Document 8J6KaozadwpRNvvdxv07ZKVK

A t ^ 3-6 "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 T a ft / 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 ok,o cr r ~ V.< p p t S'S'O <r n G< 5s 7P CO er* o GO Internal Revenue Service Circular 230 Disclosure: As 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 message 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 message are prohibited. If you received this transmission in error, please notify the sender by reply e-mail and delete the message 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 CB' f P-2 Taft/ Taft Stettinus & Hollister LLP 425 Walnut Street, Suite 1 8 0 0 /Cincinnati, OH 45202-3957 /Tel: 5)3 .3 81 .2 83 8/Fax: 513.381.0205 / www.taftlaw.com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix / Beijing Robert A. S ilott 513-357-9638 bilott@taftlaw.conn June 12, 2009 o v> Cr" t FEDERAL EXPRESS EPA Docket Center, MC 2822T U.S. Environmental Protection Agency EPA W est, Room 3334 1301 Constitution Avenue, NW Washington, D.C. 20004 ~n er o Re: Submission to IRIS and AR-226 Database For PFOA/PFOS: EPA-HQORD-2003-Q016 To IRIS Database for PFOA/PFOS: In response to the Notice issued by USEPA on February 23, 2006, regarding USE PA's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("PFOS") within the Integrated Risk Information System ("IRIS"), 71 Fed. Reg. 9333-9336 (Feb. 23, 2006), we are submitting the following additional information to USEPA for inclusion in that review, and for inclusion in the AR-226 database: 1. Fenton, S.E., et al., "Analysis of PFOA in Dosed CD-1 Mice Part 2: Disposition of PFOA in Tissues and Fluids From Pregnant and Lactating Mice and Their Pups," Reprod. Toxicol. (2009), doi:10.1016/j.reprotox.2009.02.012; 2. von Ehrenstein, O.S., et al., "Polyfluoroalkyl Chemicals 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 (PFOA) in Female CD-1 Mice: Levy Doses Induce Elevated Serum Leptin and Insulin, and Overweight in Mid Life," 304 Molecular & Cellular Endocrinology 97-105 (2009). 11434742.1 CONTAINS NO CBI 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 Werner (ATSDR)(w/ end.) (via U.S. Mail) P-3 {W1405808.1} Accepted Manuscript Title: Analysis o f PFOA in Dosed CD-I M ice Part 2: Disposition o f PFOA in tissues and fluids from pregnant and lactating mice and their pups Authors: Suzanne E. Fenton, Jessica L. Reiner, Shoji F. Nakayama, Am y D. Delinsky, Jason P. Stanko, Erin P. Hines, Sally S. W hite, Andrew B. Lindstrom, Mark J. Strynar, Syrago-Styliani E. Petropoulou PH: DOI: Reference: S0890-6238(09)00040-9 doi: 10.1016/j.reprotox.2009.02.012 RTX 6230 To appear in: Received date: Revised date: Accepted date: Reproductive Toxicology 4-2-2009 20-2-2009 25-2-2009 Please cite this article as: Fenton SE, Reiner JL, Nakayama SF, Delinsky AD, Stanko JP, Hines EP, White SS, Lindstrom AB, Strynar MJ, Petropoulou S-SE, Analysis o f PFOA in Dosed CD-I Mice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups, Reproductive Toxicology (2008), doi: 10.1016/j.reprotox.2009.02.012 This is a PD F 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. u-5: 1 Analysis o f PFOA in Dosed CD-I Mice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups. Suzanne E. Fenton"*, Jessica L. Reinerb, Shoji F. Nakayamab, Amy D, Delinsky, Jason P. Stanko", Erin P. Hines", Sally S. White"'d, Andrew B. Lindstrom6, Mark J. Strynar0, and Syrago-Styliani E. Petropouloub* *Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. ERA, 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 and Atmospheric 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 277II USA Tel: 919-541-5220 Fax:919-541-4017 E-mail: fenton.suzanne@.epa.gov 2/20/2009 Running title; PFOA disposition in lactation Abbreviations ANOVA analysis o f variance BW body weight GD gestational day LOD limit of detection LOQ limit o f quantitation MS mass spectrometer PFAA perfluoroalkyl acid PFOA perfluorooctanoic acid PFOS perfluorooctane sulfonate PND postnatal day SEM standard error of the mean UPLC ultra performance liquid chromatography 2/20/2009 3 Abstract Previous studies in mice with multiple gestational exposures to perfluorooctanoic acid (PFOA) demonstrate numerous dose dependent growth and developmental effects which appeared to worsen if offspring exposed in ulero 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 o f dose. The distribution of milk:serum PFOA varied by dose and time, but was typically in excess o f 0.20. These data suggest that milk is a substantial PFOA exposure route in mice and should be considered in risk assessment modeling designs for this compound. Key words: PFOA; serum; amniotic fluid; urine; milk; mammary gland; dosimetry; disposition . 2/20/2009 p.8 4 1 1. Introduction 2 Perfluorooctanoic acid (PFOA) is a member o f the perfluoroalkyl acid (PFAA) 3 family of man-made, fluormated organic compounds used in a number of consumer 4 goods and industrial surfactants due to their grease and water-rcpellant properties. The 5 use of PFAAs in many common applications, such as stain repellants for clothing, 6 carpeting, and upholstery, and the stability o f the carbon-fluorine bond have made them 7 ubiquitous in the environment. The predominant route o f exposure in North American 8 and European consumers is likely oral intake, including drinking water, while inhalation 9 and dermal absorption comprise routes o f lesser exposure [1-5]. 10 PFAAs are persistent, readily absorbed, not known to be metabolized, and are 11 poorly eliminated, with half-lives in humans ranging from roughly 4-8 years [2-4]. In 12 fact, the arithmetic and geometric mean half-lives of serum elimination, respectively, 13 were 5.4 years [95% confidence interval (CX), 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 contort of PFOA and related 18 chemicals was recently initiated [1], PFAAs are continually detected worldwide in both 19 human and wildlife samples [3,6-9]. A recent analysis of American Red Cross blood 20 donors indicated a reduction o f 60% in blood perfluorooctane sulfonate (PFOS) and 25% 21 in blood PFOA levels between the years 2000 and 2006[ 10]. However, while the 22 production 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 perfiuorinated compounds produced 2/20/2009 Page 4 o f 32 P -9 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 the 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 gam, 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 of the nursing offspring. White et a t [ 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 PND10 14 and PND20. In a cross-foster study utilizing CD-I mice, Wolf et al. [16] reported that 15 although in ulero exposure to 5 mgPFOA/kg BW from GD1-17 in the absence of 16 lactational exposure was sufficient to induce postnatal body weight deficits and 17 developmental delays, pup survival from birth to weaning was affected only in those both 18 in utero and lactationally exposed. Furthermore, recent studies [15] have shown that 19 unexposed neonates lactationally exposed to PFOA quickly developed mammaiy gland 20 growth deficits and that control dams nursing in wtero-exposed pups (dams exposed via 21 pup grooming) demonstrated slowed differentiation o f their own mammary glands that 22 was evident in whole mount preparations of the tissue by the 5* day of lactation. These 2/20/2009 Page 5 of 32 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 al. [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 of 12 human milk samples collected 8 from 1996-2004 in Sweden at a concentration o f492 pg/ml (0.492 ng/ml; [18], and a 9 mean 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 in the Kamnan et al. [18] study, these researchers reported a significant milk to 14 serum correlation (r2 = 0.7-0.8, /K0.05) for other PFAAs detected. Furthermore, Tao et 15 al. [19] suggested that there m aybe preferential partitioning o f 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 o f 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 '/ 2-life o f about 15 days [13], the study [22] indicated 2/20/2009 Page 6 of 32 a *ig;f*j *? 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 o f 5 PFAAs in animal studies, coupled with their detection m human cord blood and milk, 6 supports the need for examining the disposition o f PFAAs during pregnancy/lactation in 7 an appropriate animal model in order to fully establish the association between 8 prenatal/neonatal exposure and offspring effects. While other studies have examined the 9 pharmacokinetics o f PFAAs in limited contexts, little data currently exist on the 10 disposition o f PFOA in pregnant and lactating mice or their offspring. We recently 11 developed an analytical method for the analysis of PFOA in mouse serum, urine, milk, 12 mammary tissue, amniotic fluid, and pups [24]. Utilizing these methods, we report here 13 date on the distribution of PFOA in various matrices o f pregnant and lactating CD-1 14 mice, as well as die serum concentration and total body load of their offspring, following 15 a single exposure o f PFOA on GD 17. These date 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 p. 12 8 1 2, M aterials and methods 2 2,1 Chemicak 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, hi 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 (=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, PMI 18 Nutrition International LLC, Brentwood, MO) and tap water ad libitum (both contained 19 PFOA at concentrations below the LOD). Animal facilities were controlled for 20 temperature (20-24C) and relative humidity (40-60%), and kept under a 14:10-h light21 dark cycle. Mice (=25/dose group) received either 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 GDI 7. 23 2/20/2009 Page 8 of 32 p. 13 s*islla=I 9 1 2.3 Animal Assessments and Sample Collection 2 Live dam body weights were recorded on GDI 7, GDI 8 (prior to parturition), 3 PND1 (day after parturition), and PNDs 2,4, 8 ,1 1, and 18. On GDIS, 24 hr after the 4 PFOA exposure, five dams in each dose group were sacrificed and trunk blood, urine, 5 amniotic fluid (fluid immediately surrounding each fetus), and the 4th and 5th mammary 6 gland were collected. Liver weight, total number o f fetuses (live, dead, or resorbed), and 7 fetal weights were determined. One entire fetus from each litter was euthanized by 8 decapitation and quick frozen on dry ice in a 15 ml screwcap vial. Remaining fetuses 9 were quickly euthanized and discarded. The dam mammary gland, urine, and amniotic 10 fluid were kept on ice, and then frozen until assayed. The trunk blood was allowed to 11 clot; serum was collected after centrifugation and frozen until assayed. All samples were 12 kept frozen a t -80 C. 13 A similar routine was followed on PND1 (48 hr after exposure, n=5 dams/dose 14 group). Weights of the dam, pups, dam liver, and the number of live pups in each litter 15 were recorded. A single pup from each litter was weighed, euthanized, and quick frozen 16 in a collection vial (including all blood). Blood from all remaining pups in each litter was 17 pooled into a single vial, allowed to clot, and separated to serum by centrifugation. Dam 18 and pup serum, dam urine and mammary tissue were frozen until assayed. All remaining 19 litters, in all dose groups, were equalized to 10 pups each on PND1. Biological samples, 20 including a single pup and pup serum, as described for PND1, were also collected on 21 PNDs 4, .8 and 18 (n=5 dams/dose group), at the same time of day. 22 Milk collection was attempted, following administration 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 o f 32 10 1 collected on PNDs 2 ,8 ,1 1 , and 18 following a 2 hr separation of 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, ammotic 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 of urine 13 and miUfJ 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 of the original exposure, was injected and run on the UPLC-MS/MS 17 [24]. Refer to Reiner et al. [24] for method performance and quality control steps that 18 were performed to insure the precision and accuracy 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 10 o f 32 p. 15 II 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 o f each urine sample was 5 prepared and evaluated at 510 nm singly or in duplicates (five duplicates per set of 20 6 samples) according to the manufacturer's instructions. The inter-assay coefficient of 7 variation (CV) ranged from 4.0-6.8% and the intra-assay CV ranged from 0.3-16.1 %, 8 with an average of 4.9%. The assay standard accuracy ranged from 0.2-8.4%. Urinary 9 PFOA is reported as corrected for creatinine concentrations (ng PFOA/g creatinine). 10 11 2.6 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 o f 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; Caiy, NC). Statistical significance was 17 determined using a Proc GLM ANOVA, with a Dunnett's post-hoc comparison, and 18 significance was set at><0.05. 19 . 20 21 22 2/20/2009 Page 11 of 32 p. 16 iSt-. 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 lactaring mice and their offspring. The doses chosen were based on previous reports in 5 CD-I mice [3,14,16] demonstrating developmental health outcomes following multiple 6 gestational PFOA exposures. A single PFOA exposure on G D I7 did not affect the 7 number o f live fetuses (on GDI 8), implantation sites, or live-bom pups (on PND1), or 8 dam body weights (data not shown). Unlike studies using multiple gestational PFOA 9 exposures [13,25], there was no change in pup body weight, dam liver weight, and dam 10 livenBW ratios, within the PFOA dose range administered in this study (Figure 1). The 11 rise in dam livenBW ratio between GD18 and PNDl, which persisted until weaning, was 12 due to the dramatic decrease in body weight at parturition, as this single late gestation 13 PFOA exposure foiled to change mean liver weight in exposed dams, compared to control 14 values, at any time evaluated. 15 16 3.2 PFOA Concentrations Prior to Birth 17 The mean concentration of PFOA in the amniotic fluid and serum of the exposed 18 dams 24 hr after exposure is shown (Figure 2; amniotic fluid controls average 3.8 ng/ml). 19 The average concentration of PFOA detected in dam serum was about twice the amniotic 20 fluid concentration at each dose evaluated (amniotic fluid was 68.8, 51.8, and 40% of 21 dam serum levels at 0.1,1, and 5 mg PFOA/kg BW, respectively). A comparison o f the 22 amount o f PFOA in an entire GDI 8 fetus (body burden/pup+standard error of the mean . 23 [SEM]; Figure 5) to the GDIS PFOA concentration in amniotic fluid (ng/ml; assuming I 2/20/2009 Page 12 o f 32 saSS* 5 s; w m m s p. 17 13 1 ml total volume) reveals 2.3-, 3.1-, and 2.7-fold increased PFOA in the pup vs. the fluid 2 in which it was contained in utero for 0.1,3, and 5 mg/kg dose groups, respectively. 3 4 3.3 PFOA Concentrations in the Dams 5 The serum and urine PFOA concentrations were evaluated in dams that were 6 nursing litters 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-fold and 5.1-fold increases 11 between 0.1 -1.0 mg/kg and 1.0-5.0 mg/kg exposures, respectively). 12 A one-time PFOA exposure of 0.1 mg/kg produced an average dam serum PFOA 13 concentration (Figure 3A) o f 144-226 ng/tnl 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 FNED18, a time when the pups' primary caloric intake 16 came from rodent chow and riot milk. The U-shaped serum concentration curve observed 17 in the 0.1 mg PFOA/kg dose group was also present in the 1 and 5 mg/kg exposure 18 groups. 19 As shown in Figure 3 (A-C; control urine and mammary gland PFOA <LOQ), 20 although the concentrations of PFOA cannot be compared directly between serum, urine, 21 and mammary tissue, due to the difference in units, it was evident that much less PFOA 22 was being excreted in dam urine than was present in dam serum, and that mammary 23 tissue contained a considerable amount of PFOA. While a U-shaped response in dam 2/20/2009 Page 13 of 32 g* -es>tn ar ^i- * * .* r c * . _ in .i. . 1 '.,, ,. feE '~ LS S Kl Pi f?"l i? 1 5 h i '.? s = 'i I S A to ia ifc s lB 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 IS 4 (p<0.05). 5 When aspirated milk PFOA values were evaluated (Figure 3D; 1 control >LOQ), 6 a U-shaped curve over time was again evident. As depicted in Table 1, the percentage of 7 PFOA in milk (compared to serum) was substantial. Comparing the milk concentrations 8 to the closest matched dam serum concentrations (by time), the amount o f PFOA in the 9 milk consistently ranged from 1/10 to 1/2 that 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 o f 33% early and 26% late), 13 while near the peak o f lactation (PND8 and 11), the PFOA milk;serum ratio ranged from 14 11-27% (mean 17.7%). It was not possible to accurately measure the volume of milk 15 obtained at aspiration, but precise weights were compared. On PNDs 2, 8,11, and 18, the 16 average weight of milk obtained via aspiration o f control mice was 0.072,0.1906, 17 0.2547, and 0.0457 g, respectively, demonstrating over a 3.5-fold increase in weight from 18 PND2 to 11 and a 5.6-fold drop from PND11 to 18. 19 20 3.4 PFOA Concentrations in the Pups 2 1 Pup serum PFOA concentration was evaluated on PNDs l , 4, 8, and 18. In 22 comparing the average PFOA concentrations in PND1 pups vs. their respective dams 23 (Figure 4A; whole control pups and control serum < LOQ), it appeared that circulating 2/20/2009 15 1 pup serum PFOA concentrations were significantly higher than those measured in dams, 2 regardless of dose (p<0.05). Although pups possessed a substantially higher serum PFOA 3 concentration than dams, the difference in pup and dam blood volumes at those stages of 4 pup development are considerable. Regardless o f 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 rog/kg 7 and 1.0-5.0 mg/kg exposures, respectively). 8 Unlike their dams, pups did not demonstrate U-shaped serum PFOA 9 concentration curves (Figure 4B). Pup serum PFOA concentrations continued to exceed 10 the average dam serum PFOA concentrations over time, until PND18 when the pup and 11 dam concentrations approached 1:1. When the PFOA concentration (ng/g) was evaluated 12 in whole pups (pup and blood; Figure 5 left panels), a decline in PFOA concentration was 13 detected over time, across all doses. However, when the rapidly increasing body weight 14 o f the pups was taken into consideration to calculate the total amount o f PFOA in the 15 neonate (as shown in Figure 1), a completely different trend was noted (Figure 5 right 16 panels). Regardless of exposure dose, PFOA body burden (adjusted for weight) rose 17 through the peak of lactation and had begun to decline by PND18, demonstrating an 18 inverse U-shaped curve. When the administered PFOA dose and measured body burden 19 in whole pups:(body weight taken b to effect) were compared the administered 20 PFOA:measured PFOA ratio was no longer proportional throughout lactation, and unlike 21 the ratios reported for dam and pup serum PFOA. Mean body burden ratios of 13.2-fold 22 (range 11.1-17.8) and 4.3-fold (range 3.2-5.1) increases between 0.1-1.0 mg/kg and 1.0 23 5.0 mg/kg exposures, respectively, were determined. 24 2/20/2009 Page 15 of 32 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-bome (transplacental) transfer. Another important discovery 6 is the U-shaped PFOA concentration over time, regardless of dose, in the dam mammary 7 tissue, milk, and serum. This unique PFOA response was not detected in pups or pup 8 serum, and was evident to a lesser extent in the dams ' urinary excretion curves. However, 9 when PFOA body burden in whole pups was the unit o f measure, an inverse U-shaped 10 curve was apparent, and the PFOA burden of pups is proposed to increase due to milk11 borne PFOA intake. 12 The decline in concentration seen in the milk, mammary and serum U-shaped 13 curves is hypothesized to be due to hydro-dilution associated with increased blood and 14 milk volumes. Several physiological conditions are changed during lactation that have 15 been well documented in rats and directly relate to mice as their lactation period is of the 16 same length. A decrease in total plasma proteins due to increased Mood volume, cardiac 17 output, and blood flow to certain tissues, such as the mammary gland has been reported 38 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 JO [27] and the rat mammary 20 gland reaches its maximum size (as % body weight) by PND15 [26], with a steep rise in 21 size from PND5-15. Rat mammary gland blood flow and volume of milk produced are 22 directly related, when measured on PND15 [26]. Total serum proteins are lower in 23 lactating rats that those measured in non-lactating rats [27], and in humans, serum 2/20/2009 17 albumin concentration decreases during pregnancy and early lactation [28], Further, at 14 d postpartum, the cardiac output o f lactating rabbits was 30% higher than that in nonlactating animals, and the mammary gland was the only organ shown to increase in weight, relative to body weight [29]. Although a complete set o f data that could address die exact reason for the Ushaped curves during lactation was not collected in this study, the aspirated milk weights did reveal a dramatic increase in milk volume (assumed due to weight change) from PND2 up to the peak o f lactation (PND11). This dramatic change in volume (weight) may explain the decrease in milk PFOA concentration seen between PND2 and PND11. PFOA also appears to concentrate in serum and milk near, the end o f lactation (PND18, for example) when pups are eating more chow and suckle less often. Mammary gland blood flow has been reported to decrease by half in a 24 hr period, when suckling rat offspring are removed from the dam [26], and this fall in mammary blood flow is directly associated with decreased cardiac output and % blood flow used by die mammary gland. In this study a precipitous drop in weight o f milk collected between the peak o f lactation and PND18 was noted, indicating a rapid decrease in milk volume. Therefore, the Ushape of the dam PFOA curves are proposed to be driven by physiological dilution and concentration of the PFOA load over the period o f lactation, reaching the greatest dilution at or near the peak of lactation when the milk volume produced by the dam and consumed by the pups is the greatest. Increased consumption of milk up to PND11 likely directly contributed to the accumulation o f body burden in the pup over this life stage. A significant contribution o f milk-borne PFOA transfer in CD-I mice was detected in these studies. Previous reports in rats [22] and humans [18] have estimated 2/20/2009 Page 17 of 32 p. 22 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 more than 0.5 to 0.1 in mice, 3 ftepgnding 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 of increased milk:serum ratios appeared, regardless o f 8 dose, with a substantial peak in 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 (he significant PFOA concentrations in the PND 1 11 mammary gland, that substantial milk PFOA concentrations would have been evident on 12 PND1, as well, primarily due to being condensed in small milk volumes. 13 In previous reports by Lau [13], Wolf [16], White [14] and co-workers, decreased 14 body weight gain and neonatal mortality were evident on several days just after birth in 15 CD-I mouse litters gestationally exposed to 3 mg/kg PFOA and higher. In fact, in a 16 cross-foster study [16] demonstrating decreased body weight gain at 5 mg/kg from in 17 utero exposure only, significant decreases in body weight gain were detected in the 3 18 mg/kg dose group only when in utero exposed mice were also allowed to nurse from a 19 PFOA exposed dam. Even at 5 mg/kg, there was no evidence 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 = 'iz8&1sivBw^*E>cfiC83lr.l?3i; iE .M51 p. 23 19 1 outcomes suggest that the milk of gestationally PFOA-exposed mice was a major source 2 of continued exposure to this compound for the developing pups. 3 As expected, large differences in dam and pup serum PFOA concentrations from 4 those previously reported [14,16] were noticed, and those differences bring to light the 5 issue of single vs. multiple dose kinetics. As noted for PFOS, single dose kinetics may 6 differ substantially from those involving repeated doses [30]. Concentration dependent 7 changes in clearance can result in discrepancies between single and repeated dose 8 kinetics. 9 A limited number o f 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 milktserum relationship of PFOA transfer [18]. The reported 0.01 15 (l/100,b) relationship was determined from a single voluntarily contributed sample at 3 16 weeks postpartum. According to the mouse milk:serum PFOA distribution over time that 17 we report herein, the values reported in one human [18] and rats [22] may not be 18 representative of the PFOA distribution to milk throughout lactation in those species. 19 In conclusion, these studies confirmed and further defined considerable PFOA 20 exposures to mouse offspring following a single gestational exposure. They also 21 demonstrated the accumulation of chemical over time in whole pups, which likely results 22 from milk-bome 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 SXl'KW1 # -ears *"S' " .a ~ TMs a. ;jg: t. ', , * j j i L! K R i e -^ jg Sr. ^*r *- . t l i * . " ! j S . 1* JEl. ' p. 24 ? # = .'-* a^:$8:j:: 20 1 circulating serum PFOA concentrations of 44-216 ng/ml in dams and 117-326 ng/ml in 2 pups; values similar to or lower than serum PFOA concentrations 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 of 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 the National Health and Environmental Effects Research 19 Laboratory, US Environmental Protection Agency (EPA), and approved for publication. 20 Findings in this report are those of the authors and approval does not signify this report 21 reflects EPA policy. The use o f trade names or commercial products does not constitute 22 endorsement or recommendation for use. 23 24 2/20/2009 Page 20 of 32 p. 25 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/opptjntr/pfoa/pubs/pfoastewardship.htm. Accessed 2/3/2009. 5 6 [2] M E . Andersen, J.L. Butenhoff, S.C. Chang, D.G. Farrar, G.L. Kennedy, Jr., C. 7 Lau, G.W. Olsen, J. Seed and K.B. Wallace, Perfluoroalkyl acids and related chemistries8 -toxicokinetics and modes o f action, ToxicoJ 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 mid toxicological findings, Toxicol Sci, 99 12 (2007), 366-394. 13 14 [4] G.W. Olsen, J.M. Burris, D.J. Ehresman, J.W. Froehlich, A.M. Seacat, J.L. 15 Butenhoff and L.R. Zobel, Half-life of serum elimination o f perfluorooctanesulfonate, 16 perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production 17 workers, Environ Health Perspect, 115 (2007), 1298-1305. 18 19 [5] D. Trudel, L. Horowitz, M. Wormuth, 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. Gunige, 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. Olsen, 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 22 1 2 [11] J.L. Butenhoff, D.W. Gaylor, J.A. Moore, G.W. Olsen, J. Rodricks, J.H. Mandel 3 and L.R. Zobel, Characterization of risk for general population exposure to 4 perfluorooctanoate, Regul Toxicol Pharmacol, 39 (2004), 363-380. 5 6 [12] C. Lau, J.L. Butenhoff and J.M. Rogers, The developmental toxicity 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, A.M. Calafat, Z. Kuklenyik, L. Villanueva, R.D. Zehr, L. Helfant, 14 M.J. 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. Calafat, E.P. Hines, J.P. Stanko, 19 C.J. Wolf, B.D. Abbott and S.E. Fenton, Effects o f perfluorooctanoic acid on mouse 20 mammary gland development and differentiationresulting from cross-foster and 21 restricted gestational exposures, Reprod Toxicol (2008). 22 23 [16] CJ . Wolf, S.E. Fenton, J.E. Schmid, A.M. Calafat, Z. Kuklenyik, X.A. Bryant, J. 24 Thibodeaux, K.P. Das, S.S. White, C.S. Lau and B.D. Abbott, Developmental toxicity of 25 perfluorooctanoic acid in the CD-I mouse after cross-foster and restricted gestational 26 exposures, Toxicol Sci, 95 (2007), 462-473. 27 28 [17] M.K. So, N. Yamashita, S, Taniyasu, Q. Jiang, J.P. Giesy, K. Chen and P.K. Lam, 29 Health risks in infants associated with exposure to perfluorinated compounds in human 30 breast milk from Zhoushan, China, Environ Sci Technol, 40 (2006), 2924-2929. 31 32 [18] A. Karrman, I. Ericson, B. van Bavel, P.O. Damerud, M. Aune, A. Glynn, S. 33 Lignell and G. Lindstrom, Exposure of perfluorinated chemicals through lactation: levels 34 o f matched human milk and serum and a temporal trend, 1996-2004, in Sweden, Environ 35 Health Perspecl, 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) in relation to weight and size at birth, Environ 44 Health Perspect, 115 (2007), 1670-1676. 45 2/20/2009 Page 22 of 32 ?5S p i r 1i t'a== p. 27 23 1 [21] C. Fei, J.K. McLaughlin, R.E. Taronc and J. Olsen, Fetal growth indicators and 2 perfluorinated chemicals: a study in the Danish National Birth Cohort, Am J Epidemiol, 3 168 (2008), 66-72. 4 5 [22] P.M. Hinderliter, E. Mylchreest, S.A. Gannon, J.L. Butenhoff and G.L. Kennedy, 6 Jr., Perfluorooctanoate: Placental and lactational transport pharmacokinetics in rats, 7 Toxicology, 211 (2005), 139-148. 8 9 [23] J.P. Vanden Heuvel, B.I. Kuslikis, M.J, Van Rafelgbem and R.E. Peterson, Tissue 10 distribution, metabolism, and elimination o f perfluorooctanoic acid in male and female 11 rats,JB iochem 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 MJ . 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 mid C. Lau, Perfluorooctanoic acid induced 20 developmental toxicity in the mouse is dependent on expression of peroxisome 21 proliferator activated receptor-alpha, Toxicol Sci, 98 (2007), 571-581. 22 23 [26] A. Hanwell and J.L. Linzell, The effects of engorgement with milk and of 24 suckling on mammary blood flow in the rat,JP hysiol, 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, R.J. Patterson and G. Levy, Seram protein binding of drugs 30 during and after pregnancy in humans, Clin Pharmacol Ther, 28 (1980), 253-261. 31 32 [29] C.S. Jones and D.S. Parker, Mammary blood flow and cardiac output during 33 initiated involution 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 perfluorrioctane 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. Demmehnair, 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, In tJH yg 46 Environ Health, 211 (2008), 440-446. 2/20/2009 Page 23 o f 32 1 2 [33] O.S. von Ehrenstein, Fenton, Suzanne E., Kato, Kayoko, Kuklenyik, Zsuzsanna, 3 Calafat, Antonia M., Hines, Erin P ., Polyfluoroalkyl Chemicals in the Seram 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 Med, 48 (2006), 759-770. 9 10 [35] E.P. Hines, White, Sally S., Stanko, Jason P., Gibbs-Floumoy, Eugene A., Lau, 11 Christopher, Fenton, Suzanne E ., Phenotypic Dichotomy Following Developmental 12 Exposure to Perfluorooctanoic Acid (PFOA) in Female CD-I Mice: Low Doses Induce 13 Elevated Serum Leptin and Insulin, and Overweight in Mid-life, Molec Cell Endocrinol, 14 In Press (2009). 15 16 17 2/20/2009 p. 29 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 of live pups or fetuses (not shown) were also 7 unchanged by a single PFOA exposure. Data are shown as Mean + SEM or as a mean 8 ratio. 9 Figure 2. Comparison of gestation day (GD)18 dam serum and amniotie fluid PFOA 10 concentrations. PFOA concentrations were significantly higher in dam serum than 11 amniotie fluid at all doses evaluated (p<Q.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 o f dose) was also detected in 18 mammary tissue and aspirated milk. Data are shown as Mean SEM. fDenotes a single 19 reliable measurement at this time due to insufficient volumes in other dams at this dose 20 and time. 21 Figure 4. Neonatal transfer of PFOA to pups. (A) A significantly higher PFOA 22 concentration in pup vs. dam serum on PND1 was noted (p<0,05; v:v). (B) Pooled pup 23 serum PFOA concentrations did not demonstrate a U-shaped curve, but gradually 2/20/2009 Page 25 of 32 `g t i i j l S l P I p i e^sa:^3?-A'ls*i=z=:^T-i x s v Ss! 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 5. Whole pup PFOA concentrations. PFOA concentrations were measured in a 4 representative whole pup (pup and blood; ng/g; left panels) from each litter. Although 5 there is a consistent downward trend in PFOA concentration over time, the rapidly 6 increasing blood volume and body weight changes must be taken into consideration when 7 interpreting these data. Body weight-adjusted values (right panels; [ng/g PFOA 8 measures*g body weight = body burden]) demonstrate an accumulation o f exposure until 9 late in the lactational period. Data are shown as Mean SEM. 10 11 12 2/20/2009 Figure 1. (A) Dam Liver W eight W eight (g) Liver: BW Ratio (%) Control 0.1 1 PFOA Exposure (mg/kg) (B) Dam Liver:BW Ratio 7 6 5 4 Control 0.1 1 PFOA Exposure (mg/kg) 5 5 PFOA Exposure (mg/kg) GDIS DPND1 EPND4 0PND 8 BPND18 Figure 2 9000 8000 ^ 7000 Oe) 6000 w 5000 < Ou. 4000 a . 3ooo 2000 1000 0.1 5 PFOA Exposure (mg/kg) Figure 3, p. 33 P 8 W W 7 ic * i^ i* S j3S9@^@s3pg w ^M I^W m iS . m ss to !0 & im (A) Dam Serum PFOA (B) Dam Urine PFOA PNDt P N D PND4 PND8 PND16 PMD1 PMP3 PNEU PN M PW M S (C) Dam Mammary Gland PFOA PND2 PHDB PHD11 PND13 (D) Dam Mitk PFOA Time 2500 2000 r ITT Smg/kg 1500 1000* 500 0 ! Lm f s m ... . PND2 PND8 PND11 PND18 Page 29 of 32 Figure 4, (A) Dam vs. Pup Serum PFOA 18000 PND 1 Dam 16000 U) c B PND 1 Pup 14000 < 12000 Ou. 10000 L 8000 E 6000 -| E9 4000 to 2000 -J 0 0.1 1 PFOA Exposure (mg/kg) (B) Pup Serum PFOA O ver Time Postnatal Day (PND) PFOA (ng/g) o O PFOA (ng/g) S tn Tl (cS' S 00001 GDIS 1 4 S 18 Gestation (GD) and Postnatal (PND) Day o f PFOA Measurements 3 <6. Ou <on "3n GDIS 1 4 8 18 Body Burden PFOA (ng) foO <oo* Body Burden PFOA (ng) lisi 0 9 0o 0o Body Burden PFOA (ng) 0oJ <wS CfOO Tables IliS Illllp lI^ .................. . 'te /tf..... s^...'i-:^s!,,;>J.^:1.-vV!:...l................ Table 1. Milk-borne PFOA* as a percentage of dam serum concentrations over lactation. Single GD17PF0A exposure PND 1 serum PFOA comparison PND 4 serum PFOA comparison PND 2 m ilk PND 8 serum PFOA comparison PND 8 m ilk PND 8 serum PFOA comparison PN D 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% t PFOA= perfluorooctanoie add, GD-gestational day, PND=postnatal day. The milkiserum PFOA ratio reported above was calculated as: [concentration of milk PFOA/concentratlon of serum PFOA]*100=% milk:serum for each dam within a dose group. These values were averaged and reported above. e 32 o f 32 p C9>4 ffg l jdcl @B 1-8 gSSSjlffi' Reproductive Toxicotogyxxx (20095 xxx-xxx Contents llsts.aveilableat ScienceDirect Reproductive Toxicology jo u rn a l ho m e p a g e ; w w w .e ls e v le r.c o m /lo c a te /re p ro to x . lir;>n:iiuH iv.> Polyfluoroalkyl chemicals in the serum and milk of breastfeeding women O ndine S. von Ehrenstein3-*, Suzanne E. Fenton *\ Kayoko Katoc, Zsuzsanna Kuklenyikc, Antonia M. Calafatc, Erin P. H inesb'1 UCLA School ofPublicHealth. Universityo f California, bos Angeles. CA. UnitedStates 11us E nvironm ental Protection Agency, ORD,NHEERt,Reproductive Toxicology UivLcicn. KIP, H i, UnitedStares ` CentersJar DiseaseControl( f Prevention. Divisiono f Laboratory Science.NationalCenterfo r Environmental Health Atlanta. CA,UnitedStates ARTI CLE INFO Article history: Received2BJanuary2009 Receivedin revisedform27February2009 Accepted2 March2009 Availableonlinexxx Keywords: Polyfluoroalkyl chemicals Perfluotualkyi acids Perfluorooctanolcacid Perfluoroocranesulfonicacid Serum Breast milk lactation ABSTRACT Polyfluoroalkyl chemicals (PFCs) comprise a group of m an-m ade organic compounds, some of which are persistent contaminants with developmental toxicity shown in laboratory animals. There is a paucity of human perinatal exposure data. The US EPA conducted a pilot study (Methods Advancement in Milk Analysis) including 34 breastfeeding wom en in North Carolina. Milk and serum samples were collected at 2 -7 weeks and 3 -4 months postpartum : 9 PFCs w ere assessed in milk and 7 in setutn. Perfluorooctane sulfonic acid (PFOS). perfluorooctanoic acid (PFOA). perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS) were found in neatly 10056of the serum samples. PFOSand PFOAwere found at the highest concentrations. PFCs were below the Emit of detection in m ost milk samples. Serum concentra tions of PFOS. PFOAand PFHxS were lower (p <0.01) a t the second visit compared to the first v isit Living in North Carolina 10 years or longer was related to elevated PFOS. PFOA and PFNA (p < 0.03). These pilot data support the need to further explore perinatal PFCexposures and potentially related health effects, as planned in th e upcoming National Children's Study which provided the framework for this investigation. 2009 Published by Elsevier Inc. t. Introduction Polyfluoroalkyl chemicals (PFCs)comprise a large group o f man made flm arinated organic compounds used in numerous consumer products and industrial applications such as food packaging mate ria l, non-stick cookware, protective coatings fo r textiles, carpets, and paper, surface car coatings or treatm ents, as w ell as in sur factants fo r commercial and industrial applications j l ]. PFCs, and more specifically perfluoroalkyl acids (PFAAs). have been detected in w ild life , fish used fo r human consumption, and sera of humans in many different geographical areas w orldw ide [2-19]. Nation ally representative US sera biom onitoring data in subjects 12 years Abhrrrintroris: ct. confidence interval: KJR. interquartile range; bOD, limit of detection: LOQ,limit ofquantification;'Pinas, perfluoroalkyl acids: PFOSA.perflu orooctanesulfonamide; Et-PFOSA-AcOH,2-(N-ethyl-perfluorcctanesulfonamide) acetic acid; Me-PPOSA-AcOH, ?-(N-methyTpnrfiuorooctrne sulfonamido) acetic add; PFHxS, perfluorohexane so!funic'acid; PFOS. perfluorooctane sulfonic acid; PFOA. perfluorooctanoicadd; PFNA,perfluorononanoic acid; PFC.polyfluoroalkyl chemicals; WTC.WorldTradeCenter. Q2 Correspondingauthorat:UCLASchoolofPublicHealth,POBox1772.LosAnge les.CA90095-1772.UnitedSlates.Tel:*13102065324;fax:1 J 3107941805. E-mail address: ovehtenfflucla.edu(05.vonEhrenstcm). 1Currentaddress: USEnvironmentalProtectionAgency,NationalCenterforExpo sureAnalysis. Environmental MediaAssessmentCroup.MailcodeB243-01.Research TrianglePark. NC27711.UnitedStates. 0890-6238/S- seefront rnattei 2009PublishedbyElsevierInc. doi;10.imS(j.reprotox.2009.03.001 and older demonstrated widespread exposure to perfluorooctane sulfonic add (PFOS). perfluorooctanoic acid (PFOA). and perftuorononanoic acid (PFNA) during the last decade [20,211. Exposures o f iactating 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 m ilk and across the placental barrier [22-26]. Developmental and repro ductive health effects in animals, including reduced b irth w eight and gestationallength, developmental delays and structural defects especially in relation to PFOA and PFOSexposure have increasingly raised concerns, although the developmental toxicity in laboratory animals was shown at doses 100-500 times ofthose seen in human sera [2.27-29].Someexposure assessments in cord blood suggested that PFAAs can also cross the placental barrier in humans [30,31 ]. Apelberg et a). [23] recently reported average cord blood concen trations o f 4 .9 ng/ml (PFOS) and 1.6ng/m! (PFOA) (n -2 9 9 ), w hile Spliethoff et a!., reported the detection o f PFAAs in new bom blood spots confirm ing rhe transfer o f PFAAs in urero [32]. In tw o recent epidem iological studies, PFAAcord blood concen trations were related to anthropom etric indicators o f fetal grow th at b irth , and maternal pregnancy serum PFAA concentrations were associated w ith child b irth w eight [22.24], Based on the Danish National B irth Cohort, inverse associations were reported between gestational PFOA exposure and b irth weight w hile no effects were reported fo r markers o f fetal growth at birth, or postnatal develop mental milestones [24.33]. p. 38 ,<53^bjp! f c jf tf f ll -8 2 O S van Ehrettstein e t el./ Reproductive Toxicology xx x (2 0 tB ) x x x -ia x 61 Data on hum an m ilk PFC concentrations are s till sparse. The Table1 62 available data based on small samplesizes from China [34], Sweden Limitsofquantification(LOQ)inmilkandlimitsofdetection(LOD)inserum(ngiml). 63 [35], Germanyand Hungary |36], suggested detectable levels o fpre Polyfluoroalfcylchemicals ~ . ' MilkLOQ. 64 dom inantly PFOA and PFOS.The concentrations ofPFOS(131 pg/m l) 2-(N-ethyl-perfluofooctane and PFOA (43.8 pg/m l) in 45 m ilk samples collected in 2004 from sulfonamido]aceticadd ", 0.60 wom en aged 22-43 years residing in Massachusetts have been 2-(-m>thyI-perfluoTooctane . 0.60 67 reported recently [25]. Studies investigating the pa rtition o f PFCs in to m ilk are largely lacking. One earlier study in Sweden (n = 12) suggested tran sfer o f only about I %o f PFC concentration in serum sulfonamido)aceticadd Perlluorabutanesulfonate Perfliiofodcanoate. PerBuorohexahesulfonatPFHxS) . 030 0.60 ; : :' .030- . 7C in to m ilk [35]. Temporal concentration changes in serum or m ilk o f ... ..<130 71 lactating wom en are unknown, as no study has assessed concen PerfluorooctaheslfnamidefPFbsA) 0.15 72 73 tra tio n s in the same woman at tw o tim e points during lactation. To evaluate in fa n t and m aternal exposure to PFCs and to a Perfluorooctanesulfdnate(PPOS): Perfluorooetanoate(PFOA) . 0.60 030 74 range o f oth er environm ental components, as w e ll as to compare * Denotesnot measuredin serum. 75 concentrations across biological fluids J37J. the US Environmen SerumLOO 020 030 \4 ,,.a 0.10 o.ib 0.05 0.05 0,10 76 ta l Protection Agency (US EPA) conducted a p ilo t study entitled 77 M ethods Advancement fo r M ilk Analysis (MAMA). This p ito t study 2 A Analysis o f m ilk a n d serum fo r PFCs 76 was carried o u t to develop reliable collection and analysis methods 79 fo r the National Children's Study, including 100.000 children from Id serumand milk, wedetermined the concentrations of PFOS. PFOA, PFNA. PFHxS, perfluorooctane sulfonamide (PFOSA). 2-{N-methyl-perfluorooaiie sut- pre-conception to age 21 |38]. We previously reported the MAMA fonarmdo)accticacid(Me-PFOSA-AcOH),2-(N-ethyf-perfluarooccanesulfonamido) 61 findings regarding phthalates |37] and the biological components aceticadd(Et-PFOSA-AcOH): perfluorotunanesulfonicacidandperfhiorodecanolc 62 o f human m ilk 39], add were only measured in milk. The analytical method involved automated solid-phase extraction (SPE)coupled to reversed-phase high performance liquid 83 2. Materials and methods chromatography(hPLQ-tandem massspectrometry(MS/MS). Sampleswere run insingletsandwerere-analyzedonlyif thewaterand/ormatrixblankswereabove 64 2.1. S tu d y d esig n a n d papulation 3xlimit of detection (LCD).Theanalytical procedures involvingtheuseofstan dards, quality control, and blanks, as well as automatedsampleextraction were 65 66 67 86 6ft 90 a* 92 93 94 95 66 57 400 Tlicdesignof the EPAMAMAstudyand basicmethodshasbeendescribed in detail previously {391.Inbrief.34healthy.English-speakingbreastfeedingwomen between 18and38yearsofagewererecruitedvia newspaperadvertisements, uni versityemailpublicaiions.andfliersdistributedtocliniciansspecializinginwomen's healthorpediatricsbyanEPAcontractor*Wesistlnc,,ChapelHID.NC).Thequestion naireassessmentand thecollectionofmilkandserumspecimenswereconducted at theEPA`sHumanStudiesFacilityclinic(ChapelHill,NC)betweenDecember2004 andJuly2005.womenwerebreastfeedingtheirfirst,secondorthirdchildandwere not requiredto exclusivelybreastfeed for participation in this study.Thewomen donatedmilkand serumsamplesat2-7weeksflst visit:if38milk;n34serum), and at 3-4 months (2nd visit; n*20 milk; n--30 serum) postpartum.Thepartic ipation of human subjects in the MAMA studywas approvedby theinstitutional ReviewBoardsoftheUniversityofNorthCarolina'sSchool ofMedicine(IRBnumber O3-EPA-207)andtheCentersforDiseaseControlandPrevention(IRBnumber3961]. Thewomenparticipated inverbalandwritteninformedconsernprior toadminis trationofacomprehensivequestionnairewhichdidnotincludequestionsregarding theoffspringofstudyparticipants [39J. conductedaspublishedpreviously143-46].Thesamplesfrombothvisitswereana lyzedtogetherinMarch2006(serum)andNovember2006(mine). Formilk,samplepreparationwasconductedusingautomatedoff-lineSPE[431. One-mlofmilk, towhichweadded3mlof0.1Mformicacidand50pJof internal standard solution, was vortex-mixed and sonicated,andplacedona ZymarkRapid TraceStation(ZymaricCorp,,Hopkinton,MA).PFCsfrom themilkwereextractedon anOasis-HLBSPEcolumn(Waters Corporation. Milford. MA). TheSPEeluatewas evaporated at 55SC to -100pi underastream ofdry nitrogen(UHPgrade) Ina ZymarkTurbovapevaporator,and reconstitutedwith300ji!ofO.IXformic acid.The reconstituted milk extraer(-400^.1)was transferredtoapolypropyleneaucosamptervialfwtteon-llneSPE-HPLC-MS/MSanalysts.performedusingaSurveyorHPLC system{ThermoFinnigan,SanJose.CA,USA), includingonesix-portswitchingvalve (JtheodyneMX7960,Rohnert Park.CA, USA}andoneadditional Surveyor IXpump, coupled with aThermoFinniganTSQQuantumUltra criple-quadnipote massspec trometerequippedwithaheatedelectrosprayionization(HES1)interface.TheHPLC pumpoperatedat a300pl/min flowrate with20mMammoniumacetate(pH4}in water(mobilephaseA) andacetonitrile(mobile phaseB).Theextractwas injected intothe liquid chromatographsystemfor concentrationof the PFCs byon-lineSPE onaBetasflC8precolumn(3mm*10mm,5p.m;ThermoHypersit-Keystone.Belle- 2102 .2. Q jtestionnaire fonte,PA,USA),chromatographicseparationonaBetaslCBanalyticalHPLCcolumn (2.1mmx50mm.5jxm;ThermoHypersil-Keystone},anddetectionandquantifica 103 A questionnaire regarding maternal residence, occupation, and dietary and tionbynegative-ionHSJ-MS/MS. 1M lifestyle factors was administered to participants at die first clinic visit. Ques Forserum,we usedamodificationof theon-lineSPEcoupled to HPLC-MSJMS 105 tionnaireitemswereselectedtoaddress potential routesofexposure to multiple approachdescribedbefore|4?]. Briefly,weadded250p.1of0.1M formicacid and 105 environmental chemicals (phthalates, phenols. PCBs, dioxins, PFCs. persistent 25p,t ofinternal standardsolution to 100p.Iofserum, andthespikedserumwas 107 10B organic pollutants, metals,andbrominated flame retardants).The current analy sis includedthefollowingquestionsthatwerethoughttopotentiallyrelatetoPFC vortex-mixed andsonicated.ThesampleswereplacedoaSymbiosison-lineSPE system(SparkHolland. Platnsboro.NJ)forchepreenneenrrarionoftheanalyteson 108 10 exposureroutes: "Howlonghaveyoulived inNorthCarolina?" (40-42)and"Does aPolarisC18 cartridge(7pm, 10mm* Imm; Spark Holland).Theanalytes were yourhomehaveanenclosedgarageattached?".Thelatterquestionwasselectedas transferredontoaBeta&HCBHPLCcolumnOmmv50mm.5p.m;ThenroHypersil- m someapplications usedinandaroundcarscontainPFCs.e.g.,external andinternal Keystone, Bellefonte,FA), separatedbyHPLC(mobilephaseA; 20mMammonium 112 surfacecarcoatingsortreatments. acetateinwater, pH4; mobilephase B: methanol),anddetected by negative-ion Turbolonspray-MS/MSonanAPI4000massspectrometer(AppliedBiosysrems.Fos n3 2.3 . Sam ple c ollection a n d preparation terCity,CA).ReportablebreastmilkPFCconcentrationscanfallbelowtheLODdueto concentrationfactorsthatarepari oftheextractionprotocol.Thuslimitofquantify 114 US 116 Thewomen wereaskedto fast for 15hbeforesample collection. TheMAMA samplecollection proceduresfor serumandmilk werepublishedpreviously{39}. Samplingderails, includingtimeofday(between9AMand2PM)andtheamount catkm(LOQ)(3xLGD)isusedformilksamplesandLODisusedforallocherbiological media,wheresampleconcentrationis notrequired.TheLCDinserumandtheLOQ Inmilk areshown inTable 1. 117 ofbodilyfluid collected, wererecordedm.zhecollection log.Milk (90mlor -3 oz) 118 119 wasexpressed in theEPAclinic usingacommerciallyavailableelectricbreastpomp (Medela.McHenry, IL).Milkwaspumpedinto PFC-freebottlesanddivided into3 ml 2.5 Biological m arker analysis 0 aliquots in PFC-free polypropylene-tubes.Women's bloodsamples(about 20ml}, Selectedbiologiesinmilkandserumwereanalyzedforeachwomanaccordingto 121 m were collected into non-heparioked glass vacutainer tubes (Becton Dickinson. Franklin Lakes. NJ) byanEPAnurse via venipuncture. Alter 1hat roomtempera LabCorp'sstandardoperatingproceduresfortheseassaysaspreviouslyreportedin detail [39].Theassessedendpointswereinmilk: SecretoryimmunoglobulinA.pro 123 124 125 126 127 turetoallow fewdotting,blood sampleswerespunat3000rpmfor 15minat room temperaturcandserumwascollected.AHsampleswerestoredac-20Candshipped ondryicetotheCDCsDivisionofLaboratorysciences.NationalCenterforEnviron mentalHealth (Atlanta. GA)Cofanalysts.AttheCDC,all sampleswerestored ator beiow-20"C until analyzed. lactin.Tissue necrosis factor-a(TNF-o).interlcukin-5(it-6). triglycerides,glucose, andestradiol;and in serum: prolactin,imrmmoglobins.TNF-, 1L-G,triglycerides, glucose,andestradiol.InthisInvestigation,themilkand serumconcentrationofthe biological markerswereusedtoexplorepossiblerelationshipswirhthedetectable PFCs. tea 126 190 51 13? 1 134 13ft 36 13 m 140 1*1 i4a 1*3 144 145 146 147 14? 14 'SO 151 1 153 154 156 >56 157 158 158 160 '6l 165 163 164 165 166 167 166 69 70 i?l 17? 173 174 176 17* 177 176 178 ISO >81 1B2 83 104 p. 39 O S va n gftreitsfeinetof./Reproductive Toxicology x x x (2 0 0 9 )x > a -x a 3 Table2 Q3 Percentage(number)ofserumandmilksampleswithFFCs'>LODatvisit1(serum n-34: milk n- IS)andvisit 2 (serumn- 30;milkn-20) Perfloroalkylacids /, Serum>LQD&in) ; ' ) . f t - Mttk LoQSSbO p ro s ' J Visiti- ... ; Visit 2 ; -, 1 0 0 ( 3 4 ) . . V 100.(30) . . -o ' o . PFOA sin v ts(a PFHxS : Visit 1 .Visit 2 PFNA : Visit! Visit 2 , .. ' . ' . 100(34) 100(30) . 100(34) 100(30) . 97(33) . 10Q{34) . . 0 o, ... 0 .0 0 0 PFOSA Visiti Vislt2 . 44(15) 73 (22). 0 . 15(3) Me-PFOSA-AcOH : Visit i . V&!t2 . ;:. 53(18). 50 (1S) , : 5.6(1} .... ..; o. Et-PfQSA-AcOH ,:Visit .1 . . . . . .. ' . V isiti ' .o 5.6(1) .. .0- . . . . : o Ferfluorobutane sulfonate Visit! Visit 2 ` b b 0 0 Ferfluorodeeanoate Visit! Visit 2 b b 0 0 * PFOS: perFluoiooctanesulfonate; B-PFGSA-AcOH: 2-{N-ethy)-perfluorooctaie sulfonamkio) acetic acid; Me-PFOSA-AcOH: 2-(N-mechyl-perfitioroocranesulfonarmdo)acetic acid: PFHxS: perftuorohexanesulfonicacid: PFOS: pexfluorooctanyi sulfonate: PFOA: perfluorooctanoieacid;PFNArperfluorononanoicadd. b Denotesnormeasuredinserum. iub 2 .6. S m tisricol analysis me Wecalculatedthepercentageofdetectionforeachanalyre in serumandmilk, jar anddeterminedthemedian,range,mean,standarderror,andselectedpercentiles. tbb ForvaluesbelowtheLOO.valuesequaltoLOD)sqr2wereused[48,41}:.Furtheranalrss yses.including relationshipsbetweenvisitsandacrossmedia,wereconductedfor thoseanalytesforwhichthefrequencyofdetection(>LOD)was >S0%at bothvisits. FOrchosewomenwhodonated2serumsamples,themediandifferencebetweenthe concentrationsfor thesame PfCatvisit 1andvisit2wascalculatedand assessed with theWilcoxonsigned-rank test(non-parametric).Spearmancorrelationcoeffi cientsandtelatedpvalueswerecalculatedforcorrelationsbetweentheFFCsatvisit 1andvisit2,andbetween the PFCsandthebiological markers in milkandserum. Relationsbetweena priori selectedvariablesassessedbyquestionnaireandtheFFCs wereevaluated usingWilcoxon scores(rank sums)test.Thecut-ofTpoints forcat egorisingselectedvariablesweredecideda priori basedonassumptionsaccording todatapreviouslyreported(40-42],andtoachieveapproximatelyequal distribu tion of numbersofsubjectsacross categories.Two-sided pvaluesare reported.Alt analyses wereconductedwith SASversion 9(SASInstitute,Cary, NC). 3. Results The median age o f the women in this study was 31.3 years (interquartile range (fQR): 27.1-34.2 years), and the children's median ages were 5.5 weeks (LQR: 4 -6 weeks) at v is it 1 and 13 weeks (13-14 weeks) at vis it 2. Three o f the analytes, PFHxS, PFOS and PFOA, were detected in 100% o f women's serum samples at both visits, PFNA was detectable in 97% at vis it 1 and in 100% o f women's samples at v is it 2 (Table 2). In contrast, in m ilk samples o f ju s t 4 Women, only 3 o f the analytes were >L0Q: Et-PFOSAAeOH E1.0rig/ml) and Me-PFOSA-AcOH (0.7 ng/m !) were detected in 1 woman at vis it 1, and PFOSA was detected in 3 women at the 2nd v is it (0.3, 0.5, and 0.6ng/m l). The remainder o f the m ilk samples from both collections were measured and found to have concentrations <LQQ, The distribution o fPFCserum concentrations is shown in Table 3. Highest concentrations were found fo r PFOSw ith median values o f 20.0 ng/m i at the firs t v is it and 16.S ng/m l at the second visit. PFOS concentrations were alm ost six-fold higher than the concentration o fthe analyte w ith the next highestvalue, PFOA,w ith median values o f 3.5 and 2.9 ng/m l at the firs t and second visit, respectively. Median serum concentrations were significantly (p < 0.01) low er fo r PFOS, PFOAand PFHxS assessed at v is it 2 compared to the concentration assessed at v is it l, based on samples o f 30 women who donated serum samples at both visits w ith the differences shown in Table 4. Accordingly, the concentrations o f the detected serum PFCs are reported fo r each v is it (Table 3). Serum concentra tions o f the same PFC were significantly correlated between the two visits (Table 4). Due to the lim ite d number o f breast m ilk sam ples w ith detectable PFCconcentrations, we could not calculate the 190 19! 192 t9a 104 195 !B0 167 1 1M 200 1 7 203 2CU 2 206 207 Z0B 209 210 2t1 21? 213 214 2S 216 217 ?i(J 215 220 22i 223 ZM 226 226 227 2?fl Z29 230 Table 3 Distribution(mean*standarderror,median,selectedpercentiles,1Q3)ofPFCs*inserumsamplesatvisit 1(rr-34)andvisit7 (n-30)inng/ml. Mean(SEM) Kttl)percentile 25thpercentile Median 75thpercentile 90thpercentile 95thpercentile IQR PFOS Visit! 21.9(1.9) 117 13.2 20.0 30.1 37.6 45.7 16.9 : visit 2 183(1:5) 9.70 14.Q. * 16.9 22.6 30.2 35.5 8.60 PFOA Visit t 3.99(035) '1.50 .2.20 . 3.50 . . 4.60 ' 6.0 8.70 2.40 Visit 2 3.0(0.21) 1.45 .2.40 2.90 3.70 4.65 5.0 130 PFHXS -.Visit 1 ;'Visit 2 ,1.94(0.27) U70" 1.50(J22) .= 030 1.0 ' .1.55 2.40 .70 ' 1:15 ' ; 1.70 3.40 2.90 3.30 4.60 1.40 1.00 PFNA ' : Visit! , 1.22(0^2) 0.40 : 0.7: ; : 1.10 : Visita . - 1.33(0.09) y*:TC7s;: . ; 1.00 -, . .;.Ti20-. 1.60 . 2.00 USD .. .. ; .. 1.50 2.70 2.40 0.90 0.50 PFOSA Visit 1 : Visit.2 0.07(0.01) 0.09(0.01) <LOD . <lOD L0D <LOD <OD 0.10 0.10 0.10 0.10 0.1S 0.10 0.20 0D7 0.07 M^PFOSA-AcQH Visit 1 Visit 2 0.23(OjQ2) io 0.24(0,02) . <lOD . <LOD <LOD 0.20 030 0.17 0.30 030 0.40 0.40 0.50 0.16 0.16 * PFOS: Perfluorooctanesulfonate; Et-PFOSA-AcOH: 2-(N-ethyt-pwfiuoroocranfrsulfonamide)acetic acid; Me-PFOSA-AcOH: 2-[N-methyl-periluoraoctanestdfonamido) aceticadd; PFHxS: perfluorohexanesulfonicacid; PFOS:perfluorooctanyl sulfonate; PFOA: perfiuorooctanoicacid: PFNA: perfluorononanoicadd. Valuesmeasured <LOD wereimputed byLOD/sqr2. ct-af T 'o fc jilu o r t^ ^ ^ ^ ^ ^ p ^ p ^ ^ ^ r u n ^ id .m llk of-breastfel ng^weinen, p. 40 m & m ji '^p p p a jc ^ F *_ '- - * V |?j vonQ S Ehrenstein e t at, f Reproductive Toxicology wot (2009) x x x -x x x Table 4 Differenceandcorrelationin PFCserumconcentrations(ng/ml)betweenvisit one andvisit two. Tables Correlations between concentration ofPFC and interleukin-6 in serumat visit 1 [34)and2(n-30). :,;M^ahdiiFrence QJ?) 'pyaNc? : -;:bhe^on- ' pvalue ' coefficient<r* PFOS.. . PFOA . PFHxS PFNA -2-30 (-7.9.LG) -0.55 (-1.40, C.O) -0.40 ( -0 8 0 .-Q.1C) a i l (--020.050} . 0.01 ; <0.001 <0X101 0.10 ' 0*82 0.32 087 0.71 <0.001 <0.001 0.001 0.001 * Witeoxonsigned-ranktest(non-parametric)(n*30). " Spearmancorrelationcoefficienta andrelatedp value(n*30). PFOS Visit l Visit 2 PFOA Visit I Visit 2 PFHXS , . ` Correlation coefficient, a ... . r-0.21 039 -0.15 . 0,P? p value 020 0.03 0.40 0.70 Zi\ p a rtitio n coefficient from serum to m ilk, bu t can cone!ude th a t m ilk Visit 3 Visit 2 . ; -0.11 038 050 0,04 232 concentrations were notably low er than serum concentrations. PFNA' . 234 Based on self-reported data, women had lived in North Carolina visin . Visit 2 234 fo r {mean, SEM) 14.6 (1,92) years. Interestingly, women w ho had . -0,003 ^0X8 .. . IX 0.70 23$ reported liv in g in N orth Carolina fo r 10 years o r more compared *Spearmancorrelationcoefficientn and relatedpvalue. Boldedvafuessignify significantcorrelations. 236 to those w ho had reported living in N orth Carolina less than 10 237 years, had h igh er serum concentrations o f PFNA, PFOA, and PFOS 233 239 (p <0.03) (Fig. 1). Furthermore, livin g in a house w ith an enclosed garage attached as compared to living in a house w ith no enclosed Serum concentrations o fIL -6 were positively correlated w ith PFOS (p =0.03} and PFHxS (p -0 .0 4 ) at the second vis it (Table 5). 240 garage attached, suggested a relation to higher concentrations o f None o f the other selected biological markers in serum or in m ilk 241 PFHxS (ng/m l: median, iQR, vis it 1:2.2 (1.4) vs. 1.1 (0.6), p< 0.001; showed asignificant correlation w ith the PFCserum concentrations 242 v is it 2:1.5 (1.4) vs. 0-9{Q.7)p =G.03)andofPFOS(visit 1:25.4(16.9) at either collection tim e point.There was no significant relationship 243 vs. 14.4 (9.9). p-O .01; vis it 2: 212 (11.5) vs 14.5 (7.8) p - 0.1). between maternal age or parity and PFC serum concentrations in our study (data not shown). Due to the small numbers and lack o f racial diversity in this p ilo t study based on convenience sampling (only. 3 women reported themselves as Black/African-American, one as Asian and one as Hispanic), w e could not analyze PFC con centrations by ethnic group. 4. Discussion PFNA Visit 1 PFNA Visit 2 PFOA Visit 1 PFOA Visit 2 PFC by V isit Fig-1, (a)and<b)SerumconcentrationsofPFNA.PFOA.andPFOS(ng/ml)comparing livinginNorthCarolina>10to*10yearsatvisit 1andvisit2.Dataareshownasbox and whisker representations: opencircles denote meanvalueswith themedians denotedasastraight line. 'p<0.03 ia Wikoxon Scores(rank sums)tests forgroups lOyearsts.tOyearsatvisit 1andvisit2foreachPFC.Numbersorsubjectsineach group:>10years:n=16.visit 1:n-15.visit2;<10years:n -18.visit1,n15.visit2. In this p ilo t study o f healthy lactating North Carolina women. 6 o f the 7 PFCs analyzed in serum were detectable at 2-7 weeks and 3-4 months postpartum. PFOS. PFOA. PFNA, and PFHxS were 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 o f m ilk samples had detectable values o f 3 of the 9 PFCs analyzed in m ilk. Interestingly, serum levels were tower fo r PFOS, PFOA, and PFHxS at the second vis it compared to the firs t vis it, and prolonged tim e lived in North Carolina, as w ell as living in a home w ith enclosed garage attached, suggested a relation to elevated serum concentrations o f certain PFCs in our sample; however, these analyses were unadjusted and based on a small non random sample in this p ito t study and thus should be considered exploratory. We can conclude th a t postnatal exposure to PFCs via breast m ilk is like ly to be low during the tim e period captured in our investigation. Data onPFCserum concentrationsoflactating women are sparse and based on small sample sizes. Available data relevant fo r preand postnatal exposures to PFCs are summarized in Table 6. Only one earlier study assessed both serum and m ilk levels, in 12 lac tating women in Sweden, and reported sim ilar serum values to ours fo r PFOS(median: 18.7 ng/m i)and PFOA{3.8 ngjm l) w hile con centrations o f PFHxS were higher (4.0ng/m i) in the Swedish study |35J. 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 trim ester, possibly due to dilution of the PFCs w ith blood volume expansion due to pregnancy, but values in cord blood (n * 50) confirmed fetal exposures [24.33] (Table 6). The serum PFC concentrations seen in our study compare w ell w ith USserum data from NHANES 2003-2004. assessed in representative samples of 244 246 246 247 246 349 25ft 2$i ese 253 254 255 257 258 259 260 m 262 263 2M 255 266 26? 266 269 270 271 272 ??3 2>4 m 276 m 27* 279 2BQ 261 ?BT 65 254 246 266 257 p. 41 OS von Eltremlem et aI f S e p ro d u c l/v r T o x iro /o g y w o t (2009)xxx~xxx T a b le s Publisheddan onaveragePFCconcentrationsinmilk,maternalserumandcordblood. Looa^on.yeiirqfsianiplittg J ; . .. Matrix,; sample size '' d;v-;.. . Erc.cpncehlratitmMtepprted! ' " - Percentage quantified >LOD* Reference MausadiusctK.USA. 2004 tefpzfg/MiinichGermany,2005 Gyor, Hungary 1996/07 ' . MiIk;conveniencesampte,age: : PF0S:Cmean,:SD)::l3l'(ld3) pg/inl", PFOS:96% `- (25) 22-/43:years. n-45. nursingthe . PFOA: 43^(33,1 jpg/ml . PFOA:,89%. . - flrsttfme:7]-34,nursed>l:n*$ : PFHxS: 145 {13.7) pg/ml ' . ::PFNAt7i6(47d)pihhi. . : : PFHXS: 51* *PFNA: 64% . :FFHpA,PFDAFFUnDA, PFDoDA, ' PFHpX; PFOA.PFUnOAPFDoDA PFBS:aU<LOD ' PFBS: <3% / Mii|ccoaveii|ene sampUngat , PF0S(iriedian,range) - . PFOS: 100 . [36] hospital sarnplei n= 19 (Munich) - -:Milk bank he 38"( L e i p z i g ) . f ' . Munich: l13 (28-239)njg/L ' ; . PFOA: 16% . ' Mothers ofpreterm infants, n = 13 Leipzig: 123 (33-309) hg/L ; (Hungaty)at:3-7weeks : postpartum :. Hungary: 330 (96-639) ng/L PFOAall: <LOD 0 0 -4 6 0 } ng/L Zhousan.Cbina.Z004 Milk: convenience samplingat ' Rangesv(ng/L) PFOS, PFOA.PFHxS. PFNA, PFDA, Hospitalvolunteers, n - T9 PFOS: 45-360;.PFOA:47-210; PFUnDA:100% PFHxS: 4-100; PFNA: 6.3-62; PFQA:3.8-15: PFtlnDA: 7:5-56 Swedervindividiia! matched sera MOkarklserom: convenience Milk(mean.SD) ng/ml: PFOS: Milk: PFOS, PFHxS: 100%(n -12); and milk (2004); pooled composite . sample.primiparcwswomen. i t - 12 0201 (0.117); PFHxS: 0.085(0.047): . PFOSA; 67%(n-8)PFNA; 16% milk samples (1996-2004). . ; PFOSA: 0.013 (0.009};PFNA: NA; (it-2): PFOA:8%(it-T) ' , PFOAPFDA.mjn0A:;ND ;. :. ; " : V '" ' fooledanmi^compositemilk samplestn*25-90) - 1. ; Serum: PFOS.PFHxS,PFOAPFNA. PFDAPFUnDA 100%(n-12); PFOSA:75%(n-9J . Dateofmftkcollection:3weeks -Serum(mean,SO)ng/ml:PFOS: postpartum -' 20.7('105J-PFHXS:4.7(23): . PFOSA:024(0.16);PFNA;0.80 " (055); PFOA;30(1.0);PFDA:053 (0.41);PFUnOA:0.40(035) . Compositemilk ng/ml: PFOS0.209 (1996)-0.123(2004): PFHxS;OJ037 (1996>4Wn6C2004); PFOSA: <0.007(1996)-<0j007(2004); PFNA: 0J128(1996)-LD20(2004); PFOA: <0209(1996)-<0209 (2004) BaltimoreNip,USA.2004-2005 Cordblood,hospitalbased, PFOA(median,range): 1.6(0,3-71) PFOS:99% singletondeliveries(n-293) PFOS(median, range) ng/ml: 5.0 (<LOD(-02>-343) PFOA: ioo%: .KtPPPSA-ACQH,Me-PFOSA-AcOH, PFBuS;PFHpA,PFUA.PFDoA: 1-40% Denmark,'1956-2004 - Matemalplasiiiailsttrimester -Maternal;1sttrimester: PFOS (r~1399X . ' . .. (ng/ml^nean.SD):353(13.0). -Maternal, 1sttrimester: PFOS: 100%PFQA: 100%.(exceptn*l) 2ndtrimester(n-200) . . PFOA:5.6(25) ' -Matemal.irKltrimestenPFOS; 1295(11.0): PFOA:45(li9) . . Corbb!ood,n-50 : CordWood:PFOS:11.0(4.7);PFOA: Japan,2003 . . . Maternalplasma:3rdtrimester 3.7(3.4) Maternal,3rdtrimesterserum Maternal3rd trimester: PFOS: (n-TS)cordblood(n15) range'':PFOS(49-17,6ng/ml). 100%,PFOA; 20%,PFOSA; 0% PFOA(<LODto23ng/ml*PFOSA (<L0Dto<LOD) Cordbibod:PFOS(1.6-53ng/ml), Cordblood:PFOS:100%,PFOA:0%, PFOA(<LODto<LOD).PFOSA PFOSA;0 (<LODto<LOD) J PFCLODs forserum.Woodandmilk variedinthedifferent studiesasreportedintheoriginal references. 1 Noaveragesreportedbyauthors. p4) |35] [2223] 174) |30( IBS females aged 12 and above, showing m edian concentrations fo r about halfthe concentration reported for cord blood from Denmark 289 PFOS and PFOA o f 18.2 ng/m i (IQR: 12.4-27.3 ng/m i) and 3.6 ng/m l ]24], Table 6. 290 (IQR: 2.5-S.2 ng/m l), respectively [20,21 [. Based on the NHANES A few investigations o f PFCs in human m ilk have been con 291 data, nation-w ide serum concentrations dropped fo r PFOS. PFOA ducted in Sweden, China, Denmark and recently in the US(Table 6) 292 and for PFHxS between 1993/2000 and 2003/2004 w hile those for [25,34-36). Only one study assessed both serum and m ilk concen 290 PFNA increased in the same tim e period 20j. Our average levels trations and detected PFOS and PFHxS in all 12 m ilk samples at 294 are somewhat low er than reported fo r females in the US in 1989 mean concentrations o f 0.201 and 0.085 ng/m l respectively, sug 285 15] but s im ila rto other findings in samples collected between t999 gesting partitioning o f on average 1% from serum to m ilk [35]. In 298 and 2005 [6.10,20,21.50], In a recent US investigation, median cord the Chinese study, values o f PFOSand PFOAin m ilk samples (n = 19) 297 blood levels forPTOSand PFOAo f5 and 1.6 ng/rrsl, respectively, were were in the range o f0.045-0.36 and 0.047-0.21 ng/ml, respectively 298 reported ]23).This is about ath ird to a fourth (PFOS)and 50%(PFOA) [34]. M ilk concentrations are summarized in Table 6, supporting 299 o fthe concentra tions we found in maternal serum samples, and also our findings o f low er values in m ilk than in serum, as w e ll as 30(1 001 302 303 3CM 305 306 307 308 309 310 311 p. 42 RI^62SJ-8^l Z 6 l t? . Si_"g!SS / xxxO S. vo n Ehrenstefn et a t Reproductive Toxicology (2 0 0 9 ) xkk- kxx 9 313 314 315 316 31? 313 319 320 321 323 323 324 325 3 927 390 331 332 333 334 935 396 33? 336 338 340 341 342 343 344 346 346 347 3aS 34 350 95! 33 36? 958 359 360 363 364 367 36B 959 970 371 372 373 374 375 376 37? suggesting regional differences in exposure levels {25,34-38). PFAAs are strongly bound to the protein fraction o f human blood [1 0 51-5 3]. The protein concentration in human blood contains m ainly album in and fewer beta-lipoproteins and is about 3 -5 tim es higher than the protein fraction in human m ilk {casein and lactalbum in). It has been shown th a t strongly protein-bound drugs are less lik e ly to transfer to human m ilk than small non-ionic lipophilic compounds [54|. This may explain w hy PFM concentrations are m uch lower in human m ilk than in m aternal serum,although trans fe r o f PFAAs to m ilk has been observed in animal studies, albeit at m uch higher serum concentrations o f PFAAs [26}. in our study, concentrations o f PFOS, PFOA and PFHxS in serum w ere tower a t the second v is it compared to concentrations at the firs t visit. Since PFC concentrations measured in human sera have half-lives ranging between 3.4 years fo r PFOA, 4.6 years for PFOS, and 7.1 years fo r PFHxS [55], these data suggest that processes related to depuration in to breast m ilk m ight be occurring that w e could no t assess {possibly because we measured m ilk con centrations too late in lactation), o r th a t there m ight be maternal m etabolic changes during lactation that may relate to this change {i.e., changes in blood volum e, body weight, o r hepatic activities). Because PFCs are tig h tly bound to serum proteins, serum protein levels during lactation could have affected the concentrations o f PFCs in serum . Unfortunately, we did not measure serum album in to test this hypothesis. Alternatively, if PFCs pa rtition more into liv e r than serum in the course o f lactation, serum concentrations o f PFCs could be affected as w ell. The possible transfer o f PFCs to m ilk may also vary at different tim es during lactation. The nature o f the relationship between the suggested decline o f PFCvalues in serum to concentrations in m ilk are yet unclear and insufficient data exist to date to explain the relationship at this point. Few ear lie r reports suggested declines in breast m ilk during lactation fo r lip o p h ilic compounds including dioxins. PCBs, and PBDEs {56.57}. No other study to our knowledge, has investigated PFCconcentra tio n changes in serum o r m ilk over tim e during lactation assessed in the same women at tw o tim e points. However, it should be noted th a t our findings are based on a relatively sm all number o f a volun teer non-random sample o f women and need replication in a larger study for fu rth e r confirm ation. Tao er al. conducted a regression analysis of PFOS and PFOA concentrations in breast m ilk collected a t various tim e points from 25 diffe re nt women w ith in the firs t 6 months postpartum ; they concluded tha t values increased over tim e o f lactation (25).However, since these findings were based on m ilk samples o f different subjects rather than com paring changes over tim e in the same wom en, the differences may be due to intra individual variation. Our investigation suggested tha t living in N orth Carolina fo r a prolonged tim e period o f 10 years and more was related to higher serum concentrations o f PFNA, PFOA, and PFQS in our- p ilo t study. However, fu rth e r evaluation o f this explorative finding is required. Point sources may lead to elevated exposures,'as indicated by serum concentrations o fPFOAin persons livin g neara US fa c ility using and producing th is compound, tha t were notably higher than among the general US population [58}. A systematic surface water survey conducted in North Carolina showed large variation in concentra tio n ona sm all scale indicating a seriesofsource inputs around the CapeFearDrainage Basin that may potentialiy result in pockets w ith increased exposures 40}. Comparing serum PFC concentrations among donors a t the 6 American Red Cross Blood Bank locations across the US showed highest concentrations fo r PFOS and second highest fo r PFOA in Charlotte, North Carolina, in samples collected in 2000-2001 )41], w ith a substantial decline observed in samples collected in 2006 a t the same locations [42). Recently, elevated plasma concentrations especially o f PFOA. PFNA, and PFHxS have been reported fo r personnel involved in the W orld Trade Cen te r {WTC} disaster (i.e., from fire-fig htin g foams used to combat the WTC fire o r directly from the WTCs degradation) [59] further 318 supporting the notion o f source related local variations o f human exposures to certain PFCs. Women who reported living in a home 370 330 w ith an enclosed garageattached also had increasedconcentrations 381 o f PFHxS and PFOS in our sample.This may be due to certain mate 38? rials used in and around cars containing PFCs, such as post-market 383 applications of external and internal surface car coatings or treat 34 ments. However, du e to the small sample size in this p ilo t study, we 385 could not analyze the impacts o f other variables, especially socio 386 economic factors; these findings are thus explorative and should be 38? interpreted cautiously. The pro-inflam m atory cytokine 11-6 was positively correlated to PFOS and PFHxS, respectively, a t the second collection tim e point possibly indicating that certain PFAAs may be related to inflam m a tory processes.Tn line w ith these findings are recent results from 39? experim ental studies in mice, reporting suppression o f immune 993 responses follow ing exposure to PFOS in utero [60], We did not see 994 correlations w ith bthera priori selected biological markers assessed in m ilk or serum, i.e., im m unoglobulin, estradiol, prolactin orTNF- a, Rodentstudies using PFOAin concentre tions orders o fmagnitude higher than MAMA serum concentrations have shown a suppres sion o f genetic-markers o f inflam m ation after an acute exposure to 999 PFOA [61J. Because our findings are explorative, future studies may want to address the role o f chronic exposure to low dose PFCs in 400 *01 the inflam m atory process. In this p ilo t study the number ofwomen was relatively small and 40? 03 confines the investigation o f associations, possible exposure path 404 ways, and tim e trends after birth. In addition, the sample was not 405 selected randomly, thus selection bias cannot be excluded. How 406 ever. the participation o f women was unlikely to be related to PFC 4Q7 exposures o r to certain PFCexposure sources since they were most 408 like ly not aware of th e ir PFCexposures. A further lim itation o f our 40 Study is that we could not collect m ilk samples sooner after b irth in 10 view o f ethical constraints in asking fo r the colostrum m ilk. Studies in m ice measuring PFOA concentration over the course o f lactation 411 412 have shown that the peak in m ilk PFOA concentration occurs soon 413 after b irth (Fenton eta!., in this issue), a tim e that was not followed Q 3 414 in the MAMA collection scheme. Overall, the findings reported are explorative and need further evaluation. 41 41 In conclusion, although infant exposure via breast m ilk is likely <17 to be low, the cumulative daily infant intake of PFCs via breast m ilk <1 per kg body w eight could be appreciable fo r some populations or 4t$ groups (Table 5). Since toxicological and pharmacokinetic data for 420 PFCexposed infants are lacking, it is largely unknown if potential 421 health effects in infants or during childhood may be related to cur 422 rent exposure levels of PFCs. in utero exposure should continue to 23 be a concern as the MAMA serum PFAA concentrations are sim ilar 424 to values reported in tw o separate studies that have shown inverse associations between maternal serum or cord blood PFAAconcen 425 m trations and infant b irth w eight [22,24]. Tims, the findings o f this 7 p ilo t study underscore the importance o f biom onitoring maternal 426 and infant exposure to PFC as w ell as the need fo r further study <29 o f the potential human health effects o f PFCs. In the upcoming US National Children's Study [38] PFC exposures in pregnant and Iac- <30 1 tating wom en and th e ir children in North Carolina and across the 32 US w ill be further studied. 39 C onflict o f interest The authors declare that there are no conflicts of interest. 434 435 Acknowledgm ents The research in this article has been reviewed by the National Health and Environmental Effects Research laboratory, US Environ- 38 43? 43 p. 43 O S vonBtrensrcineral. /Reproductive Toxicology too. P 0 0 9 )x x x ~ x x x 7 4 mental Protection Agency(EPA),and theCentersforDiseaseControl *A9 and Prevention (CDC)anci approved for publication. Approval does 441 not signify this report reflects SPA or CDC policy. The findings in 4 this report are those of the authors and do not reflect the views of 4 the CDC.The use of trade names or commercial products does not 44 constitute endorsement or recommendation for use. 446 Thisworkwas supported mpart by the Intramural Research Pro445 gramattheEuniceffennedySfrnverNationalinstituteofChtEd Health 447 and Human Development* National Institutes of Health. Bethesda, 446 MD. 49 Partial extramural funding was provided through the rec450 ommendation of the National Children's Study Intra-Agency 4&1 Coordinating Committee. 452 The authorswould like to Richard Wang at the CDC for technical 453 assistance,Westat. Inc. recruitingstaff(Andrea Ware, Bethany Brad- 454 ford, Brian Karasek), and the US EPA nursing staff(Deb Levin. Mary 455 Ann Bassett, and Tracy Montilla). 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Available: sis sir 618 519 590 621 te sas tee 687 S30 Wt 638 533 534 5 536 537 533 39 5*0 54f 542 5*3 6*4 545 94 547 54B 5*9 550 551 658 6S3 ss* 555 55 7 5fi8 559 564 sei 5 563 564 665 SS&G76 5BB 569 570 571 572 573 57* 578 575 677 570 579 580 501 SS? 583 584 sss 565 se7 53i 5M 5S7 598 599 $00 601 p. 44 lr-8 7 I S r ~~ ' I_ " ' " a OS. vonEhrenstdn e l at. f ReproductiveToxicology x x x (2C091XXX-XXX 02 http://wu,,w,cdc.2t>v/nchs/d3t3/nhar.es/nhane5.03_Q4|lab_c_genera]cliK,pfir: 156] HooperK,Shej,SharpM,etal.Depurationofpolybrominateddiphenylethers 603 2006. (PBDEs)andpolychlorinated biphenyls (PCBs) in breast milk fromCalifornia 604 |SOJ OlsenCW. MairDC.KeagenWK.eraLPreliminaryevidenceofadeclineinper- first-timemothers(primiparae). EnvironHealthPerspect2007:115:1271-5. 605 nuoiooctanesuttbnare(PFOS)andperftuorooctanoate(PFOA)concentrationsIn 57] LaKindJS,BerlinCM,ParkCN.etal.Methodologyforcharacterizingdistributions 605 AmericanRedCrossblooddonors.Chemosphere2007;68:1OS-11. ofincrementalbodyburdensof2,3,7.8-TCDDandDDEfrombreastmilkinNorth 607 [511JonesJD.HuW,DeCoenW,etal.BindingofperfiuorinatedJauyacidstoserum Americannursinginfants.JToxicolEnvironHealthA2000;59:605-39. 606 proteins.EvnvironToxicolChem2003:22:2639-49. [53] Emmett EA,Shofer FS.ZhangH. et aL Communityexposureto perfluorooc- 60 [52] HanX.SnowTA.KemperRA,etal.Bindingofperfluoroocranoicacidtoratand tanoate: relationships betweenserumconcentrationsand exposuresources.] 010 humanplasmaproteins.ChemResToxicol2003:16:775-81. Occup Environ Med2006:48:759-70. eti 1531 Bhattacharya AA.CruneT.Curry5.Crystallographicanalysisrevealscommon [S3] Tao L. Kannan K, Aldous KM, et at. Biomonitoringof perfluorocbeimcals in 612 modesofbindingofmediumandlong-chainfattyacidstohumanserumalbu plasmaof NewYorkStatepersonnel responding ro theWorld TradeCenter 613 min.!Mol Biol2003;303:721-32. disasrer. EnvironSciTechnol 2008:42:3472-8. 614 154] AschenbrennerDS.VeableSJ,Drugtherapyinnursing.Lippinicort,Williams& [60] Keif DE. MehlmannT.Butterworth L et al. Gestational exposureto perRuo- 615 Wilkins;2008. rooctanesulfonatesuppresses immunefunction in B6C3F1 mice.Toxicol Sci 616 155] Oisen CW, Burris JM, Ehresman DJ, et al. Hail-life of serumelimination of 2008:103:77-85. 617 perfluorooctanesulfonate, perfluorohexanesulfonate. and perfluoroottanoate [61] GurugeKS,YeungLW.YbmanakaN.et al.Geneexpressionprofilesin rat liver 616 in retired fluorochemica) production workers. Environ Health Perspect treatedwithperiluomuctanoicacidEl'KIA),ToxicolSci 2006:89(1):93-107. 2007:115:1298-305. ere 20 re t see rea EM ess ere EM 635 al PoiyiJiiorpalJtyl c lie ^ i^ ^ fe A ^ is e ra rira iia rm ilk cfireistfeeS iin'g w o'm ejS 0 0 1 a _ _____ ELSEVIER M olecular and Cellular Endocrinology 304 (2009) 9 7 - lOS Contents lists available atScienceD irect Molecular and Cellular Endocrinology jo urn 31 bo mep age : www.e Isevi er.corn/!oca te //nc e . Phenotypic dichotomy following developmental exposure to perfluorooctanoic add (PFOA) in female CD-I mice: Low doses induce elevated serum leptin and insulin, and overweight in mid-life* Erin P. H in esa-*, Sally S. W h iteb, Jason P. S ta n k o \ Eugene A. Gibbs-Floumoyc, Christopher Laua, Suzanne E. Fenton3 3R eproductive Toxicology Division, Office o f Research a n d D evelopm ent, N ational H ealth a n d Environm ental E ffects Research Laboratory, U S EnyrronnienttfProtection Agency, Research TrianglePark, NC 2 7 711United States bCurriculum fitToxicology, UNC Chapel Hill, Chapel Hill, N C 275S9, United States cBiological a n d Biomedical Sciences P rogram /lm tiative fo r M axim izin g Stud e n t Diversity. UNC Chapel Hill, C hapel H ill NC27S99, U nited States A R T I C L E INFO Article history: Received27January2009 Accepted24 February2009 Keywords: 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 growth in animals exposed during feta! life. PFOA is found in the sera and tissues of wildlife and humans throughout the world, buc 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 0,0.01,0.1,0 3 , 1,3, or 5 mg PFOA/kg BW for 17 days of pregnancy or as young adults. Body weight was reduced in the highest doses on postnatal day (PND) 1 and at weaning. However, the lowest exposures (0 .0 1 -0 3 mg/kg) significantly increased body weight, and serum insulin and leptin (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 months of 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 a t high doses of PFOA in intact d e v e lo p m e n ta l exposed mice, and spleen weight was reduced in PFOA-exposed ovx mice. Brown adipose tissue weight was significantly increased in both ovx and intact mice at high PFOA doses. Liver w eight was unaffected in late life by these exposure paradigms. Finally, there was no effect ofadult expo sure to PFOAon body weight. These studies demonstrate an im portant window of exposure for low-dose effects of PFOAon body weight gain, as well as leptin and insulin concentrations in mid-life, at a lowest observed effect level o f 0.01 mgPFOA/kg 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: ANOVAanalysisofvariance:BM],bodymassindex;BW,bodyweight;CS,eight-carbon;CV.coefficientofvariation; DES,dielylstfibestrol; E2.estradiol; CD.gestationalday; Hair-life;IACUC,InstitutionalAnimalCareandUseCommittee;LH.luteinizinghormone;LOD,limitofdetection;LOQ,limitofquantitation;NHANES. National Health and Nutrition ExaminationSurvey; NMR, nuclear magneticresonance;NOAEL noobservableadverseeffect level; ovx. ovariectomized;PFAA,perfiunroalkyl add: PFOAperfluorooctanoicacid; PFOS,perfiuorooctanesulfonate; PND. postnatal day;PPAR. peroxisomeprolifeiator-activared receptots; SMR,standardized mortality ratio. Disclaimer. TheinformationinthisdocumenthasbeenfundedbytheU.S.EnvironmentalProtectionAgency.IthasbeensubjectedtoreviewbytheNational Healthand Environmental EffectsResearchLaboratoryandapprovedfor publication.Approval doesnotsignifythat thecontentsreflecttheviewsoftheAgency,nordoesmentionof tradenamesorcommeicjai productsconstituteendorsementorrecommendationforuse. * Correspondingauthor.Currentaddress:US.EnvironmentalProtectionAgency,NationalCenterforExposureAnalysis,EnvironmentalMediaAssessmentGroup. ResearchTriangle Park. NC27711.UnitedStatesTel.: *! 919541 4204;fax: +19195412985. E-moil address: hiiies.erin@epa.gov(EP. Hines). 0303-7207/5- seefrontmatter. PublishedbyElsevierIrelandLtd. do/:10.1016/j.mce.200S.02M 21 E.P. Hines et a l./Molecularand Cellular Endocrinology 304 [2009)9 7 -JOS 1. In tro d u c tio n Perfluorooctanoic add (PFOA), one o f the eight carbon (C8) perfluoroalkyl acids (PFAAs), is a synthetic, stable, persistent organic flu o rin e surfactant, used to im part w ater and grease resistance to various consumer products including non-stick pans, as sur face treatm ents fo r clothing and food wrappers, insulation and fire -fig h tin g foams. PFOA's high energy carbon-fluorine bonds are resistant to hydrolysis, photolysis and metabolism and thus it bioaccumuiates and persists w ith in biota and environm ental matrices, including w ater and soii, from the A rctic to the South Pacific (Lau et a l, 2007). This ubiquitous environm ental contam inant has an esti m ated h a lf-iife ( tjp ) in humans o f3.8 years (Olsen e t a l, 2007} and is found in production workers' sera, as w ell as those o f the general p o p u la tio n . B io-m onitoring studies show detectable levels ofPFOAin human populations. The National Health and N u trition Examination Sur vey (NHANES) reported that mean serum PFOAconcentrations are declining in the USA population, from 5.2ng/m l in 1999-2000 to 3.9 ng/m l, in 2003-2004 (Calafat e t aL. 2007). Arnsberg, Germany, an area w ith known drinking w ater PFAA contam ination, had reported PFOA mean serum levels in 2006 o f 25 ng/m l vs. 4 ng/m l in unaffected German provinces (H lzer et a l, 2008). The highest know n non-occupational PFOA exposure via drinking w ater exists in the L ittle Hocking drinking w ater d istrict where U.S. residents (Ohio and W est V irginia) have mean serum PFOA concentrations o f478 ng/m l (Em m ett et a t, 2006). Children may receive significant PFOAexposures via dietary and w ater intake. Mean serum PFAAconcentrations (such as perfluorohexane sulfonic acid) were reportedly higher in children than in adult/elderly populations (Olsen e t aL, 2004). In the L ittle Hock in g w ater d is tric t, an area o f high environm ental PFOA exposure, children age tw o to five and the elderly had significantly increased PFOA serum levels when compared w ith other age groups (Emmett e t a i, 2006). Although a bio-m onitoring study in Japan found PFOA in maternal blood, bu t not um bilical cord blood a t parturition (Inoue et a l, 2004, lim it o fquantitation [LOQ] 35.2 ng/m l), a recent U.S. study (Apelberg et a l,, 2007) o f human cord blood from term pregnancies reported relatively low levels o f PFOA (lim it o f detec tio n |LOD] 0.2 ng/m l) and another C8 compound, perfluorooctane sulfonate (PFOS). W ith in the reported study concentrations, the authors found tha t cord blood PFOA concentrations were signifi cantly negatively associated w ith b irth weight. A subsequent larger Danish study also found a significant negative correlation between m aternal plasma PFOA and b irth w eight (Fei et a l, 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 h ile high density lipoproteins have been negatively associated w ith PFOA (Olsen et a l, 2001 ). Categorical division o f workers by PFOAexposure levels showed that, although not significantly differ ent from the other categories, body mass index (BM I) was elevated in the highest PFOA category {>30 ppm and BMls >28,1995 data); th is trend was not seen in the 1993 data set (Olsen et aL, 1998). A retrospective cohort m ortality study (n > 6000) o f PFOA-exposed employees reported significantly elevated standardized m ortality ratios (SMR) in males w ith diabetes m ellitus when compared to m en residing in West Virginia (m inus the PFOA manufacturing area), Ohio, V irginia, Kentucky, Indiana, Pennsylvania. Tennessee, o r North Carolina: the SMR for PFOA workers was not significantly increased when compared to W est V irginia alone o r USA residents (DuPont, 2006). in Amsberg, Germany, PFOAwas found to have an inverse correlation w ith BMI in adults (H lzer et a t, 2008). The tjps fo r PFOA in men and women are sim ilar (Harada et al,, 2005).Untike humans, genderdifferences in PFOAclearance exist in rats (Kudo and Kawashima, 2003; Vanden Heuvel et aL, 1991). Mice are the preferred animal model fo r evaluating the effects o f PFOA on the developing fetus as they do not exhibit gender-dependent tip differences (Lau et a t, 2006). However, even in the rat model system where the female rat rapidly excretes the compound, PFOA readily crosses the placenta (H inderliter e ta l, 2005} and PFAAs are present in ra t m ilk after PFOA treatm ent (H inderliter et a l, 2005). Mice prenatally exposed to doses o f PFOA at > t mg/kg/day e xh ib it developmental to xicity including decreased litte r size, neonatal death,delayed eyeopening, grow th deficits, stunted mam m ary gland development, and early onset male puberty (Lau et a l, 2006; W hite et aL, 2007; W olfe t a l, 2007). A t higher doses and fo l low ing long-term adultexposure, cancer endpoints associated w ith PFOAexposure in ratsinclude Leydig cell adenomas, pancreatic aci nar cel! adenoma/carrinomas, mammary fibroadenomas, and liver tum ors (Biegel e t aL, 2001; Sibinski, 1987). PFOA increased estra d io l (E j) levels in male rats and FFQA-induced rodent Leydig cell tum ors are hypothesized to arise from increased estradiol levels from aromatase induction (Liu e t a t, 1996; Biegel et a l, 2001). The m ajority o f the ongoing w ork in the PFOA field has focused on the health effects follow ing developmental exposure to PFOA. This study focuses on adult latent health outcomes in female o ff spring after developmental (gestational days (GD) 1-17) vs. adult (a t 8 weeks o f age, fo r 17 days) exposure to PFOA. Ovariectomlzed siblings were utilized in our second study block to address the role o f the ovarian hormones in PFOA exposure-related health effects, as luteinizing hormone (LH)-overexpressing mice (Kero et aL, 2003) displayed several phenotypic effects resembling those in ou r preltm inaxy studies w ith PFOA. These studies address the role o f developmental exposure and ovarian hormones in adult health effects including circulating le p tin and insulin concentrations, adult body w eight, and tissue and body weights in old age. 2. Materialsandmethods 2.1. Animals Timed-pregnantCD-I mice(CharlesRiverLaboratories.Raleigh,NC)arrivedon gestational day(GD)0(spermpositive) at the US EPAwhere they wereweighed upon arrival and randomly distributed amongtreatment groups. Pregnant dams werehousedindividuallyinpolypropylenecagesandreceivedchow(LabDiet5001, PMI Nutrition international UC, Brentwood, MO)and tap water ad libitum. Two Modes of animalswereused mthesestudies. Block 1animatswere dosedwith vehicle (distilled water); 1.3, or SmgPFOA/kgbodyweight (8W) (n=5,8,7, and 5 dams, respectively); block2 animats were dosedwith vehicle. 0.01.0.1,0.3,1, or 5mgPFOA/kg (n-M dams in all groups except 5mgPFOA/kg BW. which had 10dams). PFOAexposuresareshownin thetext asmgPFOA/kg.Animal facilities were maintained on aI2:12-h light-dark cyde.at 20-24-C with 40-50%relative humidity.AnimalswerehumanelytreatedasapprovedunderNational Healthand EnvironmentalEffectsResearchLaboratoryprotocolsinaccordancewiththeUSEPA Institutional Animal CareandUseCommlttee(IACUC).Sentinel mice,housedin the sameroom,wereknowntobefreeofecto/endopairasitesandantibodiestocertain viruses fortheduration ofthesestudies. 2 2 . Dosingsolutiona n d procedures PFOA, as its ammoniumsalt (>98%pure), was acquired fromFluka Chemical (Steinhiem. Switzerland). PFOAdosingsolution was prepared freshdailyin deion ized water, and thedosing solutionwas administered atavolumeof10pt/g.Mice receivedeitherwatervehideor PFOAatam, a i. 03,1,3, orSmg/kgBWbyoral gavageoncedailyoverthedosingperiods.Thehighestdose(5mgPFOA/kg/day)was chosenbecauseitwasknowntoresultinslightlyreducedneonatalbodyweightgain withminimalpostnatal mortality(Lauetai.,2006). 2.3. Experim ental design 2.3.1, D evelopm ental exposare/intact Timed-pregnant CD-I mice(n=7-22 dams per dose group over twoblocks) received0.0.01,0.1,0,3.l. 3.or5mg/kgPFOAbyoral gavageonthemorningsofGD 1-17,Damswereweigheddailypriortodosingandthroughoutgestation.At birth, pups were individually weighedandsexed. Pups within atreatment groupwere pooledandrandomlyredistributedamongthedamsoftheir respective treatment groups, and liners were equalized to 10pups (bothgenders represented). Dams p. 47 EP. Hines e t a l / Molecular and Cellular Endocrinology304 (2009) 97-105 or PFOA Developments! DosGinOg 1-17 PFOA A3Ddousilntg wks lisssri! 3M* __ 15-16 whs j *L i gOO Birth spent Positive w an " wx Glucose tolerance test (young) 21-33whs 42Whs 11 ..... Mandibular bleeds 1I Body Mass Composition Measired 70-74WHS ji Glucose tolerance Test (old) Food intake monitoring 1 18 Months measuEr2e _____ Weightmonitored(developmentallyexposed,ovx.Intact). Fig.1. DatacollectionschematicforstudyofdevelopmentallyandadultPFQA-exposedfemalemice. 99 that deliveredsmall litters (it4 pups)wereexcluded fromthe remainderofthe study.Pupswereweanedat 3weeksofageatwhichpointfemaleswereretained andboused3-5 micepercage.Maleswereevaluatedseparately,atendpointsthat varied fromthosereportedhere. 2.32. D evelopm ental ueposure/avariettom y A subset or developmentalty exposed female siblings (OmgPFOA/kg, n-8; 0 .0 1 mgPFOA/kg, a-IS: 0.1mgPFOA/kg, n-11; 03mgPFOA/kg. n-14; 1mgPFOVkg.il-6; 5mgPFOA/kg,n=7)wereevariectomized(ovx)at21or22 days ofage.before theonsetofpuberty.Animalsweresedatedwithketamine/xylazine 87/13mg/kg Lp. respectively), their ovaries surgically removed through the abdomen,sutured, and animals were placedin warmingcages until they regained alertness.Bupienorphineanalgesic(0.05mg/kg)wasgiventwicedailyi.m.for48ii in 0.1mivolumeforpainrelief 2.33. Adult exposure A separatecohortof mice receivedPFOAstartingat 8weeks of age. for 17days (OmgPFOA/kg,n-8; 1mgPFOA/kg.n-14; 5mgPFOA/kg,n-14). 23.4. Data collection Thedata collection schemefor thesestudiesis showninFig. 1.Bloodwascol lected from the submandibularveins ofovxandintact micebetweentheagesof 21 and33weeks.Thesebleedstookplacebetween14:00and18:00,and200pi of blood(100pi ofserum)wascollectedforsubsequentanalysesofinsulinaid leptin. Femalesinail threeexposurescenarioswereweighedweeklyupto9monthsofage andthenmonthlyuntil 18months.Thenumberofintact,developmentallyexposed miceweighed weekly/monthlywas 10.25.20,11, and32,respectivelyfor0,0.01. 0.1.03.and 1.0mgPFOA/kg,Ifmice becamemoribundbeforethe studyended, they wereeuthanizedin compliancewith the protocol approvedby theUSEPA1ACUC (eariy necropsy). Date andcauseofearly morbidityor mortality was recorded if known. Ateariy necropsy(collectedwhen necessary)orat 18months,trunkblood, retroperitoneal abdominal white(foundlyingventral totheintestinesandrepro ductivetract)andinterscapuiarbrownfatpads,abnormalgrowths,andorganswere collectedfromallexposuregroups.Relativeorganweightis usedtoexpressorgan weight aspercentoftotalbodyweight.DataarereportedhereasmeanSEM. 2.4. Glucose referencerest Glucose tolerance tests were performed on two groups of intact developmentaiiy PFOA-exposed animals: old adults (17 months of age With 0, 0.1,1 or SmgPFOA/kg; n-8-13 perdosegroup)andyoungadults(15-16weeksoldwith 0.1 or 5mgPFOA/kg: n-12per dosegroup) Thenight before theassay,furwas shaved from Chelateral areaofthe towerlegtoexposethe saphenousvein andani malswereFasted.Thefollowingmorning,themicewereweighedandbloodglucose wasmeasuredbycollectingadropofblood fromeachmouseviapunctureofthe saphenousvein (ortail veinif necessaty).Theblooddropwasplacedonateststrip, andinserted intothecalibratedglucometer(AccuctiekAdvantage)forbaselineglu cosemeasurement.The miceweretheninjected i.p.witho-glucosesolution(2g/fcg bodyweight fromastocksolution), andbloodglucoseconcentrationsweremea suredat20.40,60 and129(old mice)or 180(youngmice)minutes (1-3min)after theinitial glucoseinjection 2.5. Serumlep tin Serum(10pi) collectedbymandibularvenipuncturewasassayedforleptin by radio-immunoassay(Linco ResearchSt.Charles.MO)Followingthe manufacturer's protocol(n-5,controls;n-18,0.01;n*16.0.1;n-17.03:n- 24.1mgPFOA/kg).The coefficientofvariation(CVs)forthestandards(concentrationrangeof0.2-20ng/ml) rangedfrom0 .1 XtoKOtt.ThequalitycontrolstandardstermedQC1(expectedrange 0.6-13ng/mi)andQ.C2(range1.E-3.8)hadameasuredconcentrationintheseassays of0J9 and2 .9, respectively. 2 3 Scntm insulin Sera(lOpi)collected by mandibular venipuncturewere assayedfor insulin by theultra-sensitive single molecule immunoassaybySingulex (Alameda. CA] fol lowingthe manufacturer's protocol (n-9 control, n-21, 0.01mgPfOA/kg; n=16, 0.1mgPFOA/kg: It-11,03mgPFOA/kg: n-31. t mgPFOA/kg) Sampleswere ana lyzed using a 384-well plate format with imnmdDjial capture and detection antibodiesontheSingulexErrenaequipment.TheCVsfortheassaystandards(range 19.5-5000pg/ml) werefrom3%to 17*.TheassayLODwas 16pg/ml.AHsamples were runon thesamedayand the interassayCVwas9.4*and 5.1*for the29and 1745pg/mlqualityassurancestandards,respectively. 2.7. Body m ass composition Wholebodymasscompositionwasmeasuredinlive,non-sedated42-week-okl miceusingtheBrokerMirtispecmq7.5IF50LiveMouseAnalyzer(TheWoodlands, IX) The minispec was a benchtop7.5MHz time-domain nuclear magnetic reso nance(NMR)analyzer,whichquantified bodyfat, leantissue, andfreebodyfluidin mice.TheminispecwascalibratedbyBrokerOptics,Inc.staffpriortoanimal analy sis with dailyvalidationsusingBrokerstandards.Micewereweighed andinserted intotheinstrumentforanalysis(1-2min/anhnal)Intactdevelopmentallyexposed femalemicethatunderwentbodymasscompositionanalysisincludedcontrol,031, 0.1,03.and 1mgPFOA/kg(n- 9. 23.20,11, and 32, respectively) dosegroups. It was not possible to perfotmthesemeasures withyounger micedueto equipment availability. '2.8. M easurem ent o f E t insereino f intact m ice IS months Serum Er (25pi volume) FromIS-month-old mice (intact developmentally PFDA-exposed animals) was measured with time resolved fiuoro-iriimunoassay (DELFIAEstradiol IGr, Waliac Oy. Finland) following the manufacturer's recom mendation using a VICTOR*D 1420 Muttilabel counter, PerkinElmer Precisely time-resolvedItuDrometerCPerkinEtmerlife &AnalyticalSciences;Shelton,Cr)The CVs for the standards(concentrationrangeof631-1425pg/ml) rangedfrom02* to43*. 23. Feed consum ption Feed consumption in T7-month-oId, developmentallyexposed, intact female mice(n-6 perdosegroup.0,0.1.1 and5mgPFOA/kg)wasmeasuredinmetabolic cages.Micewereallowedtoacclimate tothecagesfor1weekandfoodintakewas monitoredduringthesecondweek.Micewereindividually housedandprovided withapre-weighedamountofpowderedlabchowadlibitum.Theremainingchow wasmeasuredattheendoftheweekandthetotalamountwassubtractedfromthe startingamounttodeterminethetotal feedconsumedforeachmouseperweek. 2.10. M easurem ent o f se n t m PFOA Trunk Moodserum samples (-SOp!) fromthe female CD-I offspring at 18month necropsies orfrommiceterminatedat earlierintervals becauseof illness were transferred to theCDCfor PFOAmeasurement.SerumPFOAdetermination wasperformedasdescribedinKukienyiketal.(2005)andWhiteetal.(2009) 2.11. Statistics DatawereanalyzedusingSAS9.1SA5Inc-Cary,NC)BodyweightonPND1was evaluatedasfittermeansasthesedatawereobtainedpriortomixinglitteroffspring withinadosegroup. ' BodyweightsateachtimepointwereanalyzedwithmixedeffectsHnearmodels (SASProcMixed) toestimatemeansandstandarderrors andtest fordoseeffects separatelybyrimepoint.FOreachtimepointthemodelincludeddoseasafixedeffect p. 48 ' 100 .P.Hines et ol./ Molecular and aihtlar Endocrinology304 (2009)9 7 -J05 andcagenested within doseasarandomeffect. Pairwise t-testswerecalculated to test Toranydifferencebetweeneachtreatment group meanand thecontrol group. Repeatedmeasuresanalysisofbodyweightdatawasevaluatedtwoways.First, weights wereaveraged by animal overeight 10-weekintervals.This wasdone to decreasemissingvalues inthedata duetoanimal mortalityin latelife thatwas not equalacrosstreatment,andtoreducetheeffectoflargebodyweightvarianceslater in life.Thisdatasmoothingmethod decreaseduninformativeshort-termvariations andalsoreduced the numberofestimatedparameterstoatractablevalue. A multi variaterepeatedmeasuresanalysis(SASProcGLM)wasperformedonthesereduced data. Subsequent to asignificant finding comparisonswerecarriedout assubtests oftheoverall analysisofvariance(ANOVA)atspecifictimesordoses. Second.SAS ProcMixedwasusedtoperformaunivariaterepeatedmeasures analysisoftheweightsacrosstimeupuntil37weeks(latestweightpointatwhicbno animalshaddied).Themodelestimatedaseparatefixedquadraticcurveacrosstime foreachdosegroupandincludedarandomeffect forcagenestedwithindoseCor relationwithinanimalswasmodeledwitharandomeffectforanimalnestedwithin cageanddosein addition toanautoregressivecovariancestructurewithineachani mal. Inthisway,thecovariancematrixforeachanimal'smeasurementsincludeda constantcovariancecomponentatantimepointsinadditiontoacomponentwhich decreasedastimepointsgrewfartherapart. Tissueweight,relativetissueweight,bodycomposition,foodconsumption,and body weight measurements were analyzed using a one-way ANOVA{Dennett's post hoctests),with dosebeingtheindependentvariable.A blockingvariablewas included to adjust for the group difference. Noadjustment was made for multi ple comparisons. Glucose tolerancewas comparedat individual collection times by one-way r-testand over time by repeated measuresandareaunder thecurve comparisons according to the trapezoidal rule. Hormone (insulin. Ej and leptin) concentrations wereanalyzedusingANOVAfollowedbyTukey'spost hoctest. Mortality datawereanalyzedwithproductlimited survival estimates; tog-rank andWilcoxon tests wereusedtotestfordifferencesamongthetreatmentgroups in survivalacrosstime(SASProcLifetest).Thelevelofsignificanceforall testswas p<0.05. 3. Results 0.01 0.1 03 ] Dose PFO A (m g/kg BW) (C) 50-j * ** 3.1. Developmental exposure 3.1.1. Early and mid-life body weight effects There w ere no significant differences in live pup num ber at b irth by dose group (p<0.05) and postnatal m o rta lity was not addressed in this study as litte rs were equalized a t b irth . On post natal day (PND) 1, the average w eight o f d ie developm entally exposed 5mgPFOA/kg offspring was significantly less than con tro ls (Fig. 2A ); no other dose group demonstrated significant litte r w eight effects at PND1, A t weaning, mean female body weights were s till significa ntly decreased in the 5 mg PFOA/kg(13.9 g 0 .8 ) compared to 18.4g 0.4 in control untreated pups. A t this tim e, the 1mgPFOA/kg exposed animats were also significantly sm aller than controls (p <0.05; 16.4 g 0.3). Tim e-grouped mean body w eights o f the female offspring over th e ir life tim e are shown in Fig. 2B. Beginning at 10-13 weeks o f age, there was an increase in w eight in the 0.1 and 0.3 mgPFOA/kg groups compared to controls; by 20-29 weeks o fage,females devel opm entally exposed to PFOA showed significant dose-dependent increases in body w eight at 0.01, 0.1, and 0.3 mg PFOA/kg which extended to 40 weeks o f age in the 0.01 and 0.1 mgPFOA/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 w eights 11-15% higher than controls. Continuous analysis of repeated measures o f body w eight over tim e demonstrated th a t the five dose groups were sim ilar in inter cept using a quadratic fit; however, the 0.01, 0.1 and 0.3 groups had a significantly greater week effect than control, indicating that th e ir weights were changing at a more rapid rate than control o r 1 mg/kg. This is shown in Fig. 2D fo r weeks 6-37 (the latest w eight collection tim e point p rio r to death o fany study anim als).A ddition ally, the 0.1 m g/kg (p - 0.056) and 0.3 mg/kg (p =0.046) groups had larger negative coefficients fo r week2 (week squared), suggesting th a t th e ir w eights were starting to fa ll off more quickly at the later tim e points than the control groups (not shown). The estimated w eight curve fo r the 1 mg PFOA/kgdose group was not significantly different from the control curve. Data from 5 mg PFOA/kg exposed 11111 o.oi o.i 03 Dos*PFOA (mg/kg BW) i Elg.2. BodyweightsofdevelopmentallyPFOA-exposedfemaleoffspring Dataare shownasmeanSEMwith `p<0.05vs.control. (A)Popweightat PND1afterdevel opmentalPFOAexposure.(B)BodyweightoffemaleCD-I miceovertheirlifetime, followingdevelopmentalPFOAexposureoverSperiodsortime(period1 (0-9weeks old),period2(10-19weeksold),period3(20-29weeksold),period4(30-39weeks old).period5(40-43weeksoid),period6(50-59weeksold),peiiod7(60-59weeks old),andperiodS(70-79weeks)).(CJCroupmeanbodyweightsoffemaleoffspring at20-29weeksofagedemonstratingexcessiveweightgainatlowdoses.(D)Dosedependentquadraticregressionfit torepeatedmeasuresofbodyweightinfemale mice.Anincreasedrateofweightgainwasseenin0.01,0,1.and03mgPFOA/kgdose groupscomparedtocontrol and1rogPFOA/kg. p. 49 fl Hines et of./Molecularand CellularEndocrinology 304 (2009) 97-05 (A) 70-, 3 so-> 50- * * 101 20 40 60 Time (minutes) 180 o Dose PFOA (mg/kg BW) ** Time (minutes) Fig,3. Blood glucoseconcentrationsfollowingaglucosechallengeaftertime0in (A)young(15-16weeksold)and(6)old(70-74weeksold)femaleCD-I micethat weredevelopmentallyexposedto PFOA. Dataareshownas meanSM. mice, w hich were decreased in BW compared to control at PND1, weaning, and 18 months, are not shown. 3.1.2. Serum glucose tolerance testing Because o f the excess w eight gain in the PFOA developmen ta lly exposed mice during m id-life, various tests were conducted on these anim als (as ciose to the appropriate age as was possible) to examine the associated effects o f 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 m ice (control, 0.1,1, or 5mgPFOA/kg. p<0.05. Fig. 3). In a tim e-dependent comparison,young mice exposed to 1 mg PFOA/kg showed a nearly significant increase in blood glucose over control animals a t 20 m in post-glucose challenge (p=0.06). In old PFGAexposed m ice, although there appeared to be dose-dependent glucose insensitivity at 20 m in, this s h ift in response was not sig nificant. 3.1.3. Serum insulin and leptin Serum insulin and leptin measurements were made using blood obtained via m andibular bleeds between 21 and 33 weeks (w ith in the rim e fram e of greatest observed body w eight increases) using intact fem ale mice dosed w ith 0, 0.01,0.1,0.3, and 1 mg/kg PFOA. Insulin and leptin concentrations were significantly increased in mice developm ental^ exposed to the lowest doses o f PFOA tested (0.01 and 0.1 mg PFOA/kg). Although elevated from the control mean, le p tin concentrations were not significantly different from control a t 0 3 o r 1 mg/kg PFOA (Fig. 4). 3.1.4. Fat to lean ratio A t 42 weeks o f age, mice from block 2 (control. 0.01, 0.1, 0.3, and 1 mg PFOA/kg) were evaluated using a Bruker Optics Body Mass Analyzer, which determ ines the am ount o f fat, lean and fluid III! 0 0.01 0.1 03 DosePFOA (mg/kg) ] Fig.4. Serumleptin(A)andinsulin(B)ininitL-at21-33weeksofagef'p<0.05vs. control).Significantelevationsareseenat0.01and0.1mgPFOA/kg.Dataareshown asmeaniSEM. in live animals. There was no significant increase detected in % body fat:body weight in PFOA-exposed mice (data not shown). Deveiopmentalty exposed mice had no significant differences in fatzlean ratio across dose groups when compared to control (means ranged from 0.75% in controls to 0.9% in 0.01 and 0.1 mg PFOA/kg). Although no dose groups were significantly different from control, there was an increase above control levels o f about 12% in mean %fat:body weight ratio and 14% in mean fat: lean ratio in the dose group exhibiting the largest change in body w eight at 24 weeks (0.1 mg PFOA/kg). 3.1.4.7. Feed consumption. Feed consumption was measured in 17m onth-oid, developm ental^ exposed intact mice (0. 0.1. 1 and 5 mg PFOA/kg) and no significant differences were found across dose groups when compared to controls (mean 26 g/week con sumed: individual data not shown). 3.1.5. Late lifeorgan and body weight effects A noted loss o f animals after 36 weeks o f age was further eval uated (Fig. 5). At 51 weeks old, when there was no m ortality in controls there were 20%, 10%, 36%, and 6% m ortality rates in 0.01, 0.1.0.3, and 1mgPFOA/kg groups, respectively. By 76 weeks, there was a 40% m ortality rate in controls, and 32%, 63%, 60%, and 44%in 0.01,0.1,0.3 and 1 mgPFOA/kg groups, respectively. However, there were no significant differences between control and any treatment group at specific tim es in late life o r in survival across time. Among those mice surviving to 18 months, body w eight o f PFOA-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). A t that tim e, a ll remain ing females were necropsied. Trunk blood, tissues (affected or o f interest) and abnormal masses were collected, weighed and fixed fo r future study. Serum was collected and PFOA levels were mea sured. The m ajority o f the samples across dose groups had PFOA concentrations lower than the lim it o f detection (O.Sng/ml) w ith detectable values at maximum concentrations o f 3 3 ng/ml. and 102 EP. Hines et c l/ Molecularand CellularEndocrinology 304 (2009)97-JOS Tabic 1 MeanorrelativebodyandtissueweightsatIBmonthsofageinintactandovariectomized(ovx)femaleCD-I mice. PFOAdose . Bodyweight(g) (mg/kg) - . Abdominalwhitefei - .-: . -tatertcapatarbrown . . l/Relativespleen Weight(g) Fatweight(g) Weight(ZJF Relativeliver Weight( X f Intact Ovx Intact r Ovx Intact Ovx Intact Ovx Intact Ovx 0 54901.83 52.73*5.67 707*0.56 3.831.03 073*004 037*0.09 -039.'0,05 7 052*0.16 430*0.10 430*044 om 0.1 03 56361.48 52.613.63 63B0,41 5.081.0b 0.80*004 036*0.04 030 0.03 029*0.04 3.99* 0.11 54.80* 1.17 5276198 53tD37 4.84*0.71 ,-0.82094 0,46096 0.450.12 040098 420 0.41 56,00* 7,74 4936333 536D.S3 4370.46 '0179996 039032 045 * 0.10 033038 43o* 031 4.12035 ? 4.18021 407*022 : 1 56.15135 51.47353 5.82043* 5.82*0.77 ,0.89094* <0.69*0.06*: 0300.03 022oi)2t': 4.02* 0.10 335029**,' 3 53.694:227- 'iiiChV;-:-; 5 . . :4927 1.51* 55.135.76 DC 4.48*0,65* nc , r 1220,10 nc 5.861,67.1; 0360.05 . 0.62020 . 0.18093* nc " 032 024 021094" 338 023 437 034 nc -.-335033'. nc. Denotesnotcollectedfromthisdosegroup. * f 0.01vs.control. " p-0.05-0.07. 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 developm ental^ exposed fem ales at IS m onths when compared to controls (non-cycling: mean range across doses from 12.9 to 15.8 pg/m l). Tissue w eights from 18-m onth-old animals (in ta ct and ovx) are shown in Table 1. To determ ine i f the w eight o f fa t depots was altered in old animals due to developm ental PFOA exposures, the retroperitoneal abdominal w hite and interscapular brown fa t pads w ere collected and weighed. Abdom inal w hite fa t w eight and rela tiv e w hite fa t w eight both showed significant decreases vs. control (p <0.05) at 1 and 5 mgPFQA/kg. W hite fat weights were no t col lected fo r 3 m g/kg FF0A animals.A t 18 months, interscapularbrown fa t w eight and relative brown fa t w e igh t both showed significant increases above control(p<0.05) at 1 and 3 mgPFOA/kg. The spleen was quite variable in w eight among the different treatm ent groups, b u t there was a significant difference in spleen w eight and relative spleen w eight vs. control at 3 mgPFOAffcg (p<0.05). Finally, a t 18 m onths, no significant differences in live r w eight o r relative liver w e igh t were detected. reach statistical significance. W hen comparing the body weights o f animals in the ovx study by treatm ent group, over tim e (4 weeks to 18 months), using statistical methods consistent w ith those used fo r intact animals, there was no effect o f PFOA(Fig. 6B). Compar ison o f ovx animals to in ta ct anim als at 20-29 weeks, as shown in Fig. 6A, demonstrates an absence o f body w eight gain over con tro l in the ovx animals treated w ith PFOA. PFOA exposure did not stim uiate increased w eight gain (above tha t o f control ovx) at any developmental exposure level in the absence o f the ovaries (also seen in Fig. 6B).The ovx anim als were siblings to the intact animals in th is study. The ovx animals were also assessed at 18 months. Deveiopmen ta lly PFOA-exposed ovx animals showed no significant differences in body w eight when compared to control ovx females (con tro l m ean=52.7 5.67; highest mean, 1 m gPFOA/kg*61.53.3; V Intact SIOvx 3.1.6. Effect o f ovariectomy on tissue and body weight gain A group o f deveiopm entally PFOA-exposed animals (0,0.01,0.1, 0.3, 1, and 5 mgPFOA/kg) were ovx at weaning and th e ir body w eight gain and adult health was assessed u n til they reached 18 m onths o f age. A t m id -life the w eight o f the control ovx females was expected to be greater than th a t o f the sham-operated, intact controls (Fig. 6A; set ofbars atO m g/kg). but the variance in the ani m al weights was appreciable and therefore the differences did not o.ei o a PFOA Dose (mg/kg BW) 1 Fig. 5. Survival curves for deveiopmentally PFOA-exposed female mice (0-1mgPFOA/kg). Although a fair number of PFOA-exposedanimals dieearly, a Lifetcst(SAS)analysis detected nosignificantdecreasein timetodeath.Thereasons forearlylifemortalityareunderinvestigation. Age (weeks) Fig. 6. (A) PFOA-dependentchangesingroupmeanbodyweightofintactandovx female offspringat 20-29 weeksof age.Therewas nochangein bodyweightor ovxanimalsacrossPFOAexposures.(B)Dose-dependentquadratic regressionfit to repeated measuresof body weight in ovx female mice. Unlike intact siblings, no significant differenceswereseenbetweendosegroupsintheovxanimals. .R Wines et al./M olecular and Cellular Endocrinology 304(2009)97-105 103 As w ith inta ct siblings, the tissue weights o f ovx animals are reported in detail in Table 1. in ovx animals, neither abdominal w h ite fa t pad weight, nor relative abdominal w h ite fat pad weight, were significantly different from ovx o r inta ct control levels. This varies s lig h tly from intact siblings, where the w h ite fat pad was significantly decreased in size; although, in animals that weighed significantly less than inta ct controls. Among PFOA-exposed ovx animals, both interscapular brown fat w eight and relative brown fat w eight (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 sim ilar to the effect seen in in ta ct animals, and was significant a t the same dose. Spleen weight (data not shown) and relative spleen w eight in ovx animals was highly variable at IS months, and showed decreases, albeit not highly significant, at the 1 and 5 mgPFOA/kg doses (p=0.06 and p =0.05, respectively; Table 1). 1 and 3 mgPFOA/kg (not 5 mg/kg) were the doses in the inta ct animals showing the largest decreases in relative spleen w eight compared to controls. Finally,relative live r w eight showed no significantdifferences across dose groups when compared to ovx control. 3,1.7. Lack o f effectsfrom adult PFOA exposure A t 18 m onths o f age, body and tissue weights were recorded in adult PFOA-exposed mice. A dult PFOA exposure had no effect on term inal body or organ weights. When a comparison o f data from 18-m onth-old adult inta ct and developm ental^ exposed ani mals in the 0 ,1 and 5mgPFOA/kg dose groups was made, body weight, brown fat weight, and w hite fa t w eight o f the 1 mgPFQA/kg developm ental^ exposed animals were significantly higher than the same dose in aduit-exposed animals (data not shown). 4. Discussion These studies demonstrated the effects o f developmental PFOA exposure on CD-I female mouse body and organ w eight, as w e ll as serum le p tin and insulin in adulthood. In the developmental PFOA studies, a dose-dependent dichotom y o f phenotypes was present in intact fem ale mice; latent effects present follow ing high doses were not present in mice exposed to low-dose PFOA and vice verso. Although there was no detectable change in body weight neona tally, low-dose PFOA exposures (0.01. 0.1, or 0.3 mgPFOA/kg) led to significantly increased mean w eight and rate o f weight gain in m id -life (u p to and including37 weeks o f age) and a coincident sig nificant elevation o f serum ie ptin and in sulin values between 21 and 33 weeks (0.01 and 0.1 mgPFOA/kg). Our low-dose hormone data indicate potentially im portant m etabolic changes that m echanistically support the findings o f increased w eight in the low er dose groups. Previous dosim etry work in o u r lab has shown that in utero exposure to PFOA in the mouse translates into an extended developm ental exposure period via lactational exposure (a ll o f gestation and nearly 3 months post natally; W hite et al.. 2009: W olf et al., 2007; Fenton et al.. 2009). This long exposure may lead to reprogram m ing/m etabolic events that govern fat metabolism or appetite control. Although we were unable to perform some o f the other end points o f interest dur ing this tim e period o f greatest w eight gain, our findings relating leptin and insulin concentrations to the tim e ofoverw eightin PFOAexposed m ice support our theory. Other environm ental chemicals, termed environm ental obesogens (dietylstibestrol (DES), 20H-E2, 40H-E2, genestein and bisphenol A), have been shown to induce besity in adulthood after low-dose developmentalexposure,w hile inducing w eight loss at higher doses (G riin e t al., 2005; Newbold et a!., 2005: Miyawaki et al.. 2007) and are reviewed furthe r w ith in this issue. Serum leptin was significantly elevated in m id -life in die low dose PFOA-exposed groups. This effect occurred at the same PFOA dose range as overweight in these animals, congruent w ith a leptin-resistance mechanism o faction fo roverweight,aspreviously reported in humans (Considine e t al., 1995). Others have reported increased ie p tin w ith 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 in sulin 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, bu t not significant, at 15-16 weeks in our study), reflecting the loss o f a post-challenge peak in insulin response (reviewed in Montecucco et al., 2008). insulin resistance is known to be associated w ith excess abdominal fat in normal and overweight women (Carey etaL, 1996).High plasma levelso finsulin andglucose,due to insulin resistance,are often associated w ith type 11diabetes and metabolic syndrome in humans, and thus this effect of low-dose PFOAdevelopmental exposure and its association w ith increased serum in sulin are im p ortan t The ovx data were d iffic u lt to interpret. The lack o f additions} weight gain w ith developmental PFOA and ovx may reflect a "ceil ing effect" or that ovx-induced w eight increases may have masked any effecto fPFOA.Alternatively, asw eight gain and metabolic hor mones can be regulated by estrogens, the role o f the ovaries in developmental effects o f PFOAwas explored by using ovx animals. The potential importance o f the ovary in the effects o f PFOA was based on the observation tha t LH-transgenic (overexpressing) mice (Kero et aL, 2003) were phenotypically sim ilar to ours (increased body weight, increased brown fa t depots, and predominant ovarian cysts not discussed in this paper). We hypothesized that removal of the LH target (the ovary) in our study may reveal the mode of action for PFOAeffects fo r the increase in brown fat and possibly the exces sive w eight gain. Ovx animals typically gain body w eight in excess vs. intact animats (Kamei et a t, 2005). The critica l role o f the ovary in w eight gain o f in ta ct PFOA-exposed females beyond that o f ovx treatm ent-m atched siblings in the 0.01 and 0.1 mg PFQA/kg groups was novel and signifies the ovarian axis as a potential m ediator o f PFOA-dependent m id -life w eight changes. Another potential m ediator o f these intertw ined low-dose PFOA-induced effects is the peroxisome proiiferator-activated receptor (PPAR) activation pathway. PPAR gamma (PPAR-y) and PPARalpha (PPAR-a) are involved in lip id metabolism in adipocytes and liver/skeietal muscle, respectively (reviewed in Medina-Gomez et al., 2007; Abbott, 2009). These PPAR isoforms are known to influence lipogenesis/weight gain and have been shown to be regu lated by environm ental compounds such as trib u ty ltin (Grun et al., 2006; reviewed in this issue).W eight lossevents in leptin-deficient. obese, and insulin-resistant mouse models have coincided w ith PPAR-regulated changes in gene expression (Holvoet, 2008). A down-regulation o f PPARisoforms involved in energy expenditure, lipogenesis o r fatty acid synthesis have been reported in adipose and skeletal muscle o f ovariectomized mice (Kamei et aL, 2005). PFOA has been shown to be a PPAR activator in liv e r tissue (high doses) and ceil lines, and to be required fo r PFOA-induced devel opmental to x ic ity in mice (Takaes and Abbott, 2007; Abbott et al., 2007; Abbott, 2009). I f PPAR activation via receptor binding is a prim ary mode o f action for body w eight effects follow ing PFOA exposure, the decrease in the PPARreceptors follow ing ovariectomy and decreased circulating estrogens may explain the lack o f effect o f PFOAin ovx mice. However. PFOA-induced consequences o fPPAR activation follow ing a developmental exposure are ju s t beginning to be evaluated. A fter 40 weeks o f postnatal age. an increase in m ortality was detecced in a ll animals. There are previous reports in the literature o fincreased m ortality in non-treated CD-I mice,attributed primar 104 E.P. Hines e t aL/M olecular end Cellular Endocrinology 304 (2009) 97-105 ily to thym ic lymphomas (Son. 2004: Taddesse-Heath e t al., 2000). Because o f th is confounding circumstance, repeated measures o f body w eight were only follow ed out to 37 weeks o f age. The other h a lf o f die phenotypic dichotom y caused by devel opm ental PFOA exposure was also novel. Developmental exposure to higher doses o f PFOA (1, 3 and 5 mg PFOA/kg) led to a vastly diffe re nt phenotype from low-dose PFOA exposure. This effective PFOA dose dichotom y may m anifest its e lf in our study via unique modes o faction;the animals w ith highest dose(s) o fdevelopmental PFOAexposure have decreased early life body w eight and term inal body w eight (5 mg PFOA/kg) w ith significant decreases in w hite fat w e igh t at 18 months (1 and 5 m g PFOA/kg). significant increases in brown adipose (1 and 3 mg PFOA/kg). and significant decreases in spleen w eight (3 mgPFOA/kg) findings th a t are absent w ith the low er doses o f PFOA. Others have reported dose-dependent loss o f w h ite tis sue adiposity in adult male m ice after PFOA exposure (0.02% PFOA w eight/chow w eight, w hich translated to approxim ately 32 mg PFOA/kg BW daily) w ith fa t loss, w ith o u t fa t cell number loss, th a t is PPARy-independent w ith P-adrenergic activation (Xie et a!,, 2002). In tha t same study, investigators also reported w hite fe t and body w eight decrements a t higher doses th a t were absent at low er doses. Yang et aL (2002) showed PFOA-dependent weight loss was abrogated in PPAR-ot n u ll m ice, indicating th a t PPAR-a is a probable regulator o f w eight loss in the high dose animals. In subsequent studies, Xie et aJ. (2003) showed th a t after cessation o f exposure o f adult m ale animals to PFOA(0.02% PFOA weight/chow w eight, 32 mgPFOA/kg BW) daily fo r 7 days followed by 10 days recovery, w eight loss and w h ite adipose levels returned to base line, w hich confirm s the im portance o f developmental exposures fo r the la te n t effects reported here. In our model w ith developmen ta l PFOAexposure we seeperm anent w eightloss and w hite adipose tissue loss a t the high dose o f PFOA. However, there may be m erit in furthe r exploring these mechanisms o f action, as p-adrenergic receptor upregulation is also associated w ith increased brown fat mass in w inter-acclim ated anim ats (Feist, 1983), and this tissue was associated w ith high dose (and no t low dose) effects in both intact and ovx animals in this study. Although we suspected alleviation o f effect in the brown fat pad by elim inating the ovary (based on phenotypes in Kero et a!., 2083). significant increases in brown fat were seen a t 1 mgPFOA/kg in both intact and ovx animats. A t the IS-m onth tim e point, some endpoints remained unchanged across dose groups including live r size. Earlier w ork has shown significant hepatomegaly after developmental PFOA expo sure (1 and 3 mg PFOA/kg) observed out to at least 3 weeks after b irth (the latest tim e point evaluated: W olf et a l, 2007; W hite et al.,2007),Thetransientnatureofhepatom egaIyhasbeeniIlustrated in other acute adult exposure studies (reviewed by Lau et al.. 2007), and is fu rth e r confirm ed in these studies (intact and ovx). A final im portant com ponent o f these studies evaluated adult vs. developmental exposure to PFOA on body tissue weights. These data suggest that the tim in g o f dosing (adult vs. developmental 17-day PFOA exposure) was critica l fo r latent effects. There was no effect o f 17-day adult PFOA exposure on any endpoint in this study (early life or latent) when compared to age-matched, vehidegavaged controls. In conclusion, the tim in g and dose o f PFOA exposure fo r induc tio n o fdichotomous, persistent, adult health effects in CD-I female mice are critica l. Developmental, low-dose PFOA exposure led to increased w eight in adults, w ith increased serum insulin and leptin , a health effect not seen in high dose animals. No observable adverse effect levels (NOAEL) fo r body weight gain, serum Ieptin and insulin concentrations were not determ ined in this study; but 0.01 mg PFOA/kg had a significant im pacton these particularly sen sitive end points. The ovary appeared to play an im portant role in the overweight effect in m id -life , and it is proposed that there is a common mode o f action, potentially dysrgulation o f PPAR and its signaling through ovarian hormones, that may be responsible fo r these low-dose health effects. Further studies addressing long term PFOA-induced health outcomes in m ice should focus attention on internal dose relative to the low-dose health effects seen in this study, as w e ll as the mechanisms o faction, so that any relevance to human health effects can be addressed. A cknow ledgem ents We w ould like to thank Broker Optics, Inc. fo r the use o f the Bruker M inispec mq 7.5 LF50 Live Mouse Analyzer and Harry Xie and Basil Desousa o f Broker Optics, Inc. fo r th e ir technical assis tance. We w ould lik e to acknowledge Antonia Calafat and her labo ra to ry staff, Kayoko Kato and Zsuzsanna Kuklenyik, in the Division o f Laboratory Science, National Center fo r Environmental Health. Centers fo r Disease Controland Prevention fo r the analysis o fserum PFOA concentration from 18-m onth-oid developm ental^ exposed female m ice; Donald Doerfler, Experimental Toxicology Division. U.S. EPA, and Judy Schmid, Reproductive Toxicology Division (RTD), US. EPAfo rth e ir statisticalsupport; Deborah Best. RTD,fo r conduct ing the estradiol assays: Veronica Luzzi. David Gibson and staff at theCore Laboratory fo r Clinical Studies a tWashington University in St. Louis, MO, fo r perform ing the serum insulin assays, and finally. 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