Document 8VYDwVbq0VqxB80x0kMO01QZm

P-1 "McCrea, Deborah" <mccrea@taftlaw.com> 06/12/2009 03:54 PM To NCIC OPPT@EPA cc "Bilott, Robert A." <bilott@taftlaw.com> bcc Subject 06/12/2009 Letter To EPA Docket Center Taft / Deborah McCrea / Legal Assistant Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1800 Cincinnati, Ohio 45202-3957 Tel: 513.381.2838 Fax: 513.381.0205 www.taftlaw.com / mccrea@taftlaw.com C`.> V.-3 c ,--5 r - 'rv> Internal Revenue Service Circular 230 Disclosure: A s provided for in Treasury regulations, advice (if any) relating to federal taxes that is contained in this communication (including attachments) is not intended or written to be used, and cannot be used, for the purpose of (1) avoiding penalties under the Internal Revenue Code or (2) promoting, marketing or recommending to another party any transaction or matter addressed herein. This m essage may contain information that is attorney-client privileged, attorney work product or otherwise confidential. If you are not an intended recipient, use and disclosure of this m essage are prohibited. If you received this transm ission in error, please notify the sender by reply e-mail and delete the m essage and any attachments. From: canoncopy20a@taftlaw.com [mailto:canoncopy20a@taftlaw.com] Sent: Friday, June 12, 2009 3:56 PM To: McCrea, Deborah Subject: 06/12/2009 Letter To EPA Docket Center 2979_001.pdf CONTAINS NO CBI contains n cbi . ' i. *': V V* C t > P-3 Taft/ Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1 8 0 0 /Cincinnati, OH 45202-3957 /Tel: 5 1 3 .3 8 1 .2 8 3 8 /Fax: 5 1 3 .381.0205 / www.taftlaw.com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix / Beijing Robert A. Bilott 513-357-9638 June 12, 2009 FEDERAL EXPRESS E P A Docket Center, M C 2822T U.S. Environmental Protection Agency EP A West, Room 3334 1301 Constitution Avenue, NW W ashington, D.C. 20004 Re: Subm ission to IRIS and AR-226 Database For PFO A/PFO S: EPA-HQ - O R D -20 03-0016 ____ __________ To IRIS Database for PFO A/PFO S: In response to the Notice issued by U S E P A on February 23, 2006, regarding U S E P A 's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("P F O S ") within the Integrated R isk Information System ("IRIS"), 71 Fed. Reg. 9333-9336 (Feb. 23, 2006), we are submitting the following additional information to U S E P A for inclusion in that review, and for inclusion in the AR-226 database: 1. Fenton, S.E., et al., "Analysis of PFO A in Dosed CD-1 M ice Part 2: Disposition of PFO A in Tissues and Fluids From Pregnant and Lactating M ice and Their Pups," Reprod. Toxicol. (2009), doi:10.1016/j.reprotox.2009.02.012; 2. von Ehrenstein, O.S., et al., "Polyfluoroalkyl Chem icals in the Serum and Milk of Breastfeeding Women," Reprod. Toxicol. (2009), doi:10.1016/j.reprotox.2009.03.001; and 3. Hines, E.P., et al., "Phenotypic Dichotomy Following Developmental Exposure to Perfluorooctanoic Acid (PFO A) in Fem ale CD-1 Mice: Low Doses Induce Elevated Serum Leptin and Insulin, and Overweight in MidLife," 304 Molecular & Cellular Endocrinology 97-105 (2009). 11434742.1 CONTAINS NO CBf June 12,2009 Page 2 RABrmdm Enclosure cc: Gloria Post (NJDEP)(w/ end.) (via U.S. Mail) Helen Goeden (MDH)(w/ end.) (via U.S. Mail) Lora W erner (ATSDR)(w/ end.) (via U.S. Mail) p.4 {W1405808.1} Accepted Manuscript Title: Analysis of PFOA in Dosed CD-1 Mice Part 2: Disposition o f PFOA in tissues and fluids from pregnant and lactating mice and their pups Rcpnxti'ctrx Tpicotaiy Authors: Suzanne E. Fenton, Jessica L. Reiner, Shoji F. Nakayama, Amy D. Delinsky, Jason P. Stanko, Erin P. Hines, Sally S. White, Andrew B. Lindstrom, Mark J. Strynar, Syrago-Styliani E. Petropoulou PE: DOI: Reference: S0890-6238(09)00040-9 <kri:10.1016/j.reprotox.2009.02.012 RTX6230 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 of 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 of 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. 1 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. Delinskyc, Jason P. Stanko*, Erin P. Hines*, Sally S. White*''1, Andrew B. Lindstrom0, Mark J. Strynar*, and Syrago-Styliani E. Petropoulou" *Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. EPA, MD-67, Research Triangle Park, NC 27711, USA bOakridge Institutefo r Science and Education (ORISE)Research Participant, Human Exposure and Atmospheric Sciences Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, NC 27711, USA cHuman Exposure andAtmospheric Sciences Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, NC 27711, USA i Curriculum in Toxicology, University o fNorth Carolina, Chapel Hill, NC 27599, USA ^Current address; Division o fLaboratory Sciences, National C enterfor 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.suzannc@eoa.gov 2/20/2009 Page 1 of 32 Running title: PFOA disposition in lactation Abbreviations ANOVA analysis o f variance BW body weight GD gestational day LOD limit of detection LOQ limit o f quantitation MS PFAA mass spectrometer periluoroalkyl acid PFOA perflnorooctanoic acid PFOS perfluorooctane sulfonate PND postnatal day SEM standard error of the mean UPLC ultra performance liquid chromatography 2 2/20/2009 P-8 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 2720/2009 Page 3 of 32 P-9 4 1 1. Introduction 2 Perfluorooctanoic acid (PFOA) is a member o f the perfluoioalkyl acid (PFAA) 3 family o f man-made, fhiormated organic compounds used in a number o f 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 repellents for clothing, 6 carpeting, and upholstery, and the stability o f the carbon-fluorine bond have made them 7 ubiquitous in die environment The predominant mute of exposure in North American 8 and European consumers is likely oral intake, including drinking water, while inhalation 9 and dermal absorption comprise routes of lesser exposure [1-S). 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 o f serum 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 [4]. 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 [1], PFAAs arc 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 periluorooctane sulfonate (PFOS) and 25% 21 in blood PFOA levels between the years 2000 and 2006[10). However, while the 22 production o f 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 perihiorinated compounds produced 2720/2009 Page 4 of 32 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 o f die recent health effects research on PFOA in mice, commonly 4 associated with gestational exposures of 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, W olf 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 lactatioaally exposed. Furthermore, recent studies ( 15] have shown that 19 unexposed neonates lactationally (posed to PFOA quickly developed mammary gland 20 growth deficits and that control dams nursing in urero-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. 11 6 1 results support a role for impaired lactational development and possibly a significant 2 lactational transfer o f 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 at. {17] indicated 6 a range o f 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 o f 12 human milk samples collected 8 from 1996-2004 in Sweden 8t a concentration o f492 pg/ml (0.492 ng/ml; [18], and a 9 mean o f 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 o f 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 m the Kamnan 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 m aybe 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 o f 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 o f placental and lactational transport of PFOA in rats[22]. 22 Although female rats are known to have a serum PFOA half-life o f only a few hours [23], 23 unlike mice which have a Vi-life o f about 15 days [13], the study [22] indicated 2/20/2009 Page 6 of 32 p. 12 ffla m s m m s t 7 1 concentrations in rat milk approximately 10 times less than that o f maternal plasma and 2 that the milk concentrations were generally o f the same magnitude as the concentrations 3 in pup plasma. 4 The increasing amount o f research confirming the developmental toxicity of 5 PFAAs in animal studies, coupled with their detection m human cord blood and milk, 6 supports tile 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/nconatal 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 of PFOA in pregnant and lactating mice or their offspring. We recently 11 developed an analytical method for the analysis o f 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 of 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 of 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 8 1 2. Materials and methods 2 2 .1 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 o f 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 die National Health and 10 Environmental Effects Research Laboratory Institutional Animal Care and Use 11 Committee. Confirmed timed pregnant female CD-I mice (n=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 o f 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 (w=25/dose group) received either water 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 GD17. 23 2/20/2009 Page 8 of 32 p. 14 9 1 2.3 Animal Assessments and Sample Collection 2 Live dam body weights were recorded on GD17, GD18 (prior to parturition), 3 PND1 (day after parturition), and PNDs 2,4, 8 ,1 1, 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 IS 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 !7 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 day22 Milk collection was attempted, following administration o f oxytocin ( lU/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 'V/' ,-J.*i,-',i.-v:':<.*it Ax` ?i*'"*.''.i-n'ivV01i p. 15 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 o f 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 o f serum and amniotic fluid (50 pL used for controls), 20 pL aliquots o f 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 o f the prepared sample, depending on 16 the concentration o f 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 o f the methods used. The limit of 19 quantitation (LOQ) for these experiments were 5 ng/ml (serum), 1 ng/ml (amniotic fluid, 20 urine, milk), and 1 ng/g (whole pups, mammary tissue). 21 22 23 2/20/2009 Page tO of 32 p. 16 11 1 2.5 Urinary creatinine 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 o f 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 o f 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 Compulations and Statistics 12 Reported PFOA concentrations have been 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 /K0.05. 19 20 21 22 2/20/2009 Page 11 of 32 12 1 3. Results 2 3.1 Biological Outcomes 3 This is tiie first study to report single dose disposition of PFOA in pregnant and 4 lactating mice and their offspring. The doses chosen were based cm previous reports in 5 CD-I mice [3,14,16] demonstrating developmental health outcomes following multiple 6 gestational PFOA exposures. A single PFOA exposure on GD17 did not affect the 7 number o f live fetuses (an GDI 8), implantation sites, or live-born paps (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 liverBW ratios, within the PFOA dose range administered in this study (Figure I). The 11 rise in dam liverBW ratio between GD18 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 o f PFOA detected in dam serum was about twice the amniotic 20 fluid concenttation 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 tire 22 amount o f PFOA in an entire GDI 8 fetus (body burden/pup+standard error o f the mean 23 [SEM]; Figure 5) to the GD18 PFOA concentration in amniotic fluid (ng/mi; assuming I 2120/2009 Page 12 o f 32 p. 18 l H H 33 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 D am 5 The serum and urine PFOA concentrations were evaluated in dams that were 6 nursing litters o f approximately 10 pups (PND1 equalized; minimal pup loss over time). 7 As expected, dam sera contained the highest PFOA concentrations of 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 o f lactation (i.e., mean 9.9-fbId 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) o f 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 PND18, 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 o f 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 o f PFOA (urine) was not as pronounced as that o f serum, mammary tissue 2 demonstrated a strong U-shaped response, with the lowest concentrations measured near 3 the peak o f lactation, and a significant rise in concentration apparent again at PND 18 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 die milk concentrations 8 to die 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 o f dam serum PFOA across dose and time. 10 It appeared that the day o f 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 milk:scrum ratio ranged from 14 11-27% (mean 17.7%). It was not possible to accurately measure the volume o f milk 15 obtained at aspiration, but precise weights were compared. On PNDs 2,8 , II, and 18, the 16 average weight o f milk obtained via aspiration o f control mice was 0.072,0.1906, 17 0.2547, and 0.0457 g, respectively, demonstrating over a 3.5-fbld 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 scrum < LOQ), it appeared that circulating 2/20/2009 Page 14 of 32 p. 20 15 1 pup serum PFOA concentrations were significantly higher than those measured in dams, 2 regardless o f 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 PND 18 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 o f 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 o f 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-fo)d (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. 21 16 1 4. Discussion 2 These data confirm that on a concentration-based comparison, gestationally 3 PFOA-exposed pups exhibited a significantly larger serum PFOA load than their dam. 4 That substantial 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 o f 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 o f measure, an inverse U-shaped 10 curve was apparent, and the PFOA burden o f 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 bydro-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 Mood 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-lactatmg rats [27], and in humans, serum 2/20/2009 Page 16 of 32 p. 22 17 1 albumin concentration decreases during pregnancy and early lactation [28j. 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 o f 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 p lain the decrease in milk PFOA concentration seen between PND2 and PND11. 10 PFOA also appears to concentrate in serum and milk near.theend o f lactation (PND18, 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 fail 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 o f milk collected between the peak o f lactation 16 and PND18 was noted, indicating a rapid decrease in milk volume. Therefore, the U17 shape o f 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 o f body burden in the pup over this life stage. 22 A significant contribution o f 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 p. 23 rasjijj3 t 18 1 that the dam PFOA milk.serum distribution ratio was 0.1 and 0.01, respectively. In the 2 present study, the distribution ratio ranged from slightly mote than 0.5 to 0.1 in mice, 3 depending on dose, with the lowest doses tested demonstrating the highest ratios over 4 time. I f the milk PFOA concentrations had been measured near the peak o f lactation only 5 (days 8-11), the 0.1 milk:sera 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 o f increased milk:serum ratios appeared, regardless o f 8 dose, with a substantial peak m milk PFOA concentrations on PND2. Although volumes 9 o f 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 PND1 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-I mouse litters gestationally exposed to 3 mg/kg PFOA and higher. In fact, in a 1 & 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 o f 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. 24 '*>'>'-*.1-.'*l1`S**3*.3"JVp''?-'< 19 1 outcomes suggest that the milk o f gestationaliy 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 die 3 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 milk:serum relationship of PFOA transfer [18]. The reported 0.01 15 (1/100lb) 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 die 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 o f 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 p. 25 20 1 circulating serum PFOA concentrations o f 44-216 ng/ml in dams and 117-326 og/ml in 2 pups; values similar to or lower than serum PFOA concentrations o f children that were 3 accidentally exposed via DuPont production plant emission [34], Because o f 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 o f this manuscript. We acknowledge the excellent 17 care o f these animals by New Year Tech, Inc. (Restin, VA). The research in this article 18 has been reviewed by die National Health and Environmental Effects Research 19 Laboratory, US Environmental Protection Agency (EPA), and approved for publication. 20 Findings in this report are those o f 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. 26 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. Rutenhoff, 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 chemistries' 8 -toxicokinetics and modes o f 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. Froeblich, A.M. Seacat, J.L. 15 Butenhoff and L.R. Zobel, Half-life o f serum elimination o f perfluorooctanesulfonate, 16 perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemieal production 17 workers, Environ Health Perspect, 115 (2007), 1298-1305. 18 19 [5] D. Trudel, L. Horowitz, M. Wormutb, M. Scheringer, I.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. Taniyasu and N. 24 Yamashita, Species-specific concentrations of perfluoroalkyl contaminants in farm and 25 pet animals in Japan, Chemosphere, 73 (2008), S210-215. 26 27 [7] G.W. Oisen, H.Y. Huang, K J. Helzlsouer, K.J. Hansen, J.L. 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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 p. 27 f*W f2^ g l8S i* ; s.\ 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 JJd. Rogers, The developmental toxicity o f 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, AM . Calafat, Z. Kuklenyik, L. Villanueva, R.D. Zehr, L. Helfent, 14 MJ. Strynar, A.B. Lindstrom, J.R. Thibodeaux, C. Wood and S.E. Fenton, Gestational 15 PFOA exposure o f 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. CalafaVE.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 resultmg 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, JJ*. 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 o f perfluorinated chemicals through lactation: levels 34 o f 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] B.J. Apelberg, F.R. Witter, J.B. Herbstman, A.M. Calafat, R.U. Halden, L.L. 42 Needham and L.R. Goldman, Cord serum concentrations o f perfluorooctane sulfonate 43 (PFOS) and perfluorooctanoate (PFOA) m relation to weight and size at birth, Environ 44 Health Perspect, 115 (2007), 1670-1676. 45 2/20/2009 Page 22 of 32 p. 28 23 1 [21) C. Fei, J.K. McLaughlin, RJE. Tarone and J. Olsen, Fetal grow* indicators and 2 perfluorinated chemicals: a study in the Danish National Bird) Cohort, Am J Epidemiol, 3 168 (2008), 66-72. 4 5 [22] P.M. Hinderliter, . Mylchreest, S.A. Gannon, J.L. Butenhoff and G.L. Kennedy, 6 Jr., Perfluorooctanoate: Placenta] and lactational transport pharmacokinetics in rats, 7 Toxicology, 211 (2005), 139-148. 8 9 [23] JJ*. Vanden Heuvel, B.I. Kuslikis, MJ . Van Rafelghem and R.E. Peterson, Tissue 10 distribution, metabolism, and elimination o f perfluorooctanoic acid in male and female 11 rats, JBiochem 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 o f PFOA in dosed CD1 mice: Part 1. Methods 15 development for the analysis o f 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 2 1 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 M ed Sci, 55 (1993), 561 -564. 28 29 [28] M. Dean, B. Stock, RJf. Patterson and G. Levy, Serum protein binding of drugs 30 during and after pregnancy in humans, Clin Pharmacol Ther, 28 (1980), 253-26!. 31 32 [29] C.S. Jones and D.S. Parker, Mammaiy blood flow and cardiac output during 33 initiated involution o f 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 Perspect, 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 o f a pilot study, IntJH yg 46 Environ Health, 211 (2008), 440-446. 2/20/2009 Page 23 of 32 24 1 2 [33] O.S. vod Ehrenstein, Fenton, Suzanne E., Kato, Kayoko, Kukleayik, Zsuzsanna, 3 Calafet, Antonia M., Hines, Erin P ., Polyfhioroallcyl Chemicals in the 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 perfluorooctanoate: relationships between serum concentrations 8 and exposure sources, J Occup Environ Afed, 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 (FFOA) 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. 30 25 1 Figure legends: 2 Figure 1. Dam tissue weights and average pup weights following a single gavage PFOA 3 exposure on GD17. 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) liver:body weight ratio, and (C) pup 6 body weight over time or numbers o f live pups or fetuses (not shown) were1also 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 o f PFOA to pups. (A) A significantly higher PFOA 22 concentration in pup vs. dam serum on PNDl 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 26 1 declined over time, presumably due to dilution o f dose by increased growth-related blood 2 volume. Data are shown as Mean SEM. 3 Figure S. 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 o f exposure until 9 late in the lactational period. Data are shown as Mean SEM. 10 11 12 2/20/2009 Page 26 of 32 Figure 1. (A) Dam Liver Weight p. 32 Control 0.1 1 PFO A Exposure (mg/kg) 5 Control 0.1 1 PFO A Exposure (mg/kg) (C) Pup Body Weight 5 o> & i Control 0.1 1 PFOA Exposure (mg/kg) GD18 PND1 E3PND4 BPND8 PND18 5 Page 27 of 32 p. 33 Figure 2. 0.1 1 5 PFOA Exposure (mg/kg) Page 28 of 32 Figure 3. (A) Dam Serum PFOA PF O A (ng /m l) 0011 PH01 PM04 PND*PND1* 0D1I PMDI PMD4 PNO*PNDl* (B) Dam Urine PFOA GDI* PND1 PND4 FNDSPWH PFOA (ng/g creatinine) PN01 PND2 PND* PMDtPM>1* PNDt PND7 PNO PND*PND18 (C) Dam Mammary Gland PFOA PFOA{ng/g) PFOA (ng/ml) (D) Oam Milk PFOA PND3 R ID I PND11 PND18 Time P N 02 PM U PMD11 PND18 Page 29 of 32 p. 35 '9 itV Serum PFOA (ng/ml) Figure 4. 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 (A) Dam vs. Pup Serum PFOA 0 0.1 1 PFOA Exposure (mg/kg) (B) Pup Serum PFOA Over Time 5 1 4 8 18 1 4 8 18 Postnatal Day (PND ) 4 8 18 Page 30 of 32 Body Burden PFOA (ng) Body Burden PFOA (ng) Figure 5. PFOA(ng/g) PFOA(ng/g) 100008000' 5 mg/kg 6000 I 4000 2000 GDIS 1 4 8 18 2000 II GD18 1 4 8 18 20000 16000 12000 8000 4000 5000 1500 4000 3000 2000 1000 GDIS 1 GD18 1 8 18 400 300 200 100 PFOA (ng/g) Body Burden PFOA (ng) GD18 1 4 8 18 GD18 1 4 8 18 Gestation (GD) and Postnatal (PND) Day of PFOA Measurements Page 31 of 32 Table* IS S S S liililS S ^ ^ ^r'-iip^vi{s>5*^}iii-^a}rt.ss|5|Hif:ei^i<`miiJ',...;'.;,^jl"5.,.ff!?!ii..-.v..,;!!;J,,.ii.i.j.si,.5A.C'.'.!ii- -'!' ' l ,`J . "*&.`^ i ^ a w ^ !n^gW''*>^it8.w^H araM wwBw Table 1. Milk-borne PFOA* as a percentage of dam serum concentrations over lactation. Single GDI7 PFOA exposure PND 1 serum PFOA comparison PND 4 serum PFOA comparison PND 2 m ilk PND 8 serum PFOA comparison PND 8 m ilk PND 8 serum PFOA comparison PND 11 m ilk PND 18 serum PFOA comparison PND 18 m ilk 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, GOgestatlonal day, PND=postnatal day. The mllkiserum PPOA 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. p. 37 e 32 of 32 p. 38 BiJF-VIHR ReproductiveToxicologyxxx(2009) xxx-xxx Contents lists.available at ScienceDirect Reproductive Toxicology jo u rn a l hom epage: w w w .e ls e v ie r.c o m /lo e a ie /re p ro to x , ci Polyfluoroalkyl chemicals in the serum and milk of breastfeeding women , O ndine S. von Ehrenstein **, Suzanne E. Fenton b, Kayoko Katoc, Zsuzsanna Kuklenyikc, a A ntonia M. Calafatc, Erin P. H ines1*'1 4 JUCLA S ch o o l o jP stb lk H ealth. Universityo f C ttttfarnla, la s A ngeles. CA. UnitedStores $ us e n v iro n m en ta l P rotection A gency. OKD. NH EEItt, R eproductive Toxicology CMvitfen, RIP.NC. UnitedStores s ` C entersfo r D isease C ontrol BePrevention. D ivision ofU rbom teey Science. M atronalC enterfu r E nvironm ental H ealth A tla n ta . CA, UnitedStores ARTI CLE INFO ^ ______ iv Article M tto ry. n Received28Januaiy2009 is Receivedin revisedform27February2009 is Accepted2 March2009 m Availableonlinexxx is ____________________________ is Keywords: <7 PotyOuoroalkylchemicals is Perftuoroalkyt acids is Perfluorooctanoicacid 20 Perlluoroocianesulfonicacid ei Serum 22 Breastmilk 23 lactation ABSTRACT Potyfluoroaikyl chemicals {PFCs)comprise a group ofman-made organic compounds, some ofwhich are persistent contaminants with developmental toxicity shown in laboratory animals. There is a paucity of human perinatal exposure data. The USERAconducted a pilot study (Methods Advancement in Milk Analysis)including 34 breastfeeding women in North Carolina. Milkand serum samples were collected at 2-7 weeksand 3-4 months postpartum: 9 PFCswere assessed in milkand 7 in serum. Periluorooctane sulfonicacid (PFOSkperfluorooctanoic acid(PFOA).periluorononanoicarid (PFNA).and perfhtorohexane sulfonic add (PFHxS)were found in nearly 100%ofthe serum samples. PF05and PFOAwere found at the highest concentrations.PFCswere below the limit ofdetection in most milksamples.Serum concentra tions ofPFOS.PFOAand PFHxSwere lower(p<0.01)at the second visit compared to the first visit Living in North Carolina lOyears or longer was related to elevated PFOS.PFOAand PFNA(p <0.03).These pilot data supportthe need to further explore perinatal PFCexposuresandpotentially related health effects,as planned in the upcoming NationalChildren'sStudywhich provided the framework for this investigation 2009 Published by Elsevier Inc. 1. Introduction 2s Polyfluoroalkyl chemicals (PFCs)comprise a large groupo f man2 made fluorinared organic compounds used in numerous consumer 2v products and industrial applications such as food packaging mate2a rial, non-stick cookware, protective coatings for textiles, carpets. and paper, surface car coatings or treatments, as well as in sur3o factants fo r commercial and industrial applications ) 1 ]. PFCs, and si more specifically perfluoroalkyl acids (PFAAs}. have been detected in w ildlife, fish used foT human consumption, and sera of humans 33 in many different geographical areas worldwide (2-19). Nation34 ally representative US sera biomonitoring data in subjects 12 years Abbreviations: Cl, coniidence interval: K9Linterquartile range; 100. limit of detection: LOQ,limit of quantifkatton-lPfaas, petftuoroalkyl adds; PfOSA.perituoroocune sulfonamide: Et-PFOSA-AcOH.2-(N-etlqd-peffIuoroocunesolfbnamido) acetic acid; Me-PFOSA-AcOH. 2-fN-methyl-perflucKDOct>ne sutfonamido) acetic add; PFHxS, perfluorohexane sulfonic add; PFOS, perfluoroociane sulfonic acid: PFOAperfluorooctanoic add; PFNA, perfhtorononanoic add; PFC.polyfluoroalkyl chemicals: WTC, WorldTradeCentei. 02 * Correspondingauthorat: UCLASchoolofPublicHealth,PO9 1772.LosAngc les.CA90095-1772.UnitedSlates.TeL:13102065324; fx: r I 3107941805 E -m ail a id le ss; ovehrennurla.edu (05. vonEhrenstein). 1 Currentaddress: USEnvironmentalProtectionAgency.NationalCenter(orExpo sureAnalysis,EnvironmentalMediaAssessmentCroup.Mai)code*243-01.Research TrianglePark. NC277II. UnitedStates. 0890-023811- seefront mattei C 2009 PublishedbyElsevier Inc. doi:I0.1016j[j.reprotox.2009.03j001 and older demonstrated widespread exposure to perfluorooctane sulfonic add (PFOS). perfluoroocranoic add (PFOA), and perfluorononanoic add (PFNA) during the last decade {20,211. Exposures of lactating women and young children to PFCs have not been frequently studied, although a number of animal and recent human studies have suggested transfer to breast milk and across the placental barrier (22-26). Developmental and reproductive health effects in animals, including reduced birth weight and gestational length,developmental delays and structural defects especially in relation to PFOA and PFOSexposure have increasingly raised concerns, although the developmental toxidty in laboratory animals was shownat doses 100-500 times orthose seen in human sera (2^7-29). Someexposure assessmentsin cord blood suggested that PFAAs can also cross the placental barrier in humans (30,31 ]. Apelberg et al. [23| recently reported average cord blood concennations of4.9ng/m l (PFOS)and 1.6ng/m! (PFO A )(ti-299), while Spliethoffet aL, reported the detection ofPFAAs in new bom blood spots confirming the transfer of PFAAs in utero J32). In two recent epidemiological studies, PFAAcord blood concennations were related to anthropometric indicators of fetal growth at birth, and maternal pregnancy serum PFAAconcentrations were associated with child birth weight [22,24], Based on the Danish National Birth Cohort, inverse associations were reported between gestational PFOA exposure and birth weight while no effects were reported for markers of fetal growth at birth, or postnatal developmental milestones (24,33). ss w & re <o *' re m re # <> so 51 a * m si u p. 39 A s :*;/? 2 0.S. va n E hrenstein es o / R eproductive Toxicology XXX (2 0 0 9 ) x x x -x x x 61 Data on human milk PFC concentrations are still sparse. The Table! 62 available data based on small samplesizes from China (34}, Sweden Limitsofquantification(LOQ)inmilkandlimitsofdetection(LOD)inserum(ngjml). 63 (35}, Germanyand Hungary |36], suggested detectable levels ofpre Polyfluoroalky)chemicals MIMrtOQ SerumLOD 64 dominantly PFOA and PF0S.The concentrations ofPFOS(131 pg/ml) 2-(N-ethyLperfluciTOOCtane 65 and PFOA (4 3 .8 pg/ml) in 45 milk samples collected in 2004 from Sulfonamido) acetic add 66 women aged 22-43 years residing in Massachusetts have been 2-(fHnthyJ-perfliiorooctane . 6? 66 S reported recently [25]. Studies investigating the partition of PFCs into milk are largely lacking. One earlier study in Sweden ( n - 12) suggested transfer of only about 1%of PFC concentration in serum suMonamido)aceticadd Perfluorobutanesulfonate Perflorodcanoate. mnuofohexaheiubonat(PFHxS) 70 into milk [35 J.Temporal concentration changes in serum or milk of Perf|irohq!g>ro'(pni/)) 71 72 lactating women are unknown, as no study has assessed concen trations in the same woman at two time points during lactation. PerfluorooctaM'ifttainide(PFOSA) Perfiuorooctansulfdbare(FEOS) : Periluorooctanoate(PFOA) 73 To evaluate infant and maternal exposure to PFCs and to a 7* range of other environmental components, as well as to compare * Denotesnot measured inserum. 0.6 060 030 0.60 OJO OJO 0.15 060 OJO 020 OJO * 4.10 OJO 0.05 0l05 DIO 73 concentrations across biological fluids (37], the US Environmen 76 tal Protection Agency (US EPA) conducted a pilot study entitled 77 Methods Advancement for M ilk Analysis (MAMA). This pitot study 2/4. d n a fy sisa fadBc a n d serumfo r PFCs 7 71 60 was carried out to develop reliable collection and analysis methods for the National Children's Study, including 100.000 children from pre-conception to age 2 1 138). We previously reported the MAMA Idserum and milk, we determined the concentrations of PFOS. PFOA, PFNA. PFHxS. perfluorooctane sulfonamide (PFOSA), 2-(N-methyi-perfluorooctiTie sulfonamido)aceticacid (Me-PFOSA-AcOH). 2-(N-etbyi-perfluonooctane sulfonamido) 61 findings regarding phthalates (37) and the biological components aceticadd (Et-PFQSA-AcOH): perfluorobutane sulfonicadd and perfluorodecanok 62 o f human m ilk |39j. add were only measured in milk. The analytical method involved automated solid-phase extraction (SPE) coupled to reversed-phase high performance liquid chromatography(HPLC)-tandem mass spectrometry (MSJMS) Samples were cun 83 2. Materials anil methods in singlets and were re-analyzedonlyif the waterand/or matrix blankswere above 3xlimit ofdetection (LOD).The analytical procedures involving the use ofstan 2 .1 . S tu d y d e sig n a n d p o p u la tio n dards, quality control, and blanks, as well as automated sample extraction were conductedas published previously 143-46).Thesamples frombothvisits were ana as Thedesign or (be EPAMAMAstudy and basic methods hasbeendescribed in lyzedtogetherin Match2006(serora) andNovember2006(milk). 86 detail previously (39). Inbrief.3d healthy,English-speakingbreastfeedingwomen For milk, samplepreparation was conducted usingautomated off-line SPE[43j 67 between ISand 38yearsofagewererecruitedvianewspaperadvertisements, uni One-mlofmilk,towhichweadded 3mlor0.1Mformicacid and SOpf ofinternal versityemailpublications,andfliersdistributedtocliniciansspecializinginwomen's standard solution,was vortex-mixed and sonicated,andplacedona ZymarkRapid O health or pediatricsbyan EMcontractor(Westat Inc. ChapelHill.NOThequestion naire assessment and the collectionof milkandserumspecimenswereconducted TraceStation(ZymaikCorp-Hopkinton.MA).PFCsfromthe milkwereextractedon an Oasis-HLBSPEcolumn(Waters Corporation. Milford. MA). The SPEeluate was 9 at theEM'sHumanStudiesFacilityclinic(ChapelHilt.NC)betweenDecember2004 evaporated at 55`Cto -100pi under a stream ofdry nitrogen (UHPgrade) in a andJuly200$.womenweiebreastfeedingtbeirfirst,secondorthirdchildandwere 2ymarkTurbovapevaporator.andreconstitutedwith300p!of0.It form ic acid.The not requited to exclusively breastfeed for participation in this study. The women reconstituted milk extract(-400pi) was transferred toa polypropyleneautosam- 84 donatedmilkand serumsamplesat 2-7 weeks(1stvisit: it 18milk: n- 34serum], plervlalfortheon-lineSPE-HPLC-MS/MSanalysis.performedusingaSurveyorHPLC 93 and at 3-4 months (2nd visit: n-20 milk: n -30 serum) postpartum. The partic system(ThermoFinnigan,SanJose.CA,USA)includingonesix-portswitchingvalve 06 ipation of human subjects in the MAMAstudy was approved by the Institutional (RheodynrMX7960.Rohnert Park.CA,USA)and oneadditionalSurveyor LCpump, 97 6 0 ReviewBoardsofthe UniversityofNorthCarolina!:SchoolofMedicine(IRBnumber O3-EPA-207)and the Centersfor DiseaseControl and Prevention(IRBnumber3961). Thewomen participated inverbal and written informed consent prior to adminis- coupledwith aThermoFinniganTSQQuantumUltratriple-quadrupole massspec trometer equipped with a heated electrospray ionization(HESi) interface. The HPLC pumpoperatedat a300pl/min flowrale with20mMammoniumacetate(pH4) in >00 trationofacomprehensivequestionnairewhichdidnotincludequestionsregarding water(mobilephaseA)andacetonitrile(mobilephaseB)Theextractwasinjected the offspringofstudy participants (39). incothe liquidchromatographsystem forconcentrationofthe PFCsbyon-tineSPE ona BetasiiC8precolumn(3mm<10mm,5pm:TheimoHypersil-Keystone.Belle- 102 2 .2 . Q u estio n n a ire fonte, M. USA)chromatographic separationon a BetasiiC8analytical HPLCcolumn (2.1mmx 50rom.5pm:ThermoHypersil-Keystone)anddetection andquantifica 103 1W 1QS Aquestionnaire regarding maternal residence, occupation, and dietary and lifestyle factors was administered to participants at the first dink visit. Ques tionnaire items were selected to address potential routes ofexposure to multiple tion by negative-ion HES1-MS/MS. Forserum, we useda modificationof the on-lineSPEcoupled to HPLC-MS/MS approach described before |47) Briefly,weadded 250pi of0.1Mformicacid and 106 107 environmental chemicals (phthalates. phenols. PCBs, dioxins, PFCs. persistent organic pollutants, metals, and brominated flame retardants).The current analy 25pi of internal standard solution to 100pi ofserum, and the spiked serum was vortex-mixed and sonicated.The samples were placedon a Symbiosison-line SPE 106 sis includedthe followingquestionsthatwerethoughttopotentiallyrelateto PFC system(SparkHolland. Plainsboro, NJ)forthe preconcentration oftheanalyteson 100 exposuremutes: "Howlonghaveyoulived in NorthCarolina?"140-42)and Does a Polaris C18 cartridge (7pm, 10mm Imm: Spark Holland) The analytes were 110 yourhomehavean enclosedgarageattached?*.The latterquestionwasselectedas transferredontoaBetasiiCSHPLCcolumn(3mmx 50mm.5pm;ThermoHypersil- 111 112 some applications used inandaround carscontainPFCs,e.g.,externaland intrmal surface car coatings ortreatments. Keystone. BeUefontc. PA),separated byHPLC(mobilephase A: 20mMammonium acetate in water, pH4; mobile phase B: methanol) and detected by negative-ion Ttnbolonspray-MS/MSonanAPI4000massspectrometer(AppliedBiosystems.Fos- terCity.CA). Reportablebreast milkPFCconcentrationscan fallbelow tbe LODdueto H 3 2 .3 . Sam ple c o lle c tio n a n d prep a ra tio n concentrationfactorsthatarepart oftheextractionprotocol.Thuslimitofquantifi- cation(LOQ)(3xLOD)isusedTormilksamplesandLODisusedforallocherbiologic.! 114 The women were asked to fast for U h before sample collection. TheMAMA media,wheresample concentrationis notrequired.TheLODinserumand the LOQ US samplecollection procedures for serum and milkwere published previously (39). Inmilkareshown inTable 1. 116 Samplingdetails, includingtimeofday(between 9AMand 2PM)and theamount 117 ofbodilyfluid collected, were recorded in the collection tog.Milk(90mlor - 302) 118 wasexpressed inthe EPAclinicusingaeommerclallyavailableekciricbreast pump 2.5 Biological m arker analysis MO (Medela.McHenry.II). Milkwaspumpedin to PfC-freebottlesanddividedinto3 ml 120 aliquots in PFC-free polypropylene-tubes, women's blood samples(about 20ml). Selectedbiologiesinmilkandserumwereanalyzedforeachwomailaccordingto 121 were collected into non-heparinized glass vacutainer tubes (Becton Dickinson. LabCorp'sstandardoperating procedures fortheseassaysas previouslyreported in 122 Franklin Lakes. NJ) by an EPAnurse via venipuncture. Alter 1h at roomtempera detail(39).Theassessedendpointswereinmilk: SecretoryimmunoglobulinA,pro 123 tureto allowforclotting,blood samplesWerespunat3000rpmfor ISminat room lactin. tissue necrosis factor- (TNF-cQ.mtericukin-6(1L-6) triglycerides, glucose, 124 temperatureassdserumwascollected.Allsampleswerestoredat-20 Camishipped and estradiol; and in serum: prolactin, immunoglobins. TNF-a, 1L-6, triglycerides, 126 ondryiceto theCDCsDivisionofLaboratorySciences,NationalCenterforEnviron glucose,andestradiol,inrhisinvestigation,themilkandserumconcentrationofthe 176 7 mentalHealth (Atlanta. GA)for analysis. Atthe CDC.ail samples were scored at or below-20' Cuntil analyzed. biological markerswere used toexplore possiblerelationshipswith the detectable PFCs. IZB s 130 131 139 133 1 US 136 137 136 7 140 141 142 149 44 (46 146 47 149 149 50 T91 62 159 154 56 56 157 '5 6 15V 160 ' 61 163 163 '6 4 165 166 IS? 166 69 70 i7 i *72 *73 174 76 ?< 177 176 17 60 19 82 163 164 p. 40 O S vone h r tn tttm fal./ U p n tu a w e toxtoolo g y ta x ( 2 0 0 9 tx a - m Table 2 Q5 Percentage(number)ofserumandmilksampleswithPFCs*>LODat visit 1(serum n-34; milk n- It) andvisit2(senim n-30; mlIkn -20) PeffiuoroaUcyUcidj . Serum>L0DX(n) ,::V.MllkW*<ii) ffcs Viriti- : Visit2 100(34) 100.(30) .0 PFOA Visit! Visit.2 M)0(34) 100(30) 0 0 PFHxS : VS&1 ViSt2 100(34) 100(30) 0 c PFNA Virici Visit2 97(33) 100(34) 0 0 PFOSA Viriti Visit2 Me-PFOSA-AcOH : Viriti Writ! _ 44(15) 73(22) S3(18) 50(15) 0 15(3) 58(1) 0 Et-PfOSA-AcOH : Visitt , 0 56(1) Writ2 , .0- 0 Petfluorobutanesulfonate Visit 1 Visit2 b b 0 0 Perfluorodecanoate Viriti visita b b 0 0 PFOS: perRuorooctanesulfonate: El-PFOSA-AoOH: 2-(N-cthyl-peffluorooctanr sulfonamMo) acetic acid; Me-PFOSA-AcOH: 2-{N-mettiyl-perfluorooctane sulfonamhfo)acetic acid: PFHxS: perfluoruhexane sullbnirarid: PFOS: pcrfluonxxrany] sulfonate: PFOA: perfluorooctanoicarid; PFNA:pesfluoronoranoieacid. * Denotesnotmeasuredinserum. its ZB. Statisticalanalysis ms VWcalculated the percentage ofdetection foreachanalyte in serumand milk, isx anddetermined the median,range,mean, standarderror, andselectedpercentiles, res Forvaluesbelow rtie100.valuescr^uaitoLOO/sqr2wereused[48,49],Furtheranal, ree yses. includingrelationships betweenvisits and acrossmedia, wereconducted for thoseanalytesforwhichthe frequencyofdetection(>L0D)was >S)Rat bothvisits. Forthosewomenwhodonated2serumsamples.themediandifferencebetweenthe concentrations tor the same PFCat visit 1 and visit2wascalculatedand assessed with the WHcuxonsigned-rank test (non-parametric).Spearmancorrelation coeffi cientsand relatedpvalueswerecalculated forcorrelationsbetweenthePFCsatvisit 1and visit2,and between the PFCSandthe biologicalmarkers Inmilkand serum. Relationsbetweena prioriselectedvariaMesassessedbyquestionnaireand thePFCS wereevaluated usingWilcoton scores(rank sums)test.Thecut-offpoints torcat egorizingselectedvariableswerederided a priori basedonassumptionsaccording to data previouslyrepotted (40-42),and to achieveapproximatelyequal distribu tion ofnumbersofsubjectsacrosscategories.Two-sidedpvalues are reported.All analyses were conducted with SASversion 9(SASInstitute.Cary, NC). 3. Results The median age of the women in this study was 31.3 years (interquartile range (IQR): 27.1-34.2 years), and the children's median ages were 5.5 weeks (IQR: 4 -6 weeks) at visit I and 13 weeks (13-14 weeks) at visit 2. Three o f the analytes, PFHxS. PfOS and PFOA, were detected in 100% of women's serum samples at both visit?, PFNA was detectable in 97% at visit 1 and in 1 0 0 %of women's samples at visit 2 (Table 2). In contrast, in m ilk samples of just 4 women, only 3 of the analytes were >L0Q: Et-PFOSAAcOH (l.Ong/m l) and Me-PFOSA-AcOH (0.7ng/ro!) were detected in 1 woman at visit 1. and PFOSA was detected in 3 women at the 2nd visit (0.3, 0.5, and 0.6 ng/ml). The remainder of the milk samples from both collections were measured and found to have concentrations < LOQ, The distribution ofPFCsenim concentrations isshownin Table 3. Highest concentrations were found for PFOSwith median values of 20.0 ng/ml at the first visit and 16.9 ng/ml at the second visit. PFOS concentrations were almost six-fold higher than the concentration ofthe analyte with the next highestvalue.PFOA,with median values of 3.5 and 2.9 ng/ml at the firsc and second visit, respectively. Median serum concentrations were significantly (p < 0.01) lower for PFOS. PFOAand PFHxS assessedar visit 2 compared to the concentration assessed at visit 1 , based on samples of 30 women who donated serum samples at both visits with the differences shown in Table 4. Accordingly, the concentrations of the detected serum PFCs are reported for each visit {Table 3). Serum concentra tions o f the same PFC were significantly correlated between the two visits (Table 4). Due to the lim ited number of breast m ilk sam ples with deteccable PFCconcentrations, we could not calculate the ISO m IS2 190 19* 19S 1S9 197 m 201 x> XU 20S 209 207 210 211 21? 212 5! ?'S 21 217 2*9 220 271 722 222 22* 22S 229 227 200 libleJ Distribution(mean, standarderror, median,selectedpercentiles,XI)ofPFCs*inserumsamplesatvisit I (n- 34)andvisit2(n-30) in ng/ml. Mean (SEM) Wthpercentile 25chpercentile Median 75thpercentile 90thpercentile 95th percentile IQR PFOS V iriti 218(1.9) 117 133 208 30.1 378 45.7 16.9 Visit 2 158(13) 9.70 14J3 16S 22.6 303 35.5 8.60 PflQA VWtt 3.99(035) 1.50 230 3.50 4.60 &0 8.70 Z40 Visit 2 3-0(0^!) 1.45 2.40 2.90 3.70 4.65 5.0 130 PFHxS Visit 1 184(037) 0;70' 1-D 1-55 2.40 3.40 3.80 1.40 Visit 2 130(0.22) 03 a?o 1.15 1.70 2.90 4.60 180 PFNA Visiti 132 (OJ2) 0.40 afa 1.10 1.60 zoo 2.70 0.90 . visita 1.33(0.09) . ttTS 180 120 1J5D 180 240 0.50 PFOS visit r 0.07(001) <10D <10D <LOD aio aio 0.10 0.07 Vtsii.2 089(081) <10D <ioo 0.10 0.10 0.15 0.20 0.07 Me~PFOSA-AeOH Visit! Visit2 0.23(0X)2) 034(082) *LOD LOD <LOD <LOD 0,20 030 0.17 030 030 0.40 0.40 0.50 0.16 0.16 * PFOS: Perfluorooctanesulfonate: fit-PFOSA-AcOH: 2-(N-ethyl-perfluorooctane sulfonamide) acetic acid: Me-PFOSA-AcOH: 2-(N-methyl-perfluoiooctane sulfonamido) acetic acid: PFHxS: perfluorohexane sulfonic acid: PFOS: perfluorooctanyl sulfonate; PFOA: perfluorooctanoic acid: PFNA: perfluorononanoic arid. Values measured <LOO wereImpuced byLOD/sqr2. p. 41 as. vonEhrensnemer of./ R tprodvatvc Toxicutagyxn (2009)xx x-xxx Table4 Difference and correlation in PFCserum concentrations:(ng/ml) betweenvisit one and visit two. NtfHandifference(QJt) pyaJitf ' :.ConetaSoq . p value coefficient<r` PIOS. PFOA PFHxS PFNA -23p(-7.9tO ) -0.55 (-1.40.0.0) -0.40 (-080. --0.1f Oil (-0.20.050) < a o i: <0.001 <aom 0.10 082 082 087 071 " WUcoxonsigned-ranktest(non-parwnetrie)(s-30). Spearmancorrelation coefficientir andrelatedp value(n30). <0001 <0.001 <0001 <0001 a , partition coefficient from serum to m ilk, but can concludethat milk 2 concentrations were notably lower than serum concentrations. 23i Based on self-reported data, women had lived in North Carolina 23< for (mean, SEM) 14.6 (1.92) years. Interestingly, women who had 23s reported living in North Carolina for 10 years or more compared 2N to those who had reported living in North Carolina less than 10 237 years, had higher serum concentrations of PFNA, PFOA, and PFOS 23t (p <0.03) (Fig 1). Furthermore, living in a house w ith an enclosed 23s garage attached as compared to living in a house w ith no enclosed 240 garage attached, suggested a relation to higher concentrations of 2. PFHxS (ng/ml: median. IQR, visit 1 : 2.2 (1.4) vs. 1.1 (0.6), p< 0.001; 242 visit 2:1.5 (1.4) vs. 0.9 (0.7) p - 0.03) and of PFOS(visit 1:25.4(16.9) 24j vs. 14.4 (9.9). p -0 .0 1 : visit 2: 21.2 (11.5) vs 14.5 (7.8)p - 0.1). Tables Correlations between concentration of PFCand interleukin-6 in serum at visit t (n34)and2(n-30). Correlationcoefficient,o ' pvale PFOS Visit 1 Visit 2 -0.21 039 020 0.03 PFOA Visit 1 Visit2 -015 007 040 070 PFHXS Visit 1 Visit2 -0.11 038 050 0,04 PFNA Visit 1 'Visit2 -02HB Joos 10 0.70 ' Speamtan correlation coefficientn and related p value. Boldedvalues signify significant correlations. Serum concentrations of 1L-6 were positively correlated with PFOS (p =0.03) and PFHxS (p -0.04) at the second visit (Table 5). None of the other selected biological markers in serum or in milk showed asignificantcorrelation with the PFCserum concentrations at either collection time point.There was nosignificant relationship between maternal 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 pilot study based on convenience sampling (only 3 women reported themselves as Black/African-American, one as Asian and one as Hispanic), we could not analyze PFC con centrations by ethnic group. 2*4 245 246 247 96 249 3S0 2S 262 253 264 4. Discussion PFNA Visit 1 PFNA Visit 2 PFOA Visit 1 PFOAVisit2 PFC by Visit (b) 60 Q<10Yean NCResidan 0210 Years NCResiden 60- .6 o> 40 O 30 uQ.. -x y ~ <b 10H Visit 1 Visit 1 Visit 2 Visit 2 Ftg.1. (a)and(b)SerumconcentrationsofPFNA,PFOA.andPFOS(n*(ml)comparing 1ivinginNorthCarolina>10to <10yearsatvisit t andvisit2.Dataareshownasbox and whisker representations: o p en circles dmore mean valueswith die medians denotedasa straightline.'p<0O3 inWikoxonScores(ranksums)tests lor groups :>10yeaisu.=t0yearsat visit Iandvisit2foreachPFC.Numbersofsubjectsineach group: >10years: u -16.visit 1: n -15.visit2: <10years: n -18,visit 1.n -15,visit2. In this pilot study of healthy lactaring North Carolina women. 6 of the 7 PFCs analyzed in serum were detectable at 2-7 weeks and 3-4 months postpartum. PFOS. PFOA. PFNA,and PFHxSwere found in nearly 100% of the serum samples. PFOS, followed by PFOA and PFHxS were the compoundsdetected at the highest concentrations. Only a small proportion of milk samples had detectable values of 3 of the 9 PFCs analyzed in milk. Interestingly, serum levels were lower for PFOS. PFOA, and PFHxS at the second visit compared to the first visit, and prolonged time lived in North Carolina, as well as living in a homewith enclosed garage attached, suggesteda relation to elevated serum concentrations of certain PFCs in our sample; however, theseanalyses were unadjusted and based o n a small nonrandom sample in this pilot study and thus should be considered exploratory. We can conclude that postnatal exposure to PFCs via breast milk is likely to be low during the rime period captured in our investigation. Data onPFCserum concentrationsoflactaring women are sparse and based on small sample sizes. Available data relevant for preand postnatal exposures to PFCs are summarized in Table 6 . Only one earlier study assessed both serum and milk levels, in 12 Iso lating women in Sweden, and reported similar serum values to ours for PFOS(median: 18.7 ng/ml) and PFOA(3 .8 ng/ml) while con centrations ofPFHxS were higher (4.0 ng/ml) In the Swedish study 135J. Based on data from the Danish National Birth Cohort, prena tal maternal 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 lower in the second than in the first trimester, possibly due to dilution of the PFCs with blood volume expansion due to pregnancy, but values in cord blood (n> 50) confirmed fetal exposures (24331(Tibie 6 ). The serum PFC concentrations seen in our study compare well with USserum data from NHANES 2003-2004. assessed in representative samples of 255 756 267 rae 259 260 2*3 26? 763 2*4 265 266 967 26 269 270 771 772 773 y>4 275 276 277 776 77 2B0 241 26? 263 264 765 266 767 p. 42 h W f ^ m T J t l a 3 im ;u~ OS ven Ehrwltin et aL/ Xtpmdiialiie ToxicologyJUtx(2009)But-joot Tablee Publisheddan oa v e n g e pfcconcentrationsh>milk, maternalserumandcordblood. Lodidon,yeirbiMHiplin* ! Matrix.: samplesize'' : ' 'I" ' PFCconcentrationas reported Massachusetts. USA,2004 'lO^'sowrelenc'samptel'ace: PF05(irjeaa,SD):131(103)p$/ml 22r43yers.a-43. nosingthe PFpA:43X(33.1)pg/tnl . Brsttliiw:n"34.nur*ed>l:ng PHxS: 14X(li?)p|/mt . PfNA:7.26C4.70)p*/ml . . . fWpA,PFBA,PfUnHA,PFDoDA. \ ... Is^^M unidi Oenmiy. 2006 Milfccoomnjeneesampling hwpitalatopies; n*19 (Munfch)- FTOS:akeLOD PfOS(medjan,range) CyntiHungary1996/97 ' ' Munich: fl3(28-239)ng/l. MtMSFrtbfpreterinhtbnts.n -13 : Leipzig: 123(33-3d9)bii/l (Hungity)MX-;?weeks . postpartum Hungary:330(96-639)ng/L PFOA.41; <UX>(<(00-460) ng/L 2fcxisat>,China,2004 Milk: co n ven ien c e samplinga t Ranges*(ng/L.) hospitalvolunteers,n>19 PFOS;45-360;PFOA:47-210: PFHxS:4-00; FfMA:63-62! PPDA:3X^15; PFUnDA: 7X-56 Sweden,mcfivlchulmatchedsera Mikandseruin:convenience MHk(mn,SD)ng/ml: PPOS: zndjiiilk(2004); pooledcomposite Simpleprimipan?urptnen.n*12 0L2O7(CX1T7);PFHxS:0BX3(0.047); nUksamples0996-2004). . PFOSA:0X13(0X09);PFNA:NA; i ; p K ^ p H v v f p u n D f c H P ~ : PooledaniHialcaitnpodieteilk 1 samples(n-25-90) Baltimore. Mp, USA. 2004-2005 ............. Daleof.mOkcollectioa:3weeks postpartum Cord blood, hospital based. singlea)ndeliveries(n-293) Serum(mean.SD)ag/tnJ:FFaS: 2D7(TOlSE'PFHiS; 4.712X): PFOSA:024 (0.16);FFMA:080 (055); PFO:3XCL0);PISA:053 : 0l);PFVnqA:1146{O35f Compos!te milVng/ml: PFOS0.209 (1996)4.123 (2004); PfHxS;0X37 (1998>0X16(2004); PFOSA: 0X07 (1996)-<0X07(2004): PFNA:0X29(1996V0XQ0(2004); PFOA: OX09{1996)-0209 (2004) PFOA(median,range): L6(03-7.1) PFOS(median.range) ng/ml: SX (<L0D(-(L2)-34X) Denmark. 1996-2004 /apan. 2003 Maternal plajrna:1s trimester (n>399Xv . . y;--." 2ndtrimester(n-200) . Cordblood n-50 Maternalplasma: 3rdtrimester ( -1 5 ) cord Wood(n -15) Maternal.1sttrimester: PFOS (ng/ndpiiean.SD):353(13.0). PF0A.K6(2X) Matemal,2ndtrimesterPFOS: : 29X(11X); PFOA:4X(IX) Cordblood: PFOS:11.0(4.7);PFOA: 3.7(34) Maternal, 3rd trimester serum tense*: PfOS(4.9-17.6ng/ml). PFOA(<UM>CO23 ng/ml),PFOSA (<LOD.te.<LOD) Coidblood: PfOS(1X-53ng/ml). PFQA(<L0Dto LOO).PFOSA (<LODto <LOD) J PFCLODsfor serum, bloodandmilkvariedothedifferent studiesasreponedinthe originalreferences. * fanaveragesreponed byauthors. Percentage quantified>LOD* >FOS:96X PTOA:.89X PFHxS:51* PFNA:64* PFHpAPFDA.PFUnDA,PFDoDA, PFBS:<8* pros: wok PFOA: 16* PFOS,PFOA,PFHxS.PFNA.PFDA. PFUnDA:100* Milk; PFOS.PFHxS: 100X(n-12); PFOSA:0/1 (n- 8XPFNA: 16% (n-2);PFA:*{n-T) Snim:lWS.PFHxS, PFOMTOA. PPM,PFUnDA: 100*(n -12); PFOSA:7SX'(lt-9) PFOS:99* PFOA: I00X: H?PFQSA-AeQH.Me-PFOSA-AcOH. PF9uS.PFHpA.PfUA. PFDoA: 1-40* Maternal, 1sttrimester PFOS: IDO*.PFOA: >00*(exceptn-1) Maternal, 3rdtrimester PFOS: 100*.PFOA:20, PFOSA:0* Cordblood: PFOS: 100*.PR)A:0*. PFOSA:0* Reference 125) |36) |34| 1351 [2233 J |24j |30J 2B8 females aged 1 2 and above, showing median concentrations for about halfthe concentration reported for cord blood from Denmark 8M PFOS and PFOA of 18.2 ng/ml (IQR: 12.4-273 ng/ml) and 3.6ng/ml 1241.Table 6 . 290 (IQR: 2.5-5.2 ng/ml), respectively [20.21 ]. Based on the NHANES A few investigations of PFCs in human milk have been con- 291 data, nation-wide serum concentrations dropped for PFOS, PFOA ducted in Sweden, China, Denmark and recently in the US(Table 6 ) 292 and for PFHxS between 1999/2000 and 2003/2004 while those for 125,34-36}. Only one study assessed both serum and milk concen- 293 PFNA increased in the same time period |20[. Our average levels nations and detected PFOS and PFHxS in all 1 2 milk samples at 294 are somewhat lower than reported for females in the US in 1989 mean concentrations of 0301 and 0.085 ng/ml respectively, sug- 295 [5] but sim ilarto other findings in samples collected between 1999 gesting partitioning of on average 1% from serum to milk [35], In 29 and 2005 [6.10.2031,50]. In a recent US investigation, median cord the Chinese study, values o fPFOSand PFOAin milk samples (n = 19) 29? blood levels for PFOSand PFOAof5 and 13 ng/ml, respectively,were were in the range o f0.045-036 and 0.047-0.21 ng/ml, respectively 3M reported [23 ].This isaboutathird to a fourth (PFOS)and 50%(PFOA) [34). Milk concentrations are summarized in Table 6 , supporting 209 ofthe concentrations we found in maternal serum samples, and also our findings of lower values in milk than in serum, as w ell as s > 2 m xw m m oar or om no *< p. 43 6 O S. von Ehrenstetn e t a l f ReproductiveTowrofogyxxx(2 0 0 9 ) xx x-xxx 9? suggesting regional differences in exposure levels [25,34-36). the WTC fire or directly from the WTC's degradation) [59] further 3 J 313 PFAAs are strongly bound to the protein fraction of human blood supporting the notion of source related local variations of human 8 314 [10.51-53]. The protein concentration in human blood contains exposures to certain PFCs. Women who reported living in a home 916 mainly album)n and fewer beta-lipoproteins and is about 3-5 times w ith anenclosedgarageattached alsohad increased concentrations mo 381 916 higher than the protein fraction in human mitk (casein and lactal- o f PFHxS and PFOS in our sample. This may be due to certain mate 317 bum in). It has been shown that strongly protein-bound drugs are rials used in and around cars containing PFCs, such as post-market 916 less likely to transfer to human m ilk than small non-ionic lipophilic applications of external and internal surface car coatings or treat 383 36 91 compounds [5-4). This may explain why PFM concentrations are ments. However, due to the small sample size in this pilot study, we 366 990 much lower in human milk than in maternal serum,although trans could not analyze the impacts of other variables, especially socio 366 321 fer o f PFAAs to milk has been observed in animal studies, albeit at economic factors; these findings are thus explorative and should be 367 322 much higher serum concentrations of PFAAs [26). inteipreted cautiously. 360 323 In our study, concentrations of PFOS, PF0A and PFHxS in serum The pro-inflammatory cytokine IL-6 was positively correlated to 324 were tower at the second visit compared to concentrations at the PFOS and PFHxS, respectively, at the second collection time point 368 390 325 first visit Since PFC concentrations measured in human sera have possibly indicating that certain PFAAs may be related to inflamma 391 926 half-lives ranging between 3.4 years for PFOA, 4.6 years for PFOS, tory processes. In line w ith these findings are recent results from 36? 327 and 7.1 years for PFHxS [55], these data suggest that processes experimental studies in mice, reporting suppression of immune 363 926 related to depuration into breast milk might be occurring that responses M ow ing exposure to PFOS in tero [60]. We did not see 394 9 we could not assess (possibly because we measured milk con correlations with othera priori selected biological markersassessed 395 390 centrations too late in lactation), or that there might be maternal in milk or serum, i.e., immunoglobulin, estradiol, prolactin orTNF- 386 391 metabolic changes during lactation that may relate to this change a. RodentstudiesusingPFOAin concentrations orders ofmagnitude 397 392 (i.e,, changes in blood volume, body weight, or hepatic activities). higher than MAMA serum concentrations have shown a suppres 386 333 Because PFCs ate tightly bound to serum proteins, serum protein sion ofgenetic markers of inflammation after an acute exposure to 399 334 levels during lactation could have affected the concentrations of PFOA [61 ]. Because our findings are explorative, future studies may 400 395 PFCs in serum. Unfortunately, we did not measure serum albumin want to address the role of chronic exposure to low dose PFCs in 40 396 to test this hypothesis. Alternatively, if PFCs partition more into the inflammatory process. 07 337 liver than serum in the course of lactation, serum concentrations In this pilot studythe number ofwomen was relatively small and 03 336 o f PFCs could be affected as well. The possible transfer of PFCs to confines the investigation of associations, possible exposure path 96 m ilk may also vary at different times during lactation. The nature ways, and time trends after birth. In addition, the sample was not 05 340 o f the relationship between the suggested decline of PFC values in selected randomly, thus selection bias cannot be excluded. How 406 341 serum to concentrations in milk are yet unclear and insufficient ever, the participation of women was unlikely to be related to PFC 407 342 data exist to date to explain the relationship at this poinr. Few ear exposures or to certain PFC exposure sources since they were most 408 349 lier reports suggested declines in breast milk during lactation for likely not aware of their PFCexposures. A further limitation of our 40 344 lipophilic compounds including dioxins. PCBs. and PBDEs [56.57]. study is that we could not collect milk samples sooner after birth in 10 346 No other study to our knowledge, has investigated PFC concentra view ofethical constraints in asking for the colostrum milk. Studies 4)1 346 tion changes in serum or milk over time during lactation assessed in mice measuring PFOA concentration over the course of lactation 41? 347 in the same women at two time points. However, it should be noted have shown that the peak in milk PFOA concentration occurs soon 413 348 that our findings are based on a relatively small number of a volun after birth (Fenton et aL, in this issue), a time that was not followed Q 3 414 34 teer non-random sample ofwomen and need replication in a larger in the MAMA collection scheme. Overall, th e findings reported are 4:5 350 study for further confirmation. Tao et al. conducted a regression explorative and need further evaluation. 46 361 analysis of PFOS and PFOA concentrations in breast milk collected In conclusion, although infant exposure via breast milk is likely 417 352 a t various tim e points from 25 different women within the first to be low, the cumulative daily infant intake of PFCs via breast milk 416 369 6 months postpartum; they concluded that values increased over per kg body weight could be appreciable for some populations or 41 364 tim e of lactation [25]. However, since these findings were based on groups (Table 6). Since toxicological and pharmacokinetic data for 470 365 m ilk samples of different subjects rather than comparing changes PFC exposed infants are lacking, it is largely unknown if potential 4?1 366 over time in the same women, the differences may be due to intra- health effects in infants or during childhood may be related to cur 4?? 357 individual variation. rent exposure levels of PFCs. in utero exposure should continue to 423 368 Our investigation suggested that living in North Carolina for a be a concern as the MAMA serum PFAA concentrations are similar 43* 369 prolonged tim e period of 10 years and more was related to higher to values reported in two separate studies that have shown inverse 426 360 serum concentrations of PFNA, PFOA, andPFQS in our pilot study. associations between maternal serum or cord blood PFAAconcen 76 961 However, further evaluation of this explorative finding is required. trations and infant birth weight [22,24). Thus, the findings of this 427 36? Pointsources maylead to elevated exposures.asindicated by serum pilot study underscore the importance of biomonitoring maternal 426 363 concentrations ofPFOA in persons living neara US facility using and and infant exposure to PFC as well as the need for further study 4? 364 producing this compound, that were notably higher than among of the potential human health effects of PFCs. In the upcoming US 430 90S the general US population [58). A systematic surface water survey National Children's Study [38] PFC exposures in pregnant and tac- 431 306 conducted in North Carolina showed large variation in concentra tating women and their children in North Carolina and across the 43? 367 tion on a small scale indicating a seriesof source inputs around the US w ill be further studied. 433 366 CapeFearDrainage Basinthat may potentially result in pockets with 369 370 increased exposures {40]. Comparing serum PFC concentrations among donors at the 6 American Red Cross Blood Bank locations Conflict o f interest 34 371 across the US showed highest concentrations for PFOS and second 372 highest for PFOA in Charlotte. North Carolina, in samples collected The authors declare that there are no conflicts of interest. 435 373 in 2000-2001141]. with a substantial decline observed in samples 374 collected in 2006 at the same locations [42). Recently, elevated Acknowledgments 436 376 plasma concentrations especially of PFOA. PFNA, and PFHxS have 376 b e en reported for personnel involved in the World Trade Cen The research in this arricie has been reviewed by the National 437 377 ter (WTC) disaster (i.e,, from fire-fighting foams used to combat Health and Environmental Effects Research Laboratory. US Environ- 436 OS von fkrcmMn etoLfReproductiveTbdcotogyxxx (200&)xa~xxx 7 mentalProtection Agency(EPA).and the Centersfor DiseaseControl 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 of 44 the CDC The use or trade names or commercial products does not 44< constitute endorsement or recommendation for use. 44 Thisw ork was supported in part by the Intramural Research Pro44 gram atd ie EuniceKennedy ShriverNational Institute ofChild Health 447 and Human Development, National Institutes of Health. Bethesda. 44 MD. 44 Partial extramural funding was provided through the reciso ommendation o f the National Children's Study Intra-Agency 4st Coordinating Committee. 49 The authors would like to Richard Wang at the CDCfor technical 4 assistance,W esut, Inc. recruitingstaff(AndreaWare. Bethany Brad44 ford. Brian Karasek). and the US EPA nursing staff(Deb Levin. Mary 4 Ann Bassett, and Tracy Montilla). 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EnvironSdTerhnol 2008:42:3472-8. s 0)4 154] Aschtnbtenner DS.VeableSJ.Drugtherapyin nursing. Lippinicott.Williams& [60] Keil DE. Mehlnunn T. Butterworth L et aL Gestational exposure to perduo- 19 Wilkins; 2009. rooctane sulfonate suppresses immune function in B6C3H mice. Toxicol Sci ss? 10 155) Olsen CW. Burris JM. Ehresman Q). et a). Half-life of serum elimination of 2008;103:77-85. 6 017 perfluorooctanesuHonate. perAuorohexanesulfonate. and perfluorooctanoate [61] GurugeKS.YeungLW.Yamanaka N.et al. Geneexpressionprofiles in rat liver km 18 in retired fluorachemical production workers. Environ Health Perspect treated withperfluorooctanoicacidO'FOA).ToxicolSci2006:89(1)^3-107. gb 2007:115:1298-305. p. 46 ELSEVIER Molecular and Cellular Endocrinology 304 (2009)97-105 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology JournalW orn,epage: w w w .e lse 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. H ines4'*, Sally S. W hitebJ a s o n P. Stanko4, Eugene A. Gibbs-F1oumoyc, Christopher Lau4, Suzanne E, Fenton4 JR ep ro d u ctive ToxicologyD ivision, O ffice o f R esettrek andD evelo p m en t, N otional H ealth a n d E n vironm ental E ffects R esearch L aboratory, U S E n v iro n m en ta l P rotection A gency, Research Triangle P ark, N C 27711 U nited S ta les b C urriculum inToxicology, UNC C hapel H ill, C hapel H ill, NC 27599, UnitedStates cB iologicala n d B iom edical Sciences P rogram JInitiative fo r M a xim isin g S tu d e n t D iversity. UNC C hapel H ill, C hapel H ilt NC 2 7 5 9 9 , U nited S ta te s ART ICL E INFO A n k le h isto ry: Received 27 January 2009 Accepted 24 February2009 K eyw ords: PFOA Overweight Leptin Developmental exposure Obesity Ovariectomy 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 grow th fn animals exposed during fetal lift. PFOA is found in the sera and tissues of wildlife and humans throughout the world, but is especially high in the sera of children compared to adults. These studies in CD-I mice aim to determine the latent health effects of PFOA following: (1) 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 , or5mgPFQA/kgBW for 17 days of pregnancy or as young adults. Body weight was reduced in the highest doses on postnatal day (PND) 1 and a t weaning. However, the lowest exposures (0 .0 1 -0 3 mg/kg) significantly increased body weight, and serum insulin and leptin (0.01-0.1 mg/kg) in mid-life after developmental exposure. PFOA exposure combined w ith ovx caused no additional increase in mid-life body w eight At 18 m onths o f age, the effects of in utero PFOA exposure on body w eight were no longer detected. White adipose tissue and spleen weights w ere decreased at high doses of PFOA in intact developm ental^ exposed mice, and spleen w eight w as reduced in PFQA-exposed ovx mice. Brown adipose tissue weight was significantly increased in both ovx and intact mice at high PFOA doses. Liver w eight w as unaffected in late life by these exposure paradigm s Finally, there was no effect ofadult expo sure to PFOA On body weight. These studies dem onstrate an im portant window of exposure for low-dose effects o f PFOA on body weight gain, as well as leptin and insulin concentrations in mid-life, at a lowest observed effect level of 0.01 mgPFOAfkg BW. The mode of action of these effects and its relevance to hum an health remain to be explored. Published by Elsevier Ireland Ltd. Abbreviations: ANOVA.analysisofvariance;BM1.bodymass index; BW,bodyweight;C8.eight-carbon;CV.coefficientofvariation; DES,dietybtilbestrol; Ej.estradiol; CD,gestationalday;t1n,Half-Mfe;IACUC,InstitutionalAnimalCareandUseCommittee;LH.luteinizinghormone; LOD.limitordetection;UOQ,limitofquantitation; NHANES. NationalHealth and NutritionExaminationSurvey;NMR.nuclearmagneticresonance;NOAELnoobservableadverseeffectlevel;ovx.ovariectomieed; PFAA,perttuoroalkyt add; PFOAperfluorooctanoic acid; PFOS.perfluorooctanc sulfonate; PND. postnatal day; PPAR, peroxisome prolifictor-activated receptors; SMR,standardized mortality ratio. * D isclaim er. TheInformationin thisdocument has beenfundedbythe IIS. EnvironmentalProtectionAgency.It has beensubjectedto reviewbytheNationalHealthand Environmental EffectsResearchLaboratoryandapproved forpublication.Approval docs notsignifythat thecontentsreflect theviewsoftheAgency,nordoesmentionof trade names orcommercial productsconstituteendorsementorrecommendationforuse. Correspondingauthor. Currentaddress; U S. EnvironmentalProtectionAgency,NationalCenter forExposureAnalysis.EnvironmentalMediaAssessment Croup. ResearchTrianglePart. NC27711.UnitedStates.TeL:1 919541 4204rfax; *1 919541 2985. E-mailaddress: hihes.erinffepa.gov(EP.Hines) 0303-7207jS - secfrontmatter. PublishedbyElsevier Ireland Ltd. dor:10.1016/j.mce.2009.027)21 p. 47 98 EJ>.Hints t l t L f Meltcubr omtCtthihsrEndocrinology 304 (2009) 97-J05 1. Introduction Perfluorooctanoic add (PFOA), one ofthe eight carbon (C8) perfluoroalkyl acids (PFAAs). is a synthetic, stabte, persistent organic fluorine surfactant used to impart water and grease resistance to various consumer products including non-stick pans, as sur face treatments for clothing and food wrappers, insulation and fire-fighting foams. PFOA's high energy carbon-fluorine bonds are resistantto hydrolysis,photolysis and metabolism and thus it bioaccumuiates and persists within biota and environmental matrices, including w ater and soil, from the Arctic to the South Pacific(Lau et a l, 2007). This ubiquitous environmental contaminant has an esti mated half-life (ttp ) in humans of3.8 years (Olsen et al-, 2007) and is found in production workers' sera, aswell as those ofthe general population. Bio-monitoring studiesshow detectablelevels ofPFQAin human populations. The National Health and Nutrition Examination Sur vey (NHANES) reported that mean serum PFOAconcentrations are declining in the USA population, from 5.2 ng/ml in 1999-2000 to 3.9 ng/ml, in 2003-2004 (Calafat et aL, 2007). Arnsberg. Germany, an area w ith known drinking water PFAA contamination, had reported PFOA mean serum levels in 2006 of 25ng/ml vs. 4 ng/ml in unaffected German provinces (Hfilzer et aL, 2008). The highest known non-occupationa! PFOA exposure via drinking water exists in the little Hocking drinking water district where US. residents (Ohio and W est Virginia) have mean serum PFOA concentrations o f478 ng/ml (Emmett et aL. 2006). Children may receive significantPFOAexposures via dietary and water intake. Mean serum PFAAconcentrations (such as perfluorohexane sulfonic acid) were reportedly higher in children than in adult/eiderty populations (Olsen et aL, 2004). In the Little Hock ing water district, an area of high environmental PFOA exposure, children age two to five and the elderly had significantly increased PFOA serum levelswhen compared w ith other age groups (Emmett et al.,2006). Although a bio-monitoring study in Japan found PFOA in maternal blood, but not umbilical cord blood at parturition (Inoue et al.. 2004, lim it ofquantitation [LOQ] 35.2 ng/ml). a recent US. study (Apelberg et al- 2007) of human cord blood from term pregnancies reported relatively low levels ofPFQA (lim it of detec tion |L0DI 0.2 ng/ml) and another C8 compound, perfluorooctane sulfonate (PFOS). W ithin the reported study concentrations, the authors found that cord blood PFOA concentrations were signifi cantly negatively associated w ith birth weight.A subsequent huger Danish study also found a significant negative correlation between maternal plasma PFOA and birth weight (Pet 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 worker populations, serum cholesterol and triglycerides have been positively associated w ith PFOA exposure w hile high density lipoproteins have been negatively associated w ith PFOA (Olsen et al.. 2001). Categorical division of workers by PFOA exposure levels showed that, although notsignificantlydiffer ent from the other categories, body mass index (BM I) was elevated in the highest PFOA category (>30ppm and BMls >28,1995 data); this trend was not seen in the 1993 data set (Olsen et aL. 1998). A retrospective cohort mortality study (n > 6000) of PFQA-exposed employees reported significantly elevated standardized mortality ratios (SMR) in mates with diabetes mellitus when compared to men residing in West Virginia (minus the PFOA manufacturing area), Ohio, Virginia, Kentucky. Indiana. Pennsylvania. Tennessee, or North Carolina; the SMR for PFOA workers was not significantly increased when compared to West Virginia alone or USA residents (DuPont, 2006). ln Arnsberg, Germany. PFOA was found to have an inverse correlation w ith BMI in adults (Hlzer et at.. 2008). The t , p s for PFOA in men and women are similar (Harada et al.. 2005). Unlike humans, genderdifferences in PFOAclearance exist in rats (Kudo and Kawashima. 2003; Vanden Heuvelet aL, 1991). Mice are the preferred animal model for evaluating the effects of PFOA on the developing fetus as they do not exhibit gender-dependent tjp differences (Lau et aL, 2006). However, even in the tat model system where the female tat rapidly excretes the compound, PFOA readily crossesthe placenta (Hinderiiter et aL, 2005) and PFAAsare present in rat milk after PFOA treatment (Hinderiiter et a l- 2005). Mice prenatally exposed to doses of PFOA at >1 mg/kg/day exhibit developmental toxicity including decreased litter size, neonatal death,delayedeyeopening,growth deficits, stunted mam mary gland development, and early onset male puberty (Lau et al,, 2006; W hite eta l, 2007; W olfet a l- 2007). At higher doses and fol lowing long-term adultexposure,cancerendpoints associated with PFOAexposure in ratsinclude Leydig cell adenomas,pancreatic aci nar cell adenoma/carrinomas, mammary fibroadenomas, and liver tumors (Biegel et aL, 2001; Sibinski, 1987). PFOA increased estra diol (E j) levels in male rats and PFQA-induced rodent Leydig cell tumors are hypothesized to arise from increased estradiol levels from aromatase induction (Liu et aL, 1996; Biegel et al., 2001). The majority ofdie ongoing work in the PFOA field has focused on the health effects following developmental exposure to PFOA. This study focuses on adult latent health outcomes in female off spring after developmental (gestational days (GD) 1-17} vs. adult (at 8 weeks of age, for 17 days) exposure to PFOA. Ovariectomized siblings were utilized in our second study block to address the role of the ovarian hormones in PFOA exposure-related health effects, as luteinizing hormone (LH)-overexpressing mire (Kero et aL. 2003) displayed several phenotypic effects resembling those in our preliminary studies with PFOA.These studies address the role of developmental exposure and ovarian hormones in adult health effects including circulatingleptin and insulinconcentrations,adult body weight, and tissue and body weights in old age. 2. Material*and methods 2 1 . A nim ats Timed-pregnantCD-I mice(CharlesRiverLaboratories,Raleigh,NC]arrivedon gestational day(GD)0 (sperm positive) at the USEPAwhen they were weighed upon arrival and randomly distributed among treatment groups. Pregnant dams were housedindividuallyin polypropylenecages end receivedchow(LabOlet S001, PM! Nutrition international LLCBrentwood, MO) and tap water ad U btirni. Two blocks o f animals were used hi these studies. Block 1animals were dosed with vehicle (distilled water! I or SmgPFDA/kg body weight (BW)(n-S, *. 7. and S dams, respectively); block 2 animals were dosed with vehicle, (U)L0.1,03.1, or 5mgPF0Ajkg (n*14 dams in all groups except 5mgPFOA/kgBW.which had 10dams! PFOAexposuresare shown lo the text as mgFFOA/kg.Animal facilities were maintained on a I2:12-b light-dark cycle, at 20-24-Cwith 40-50%relative humidity.Animalswerehumanelytreated as approvedunderNationalHealthand Environmental Effects ResearchLaboratoryprotocolsinaccordance with the USEPA InstitutionalAnimal Careand UseCommittee (IACUC!Sentinel mice, housed in the sameroom,wereknowntobefreeofectofendoparasltesandantibodiesto ceitain viruses for the duration ofthese studies. 2 2 . Dosingsafurion and procedures PFOA. as its ammonium salt (>98Xpure), was acquired from Fiuka Chemical (Stefnhiem,Switzerland! PFOAdosingsolution wasprepared freshdailyIndeion izedwater, and the dosingsolutionwasadministered atavolume of10pJ/g.Mice receivedeither water vehicleor PFOAat 0.01.0.1,03,1,3, or Smgjkg BWbyoral gavageoncedailyoverthedosingperiods.Thehighestdose(5mgPFQAJkg/day)was chosenbeeaaiseit wasknowntoresult inslightlyreduced neonatalbodyweightgain with minimalpostnatal mortality(Lauetal..2006! 23. E xperim ental design 23.1. D erela p m en ta l e x p a su n /m u c t Timed-pregnant CD-I mice (n-7-22 dams per dose group over two blocks) receivedO.OjOI.O.1,0.3, 1.3,or5mg/kgPFOAbyoralgavageonthe roomingsofCD t -17. Damswereweigheddailyprior to dosing and throughout gestation. Atbirth, pups were tndMdualiy weighed and sexed. Pups within a treatment group were pooled and randomlyredistributed amongthe dams oftheir respective treatment groups, and litters were equalized to to pups (both genders represented! Dams p. 48 EJ>.Hinesel aL/Molecularand CellularEndocrinology304 (2009) 97-70S SS- PFOA PFOA Devetopmwtal Atfuft Dosing Dosing GO 8 1-17 wks 3*rfc* 15-16 wfcs F3 CDO Blr1h Sperm Positive M * an JVX | Glucose tolerance test tyoung) 21-33wks 1 .. Mandibular bleeds 42wkl I1 Body Mass Composition Measured 70-74Wks 1I Glucose tolerance Test (old) Food Intake monitoring 1 It Months E2 BMtttttt -----------....welgfn monttwed(developmental!/exposed,ovxgjntact)___________ Fie.1. Datacollectionschematicforstudyofdevelopmental^andadult PFOA-exposedfemalemice. that deliveredsmall litters (n<4 pups) were excluded bomthe remainder ofthe study:Pupswere weaned at 3 weeksofate at which point females were retained andhoused 3-5 micepercaje. Maleswereevaluated separately, atendpointsthat varied fromthosereportedhere. 2 X 2 . D evelo p m en ta l ejrp a su re/m m riea a m y A subset of devclopmentally exposed female siblings (OmgPFQA/kg, n-8: 0.01mgPFQA/kg, n-15; 0.1mgPFQA/kg, n-11; 03 mgPFDA/kg. n-14; 1mgPFQA/kg.n*6:5mgPFQA/kg,n- 7)wereavariectomlzed(ovx)at21or22days ofage,before the onset ofpuberty: Animalswere sedated with ketamine/xylazbie (87/13mg/kg Lp, respectively), their ovaries surgically removed through the abdomen,sutured, and animals were placed in wanningcages until they regained alertness. Buprcnoiphineanalgesic(0-05mg/kg)wasgiventwicedailyim. for48ft in 03mlvolumeforpain relief 2 3 3 . M u lt exposure AseparatecohortofmicereceivedFFQAstarting at8weeks ofage, for17days (OmgPFOA/kg.n-S; 1mgPFQA/kg.n-14; 5mgPFOA/kg,n-14). 2 .3 .4 . D ata co llectio n Thedata collection schemefor these studiesis shown inFig. 1. Bloodwascol lected bom the submandibularveins ofovxand intact micebetween the ages of 21 and 33weeks.These bleeds took placebetween 14:00and 18:00; and 200pi of blood(100pi ofserum)wascollected forsubsequentanalysesofInsulinandleptm. Femalesinall threeexposurescenarioswereweighedweeklyupto9monthsofage and thenmonthlyuntil 18months.Thenumberoflmact.developmeutallyexposed miceweighed weeldy/monthly was 10.25.20.11. and 32. respectivelyfor 0.0.01. 0.1.03.and Idling PFOA/kg.Ifmicebecamemoribundbeforethe studyended, they wereeuthanizedin compliancewith die protocol approved by die USEPA1ACUC (early necropsy): Date and cause ofearly morbidity or mortality was recorded if known.Atearly necropsy(collectedwben necessary)orat 18months,trunkblood, retroperitoneal abdominal white (found lyingventral to the intestines and repro ductivetract) andinlerscapularbrown fatpads, abnormalgrowths,and organswere collected bom anexposuregroups.Relativeorganweight is usedto expressorgan weight aspercentoftotal bodyweight. Dataarereported hereasmeanSEM. 2.4. G/ucosetolerancelest Glucose tolerance tests were performed on two groups of intact develop mentally PFOA-exposed animals: old adults (17 months of age with 0, 0.1,1 or 5mgPFOA/kg; n-8-13 per dose group) and youngadults (IS-16weeks old with Q. l or 5mgPFOA/kg; n-12 per dose group) The night before the assay, fur was shaved fromthe lateralareaofthe lowerkg toexposethe saphenousveinandani malswerefasted.Thefollowingmorning,the micewereweighedandbloodglucose was measured by collecting a drop of blood fromeach mouseviapuncture ofthe saphenousvein (ortailveinifnecessary).Theblooddropwasplacedonateststrip, andinserted intothecalibratedgluGOtneCer(AccuchekAdvantage)forbaselineglu cosemeasurement.The micewere then injected up. with o-glucose solution(2g/kg bodyweight from a stock solution), and bloodglucose concentrationswere mea sured at20.40, GOand 120(old mice)or 180(youngmice) minutes (1-3 min)after theInitialglucose injection. 2.5L Serumlep tin Serum(10pi) collected bymandibular venipuncture wasassayed forleptin by tadis-immunoassay(Unco Research,St.Charles.MO)followingthe manufacturer's protocol(n- 5,controls:n-18,0:01: n-16.0.1;n- ii. 03:n- 24.1mgPFQA/kg)The coeffiefentofvariation(CVs)forthestandards(concentrationrangeof0.2-20ng/mlj rangedfrom0.1%to8.0%.ThequalitycontrolstandardstermedQC1 (expectedrange 0.6-13ng/ml)andQC2(range 1.8-33)hadameasuredconcentrationintheseassays of03 and23. respectively. 2.6 Serum insulin Sera(lOpI)collected bymandibularvenipuncturewere assayedforinsulin by the ultra-sensitive single molecule Immunoassay bySingulex (Alameda. CA) fol lowing the manufacturer's protocol (n-9 control, n-21, 001 mgPFOA/kg; n-16, 0.1mgPFQA/kg: n-11.03mgPFQA/kg: n>31.1mgPFQA/kg) Samples were ana lyzed using a 384-wefl plate format with monoclonal capture and detection antibodiesontheSingulexFrrenaequipment.TheCVsfortheartaystandards(range 19.5-5000pg/ml) were from3%to 17%.TheassayPODwas 16pg/ml. Alt samples were ranon the samedayand the mterassayCVwas94%and 5.1%for the29and 1745pg/mlqualityassurancestandards, respectively. 2 .7 . B ody m a ss co m position Whole body mass compositionwas measured in live,non-sedated 42-week-old miceusingthe BrokerMinispecmq7.5IF50LiveMouseAnalyzer(TheWoodlands. IX) The minispec was a benchtop 73MHztime-domain nuclear magnetic reso nance(NMR)analyzer,which quantified body fat. leantissue, and free bodyfluid in mice.TheminispecwascalibratedbyBroketOptics.Inc.staffpriortoanimal analy siswithdailyvalidationsusingBrokerstandards.Micewereweighedand inserted intothe instrumentforanalysis(1-2min/anhnal)Intactdevetopmeotallyexposed femalemicethatunderwentbodymasscompositionanalysisincludedcontrol.0.01. 0.1,03. and 1mgPFQA/kg(n-9. 23. 20,11. and 32. respectively) dose groups. It wasnot possibletoperformthese measureswithyoungermicedue toequipment availability. 2.8. M easurem ent ofEarnserumo fin ta c t m ice a t 18 months Serum Ej (25pi volume) from 18-month-old mice (intact developmentafly PFOA-exposed animals) was measured with time .resolved nuofo-unmunoassay (DELFIAEstradiol Kit, Wallac Oy. Finland) following the manufacturer's recom mendation using a VICTOR3D 1420 Multlabel counter. PeritfnElmer Precisely time-resolved fluorometer(PerkinElmerlife h AnalyticalSciences,Shelton,CT)The CVsfor the standards(concentration rangeof631-1423pg/ml) rangedfrom02% to 43%. 2.9. fe e d consum ption Feed consumption in 17-month-old, developmental!/ exposed, intact female mice(n-6 perdosegroup:0,0.1.1 and 5mgPFOA/kg)was measuredinmetabolic cages.Micewereallowed toacclimate tothecagesfor1weekandfoodintakewas monitored during the secondweek. Micewere individually housed and provided withapie-weighedamountofpowdered labchow1hbitum .Theremainingchow wasmeasuredattheendoftheweekandthetotalamountwassubtracted fromthe startingamounttodeterminethe total feedconsumed foreachmouseperweek. 2 JO . M ea su rem en t o fseru m PFOA Trunk blood serum samples (-50 p.1) from the female CD-I offspring at 18month necropsies or from mice terminated at earlier intervals because of illness were transferred to the CDCfoe PFOAmeasurement- Serum PFOAdetermination wasperformedasdescribedin Kutdenyikel al.(2005)andWhiteetal.(2009). 2.11. S ta tistic s Data were analyzed usingSAS9.1 (SASInc-Cary. NC)Bodyweight on PND1was evaluatedas littermeansasthesedatawereobtainedpriortomixinglitteroffspring within a dose group. Bodyweightsateachtimepoint wereanalyzedwith mixedeffectslinearmodels (SASProc Mixed)to estimate means and standard errors and test for dose effects separatelybyrimepoint.Foreachtimepointthemodelincludeddoseasafixedeffect I p. 49 100 E f. Hines et o l / Molecularant Cdhifar Endocrinology 304 (2009)97-JOS andcagenested withindoseas arandomeffect Pairwisec-testswerecalculated to testToranydifferencebetween eachtreatmentgroupmeanand thecontrol(roup. Repeated measuresanalysisofbodyweightdata was evaluated twoways. First weights were averaged by animal over eight 10-week intervals. This was done to decreasemissingvaluesinthe data doetoanimalmonatttyInlateMethatwasnot equal across treatment and to reducethe effect oflargebodyweight varianceslater hr life.Thisdam smoothingmethod decreased uninformativeshort-term variations andalsoreducedtbe numberDfestimatedparametersto atractablevalue Amulti variaterepeated u)easuresanalysis(SASProcGlM}wasperformedonthesereduced data. Subsequent to asignificantfinding,comparisonswerecarriedoutassubtests ofthe overallanalysisofvariance(ANOVA)atspedfictimesordoses. Second. SASProeMixed was osed to performa univariate repeated measures analysisoftheweightsacrosstimeupuntil37weeks(latestweightpointatwhichno animalshaddied).Themodelestimatedaseparatefetedquadraticcurveacrossdme foreachdosegroup andnduded arandomeffectItercagenested withindose.Cor relation withinanimalswasmodeledwitha randomeffect foranimal nestedwithin cageanddosein additiontoanautoregressivecovariancestructurewithineachani mal. Inthis way. thecovariancematrix loreachanimal'smeasurementsincludeda constantcovariance componentat ad timepoints in addition toa componentwhich decreasedas timepointsgrewfartherapart. Tissue weight, relative tissue weight,bodycomposition, food consumption,and body weight measurements were analyzed using a one-way ANOVA(Dnnnetfs post hoctests),with dosebeingthe independentvariable.Ablockingvariablewas included to adjust for the group difference. Noadjustment was made fo r multi ple comparisons. Chrcose mleiance was compared at Individual collection times by one-way t-test and over time by repeated measures and area under the curve comparisons according to the trapezoidal rale. Hormone (insulin, Ea and leptin) concentrations were analyzed usingANOVAfollowed byTukey*spost hoctest. Mortalitydata woeanalyzedwithproductlimitedsurvivalestimates; tog-rank and Wilcoxon tests were used to test fordifferencesamong the treatmentgroups in survivalacross time(SASProcUfetest). Thelevelofsignificance forall testswas p<0.0S. o jm u 0 3 i DosePFOA (mg/kg SW) s 3. R esults 3.3. D evelopm ental exposure 3.3.1. Early a n d m id-life body weight effects There were no significant differences in live pup number at birth by dose group (p<0.05) and postnatal mortality was not addressed in this study as litters were equalized at birth. On post natal day (PND) 3, the average weight of die developmentally exposed 5 mg PFOA/kg offspring was significantly less than con trols (Fig. 2A): no other dose group demonstrated significant litter weight effects at FNDl. At weaning, mean female body weights were still significantly decreased in the 5 mg PFOA/kg(13.9 g 0.8) compared to 18.4 g 0.4 in control untreated pups. At this time, the 1mg PFOA/kg exposed animals were also significantly smaller than controls(p<0.05; 36.4g 03 ). Time-grouped mean body weights o f the female offspring over their lifetim e are shown in Kg. 2B. Beginning at 10-19 weeks of age. there was an increase in weight in the 0.1 and 0 3 mgPFOA/kg groups compared to controls;by20 -29weeks ofage,femalesdevelopmentally exposed to PFOA showed significant dose-dependent increases in body weight at 0.01, 0.1, and 0.3 mg PFOA/kg which extended to 40 weeks of age in the 0.01 and 0.1 mg PFOA/kg when compared w ith control (p<0.05). This is specifically shown at 20-29 weeks (Fig. 2C), where the 0.01-03 mg PFOA/kg groups had average weights 11--15% higher than controls. Continuous analysis of repeated measures of body weight over time demonstrated that the five dose groups were similar in inter cept using a quadratic fit; however, the 0.01, 0.1 and 0 3 groups had a significantly greaterweek effect than control, indicating that their weights were changing at a more rapid rate than control or 1 mg/kg. This is shown in Fig. 2D for weeks 6-37 (the latest weight collection tim e point priorto death ofanystudyanimals);Addition ally, the 0.1 mg/kg (p - 0.056) and 0 3 mg/kg (p 0.046) groups had larger negative coefficients for week2 (week squared), suggesting that their weights were starting to fail o ff more quickly at the later time points than the control groups (not shown). The estimated weight curve for the 1 mg PFOA/kgdosegroup was not significantly different from the control curve. Data from 5 mg PFOA/kg exposed Ml 4 03 DosePFOA (mg/kg BW) 1 tig.2. Bodyweights ofdeveiopmenuftyPFOA-exposedfemaleoffspring Dataare shownasmean4:SEMwith> <0.05vs.control.(A)Pupweightat FND1 afterdevel opmentalPFOAapowre.(B) BodyweightoffemaleCD-I miceovertheirlifetime, followingdevelopmentalPFOAetposureoverSperiodsottime[period1 (0-9weeks old);period2(10-19weeksold), period 3 (20-29weeksoitf).periods (30-39weeks old),period5(40-49weeksold).period6(50-39weeksold),pcriod7(60-69weeks old),andperiod*(70-79weeks)).(OGroupmeanbodyweightsoffemaleoffspring at 20-29 vreeks of age demonstrating excessive weight p i n at low doses. (D) Dose- dependentquadraticregression lit to repeated measuresofbodyweightinfemale mice.Anincreasedrateofweightgainwasseenin0.01.Ol.and03 mgFFQA/kgdose gtoopscomparedtocontrol and 1mgPFOA/kg. E.P. mues etat/M olecularand CellularEndocrinology 304(2009) 97-105 101 (B) 600-, Time (minutes) Bg.3. Blood glucoseconcentrationsfollowinga glucosechallengeaftertime0 in (A)young(15-16 weeksold)and(B)old(70-74weeksold)femaleCD-I micethat weredevelopmentallyexposedto PfOA.Dataareshownas mcaniSEM. mice, which were decreased in BW compared to control at PND1. weaning, and 18 months, are not shown. 3.1.2. S eru m glucose tolerance testing Because or the excess weight gain in the PFOA developmen tal))! exposed mice during mid-life, various tests were conducted on these animals (as close to the appropriate age as was possible) to examine the associated effects of these changes. No significant differences were detected in baseline glucose or serum glucose area under the curve in response to a glucose challenge in young or old mice (control, 0.1, 1, or 5 mgPFOA/kg. p<0.05. Fig. 3). In a time-dependentcomparison, young mice exposed to 1 mgPFOA/kg showed a nearly significant increase in blood glucose over control animals at 20 min post-glucose challenge (p -0.06). In old PFQAexposed mice, although there appeared to be dose-dependent glucose insensitivity at 20 min, this shift in response was not sig nificant. 3.13. Seru m insulin and Ieptin Serum insulin and leptin measurements were made using blood obtained via mandibular bleeds between 21 and 33 weeks (within the time frame of greatest observed body weight increases) using intact female mice dosed w ith 0.0.01,0.1,0.3, and 1mg/kg PFOA. Insulin and leptin concentrations were significantly increased in mice developmental^ exposed to the lowest doses of PFOAtested (0.01 and 0.1 mg PFOA/kg). Although elevated from the control mean, leptin concentrations were not significantly different from control at 0 3 or 1 mg/kg PFOA (Fig. 4). 3.1.4. Fat to lean ratio At 42 weeks of age, mice from block 2 (control, 0.01, 0 .1 , 0.3, and 1 mg PFOA/kg) were evaluated using a Broker Optics Body Mass Analyzer, which determines the amount of fat, lean and fluid o Ml 6.1 <u Dose PPOA (mg/kg) ] Fig.4. Serumleptin(A)and insulin(B)inmice 21-33 weeksofagefp < 0 .0 5 vs. control).Significantelevationsareseenat0.01 and0.1mgPFOA/kg.Dauareshown asmeanSEM. in live animals. There was no significant increase detected in % body fat:body weight in PFOA-exposed mice (data not shown). Developmental^ exposed mice had no significant differences in fatilean ratio across dosegroups when compared to control (means ranged from 0.75% in controls to 0.9% in 0.01 and 0.1 mgPFOA/kg). Although no dose groups were significantly different from control, there was an increase above control levels of about 12% in mean %fat:body weight ratio and 14%in mean fatilean ratio in the dose group exhibiting the largest change in body weight at 24 weeks (0.1 mgPFOA/kg). 3.1.4.1. Feed consumption. Feed consumption was measured in 17month-old, developmentally exposed intact mice (0, 0.1. 1 and 5 mgPFOA/kg) and no significant differences were found across dose groups when compared to controls (mean 26g/week con sumed: individual data not shown). 3.7.5. Late h/e organ and body w eig h t effects A noted loss of animals after 36 weeks of age was further eval uated (Fig. 5). At 51 weeks old, when there was no mortality in controls there were 20%, 10%. 36%. and 6% mortality rates in 0.01, 0.1,0.3, and 1mgPFOA/kg groups, respectively. By 76 weeks, there was a 40% mortality rate in controls, and 32%, 63%, 60%, and 44%in 0.01.0.1,0.3 and 1 mgPFOA/kggroups, respectively. However, there were no significant differences between control and any treatment group at specific times in late life or in survival across time. Among those mice surviving to 18 months, body weight of PFQA-exposed females was no longer elevated compared to con trols. Furthermore, a significant decrease in body weight at the 5 mg PFOA/kg dose was noted (Table 1). At that time, all remain ing females were necropsied. Trunk blood, tissues (affected or of interest) and abnormal masses were collected, weighed and fixed for future study. Seram was collected and PFOA levels were mea sured. The majority of the samples across dose groups had PFOA concentrations lower than the lim it of detection (0.5ng/mt) with detectable values at maximum concentrations of 35 ng/ml. and 102 Eft fffnesef e t /Molecular and GcNuhrEndocrinology 304 (2009)97-105 Tabic 1 Meanor Rbthe bodyandtissueweightsat IBmonthsofageinintact andovariectomlzed(ovx)femaleCD-I mice. PFOAdose Bodywelghl(it) .. (m*/kg) ; : s ::;`A Ovx VMomlnainMteiErt toterscapuUrbrown ; : ;> :,.r V. wwiht.(fj:;... . 7-v / 7.<, Ovx Relativespleen Relathreliver :, WMgjtiffl1. -a if f ,,` - J j i i c l - T O y x . . j v ^ a c r ; "______.Owe o r- 0.01 0.1 03 1' 3 5. S4$0'rti3 '/5735JS7; 037101 039.1-OIB ;p 3 p - M 6 \- 4 3 0 * (UO . 490*044 sM-'- 'a36;oo4:. \ \ o M S a & t g g * ' > # d i i i i a s . S4J60`llT7V 56.00*134 M m'U ia id ife io la m ,.-,... 596*063 045*032: ; <: 4 J 8 * 0 2 t O ^ i ' M /jjiS S o j . '.:4 3 o; 3 1 4371022 .56.15 * `t35 61^*553;' i d i o t e 5B2p.77'i;-ia9:*.Q ra s a o p p ,,- ..0 3 0 1 ^003 i o j a * o r i v i m V o i o -3 5 5 * 0 2 ?.' .227 nel .DC.:''' .. k " ` ` n e \V ''-'' 4937*151* 551315.76 io fc s *. $61167 ^ b jf c * ofis 02ill20 i 0 5 2 * 0 3 4 H - : ^ l i ^ ^ : :4 3 ^ 0 a 4 - 355*033 nc. Denotesnot collectedfromthiidosegroup. * p<031 vs.control. p-035-037. c Relativeweight(organweightaspercentofbodyweight). there was no significant difference in serum PFOA concentrations across dose groups (data not shown). There were no significant differences in serum estradiol levels in developmentally exposed females at 18 months when compared to controls (non-cycling: mean range across doses from 12.9 to 15.8 pg/snl). Tissue weights from 18-month-old animals (intact and ovx) are shown in Table 1. To determine if the weight of fat depots was altered m old animals due to developmental PFQA exposures, the retroperitoneal abdominal white and interscapular brown fat pads were collected and weighed. Abdominal white fatweight and rela tive white fat weight both showed significant decreases vs. control (p < 0.05) at 1 and 5 mgPFQA/kg. W hite fat weights were not col lected for3 mg/kg PFOAanimals.At 18 months, interscapularbrown fat weight and relative brown fat weight both showed significant increases above control(p<0.05) at 1 and 3 mgPFQA/kg.The spleen was quite variable in weight among the differenttreatment groups, but there was a significant difference in spleen weight and relative spleen weight vs. control at 3 mg PFQA/kg (p <0.05). Finally, at 18 months, no significant differences in liver weight or relative liver weight were detected. reach statistical significance. When comparing the body weights of animals in the ovx study by treatment group, over time (4 weeks to 18 months), using statistical methods consistent w ith those used for intact animals, there was no effect of PFOA(Fig. 6B). Compar ison of ovx animals to intact animals at 20-29 weeks, as shown in Fig. 6A. demonstrates an absence o fbody weight gain over con trol in the ovx animals treated w ith PFOA. PFQAexposure did not stimulate increased weight gain (above that of control ovx) at any developmental exposure level in the absence of the ovaries (also seen in fig SB).The ovx animals were siblings to the intactanimals in this study. The ovx animals were also assessed at 18 months.Developmentally PFOA-exposed ovx animals showed no significant differences in body weight when compared to control ovx fem a les (con trol mean52.75.67; highest mean. 1 mgPFOA/kg61.533; Table 1). 3.1.6. Effect o f ovariectom y on tissue a n d b o d y w eig h t gain A group o f developmental^ PFOA-exposed animals (0.0.01.0.1, 0.3. 1. and 5mgPF0A/kg) were ovx at weaning and their body weight gain and adult health was assessed until they reached 18 months of age. At mid-life the weight of the control ovx females was expected to be greater than that o f the sham-operated, intact controls (Fig. 6A; set ofbars at 0 mg/kg), but the variance in the ani mal weights was appreciable and therefore the differences did not o o.et <u as t PFOA Dose(mg/kg BW) Fig. 5. Survival curves for developmenttty PFOA-exposed female mice (0-1 mgPFOA/kgXAlthough fair number of PFOA-exposed animals die early, a lifelest(SAS)analysisdetectednosignificanedecreasein Urnetodeath.Thereasons for early life mortality are under Investigatimi. Fig. 6. (A)PFQA-depcndencchangesingroup meanbodyweight ofintactandovx female offspring at 20-29 weeksof age. There was no change in bodyweight of ovxanimalsacrossPFQAexposures.(B)Dose-dependentquadratic regressionfitto repeated measures of body weight in ovx female mice. Unlike intact siblings, no significant differenceswere seen betweendnsegroupsinthe ovxanimals. P. Nines et uL/Molecular and Cellular Endocrinology304 (2009)97-105 103 As w ith intact siblings, the tissue weights of ovx animals are reported in detail in Table 1. In ovx animals, neither abdominal white fat pad weight, nor relative abdominal white fat pad weight, were significantly different from ovx or intact control levels. This varies slightly from intact siblings, where the white fat pad was significantly decreased in size; although, in animals that weighed significantly less than intact controls. Among PFOA-exposed ovx animals, both interscapular brown fat weight and relative brown fat weight (data not shown) showed significant increases above control ovx levels at 1 mgPFOA/kg (p <0.05); no other dose groups showed a significant increase. This is similar to the effect seen in intact animals, and was significant at the same dose.Spleen weight (data not shown) and relative spleen weight in ovx animals was highly variable at 18 months, and showed decreases, albeit not highly significant, at the 1 and 5 mgPFOA/kg doses (p -0 .0 6 and p - 0.05, respectively: Table 1). 1 and 3 mgPFOA/kg (not 5 mg/kg) were the doses in the intact animals showing the largest decreases in relative spleen weightcompared to controls. Finally,relative liver weight showed no significantdifferences across dose groups when compared to ovx control. 3.1.7. Lack o f effectsfro m a d u lt PFOA exposure At 18 months of age, body and tissue weights were recorded in adult PFOA-exposed mice. Adult PFOA exposure had no effect on terminal body or organ weights. When a comparison of data from 18-month-old adult intact and developmental^ exposed ani mals in the 0, 1 and SmgPFOA/kg dose groups was made, body weight, brown fat weight, and white fat weight ofthe 1 mgPFQA/kg developmental^ exposed animals were significantly higher than the same dose in adult-exposed animals (data not shown). 4. Discussion These studies demonstrated the effects of developmental PFOA exposure on CD-I female mouse body and organ weight, asw ell as serum leptin and insulin in adulthood, lit the developmental PFOA studies, a dose-dependent dichotomy of phenotypes was present in intact female mice; latent effects present following high doses were not present in mice exposed to low-dose PFOAand vice versa. Although there was no detectable change in body weight neonataliy, low-dose PFOA exposures (0.01. 0.1, or 0 3 mgPFOA/kg) led to significantly increased mean weight and rate of weight gain in mid-life (up to and including37 weeks of age) and a coincident sig nificant elevation o f serum leptin and insulin values between 21 and 33 weeks(0.01 and 0.1 mgPFOA/kg). Our tow-dose hormone data indicate potentially important metabolic changes that mechanistically support the findings of increased weight in the lower dose groups. Previous dosimetry work in our lab has shown that in utero exposure to PFOA in the mouse translates into an extended developmental exposure period via lactational exposure (ail ofgestation and nearly 3 months postnatally; W hite et al,, 2009: W olf et a!., 2007; Fenton et al.. 2009). This long exposure may lead to reprogramming/metabolic events that govern fat metabolism or appetite control Although we were unable to perform some of the other end points of interest dur ing this tim e period of greatest weight gain, our findings relating leptin and insulin concentrations to the time ofoverweightin PFOAexposed mice support our theory. Other environmental chemicals, termed environmental obesogens (dietylstibestrol (DES), 20H-E2, 40H'E2. genestein and bisphenol A), have been shown to induce obesity in adulthood after low-dose developmentalexposure,while inducing weight loss at higher doses (Griin et a l, 2006; Newbold et al, 2005; Miyawaki etal.. 2007) and are reviewed further within this issue. Serum leptin was significantly elevated in m id-life in the lowdose PFOA-exposed groups. This effect occurred at the same PFOA dose range as overweight in these animals, congruent with a leptin-resistancemechanism ofaction foroverweight,aspreviously reported in humans (Considine et a l, 1996). Others have reported increased leptin with developmental exposure to environmental obesogens including DES(Newbold et al.. 2007). Low-dose (0.01 and 0.1 mg PFQA/kg)developmentalPFOAexpo sure that led to increased serum leptin and body weight also increased insulin values at 21-33 weeks. This suggests that the insulin resistance mechanistic pathway could also be affected and play a role in developmental PFOA exposure-induced overweight in mice, in an insulin resistance scenario, there are raised plasma glucose levels (elevated, but not significant, at 15-16 weeks in our study), reflecting the loss of a post-challenge peak in insulin response (reviewed in Montecucco et al,, 2008). Insulin resistance is known to be associated with excess abdominal fat in normal and overweightwomen (Carey etaL, 1996).High plasmalevelsofinsulin andglucose,dueto insulin resistance,are often associated with type II diabetes and metabolic syndrome in humans, and thus this effect of low-dose PFOAdevelopmental exposure and its association with increased serum insulin are important The ovx data were difficult to interpret The lack of additional weight gain with developmental PFOAand ovx may reflect a "ceil ing effect" or that ovx-induccd weight increases may have masked any effecto fPFOA.Alternatively, as weight gain and metabolic hor mones can be regulated by estrogens, the role of the ovaries in developmental effects o f PFOAwas explored by using ovx animals. The potential importance of the ovary in the effects of PFOA was based on the observation that LH-transgenic (overexpressing)mice (Kero et aL. 2003) were phenotypicafiy simitar to ours (increased bodyweight, increased brown fat depots, and predominant ovarian cystsnot discussed in this paper). We hypothesized that removal of the LHtarget(the ovary) in our study may revealthe mode of action for PFOAeffects forthe increase in brown fatand possiblythe exces sive weight gain. Ovx animals typically gain body weight in excess vs. intact animals (Kamei et a l, 2005). The critical role of the ovary in weight gain of intact PFOA-exposed females beyond that of ovx treatment-matched siblings in the 0.01 and 0.1 mg PFQA/kg groups was novel and signifies the ovarian axis as a potential mediator of PFOA-dependent mid-life weight changes. Another potential mediator of these intertwined low-dose PFOA-induced effects is the peroxisome proliferator-activated receptor (PPAR) activation pathway. PPAR gamma (PPAR-'y) and PRARalpha (PPAR-a) are involved in lipid metabolism in adipocytes and liver/skeietal muscle, respectively(reviewed in Medina-Gomez et a l, 2007; Abbott, 2009). These PPAR isofbrms are known to influence iipogenesis/weightgain and have been shown to be regu lated by environmental compounds such as tributyitin (GrOn et al, 2006: reviewed in this issuei Weightlosseventsin leptin-deficient. obese, and insulin-resistant mouse models have coincided with PPAR-regulated changes in gene expression (Holvoet, 2008). A down-regulation ofPPARisoforms involved in energy expenditure, lipogenesis or fatty acid synthesis have been reported in adipose and skeletal muscle of ovarieetomized mice (Kamei 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 opmental toxicity in mice (Takacs and Abbott, 2007; Abbott et al., 2007; Abbott, 2009). if PPAR activation via receptor binding is a primary mode of action for body weight effects following PFOA exposure, the decrease in the PPARreceptorsfollowing ovariectomy and decreased circulating estrogens may explain the lack of effect ofPFOAin ovx mice. However.PFOA-induced consequencesofPPAR activation following a developmental exposure are just beginning to be evaluated. After 40 weeks of postnatal age. an increase in mortality was detected in all animals. There are previous reports in the literature ofincreased mortality in non-treated CD-I mice,attributed primar 104 EJi Hbws et qL/Motoculorend CetMarEndocrinohgy 304 (2009) 97-105 ily to thymic lymphomas (Son, 2004: Taddesse-Heath et at., 2000). Because of this confounding circumstance, repeated measures of body weight were only followed out to 37 weeks of age. The other half o f the phenotypic dichotomy caused by devel opmental PFOA exposure was also novel Developmental exposure to higher doses o f PFOA (1, 3 and 5 mg PFOA/kg) led to a vastly different phenotype from low-dose PFOA exposure. This effective PFOA dose dichotomy may manifest itself in our study via unique modesofaction;the animals w ith highestdose(s) erfdevelopmental PFOAexposure have decreased early life bodyweight and terminal body weight (5 mgPFOA/kg) w ith significant decreases in white fat weight at 18 months ( l and 5 mg PFOA/kg). significant increases in brown adipose (1 and 3 mgPFOA/kg). and significant decreases in spleen weight (3 mgPFOA/kg) findings that are absent with the lower doses of PFOA. Others have reported dose-dependent loss of white tis sue adiposity in adult male mice after PFOA exposure (0.02X PFOA weight/chow weight, which translated to approximately 32 mg PFOA/kg BW daily) w ith fat loss, without fat cell number loss, that is PPARy-independent w ith p-adreneigic activation (Xie et a l, 2 002). In that same study, investigators also reported white fat and body weight decrements at higher doses that were absent at lower doses. Yang et aL (2002) showed PFOA-dependent weight loss was abrogated in PPAR-a null mice, indicating that PPAR-a is a probable regulator of weight loss in the high dose animals. In subsequentstudies, Xie et al. (2003) showed that after cessation of exposure of adult mate animals to PFOA(0.02% PFOAweight/chow weight; 32 mg PFOA/kg BW) daily for 7 days followed by 10 days recovery, weight kiss and w hite adipose levels returned to base line, which confirms the importance of developmental exposures for the latent effects reported here. In our model w ith developmen tal PFOAexposure we seepermanentweight lossand white adipose tissue loss a t the high dose of PFOA. However, there may be merit in further exploring these mechanisms of action, as ^-adrenergic receptor upregulation is also associated w ith increased brown fat mass in w inter-acclimated animals (Feist, 1983), and this tissuewas associated w ith high dose (and not low dose) effects in both intact and ovx animats in this study. Although we suspected alleviation of effect in the brown fat pad by eliminating the ovary (based on phenotypes in Kero et aL. 2003), significant increases in brown fat were seen at 1 mg PFOA/kg in both intact and ovx animals. At the 18-month time point, some endpoints remained unchanged across dose groups including liversbe. Earlier work has shown significant hepatomegaly after developmental PFOA expo sure (1 and 3 mgPFOA/kg) observed out to at least 3 weeks after birth (the latest time point evaluated: W olf et al., 2007; White et al., 2007).The transientnature ofhepatomegalyhasbeen illustrated in other acute adult exposure studies (reviewed by Lau et al.. 2007), and is further confirmed in these studies (intact and ovx). A final important component of these studies evaluated adult vs. developmental exposure to PFOAon body tissue weights. These data suggest that the timing of dosing (adult vs. developmental 17-day PFOA exposure) was critical for latent effects. There was no effect of 17-day adult PFOA exposure on any endpoint in this study(early life orlatent) when compared to age-matched,vehidegavaged controls. In conclusion, the timing and dose o f PFOA exposure for induc tion ofdichotomous, persistent, adult health effects in CD-I female mice are critical. Developmental, low-dose PFOA exposure led to increased weight in adults, w ith increased serum insulin and leptin, a health effect not seen in high dose animals. No observable adverse effect levels (NOAEL) for body weight gain, serum Ieptin and insulin concentrations were not determined in this study: but 0.01 mg PFOA/kg had a significant impacton these particularly sen sitive end points. The ovary appeared to play an important rote in the overweight effect in m id-life, and it is proposed that there is a common mode of action, potentially dysregulation of PPAR and its signaling through ovarian hormones, that may be responsible for these low-dose health effects. Further studies addressing long term PFOA-inducedhealth outcomes in mice should focusattention on internal dose relative to the low-dose health effects seen in this study, asw ell as the mechanisms ofaction, so that any relevance to human health effects can be addressed. Acknowledgem ents We would like to thank Broker Optics. Inc for the use of the Broker M inispec mq 7.5 LF50 Live Mouse Analyzer and Harry Xie and Basil Desousa o f Broker Optics, In c for their technical assis tance.We would like to acknowledge Antonia Calafat and her labo ratory staff, Kayoko Kato and Zsuzsanna Kuklenyik, in the Division of Laboratory Science, National Center for Environmental Health, Centers forDiseaseControland Prevention forthe analysisofserum PFOA concentration from 18-montb-oid developmentally exposed female mice; Donald Doerfler, Experimental Toxicology Division, US. EPA, andJudy Schmid. Reproductive Toxicology Division (RTD). US. EPAfortheir statisticalsupport: Deborah Best RTD.forconduct ing the estradiol assays; Veronica Luzzi. David Gibson and staff at the Core Laboratory forClinkal StudiesatWashington University in S t Louis, MO, for performing the serum insulin assays, and finally. Dr. David Kurtz and the technical staff at New Year Tech, In c for their exceptional animal care during these lengthy studies. 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