Document e3yv5ebYnD22oaB41RB03RLm

Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1800/Cincinnati, OH 45202-3957 / Tel: 513.381.2838/Fax: 513.381.0205 /wvwv.taftlaw.com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix / Beijing Robert A. Bilott 513.357.9638 bilott@taftlaw.com FYI-00-001378 OUD June 12, 2009 ^ / 2 . 3 _ ^ ' 3 ? 0 0 - / CO ~~ipr* TSCA Confidential Business Information Center (7407M) EPA East - Room 6428, Attn: Section 8(e) & FYI U.S. Environmental Protection Agency 1200 Pennsylvania Avenue, NW Washington, DC 20460-0001 f f E 'IJ " 2 Re: Submission To TSCA 8(e)/FYI Database Re: PFQA/PFOS To TSCA 8(e)/FYI Database: We are hereby providing the following information for inclusion in the TSCA 8(e)/ FYI databases with respect to PFOA/PFOS: 1. Fenton, S.E., et al., "Analysis of PFOA in Dosed CD-1 Mice Part 2: 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: Low Doses Induce Elevated Serum Leptin and Insulin, and Overweight in MidLife," 304 Molecular & Cellular Endocrinology 97-105 (2009). 85090000010 RAB:mdm Enclosure 11434740.1 o o n im n s n o c b "^>0 ( 7 3 ft/ itinius & Hollister LLP Inut Street, Suite 1800 ati, Ohio 45202-3957 06 r u '^ "u s POSTAGE 016H16504933 $02.419 06/12/2009 Mailed From 45202 US POSTAGE TAFT STETTINUJS & HOLLISTER LLP ROBERTA. BILOTT TSCA Confidential Business Information Center (74 07 M ) ERA East - Room 6428, Attn: Section 8(e) S FYI U.S. Environmental Protection Agency 1200 Pennsylvania Avenue, NW Washington, DC 20460-0001 20460000199 P-3 y Accepted Manuscript Title: Analysis of PFOA in Dosed CD-I M ice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups Authors: Suzanne E. Fenton, Jessica L. Reiner, Shoji F. Nakayama, Amy D. Delinsky, Jason P. Stanko, Erin P. Hines, Sally S. White, Andrew B. Lindstrom, Mark J. Strynar, Syrago-Styliani E. Petropoulou 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 o f PFOA in tissues and fluids from pregnant and lactating mice and their pups. Reproductive Toxicology (2008), doi: 10.1016/j.reprotox.2009.02.012 This is a PDF file o f an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Analysis of PFOA in Dosed CD-I Mice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups. Suzanne E. Fenton**, Jessica L. Reinerb, Shoji F. Nakayama\ Amy D. Delinskyc, Jason P. Stanko*, Erin P. Hines*, Sally S. White*'*1, Andrew B. Lindstrom', Mark J. Strynar', and Syrago-Styliani E. Petropouk>ub* *Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, ORD. U.S. EPA, MD-67, Research Triangle Park, NC2771J, USA hOakridge 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 dCurriculum in Toxicology, University o fNorth Carolina, Chapel Hill, NC 27599, USA "Current address: Division o fLaboratory Sciences, National Centerfo r Environmental Health, Centersfo r Disease Control and Prevention, Atlanta, GA 30341, USA *Corresponding author and address: Suzanne E. Fenton, Ph.D. U.S. Environmental Protection Agency Mail Drop 67 Research Triangle Park, NC 27711 USA Tel: 919-541-5220 Fax:919-541-4017 E-mail: fenton.suzanne@epa.eov 2/20/2009 Page 1 of 32 Running title: PFOA disposition in lactation Abbreviations ANOVA analysis o f variance BW body weight GD gestational day LOD limit of detection LOQ limit of quantitation MS mass spectrometer PFAA periluoroalky] acid PFOA perfhiorooctanoic acid PFOS perfluorooctane sulfonate PND postnatal day SEM standard error of the mean UPLC ultra performance liquid chromatography 2 2/20/2009 Page 2 of 32 Abstract Previous studies in mice with multiple gestational exposures to periluorooctanoic acid (PFOA) demonstrate numerous dose dependent growth and developmental effects which appeared to worsen if offspring exposed in utero nursed from PFOA-exposed dams. To evaluate the disposition of PFOA in the pregnant and lactating dam and her offspring, time-pregnant CD-I mice received a single 0 ,0 .1 ,1 , or 5 mg PFOA/kg BW dose (N=25/dose group) by gavage on gestation day 17. Maternal and pup fluids and tissues were collected over time. Pups exhibited significantly higher serum PFOA concentrations than their respective dams, and their body burden increased after birth until at least 8 days old, regardless o f dose. The distribution of milk:serum PFOA varied by dose and time, but was typically in excess of 0.20. These data suggest that milk is a substantial PFOA exposure route in mice and should be considered in risk assessment modeling designs for this compound. Key words: PFOA; serum; amniotic fluid; urine; milk; mammary gland; dosimetry; disposition 2/20/2009 W BM I P-7 4 1 1. Introduction 2 Perfluorooctanoic acid (PFOA) is a member o f the perfluoroalkyl acid (PFAA) 3 family of man-made, fluorinated organic compounds used in a number of consumer 4 goods and industrial surfactants due to their grease and water-repellant properties. The 5 use of PFAAs in many common applications, such as stain repellants for clothing, 6 carpeting, and upholstery, and the stability of the carbon-fluorine bond have made them 7 ubiquitous in the environment. The predominant route of exposure in North American 8 and European consumers is likely oral intake, including drinking water, while inhalation 9 and dermal absorption comprise routes of lesser exposure [1-5]. 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 (Cl), 3.9-6.9] and 4.8 years (95% Cl, 4.0-5.8) 14 for PFOS; 8.5 years (95% Cl, 6.4-10.6) and 7.3 years (95% Cl, 5.8-9 2) for PFHS; and 15 3.8 years (95% Cl, 3.1-4.4) and 3.5 years (95% Cl, 3.0-4.1) for PFOA [4], 16 These characteristics led to increased concern for the potential health risks of 17 PFAAs and a program to reduce product and emission content of PFOA and related 18 chemicals was recently initiated [1], PFAAs 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 of 60% in blood perfluorooctane sulfonate (PFOS) and 25% 21 in blood PFOA levels between the years 2000 and 2006[ 10], However, while the 22 production of and potential for human and wildlife exposures to certain PFAAs has been 23 reduced in the US in recent years, it is not clear that perfluorinated compounds produced 2/20/2009 Page 4 of 32 p. 8 *3? j ? **ji: = * .,_ V - t-S&E!rl5f- 5 1 in other countries will not continue to replace them in the US marketplace or in the 2 contribution to worldwide exposure. 3 Much of the recent health effects research on PFOA in mice, commonly 4 associated with gestational exposures o f 0.01-5 mg PFOA/kg BW, has focused on 5 developmental toxicities such as decreased maternal weight gain, reduced neonatal 6 survival and body weight, as well as later life effects such as pubertal delays, mammary 7 gland abnormalities, and excessive weight gain [2,11 -16], Early postnatal adverse health 8 observations prompted studies examining the effect of PFOA on maternal lactation and 9 health effects o f the nursing offspring. White et al. [ 14) described reduced epithelial 10 differentiation on postnatal day (PND) 10 in mammary glands of CD-I mouse dams 11 exposed to 5 mg PFOA/kg BW from GD I-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 utero exposure to 5 mgPFOA/kg BW from GDI-17 in the absence of 16 lactational exposure was sufficient to induce postnatal body weight deficits and 17 developmental delays, pup survival from birth to weaning was affected only in those both 18 in utero and lactationally exposed. Furthermore, recent studies [ 15] have shown that 19 unexposed neonates lactationally exposed to PFOA quickly developed mammary gland 20 growth deficits and that control dams nursing in ufero-exposed pups (dams exposed via 21 pup grooming) demonstrated slowed differentiation of their own mammary glands that 22 was evident in whole mount preparations of the tissue by the 5* day of lactation. These 2/20/2009 Page 5 of 32 . i -(V^; ;"^5T rri^v S .T = ^es^f3 ?'.v ^X 'L lt-X -a^W * - x x 't s ~i P-9 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 of 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. Ineast 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 Kanrnan 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 may be preferential partitioning of PFOA to milk compared 16 to other PFAAs and also indicated that women who were nursing for the first time 17 exhibited 49% higher concentrations of PFOA in breast milk than women who had 18 nursed previously, although inter-individual variation, daily milk output and milk protein 19 concentration were not taken into consideration. The only study that has evaluated the 20 distribution coefficient o f PFCs between blood and milk in animal models was a 21 pharmacokinetic study o f placenta] and lactational transport of PFOA in rats[22]. 22 Although female rats are known to have a serum PFOA half-life of only a few hours [23], 23 unlike mice which have a '/5-life of about 15 days 113], the study [22] indicated 2/20/2009 Page 6 of 32 7 1 concentrations in rat milk approximately 10 times less than that of maternal plasma and 2 that the milk concentrations were generally of the same magnitude as the concentrations 3 in pup plasma. 4 The increasing amount of research confirming the developmental toxicity of 5 PFAAs in animal studies, coupled with their detection in human cord blood and milk, 6 supports the need for examining the disposition of PFAAs during pregnancy/lactation in 7 an appropriate animal model in order to fully establish the association between 8 prenatal/neonatal exposure and offspring effects. While other studies have examined the 9 pharmacokinetics 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 o f PFOA in mouse serum, urine, milk, 12 mammary tissue, amniotic fluid, and pups [24], Utilizing these methods, we report here 13 data on the distribution o f PFOA in various matrices o f pregnant and lactating CD-1 14 mice, as well as the serum concentration and total body load of their offspring, following 15 a single exposure o f PFOA on GD 17. These data will allow us to reduce the 16 uncertainties in risk assessment for this particular PFAA. 17 18 19 20 21 22 23 24 2/20/2009 Page 7 of 32 p. 11 8 1 2. Materials and methods 2 2.1 Chemicals 3 PFOA (ammonium salt; >98% pure), used in animal exposures, was purchased 4 from Fhika Chemical (Steinheim, Switzerland). PFOA was completely dissolved by 5 agitation in deionized tap water, in which PFOA was below the level of detection (LOD 6 0.5 ng/L for water), and prepared fresh just prior to use. 7 8 2.2 Animals 9 All animal studies were conducted as approved by the National Health and 10 Environmental Effects Research Laboratory Institutional Animal Care and Use 11 Committee. Confirmed timed pregnant female CD-I mice (n=100) were purchased from 12 Charles River Laboratories (Raleigh, NC). Pregnant mice were received at the U.S. 13 EPA's Laboratory Animal Care facility on gestation day (GD) 14 (day of sperm-positive 14 designated as GDO). Upon arrival, mice (approximately 12 weeks old) were weighed and 15 randomly distributed among PFOA treatment groups. They were housed individually in 16 polypropylene cages with Alpha-dri (Shepherd Specialty Papers, Kalamazoo, MI) 17 bedding and nesting materials. They were provided pelleted chow (LabDiet 5001, PM1 18 Nutrition International LLC, Brentwood, MO) and tap water ad libitum (both contained 19 PFOA at concentrations below the LOD). Animal facilities were controlled for 20 temperature (20-24C) and relative humidity (40-60%), and kept under a 14:10-h light21 dark cycle. Mice (n=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. 12 9 1 2.3 Animat Assessments and Sample Collection 2 Live dam body weights were recorded on GDI7, GDI 8 (prior to parturition), 3 PND1 (day after parturition), and PNDs 2,4, 8 ,1 1, and 18. On GD18,24 hr after the 4 PFOA exposure, five dams in each dose group were sacrificed and trunk blood, urine, 5 amniotic fluid (fluid immediately surrounding each fetus), and the 4th and 5th mammary 6 gland were collected. Liver weight, total number o f fetuses (live, dead, or resorbed), and 7 fetal weights were determined. One entire fetus from each litter was euthanized by 8 decapitation and quick frozen on dry ice in a 15 ml screwcap vial. Remaining fetuses 9 were quickly euthanized and discarded. The dam mammary gland, urine, and amniotic 10 fluid were kept on ice, and then frozen until assayed. The trunk blood was allowed to 11 clot; serum was collected after centrifugation and frozen until assayed. All samples were 12 kept frozen at -80 C. 13 A similar routine was followed on PND1 (48 hr after exposure, n=5 dams/dose 14 group). Weights of the dam, pups, dam liver, and the number of live pups in each litter 15 were recorded. A single pup from each litter was weighed, euthanized, and quick frozen 16 in a collection vial (including all blood). Blood from all remaining pups in each litter was 17 pooled into a single vial, allowed to clot, and separated to serum by centrifugation. Dam 18 and pup serum, dam urine and mammary tissue were frozen until assayed. All remaining 19 litters, in all dose groups, were equalized to 10 pups each on PND1. Biological samples, 20 including a single pup and pup serum, as described for PND1, were also collected on 21 PNDs 4, .8 and 18 (n=5 dams/dose group), at the same time of day. 22 Milk collection was attempted, following administration o f oxytocin (lU/ml, i.p., 23 20 min prior to milking) on both GD 18 and PND 1, but was unsuccessful. Milk was 2/20/2009 Page 9 of 32 10 ] collected on PNDs 2, 8,11, and 18 following a 2 hr separation of the pops from the dam 2 and an oxytocin stimulus. The milk was vacuum aspirated using low, pulsatile pressure, 3 into a pre-weigbed microcentrifiige tube. Collected milk was weighed and frozen until 4 analyzed. Biological samples including urine, dam and pup serum, amniotic fluid, 5 mammary tissue, whole pup, and milk were analyzed for PFOA using the methods 6 described briefly below and in our companion paper [24]. 7 8 2.4 PFOA sample analyses 9 Briefly, the analysis of PFOA was performed using a Waters AcquityTM Ultra JO Performance liquid chromatography system interfaced with a Waters Quattro Premier XE 11 triple quadrupole mass spectrometer (UPLC-MS/MS) (Waters, Milford, MA). Either 25 12 or 50 pL of serum and amniotic fluid (50 pL used for controls), 20 pL aliquots o f urine 13 and milk, and 300 pL o f pup or mammary tissue homogenates were utilized as starting 14 material for these analyses. Samples were extracted, purified, and concentrated or diluted 15 exactly as described by Reiner et al. [24], 10-40 pL o f the prepared sample, depending on 16 the concentration of the original exposure, was injected and run on the UPLC-MS/MS 17 [24], Refer to Reiner et al. [24] for method performance and quality control steps that 18 were performed to insure the precision and accuracy of the methods used. The limit of 19 quantitation (LOQ) for these experiments were 5 ng/ml (serum), 1 ng/mi (amniotic fluid, 20 urine, milk), and 1 ng/g (whole pups, mammary tissue). 21 22 23 2/20/2009 Page 10 of 32 s 5..asS5s~g3?j>fl: p. 14 11 1 2.5 Urinary creatinine measures 2 Creatinine concentrations were measured as a basis to evaluate PFOA in mouse 3 urine. The QuantiChrom creatinine assay (BioAssay Systems, Hayward, CA) exhibited 4 an LOD o f 0.10 ng/ml and was linear up to 300 ng/ml. Thirty pi 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 of the tissue evaluated 14 (ng/g; mammary tissue and whole pups). Serum, amniotic fluid, milk and urinary 15 concentrations are reported as ng/ml. Averages, proportions, and statistical comparisons 16 were calculated with SAS 9.1 (SAS Institute; Cary, NC). Statistical significance was 17 determined using a Proc GLM ANOVA, with a Dunnett's post-hoc comparison, and 18 significance was set at k O.05. 19 20 21 22 2/20/2009 Page 11 of 32 p. 15 12 1 3. Results 2 3.] Biological Outcomes 3 This is the first study to report single dose disposition of PFOA in pregnant and 4 lactating mice and their offspring. The doses chosen were based on previous reports in 5 CD-I mice [3,14,16] demonstrating developmental health outcomes following multiple 6 gestational PFOA exposures. A single PFOA exposure on GDI 7 did not affect the 7 number of live fetuses (on GDI 8), implantation sites, or live-bom pups (on FND1), or 8 dam body weights (data not shown). Unlike studies using multiple gestational PFOA 9 exposures {13,25], there was no change in pup body weight, dam liver weight, and dam 10 liver:BW ratios, within the PFOA dose range administered in this study (Figure 1). The 11 rise in dam livertBW ratio between GDI 8 and PND1, which persisted until weaning, was 12 due to the dramatic decrease in body weight at parturition, as this single late gestation 13 PFOA exposure failed to change mean liver weight in exposed dams, compared to control 14 values, at any time evaluated. 15 16 3.2 PFOA Concentrations Prior to Birth 17 The mean concentration of PFOA in the amniotic fluid and serum of the exposed 18 dams 24 hr after exposure is shown (Figure 2; amniotic fluid controls average 3.8 ng/ml). 19 The average concentration of PFOA detected in dam serum was about twice the amniotic 20 fluid concentration at each dose evaluated (amniotic fluid was 68.8, 51.8, and 40% of 21 dam serum levels at 0.1, 1, and 5 mg PFOA/kg BW, respectively). A comparison of the 22 amount of PFOA in an entire GDI 8 fetus (body burden/pup+standard error of the mean 23 [SEM]; Figure 5) to the GD18 PFOA concentration in amniotic fluid (ng/ml; assuming 1 2/20/2009 Page 12 of 32 p. 16 13 1 ml total volume) reveals 2.3-, 3.1-, and 2.7-fold increased PFOA in the pup vs. the fluid 2 in which it was contained in utero for 0.1,1, and 5 mg/kg dose groups, respectively. 3 4 3.3 PFOA Concentrations in the Dams 5 The serum and urine PFOA concentrations were evaluated in dams that were 6 nursing litters o f approximately 10 pups (PND1 equalized; minimal pup loss over time). 7 As expected, dam sera contained the highest PFOA concentrations o f any matrix 8 evaluated, regardless o f dose (Figure 3; all serum controls <LOQ). The rise in circulating 9 serum PFOA with increasing dam exposures was proportional to the change in dose 10 delivered, regardless of stage of lactation (i.e., mean 9.9-fold and 5.1-fold increases 11 between 0.1 -1.0 mg/kg and 1.0-5.0 mg/kg exposures, respectively). 12 A one-time PFOA exposure of 0.1 mg/kg produced an average dam serum PFOA 13 concentration (Figure 3A) of 144-226 ng/ml at 24 and 48 hr after exposure, respectively, 14 which was reduced to an average of 44 ng/ml near the peak of lactation (PND8), and had 15 risen to a mean of 123 ng/ml by PND18, a time when the pups' primary caloric intake 16 came from rodent chow and not milk. The U-shaped serum concentration curve observed 17 in the 0.1 mg PFOA/kg dose group was also present in the 1 and 5 mg/kg exposure 18 groups. 19 As shown in Figure 3 (A-C; control urine and mammary gland PFOA <LOQ), 20 although the concentrations 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 o f PFOA. While a U-shaped response in dam 2/20/2009 Page 13 of 32 14 1 excretion o f PFOA (urine) was not as pronounced as that of serum, mammary tissue 2 demonstrated a strong U-shaped response, with the lowest concentrations measured near 3 the peak of lactation, and a significant rise in concentration apparent again at PND 18 4 (p<0.05). 5 When aspirated milk PFOA values were evaluated (Figure 3D; 1 control >LOQ), 6 a U-shaped curve over time was again evident. As depicted in Table 1, the percentage of 7 PFOA in milk (compared to serum) was substantial. Comparing the milk concentrations 8 to the closest matched dam serum concentrations (by time), the amount o f PFOA in the 9 milk consistently ranged from 1/10 to 1/2 that of dam serum PFOA across dose and time. 10 It appeared that the day of lactation on which milk PFOA was measured had an important 11 influence on this relationship. The milk:serum PFOA ratio was greatest in early and late 12 lactation (PND2 and PND18), ranging from 15-56% (means 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, I I , and 18, the 16 average weight of milk obtained via aspiration of control mice was 0.072,0.1906, 17 0.2547, and 0.0457 g, respectively, demonstrating over a 3.5-fold increase in weight from 18 PND2 to 11 and a 5.6-fold drop from PND11 to 18. 19 20 3.4 PFOA Concentrations in the Pups 21 Pup serum PFOA concentration was evaluated on PNDs 1,4, 8, and 18. In 22 comparing the average PFOA concentrations in PND1 pups vs. their respective dams 23 (Figure 4A; whole control pups and control serum < LOQ), it appeared that circulating 2/20/2009 Page 14 of 32 p. 18 15 1 pup serum PFOA concentrations were significantly higher than those measured in dams, 2 regardless of dose (p<0.05). Although pups possessed a substantially higher serum PFOA 3 concentration than dams, the difference in pup and dam blood volumes at those stages of 4 pup development are considerable. Regardless of those differences, heightened 5 circulating PFOA in pup sera reflected increased exposures, proportional to dose 6 throughout lactation (i.e., mean 10.4-fold and 4.3-fold increases between 0.1-1.0 mg/lcg 7 and 1.0-5.0 mg/kg exposures, respectively). 8 Unlike their dams, pups did not demonstrate U-shaped serum PFOA 9 concentration curves (Figure 4B). Pup serum PFOA concentrations continued to exceed 10 the average dam serum PFOA concentrations over time, until PND18 when the pup and 11 dam concentrations approached 1:1. When the PFOA concentration (ng/g) was evaluated 12 in whole pups (pup and blood; Figure 5 left panels), a decline in PFOA concentration was 13 detected over time, across all doses. However, when the rapidly increasing body weight 14 of the pups was taken into consideration to calculate the total amount of PFOA in the 15 neonate (as shown in Figure 1), a completely different trend was noted (Figure 5 right 16 panels). Regardless of exposure dose, PFOA body burden (adjusted for weight) rose 17 through the peak of lactation and had begun to decline by PND 18, demonstrating an 18 inverse U-shaped curve. When the administered PFOA dose and measured body burden 19 in whole pups (body weight taken into effect) were compared the administered 20 PFOArmeasured PFOA ratio was no longer proportional throughout lactation, and unlike 21 the ratios reported for dam and pup serum PFOA. Mean body burden ratios of 13.2-fold 22 (range 11.1-17.8) and 4.3-fold (range 3.2-5.1) increases between 0.1-1.0 mg/kg and 1.023 5.0 mg/kg exposures, respectively, were determined. 24 2/20/2009 Page 15 of 32 p. 19 16 1 4. Discussion 2 These data confirm that on a concentration-based comparison, gestationally 3 PFOA-exposed pups exhibited a significantly larger semrn PFOA load than their dam. 4 That substantia] serum PFOA load in pups was evident 24 hr after a single exposure, and 5 was apparently due to blood-borne (transplacental) transfer. Another important discovery 6 is the U-shaped PFOA concentration over time, regardless of dose, in the dam mammary 7 tissue, milk, and serum. This unique PFOA response was not detected in pups or pup 8 serum, and was evident to a lesser extent in the dams' urinary excretion curves. However, 9 when PFOA body burden in whole pups was the unit of measure, an inverse U-shaped 10 curve was apparent, and the PFOA burden of pups is proposed to increase due to milk11 borne PFOA intake. 12 The decline in concentration seen in the milk, mammary and serum U-shaped 13 curves is hypothesized to be due to hydro-dilution associated with increased blood and 14 milk volumes. Several physiological conditions are changed during lactation that have 15 been well documented in rats and directly relate to mice as their lactation period is of the 16 same length. A decrease in total plasma proteins due to increased blood volume, cardiac 17 output, and blood flow to certain tissues, such as the mammary gland has been reported 18 in rats [26, 27]. Elevated blood volume is due to increased plasma volume [27], Milk 19 yield (g/hr) in rats was reported to reach its peak by PND 10 [27] and the rat mammary 20 gland reaches its maximum size (as % body weight) by PND15 [26], with a steep rise in 21 size from PND5-15. Rat mammary gland blood flow and volume of milk produced arc 22 directly related, when measured on PND15 [26]. Total serum proteins are lower in 23 lactating rats that those measured in non-lactating rats [27], and in humans, serum 2/20/2009 Page 16 of 32 p. 20 17 1 albumin concentration decreases during pregnancy and early lactation [28]. Further, at 14 2 d postpartum, the cardiac output o f lactating rabbits was 30% higher than that in non3 lactating animals, and the mammary gland was the only organ shown to increase in 4 weight, relative to body weight [29]. 5 Although a complete set of data that could address the exact reason for the U6 shaped curves during lactation was not collected in this study, the aspirated milk weights 7 did reveal a dramatic increase in milk volume (assumed due to weight change) from 8 PND2 up to the peak of lactation (PND 11). This dramatic change in volume (weight) 9 may explain the decrease in milk PFOA concentration seen between PND2 and PND 11. 10 PFOA also appears to concentrate in serum and milk near the end o f lactation (PND18, 11 for example) when pups are eating more chow and suckle less often. Mammary gland 12 blood flow has been reported to decrease by half in a 24 hr period, when suckling rat 13 offspring are removed from the dam [26], and this fall in mammary blood flow is directly 14 associated with decreased cardiac output and % blood flow used by the mammary gland. 15 In this study a precipitous drop in weight of milk collected between the peak of lactation 16 and PND18 was noted, indicating a rapid decrease in milk volume. Therefore, the U17 shape o f the dam PFOA curves are proposed to be driven by physiological dilution and 18 concentration of the PFOA load over the period of lactation, reaching the greatest dilution 19 at or near the peak of lactation when the milk volume produced by the dam and 20 consumed by the pups is the greatest. Increased consumption of milk up to PND11 likely 21 directly contributed to the accumulation of body burden in the pup over this life stage. 22 A significant contribution of milk-borne PFOA transfer in CD-I mice was 23 detected in these studies. Previous reports in rats [22] and humans [18] have estimated 2/20/2009 Page 17 of 32 n ? 5s. v sit i-V V fiJv W r--tr. p. 21 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 depending on dose, with the lowest doses tested demonstrating the highest ratios over 4 time. If the milk PFOA concentrations had been measured near the peak of lactation only 5 (days 8-11), the 0.1 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 milkrserum ratios appeared, regardless of 8 dose, with a substantial peak in milk PFOA concentrations on PND2. Although volumes 9 of milk large enough to perform analytical measures prior to PND2 were not able to be 10 obtained, we suspect, based on the significant PFOA concentrations in the PND1 11 mammary gland, that substantia] milk PFOA concentrations would have been evident on 12 PND 1, 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 of decreased pup body 20 weight or neonatal mortality in the current study, following a single gestational PFOA 21 exposure. Our PFOA measurements in whole pups indicate that the PFOA body burden 22 accumulates in early life, and begins a decline as pups mature, open their eyes, and begin 23 to eat chow and drink water. Our data and those demonstrating deleterious health 2/20/2009 Page 18 of 32 outcomes suggest that the milk o f gestationally PFOA-exposed mice was a major source of continued exposure to this compound for the developing pups. As expected, large differences in dam and pup serum PFOA concentrations from those previously reported [14,16] were noticed, and those differences bring to light the issue of single vs. multiple dose kinetics. As noted for PFOS, single dose kinetics may differ substantially from those involving repeated doses [30]. Concentration dependent changes in clearance can result in discrepancies between single and repeated dose kinetics. A limited number of epidemiological studies have revealed associations between health outcomes (birth weight, head circumference) and cord blood or maternal serum PFOA concentrations in humans [20, 21], while other studies failed to detect associations with later developmental milestones in infants [31]. Several studies have now measured PFAAs in human milk [17-19, 32, 33], however only one study has been able to approximate the milk:serum relationship of PFOA transfer [18]. The reported 0.01 (1/100,h) relationship was determined from a single voluntarily contributed sample at 3 weeks postpartum. According to the mouse milk:serum PFOA distribution over time that we report herein, the values reported in one human [18] and rats [22] may not be representative of the PFOA distribution to milk throughout lactation in those species. In conclusion, these studies confirmed and further defined considerable PFOA exposures to mouse offspring following a single gestational exposure. They also demonstrated the accumulation o f chemical over time in whole pups, which likely results from milk-borne PFOA, an exposure that had previously been incompletely assessed in other species. A single 0.1 mg/kg PFOA exposure to a pregnant mouse induced 2/20/2009 sssJggi>? fefS4 *2: g-Sf X 1'.^ ^ p. 23 Ssfc^;;:- 20 1 circulating serum PFOA concentrations of 44-216 ng/ml in dams and 117-326 ng/ml in 2 pups; values similar to or lower than serum PFOA concentrations of children that were 3 accidentally exposed via DuPont production plant emission [34], Because 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 of these animals by New Year Tech, Inc. (Restin, VA). The research in this article 18 has been reviewed by the National Health and Environmental Effects Research 19 Laboratory, US Environmental Protection Agency (EPA), and approved for publication. 20 Findings in this report are those of the authors and approval does not signify this report 21 reflects EPA policy. The use of trade names or commercial products does not constitute 22 endorsement or recommendation for use. 23 24 2/20/2009 Page 20 of 32 p. 24 21 1 References 2 {1] U.S. Environmental Protection Agency, Announcement of the 2010/15 PFOA 3 Stewardship Program by Administrator Stephen L. Johnson. (2006) Available at 4 http://www.epa.gov/opptintr/pfoa/pubs/pfoastewardship.btm. 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 of action, Toxicol Sci, 102 (2008), 3-14. 9 10 (3] C. Lau, K. Anitole, C. Hodes, D. Lai, A. Pfahles-Hutchens and J. Seed, 11 Perfluoroalkyl acids: a review of monitoring and toxicological findings, Toxicol Sci, 99 12 (2007), 366-394. 13 14 [4] G.W. Olsen, J.M. Burris, D.J. Ehresman, J.W. Froehlich, A.M. Seacat, J.L. 15 Butenhoff and L.R. Zobel, Half-life of serum elimination o f perfluorooctanesulfonate, 16 perfluorohexanesuifonate, and perfhiorooctanoate in retired fluorochemical production 17 workers, Environ Health Perspect, 115 (2007), 1298-1305. 18 19 [5] D. Trudel, L. Horowitz, M. Wormuth, M. Scheringer, l.T. Cousins and K. 20 Hungerbuhler, Estimating consumer exposure to PFOS and PFOA, Risk Anal, 28 (2008), 21 251-269. 22 23 [6] K.S. Guruge, P.M. Manage, N. Yamanaka, S. Miyazaki, S. 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. Butenhoff and J.H. 28 Mandel, Historical comparison o f perfluorooctanesulfonate, perfhiorooctanoate, and 29 other fluorochemicals in human blood, Environ Health Perspect, 113 (2005), 539-545. 30 31 [8] L. Tao, K. Kannan, N. Kajiwara, M.M. Costa, G. Fillmann, S. Takahashi and S. 32 Tanabe, Perfluorooctanesulfonate and related fluorochemicals in albatrosses, elephant 33 seals, penguins, and polar skuas from the Southern Ocean, Environ Sci Technol, 40 34 (2006), 7642-7648. 35 36 [9] L.W. Yeung, M.K. So, G. Jiang, S. Taniyasu, N. Yamashita, M. Song, Y. Wu, J. 37 Li, J.P. Giesy, K.S. Guruge and P.K. Lam, Perfluorooctanesulfonate and related 38 fluorochemicals in human blood samples from China, Environ Sci Technol, 40 (2006), 39 715-720. 40 41 [10] G.W. Olsen, D.C. Mair, T.R. Church, M.E. Ellefson, W.K. Reagen, T.M. Boyd, 42 R.M. Herron, Z. Medhdizadehkashi, J.B. Nobiletti, J.A. Rios, J.L. Butenhoff andL.R. 43 Zobel, Decline in perfluorooctanesulfonate and other polyfluoroalkyl chemicals in 44 American Red Cross adult blood donors, 2000-2006, Environ Sci Technol, 42 (2008), 45 4989-4995. 2/20/2009 Page 21 of 32 p. 25 'V 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 perfhiorooctanoate, Regul Toxicol Pharmacol, 39 (2004), 363-380. 5 6 [12] C. Lau, J.L. Butenhoff and J.M. Rogers, The developmental toxicity of 7 perfluoroalkyl acids and their derivatives, Toxicol Appl Pharmacol, 198 (2004), 231-241. 8 9 [13] C. Lau, J.R. Thibodeaux, R.G. Hanson, M.G. Narotsky, J.M. Rogers, A.B. 10 Lindstrom and M.J. Strynar, Effects of perfluorooctanoic acid exposure during pregnancy 11 in the mouse, Toxicol Sci, 90 (2006), 510-518. 12 13 [14] S.S. White, A.M. Calafat, Z. Kuklenyik, L. Villanueva, R.D. Zehr, L. Helfant, 14 M J . 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 of perfluorooctanoic acid on mouse 20 mammary gland development and differentiation resulting from cross-foster and 21 restricted gestational exposures, Reprod Toxicol (2008). 22 23 [16] C.J. Wolf, S.E. Fenton, J.E. Schmid, A.M. Calafat, Z. Kuklenyik, X.A. Bryant, J. 24 Thibodeaux, K.P. Das, S.S. White, C.S. Lau and B.D. Abbott, Developmental toxicity of 25 perfluorooctanoic acid in the CD-I mouse after cross-foster and restricted gestational 26 exposures, Toxicol Sci, 95 (2007), 462-473. 27 28 [17] M.K. So, N. Yamashita, S. Taniyasu, Q. Jiang, J.P. Giesy, K. Chen and P.K. Lam, 29 Health risks in infants associated with exposure to perfluorinated compounds in human 30 breast milk from Zhoushan, China, Environ Sci Technol, 40 (2006), 2924-2929. 31 32 [18] A. Karrman, I. Ericson, B. van Bavel, P.O. Damenid, M. Aune, A. Glynn, S. 33 Lignell and G. Lindstrom, Exposure of perfluorinated chemicals through lactation: levels 34 of matched human milk and serum and a temporal trend, 1996-2004, in Sweden, Environ 35 Health 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 senim concentrations of perfluorooctane sulfonate 43 (PFOS) and perfhiorooctanoate (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 p. 26 -m rssr-'.rte ilW/j 23 1 [21] C. Fei, J.K. McLaughlin, R.E. Tarone and J. Olsen, Fetal growth indicators and 2 perfluorinated chemicals: a study in the Danish National Birth Cohort, Am J Epidemiol, 3 168 (2008), 66-72. 4 5 [22] P.M. Hinderliter, E. Mylchreest, S.A. Gannon, J.L. ButenhofT 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, MJ . Van Rafelghem and R.E. Peterson, Tissue 10 distribution, metabolism, and elimination of perfluorooctanoic acid in male and female 11 rats, J Biochem Toxicol, 6 (1991), 83-92. 12 13 [24] J.L. Reiner, S.F. Nakayama, A.D. Delinsky, J.P. Stanko, S.E. Fenton, A.B. 14 Lindstrom and M.J. Strynar, Analysis of PFOA in dosed CD1 mice: Part 1. Methods 15 development for the analysis of tissues and fluids from pregnant and lactating mice and 16 their pups, Reprod Toxicol (2008). 17 18 [25] B.D. Abbott, C.J. Wolf, J.E. Schmid, K.P. Das, R.D. Zehr, L. Helfant, S. 19 Nakayama, A.B. Lindstrom, M.J. Strynar and C. Lau, Perfluorooctanoic acid induced 20 developmental toxicity in the mouse is dependent on expression of peroxisome 21 proliferator activated receptor-alpha, Toxicol Sci, 98 (2007), 571-581. 22 23 [26] A. Hanwell and J.L. Linzell, The effects of engorgement with milk and of 24 suckling on mammary blood flow in the rat, J Physiol, 233 (1973), 111-125. 25 26 [27] K. Suzuki, H. Hirose, R. Hokao, N. Takemura and S. Motoyoshi, Changes of 27 plasma osmotic pressure during lactation in rats, J Vet Med Sci, 55 (1993), 561-564. 28 29 [28] M. Dean, B. Stock, RJ . Patterson and G. Levy, Serum protein binding of drugs 30 during and after pregnancy in humans, Clin Pharmacol Ther, 28 (1980), 253-261. 31 32 [29] C.S. Jones and D.S. Parker, Mammary blood flow and cardiac output during 33 initiated involution o f the mammary gland in the rabbit, Comp Biochem Physio! A Comp 34 Physio!, 91 (1988), 21-25. 35 36 [30] L.A. Harris and H.A. Barton, Comparing single and repeated dosimetry data for 37 perfluorooctane sulfonate in rats, Toxicol Lett, 181 (2008), 148-156. 38 39 [31] C. Fei, J.K. McLaughlin, R.E. Tarone and J. Olsen, Perfluorinated chemicals and 40 fetal growth: a study within the Danish National Birth Cohort, Environ Health Perspect, 41 115(2007), 1677-1682. 42 43 [32] W. Volkel, O. Genzel-Boroviczeny, H. Demmelmair, C. Gebauer, B. Koletzko, 44 D. Twardella, U. Raab and H. Fromme, Perfluorooctane sulphonate (PFOS) and 45 perfluorooctanoic acid (PFOA) in human breast milk: results of a pilot study, Int J Hyg 46 Environ Health, 211 (2008), 440-446. 2/20/2009 Page 23 of 32 24 1 2 [33] O.S. von Ehrenstein, Fenton, Suzanne E., Kato, Kayoko, Kuklenyik, Zsuzsanna, 3 Calafat, Antonia M., Hines, Erin P ., Polyfluoroalkyl Chemicals in the Serum and Milk of 4 Breastfeeding Women Reprod Toxicol, In Press (2009). 5 6 [34] E.A. Emmett, F.S. Shofer, H. Zhang, D. Freeman, C. Desai and L.M. Shaw, 7 Community exposure to perfluorooctanoate: relationships between serum concentrations 8 and exposure sources, J Occup Environ Med, 48 (2006), 759-770. 9 10 [35] E.P. Hines, White, Sally S., Stanko, Jason P., Gibbs-Floumoy, Eugene A., Lau, 11 Christopher, Fenton, Suzanne E ., Phenotypic Dichotomy Following Developmental 12 Exposure to Perfluorooctanoic Acid (PFOA) in Female CD-I Mice: Low Doses Induce 13 Elevated Serum Leptin and Insulin, and Overweight in Mid-life, Molec Cell Endocrinol, 14 In Press (2009). 15 16 17 2/20/2009 Page 24 of 32 p. 28 fSssii 25 1 Figure legends; 2 Figure I. Dam tissue weights and average pup weights following a single gavage PFOA 3 exposure on GDI7. PFOA was without effect on several biological end points (p>0.05), 4 such as dam body weight measured on several postnatal days (PND) and on gestation day 5 (GD)18 (not shown). (A) Dam liver weight, (B) liverrbody weight ratio, and (C) pup 6 body weight over time or numbers of live pups or fetuses (not shown) were also 7 unchanged by a single PFOA exposure. Data are shown as Mean + SEM or as a mean 8 ratio. 9 Figure 2. Comparison of gestation day (GD)18 dam serum and amniotic fluid PFOA 10 concentrations. PFOA concentrations were significantly higher in dam serum than 11 amniotic fluid at all doses evaluated (p<0.05). Data are shown as Mean + SEM. 12 Figure 3. PFOA concentrations in exposed dams. PFOA concentrations were measured 13 in dam serum (A; ng/ml), urine (B; ng/g creatinine), and mammary tissue (C; ng/g tissue 14 weight) on postnatal days (PND) 1,4,8 and 18. PFOA concentrations were measured in 15 aspirated milk samples collected on PNDs 2, 8, 11, and 18 (D; ng/ml). Although panels 16 A-C and B-D cannot be directly compared (due to different units), the U-shaped 17 concentration curve present in dam serum (regardless of dose) was also detected in 18 mammary tissue and aspirated milk. Data are shown as Mean + SEM .fDenotes a single 19 reliable measurement at this time due to insufficient volumes in other dams at this dose 20 and time. 21 Figure 4. Neonatal transfer of PFOA to pups. (A) A significantly higher PFOA 22 concentration in pup vs. dam serum on PND1 was noted (p<0.05; v:v). (B) Pooled pup 23 serum PFOA concentrations did not demonstrate a U-shaped curve, but gradually 2/20/2009 Page 25 of 32 26 1 declined over time, presumably due to dilution of dose by increased growth-related blood 2 volume. Data are shown as Mean + SEM. 3 Figure 5. Whole pup PFOA concentrations. PFOA concentrations were measured in a 4 representative whole pup (pup and blood; ng/g; left panels) from each litter. Although 5 there is a consistent downward trend in PFOA concentration over time, the rapidly 6 increasing blood volume and body weight changes must be taken into consideration when 7 interpreting these data. Body weight-adjusted values (right panels; [ng/g PFOA 8 measures*g body weight = body burden]) demonstrate an accumulation o f exposure until 9 late in the lactational period. Data are shown as Mean + SEM. 10 11 12 2/20/2009 Page 26 of 32 V iTI c <K; *iS-i KSf srt S S : i ? H 5 ** i*4: Figure 1. (A) Dam Liver Weight p. 30 Weight (g) Liver: BW Ratio (%) Control PFOA Exposure (mg/kg) 5 15(B) Dam Liver:BW Ratio ,B, iImll v Inwg%mBm $* $! Control 0.1 1 PFOA Exposure (mg/kg) (C) Pup Body Weight Control 0.1 1 PFOA Exposure (mg/kg) GP18 DPND1 DPNP4 0PND8 PND18| Page 27 of 32 Figure 2 9000 8000 G DIS Amniotic Fluid B GD18 Dam Serum ^ 7000 'cS) 6000 5000 < O 4000 U. O. 3000 2000 1000 0 0.1 1 PFOA Exposure (mg/kg) Hlliii sasggffpK asSi ? i i ^ E l S i : Figure 3. (A) Dam Serum PFOA p. 32 G o ii m o i p n m m o t n e o i (B) Dam Urine PFOA GDI PND1 PM M P N D tP N D It (C) Dam Mammary Gland PFOA (D) Dam Milk PFOA Tim e PND2 PHD% PHD11 PND1B Page 29 of 32 Figure 4 (A) Dam vs. Pup Serum PFOA Serum PFOA (ng/ml) PFOA Exposure (mg/kg) (B) Pup Serum PFOA Over Time P o stnatal Day (PN D ) O 03 > (O PFOA (ng/g) 0 (C/>D PFOA (ng/g) Ii PFOA (ng/g) C 3 cn 6D18 1 4 8 18 GD18 Body Burden PFOA (ng) Body Burden PFOA (ng) p#r W fft If p. 34 Page 31 of 32 Tables ,i;>j i yiiiiV -i iv^TS\>i ^ i\J'i^KiCi ii'ij.ilJ n;\ !"!;:,V i|lIillli|l r:tV1* *>1>\'`Ai0i^iiWi Table 1. Milk-borne PFOAt as a percentage of dam serum concentrations over lactation. Single GD17PF0A exposure PND 1 serum PFOA comparison PND 4 serum PFOA comparison PND 2 milk PND 8 serum PFOA comparison PND 8 milk PND 8 serum PFOA comparison PND 11 milk PND 18 serum PFOA comparison PND 18 milk 0.1 mg PFOA/kg 15% 31% 27% 11% 36% 1.0 mg PFOA/kg 37% 56% 21% 21% 24% 5.0 mg PFOA/kg 25% 36% 13% 13% 18% tPFOA* perfluorooctanoic acid, GDagestational day, PND*postnatal day. The milk:serum PFOA ratio reported above was calculated as: [concentration of milk PFOA/concentratlon of serum PFOA]*100*% milk:serum for each dam within a dose group. These values were averaged and reported above. p. 35 a 32 of 32 - jQ fU lode jff*?S2311-8 '- 5 * Reproductive Toxicology xxx (2009) xxx-xxx Contents lists available at ScienceDirect Reproductive Toxicology jo u rn a l h o m ep ag e: w w w .e ls e v ie r.c o m /lo c a te /re p ro to x p. 36 , <ii Polyfluoroalkyl chemicals in the serum and milk of breastfeeding women r Ondine S. von Ehrensteina- \ Suzanne E. Fenton**, Kayoko Katoc, Zsuzsanna Kuklenyikc, a Antonia M. Calafatc, Erin P. Hines**-1 4 1UCLA School o f Public H ealth. University o f California. Los Angeles, C l United States e *U S EnvwonmeiMrf Protection Agency. ORD. NHEEM, Reproductive Toxicology Division, KTP. NC. U nited Slates s ` Centers fo r Disease Control & Prevention. Division o f Laboratory Science. Notional Crmfrfor Environmental Health Atlanta. CA. United Slates s ARTICLE INFO ____________ _______ __ to Article history: 11 Received 28 January 2009 is Received in revised form 27 February 2009 13 Accepted 2 March 2009 it Available online xxx *s ____________ .______________________ if. Keywords: i t Polyfluoroalkylchemicals in Periluoroalkyi acids < Perfluotooctanoicacid ao Perfluorooctane sulfonic acid 21 Serum 22 Breast milk 23 Lactation ABSTRACT PoiyOuoroalkyl chemicals (PFCs) com prise a group of m an-m ade organic com pounds, som e of w hich are persistent contam inants w ith developmental toxicity show n in laboratory animals. There is a paucity o f hum an perinatal exposure data. The US EPA conducted a pilot study (M ethods Advancement in Milk Analysis) including 34 breastfeeding w om en in North Carolina. Milk and serum samples were collected al 2 -7 w eeks and 3 -4 m onths postpartum ; 9 PFCs w ere assessed in milk and 7 in serum . Perfluorooctane sulfonic a d d (PFOS). perfluorooctanoic acid (PFOA). perfluorononanoic a d d (PFNA).and perfluorohexane sulfonic a d d (PFHxS) w ere found in neatly I00X of the serum sam ples. PFOS an d PFOA w ere found a t th e highest concentrations. PFCs w ere below th e limit of detection in m ost milk sam ples. Serum concentra tions o f PFOS. PFOA and PFHxS w ere low er (p <0.01) at the second visit com pared to the first visit. Living in N orth Carolina 10 years o r longer w as related to elevated PFOS. PFOA and PFNA (p < 0.03). T hese pilot d a ta support the need to further explore perinatal PFC exposures and potentially related health effects, as planned in the upcoming National Children's Study w hich provided the framework for this investigation. 2009 Published by Elsevier Inc. 24 1. Introduction 2s Polyfluoroalkyl chemicals ( PFCs)comprise a large group of man2s made fluorinated organic compounds used in numerous consumer 2 7 products and industrial applications such as food packaging mate28 rial, non-stick cookware, protective coatings for textiles, carpets. 29 and paper, surface car coatings or treatments, as welt as in surao factants fo r commercial and industrial applications |1 ). PFCs. and si more specifically perfluoroalky! adds (PFAAs). have been detected 32 in w ildlife, fish used for human consumption, and sera of humans 33 in many different geographical areas worldwide |2 -1 9 ). Nation34 ally representative US sera biomonitoring data in subjects 12 years Abbreviations: Cl. confidence interval: 1QR. interquartile range: tDO. limit of detection; LOQ. lim it of quantification:.Pbas. periluoroalicyf acids; PFOSA. perfhiorooctane sulfonamide: Et-PFOSA-AcOH. 2-{N-ethyt-perfluorooctane sulfonamido) acetic acid; Me-PfOSA-AcOH, 2-(N-methyl-perfluocooctane sulfonamido) acetic acid: PfHxS, perfluorohexane sulfonic acid; PFOS. perfluorooctane sulfonic acid; PFOA. perfluorooctanoic acid; PFNA. peefluorononanok acid; PFC. polyfluoroalkyi chemicals; WTC, World Trade Center. Q 2 * Corresponding author at: UCLA School of Public Health. PO Box 1772. Los Ange tes.CA90095-1772. United Slates. Tel.: *1 310 206 5324: fax: -*I 310 794 1805 E mail address: ovehren9urta.edu (O.S. von Chrenstein). 1Current address: USEnvironmentalProtectionAgency.NationalCenter for Expo sureAnalysis. EnvironmentalMediaAssessmentGroup.Mai) codeB243-01. Research Triangte Park. NC27711. United States. 0890-6238/S - see front matter C 2009 Published by Elsevier Inc. dor:10.1016ft.reprotojL2009.03.001 and older demonstrated widespread exposure to perfluorooctane sulfonic arid (PFOS). perfluorooctanoic acid (PFOA). and perfluorononanoic acid (PFNA) during the last decade [20.211. Exposures of lariating 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 [22-26J. Developmental and reproductive health effects in animals, including reduced birth weight and gestational length.developmental delays and structural defects especially in relation to PFOA and PFOSexposure have increasingly raised concerns, although the developmental toxicity in laboratory animals was shownat doses 100-500 times of those seen in human sera [2,27-29|.Som e exposure assessments incord blood suggested that PFAAs can also cross the placental barrier in humans [30.311. Apelberg et al. [23[ recently reported average cord blood concentrations of 4.9ng/mt (PFOS) and 1.6ng/m! (PFOA) ( n - 299). w hile Spliethoff et al.. reported the detection of PFAAs in new bom blood spots confirming the transfer of PFAAs in urero [32J. In two recent epidemiological studies. PFAA cord blood concentrations were related to anthropometric indicators o f fetal growth at birth, and maternal pregnancy serum PFAA concentrations were associated w ith child birth weight [22.24], Based on the Danish National Birth Cohort, inverse associations were reported between gestational PFOA exposure and birth weight while no effects were reported for markers of fetal growth at birth, or postnatal developmental milestones [2433). m v w .*> 40 .> .? 43 *. .s , 41 4 51 s? 53 u v. n u M p. 37 - * '' ' ' V -' "' V" ;V: " y ;-:vvvg^r ' 2 O.S. von Ehrenstem e t o t f Reproductive Taxicotogy *xx (2009) xxx-xxx ft 63 64 66 66 67 66 69 70 71 72 73 7* 75 76 77 76 79 60 61 82 83 64 66 86 67 66 69 60 1 97 S3 94 95 06 97 96 99 too Oi 0? 103 04 105 106 107 108 100 110 m t2 113 m 115 l6 117 116 119 120 121 12? 123 124 126 176 127 Data on human m ilk PFC concentrations are still sparse. The available data based on small sample sizes from China (34), Sweden (35), Germany and Hungary |36], suggested detectable levels ofpre dom inantly PFOA and PFOS.The concentrations ofPFOS(131 pg/m l) and PFOA (4 3 .8 pg/m l) in 45 m ilk samples collected in 2004 from wom en aged 2 2 -4 3 years residing in Massachusetts have been reported recently (25 ). Studies investigating the partition of PFCs into m ilk are largely lacking. One earlier study in Sweden (n = 12) suggested tran sfer of only about 1%o f PFC concentration in serum into m ilk (35). Temporal concentration changes in serum or m ilk of lactating wom en are unknown, as no study has assessed concen trations in the same woman at tw o tim e points during lactation. To evaluate infant and maternal exposure to PFCs and to a range of other environmental components, as well as to compare concentrations across biological fluids (37). the US Environmen ta l Protection Agency (US EPA) conducted a pilot study entitled Methods Advancement for M ilk Analysis (MAMA). This pilot study was carried our to develop reliable collection and analysis methods fo r the National Children's Study, including 100.000 children from pre-conception to age 21 (38). W e previously reported the MAMA findings regarding phthalates (37) and the biological components o f human m ilk |39). 2 . Materials and methods 2.1. S tu d y design a n d population The design o f the CPA MAMA study and basic methods has been described in deutt previously (39). bi brief. 34 healthy. English-speaking breastfeeding women between 18 and 38 years of age were recruited via newspaper advertisements, uni versityemail publications,and fliersdistributed tocliniciansspecializing in women's health or pediatrics byan EPAcontractor(Westat tnc,,Chapel Hill. NC).The question naire assessment and the >1lection of milk and serum specimens were conducted at the EPA'sHuman Studies Facility d in k (Chapel H ilt NC)between December 2004 and July2005. Women were breastfeeding their first, secondor third child and were not required to exclusively breastfeed for participation in this study. The women donated milk and serum samplesat 2-7 weeks (1st visit: n - 18 milk: n -3 4 serum), and at 3-4 months (2nd visit: n -2 0 milk; n - 30 serum) postpartum. The partic ipation of human subjects in the MAMA study was approved by the Institutional Review Boards of the University of North Carolina's School of Medkine (IRB number 03-EPA-20?) and the Centers for DiseaseControl and Prevention (IRB number 3961). The women participated in verbal and written informed consent prior to admmistrationofacomprehensivequesckHinaire which did not include questions regarding the offspring of study participants (39). 2.2. Questionnaire A questionnaire regarding maternal residence, occupation, and dietary and lifestyle factors was administered to participants at the first dink visit. Ques tionnaire items were selected to address potential routes of exposure to multiple environmental chemicals (phthalates. phenols. PCBs. dioxins. PFCs. persistent organk pollutants, metals, and brominated flame retardants). The current analy sis included the following questions that were thought to potentially relate to PFC exposure routes: "How long have you lived in North Carolina?" |40-42| and "Does your home have an enclosed garage attached?".The latter question was selected as some applications used in and around cars contain PFCs.eg., external and internal surface car coatings or treatments. 2 3 . Sample collection andpreparation The women were asked to fast for 1.5b before sample collection. The MAMA sample collection procedures for serum and milk were published previously |39|. Sampling details, including time of day (between 9 AM and 2 PM) and the amount o f bodily fluid collected, were recorded in the collection log Milk (90ml or -3 oz) was expressed in the EPAclinic using a commercially available electric breast pump ( Medela. McHenry. II). M ilk was pumped into PFC-free bottles and divided into 3 ml aliquots in PFC-free polypropylene-tubes. Women's blood samples (about 20mi), were collected into non-heparinized glass vacutainer tubes (Beeron Dickinson. Franklin Lakes. NJ) by an EPA nurse via venipuncture. After 1h at room tempera ture to allow for clotting, blood samples were spun at 3000rpm fox is min at room temperature and serum wascollected.Allsampleswerescoredat -2 0 Candshipped on dry iceto the CDCs Division of Laboratory Sciences. NationalCenter for Environ mental Health (Atlanta. GA) for analysis. At the CDC. alt samples were stored at or below -2 0 'C until analyzed. Table 1 Umits ofquantification (LOQ)in milk andlimits ofdetection (LOD)in scnim(ng/ml). Potyftuoroalkyl chemicals 2-(N-ethyl-perfluorOoctane sulfbnamido) acetk acid 2-<N-methyJ-perfkiorooctane sulfonamklo) acetic add Perfluorobutane sulfonate Petfluorodecanoate Peritaorohexairk sulfonate(PFHxS) PerfhjotononanoKe (PFNA) Peffluorooctanestilfoaamkle (PFQSA) Perfluorooctane sulfonate(PF05) fletfluorooctanoate (PFOA) Milk LOQ 0.60 0.60 0J0 0.60 0J0 OJO 0J5 060 OJO Serum LOD 0.20 OJO '* 0.10 OJO 0.05 0.05 0.10 * Denotes not measured in serum. 2.4. Anc/ysis o f m ilk and serumfo r PFCs In serum and milk, we determined the concentrations of PFOS. PFOA. PFNA. PFHxS. perfluorooccane sulfonamide (PFQSA). 2-<N-methyl-perfluorooctane sulfonamido)acetk acid (Me-PF05A-AcOH)2-{N-ethy1-perAooroocunc sulfonamkfo) acetic add (Ec-PFOSA-AcOH): perfluorobutane sutfbnk acid and perfluorodecanok add were only measured in milk. The analytical method involved automated solid-phase extraction (SPE) coupled to reversed-phase high performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS). Samples were run in singlets and were re-analyzed only if the water and/or matrix blanks were above of3 x lim it of detection (LOD). The analytical procedures involving the use stan dards. quality control, and blanks, as well as automated sample extraction were conducted as published previously (43-46). The samples from both visits were ana lyzed together in March 2006 (serum) and November 2006 (mflk). For milk, sample preparation was conducted usingautomated off-line SPE|43J. One-ml of milk, to which we added 3 ml of 0.1 M formic acid and 50 pJ of internal standard solution,was vortex-mixed and sonicated, and placed on a Zymark Rapid Trace Station (Zymark Corp. Hopkinlon. MA). PFCsfrom the milk were extracted on an Oasts-KLB SPE column (waters Corporation. Milford. MA). The SPE eluate was evaporated at 55 C to --lOOpJ under a stream of dry nitrogen (UHP grade) in a Zymark Turbovapevaporator, and reconstituted with 300 pJo f0.1X formic acid.The reconstituted milk extract (-4 0 0 pJ) was transferred to a polypropylene aurosaro piervial for rheon-line SPE-HPIC-MS/MS analysis,performed usinga SurveyorHPIC system (ThermoFinnigan.SanJose.CA. USA), including one six-port switching valve (Rheodyne MX7960. Rohnert Park. CA. USA) and one additional Surveyor LCpump, coupled with a ThermoFinnigan TSQQuantum Ultra triple-quadrupole mass spec trometer equipped with a heated electrospray ionization(HES1)interface.The HPLC pnmp operated at a 300 pl/min flow rate with 20 mM ammonium acetate ( pH 4 ) m water (mobile phase A) and acetonitrile (mobile phase B). The extract was injected into the liquid chromatograph system for concentration of the PFCs byon-line SPE on a BetasilC8 precolumn (3 mm < 10 mm. S pm: ThermoHypersd-Keystone. Belle* fonte. PA.USA),chromatographic separation on a BetasilC8 analytical HPLCcolumn (2.1 mm x 50 mm. 5 pm: ThermoHypersil-Keystone). and detection and quantifica tion by negative-ion HESI-MS/MS. For serum, we used a modification of the on-line SPEcoupled to HPLC-MS/MS approach described before (47). Briefly, we added 250 p i of 0.1 M formic acid and 25 p i of internal standard solution to 100 p i of serum, and the spiked serum was vortex-mixed and sonicated. The samples were placed on a Symbiosis on-line SPE system (Spark Holland. Ptainsboro. NJ) for the preconcentration of the analytes on a Polaris CIS cartridge (7 pm. 10mm * I mm: Spark Holland). The analytes were transferred onto a BetasilC8 HPLCcolumn (3 mm * SOmm. 5 pm; ThermoHypersilKeystone. Beflefonte. PA), separated by HPLC (mobile phase A: 20mM ammonium acetate in water. pH 4; mobile phase B: methanol), and detected by negative-ion Tiivbofonspray-MS/MS onanA n 4000 massspectrometer(Applied Biosysrems.fos ter CHy.CA).Reportablebreast milk PFCconcentrationscanfall below the LODdue to concentration factors that are part of the extraction protocol. Thus limit of quantificatk>n(LOQ)(3xLOD)isusedfor milk samplesand LODisused for all ocher biological media, where sample concentration is not required. The LOD in serum and the LOQ in milk are shown in Table 1. 2.S Biological marker onafysis Selectedbiologies in milk andserum were analyzed foreachwomanaccordingto LabCorp's standard operating procedures for these assays as previously reported in detail {39). The assessed endpoints were in milk: Secretory immunoglobulin A. pro lactin. tissue necrosis factor-a (TNF-a). interleukin-6 (H.-6). triglycerides, glucose, and estradiol; and in serum: prolactin, immunoglobins, TNF-a. 1L-6. triglycerides, glucose,and estradiol In this investigation,the milk and serum concentration ofthe biological markers were used to explore possible relationships with the detectable PFCs. 126 126 3 31 132 133 134 135 136 137 136 >39 140 41 142 143 144 146 46 *47 146 <46 >50 15 <S2 S3 *54 >55 >66 57 56 168 <60 161 162 163 164 65 66 167 66 *69 70 7i 17? 173 174 7fi 74 177 17 17 166 10 87 63 64 O S von Ebrrrotem of./Reproductive Toxirofogyxxx(2009)xxx- m x 3 T ib i 2 Q5 percentage (number) ofscrum and milk simples w ith PFCsJ>LODal visit 1 [scrum n - 34; m ilk n - 18)and visit 2 (scrum n - 30: milk n - 20). Perftuoroalkyt.atids Serum >LOD%(n) MUk> LOQS(b) pros Visit 1 Visit 2 100(34) Ib 0(30 ) 0 0 PFOA y is tn Visit 2 100(34) 100(30) 0 0 PFHxS visit l Visit 2 100(34) 100(30) 0 0 PFNA Visit 1 V bil 2 9 7 (3 3 ) 100(34) 0 0 PFOSA Visit 1 :v i$ it2 44 {)5 ) 7 3 (2 2 ) 0 15(3) Me-PFOSA-AcOH Visit 1 Visit 2 5 3 (1 8 ) 5 0 (1 5 ) S 6(1) 0 Et-PFOSA-AcOH V isit! ..Visit 2 0 5-6 (1) 00 Periluorobutane sulfonate Visit 1 Visit 2 9 9 0 0 perfluorodecanoate V iS itl Vfefr 2 b b 0 0 * PFOS: perfhiocooctane sulfonate; Et-PFOSA-AcOH; 2-{N-rtttyl-pcrfluoroocune sulfonamklo) acetic acid; Me-PFOSA-AcOH: 2-{N-methyl-perfluorooctane suifooamido) acetic acid; PFHxS: perfluorohexane sulfonic add; PFOS: perfluorooctanyl sulfonate; PFOA: perfluorooctaaokr add: PFNA: perfluorononanoic and. * Denotes nor measured in serum. i85 2 6 . Statistical analysis iso We calculated the percentage of defection for each analyte in serum and millr. 187 and determined the median, range, mein, standard error, and selected percentiles. 188 Forvalues below the LOD.valuesequal to LOD/sqr2 were used [48,49f Furtheranil188 yses. including relationships between visits and across media, were conducted for thoseanalytes lor which the frequency ofdetection(>L0D) was >60kat both visits. Forthosewomenwhodonated2 serum samples,the median difference between the concentrations for the same PPCat visit 1 and visit 2 was calculated and assessed with the Wilcotconsigned-rank test (non-parametric).Spearman correlation coeffi cients and related p valueswere calculated for correlations between the PFCsat visit 1 and visit 2. and between the PFCsand the biological markers in milk and serum. Relations betweennprioriselected variablesassessedbyquestionnaireand the PFCs were evaluated using Wilcotnn scares (rank sums) test. The cut-off points for cat egorizing selected variables were decided a priori based on assumptions according to data previously reported (40-42), and to achieve approximately equal distribu tion of numbers of subjects across categories.Two-sided p values are reported. All analyses were conducted with SASverskm 9 (SASInstitute. Cary. NC). 3. Results The median age o f the women in this study was 31.3 years (interquartile range (IQR): 27.1-34.2 years), and the children's median ages were 5.5 weeks (IQR: 4 -6 weeks) at visit 1 and 13 weeks (13-14 weeks) at visit 2. Three of the analytes. PFHxS, PFOS and PFOA. were detected in 100* o f women's serum samples at both visits, PPNA was detectable in 9 7 * at visit 1 and in 100* of women's samples at visit 2 (Table 2). In contrast, in m ilk samples o f just 4 women, only 3 o f the analytes were >L0Q: Et-PFOSAAcOH (l.O ng/m l) and Me-PFOSA-AcOH (0.7 ng/m l) were detected in 1 woman at visit 1. and PFOSA was detected in 3 women at the 2nd visit (0 3 . 0.5. and 0.6 ng/ml). The remainder of the m ilk samples from both collections were measured and found to have concentrations < LOQ. The distribution o fPFCserum concentrations isshown in Table 3. Highest concentrations were found for PFOS w ith median values of 20.0 ng/ml at the first visit and 16.9 ng/ml at the second visit. PFOS concentrations were almost six-fold higher than the concentration o fthe analyte w ith the next highestvalue, PFOA.w ith median values o f 3.5 and 2.9 ng/ml at the first and second visit, respectively. Median serum concentrations were significantty (p < 0 .0 1 ) lower for PFOS. PFOAand PFHxS assessed at visit 2 compared to the concentration assessed at visit 1. based on samples of 30 women who donated serum samples at both visits w ith the differences shown in Table 4. Accordingly, the concentrations of the detected serum PFCs are reported for each visit (Table 3). Serum concentra tions of the same PFC were significantly correlated between the two visits (Table 4). Doe to the lim ited number of breast m ilk sam ples w ith detectable PFCconcentrations, we could not calculate the 190 191 192 190 19* 195 >0 197 96 190 200 1 202 203 204 205 206 207 208 209 210 ?M 212 213 214 25 21 2 i7 ?i9 220 221 222 223 224 225 226 227 228 229 230 1*M e3 Distribution (mean, standard error, median, selected percentiles. IQR) of PFCs* in serum samples at visit I ( n- 34)and visit 2 (n - 30) m ng/ml. MeanfSEM) 10th percentile 25th percentile Median 75th percentile 90th percentile 95th percentile IQR PFOS Visit l 21.9(1.9) 1L7 13.2 20.0 30.1 37j6 45.7 16.9 Visit 2 1 8 .8 (1 5 ) 9.70 14.0 16.9 22.6 30.2 35.5 8.60 PFOA Visit 1 3 .9 9 (0 3 5 ) J.50 2-20 330 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 1 9 4 (0 3 7 ) 0.70 I jO 1.55 2.40 3.40 3.80 1.40 Visit 2 1 .5 0 (0 2 2 ) 0.50 0:70 1.15 1.70 2.90 4.60 1.00 PFNA V isit! 1.22 (0J2) 0.40 0.70 1.10 130 2.00 2.70 030 Visit 2 1.33 (0.09) 0.75 1.00 1:20 1.50 1.90 2.40 0 3 0 PFOSA Visit 1 0.07(0.0!) <LOD <L0D <LOO 0.10 a io 0.10 007 Visit 2 0.09(0.01) <LOD <L0D 0.10 0.10 0.15 0.20 0-07 Me-PFOSA-AcOH V isit! 0.23 <0.02) <LOD <LOD 0.20 030 0.30 0.40 0.16 Visit 2 0.24 (0.02) <L0D <LOD a n 030 0.40 0.50 0.16 1 PFOS: Perfluoroociane sulfonate; Et-PFOSA-AcOH: 2-(N-ethyl-perfluorooctane sulfonamide) acetic acid; Me-PFOSA-AcOH. 2-{N-methy! peifluoroociane sul(onamido) acetic acid; PFHxS: perfluorohexane sulfonic acid; PFOS: perfluorooctanyl sulfonate; PFOA: perfluorooctanoic acid; PFNA: perfluorononanoic acid. Values measured <tOO were imputed by LOD/sqr2. 4 OS. von Ehrm stein rf a t / f t t p m h u m e Toxicology xxx (2009) x xx -rx x Table 4 concentrations (ng/m l) between visit one Tables Correlations between concentration of PFC and interleukin-6 in serum at visit 1 (n - 34) and 2 (n - 30). Median difference (IQR) * ' p value* CombbQ coefficient a ' pros PFOA PFHxS PFNA - 2 3 0 (- 7 j9.1jB) -0 .5 5 (-1 .4 0 .0 .0 ) -0 .4 0 (-0 8 0 .-0 .1 0 ) 0.11 (-0.20 ,0.50 ) <0.01 <0.001 <0.001 0.10 OB2 082 0fi7 0.71 Wikaxon signed-rank test (noi*-param etric)(n-30) p value <aooi <0.001 <0001 <0.001 PFOS Visit 1 Visit 2 PFOA V isit! Visit 2 Corrlation coefficient, o ' -o a t 039 -OHS 007 p value 030 003 040 070 Spearman correlation coefficient <r and related p value( 30). PFHxS Visit t -0.11 050 Visit 2 231 partition coefficient from serum to m ilk, but can conclude that m ilk 232 concentrations were notably lower than serum concentrations. PFNA Visit 1 233 Based on self-reported data, women had lived in North Carolina Visit 2 038 -0 003 -OJDB 004 I jO 0.70 234 for (mean, SEM) t4 .6 (1 5 2 ) years. Interestingly, women who had ' Spearman correlation coefficient <r and related p value. Bolded values signify 235 reported living in North Carolina for 10 years o r more compared significant correlations. 236 to those who had reported living in North Carolina less than 10 237 years, had higher serum concentrations o f PFNA, PFOA, and PFOS 236 (p < 0.03) (Fig. 1). Furthermore, living in a house w ith an enclosed Serum concentrations of 1L-6 were positively correlated with 238 garage attached as compared to living in a house w ith no enclosed PFOS (p =0.03) and PFHxS (p -0 .0 4 ) at the second visit (Table 5). 24 garage attached, suggested a relation to higher concentrations of None of the other selected biological markers in serum or in milk 741 PFHxS (ng/m l; median. IQR, visit 1: 2.2 (1.4) vs. 1.1 (0.6). p < 0.001; showed a significant correlation w ith the PFCserum concentrations 242 visit 2:1.5 (1.4) vs. 0.9 (0.7) p - 0.03) and o f PFOS (visit 1; 25.4(16.9) at either collection time point. There was no significa nt relationship 243 vs. 1 4 .4 (9 5 ).p -0 .0 1 ; visit 2: 21.2 (115) 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 of racial diversity in this pitot study based on convenience sampling (only .3 women reported themselves as Black/African-American, one as Asian and one as Hispanic), we could not analyze PFC con centrations by ethnic group. 2*4 246 2*6 247 246 24 260 26* 262 263 264 4. Discussion PFNA Visit 1 PFNA Visit 2 PFOA Visit 1 PFOA Visit 2 PFC by Visit (b) 60 O <10 Years NC Residence D >10 Years NC Residence 50- 40- in O 30 uQ.. E i2<nD 20- 10- 0 1 X Visit 1 Visit 1 Visit 2 Visit 2 F t*. 1. (a)and ( b)Serum concentratk>nsoTPFNA. PFOA.and PFOS(ng/mDcompaiing living in North Carolina > 10 to <1Cyears at visit I and visit 2. Data are shown as box and whisker representations: open tittles denote mean values with the medians denoted asa straight line, p <0.03 in Wikoxon Scores (rank sums) tests for groups tfOOyeatsvs. <10 years at visit I and visit 2 for each PFC.Numbers of subjectsin each group: >M)years: n 16. visit l: n* IS. visit 2: <10 years: n-1 8. visit 1.n* 15. visit 2. In this pilot study of healthy lactating North Carolina women. 6 of the 7 PFCs analyzed in serum were detectable at 2 -7 weeks and 3-4 months postpartum. PFOS. PFOA. PFNA. and PFHxS were found in nearly 100% o f the serum samples. PFOS. followed by PFOA and PFHxS were the compounds detected at the highest concentrations. Only a small proportion of m ilk samples had detectable values of 3 of the 9 PFCs analyzed in m ilk. Interestingly, serum levels were lower for PFOS. PFOA, and PFHxS at the second visit compared to the first visit, and prolonged tim e lived in North Carolina, as w ell as living in a home w ith enclosed garage attached, suggested a relation to elevated serum concentrations of certain PFCs in our sample; however, these analyseswere unadjusted and based on a small nonrandom sample in this pilot study and thus should be considered exploratory. We can conclude that postnatal exposure to PFCs via breast milk is likely to be low during the rime period captured in our investigation. Data on PFCserum concentrationsof lactating women are sparse and based on small sample sizes. Available data relevant for preand postnatal exposures to PFCs are summarized in Table 6. Only one earlier study assessed both serum and m ilk levels, in 12 lactaring women in Sweden, and reported similar serum values to ours for PFOS(median: 18.7 ng/m l) and PFOA(3.8 ng/m l) w hile con centrations of PFHxS were higher (4.0 ng/m l) in the Swedish study |35). Based on data from the Danish National Birth Cohort, prena tal maternal serum concentrations appeared to be higher for PFOS and PFOA in Denmark than seen postpartum in our study. Interest ingly, in the Danish study, concentrations were lower in the second than in the first trimester, possibly due to dilution of the PFCs w ith blood volume expansion due to pregnancy, but values in cord blood (n - 50) confirmed fetal exposures (24.331 (Table 6)- The serum PFC concentrations seen in our study compare w ell w ith US serum data from NHANES 2003-2004. assessed in representative samples of 266 7S6 267 266 269 760 26* 262 263 26* 266 266 26? ?66 269 270 771 272 773 274 216 776 277 778 77B 780 ?i 787 783 784 266 286 287 p. 40 0 5 . von Ebrensfein el o f / Reproductive Toxicology xxx (2009) xxx-xxx 5 T ab le 6 Published data on average PFCconcentrations in milk, maternal serum and cord blood. Location,year of sampling Matrix, studypopulatipiuand sample size ' PFCconcentration as reported Massachusetts. USA. 2004 loip2lg /M u nkiiG enn an y.2006 Cyor.Hun&ary 1996/97 Milk:convenience sample, age: 22-43 years. n -4 5 , nursingthe ttrstthne: it - 34, nursed 1; it--8 . Milk: convenience sampling at ; hospital samples, -1 9 (M uoidt) . MiDcbank. n -3 8 (Leipzig) Mothers ofpreterm infants, it -1 3 (Hungary) * 3 - 7 weeks postpartum PFQS(mran.S0k 131(103)pg/ml PFOA: 4 3 * (33.1)pg/tnl PFHxS: 145(13-7)pg/m l PFNA: 7 2 6 (4.70)pg/m l . PFHpA. PFOA. PFUnDA. PfOoDA PFBS:aU<lOD PFOS(median, range) Munich: 113 (28-239)ng/L Leipzig: 123 (33-309) ng/l Hungary. 330 (96-639) ng/l PFOA. alb <10D (<LOD-460) ng/L Zbousan, China.2004 Milk: convenience sampling at hospital volunteers, n-1 9 Sweden, ndrrfduat matched sera andm ilk (2004); pooled composite imUc samples (1996-2004). Milk and scram: convenience . sample primiparouswomen, n=12 I . - ". , . . ; . ' : ' Pooled annualcomposite milk samples (n -2 5 -9 0 ) - Ranges*(ng/L) PFOS:45-360; PFOA:47-210; PFHxS: 4-100; PFNA: 6 3-62; PFOA.-33-15; PFUnDA: 7.6-S6 M ilk (mean. SD)ngfmb PFOS: 0201 (0.117); PFHxS:0.085 (0.047): PFOSA: 0.013 (0.009); PFNA: NA; PFOA. PFDA. PFUnDA; NO Baltimore. MO. USA. 2004-2005 Daceof m ilk collection: 3 weeks postpartum Cord blood, hospital based, singleton deliveries (n -2 9 3 ) Serum (mean,SD) ng/ml: PFOS: 20.7 (105 )ePFHxS: 4.7 (2 5k PFOSA:0 2 4 (0.16k PFNA: 080 (0 55k PFOA: 3 3 (tO k PFOA: 053 (0.41); PFUnDA: 0.40(035) Composite mBk ng/ml: PfOS0209 (1996)-0.123 (2004k PFHxS: 0537 (1996HU016 (2004k PFOSA: <0507 (1996)-<0507 (2004): PFNA: 0528 (I9 96)-052 0 (2004): PFOA: <0209(1996)-<0209 (2004) PFOA(median. langek ! (0 3 -7 .t) PFOS(median, range) ng/ml: 5.0 (<U )D (-02>-343) Denmark. 1096-2004 Japan. 2003 Maternal plasma; 1st trimester (H-1399X 2nd trimester (n -200) Cord Wood.n - 50 Maternal plasma; 3rd trimester (n -1 5 ) cord blood (n -1 5 ) Maternal. 1st trimester: PFOS (ng/mlxnean. SDk 353 (135X PFOA: 5 5 (2 5 ) Maternal 2nd trimestet-.PFOS: 295 (11.0k PFOA: 4.5(15) Cord blood: PFOS: 115(4.7) PFOA: 3 7 (3.4 ) Maternal 3rd trimester serum range*: PFOS(45-17.6 ng/ml) PFOA(<tOP to 2 3 ng/ndk PFOSA (<L0D to <lOD) Cord blood: PFOS(1 5 -5 3 ng/m l) PFOA(<LOD to <L0D) PFOSA (<LOD to <LOO) 1 RFCLODs (or setutn. blood and milk varied in (he dillerenl studies as reported in the original references. * No averages reported by authors. Percentage quantified >lOD* PFOS: 96X PFOA: 89X PFHxS: 51* PFNA: 64X PFHpA. PFDA. PFUnDA. PFDoOA. PFBS; <8% PFOS: 100X PFOA: 16* PFOS.PFOA. PFHxS. PFNA. PFDA. PFUnDA: 100* Milk: PFOS. PFHxS: 100 ( i t - 12). PFOSA: 67X (n- PFNA: 16% (n-2);PFO A ;B X[-T) Seram: PFOS. PFHxS. PFOA.PFNA. PFDA, PFUnDA: 1 0 0 *(n -12); PFOSA: 7 5 *(n -9 ) PFOS: 99 PFOA: 100X: Et-PFOSA-AcOH. Me-PFOSA-AeOH. PFBuS.PFHpA. PFUA. PFOoA: 1-40 Maternal 1st trimester: PFOS: 100. PFOA: WO* (except n-1 ) Maternal, 3rd trimester PFOS: 100X, PFOA: 20X. PFOSA: OX Cord blood: PFOS: 100X.PFOA: OX. PFOSA: OX Reference (25J |361 1341 |35| 12223) (24) |3 0 | zm females aged 12 and above, showing median concentrations for about halfthe concentration reported for cord blood from Denmark MS PPOS and PFOA of 18.2 ng/mi (IQR: 12.4-27.3 ng/m l) and 3.6ng/m l 124), Table 6. 75 (IQR: 2 .5 -5 2 ng/m l), respectively [20.21], Based on the NHANES A few investigations of PFCs in human milk have been con- 1 data, nation-w ide serum concentrations dropped for PFOS. PFOA ducted in Sweden.China, Denmark and recently in the US (Table 6 ) 7 and for PFHxS between 1999/2000 and 2003/2004 w hile those for 25.34-36|. Only one study assessed both serum and milk concen- 773 PFNA increased in the same tim e period |20 |. Our average levels trations and detected PFOS and PFHxS in all 12 milk samples at 79* are somewhat lower than reported for females in the US in 1989 mean concentrations of 0.201 and 0.085 ng/ml respectively, sug- 7 [5| but sim ilarto other findings in samples collected between 1999 gesting partitioning of on average 1 * from serum to m ilk (35J. In 799 and 2 0 0 5 16.10,20.21.501. In a recent US investigation, median cord the Chinese study, values of PFOSand PFOAin m ilk samples (it = 19) 757 blood levels for PFOSand PFOAof5 and 1.6 ng/ml, respectively, were were in the range o f0.045-0.36 and 0.047-0.21 ng/ml, respectively 7 reported [23J.This is about a third to afourth (PFOS) and 5 0 * (PFOA) 134], M ilk concentrations are summarized in Table 6, supporting 719 ofthe concentrations we found in maternal serum samples, and also our findings of lower values in milk than in serum, as w ell as , xi xa x>* dm m w m a>o an p. 41 G.$i0<tei ; 5R^pfl-S =l 'y ?- : ` ^ _~ s O.SL von Ehrensteln e t o L f Reproductive Toxicology xxx (2009) xxx-xxx 3t2 913 314 315 916 31? 918 319 32 323 323 324 325 326 32? 328 329 930 331 332 333 334 335 336 33? 338 338 30 341 34? 343 344 345 346 34? 348 349 350 351 3S? 353 354 355 350 357 350 359 360 361 36? 363 364 365 366 367 368 369 370 371 372 373 374 375 378 377 suggesting regional differences in exposure levels 125.34-36). PFAAs are strongly bound to the protein fraction o f human blood [10.51-53]. The protein concentration in human blood contains m ainly album in and fewer beta-lipoproteins and is about 3 -5 times higher than the protein fraction in human m ilk (casein and lactalbum in). It has been shown that strongly protein-bound drugs are less likely to transfer to human m ilk than small non-ionic lipophilic compounds [54 |. This may explain why PFAA concentrations are much lower in human m ilk than in maternal serum, although trans fer o f PFAAs to m ilk has been observed in animal studies, albeit at much higher serum concentrations of PFAAs (26). In our study, concentrations of PFOS, PFOA and PFHxS in serum w ere lower at the second visit compared to concentrations at the first visit. Since PFC concentrations measured in human sera have half-lives ranging between 3.4 years for PFOA, 4.6 years for PFOS. and 7.1 years for PFHxS [55], these data suggest that processes related to depuration into breast m ilk might be occurring that w e could not assess (possibly because we measured m ilk con centrations too late in lactation), or that there might be maternal metabolic changes during lactation that may relate to this change (i.e .. changes in blood volume, body weight, or hepatic activities). Because PFCs are tightly bound to serum proteins, serum protein levels during lactation could have affected the concentrations of PFCs in serum. Unfortunately, we did not measure serum albumin to test this hypothesis. Alternatively, if PFCs partition more into liver 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 times during lactation. The nature o f the relationship between the suggested decline of PFC values in serum to concentrations in m ilk are yet undear 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 for lipophilic compounds including dioxins. PCBs. and PBDEs (56.57). No other study to our knowledge, has investigated PFC concentra tion changes in serum or m ilk over tim e during lactation assessed in the same women at two time points. However, it should be noted that our findings are based on a relatively small number of a volun teer non-random sample of women and need replication in a larger study for further confirmation. Tao et al. conducted a regression analysis of PFOS and PFOA concentrations in breast m ilk collected a t various tim e points from 25 different women w ithin the first 6 months postpartum; they concluded that values increased over tim e of lactation |25). However, since these findings were based on m ilk samples of different subjects rather than comparing changes overtim e in the same women, the differences may be due to intraindividual variation. Our investigation suggested that living in North Carolina for a prolonged rim e period of 10 years and more was related to higher serum concentrations of PFNA, PFOA. and PFOS in our pilot study. However, further evaluation of this explorative finding is required. Point sources may lead to elevated exposures,as indicated by serum concentrations ofPFOA in persons living neara US facility using and producing this compound, that were notably higher than among the general US population [58). A systematic surface water survey conducted in North Carolina showed large variation in concentra tion on a sm all scale indicating a series o f source inputs around the Cape Fear Dra inage Basin that may potentially result in pocketsw ith increased exposures |40|. Comparing serum PFC concentrations among donors at the 6 American Red Cross Blood Bank locations across the US showed highest concentrations for PFOS and second highest for PFOA in Charlotte. North Carolina, in samples collected in 2000-2001 [41 ]. w ith a substantial decline observed in samples collected in 2006 at the same locations (42). Recently, elevated plasma concentrations especially of PFOA. PFNA. and PFHxS have been reported for personnel involved in the World Trade Cen ter (WTC) disaster (i.e., from fire-fighting foams used to combat the WTC fire or directly from the W TCs degradation) |59J further 378 supporting the notion o f source related local variations of human 379 exposures to certain PFCs. Women who reported living in a home w ith an enclosed garageattached also had increased concentrations 380 38t of 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 384 ments. However, due to the small sample size in this pilot study, we 395 could not analyze the impacts of other variables, especially socio 386 economic factors; these findings are thus explorative and should be 387 interpreted cautiously. 388 The pro-inflammatory cytokine IL-6 was positively correlated to 389 PFOS and PFHxS, respectively, at the second collection time point 390 possibly indicating that certain PFAAs may be related to inflamma 391 tory processes. In line w ith these findings are recent results from experimental studies in mice, reporting suppression of immune 382 393 responses following exposure to PFOS in utero (60). We did not see 394 correlations w ith othera priori selected biological markers assessed 395 in m ilk or serum. i.e_ immunoglobulin, estradiol, prolactin or TNF- 396 a . Rodentstudies using PFOAin concentrations orders ofmagnitude 397 higher than MAMA serum concentrations have shown a suppres 396 sion of genetic markers o f inflammation after an acute exposure to 399 PFOA (61). Because our findings are explorative, future studies may 400 want to address the role of chronic exposure to low dose PFCs in 40 the inflammatory process. 40? In this pilot study the number ofwomen was relatively small and confines the investigation of associations, possible exposure path 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 of women was unlikely to be related to PFC 407 exposures or to certain PFC exposure sources since they were most 406 likely not aware of their PFC exposures. A further lim itation of our 40 study is that we could not collect milk samples sooner after birth in 40 view of ethical constraints in asking for the colostrum milk. Studies 4H in mice measuring PFOA concentration over the course of lactation 4? have shown that the peak in milk PFOA concentration occurs soon 413 after birth (Fenton et aL. in this issue), a time that was not followed Q3 44 in the MAMA collection scheme. Overall, the findings reported are 45 explorative and need further evaluation. 416 In conclusion, although infant exposure via breast m ilk is likely 4? to be low, the cumulative daily infant intake of PFCs via breast milk 4*8 per kg body weight could be appreciable for some populations or 41 groups (Table 6). Since toxicological and pharmacokinetic data for 470 PFC exposed infants are lacking, it is largely unknown if potential 431 health effects in infants or during childhood may be related to cur 4?? rent exposure levels of PFCs. In utero exposure should continue to *23 be a concern as the MAMA serum PFAA concentrations are similar 47* to values reported in two separate studies that have shown inverse 475 associations between maternal serum or cord blood PFAA concen 4?6 trations and infant birth weight (22.24). Thus, the findings o f this 477 pilot study underscore the importance o f biomonitoring maternal 428 and infant exposure to PFC as w ell as the need for further study 47 of the potential human health effects of PFCs. In the upcoming US 430 National Children's Study (38) PFC exposures in pregnant and lac- 431 tating women and their children in North Carolina and across the 43? US w ill be further studied. 433 Conflict o f interest The authors declare that there are no conflicts of interest. 434 438 A cknow ledgm ents The research in this article has been reviewed by the National Health and Environmental Effects Research Laboratory. US Environ- 43C *37 43 p. 42 ./-Vi:;' O S. von Ebrensfeto et at/R ep ro d u c tiv e Taxkotogy x xx (2009) xxx-xxx 1 4 mental Protection Agency(EPA). and the Centers for Disease Control 440 and Prevention (CDC) and approved for publication. Approval does 441 not signify this report reflects EPA or CDC policy. The findings in 442 this report are those of the authors and do not reflect the views of 443 the CDC The use of trade names or commercial products does not 44 constitute endorsement or recommendation for use. 446 Thisw ork was supported in part by the Intram ural Research Pro- 44 gram at th e Eunice K ennedy Shriver National Institute o fChild Health 447 and Human Development, National Institutes o f Health, Bethesda, 448 M D. 44 Partial extram ural funding was provided through the rec- 4so ommendation o f the National Children's Study Intra-Agency 451 Coordinating Comm ittee. 45? The authors would like to Richard Wang at the CDC for technical 453 assistance,W estat, Inc. recruiting staff(Andrea Ware. Bethany Brad- 464 ford, Brian Karasek). and the US EPA nursing staff(Deb Levin, Mary 455 Ann Bassett, and Tracy M ontilla). Finally we would like to thank the 456 MAMA participants, without whom none of this would have been 457 possible. 458 References 459 (1) 3M Company Fhiorochemkal use. distribution and release overview. EPA 460 Docket OPP7-2002-0043; 1999. 461 12) LauC.AnitoleK.HodesC.et a).Perfluoroalkyl acids: a review ofmonitoringand 46e toxicological findings.Toxicol Sri 2007;99:366-94. 463 |3| Olsen GW.Church TR. Miller JP. et al. Prrfluorooctanesulfcmateand ocher ftuo464 rochemicals in the serum of American Red Cross adult blood donors. Environ 465 Health Perspect 2003;111:1892-901. 466 (4) Olsen GW. Logan PW. Hansen KJ, et at. An occupational exposure assessment 467 of a perflooroocranesulfonyi fluoride production site: biomomtoring. A1HA j 468 (Fairfax.VA) 2003:64:651 -9. 40 |S| Olsen CW.Huang HY. HelzlsouerK).et aL Historical comparison ofperRuorooc470 unesutfonate. perfluorooctanoaie. and other fiuorochemirals In human blood. 4 7 Environ Health Perspect 2005;113:539-45. 472 (6) Olsen CW. Mair DC. Church TR. et al. Decline in perfHiorooctanesulfdnatc and 473 ocher potyfluoroaHcyi chemicals in American Red Cross adult blood donors. 474 2000-2006. Environ SciTechnol 2008:42:4989-95. 475 |7) Ye X. Schoenfuss HL Jahns ND. et al. Perfluorinated compounds in common 47e carp {Cyprinus carpio) fillets from the tipper Mississippi River. Environ Int 477 2008;34:932-8. 476 [8} Hart K. Kannan K. IsobeT. et a). Time trends and transplacental transfer of per47o fluorinated compounds in melon-headed whales stranded along the Japanese 480 coast in 1982,2001/2002. and 2006. Environ SciTechno! 2008;42:7132-7. si Q 4 (9} Hart K. G ill VA. Kannan K_ Temporal trends (1992-2007) of perfluorinated 482 chemicals in northern sea otters (Bnhydra Jurrfe kenyoni) from South-Centra! 463 Alaska. Arch Environ Contam Toxicol 2008. 84 110| Calafat AM. Kuklenyik Z. Caudill SP.et al. Perftuorochemicats in pooled serum 85 samples from United States residents in 2001 and 2002. Environ Sci Techool 86 2006;40:2128-34. 87 ( l l | Ehrrsman DJ,FroehlichJW.OlsenGW. et aLComparisonofhuman whole blood, as plasma, and serum matrices for the determination of perfluorooctanesulfonate an (PfOS). perfluoroocranoate (PFOA). and other Quorochemicab. Environ Res 490 2007;103:176-84. 4 {12) Culkowska A Jiang Q. So MK, et al. Persistent perfluorinated acids in seafood 9? collected from two cities ofChina. Environ Sci Techno! 2006;40:3736-4L 493 (13) bhibashi H. Iwaca H. Kim EY. et a l Contamination and effects of perfluoro494 chemicals in Baikal seal (Puso birico). 1. Residue level, tissue distribution, and 95 temporal trend. Environ SciTechnol 2008;42:2295-301. 96 (14) Kannan K. Tao L Sinclair E. et aL Perfluorinated compounds in aquatic organ497 isms at various trophic levels in a Great Lakes food chain. Arch Environ Contam 498 Toxicol2005;48:559-66. 99 (15) Kim SK. Kannan K. Perfluorinated acids in air. rain. snow, surface runoff, and soo takes: relative importanceofpathways tocontaminationofurbantakes. Environ sot Sri Technol 2007;41:8328-34. 602 ( 16) Otivero-VerbelJ. Tao L.Johnson-RestrepoB.etaL Perfluorooctanesulfonate and eoa related fluorochemkab in bioJogkralsamples from the north coast ofColombia. 50 Environ PoiliH 2006:142367-72. 505 [ 17) Senthilkumar K.Ohi E.SajwanK,et aLPerfluorinated compounds in river water. so6 river sediment,market fish,and wildlife sampleshornJapan.Bull Environ Con507 tam Toxicol 2007;79:427-31. so (18) Wei S. Chen LQ, Taniyasu S. el aL Distribution of perfluorinated com609 pounds in surface seawaters between Asia and Antarctica. Mar PoHut Bull Sio 2007:54:1813-8. 5i> 119) Yeung LW. Miyake Y.Taniyasu S .etal Perfluorinated compounds and total and si? extractable organic fluorine in human blood samples from China. Environ Sci s3 Technol 2008:42:8140-5. si (20) Calafat AM. Wong LY, Kuklenyik 2. et al. Polyfluoroalkyl chemicals in the US. Sis population: data from the National Health and Nutrition Examination Survey (NHANES) 2003-2004 and comparisons with NHANES 1999-2000. Environ Health Perspect 2007; 115:1596-602. [211 Calafat AM, Kuklenyik Z. Reidy JA et al. Serum concentrations of II potyfluoroaJkyt compounds in the U.S. population: data from the national health and nutrition examination survey (NHANES). Environ Sci Technol 2007;41:2237-42. (22) ApelbergBJ.W iner FR. HerbstmanJB.etat.Cord serumconcencralionsofpcrflu' orooctanesulfonate(PFOS)andperfluorooctanoace(PFQA)in relation toweight and size at birch. Environ Health Perspect 2007;l15:1670-& (23) Apelberg 8). Goldman LR. Calafat AM. et aL Determinants of fetal exposure to polyfluoroalkyl compounds in Baltimore. Maryland. Environ Sci Technol 20 07;4t389l-7. (24) FtfC.McLaughlinJICTarone RE,eca i Perfluorinated chemicalsandfeta!growth: a study within the Danish National Birds Cohort. Environ Health Perspect 2007;115:1677-82. |25) Tao L Kannan K, Wong CM. et aL Perfluorinated compounds in human milk from Massachusetts.U. 5. A Environ Sci Technol 2008:42:3096-101. (26) Hinderlfter PM. Myfchreesi E. Gannon SA, et aL Perfluorooctanoaie: pla cental and lactational transport pharmacokinetics in rats. Toxicology 2005:2)1:139-48. (27) Lau C. ButenhoffJL Rogers JM. The developmental toxicity of perfluoroalkyl acids and their derivatives.ToxicolApp) Pharmacol 2004;198:231-41. (28) LauC.ThibodeauxJR,Hanson RG.et at. Effects of perfluorooctanok acid expo- sure during pregnancy in the mouse.ToxicolSci2006:90:510-8. (29) WolfCJ.FentonSE.SchmidJE.etal. Developmentaltoxickyofperfluoroocianoic arid in the CD-I mouse after cross-foster and restricted gestational exposures. Toxicol Sd 2007;95:462-73. 130] lnoueK.OkadaF.IioR.etal.Perfloorooctaoe$uJfonate(PFQ5)andrelated perfhi- orinated compounds in human maternal and cord blood samples: assessment of PTOSexposure in a susceptible populationduring pregnancy.Environ Health Perspect 2004;112:1204-7. (31) Midasch O. Drexler H. Hart N. et al. Transplacental exposure o f neonates to perfluorooctanesulfonate and perfluorooctanoaie: a pilotsrudy. Int ArchOccup Environ Health 2007;80:643-8. (32) SpBethoffHM.TaoLShavetSM.etal. Useof newborn screeningprogram blood spots for exposureassessment: declining levels ofperluorinatedcompounds in New York Stare infants. Environ Sd Technol 2008:42:5361-7. (33| FeiC,McLaughlinJK. UpworthLet aL Prenatal exposureto perfluorooctanoaie (PPM )and perfluoFOocranesuUbnate(PFOS)and maternally reported develop mental milestones in infancy. Environ Health Perspect 2008;t16:1391-S. (34) So MK, Yamashha N. Taniyasu S, et al. Health risks in infants associated with exposure to perfluorinated compounds m human breast milk horn Zhoushan. China. Environ SciTechnol 2006;40:2924-9. (35) KarrmanA EricsonLvan BB.etaL Exposure ofperfluorinatedchemicals through lactation: levels of matched human milk and serum and a temporal trend. 1996-2004. in Sweden. Environ Health Perspect 2007;115:226-30. (36) Vofke! W. GeiueLBorovkzeny O. Demmclmair H. ct aL Perfluorooctanc sutphonate (PFOS) and perfluorooctanok: acid (PFOA) in human breast milk: results ofa pilot study. Int J Hyg Environ Health 2008:211:440-6. |37) Hines EP. Calafat AM. Sifva MJ. et al. Concentrations of phthalate metabolites in milk, urine, salvia and serum of lactaring North Carolina women. Environ Health Perspectdoi:1tU289/ehp.11610 available via http://dx.doi.org/ (Online 22 August 2008), (38| Landrigan Pf. Trasande L. Thorpe IE. ct al. The National Chiktien's Study: a 21-year prospective study of 100.000 American children. Pediatrics 2006;118:2173-86. |39| Hines EP. Rayner JL Barbee R. et a t Assays for endogenous components of human milk: comparison of flesh and frozen samples and corresponding ana lytes in serum.) Hum Lao 2007.23:144-56. (40) Nakayaroa S, Strynar MJ. Hetfanr L et al. Perfluorinated compounds in The cape fear drainage basin in North Carolina. Environ Sci Technol 2007;41(15J: 5271-6. (411 Olsen CW. Church TR, Miller JP. et a). Perfluorooctanesulfoate ad other fluororhemicah in the serum of American Red Cross Adult Blood Donors. Environ Health Perspect 2003;111:1892-901. (42) Olsen GW. Mak DC. Church TR. et a t Decline in peiftuoroctanesulfonate and other polyfluoroalsfcy chemicals in American Red CToss adult blood donors. 2000-2006. Environ SciTechnol 2008;42:4989-95. (431 KuklenyikZ.ReichJA,TuUyJS,et aLAutomated solid-phaseextraction and mea surementofperfluorinatedorganic acidsand amidesin humanserumand milk. Environ SciTechnol 2004;38:3698-704. (44) CDC Third national report on human exposure to environmental chemicals. http://www.cdcgov/exposurefepoft/; 2005. |4S] Taylor JK Quality assurance of chemical measurements. Chelsea. MI: lewis Publishers; 1987. 146) Van LeeuwenSPf, Karrman A van Bavel B. DeBoerJ. Lindstrom C. Struggle for quality in determinationof perfluorinated contaminants in environmentaland human samples. Environ So Technol 2006;40:7854-60. (47) Kuklenyik Z. Needham LL Calafat AM. Measurement of 18 perfluorinated organic acidsand amides in human serum usingon-line solid-phaseextraction. Anal Chcro 2005;77:6085-91. (48) Homung RW. Reed LD. Estimation of average concentraion in the presence of nondetecuble values.Appi Occup Environ Hyg 2008:46-51. (49) CDC (Centers for Disease Control and Prevention) General documen tation on laboratory data. General Information about the NHANES 2003-2004 laboratory methodology and public Clara files. Available: 51 661167 519 570 521 52? 523 524 525 26 627 S?* 629 530 531 632 533 534 535 S3 S37 S3 539 50 S 4t. S2 5*3 644 5*5 64 547 54 & 550 551 662 553 55* sss 556 55? 56 55 560 561 56? 563 564 565 56 56? 56 56 570 571 572 573 574 575 57 571 57 57 580 1 58? 563 584 585 556 56? 586 589 590 SOv 582 593 504 505 596 507 59 59 0 0 fiO> p. 43 8 as . von Ehrautein rt at / Reproductive Toxicologyxxx (2009)joa-xxx 602 http://www.cdc.gov/nchs/d3ia/nlunes/nhanes_03jD4/tab.c_generaldoc.pdf; 603 2006. 0 [50} OlseoGW, Mair DC. Reagen WK. et a l Preliminary evidence ofadcrtine in per605 fluorooetanesulfooate(PfOS)and perftuorooctanoale(PFOA)concentrations m 606 American Red Cross blood donors. Chemosphere 2007;68:105-11. a? (s i) janes }D.Hu W . De CoenW.et al, Binding ofperfluorinated fatty adds to serum sot proteins. Evnvkon Toxicol Chem 2003;22:2639-49. 6oo {52} Han X, Snow TA. Kemper RA.eial. Binding oTperfluorooctanoic acid to rat and oio human plasma proteins. Chem ResToxicol 2003:16:775-61. on [53| Shattacharya AA. Grime T. Curry S. Crystallographic analysis reveals common 612 modeso f binding of medium and long-chain fatty adds to human serum albu613 min.J Mol (hoi 2003;303:72t-32. si }S4| Aschenbrenner DS.Veable Sj. Drug therapy in nursing. Uppinicott. Williams & 6i5 Wilkins; 2008. i6 155} Olsen CW. Burris JM. Ehresman DJ. et al. Half-lile of serum elimination of 6i7 perfluorooctanesulfonate. perfloorobexanesulfonate. and perftuorooctanoate c ii in retired fluorochemical production workers. Environ Health Perspect 2007:11S: 1298-305. f56} Hooper K, Shej. Sharp M. et aL Depuration ofpolybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in breast milk from O H ornu first-time mothers (primiparae). Environ Health Perspect 2007:115:1271-5. |57] LaKtndJS.8ertinCM.ParkCN.etaL Methodologyforcharacterizingtfiscributioos ofincrementalbodyburdensof23.7JB-TC00 andDOEfrombreastm ik m North American nursing Infants.JToxicol Environ Health A 200059:605-39. }58| Emmett EA, Sbohr FS. Zhang H. et aL Community exposure to perftuorooc~ tanoate: relationships between serum concentrations and exposure sources, j Occup Environ Med 2006:48:759-70. |S9) Tao U Kannan K. Aldous KM. et ai. BiomooRoring of perfluorocheimcab m plasma o f New York State personnel responding to (he World Trade Center disaster. Environ SciTechnol 2008;42:3472-8. (60) Keil PC, Mehlmann T. Butterworth L et aL Gestational exposure to perfhiO' rooctane sulfonate suppresses immune function in 86C3P1 mice. Toxicol Sci 2008:103:77-85. {61} Guruge KS. Yeung LW. Yamanaka N. et al. Gene expression profiles in rat liver Heated with perftuorooctanoic acid (PF0A). Toxicol Sci 2006;89(1).*93-107. nr 620 eat 6 6 62 625 cm 627 62 62 630 n< s 633 63 635 ELSEVIER M olecular and CeHular Endocrinology 304 (2009)97-105 C ontents lists available at ScienceDirect Molecular and Cellular Endocrinology jo u rn a l h o m e p a g e : w w w .e lse v ie r.c o m /lo c a te /m c e Phenotypic dichotomy following developmental exposure to perfluorooctanoic acid (PFOA) in female CD-1 mice: Low doses induce elevated serum leptin and insulin, and overweight in mid-life* Erin P. Hines1'*, Sally S. W hiteb, Jason P. Stanko1, Eugene A. Gibbs-Floumoyc, Christopher Laua, Suzanne E. Fenton1 * Reproductive Toxicology Division. Office o f Research a n d Development. National Health and Environmental Effects Research laboratory, US. Environm ental ProtectAgency. Reseordr Triongle Park. NC 27711. United Stoles bcurriculum in Toxicology. UNC Chapel Hill. Chapel H ill N C 27599. United States cBiological a n d Biomedical SciencesProgramftnitiatrve fo r M axim izing Student Diversity. UNC Chapel H ill Chapel H ill NC27599. United States ARTICLE INFO Article history: Received 27 January 2009 Accepted 24 February 2009 Keywords: PFOA Overweight Leptin Developmental exposure Obesity Ovariectomy ABSTRACT The synthetic surfactant, perfluorooctanoic acid (PFOA) is a proven developm ental toxicant in mice, caus ing pregnancy loss, increased neonatal m ortality, delayed eye opening, and abnormal m am m ary gland grow th in anim als exposed during fetal life. PFOA is found in th e sera and tissues of wildlife an d hum ans throughout the world, but is especially high in th e sera o f children com pared to adults. These studies in CD-I m ice aim to determ ine th e latent health effects of PFOA following: (1) an in m en exposure, (2) an in utero exposure followed by ovariectomy (ovx). or (3) exposure as an adult. Mice w ere exposed to 0 .0 .0 1 .0 .1 ,0 .3 .1 .3 . or SmgPFQA/kg BW for 17 days of pregnancy or as young adults. Body w eight w as reduced in th e highest doses on postnatal day (PND) 1 and at w eaning. However, th e lowest exposures (0 .0 1 -0 3 mg/kg) significantly increased body weight, and serum insulin and leptin (03)1-0.1 mg/kg) in m id-life after developm ental exposure. PFOA exposure com bined w ith ovx caused no additional increase in m id-life body w eig h t At 18 m onths of age, the effects of in utero PFOA exposure on body w eight w ere no longer detected. W hite adipose tissue and spleen w eights w ere decreased a t high doses of PFOA in intact developm ental^ exposed mice, and spleen weight w as reduced in PFOA-exposed ovx mice. Brown adipose tissue w eight was significantly increased in both ovx and intact mice at high PFOA doses. Liver w eight w as unaffected in late life by these exposure paradigms. Finally, there was no effect o f ad u lt expo sure to PFOA on body weight. These studies dem onstrate an im portant w indow of exposure for low -dose effects o f PFOA on body weight gain, as well as leptin and insulin concentrations in m id-life, a t a lowest observed effect level o f 0.01 mgPFOA/kg BW. The m ode of action of these effects and its relevance to hum an health remain to be explored. Published by Elsevier Ireland Lid. Abbreviations: ANOVA. analysis of variance; BMI. body mass index: BW. body weight; C8. eight-carbon; CV, coefficient of variation; DE5. dietylstilbestrof; t j. estradiol; CD,gestational day; r,B. Half-life; IACUC.InstitutionalAnimalCaie and Use Committee; LH. luteinizing hormone; IOD. limit ofdetection: LOQ,limit ofquantitation: WANES. National Health and Nutrition Examination Survey; NMR. nuclear magnetic resonance: NOAEL no observable adverse effect level; ovx. ovarieclomized; PFAA.perfluoroalkyl acid; PFOA, perfluorooctanoic acid; PFOS, perfluoiooctane sulfonate; PND. postnatal day; PPAR. peroxisome proliferatot-activated receptors: SMR. standardized mortality ratio. Disclaimer: Hie information in ibis document has been funded by the US. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratoryand approved for publication. Approval does not signify that the contents reded the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. * Corresponding author. Current address; US. Environmental Protection Agency. National Center for Exposure Analysis. Environmental Media Assessment Croup. ResearchTriangle Park. NC27711. United Slates. Tel.: +1 919 541 4204ffax: *1 919 541 2985. E-moil address: hines.erin#epagov (E.P.Hines) 0303-7207fS - see front mailer. Published by Elsevier Ireland Ltd. doi:10.t016/j.mce.2009.02.021 98 EJ>. H in a e t o L / Molecular and Cellular Endocrinology 304 (2009) 9 7 -MS 1. Introduction Perfluorooctanoic acid (PFOA). one o f the eight carbon (C8) perfluoroalkyl acids (PFAAs). is a synthetic, stable, persistent organic fluorine surfactant, used to im part water and grease resistance to various consumer products including non-stick pans, as sur face treatments for clothing and food wrappers, insulation and fire-fighting foams. PFOA's high energy carbon-fluorine bonds are resistant to hydrolysis, photolysis and metabolism and thus it bioaccumulates and persists w ithin biota and environmental matrices, including w ater and soil, from the Arctic to the South Pacific (Lau et a ), 2007). This ubiquitous environmental contaminant has an esti mated half-life (t1/2) in humans of 3.8 years (Olsen et a l, 2007) and is found in production workers' sera, as w ell as those of the general population. Bio-monitoring studies show detectable levels ofPFOA in human populations. The National Health and Nutrition Examination Sur vey (NHANES) reported that mean serum PFOA concentrations are declining in the USA population, from 5.2 ng/ml in 1999-2000 to 3.9ng/m l, in 2003-2004 (Calafat et a l, 2007). Arnsberg. Germany, an area w ith known drinking w ater PFAA contamination, had reported PFOA mean serum levels in 2006 o f 25 ng/ml vs. 4 ng/ml in unaffected German provinces (Hdlzer et a l, 2008). The highest known non-occupational PFOA exposure via drinking water exists in the Little Hocking drinking w ater district where U.S. residents (Ohio and W est Virginia) have mean serum PFOA concentrations o f 478 ng/m l (Emm ett et a l, 2006). Children may receive significant PFOAexposures via dietary and w ater intake. Mean serum PFAAconcentrations (such as periluorohexane sulfonic acid) were reportedly higher in children than in adult/elderly populations (Olsen et a l, 2004). In the Little Hock ing water district, an area of high environmental FTOA exposure, children age tw o to five and the elderly had significantly increased PFOA serum levels when compared w ith other age groups (Emmett et a l, 2006). Although a bio-monitoring study in Japan found PFOA in maternal blood, but not umbilical cord blood at parturition (Inoue et a l, 2004. lim it of quantitation |LOQJ 35.2 ng/ml). a recent U.S. study (Apelberg et a l, 2007) of human cord blood from term pregnancies reported relatively low levels of PFOA (lim it of detec tion |LOD] 0.2 ng/m l) and another C8 compound, perfluorooctane sulfonate (PFOS). W ithin the reported study concentrations, the authors found that cord blood PFOA concentrations were signifi cantly negatively associated w ith birth weight. A subsequent larger Danish study also found a significant negative correlation between maternal plasma PFOA and birth weight (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 hile high density lipoproteins have been negatively associated w ith PFOA (Olsen et a ), 20 0 1 ) Categorical division o f workers by PFOAexposure levels showed that, although not significantly differ ent from the other categories, body mass index (B M I) was elevated in the highest PFOA category (>30ppm and BMls >28.1995 data); this trend was not seen in the 1993 data set (Olsen et a l, 1998). A retrospective cohort m ortality study (n > 6000) of PFOA-exposed employees reported significantly elevated standardized mortality ratios (SMR) in males w ith diabetes mellitus when compared to men residing in West Virginia (minus the PFOA manufacturing area). Ohio. Virginia. Kentucky. Indiana. Pennsylvania, Tennessee, or North Carolina; the SMR for PFOA workers was not significantly increased when compared to West Virginia alone or USA residents (DuPont. 2006). In Arnsberg. Germany. PFOA was found to have an inverse correlation w ith BMI in adults (Hdlzer et aL, 2008). The r,ps for PFOA in men and women are sim ilar (Harada et a l, 2005). Unlike humans, gender differences in PFOAclearance exist in rats ( Kudo and Kawashima. 2003; Vanden Heuvel et a l, 1991). Mice are the preferred animal model for evaluating the effects o f PFOA on the developing fetus as they do not exhibit gender-dependent rt p differences (Lau et aL. 2006). However, even in the rat model system where the female rat rapidly excretes the compound. PFOA readily crosses the placenta (H inderliter et a l, 2005) and PFAAs are present in rat m ilk after PFOA treatm ent (Hinderliter et a l, 2005). Mice prenatally exposed to doses of PFOA at >1 mg/kg/day exhibit developmental toxicity including decreased litter size, neonatal death, delayed eye opening,growth deficits,stunted mam mary gland development, and early onset male puberty (Lau et a l, 2006; W hite et a l, 2007; W olf et a l, 2007). At higher doses and fol lowing long-term adult exposure, cancer endpoints associated w ith PFOA exposure in rats include Leydig cell adenomas, pancreatic aci nar cell adenoma/carcinomas, mammary fibroadenomas, and liver tumors (Biegel et aL, 2001; Sibinski, 1987). PFOA increased estra diol (E2) levels in male rats and PFOA-induced rodent Leydig cell tumors are hypothesized to arise from increased estradiol levels from aromatase induction (Liu et a l, 1996; Biegel et a l, 2001). The majority of the ongoing work in the PFOA field has focused on the health effects following developmental exposure to PFOA. This study focuses on adult latent health outcomes in female off spring after developmental (gestational days (GD) 1-17) vs. adult (at 8 weeks o f age. for 17 days) exposure to PFOA. Ovariectomized siblings were utilized in our second study block to address the role of the ovarian hormones in PFOA exposure-related health effects, as luteinizing hormone (LH)-overexpressing mice (Kero et a l, 2003) displayed several phenotypic effects resembling those in our preliminary studies w ith PFOA. These studies address the role of developmental exposure and ovarian hormones in adult health effects including circulating leptin and insulin concentrations,adult body weight, and tissue and body weights in old age. 2. Materials and methods 2.1. Animals Timed-pregnant CD-I mice (Charles River Laboratories. Raleigh. NC) arrived on gestational day (GD)0 (sperm positive) at the US EPA where they were weighed upon anival and randomly distributed among treatment groups. Pregnant dams were housed individually in polypropylene cages and received chow (LabDiet 5001. PMI Nutrition International LLC. Brentwood. MO) and tap water ad fibilvm. Two blocks of animats were used in these studies. Block 1 animals were dosed wkh vehicle (distilled waterX 13. or 5 mgPFOA/kg body weight (BW) (n -S , 8. 7. and 5 dams, respectively); block 2 animats were dosed with vehide. 0.01. 0.1. 0 3 .1 . or 5 mgPFOA/kg ( M dams in all groups except 5 mgPFOA/kg BW. which had 10 dams). PFOA exposures are shown in the text as mgPFOA/kg. Animal facilities were maintained on a l2:12-h light-dark cycle, at 20-24 "C with 40-50% relative humidity. Animals were humanely treated as approved under National Health and Environmental Effects ResearchLaboratory protocols m accordance with the USEPA institutional Animal Care and Use Committee (1ACUC). Sentinel mice, housed in the same room, were known to be free ofecto/endoparasites and antibodies to certain viruses for the duration of these studies. 2 2 . Dosing solution and procedures PFOA. as its ammonium salt (>98% pure), was acquired from Ftuka Chemical (Stemhiem, Switzerland). PFOA dosing solution was prepared fresh daily in deion ized water, and the dosing solution was administered at a volume of 10 pi/g. Mice received either water vehicle or PFOA at 0.01.0.1.03.1.3. or 5 mg/kg BW by oral gavageoncedaily over the dosingperiods.The highest dose(5 mg PFOA/kg/day)was chosen because it was knownto result in slightly reduced neonatal bodyweight gain with minimal postnatal mortality (Lau et al.. 2006). 2 3 . Experimental design 23.1. Developmental exposure/mtact Timed-pregnant CD-I mice (n * 7-22 dams per dose group over two blocks) received 0.0.01.0.1.03.1.3. or 5 mg/kg PFOA by oral gavageon the mornings ofGD 1-17. Dams were weighed daily prior to dosing and throughout gestation. At birth, pups were individually weighed and sexed. Pups within a treatment group were pooled and randomly redistributed among the dams of their respective treatment groups, and litters were equalized to 10 pups (both genders represented). Dams tp . Hines f i a l I M olecular and Cellular Endocrinology 304 (2009) 97-105 or PFOA Developmental PFOA Adult Dosing Dosing GO 9 1-17 wfcs 3 *k t i|i|5M==nj ji P 16-16 wki I l Glucose CDO B,nh tolerance P1 ?osTMMve V * an ^ wx lest (young) 21-33 wk* I_ ! Mandftxdar bleeds 42wfcs |I Body Mass Composition Meastsed 70-74 wks lI Clueose tolerance Test (old) Food intake m onitoring 1 It Months E2 measure ...Weight monitored (developmentally exposed, ovx Untoci).. Fig. 1. Data collection schematic for study ofdevelopmentally and adult PFOA-exposed female mice. 99 that delivered small litters (n<4 pups) were excluded from the remainder of the study. Pups were weaned at 3 weeks of age at which point females were retained and boused 3 -5 mice per cage. Males were evaluated separately, at end points that varied from those reported here. 2 3 2 Developmental exposure/ovariectomy A subset of developm ental exposed female siblings (0 mgPFOA/kg. n -8 ; 0.01 mgPFOA/kg. n -15; 0.1 mgPFQA/kg. n - lt ; 03 mgPFOA/kg. n-14; 1 mgPFQA/kg.n 6; 5 mgPFOA/kg.n*=7) were ovariectomized (ovx)at 21 or 22 days of age, before the onset of puberty. Animals were sedated with ketamine/xylazine (87/13 mg/kg up., respectively), their ovaries surgically removed through the abdomen, sutured, and animals were placed in warming cages until they regained alertness. Buprenorphine analgesic (0.05 mg/kg)was given twice daily un. for 48 h in 0.1 ml volume for pain relief 2 3 3 . Adult exposure A separate cohort of mice received PFOAstarting at 8 weeks of age. for 17 days (Omg PFOA/kg. n - 8; 1 mg PFQA/kg. n = 14; 5 mgPFQA/kg. n - 14). 23.4. Data collection The data collection scheme for these studies is shown in Fig. 1. Blood was cot* lected from the submandibular veins of ovx and intact mice between the ages of 21 and 33 weeks.These bleeds took place between 14:00 and 18:00. and 200 pJ of blood (100 pJ of serum)was collected for subsequent analysesofinsulin and leptin. Females in all three exposure scenarios were weighed weekly up to9 months ofage and then monthly until 18 months.The number of intact,developmental^ exposed mice weighed wrekly/monthly was 10, 25.20.11. and 32. respectively for 0.0.01. 0.1.03, and 1.0 mg PFQA/kg. If mice became moribund before the study ended, they were euthanized in compliance with the protocol approved by the US EPA IACUC (early necropsy) Date and cause of early morbidity or mortality was recorded if known. At early necropsy (collected when necessary)or at 18 months, trunk blood, retroperitoneal abdominal while (found lying ventral to the intestines and repro ductivetract) and interscapular brown for pads, abnormalgrowths,andorganswere collected from all exposure groups. Relative organ weight is used to express organ weight as percent of total body weight. Data are reported here as mean SEM. 2.4. Glucose tolerance test Glucose tolerance tests were performed on two groups of intact developmentally PFOA-exposed animals: old adults (17 months of age with 0, 0.1, 1 or SmgPFQA/kg; n - 8-13 per dose group) and young adults (15-16 weeks old with 0.1 or 5 mg PFOA/kg: n -1 2 per dose group) The night before the assay, fur was shaved from the lateral area of the lower leg to expose the saphenous vein and ani malswere fasted.The following morning,the mice were weighed and blood glucose was measured by collecting a drop ol blood from each mouse via puncture of the saphenous vein (or tad vein if necessary)The blood drop was placedon a test strip, and inserted into the calibrated glucometer (Accuchek Advantage)for baseline glu cose measurement. The mice were then injected i.p. with D-ghicose solution (2 g/kg body weight from a stock solution) and blood glucose concentrations were mea sured at 20.40.60 and 120 (old mice)or 180(young mice)minutes ( 1-3 m in)after the initial glucose injection. 2 3 . Serum leptin Serum (10 p i) collected by mandibular venipuncture was assayed for leptin by radio-immunoassay (Unco Research. St. Charles. MO) following the manufacturer's protocol (n 5. controls: n- 18.0.01: n* 16,0.1: n -1 1 .0 3 : n- 24.1 mgPFOA/kg)The coefficient of variation (CVs)for the standards (concentration rangeof0 3 -2 0 ng/ml) ranged from 0.1%to 8.0%.The quality controlstandards termed QC1 (expected range 0.6-13 ng/ml)and QC2(range 1.8-3.8)had ameasuredconcentration in theseassays of 0.9 and 2.9. respectively. 2.6. Serum insulin Sera (lO p I) collected by mandibular venipuncture were assayed for insulin by the ultra-sensitive single molecule immunoassay by Singulex (Alameda. CA) fol lowing the manufacturer's protocol (n - 9 control. n21.0.01 mgPFOA/kg; n -16. 0.1 mgPFOA/kg: n - 11. 03 mgPFOA/kg: n-31. 1mgPFQA/kg) Samples were ana lyzed using a 384-well plate format with monoclonal capture and detection antibodies on the Singulex ErrenaequipmentTheCVsfor the assaystandards(range 193-5000pg/mt) were from 31 to 17%. The assay LOD was )6pg/mL AB samples were run on the same day and the mterassay CV was 9.4% and 5.1% for the 29 and 1745pg/ml quality assurance standards, respectively. 2.7. Body mass composition Whole body masscomposition was measured m live, iron-sedated 42-week-old mice using the Broker Minispec mq 73 LF50 Live Mouse Analyzer(The Woodlands. TX) The minispec was a benchtop 73 MHz time-domain nuclear magnetic reso nance (NMR) analyzer, which quantified body (at, lean tissue,and free body fluid in mice. The minispec was calibrated by Broker Optics.Inc. staffprior to animal analy sis with dally validations using Broker standards. Mice were weighed and inserted into the instrument for analysis(1 -2 mm/animal) Intact devetopmemaDy exposed female truce that underwentbody mass composition analysisincluded control,0.01. 0.1. 0 3 . and 1 ragPFOA/kg (n -9 . 23. 20. 11, and 32, respectively) dose groups. It was not possible to perform these measures with younger mice due to equipment availability. 2.8. Measurement o f Ej in serum o f intact mice ot 18 months Serum E2 (25 p i volume) from 18-montlt-old mice (intact developmentally PFOA-exposed animals) was measured with time resolved Ouoro-immunoassay (DELFIA Estradiol Kit, WaHac Oy. Finland) following the manufacturer's recom mendation using a VICTOR2D 1420 Multilabel counter. PerkinElmer Precisely time-resolved fluorometer(PerkinElmer Lifeft AnalyticalSciences,Shelton.CT)Th* CVs for the standards (concentration range of 631-1423 pg/ml) ranged from 02% to 4JSX. 2 3 . Feed consumption Feed consumption rn 17-momh-okt. developmentally exposed, intact female mice (n - 6 per dose group, 0 ,0.1.1 and 5 mgPFQA/kg) was measured in metabolic cages. Mice were allowed to acclimate to the cages for 1 week and food intake was monitored during the second week. Mice were individually boused and provided with a pre-weighed amount of powdered tabchow od Kbitum, The remaining chow was measured at the end ofthe week and the totalamount was subtracted from the starting amount 10 determine the total feed consumed for each mouse per week. 2JO. M easurem ent o f serum PFOA Trunk blood serum samples (-5 0 p i) from the female CD-I offspring at 18month necropsies or from mice terminated at earlier intervals because of Hlness were transferred to the CDC for PFOA measurement. Serum PFOA determination was performed as described in Kutdenyik el aL(2005) and White et aL(2009) 2J1. Statistics Data were analyzed usingSAS9.1 (SASInc.. Cary.NC) Body weight on PND1 was evaluated as liner meansas thesedata were obtained prior to mixing litter offspring within a dose group. Bodyweights at each time point were analyzed with mixedeffects linearmodels (SAS Proc Mixed) to estimate means and standard errors and test for dose effects separatelybyrimepoint. Foreach time pointthemodelincludeddoseasafixed effect p. 47 100 EP. Hines e l oL / Molecular and Cellular Endocrinology 304 (2009) 9 7 -70S and cage nested within dose as a random effect. Pairwise t-rests were calculated to test for any difference between each treatment group mean and the control group. Repeated measures analysis ofbody weight data was evaluated two ways. First, weights were averaged by animal over eight lO week intervals. This was done to decrease missing values in the data due to animal m orality in late life that was not equal across treatment, and to reduce the effect oflarge body weight variances later in life. This data smoothing method decreased uninformative short-term variations and also reduced the number ofestimated parameters to a tractable value. A multi variate repeated measuresanalysis (SASProcGLM)was performed onthese reduced data. Subsequent to a significant finding, comparisons were carried out as subtests of the overallanalysis ofvariance (ANOVA) at specific times or doses. Second. SAS Proc Mixed was used to perform a univariate repeated measures analysisoftheweights acrosstime upuntil 37weeks(latestweight pointatwhich no animals had died).The modelestimated a separate fixedquadraticcurve acrosstime for each dosegroup and included a random effect for cage nested within dose. Cor relation within animals was modeled with a random effect for animal nested within cage and dose in adefition to an autoregressive covariance structure within eachani mal. In this way. the covariance matrix for each animal's measurements included a constantcovariance component at all time points in addition to a component which decreased as tim e points grew farther apart. Tissueweight,relative tissue weight,body composition, food consumption,and body weight measurements were analyzed using a one-way ANOVA (Dunnctt's post hoc tests), w ith dose being the independent variable. A blocking variable was included to adjust for the group difference. No adjustment was made for multi ple comparisons. Glucose tolerance was compared at individual collection times by one-way r-test and over time by repeated measures and area under the curve comparisons according to the trapezoidal rule. Hormone (insulin. Ez and leptin) concentrations were analyzed usingANOVA followed byTukey*s post hoc test. Mortality data were analyzed wkh product limited survival estimates; log-rank and Wikoxon tests were used to test for differences among the treatment groups in survival across time (SASProc Lifelest)The level of significance for all tests was p<0.OS. (A) 1-75 1.7 3 Xj6 1.55 SL *5 13 1.45 OS u .01 0.1 u Dos* PFOA (mg/kg BW) Period 3. Results 3.1. D evelopm ental exposure 3.1.1. E arly a n d m id -life b o d y w e ig h t effects There w ere no significant differences in live pup number at birth by dose group (p <0.05) and postnatal m ortality was not addressed in this study as litters were equalized at birth. On post natal day (PND) 1. the average weight o f the devetopmentally exposed SmgPFOA/kg offspring was significantly less than con trols (Fig. 2A ); no other dose group demonstrated significant litter weight effects at PND1. At weaning, mean female body weights were still significantly decreased in the 5 mg PFOA/kg (13.9 gdfc0.8) compared to 18.4 g 0.4 in control untreated pups. At this tim e, the 1 mg PFOA/kg exposed animals were also significantly smaller than controls (p< 0.05; 16.4g 0.3). Time-grouped mean body weights o f the female offspring over their lifetim e are shown in Fig 2B. Beginning at 10-19 weeks of age. there was an increase in weight in the 0.1 and 0 3 mg PFOA/kg groups compared to controls; by2 0 -2 9 weeks ofage, females devel opm ental^ exposed to PFOA showed significant dose-dependent increases in body weight at 0.01. 0.1. and 0.3 mg PFOA/kg which extended to 40 weeks o f age in the 0.01 and 0.1 mg PFOA/kg when compared w ith control (p 5 0.05). This is specifically shown at 2 0 -2 9 weeks (Fig. 2C). where the 0.01 -0 3 mg PFOA/kg groups had average weights 11-15% higher than controls. Continuous analysis o f repeated measures o f body weight over tim e demonstrated that 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 their weights were changing at a more rapid rate than control or 1 mg/kg. This is shown in Fig. 2D for weeks 6-37 (the latest weight collection tim e point prior to death ofany study animals). Addition ally. the 0.1 mg/kg (p = 0.056) and 0.3 mg/kg (p = 0.046) groups had larger negative coefficients for week2 (week squared), suggesting that their weights were starting to fall o f f more quickly at the later tim e points than the control groups (not shown). The estimated weight curve for the 1 mg PFOA/kg dose group was not significantly different from the control curve. Data from 5 mg PFOA/kg exposed .. ................................................................... Dose PFOA (mg/kg BW) Hg. 2. Body weights o f developmentally PFQA-exposed female offspring. Data are shown asmean SIM with > <OJOi vs. control.(A)Pupweight at PND1 after devel opmental PFOAexposure. (B) Body weight of female CD-I mice over their lifetime, followingdevelopmentalPFOA exposureover8 periodsoftime [period 1(0 -9 weeks old),period 2 (10-19 weeks old) period 3 (20-29 weeks old) period 4 (30-39weeks old) period S(40-49 weeks old),period 6 (50-59 weeksold) period7 (GO-69weeks old) and period 8 (7 0 -7 9 weeks)) (C)Group mean bodyweights of female offspring at 20-29 weeks ofage demonstrating excessive weight gain at low doses. (D) Dosedependent quadratic regression fit to repeated measures of body weight in female mice.An increased rate ofweight gain was seen in 0.01.0.1, and 0.3 m g PFOA/kgdose groups compared to control and 1 mg PFOA/kg. p. 48 EJ* Hines e t a 1 / Molecular anti CeButar Endocrinology 304 (2 0 0 9 )97-105 101 ( A) SOD-, 450400- O 350 300- ? S 2005 15*- 0 (B ) 600-j T im e (m in u trs ) T im e (m m o le s ) Fig. 3. Blood glucose concentrations following a glucose challenge after time 0 in (A) young (15-16 weeks old) and (B) old (70-74 weeks old) female CD-I mice that were developmentally exposed to PFOA. Data are shown as meanSEM. mice, w hich were decreased in BW compared to control at PND1, weaning, and 18 months, are not shown. 3.1.2. S e r u m g lu co se tolerance testin g Because of the excess weight gain in the PFOA developmen tally exposed mice during m id-life, various tests were conducted on these animals (as close to the appropriate age as was possible) to examine the associated effects of these changes. No significant differences were detected in baseline glucose or serum glucose area under the curve in response to a glucose challenge in young or old m ice (control. 0.1.1. or 5 mg PFOA/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 min post-glucose challenge (p =0.06). In old PFOAexposed mice, although there appeared to be dose-dependent glucose insensitivity at 20 min, this shift in response was not sig nificant. 3.1.3. S e r u m in sulin a n d leptin Serum insulin and leptin measurements were made using blood obtained via mandibular bleeds between 21 and 33 weeks (w ithin the tim e fram e of greatest observed body weight 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 developmentally exposed to the lowest doses of PFOA tested (0.01 and 0.1 mg PFOA/kg). Although elevated from the control mean, leptin concentrations were not significantly different from control at 0 3 or 1 mg/kg PFOA (Fig. 4). 3.1.4. Fat to lean ratio At 42 weeks of age. mice from block 2 (control, 0.01. 0.1, 0.3. and 1 mg PFOA/kg) were evaluated using a Broker Optics Body Mass Analyzer, which determines the amount of fat. lean and fluid (B ) 3500-j i= o.oi o.i oj Dose PFOA (mg/kg BW ) 1* * I IIm i 9-01 0.1 9 3 Dose PFOA (mg/kg) 1 Fig.4. Serum leptin (A) and insulin (B) in mice at 21-33 weeks o f age (*p <0.05 vs. control) Significant elevations are seen at 0.01 and 0.1 mgPFOA/kg Data are shown as meanzfcSEM. in live animals. There was no significant increase detected in X body fatrbody weight in PFOA-exposed mice (data not shown). Developmentally exposed mice had no significant differences in fat:lean 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 %fatrbody weight ratio and 14% in mean fatrlean ratio in the dose group exhibiting the largest change in body weight at 24 weeks (0.1 mg PFOA/kg). 3.1.4. J. Feed consum ption. Feed consumption was measured in 17month-old, developmentally 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 26g/week con sumed: individual data not shown). 3.J.5. Late life organ an d b o d y w e ig h t effects A noted loss of animals after 36 weeks of age was further eval uated (fig . 5). At 51 weeks old. when there was no mortality in controls there were 20%, 10%. 36%. and 6% m ortality rates in 0.01, 0 .1 .0 3. and 1 mg PFOA/kg groups, respectively. By 76 weeks, there was a 40% mortality rate in controls, and 32%. 63%. 60%. and 44% in 0.01,0.1.0.3 and 1 mgPFOA/kg groups, respectively. However, there were no significant differences between control and any treatment group at specific times in late life or in survival across time. Among those mice surviving to 18 months, body weight of 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). At that time, all remain ing females were necropsied. Trunk blood, tissues (affected or o f interest) and abnormal masses were collected, weighed and fixed for future study. Serum was collected and PFOA levels were mea sured. The majority of the samples across dose groups had PFOA concentrations lower than the lim it of detection (0.5ng/m !) with detectable values at maximum concentrations of 3 3 ng/ml, and p. 49 * 102 E.P. Hines e t cl. / M olecularand CeHulor Endocrinology 304 (2009) 97-105 Table 1 Mean or relative body and tissue weights at IS months ofage in intact and ovariectomued (ovx) female CD-I mice. PFOA dose Body weight (g) (mgfrg) ' Abdominal white fat W right (g )-. Interscapular brown Fat weight<g) Relative spleen Weight ( X f in ta c t. Ovx Intact. . O vx , . Intact Ovx . Intact Oyx o r :--. .5 4 5 0 *1 1 1 3 52.73*5.67; o.oi 56 56 *1 1 4 3 5 2 .6 1 *3 *3 0.1 S 4 5 0 ll7 5 2 7 6 *1 9 8 o i 5 6 3 0 * 1,74 4 9 3 6 *3 3 3 1 56J5 i 135 61.47*353 3 5 3 5 9 * 2 2 7 lie. 5 49 37 *1 5 1 * 55.13*5.76 7 0 7 *0 5 6 3A 3U >3 . 0 7 3 * 0 0 4 0 3 7 *0 0 9 039 * 0 0 5 0520.16 658*6 .41 ; 5 5 8 *1 5 0 .! *0 3 o *;d o 4 : 0 3 6 a b 4 ;: 0301*033 0 2 9 *0 5 4 5 5 1 1 0 3 7 4 3 4 * 0 3 1 ., 082 * 0 5 4 0 .4 6 *0 5 6 045 * 012 0 4 0 *6 5 8 5 5 6 *0 5 3 4 5 7 *0 4 6 079 * 65 6 0 3 9 *5 5 2 035 * (ElO 0 3 3 0 jD8 5 3 2 *0 .4 3 * 5 3 2 *0 .7 7 7- .03 9 * 054* 0 5 9 *0 5 6 * 0 3 0 *0 .0 3 - 0 2 2 *0 5 2 * / nc . 122 * 0.10*: nc 0 .1 8 *0 0 3 * Jic :. vV -\" 4 .4 8 * 05S* 5.86 1 -6 7 ' 0 3 6 * 0.05 : 0 3 2 *0 3 0 052 * 024 0 2 1 *0 5 4 * Relative liver W right( x y Intact. 4120*0.10 ~339 0.11 -420 *0 4 1 430 * 031 4j02 * Olio 358 * 023 43 7*0 34 .Oyx 43 0*6 .44 1 2 *0 3 5 4.18*021 4 5 7 *0 2 2 3 5 5 *0 2 9 * 'nc'-. -3 5 5 *0 3 3 nc. Denotes not collected from this dose group. * p<051 vs. control. * p - 0.05-0.07. c Relative weight (organ weight as percent of body weight). 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 females at 18 months when compared to controls (non-cycling; mean range across doses from 12.9 to 15.8 pg/ml). Tissue weights from 18-month-old animals (intact and ovx) are shown in Table 1. To determine if the weight o f fat depots was altered in old animals due to developmental PFOA exposures, the retroperitoneal abdominal w hite and interscapular brown fat pads w ere collected and weighed. Abdominal w hite fat weight and rela tive w hite fat weight both showed significant decreases vs. control (p < 0 .0 5 ) at 1 and 5 mgPFOA/kg. W hite fat weights were not col lected for 3 m g/kg PFOAanimals.At 18 months, interscapularbrown fat weight and relative brown fat weight both showed significant increases above control (p <0.05) at 1 and 3 mg PFOA/kg The spleen was quite variable in weight among the different treatment groups, but there was a significant difference in spleen weight and relative spleen w eight vs. control at 3 mg PFOA/kg (p <0.05). Finally, at 18 months, no significant differences in liver weight or relative liver w eight were detected. reach statistical significance. W hen comparing the body weights of animals in the ovx study by treatm ent group, over time (4 weeks to 18 months), using statistical methods consistent w ith those used for intact animals, there was no effect erf PFOA (Fig. 6B). Compar ison of ovx animals to intact animals at 20-29 weeks, as shown in Fig. 6A, demonstrates an absence of body weight gain over con trol in the ovx animals treated w ith PFOA. PFOA exposure did not stimulate increased weight gain (above that o f control ovx) at any developmental exposure level in the absence of the ovaries (also seen in Fig. 6B). The ovx animals were siblings to the intact animals in this study. The ovx animals were also assessed at 18 months. Developmen tally PFOA-exposed ovx animals showed no significant differences in body weight when compared to control ovx females (con trol mean - 52.7 5.67; highest mean. 1 mgPFOA/kg-61.5 3 3 ; Table 1). 3.1.6. E ffect o f ova riecto m y on tissu e a n d b o d y w e ig h t gain A group o f developmentally PFOA-exposed animals (0.0.01,0.1, 0.3, 1. and 5 mg PFOA/kg) were ovx at weaning and their body weight gain and adult health was assessed until they reached 18 months of age. At m id-life the w eight o f the control ovx females was expected to be greater than that of the sham-operated, intact controls (Fig. 6A; set o fbars at 0 m g/kg), but the variance in the ani m al weights was appreciable and therefore the differences did not 0 M l 0.1 0L3 | PFOA Dose (m g/kg BW") Fig. 5. Survival curves for developmentally PFOA-exposed female mice (0-1 mgPFOA/kg). Although a fair number of PFOA-exposed animals die early, a Lifetest (SAS)analysts detected no significant decrease in time to death.The reasons h r early life mortality are under investigation. Fig- 6. (A) PFOA-dependent changes in group mean body weight of intact and ovx female offspring at 20-29 weeks of age. There was no change in body weight of ovx animals across PFOAexposures. (B) Dose-dependenl quadratic regression fit to repealed measures of body weight in ovx female mice. Unlike intact siblings, no significant differences weie seen between dose groups in the ovx animals. E.P. Hines e l a t/M o le c u la r a n d Cellular Endocrinology 3 0 4 (2 0 0 9 )9 7 -1 0 5 103 As w ith intact siblings, the tissue weights of ovx animals are reported in detail in Table 1. In ovx animals, neither abdominal w hite fat pad weight, nor relative abdominal w hite fat pad weight, were significantly different from ovx or intact control levels. This varies slightly from intact siblings, where the w hite fat pad was significantly decreased in size; although, in animals that weighed significantly less than intact controls. Among PFOA-exposed ovx animals, both interscapular brown fat weight and relative brown fat 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 intact animals, and was significant at the same dose. Spleen weight (data not shown) and relative spleen weight in ovx animals was highly variable at 18 months, and showed decreases, albeit not highly significant, at the 1 and 5 mgPFOA/kg doses (p -0 .0 6 and p =0.05, respectively; Table 1). 1 and 3 mgPFOA/kg (not 5m g/kg) were the doses in the intact animals showing the largest decreases in relative spleen weight compared to controls.Finally, relative liver weight showed no significant differences across dose groups when compared to ovx control. 3.1.7. Lack o f e ffects fr o m adult PFQA exposure At 18 months o f age. body and tissue weights were recorded in adult PFOA-exposed mice. Adult PFOA exposure had no effect on term inal body or organ weights. When a comparison of data from 18-m onth-old adult intact and developmentally exposed ani mals in the 0. 1 and 5 mg PFOA/kg dose groups was made, body weight, brown fat weight, and w hite fat weight of the 1 mgPFOA/kg developmentally exposed animals were significantly higher than the same dose in adult-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 weight, as w ell as serum leptin and insulin in adulthood. In the developmental PFOA studies, a dose-dependent dichotomy of phenotypes was present in intact fem ale mice; latent effects present following high doses were not present in mice exposed to low-dose PFOA and vice versa. Although there was no detectable change in body weight neonatally. low-dose PFOA exposures (0.01. 0.1, or 0.3 mgPFOA/kg) led to significantly increased mean weight and rate of weight gain in m id-life (u p to and including 37 weeks o f age) and a coincident sig nificant elevation of serum leptin and insulin values between 21 and 33 weeks (0.01 and 0.1 mg PFOA/kg). Our low-dose hormone data indicate potentially important metabolic changes that mechanistically support the findings of increased weight in the lower dose groups. Previous dosimetry work in our lab has shown that in u tero exposure to PFQA in the mouse translates into an extended developmental exposure period via lactational exposure (all o f gestation and nearly 3 months postnatally; W hite et al.. 2009; W olf et al., 2007; Fenton et al,, 2009). This long exposure may lead to reprogramming/metabolic events that govern fat metabolism or appetite control. Although we were unable to perform some of the other end points o f interest dur ing this tim e period of greatest weight gain, our findings relating leptin and insulin concentrations to the time ofoverweight in PFOAexposed mice support our theory. Other environmental chemicals, termed environmental obesogens (dietylstibestro! (DES), 20H-E2, 40H-E2, genestein and bisphenol A), have been shown to induce obesity in adulthood after low-dose developmentalexposure,w hile inducing w eight loss at higher doses (Grun et al., 2006; Newbold et al.. 2005; Miyawaki et al.. 2007) and are reviewed further w ithin this issue. Serum leptin was significantly elevated in m id-life in the lowdose PFOA-exposed groups. This effect occurred at the same PFOA dose range as overweight in these animals, congruent w ith a leptin-resistance mechanism ofaction for overweight,aspreviously reported in humans (Considine et al., 1996). Others have reported increased leptin w ith developmental exposure to environmental obesogens including DES (Newbold et al.. 2007). Low-dose (0.01 and 0.1 mg PFOA/kg) developmental PFOAexpo sure that led to increased serum leptin and body weight also increased insulin values at 21-33 weeks. This suggests that the insulin resistance mechanistic pathway could also be affected and play a role in developmental PFOA exposure-induced overweight in mice. In an insulin resistance scenario, there are raised plasma glucose levels (elevated, but not significant, at 15-16 weeks in our study), reflecting the loss 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 (Careyet a l, 1996). High plasma levelso finsulin and glucose,due to insulin resistance,are often associated w ith type II diabetes and metabolic syndrome in humans, and thus this effect of low-dose PFOAdevelopmental exposure and its association w ith increased serum insulin are important. The ovx data were difficult to interpret. The lack of additional weight gain w ith developmental PFOA and ovx may reflect a 'ce il ing effect" or that ovx-induced weight increases may have masked any effect o fPFOA. Alternatively, as weight gain and metabolic hor mones can be regulated by estrogens, the role of the ovaries in developmental effects of PFOA was explored by using ovx animals. The potential importance o f the ovaty in the effects of PFQA was based on the observation that LH-transgenic (overexpressing) mice (Kero et a l, 2003) were phenotypically similar to ours (increased body weight, increased brown fat 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 for the increase in brown fat and possiblythe exces sive weight gain. Ovx animals typically gain body weight in excess vs. intact animals (Kamei et aL. 2005). The critical role of the ovary in weight gain of intact PFOA-exposed females beyond that of ovx treatment-matched siblings in the 0.01 and 0.1 mg PFOA/kg groups was novel and signifies the ovarian axis as a potential mediator of PFOA-dependent m id-life weight changes. Another potential mediator of these intertwined low-dose PFOA-induced effects is the peroxisome proliferator-activated receptor (PPAR) activation pathway. PPAR gamma (PPAR-y) and PPARalpha (PPAR-o) are involved in lipid metabolism in adipocytes and liver/skeletal muscle, respectively(reviewed in Medina-Gomez et a l, 2007; Abbott, 2009). These PPAR isoforms are known to influence lipogenesis/weight gain and have been shown to be regu lated by environmental compounds such as tributyltin (Grun et a l, 2006; reviewed in this issue) Weight loss events in leptin-deficient, obese, and insulin-resistant mouse models have coincided w ith PPAR-regulated changes in gene expression (Holvoet, 2008). A down-regulation of PPAR isoforms involved in energy expenditure, lipogenesis or fatty acid synthesis have been reported in adipose and skeletal muscle of ovariectomized mice (Kamei et aL, 2005). PFOA has been shown to be a PPAR activator in liver tissue (high doses) and cell lines, and to be required for PFOA-induced devel opmental toxicity in mice (Takacs and Abbott. 2007; Abbott et a l, 2007; Abbott. 20 09 ) If PPAR activation via receptor binding is a primary mode of action for body weight effects following PFOA exposure, the decrease in the PPARreceptors following ovariectomy and decreased circulating estrogens may explain the lack of effect of PFOAin ovx mice. However. PFOA-induced consequences of PPAR activation following a developmental exposure are just beginning to be evaluated. After 40 weeks of postnatal age. an increase in m ortality was detected in all animals. There are previous reports in the literature ofincreased m ortality in non-treated CD-I mice, attributed primar 104 EJ*. Hines e t o L / Molecular a n d Cellular Endocrinology 304 (2009) 9 7 - JOS ily to thymic lymphomas (Son, 2004; Taddesse-Heath et al.. 2000). Because of this confounding circumstance, repeated measures of body weight were only followed out to 37 weeks of age. The other half o f the phenotypic dichotomy caused by devel opmental PFOA exposure was also novel. Developmental exposure to higher doses of PFOA (1, 3 and 5mgPFOA/kg) led to a vastly different phenotype from low-dose PFOA exposure. This effective PFOA dose dichotomy may manifest itself in our study via unique modes ofaction; the animals w ith highest dose(s) ofdevelopmental PFOA exposure have decreased early life body weight and terminal body w eight (5 mgPFOA/kg) w ith significant decreases in w hite fat weight at 18 months (1 and 5 mgPFOA/kg). significant increases in brown adipose (1 and 3 mgPFOA/kg), and significant decreases in spleen weight (3 mgPFOA/kg) findings that are absent w ith the low er doses of PFOA. Others have reported dose-dependent loss of w hite tis sue adiposity in adult male mice after PFOA exposure (0.02X PFOA weight/chow w eight, which translated to approximately 32 mgPFOA/kg BW daily) w ith fat loss, w ithout fat cell number loss, that is PPAR-y-independent w ith (3-adrenergic activation (Xie et a l, 2002). In that same study, investigators also reported w hite fa t and body weight decrements at higher doses that were absent at lower doses. Yang et al. (2002) showed PFOA-dependent weight loss was abrogated in PPAR-a null mice, indicating that PPAR-a is a probable regulator of weight loss in the high dose animals. In subsequent studies. Xie et al. (2003) showed that after cessation o f exposure o f adult male animals to PFOA (0.02% PFOA weight/chow w eight. 3 2 mgPFOA/kg BW) daily for 7 days followed by 10 days recovery, weight loss and w hite adipose levels returned to base line. which confirms the importance o f developmental exposures for the latent effects reported here. In our model w ith developmen tal PFOA exposure we see permanent weight loss and w hite adipose tissue loss a t the high dose of PFOA. However, there may be merit in further exploring these mechanisms of action, as (i-adrenergic receptor upregulation is also associated w ith increased brown fat mass in winter-acclim ated animals (Feist, 1983), and this tissue was associated w ith high dose (and not low dose) effects in both intact and ovx animals in this study. Although we suspected alleviation of effect in the brown fat pad by eliminating the ovary (based on phenotypes in Kero et a l, 2003). significant increases in brown fat were seen at 1 mgPFOA/kg in both intact and ovx animals. At the 18-month tim e point, some endpoints remained unchanged across dose groups including liver size. Earlier work has shown significant hepatomegaly after developmental PFOA expo sure (1 and 3mgPFOA/kg) observed out to at least 3 weeks after birth (the latest tim e point evaluated; W olf et a l, 2007; W hite et a l, 2007).Thetransient nature ofhepatomegaly has been illustrated in other acute adult exposure studies (reviewed by Lau et a l, 2007). and is further confirmed in these studies (intact and ovx). A final im portant component of these studies evaluated adult vs. developmental exposure to PFOA on body tissue weights. These data suggest that the tim ing of dosing (adult vs. developmental 17-day PFOA exposure) was critical for latent effects. There was no effect of 17-day adult PFOA exposure on any endpoint in this study (early life or latent) when compared to age-matched, vehidegavaged controls. In conclusion, the tim ing and dose of PFOA exposure for induc tion of dichotomous, persistent, adult health effects in C D -I female mice are critical. Developmental, low-dose PFOA exposure led to increased weight in adults, w ith increased serum insulin and leptin, a health effect not seen in high dose animals. No observable adverse effect levels (NOAEL) for body weight gain, serum leptin and insulin concentrations were not determined in this study; but 0.01 mg PFOA/kg had a significant impact on these particularly sen sitive end points. The ovary appeared to play an important role in the overweight effect in m id-iife. and it is proposed that there is a common mode of action, potentially dysregulation of PPAR and its signaling through ovarian hormones, that may be responsible for these low-dose health effects. Further studies addressing long term PFOA-induced health outcomes in mice should focus attention on internal dose relative to the low-dose health effects seen in this study, as w ell as the mechanisms of action, so that any relevance to human health effects can be addressed. Acknowledgements W e would like to thank Broker Optics. Inc. for the use of the Broker Minispec mq 7.5 LF50 Live Mouse Analyzer and Harry Xie and Basil Desousa o f Broker Optics, In c for their technical assis tance. W e would like to acknowledge Antonia Calafat and her labo ratory staff, Kayoko Kato and Zsuzsanna Kuklenyik. in the Division of Laboratory Science. National Center for Environmental Health. Centers for Disease Control and Prevention for the analysis o fserum PFOA concentration from 18-month-old developmental^ exposed female mice; Donald Doerfler, Experimental Toxicology Division. US. EPA. and Judy Schmid, Reproductive Toxicology Division (RTD). US. EPAfor their statisticalsupport; Deborah Best. RTD.for conduct ing the estradiol assays; Veronica Luzzi. David Gibson and staff at the Core Laboratory for Clinical Studies at Washington University in St. Louis. MO. for performing the serum insulin assays, and finally. Dr. David Kurtz and the technical staff at New Year Tech. In c for their exceptional animal care during these lengthy studies. Thanks to Retha Newbold. NIEHS. and Rob Ellis-Hutchings. Dow Chemical. Midland. M I. for their constructive input on this manuscript. References Abbott BD,, 2009. Review of the expression of peroxisome proTiferators-activated receptors alpha (PPARaX beta (PPARfS). and gamma (PPARy) in rodent and human development. Reprod. ToxkoL. doi:10.1016/j.reprotax^008.10.001, Epub. Abbott. B.D, Wolf. C.J, Schmid. JJL. Das. ICR. Zehr. R.D.. Laurence. H,, Nakayama. S , Lindstrom. A B , Strynar. M.J,, Lau. OS.. 2007. Perfluoroctaook acid (PFOA)mduced developmental toxicity in the mouse is dependent on expression of peroxisome profiferator activated receptor-alpha (PPAR-aX ToxkoL Set 98 (2). 571-581. Apetberg.B.J,W itter. F.R, Herbstman.JJL.Calafat.A.M, Halden. R.U,,Needham.LL, Goldman. LR , 2007. Cord sensm concentrations of perfluorooctane sulfonate (PFOS) and perftuorooctanoate (PFOA) m relation to weight and size at birth. Environ. Health Perspect. 115.1670-1676. Bieget. LB , Hortt. M .E, Frame. S.R, O'Connor. J.C, Cook. J.C, 200). Mechanisms of extrahepatk tumor induction by peroxisome proliferators m male CD rats. ToxkoL ScL60 (IX 44-55. Calafat. A Jti, Wong. LY, Kuklenyik. Z , Retdy.JA . Needham. LL, 2007. Polyftuoroalkyl chemicals m the US. population: data from the National Health and Nutrition Examination Survey (NHANES) 2003-2004 and comparisons with NHANES 1999-2000. Environ. Health Perspect 115 (11X1596-1602. Carey.D.G,Jenkins.A 4 ,CampbellLV ,Freund.J,Chisholm.D.J, 1996.Abdominalfat and insulin resistance in normal and overweight women: direct measurements reveal a strong relationship hi subjects at both low and high risk of NIDDM. Diabetes 45 (5). 633-638. Considtne. R.V, Siitha. M X . Hetman. MJ_. Kriaudunas, A , Stephens. T.W, Nyce. MJL, Ohannesian.JP, Marco.C.C, McKee, LJ, Bauer.TL , Caro.JT, 1996. Seram immunoreactive-lepttn concentrations in normal-weight and obese humans. N. EngLJ.Med. 334 (5). 292-295. DuPont 2006. Ammonium perftuorooctanoate: phase IL Retrospective cohort mor tality analysesrelated toa serum biomarkerofexposurein a polymer production plant US ERAAdministrative Record. 8EHQ-0381-0394. Emmett. EA . Shofer. F5. Zhang. H , Freeman. D , Dcsai. C, Shaw. LM , 2006. Community exposure to perfluorooctanoate: relationships between serumcon centrations and exposure sources.J. Occup. Environ. Med. 48 (BX 759-770. Fei. C , McLaughlin. J.K, Tarone. R.E, Olsen,j , 2007. PerAuorinated chemicals and fetal growth: a study within the Danish National Birth Cohort. Environ. Health Perspect. 115,1677-1682. Fenton. S.E, Remer,JJ_. Nakayama. S.F, Delinsky. A D , Startko.J.P,, Hines. E.P, Lind strom. A.B, Strynar. M .j, Petropouloo. S. Analysis of PFOA in mke part 2: disposition of PFOA In biologkal samples collected from pregnant and lactating mke and their pups. Reprod.Toxicol, 2009. doi:l0.10l6/j.reprotox^009i)2.012. epub. Feist. D.D, 1983. Increased beta-adrenergk receptors in brown fat of winteracchmatued Alaskan voles. Am. J. Physiol. Regtri. Integr. Comp. Physiol. 245. R357-R363. EP. Hines er a t f Molecular and Cellular Endocrinology 304 (2009) 97-105 105 Grn. F, Watanabe, H , Zamanian. E Maeda, L , Arima. K , Cubacha. R, Gardiner. D M , Kanno.J,, Iguchi. T,, Bhimberg, B, 2006. Endocrine-dbrupting organotin compounds are potent inducen ofadipogenesis in vertebrates.MoL EndocrinoL 2 0 (9 X 2 1 4 1 -2 1 5 5 . Harada. IC. (none. K , Morkawa. A , Yoshmaga. T , Saito, N , Koizumi, A , 2005. Renal clearance of perfluorooctane sulfonate and perfluorooctanoate In humans and their species-specific excretion. Environ. Res. 99,253-261. Hinderliter,P.M.,Mykhreest.E,Gannon.SA ,Burenhoff.XU Kennedy.Gi_.2005. Perfiuorooctanoate: placental and lactational transport pharmacokinetics in rats. Toxicology 211.139-148. Holvoet, P, 2008. Relations between metabolic syndrome, oxidative stress and inflammation and cardiovascular disease. Verb. K Acad. Geneeskd Belg. 70. 193-219. Hlzer.J, Midasch. 0,, Rauchfass,1C,Kraft, MmReupert, 1C,Angerer,J, Kleeschulte.P, Marschalt, N , Wilhelm, M , 2008. Biomonitoring of perfluorinated compounds in children and adults exposed to perfluorooctanoate-contaminated drinking water. Environ. Health Perspect. 116 (5X 651-657. lnoue, IC. Okada, F.Ito, IC. KaloS, Sasaki, S, Nakapma, S, UnoA , Sago.Y , Sata, F,, Yoshimura, Y . Klshi. R, Nakazawa H , 2004. Perfluorooctane sulfonate (PFOS) and related perfluorinated compounds in human maternal and cord blood sam ples:assessmentofPFOSexposurein a susceptible population duringpregnancy. Environ. Health Perspect. 112 (11X 1204-1207. Kamel.Y,, Suzuki. M , Miyazaki, H , Tsuboyama-Kasaoka. N,,W ,J, Ishimi. Y , Ezaki. 0.. 2005. Ovariectomy in mice decreases lipid metabolism-related expression in adiposetissue andskeletalmusclewith increased bodyfatJ.Nutr.SeiVUamiooL (Tokyo) 51.110-117. Kero. j.T . Savontaus. E , Mikola. M , Pesonen. U,, Koulu. M , Keri, R A , Nikon. Poutanen. M , Huhtaniemi LR, 2003. Obesity in transgenic female mice with constitutivelyelevatedluteinizing hormone secretion.Am.J. Physiol Endocrinol Metah. 285, E812-E81S. Kudo. N , Kawashima.Y,,2003. Toxicity and toxicokinetics of perfluorooctanok arid in humans and animals.X Toxicol Sri. 28.49-57. Kuklenyik, I , Needham. LL. C alahl A M . 2005. Measurement of 18 perfluorinated organic acids and amides in human serum using on-line soKdphase extraction. Anal. Chem. 77,6085-6091. Lau. C . Ailte. IC. Hodes, C . Lai. D , Pfahles-Hutchens, A , Seed. J, 2007. Perfluoroalkyf acids: a review of monitoring and toxicological findings.Toxicol. Sri. 99 (2 X 3 6 6 -3 9 4 . Lau. C , Thibodeaux.JJI Hanson. H C . Narotsky. M .C, Rogers.X M , Lindstrom. AB, Strynar. 2006. Effects ofperfluorooctanok acid exposure during pregnancy in the mouse.Toxkol Sri. 90 (2X 510-518. Liu.R.C,HurtI.MJE,Cook.XC,BiegelLB, 1996.Effectofthe peroxisome proliferator. ammonium perfluorooctanoate(C8Xon hepaticaromataseactivity in adult male CRJrCDBR(CD) rats. Fundara AppLToxicoL 30 (2X 220-228. Medina-Gomez. G , Gray. S, Vidal-Puig, A , 2007. Adipogenesis and hpotoxkity: role of peroxisome profiferator-activated receptor gamma (PPARgamma) and PPARgammacoactivalor-1 (PGC1X Publk Health Nutr. 10. 11321137. Miyawaki.J, Sakayama. K.. Kato. H,, Yamamoto, H , Masuno, H , 2007. Perinatal and postnatal exposure to bisphenol A increases adipose tissue mass and serum cholesterol level in mke. X Atherosder. Tbromb. 14 (5X 245-252. Monlecucco. F,, Steffens, S,, Mach. F,, 2008. Insulin resistance: a proinflamina tory state mediated by lipid-induced signaHng dysfunction and involved in atherosclerotic plaque instability. Mediators fnflamm, dot:10.1155/2008/ 767623. Newbold, H R , PadDla-Banks, E, Snyder, R.J,Jefferson. W H . 2005. Developmental exposure to estrogenic compounds and obesity. Birth Defects Res. Part A 73. 478-480. Newbold, RJt, PadMa-Banks, E , Snyder, E X Phillips, T M , Jefferson. W JI, 2007. Developmental exposure to endocrine disruptors and the obesity epidemk. Reprod. Toxkol 23 (3). 290-296. Olsen.G.W,, Gilliland.FJX, Buriew. M M , Burris.JM,Mandet.J.S,Mandet,XH, 1998. An epidemiologk investigation of reproductive hormones in men with occu pational exposure to perfluorooctanok arid. J. Occup. Environ. Med. 40 (7X 614-622. Olsen.C.W..Oiurch.TR, Hansen. K.J, Burris,XM , Butenhoff.XU MandelXH, 2004. Quantitative evaluation of perfluorooctanesulfonate (PFOS) and other ftuorocbemkals In the serum of dtifdren.X Child Health 2.53-76. Olsen.G.W , Buriew, M Jtl, Burris.X M , MandelXH, 2001.A longitudinal analysis of serum perfluorooctanesulfonate (PFOS)andperfluorooctanoate(PRM)leveb in relation to Kpid and hepatic cKnkal chemistry test results from male employee participants ofthe 1994(95,1997,and 2000 fluorochemkal medicalsurveillance program. 3M Company. Final report. October 11,2001. US EM Administrative Record. AJt-226-1088. Olsen. G.W , Burris,XM , Ehresman. D.J, Froehlich.XW.. Seacat. A M , ButenhofT.XL, Zobel LR , 2007. Half-life of serum elimination of perfluorooctanesulfonate. perfluorohexanesulfonate. and perfluorooctanoate in retired fluorochemkal production workers. Environ. Health Perspect 115(9X 1298-1305. SibinsklLX. 1987.Two-yearoral(diet) toxicity/carcinogenicity studyofftuorochemkal FC-143 (perfluorooctane ammonium carboxytate) in rats. Report prepared for 3M. St. Paul Minnesota by Riker Laboratories Inc. Study No. 0281CR0012: 8EHQ-1087-0394. October 16.1987. Son. W.*C, 2004. Early occurrence of spontaneous tumors in CD-I mke and Sprague-Dawley rats.Toxkol. Pathol 32.371-374. Taddesse-Heath. L. Chattopadhyay. SJC, Diflebay, DE. Lander. M Jt, Nagasbfar. Z , Morse 3rd, H X , Hartley.J.W , 2000. Lymphomas and high-level expression of murine leukemia viruses m CFW m ke.X Virol.74 (l$X 6832-6837. Takacs. M X , Abbott, B i), 2007. Activation of mouse and human peroxisome proKferator-activated receptors(alpha.beta/ddta.gamma)by perfluorooctanok add and perfluorooctane sulfonate. ToxkoL So.95.108-117. Vanden Heuvel,X*Kuslflds.B ,Van Refelghem, M , Peterson. E . 1991.Tissuedistribu tion, metabolism and elimination of perfluorooctanok acid.J. Biochem.ToxicoL 6 .8 3 -9 2 . White.S 5, Calafat.A M , Kuklenyik, Z , Villanueva, L, Zehr. R.D, HeHint. L, Strynar. M.X. Lindstrom. A B , Thibodeaux.JJL. Wood. C, Fenton. S i, 2007. Gestational PFOAexposure ofmice is associated with altered mammarygland development in dams and female offspring. Toxkol ScL 96 (IX 133-144. W hite. S S , Kato. K , Jia. L T , Basden. B.J, C a la fa tA M , Hines. EJL SUnko. XP,W o(f, CX. A bbott, B.O., Fenton. S i. 2009. Effects o f perfluorooctanok acid on mouse m am m ary gland developm ent and d iffe re n tia tio n result ing horn cross-foster and re stricte d gestational exposures. Reprod. T a xie d , doi:10.1016/Xreprotax-2008.11.054. Epub. W ott CJ, Fenton. SB, Schmid. XE, Calafat. A M , Kuklenyik. Z . Bryant. X A , Thi bodeaux. X* Das, HP,. White. S 5 , Lau. CS, Abbott. B D , 2007. Developmental toxicity of perfluorooctanok arid in the CD-I mouse after cross-foster and restricted gestational exposures. Toxicol. Sri. 95.462-473. Xle,Y^Yhng.Q , Nelson.B.D,DePierre.XW,2002.Characterization ofthe adiposetis sueatrophy induced by peroxisome prollferators in mice.Lipids37 (2X139-146 Xie. Y , Yang. Q , Nelson. B i), DePierre.XW, 2003. The relationship between fiver peroxisome proliferation and adipose tissue atrophy induced by peroxisome proKferator exposure and withdrawal in mke. Biochem. PharmacoL 66 (5X 749-756 Yang. 0 Xie, Y , AJexson. S i, Nelson, BJ>, DePierre, XW, 2002. Involvement of the peroxisome proKferator-activated receptor alpha in the immunomodulation caused by peroxisome proKferators in mke. Biochem. PharmacoL 63 (10X 1893-1900.