is a synthetic, stable, persistent organic fluo rine surfactant, used to im p art w ater and grease resistance to various consum er products including non-stick pans, as sur face treatm ents fo r clothing and food w rappers, insulation and fire -fig h tin g foams. PFOA's high energy carbon-fluorine bonds are resistant to hydrolysis, photolysis and m etabolism and thus it bioac cum ulates and persists w ith in biota and environm ental matrices, including w a te r and soil, from the Arctic to the South Pacific (Lau et at., 2 0 0 7). This ubiquitous environm ental contam inant has an esti m ated h a lf-life { i p ) in humans o f 3.8 years (Olsen e t a l, 2007) and is found in production w orkers' sera, as w e ll as those o f the general p o p u la tio n . B io-m onitoring studies show detectable levels ofPFOAin human populations. The N ational H ealth and N utrition Exam ination Sur vey (NHANES) reported that m ean serum PFOA concentrations are declining in th e USA population, from 5.2 ng/m l in 1 9 9 9-2 0 0 0 to 3.9 ng/m l. in 2 0 0 3 -2 0 0 4 (C alafat e t a]., 2007)lArnsberg, Germany, an area w ith know n drinking w ater PFAA contam ination, had reported PFOA mean serum levels in 2 006 o f 25 ng/m l vs. 4 ng/m l in unaffected Germ an provinces (H & lzer et a l, 2 0 0 8 ). The highest know n non-occupational PFOA exposure via drinking w ater exists in th e L ittle Hocking drinking w a te r district w here U.S. residents (O hio and W est V irg in ia ) have mean serum PFOA concentrations o f 478 ng/m l (E m m ett et a l, 2 0 0 6 ). Children m ay receive significant PFOA exposures via dietary and w ater intake. M ean serum PFAA concentrations (such as perfluorohexane sulfonic acid) w ere reportedly higher in children than in ad u lt/eld eriy populations (Olsen et al., 2004). In the L ittle Hock ing w ater d is tric t, an area o f high environm ental PFOA exposure, children age tw o to five and the elderly had significantly increased PFOA semm levels w hen compared w ith other age groups (Em m ett e t al.. 2 0 0 6 ). Although a bio-m onitoring study in japan found PFOA in m aternal blood, b ut not um bilical cord blood at parturition (Inoue et al.. 2 004. lim it o f quantitation |LOQ] 35.2 ng/m l), a recent U.S. study (A pelberg et al., 2 0 0 7 ) o f hum an cord blood from term pregnancies reported relatively low levels o f PFOA (lim it o f detec tio n |LOD) 0 .2 n g /m l) and another C8 compound, perfluorooctane sulfonate (PFOS). W ith in the reported study concentrations, the authors found that cord blood PFOA concentrations were signifi cantly negatively associated w ith b irth w eight. A subsequent larger Danish study also found a significant negative correlation between m aternal plasm a PFOA and b irth w eight (Fei et al.. 2007). There have been no consistent adverse health effects associated w ith occupational exposure to PFOA, in fact, the studies to date are contradictory. In w orker populations, serum cholesterol and triglycerides have been positively associated w ith PFOA exposure w h ile high density lipoproteins have been negatively associated w ith PFOA (O lsen et al., 2001). Categorical division o f workers by PFOA exposure levels showed th at, although not significantly differ en t from the oth er categories, body mass index (B M I) was elevated in the highest PFOA category (>30p pm and BMIs >28,1995 data): this trend was not seen in the 1993 data set (Olsen et al.. 1998). A retrospective cohort m o rtality study (n > 6 0 0 0 ) o f PFOA-exposed employees reported significantly elevated standardized m ortality ratios (SM R) in males w ith diabetes m ellitus w hen compared to m en residing in W est Virginia (m inus the PFOA manufacturing area), Ohio, V irg in ia, Kentucky. Indiana. Pennsylvania, Tennessee, or North Carolina; the SMR for PFOA workers was not significantly increased w hen com pared to W est V irginia alone or USA residents (DuPont. 2 0 0 6 ). In Arnsberg. Germany, PFOA was found to have an inverse correlation w ith BMI in adults (H olzer et al.. 2008). The r,p s fo r PFOA in men and w om en are sim ilar (Harada et a l, 2005). U nlike hum ans, gender differences in PFOA clearance exist in rats (Kudo and Kawashima. 2003; Vanden Heuvel e t a l, 1991). M ice are the preferred anim al m odel for evaluating the effects o f PFOA on the developing fetus as they do not exhibit gender-dependent t i p differences (Lau et a l, 2 0 0 6). However, even in the rat model system w here the female rat rapidly excretes the compound. PFOA readily crosses the placenta (H in d e rlite r e t al.. 2 0 0 5) and PFAAs are present in rat m ilk after PFOA treatm ent (H in d erliter et al., 2 0 0 5). M ice prenatally exposed to doses o f PFOA at >1 m g/kg/day exhib it developm ental toxicity including decreased litte r size, neonatal death, delayed eye opening, grow th deficits, stunted m am m ary gland developm ent, and early onset m ale puberty (Lau et a l, 2 0 0 6; W h ite et al., 2007; W o lf et a l, 2 0 0 7 ). A t higher doses and fol low ing long-term adult exposure, cancer endpoints associated w ith PFOA exposure in rats include Leydig cell adenomas, pancreatic aci nar cell adenoma/carcinomas, m am m ary fibroadenomas, and liver tum ors (Biegel et aL, 2001; Sibinski, 1987). PFOA increased estra diol (E2) levels in m ale rats and PFOA-induced rodent Leydig cell tum ors are hypothesized to arise from increased estradiol levels from aromatase induction (Liu e t a l, 1996; Biegel et a l,2 0 0 1 ). The m ajority o f the ongoing w ork in the PFOA field has focused on the health effects follow ing developm ental exposure to PFOA. This study focuses on adult laten t health outcomes in fem ale off spring after developm ental (gestational days (G D ) 1 -1 7 ) vs. adult (a t 8 weeks o f age. for 17 days) exposure to PFOA. Ovariectom ized siblings w ere utilized in our second study block to address the role o f the ovarian hormones in PFOA exposure-related health effects, as luteinizing horm one (LH)-overexpressing mice (Kero et a l, 2 0 0 3 ) displayed several phenotypic effects resem bling those in our prelim inary studies w ith PFOA. These studies address the role o f developm ental exposure and ovarian hormones in adult health effects including circulating leptin and insulin concentrations, adult body w eight, and tissue and body w eights in old age. 2. M a te ria ls and m ethods 2.7. Animo/s Tim ed-pregnant CD-I mice (Charles River Laboratories. Raleigh. NC) arrived on gestational day (GD)0 (sperm p ositive ) at the US EPA w here they were w eighed upon a rriva l and random ly d istrib u te d am ong treatm ent groups. Pregnant dams w ere housed individtiaR y Hi polypropylene cages and received chow (U bD iet 5001. PM! N u tritio n International LLC. Brentwood. M O) and tap w ater ad Bbitum. Two blocks o f anim als were used in these studies. Block 1 anim als were dosed w ith vehicle (d is tille d w ater), I. 3. o r SmgPFQA/kg body w eight (BW ) (n -5 . 8. 7. am i 5 dam s, respectively); block 2 anim als w ere dosed w ith vehide. 0J)1. 0.1. 0 3 .1 . o r 5mgPFOA/kg ( o - 14 dams in a ll groups except 5 mgPFOA/kg BW. w hich had 10 dams). PFOA exposures are shown in the te xt as mg PFOA/kg. A nim al fa cilitie s were m aintained on a I2:12-h lig h t-d a rk cycle, a t 20-24 "C w ith 40-50% relative h u m id ity. Anim als were hum anely treated as approved under N ational H ealth and Environm ental Effects Research Laboratory protocols in accordance w ith the US EPA In stitu tio n a l Anim al Care and Use Com m ittee (IACUC). S entinel m ice, housed in the same room , were known to be free o f ecto/endoparasites and antibodies to certain viruses fo r the duration o f these studies. 2 3. Dosing solution and procedures PFOA. as its am m onium salt (>98% pure), was acquired from Fluka Chem ical (Stem hiem . S w itzerland). PFOA dosing so lu tio n was prepared fresh d a ily in deion ized w ater, and the dosing solution was adm inistered at a volum e o f 10 p i/g . M ice received e ithe r w ater vehicle o r PFOA a t 0 .0 1 .0 .1 .0 3 .1 . 3. o r 5m g/kg BW by oral gavage once d a ily over the dosing periods.The highest dose (5 mg PFOA/kg/day)was chosen because it was know n to re sult in s lig h tly reduced neonatal body w eight gain w ith m inim a l postnatal m o rta lity (Lau et al.. 2006). 2 3. Experimental design 23. 7. Developmental exposure/mtoct Tim ed-pregnant CD-I mice (n 7 -2 2 dams per dose group over tw o blocks) received 0.0.01.0.1.0.3.1.3. or 5 m g/kg PFOA by oral gavage on the m ornings o f GD 1-17. Dams w ere weighed daily p rio r to dosing and throughout gestation. Al b irth , pups were in d ivid u a lly weighed and scxed. Pups w ith in a treatm ent group were pooled and random ly redistributed among th e dams of th e ir respective treatm ent groups, and lin e rs were equalized to 10 pups (both genders represented). Dams EJ*. Hines t a l / Molecular a n d Cellular Endocrinology 304 (2009) 9 7 - 70S or PFOA PFOA Developmental Adult Dosing GO 1-17 Dosing 8 wks 3 **> |nH| P GDO B*r* ' . an ? nx 16-16 w its 1 Glucose tolerance lest (young) 21-33 wks 11 MandBular bleeds 42 wfcs I 1 Body Mass Composition Measued 70-74 wks 1 1 Glucose tolerance Test (old) Food Intake m onitoring 1 1* Months E2 m asure _________ .....W eifpit monitored (developm ental exposed, ovx Intact)..___ _________ F ig . 1. Data co lle ctio n schem atic fo r study o f devetopmentaRy and a du lt PFOA-exposed fem ale mice. 99 th a t d elive re d sm a ll litte rs (n < 4 pups) w ere excluded from the rem ainder o f the study. Pups w ere weaned a t 3 weeks o f age at w hich p o in t fem ales w ere retained and housed 3 -5 m ice p e r cage. M ales w ere evaluated separately, a t end p oints that varied from those reported here. 2.32. Developm ents! exposure/ovariectomy A subset o f developm entally exposed fem ale siblings (OmgPFOA/kg, n - 8 ; 0.01 m gPFOA/kg. n - lS ; 0.1 mgPFQA/kg. n -1 1 ; 0 3 mgPFOA/kg, n -1 4 ; 1 m g PFOA/kg, n - 6 ; 5 m g PFOA/kg. n = 7 ) w ere avariectom tzed (o vx) at 21 o r 22 days o f age. before the onset o f puberty. Anim als w ere sedated w ith ketam ine/xylazine (87/13 m g/kg L p , respectively), th e ir ovaries su rgica lly rem oved through the abdom en, su tu re d , and anim als w ere placed in w arm ing cages u n til they regained alertness. Bupvenorphine analgesic (0.05 m g/kg) was given tw ice d a ily i.m . fo r 48 h in 0.1 m l vo lu m e fo r pain re lie f. 2.33. A dult exposure A separate co ho rt o f m ice received PFOA sta rtin g a t 8 weeks o f age. fo r 17 days (0 mg PFOA/kg. n = 8 ; 1 m g PFOA/kg. n = 14; 5 mg PFQA/kg. n -1 4 ). 23.4. Data coRecrion The data co lle ctio n scheme fo r these studies is show n in Fig. |. Blood was col lected fro m th e subm andibular veins o f ovx and in ta ct m ice between the ages o f 21 and 33 w eeks. These bleeds took place between 14:00 and 18:00. and 200 p i o f Mood (100 p.1 o f serum ) was collected fo r subsequent analyses o f in su lin and te p tin . Females in a ll three exposure scenarios w ere w eighed w eekly up to 9 m onths o f age and then m o n th ly u n til 18 m onths. The num ber o f in ta ct, developm entally exposed m ice w eighed w ee kly/m o n th ly was 10. 2 5 .2 0 .1 1 . and 32. respectively fo r 0.0.01. 0.1.03. and l.O m g PFOA/kg. If m ice became m oribund before the study ended, they w ere euthanized in com pliance w ith the protocol approved by the US EPA IACUC (e arly necro p sy) Date and cause o f early m o rb id ity o r m o rta lity was recorded if know n. A t e a rly necropsy (collected w hen necessary) o r a t 18 m onths, tru n k blood, re tro pe rito n ea l abdom inal w hite (found lyin g ve ntra l to the intestines and repro ductive tra c t) and interscapular brow n fa t pads, abnorm al grow ths, and organs w ere collected fro m a ll exposure groups. Relative organ w eight is used to express organ w eight as percent o f to u t body w e ig h t. Data are reported here as mean SEM. 2.4. Glucose tolerance test Glucose tolerance tests w ere perform ed on tw o groups o f in ta ct develop m entally PFOA-exposed anim als: o ld adults (17 m onths o f age w ith 0. 0.1, 1 or 5 mg PFQA/kg; n - 8 -13 per dose g rou p ) and young a dults (15-16 weeks o ld w ith 0 ,1 o r 5 m g PFOA/kg; n -1 2 per dose g ro u p ) The n ig h t before the assay, fu r was shaved fro m th e latera l area o f the low er leg to expose the saphenous vein and ani mals w ere fasted.T he fo llo w in g m o rnin g , the m ice w ere weighed and blood glucose was m easured by co lle ctin g a drop o f blood from each mouse via puncture o f the saphenous v e in (o r taH ve in if necessary).The blood d rop was placed on a test s trip , and inserted in to the calibrated glucom eter (Accuchek Advantage) fo r baseline glu cose m easurem ent. The m ice w ere then injected i.p . w ith o-gtucose so lu tio n (2 g/kg body w e ig h t from a stock s o lu tio n ) and blood glucose concentrations w ere mea sured at 2 0 .4 0 .6 0 and 120 (o ld m ice) o r 180 (young m ice) m inutes ( 1 -3 m in ) after the in itia l glucose in je ctio n . 2 3. Serum leptin Serum (1 0 p.1) collected by m andibular venipuncture was assayed fo r te p tin by radio-im m unoassay (Linco Research. St. Charles. M O ) fo llo w in g the m anufacturer's protocol (n = 5. co n tro ls; * 18.0.01; n - 16.0.1; n - 1 1.0 3; n - 24,1 mg PFOA/kg)The coefficient o f va ria tio n (CVs) fo r the standards (concentration range o f0 3 -2 0 n g /m l) ranged from 0.1 %to 8.0%.The q u a lity co n tro l standards term ed QCl (expected range 0.6 -1 3 n g/m l)a n d QC2 (range 1.8-3.8) had a measured concentration in these assays o f 0.9 and 2.9. respectively. 2.6. Serum insulin Sera (10 p j) collected by m andibular venipuncture w ere assayed fo r in s u lin by the ultra-sensitive single m olecule immunoassay by Stngulex (Alam eda, CA) fo l low ing the m anufacturer's p roto col (n 9 control, n -2 1 . 0.01 mgPFOA/kg; n - 16. 0.1 mgPFOA/ltg: n 11. 0 3 m gPFOA/kg; n - 3 1 .1 mgPFOA/kg) Samples w ere ana lyzed using a 384-w e ll plate fo rm a t w ith m onoclonal capture and detection antibodies on the Singulex Errena equipm ent.The CVs fo r the assay standards (range l9 5 -5 0 0 0 p g |m l) w ere from 3% to 17%. The assay LOD was 16pg/m ). AH samples were run on the same day and the interassay CV was 9.4% and 5.1% fo r the 29 and 1745 pg/m t quahty assurance standards, respectively. 2.7. Body mass composition W hole body mass com position was measured in live , non-sedated 42-w eek-old m ice using the Broker M intspec m q 7 5 LF50 Live Mouse A nalyzer (The W oodlands, T X ) The m inispec was a benchtop 75 MHz tim e-dom ain nuclear m agnetic reso nance (NM R) analyzer, w hich q ua n tifie d body fa t, lean tissue, and free body flu id in m ice. The m intspec was calibrated by Broker Optics. Inc. s ta ffp rio r to anim al analy sis w ith d a ily validations using B roker standards. Mice w ere weighed and inserted in to the instrum ent fo r analysis (1 -2 m in/an im a !) Intact developm entally exposed fem ale m ice th a t underw ent body mass com position analysis included co ntro l. 0.01. 0.1. 0 3 , and 1 mgPFOA/kg (n -9 . 23. 20, 11. and 32, respectively) dose groups. It was not possible to perform these measures w ith younger m ice due to equipm ent a vailability. 2.8. Measurement o f Ej in serum o f intact mice at 18 months Serum E2 (25 pci volum e) from 18-m onth-old mice (in ta c t developm entally PFOA-exposed anim als) was measured w ith tim e resolved fluoro-im m unoassay (DELF1A Estradiol K it. W altac Oy. Finland) follow ing the m anufacturer's recom m endation using a VICTOR2D 1420 M u ltila be l counter. PerfcmElmer Precisely tim e-resolved fkuorom eter (PerkinE lm er Life ft A nalyticalSciences. Shelton.CT)>The CVs fo r the standards (concentration range o f 6 5 1 -1 4 25 p g /m l) ranged from 02% to 45%. 25. Feed consumption Feed consum ption m 17-m onth-old. developm entally exposed, in ta ct female m ice (n * 6 per dose group. 0 .0 .1 .1 and 5 mg PFOA/kg) was measured in m etabolic cages. M ice were allow ed to acclim ate to the cages for 1 week and food intake was m onitored during the second w eek. M ice were in d ivid u a lly housed and provided w ith a pre-w eighed am ount o f pow dered lab chow od libitum . The rem aining chow was measured at the end o f the week and die to ta l am ount was subtracted from the sta rtin g am ount to determ ine the to ta l feed consumed fo r each mouse per week. 2JO. Measurement o fserum PFOA Trunk Wood serum samples (-5 0 p i) from the fem ale CD-1 offspring at 18m onth necropsies o r from m ice term inated at earlier intervals because o f Alness were transferred to the CDC fo r PFOA measurement. Serum PFOA determ ination was perform ed as described in K uklenyik et aL (2005) and W h ite et aL (2 0 0 9 ) 2 J I. Statistics Data were analyzed using SAS 9.1 (SAS Inc.. Cary. NC) Body w eight on PND1 was evaluated as fitte r means as these data w ere obtained p rio r to m ixing litte r offspring w ith in a dose group. Body w eights at each tim e p o in t were analyzed w ith m ixed effects lin e a r m odels (SAS Proc M ixed) to estim ate means and standard errors and te st fo r dose effects separately by tim e point. Foreach tim e p o in t the model included dose as a fixed e ffee1 p. 47 r 100 EP. Hines e t a l / Molecular and Cellular Endocrinology 304 (2009) 9 7 - IOS and cage nested w ith in dose as a random effect. Pairw ise r-rests w ere calculated to te st fo r any diffe re nce betw een each treatm ent group mean and the co n tro l g rou p Repeated m easures analysis o f body w eight data was evaluated tw o ways. F irst, w e ig h ts w ere averaged by anim al over eight lO week interva ls. This was done to decrease m issin g values in th e data due to anim al m o rta lity in late Hfe th a t was not equal across tre a tm e n t, and to reduce the effe ct o f large body w eig h t variances later in life . T his data sm oothing m ethod decreased u nin fo rm a tive sh ort-te rm variations and also reduced the num ber o f estim ated param eters to a tractable value. A m u lti va ria te repeated m easures analysis (SAS Proc GLM) was perform ed on these reduced data. Subsequent to a sig n ifica n t fin d in g , com parisons w ere carried o u t as subtests o f th e o ve ra ll analysis o f variance (ANOVA) at specific tim es o r doses. Second, SAS Proc M ixed w as used to perform a u niva ria te repeated measures analysts o f the w e ig h ts across tim e up u n til 37 weeks (la te st w e ig h t p o in t a tw hich no anim als had die d ).T h e m odel estim ated a separate fixed quadratic curve across tim e fo r each dose g ro u p and included a random effect fo r cage nested w ith in dose. Cor re la tio n w ith in anim als was m odeled w ith a random e ffe ct fo r anim al nested w ith in cage and dose in a d d itio n to an autoregressive covariance stru ctu re w ith in each ani m a l. In th is w ay, th e covariance m a trix fo r each anim al's m easurem ents included a constantcovariance com ponent a t a ll tim e poin ts in a d d itio n to a com ponent w hich decreased as tim e p o in ts grew fa rth e r apart. Tissue w e ig h t, re la tive tissue w eig h t, body com position, food consum ption, and body w e ig h t m easurem ents w ere analyzed using a one-w ay ANOVA (D unnett's post hoc tests), w ith dose being the independent variable. A blocking variable was inclu d ed to a d ju st fo r th e group difference. No adjustm ent was made lo r m u lti ple com parisons. Glucose tolerance was com pared a t in d ivid u a l co lle ctio n tim es by one-w ay t-te s t and over tim e by repeated measures and area under the curve com parisons according to th e trapezoidal ru le . Horm one (in s u lin . Ez and le p tin ) concentrations w ere analyzed using ANOVA follow ed by Tukey's post hoc test. M o rta lity data w ere analyzed w ith product lim ite d su rviva l estim ates: tog-rank and W ilcoxon tests w ere used to test fo r differences am ong th e treatm ent groups in su rviva l across tim e (SAS Proc Lifetest). The level o f significance fo r a ll tests was p<0J0S. (A ) 1*75 5 -a ? 1.45 05 13 M l 0.1 3 I Dose PFOA (tng/kg BYV) Period 3. Results 3.1. D e ve lo p m e n ta l exposure 3.1.1. E arly a n d m id -life b o d y w e ig h t effects There w ere no significant differences in live pup num ber at b irth by dose group (p < 0 .0 5 ) and postnatal m o rta lity was not addressed in this study as litters w ere equalized a t b irth. On post natal day (P N D ) 1, the average w eight o f the developm entally exposed 5m gPFO A/kg offspring was significantly less than con trols (Fig. 2 A ): no other dose group dem onstrated significant litte r w eigh t effects at PND1. A t w eaning, mean fem ale body weights w ere s till significantly decreased in the 5 mg PFOA/kg (13.9 g 0 .8 ) com pared to 18.4 g 0.4 in control untreated pups. A t this tim e, the 1 m g PFOA/kg exposed anim als w ere also significantly sm aller than controls (p < 0 .0 5; 16.4 g 0.3). Tim e-grouped mean body w eights o f the fem ale offspring over th e ir life tim e are shown in Fig. 2B. Beginning a t 1 0 -1 9 weeks o f age, there w as an increase in w eight in the 0.1 and 0 3 mg PFOA/kg groups com pared to controls; b y 2 0 -2 9 weeks o fage. fem ales devel opm entally exposed to PFOA showed significant dose-dependent increases in body w eight at 0.01. 0.1. and 0.3 mg PFOA/kg which extended to 4 0 weeks o f age in the 0.01 and 0.1 mg PFOA/kg when com pared w ith control (p < 0 .0 5 ). This is specifically shown at 2 0 -2 9 w eeks (Fig. 2C). w here the 0.01 - 0 3 m g PFOA/kg groups had average w eights 11-15% higher than controls. Continuous analysis o f repeated measures o f body w eight over tim e dem onstrated th a t the five dose groups w ere sim ilar in inter cept using a quadratic fit; however, the 0.01, 0.1 and 0.3 groups had a significantly greater w eek effect than control, indicating that th e ir w eights w ere changing at a more rapid rate than control or 1 m g/kg. This is shown in Fig. 2D for weeks 6 -3 7 (th e latest w eight collection tim e point p rior to death o fany study anim als). A ddition ally, the 0.1 m g/kg (p = 0 .0 56 ) and 0.3 m g/kg (p = 0 .0 4 6 ) groups had larger negative coefficients for w eek2 (w eek squared), suggesting th at th eir w eights w ere starting to fall o ff m ore quickly at the later tim e points than the control groups (not show n). The estim ated w eight curve fo r the 1 mg PFOA/kg dose group was not significantly d ifferent from the control curve. Data from 5 mg PFOA/kg exposed Fig. 2. Body w eights o t devetopm entally PFQA-exposed fem ale o ffsp rin g Data are shown as mean SEM w ith 'p <0.05 vs. co ntro l. (A ) Pup w eight a t PND1 a lte r devel opm ental PFOA exposure. (8 ) Body w eight o ( fem ale CD-I m ice over th e ir life tim e , fo llo w in g developm ental PFOAexposure over 8 periods o ftim e [p e rio d 1 (0 -9 weeks oldX period 2 (10-19 weeks old), period 3 (2 0 -2 9 weeks o ld ) period 4 (3 0 -3 9 weeks o ld ), period 5 (4 0 -4 9 weeks o ld ),period 6 (5 0 -5 9 weekso ld ), period 7 (6 0 -6 9 weeks o ld ), and period 8 (7 0 -7 9 weeks)). (C) G roup mean body w eights o f fem ale offspring at 2 0-2 9 weeks o f age dem onstrating excessive w eight p in at low doses. ( D) Dosedependent quadratic regression lit to repeated measures o f body w eight in fem ale mice. An increased rate o f w eight gain was seen in 0.01.0.1. and 0.3 m g PFOA/kg dose groups com pared to co n tro l and 1 m g PFOA/kg. p. 48 EJ> H in a e l a l / Molecular and Cellular Endocrinology 304 (2009) 97-105 (A ) 78- ** 3 60- IIIE ft So 0 (B ) 0 0 ~ 3000^ 2500- 3 2600- g 1500- 0.01 0.1 03 Dose PFO A (m g/kg B W ) ** 101 Fig. 3. B lood glucose concentrations fo llo w in g a glucose challenge a fte r tim e 0 m (A ) young (1 5 -1 6 weeks o ld ) and (B ) o ld (7 0 -7 4 weeks o ld ) fem ale C D -I m ice th a t w ere d e ve lo p m e n ta l^ exposed to PFOA Data are show n as mean SEM. m ice, w h ich w ere decreased in BW com pared to control at PND1. w eaning, and 18 m onths, are not shown. 3.1.2. S e r u m glucose tolerance testin g Because o f the excess w eigh t gain in the PFOA developm en- tally exposed mice during m id -life, various tests w ere conducted on these anim als (as close to the appropriate age as was possible) to exam ine the associated effects o f these changes. No significant differences w ere detected in baseline glucose or serum glucose area u nd er the curve in response to a glucose challenge in young or old m ice (control, 0 .1 .1 . or 5m gPFO A/kg, p < 0.05. Fig. 3 ). In a tim e-dependent com parison, young m ice exposed to 1 mg PFOA/kg showed a nearly significant increase in blood glucose over control anim als a t 20 m in post-glucose challenge ( p - 0 .0 6). In old PFOAexposed m ice, although there appeared to be dose-dependent glucose in sen sitivity at 20 m in. this shift in response was not sig nificant. 3.1.3. S e r u m insulin a n d lep tin Serum insulin and leptin measurem ents w ere made using blood obtained v ia m andibular bleeds betw een 21 and 33 weeks (w ith in the tim e fram e o f greatest observed body w eight increases) using intact fem ale mice dosed w ith 0. 0.01, 0.1, 0.3. and 1 mg/kg PFOA. Insulin and leptin concentrations w ere significantly increased in mice developm entally exposed to the low est doses o f PFOA tested (0.01 and 0.1 mg PFOA/kg). Although elevated from the control mean, le p tin concentrations w ere not significantly different from control a t 0 3 or 1 m g/kg PFOA (Fig. 4 ). 3.1.4. Fat to lean ratio At 42 weeks o f age, m ice from block 2 (control. 0.01, 0.1, 0.3. and 1 mg PFOA/kg) were evaluated using a Bruker Optics Body Mass A nalyzer, w hich determ ines the am ount o f fat. lean and fluid 0 Dose PFOA (m g/kg) F ig . 4 . Serum lep tin (A ) end in su lin (B ) in m ice at 21-33 weeks o f age ( p <0.05 vs. co n tro l). S ignificant elevations are seen at 0.01 and 0.1 m g PFOA/kg. Data are shown as mean SEM. in live anim als. There was no significant increase detected in % body fatibody w eight in PFOA-exposed m ice (data not shown). Developm entally exposed mice had no significant differences in fat:lean ratio across dose groups w hen compared to control (means ranged from 0.75% in controls to 0.9% in 0.01 and 0.1 mg PFOA/kg). Although no dose groups w ere significantly d ifferent from control, there was an increase above control levels o f about 12% in mean % fatrbody w eight ratio and 14% in mean fatilean ratio in the dose group exhibiting the largest change in body w eight at 24 weeks (0.1 mg PFOA/kg). 3.1.4.1. Feed consum ption. Feed consumption was measured in 17m onth-old, developm entally exposed intact mice (0. 0.1. 1 and 5 mg PFOA/kg) and no significant differences w ere found across dose groups w hen compared to controls (m ean 26g/w eek con sum ed: individual data not shown). 3.1.5. Late lij organ an d b o d y w e ig h t effects A noted loss o f animals after 36 weeks o f age was further eval uated (Fig. 5). At 51 weeks old. w hen there was no m ortality in controls there were 20%, 10%. 36%. and 6% m o rtality rates in 0.01, 0 .1 .0.3 , and 1 mg PFOA/kg groups, respectively. By 76 weeks, there was a 40% m ortality rate in controls, and 32%, 63%. 60%. and 44% in 0.01,0.1.0.3 and 1 mg PFOA/kg groups, respectively. However, there w ere no significant differences between control and any treatm ent group at specific tim es in late life or in survival across tim e. Among those mice surviving to 18 months, body w eight o f PFOA-exposed females was no longer elevated compared to con trols. Furtherm ore, a significant decrease in body weight at the 5 mg PFOA/kg dose was noted (Table 1). At that tim e, all rem ain ing females were necropsied. Trunk blood, tissues (affected or o f interest) and abnormal masses w ere collected, weighed and fixed for future study. Serum was collected and PFOA levels were mea sured. The m ajority o f the samples across dose groups had PFOA concentrations lower than the lim it o f detection (0.5 ng/m l) w ith detectable values at m axim um concentrations o f 3 5 ng/m l. and 102 E-P Hines e l at. / M olecular a n d Cellular Endocrinology 304 (2009) 9 7 - 705 Table 1 M ean o r re la tive body and tissue w eights a t IB m onths o f age in in ta ct and ovariectom ized (ovx) fem ale C D -I mice. PFOA dose Body w eight (g ) (m g /kg ) Abdom inal w h ite fa t W eight (g ) . Interscapular brow n Fat w eight (g ) Relative spleen W eight {% y In ta c t O vx In ta c t. - Ovx In ta ct Ovx ; Inta ct O vx 0 0.01 0.1 03 1 3 5 . 5 4 3 0 * 133 5 3 7 3 *5 3 7 7 .0 7 *0 5 6 3 8 3 *1 .0 3 0.73 * * 0 4 0 3 7 * 0 8 9 5 6 3 8 * 1 :4 8 52.6 1 3 63 6 8 8 * 0 > i: 5 3 6 *1 .0 0 0 3 0 * 0 0 4 0 3 6 *0 3 4 5 4 0 0 * 1 1 7 5 3 7 6 *1 9 8 5 3 1 * 0 3 7 4 3 4 *0 7 1 0 8 2 0 0 4 0 .4 6 *0 3 6 . 5 6 .0 0 * 1*74 4 *3 6 * 3 .5 3 5 3 6 * 0 3 3 4 3 7 *0 .4 6 - 0 7 9 * 0 0 6 0 3 9 * 0 0 2 5 6 3 5 1 3 5 61.47 3.53 5 3 2 *0 .4 3 * 5 8 2 *0 .7 7 .0 8 9 * 0 0 4 * 0 8 9 *0 8 6 5 3 3 9 * 2 3 7 nc. .nc. nc ' . .1 2 2 * 0.10* nc 4 9 3 7 * 131* 5 5.1 3*5 .7 6 4 .4 8 *0 .6 5 * 5 3 6 * 1 3 7 0 3 6 * 6 .0 5 0 8 2 *0 .2 0 0 3 9 *0 0 5 036 * 003 0.45 * 0.12 0 .4 5 *0 .1 0 0 3 0 *0 8 3 D jS '0 O 3 * 082 * 034 0 82 0.16 039 *0 04 040 *0 08 033 *0 08 0 3 2 *0 0 2 * -DC -.v " 0 21*004" nc. Denotes not collected from th is dose group. ` p<0.01 vs.c o n tro l. " p - 0.05-0.07. c R elative w e ig h t (organ w eig h t as percent o f body w eight). Relative liv e r W eight (X)* In ta c t 4 3 0 * 0.10 3 3 9 * 6.11 420 *0 4 1 430 * 031 402 * 010 338 * 023 437 * 034 Ovx 4 3 0 *0 34 4:12*0^35 4 .1 8 *0 2 1 407 *0 22 3 35 *0 29 " nc 3 35*033 th e re was no significant difference in serum PFOA concentrations across dose groups (data not show n). There w ere no significant differences in serum estradiol levels in developm ental^ exposed fem ales at 18 m onths w hen com pared to controls (non-cycling; m ean range across doses from 12.9 to 15.8p g /m l). Tissue w eights from 18-m onth-old anim als (in ta ct and ovx) are show n in Table 1. To determ ine i f the w eight o f fat depots was altered in o ld anim als due to developm ental PFOA exposures, the retro perito neal abdom inal w hite and interscapular brown fat pads w ere collected and w eighed. Abdom inal w hite fa t w eigh t and rela tiv e w h ite fa t w eight both showed significant decreases vs. control (p < 0 .0 5 ) at 1 and 5 mg PFOA/kg. W h ite fat w eights w ere not col lected fo r 3 m g/kg PFOA anim als. A t 18 m onths, interscapular brown fa t w eight and relative brown fa t w eigh t both showed significant increases above control (p < 0 .0 5 ) at 1 and 3 mg PFOA/kg. The spleen w as quite variable in w eight among th e different treatm ent groups, b ut there w as a significant difference in spleen w eight and relative spleen w e ig h t vs. control at 3 mg PFOA/kg (p < 0 .0 5 ). Finally, at 18 m onths, no significant differences in liver w eight or relative liver w eig h t w ere detected. reach statistical significance. W hen comparing the body weights o f anim als in the ovx study by treatm ent group, over tim e (4 weeks to 18 m onths), using statistical methods consistent w ith those used for intact anim als, there w as no effect o f PFOA (Fig. 6B). Compar ison o f ovx anim als to intact anim als at 2 0 -2 9 weeks, as shown in Fig. 6A. demonstrates an absence o f body w eight gain over con tro l in the ovx anim als treated w ith PFOA. PFOA exposure did not stim ulate increased w eight gain (above that o f control ovx) at any developm ental exposure level in the absence o f the ovaries (also seen in Fig. 6B). The ovx anim als w ere siblings to the intact animals in this study. The ovx anim als w ere also assessed at 18 months. Developmen tally PFOA-exposed ovx anim als showed no significant differences in body w eight w hen com pared to control ovx females (con trol mean *5 2 .7 5 .6 7 ; highest mean. 1 mg PFOA/kg=61.5 3 3 ; Table 1). 3.1.6. Effect o f ovariectom y on tissu e a n d b o d y w eight gain A group o f developm ental^ PFOA-exposed anim als (0 .0 .0 1 ,0 .1 . 0.3, 1. and 5 mg PFOA/kg) w ere ovx at w eaning and th e ir body w eigh t gain and adult health was assessed u n til they reached 18 m onths o f age. A t m id -life the w eigh t o f the control ovx females was expected to be greater than th a t o f the sham -operated, intact controls (Fig. 6A; set o f bars at 0 m g/kg), but the variance in the ani m al weights was appreciable and therefore the differences did not 0 0 .# l 0.1 0 3 I PFOA Dose (m g/kg BW ) Fig. 5. S urvival curves fo r developm ental^ PFOA-exposed fem ale mice (0 -1 mg PFOA/kg). Although a fa ir num ber o f PFOA-exposed anim als die early, a U fetest (SAS) analysis detected no sign ifica nt decrease in tim e to death. The reasons fo r early life m o rta lity are under investigation. Fig. 6. (A ) PFOA-dependent changes in group mean body w eight o f intact and ovx fem ale offspring at 20-29 weeks o f age. There was no change in body w eight o f ovx anim als across PFOA exposures. (B ) Dose-dependent quadratic regression fit to repealed measures o f body w eight in ovx fem ale mice. U nlike intact siblings, no significant differences were seen between dose groups in the ovx animals. E.P. Hines et a l / Molecular a n d Cellular Endocrinology 304 (2009) 9 7 - 705 103 As w ith intact siblings, the tissue w eights o f ovx anim als are reported in detail in Table 1. In ovx anim als, neither abdom inal w h ite fat pad w eight, nor relative abdom inal w h ite fat pad w eight, w ere significantly d ifferent from ovx o r intact control levels. This varies sligh tly from intact siblings, w here the w h ite fat pad was significantly decreased in size; although, in anim als th a t weighed significantly less than intact controls. Am ong PFOA-exposed ovx anim als, b oth interscapular brow n fat w eight and relative brown fat w eig h t (data not show n) showed significant increases above control o vx levels a t 1 mg PFOA/kg (p < 0 .0 5 ); no other dose groups showed a significant increase. This is sim ilar to the effect seen in in tact anim als, and was significant at the same dose. Spleen w eight (data not show n) and relative spleen w eigh t in ovx anim als was highly variable at 18 m onths, and showed decreases, albeit not highly significant, at the 1 and 5 mg PFOA/kg doses (p -0 .0 6 and p = 0 .0 5 , respectively; Table 1). 1 and 3 m g PFOA/kg (not 5m g /kg) w ere the doses in the intact anim als showing the largest decreases in relative spleen w eight compared to controls. Finally, relative liver w eigh t show ed no significant differences across dose groups w hen com pared to ovx control. 3.1.7. L a ck o f e ffects fr o m a d u lt PFOA exposure A t 18 m onths o f age. body and tissue w eights w ere recorded in adult PFOA-exposed m ice. A dult PFOA exposure had no effect on term in al body or organ w eights. W hen a comparison o f data from 18-m onth-old adult intact and developm entally exposed ani m als in th e 0, 1 and 5 mg PFOA/kg dose groups was m ade, body w eight, brow n fat w eight, and w h ite fat w eigh t o f the 1 mg PFOA/kg developm entally exposed anim als w ere significantly higher than the same dose in adult-exposed anim als (data not shown). 4. Discussion These studies dem onstrated the effects o f developm ental PFOA exposure on C D -I fem ale mouse body and organ w eight, as w e ll as serum le p tin and insulin in adulthood. In the developm ental PFOA studies, a dose-dependent dichotom y o f phenotypes was present in intact fem ale mice; latent effects present follow ing high doses w ere not present in m ice exposed to low -dose PFOA and vice versa. Although there was no detectable change in body w eight neonatally, low -dose PFOA exposures (0.01. 0.1, or 0.3 m g PFOA/kg) led to significantly increased m ean w eight and rate o f w eight gain in m id -life (u p to and including 37 weeks o f age) and a coincident sig n ificant elevatio n o f serum lep tin and insulin values betw een 21 and 33 w eeks (0.01 and 0.1 mg PFOA/kg). Our tow -dose horm one data indicate potentially im portant m etabolic changes th at m echanistically support the findings of increased w eight in the low er dose groups. Previous dosim etry w ork in o u r lab has shown th a t in utero exposure to PFOA in the mouse translates into an extended developm ental exposure period via lactational exposure ( all o f gestation and nearly 3 months postnatally; W h ite et al.. 2009; W o lf et al., 2007: Fenton et al.. 2009). This long exposure may lead to reprogram m ing/m etabolic events th at govern fat m etabolism o r appetite control. Although we w ere unable to perform some o f the other end points o f interest dur ing this tim e period o f greatest w eight gain, our findings relating leptin and insulin concentrations to the tim e o foverw eight in PFOAexposed m ice support our theory. O ther environm ental chemicals, term ed environm ental obesogens (dietylstibestrol (DES). 2 OH-E2 , 4 OH-E2 . genestein and bispheno! A ), have been shown to induce obesity in adulthood after low -dose developm ental exposure,w hile inducing w eigh t loss at higher doses (G riin et al., 2006; Newbold e t al., 2 0 0 5; M iyaw aki et al.. 2 0 0 7 ) and are review ed furth er w ith in this issue. Serum leptin was significantly elevated in m id -life in the low dose PFOA-exposed groups. This effect occurred at the same PFOA dose range as overw eight in these anim als, congruent w ith a leptin-resistance mechanism o faction for overw eight,as previously reported in humans (Considine et al.. 1996). Others have reported increased leptin w ith developm ental exposure to environm ental obesogens including DES (New bold e t al., 2007). Low-dose (0.01 and 0.1 m g PFOA/kg) developm ental PFOA expo sure that led to increased serum leptin and body w eight also increased insulin values a t 2 1 -3 3 weeks. This suggests th at the insulin resistance mechanistic pathway could also be affected and play a role in developm ental PFOA exposure-induced overweight in mice. In an insulin resistance scenario, there are raised plasma glucose levels (elevated, but not significant, at 1 5-16 weeks in our study), reflecting the loss o f a post-challenge peak in insulin response (review ed in Montecucco et al., 2008). Insulin resistance is known to be associated w ith excess abdom inal fat hi norm al and overw eight wom en (Ca rey e t al.. 1996). High plasma levels o finsulin and glucose, due to insulin resistance, are often associated w ith type II diabetes and m etabolic syndrome in humans, and thus this effect of low -dose PFOA developm ental exposure and its association w ith increased serum insulin are im portant. The ovx data w ere d ifficu lt to interpret. The lack o f additional w eight gain w ith developm ental PFOA and ovx m ay reflect a ' ceil ing effect* o r th at ovx-induced w eight increases may have masked any effect o f PFOA. A lternatively, as w eight gain and m etabolic hor mones can be regulated by estrogens, the role o f the ovaries in developm ental effects o f PFOA was explored by using ovx anim als. The potential im portance o f the ovary in the effects o f PFOA was based on the observation th at LH-transgenic (overexpressing) m ice (Kero et a l, 2 0 0 3 ) w ere phenotypically sim ilar to ours (increased body w eight, increased brown fat depots, and predom inant ovarian cysts not discussed in this paper). W e hypothesized th at removal of the LH target (th e ovary) in o u t study may reveal the mode of action for PFOAefTects for the increase in brown fat and possibly the exces sive w eight gain. Ovx anim als typically gain body w eight in excess vs. intact anim als (Kam ei et al., 2005). The critical role o f the ovary in w eight gain o f intact PFOA-exposed females beyond that o f ovx treatm ent-m atched siblings in the 0.01 and 0.1 mg PFOA/kg groups was novel and signifies the ovarian axis as a potential m ediator of PFOA-dependent m id -life w eight changes. Another potential m ediator o f these intertw ined low-dose PFOA-induced effects is the peroxisome proliferator-activated receptor (PPAR) activation pathway. PPAR gamma (PPAR-y) and PPAR alpha (PPAR-a) are involved in lipid m etabolism in adipocytes and liver/skeletal muscle, respectively (review ed in M edina-Gom ez et a l, 2007; Abbott. 2 0 0 9 ). These PPAR isoforms are known to influence lipogenesis/w eight gain and have been shown to be regu lated by environm ental compounds such as trib u tyltin (G rn et a l, 2006; review ed in this issue) W eight loss events in leptin-deficient. obese, and insulin-resistant mouse models have coincided w ith PPAR-regulated changes in gene expression (Holvoet. 2008). A dow n-regulation o f PPAR isoforms involved in energy expenditure, lipogenesis or fatty acid synthesis have been reported in adipose and skeletal muscle o f ovariectom ized mice (Kam ei et aL. 2005). PFOA has been shown to be a PPAR activator in liver tissue (high doses) and cell lines, and to be required for PFOA-induced devel opm ental toxicity in mice (Takacs and Abbott. 2007; Abbott et a l, 2007; Abbott, 2009). If PPAR activation via receptor binding is a prim ary m ode o f action for body w eight effects follow ing PFOA exposure, the decrease in the PPAR receptors follow ing ovariectom y and decreased circulating estrogens may explain the lack o f effect of PFOA in ovx m ice. However. PFOA-induced consequences of PPAR activation follow ing a developm ental exposure are just beginning to be evaluated. After 4 0 weeks o f postnatal age. an increase in m ortality was detected in all anim als. There are previous reports in the literature of increased m o rtality in non-treated C D -I mice, attributed prim r- 104 E f. Hines e t o t / Molecular a m i Cellular Endocrinology 304 (2 0 0 9 ) 97-105 ily to thym ic lymphomas (Son, 2 0 0 4 : Taddesse-Heath et al.. 2 0 0 0). Because o f th is confounding circum stance, repeated measures of body w eight w ere only follow ed out to 37 weeks o f age. The o th er h alf o f the phenotypic dichotom y caused by devel opm ental PFOA exposure was also novel. Developm ental exposure to higher doses o f PFOA (1. 3 and 5m gP F0A /kg) led to a vastly d iffe re n t phenotype from low -dose PFOA exposure. This effective PFOA dose dichotom y may m anifest its e lf in our study via unique modes o faction; th e anim als w ith highest dose(s) o fdevelopm ental PFOA exposure have decreased early life body w eight and term inal body w eigh t (5 mg PFOA/kg) w ith significant decreases in w hite fat w eigh t at 18 months (1 and 5 m g PFOA/kg), significant increases in brow n adipose (1 and 3 m g PFOA/kg), and significant decreases in spleen w eight (3 mg PFOA/kg) findings th at are absent w ith the low er doses o f PFOA. Others have reported dose-dependent loss o f w hite tis sue adiposity in adu lt m ale m ice a fte r PFOA exposure (0.02% PFOA w eight/chow w eight, w hich translated to approxim ately 32 mg PFOA/kg BW d aily ) w ith fat loss, w ith o u t fat cell num ber loss, th a t is PPAR-y-independent w ith P-adrenergic activation (Xie et a l, 2 0 0 2 ). In th at same study, investigators also reported w hite fa t and body w eight decrem ents at higher doses th a t w ere absent a t low er doses. Yang et al. (2 0 0 2 ) showed PFOA-dependent w eight loss was abrogated in PPAR-a n u ll m ice, indicating th a t PPAR-a is a probable regulator o f w eigh t loss in the high dose anim als. In subsequent studies, X ie e t al. (2 0 0 3 ) showed th at a fter cessation o f exposure o f adu lt m ale anim als to PFOA (03)2% PFOA w eight/chow w eigh t. 32 m g PFOA/kg BW ) d aily fo r 7 days follow ed by 10 days recovery, w eight loss and w h ite adipose levels returned to base lin e . w hich confirm s th e im portance o f developm ental exposures fo r the laten t effects reported here. In our m odel w ith developm en ta l PFOA exposure w e see perm anent w eight loss and w h ite adipose tissue loss a t the high dose o f PFOA. How ever, there m ay be m erit in fu rth er exploring these m echanisms o f action, as P-adrenergic receptor upregulation is also associated w ith increased brown fat mass in w inter-acclim ated anim als (Feist, 1983), and this tissue was associated w ith high dose (and not low dose) effects in both intact and ovx anim als in this study. Although w e suspected alleviation o f effect in th e brown fat pad by elim inating the ovary (based on phenotypes in Kero et a l, 2 0 0 3 ). significant increases in brown fat w ere seen a t 1 mg PFOA/kg in both intact and ovx anim als. At the 18-m onth tim e point, some endpoints rem ained unchanged across dose groups including liv e r size. E arlier w ork has shown significant hepatom egaly a fte r developm ental PFOA expo sure (1 and 3m gPFO A/kg) observed out to at least 3 weeks after b irth (th e latest tim e point evaluated; W o lf e t a l, 2007; W h ite et aI , 2007). The transient nature o f hepatom egaly has been illustrated in other acute adult exposure studies (review ed by Lau et a l, 2007), and is fu rth e r confirm ed in these studies (in tact and ovx). A final im portant com ponent o f these studies evaluated adult vs. developm ental exposure to PFOA on body tissue w eights. These data suggest that the tim ing o f dosing (ad u lt vs. developm ental 17-day PFOA exposure) was critical fo r laten t effects. There was no effect o f 17-day adult PFOA exposure on any endpoint in this study (early life or la te n t) w hen com pared to age-m atched, vehiclegavaged controls. In conclusion, the tim ing and dose o f PFOA exposure for induc tion o f dichotom ous, persistent, adult health effects in C D -I fem ale m ice are critical. Developm ental, low -dose PFOA exposure led to increased w eight in adults, w ith increased serum insulin and leptin , a health effect not seen in high dose anim als. No observable adverse effect levels (NOAEL) fo r body w eight gain, serum leptin and insulin concentrations w ere not determ ined in this study; but 0.01 mg PFOA/kg had a significant im pact on these particularly sen sitive end points. The ovary appeared to play an im portant role in the overw eight effect in m id -life , and it is proposed that there is a com mon m ode o f action, potentially dysrgulation o f PPAR and its signaling through ovarian hormones, that m ay be responsible for these low -dose health effects. Further studies addressing long term PFOA-induced health outcomes in mice should focus attention on internal dose relative to th e low-dose health effects seen in this study, as w ell as the mechanisms o f action, so th at any relevance to hum an health effects can be addressed. A cknow ledgem ents W e w ould like to thank Broker Optics. Inc. for th e use o f the Broker M inispec mq 7.5 LF50 Live Mouse Analyzer and H arry Xie and Basil Desousa o f Broker Optics. In c for th e ir technical assis tance. W e w ould lik e to acknowledge Antonia Calafat and her labo ratory stafT, Kayoko Kato and Zsuzsanna Kuklenyik, in the Division o f Laboratory Science. National Center for Environm ental Health. Centers for Disease Control and Prevention for the analysis o f seram PFOA concentration from 18-m onth-old developm ental^ exposed fem ale m ice; Donald Doerfler, Experim ental Toxicology Division. U.S. EPA. and Judy Schmid, Reproductive Toxicology Division (R ID ). US. EPA for th e ir statistical support: Deborah Best, RTD.for conduct ing the estradiol assays; Veronica Luzzi. David Gibson and stafT at the Core Laboratory fo r Clinical Studies a t W ashington University in S t Louis. M O , fo r perform ing the serum insulin assays, and finally. D r. David Kurtz and the technical staff at New Year Tech, In c for th e ir exceptional anim al care during these lengthy studies Thanks to Retha New bold. NIEHS.and Rob Ellis-Hutchings. Dow Chemical. M idland. M I. for th e ir constructive input on this m anuscript. References Abbott BjD.. 2009. 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