Document vV7VZrD2o2XOqYj2JyDz7LXpm

Aezatr3155 mEaan August 4, 2010 EPA Docket Center, MC 2822T U.S. Environmental Protection Agency [EPA West, Room 3334 1200 Pennsylvania Avenue, NW `Washington, D.C. 20460-0001 Re: Submission to IRIS and AR-226 Database For PFOA/PFOS: EPA-HQ- 'ORD-2003-0016 ------"_-- To IRIS Database for PFOA/PFOS: In response to the Notice issued by USEPA on February 23, 2006, regarding USEPA's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("PFOS") within the Integrated Risk Information System ("IRIS"), 71 Fed. Reg. 9333-9336 (Feb. 23, 2006), we are submitting the following additional information to USEPA for inclusion in that review, and for inclusion in the AR-226 database: 1. Stone, KL., "A Review of Perfluorooctanoic Acid Carcinogenicity and Application to Human Risk," (Indiana Univ. Masters Thesis) (Submitted May 2010, posted on-line July 2010). (Td obert A. Bilott RAB:mdm Enclosure cc: Gloria Post (NJDEP)(w/ encl.) (via U.S. Mail) Helen Goeden (MDH)(w/encl.) (via U.S. Mail) Lora Wemer (ATSDR)(w/ encl.) (via U.S. Mail) CONTAINS NO CBI p2 A REVIEW OF PERFLUOROOCTANOIC ACID CARCINOGENICITY AND APPLICATION TO HUMAN RISK Kenneth Lee Stone SubmittedintopatrhtieaflacfuulltfyiollfmetnhteofUtnhieverresqiutiyrGermaednutaste School inthe DepartmentMoaffsotrPehtrhoaerfmSdaeccgiroeelneocgey and Toxicology Indiana University May 2010 p3 fulfilAlcmcenetpotfetdhbeyrtehqeuiFraecmuelnttyosffoIrntdhieandaegUrneievoerfsiMtays,tienrpoafrtSiaclience. CMaosmtmeirt'tseeT:hesis James E. Klaunig, Ph.D, Chair Lisa M. Kamendulis, Ph.D. Barbara Hocevar, Ph.D. Ix ACKNOWLEDGEMENTS T would like to thank the people who have influenced and inspired me. Without their help1 could not have succeed in my academic career and this thesis would not have been possible. specifically want to thank my advisor, Prof. James E. Klaunig, for his guidance throughout my graduate career. His Introduction to Toxicology class inspired me to seek my Master's degree where his life stories about his experiences as a Toxicologist captivated my interests and propelled me to continue my education. | could not have accomplished my goal without his support and recommendation to the graduate program. 1 would also li0tkhaenk Dr. Lisa M. Kamendulis for her support and for serving as my co-advisor. She has always been accessible and has regularly taken time outof her schedule to offer her asistance and hercarto listen to my concerns. Her lectures will be remembered as engaging and motivational and her hard work and dedication have served as an example worthyof emulation. Dr. Barbera Hocevar also deserves special thanks as she has served on my thesis `committee and has given valuable guidance and direction to me without which this thesis could not have been completed My deepest gratitude goes to my wife, Jannie Stone, for her unwavering love and support throughout this entire process; I could not heve achieved success without her. She is my soul mate, partner and friend and has never left my side. | am also indebted to my father, Kenneth Stone, for his example of Christian faith and work ethic. He worked tirelessly to support the family and spared no effort to provide the best possible home. ps `Words cannot describe my gratitude for my mother, Janet Stone, who is simply the best mother a child could be given by God. I remember many sacrifices my mother has made: for me, often going without simply to provide for me. I remember her constant support and encouragemeantd the strengthof her Christian faith, Furthermore, my children Sam Stone, Lindsay Stone and Misty Johnson have completed my life. 1 am grateful for the love and joy they have given me and their encouragement which has motivated me to continue. I also want to express my gratitude to God for giving me my grandchildren, Ronni, Cole and Camryn Johnson, whose smile alone brightens my day and makes me forget my struggles. 1also want to thank of my sister, Karen, forbingthe best sister a brother could ask for. She has helped our family out more times than I can remember. Last but not least, my greatest thanks belong to God. He has walked with me throughout my life and has stood by me during my most challenging times. God, you have truly blessed me end made my Ife joyful and complete. 1 owe everythintgo you pe `TABLE OF CONTENTS LIST OF TABLES sms A LIST OFFIGUREvSv ix LIST OF ABBREVIATIONS. x 1. INTRODUCTION wr: | A Tomar PSS mmm B. Environmental PIESeNce ocr rme md l 1H 2) PTOA 200A IHS BWR w mmmmo mmmmmmmmmmmmmmmmonm-- 3) PFOA i the EAVIIONMENt ns 7 C. CheandmPhyisicacl PRaOPERltES .......ruvuvvovvorsorssso 8 ILTOXIINCANIIMATLS.Y.. . cute oxicity... nsm-------------- 10 AA T e B. Subhronic TOXIC v wmmm--------1 C. MUBGEIGHY rss 14 13. MECHANISMS OF CARCINOGENICITY cr covrssnsssnnns 19 Ar LVET rss wasn 1) RERClivE OXYEE SPECIES. 19 2). Mitochondrial BIOBENESIS rrr 21 3) PPAR AGONIST. ssi 2 7 4) CAR ACHVBION. ovr son 2h O5) EGtIroCgeInRleRSbOVNGv wmmms mmmmms semmmmmmmm--3824 7) immunomodulation. ........ wsdl LE 1) Inhibition OfTeSIOSIETONE BiOSYNMRESES...vvcrvvvvrorrnnns 28 2) ESUAdOl MOGUIRON. rss 29 C. PaRCIEES LC ------ wm ----we-- ssm- ommm--50 IV. HUMAN RELEVANCE cs ------s ------: -533 Ae MV rmenmmm 1) ROASR GeCnHeVraBtIiOoNn......ccrrssnssssssswsosm ns--h 2) C A 2 CCI mmmmmmmsm------ 3 4) ESTOGENC SIGAANNG vs mmss m---- 35 DIR m lh msmmm IB. 6)TPWPARt G AGu OISm c mi v ess ----T36 1) Estradiol MOAUIiON.....v S----- V. EPIDEMIOLOGICALSTUDIES... 40 VI. DISCUSNSDSSUIMMOARNY commend] [x] VIL REFERENCES c.coovoemmmsnsssmsmmssmessssmssssmmmsmmsmmmsnonn CURRICULUM VITAE po LIOF TSABLTES Table 1.1: Table 1.2: Table 2.1: Table2.2: Table23: Table 24: SSeurmummarCoynocfenGteroamteitorniscinMUenainteadndStaRtaensgPeoOPfUPISFHOOANS............rocvd BSOumRmEarRyrocf aSecrsuemmPmFsOAsmComnrcemnstrmamtisonssa(mngs/mmlm)sian sShmeenymaenmgsChminmao. ml Summary of PFOA LDSOVANES... 0 L SummaryofE the PFOA levels found in surviving | SummaryoftheEffects ofAPFOExposurein MaleRas................|7 Summaryof the Tumor Induction FrequencyofAPFO in RES...........18 p.10 LIST OF FIGURES Figure 3.1: Figure 32: Figure 33: Summaryof therolesofoxidative stress in CATGIOgENESis......... mn Tlustration detailing the proposed MOA for PFOA [PANCIERtC ACHATCell MONGENESS....vvvrvivsnsnrn 3] SummaryofPossible Mechanisms for PFOA `CAICINOGERICIY in ROGERS... 32 ix ptt APFO CAR CAT ck cH DMSO EPA ER ERK FSH GR cic HPT Let LH MOA ND 8OHIG PACT PB PCPLC LIST OF ABBREVIATIONS Ammonium Perfluorooctancate Constitutive Androstane Receptor Catalase Cholesteystokinin Chinese Hamster Ovary Dimethyl Sulfoxide Environmental Protection Agency Estrogen Receptor Extracellular Receptor Kinase. Follicle-Stimulating Hormone: Glutathione Reductase Gap Junctional Intracellular Communication Hypothalamic Pituitary Testicular Leydig Cell Tumors Luteinizing Hormone: Mode of Action None Detected 8-Hydroxydeoxyguanosine Pancreatic Acinar Cell Tumors Phenobarbital Phosphatidylcholine-specific Phospholipase C p12 PECs PEDA PFOA PFOS PHA Ld PPAR ROS sop wy Perfluorochemicals Perfluorodecanoic Acid Perfluorooctancic Acid Perfluorooctanc Sulfonate Provisional Health Advisories Peroxisome Proliferator Peroxisome Proliferator-Activated Receptor Alpha Reactive Oxygen Species Superoxide Dismutase Wyeth 14,643 p13 1. INTRODUCTION Perfluorooctanoic acid (PFOA) is a synthetic organic chemical that consists of an 8 carbon alkyl chain witha terminal carboxyl group in which the carbon-hydrogen bonds. have been replaced with carbon-fluorine bondsexceptat the terminal carboxyl end. This perfluoralkyl carboxylate is a contemporary synthetic. chemical that does not occur naturally in the environment [1] and has only seen widespread use within the last SO years. Ammonium Perfluorooctanoate (APFO) is the ammonium salt derivative of PFOA and is used in the manufacturing process and readily disassociates into PFOA in the human body and environment. The data on APFO/PFOA can be used interchangeably since, in the presenceof water, APFO readily dissociates into the PFOA anion (2). PFOA is used in the manufacture of fluoropolymers and fluoroclastomers and is present as a component of some of the top-antirefective coating materials in use today. Fluoropolymers have properties that are useful in the manufacturing and textile industry such as fire resistance and their ability to repel oil, tain, grease and water which makes them ideal for creating a non-stick surface for cookware and protective coatings on clothing and carpeting and are also valuable to the aerospace industry as well. Fluoroelastomers are a family of synthetic rubbers that can be repeatedly stretched and stil eturn to their original shape, such as Viton. `Sinclair et al. found measurable PFOA released from several brands of nonstick cookware when heated which suggests that residual PFOA from the manufacturing process may remain on the surface and can be off-gassed when heated at normal cooking temperatures. This study also found that 1 p14 PFOA was present inthe vapors released from prepackaged microwave popcorn bags that are impregnated with PFOA to improve oil and fie resistance during microwaving. [3] Since there has been recent progress in understanding the developmental, tumorigenic and other adverse effects seen in laboratory animals exposed to PFOA, the purpose of this review is to provide an overvievw of the tumorigenic modes of action of PFOA that result in the tumor triad seen inrodentsand to determine the relevanceofthese studies to human risk. A. Environmental Persistence "The hydrogen-fluorine bonds found in PFOA are extremely stable and as a result PFOA is resistant to hydrolysis, photolysis, biodegradation, and metabolism leading to a high degree of environmental persistence and bioaccumulation. In January 2006, the Environmental Protection Agency (EPA) initiated the 2010/15 PFOA Stewardship Program in which the cight major companies in the industry committed voluntarily to reduce facility emissions and product content of PFOA and related chemicals on a global basis by 95 percent no later than 2010, and to work toward climinating emissions and product content ofthese chemicals by 2015. B. Environmental Presence The environmental persistence of PFOA has culminated in the ubiquitous presence of PFOA in the environment with bioaccumulation in many species worldwide, including humans. We will discuss the results of recent studics that have shown the presenceofPFOA in human, wildlife andthe environment. 2 p15 1) PFOA in Humans Recent studies of human populations in the United Sates have demonstrated the presence of PFOA in similar concentrations in the serum of children, adults and the elderly in the general population as well as in higher concentrations in individuals that were occupationally exposed to PFOA as summarized in Table 1.1. A study by Olsen ct al. of 645 serum samples collected in 2001 from adults in the cities of Los Angeles, Boston, Minneapolis-St Paul, Charlotte, Portland, and Hagerstown found PFOA levels in serum that ranged from 1.4 ng/mi to 56.1 ng/ml witah geometric mean for all samples of 4.6 ng/ml [4]. In this study the geometric mean PFOA levels were 4.9 ng/ml for males and 4.2 ng/ml for females. Serum collected in 2001 from 238 elderly adults (age 65-96 Years) in Seatle also demonstrated PFOA concentrations in the range of 1.4 ng/ml to 16.7 ng/ml with a geometric meanofall samplesof4.2 ng/ml. Olsen eta. [5] also measured PFOA in serum collected from 598 children ages 2-12 at concentrations ranging from 1.9 ng/ml- 56.1 ng/ml in samples collected in 1994 and 1995. The geometric mean for all participants was 4.9 ng/ml with a geometric mean of 5.2 n/m for meles and 4.7 ng/ml for females. In a 2000 study by Olsen ct al. of PFOA levels in the serum of occupationally exposed workers, Olsen determined that PFOA concentrations were over 300 times higher in the serum of workers at a 3MDecaturAlabama plant than thatofthe general population (6). Olsen reporteda geometric mean of 1130 ng/ml from a sample of 263 Decatur employees. Serum samples collected from 2,094 members of the general US population in 2003-2004 for the National Health and Nutrition Examination Survey (NHANES) also indicated the presence of PFOA at a mean concentration of 3.9 ng/ml. "This is a decrease from the mean PFOA concentrationof 5.0 ng/ml found in the serum of 3 p16 1562 participants in the 1999-2000 NHANES. It is also important to note that PFOA was quantifiable in 99.7%ofthe U.S. population tested in this study [7]. Somes| Number| Losses | Denogrphic | Mean | (5 |Referee Tab 11 TE ae aT TET `Summary of Geometric Mean and Range of PFOA Serum Concentrations in United States Trad FE Lamon Populations ERCHES Adult P`oApduullattion 2001 109 on | 2001 2001 107 Boston, Ma | PoGpeunelraatlion Mpls-St.Paul, Adult epuiton rss[oom Adit CharlottNeC,| General Population Adult pPepvinion Portland, OR | GeneralEk i- ] 5 19 | " os 63 36 19- [1py | 290 - 21 16.37 | 41 | 2001 238 w o m Seattle, WA PoPoppeulation oo Elderly Adult CPephe ie | General 42 fw| 14 :16. 7 55 om Ato [posse|PPoeppuliantion -ES- |o 2000 263 Decatur, AL Exponte | Occupational | 1,130 -12,700 2109050. [utPsoopulaion [|`SGAdEuml1t EL [0[5w7o| r | 2003- us Adult *3.6- SianPsoopnufladteinoens e|lpopuriaantgi.on [|i ` pA? The global presence of PFOAS in the human population has been evidenced by detection of PFOA in the blood of human adults in Columbia, Poland, Belgium, Indis, Korea, Japan, Sweden, China, Australia, Germany and Spain [9-14]. Results found in a recent study by Jin et al. on the serum levels taken in 1987, 1990, 1999 and 2002 from students, saff and faculty members at China Medical University in Shenyang China arc indicative of the exposure to PFOA in the general population that is not occupationally exposed 10 PFOA [10]. Table 2.1 summarizes the significant increase of PFOA levels seen in the Shenyang China general population from 1987-2002 T`Saubmlmea1r2y of Serum PFOA Concentrations (ng/ml) in Shenyang China Population [isso Tes TTooe lootsa | [2002 [ie 30 Jozuses | `The marked increaseof PFOA levels present in the bloodofoccupational exposed workers in the United States was reproduced in a study of exposed workers in Antwerp Belgium. A study performed in 2000 by Olsen et al. [4] found the geometric mean level of PFOA in 255 occupationally exposed Antwerp workers to be 440 ng/ml with a range of 40- 6,240 ng/ml as compared to the rangeof 4.5-27 ng/ml found in a sampleof20 `membersofthe general Belgium population (11). 5 p18 2) PFOA in Wildiife The environmental persistence of PFOA and its global presence has triggered studies for detection of the compound in several species of wildlife across the globe. PFOA has been found inthe seraofcaptive wildlife species Bengal tigers (Panthera tigris tigris) and African lions (Panthera leo Linnaeus) from Harbin Wildiife Park, Heilongjiang Province, in China [15]. PFOA was found in 175 samples of liver and blood of blucfin wna (Thunnus thynnus), swordfish (Xiphias gladius), common cormorants (Phalacrocorax carbo), bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalbs), common dolphins (Delphinus delphi), fin whales (Balenoptera physalus), and long- finned pilot whales (Globicephala melas) from the Italian coast of the Mediterrencan Sea and in livers of ringed seals (Phoca hispida), gray seals (Halichoerus grypus), white-tailed sea eagles (Haliacetus albiclla), in Atlantic salmon (Salmo salar) from coastal areasofthe Baltic Sea [16]. PFOA has been found in the plasma of 73 loggerhead sea turtles (Caretta caretta) and 6 Kemp's ridley sea turtles (Lepidochelys kempii) captured from inshore waters of Core Sound, North Carolina (NC), and offshore watersofSouth Carolina, Georgia, and Florida. [17]. PFOA has even been detected in the serum of the giant panda and the red panda from 200s and animal parks from six provinces in China and has also been detected in all specimens of European Beaver (Castor fiber) liver, in whole blood of Cod (Gadus morhua), Velvet Scoter (Melanitta fusca), Eider Duck (Sommateria mollisima), Long-tailed Duck (Clangula hyemalis), Razorbill (Alea tords), Red-throated Diver (Gavia sellata) sampled in Poland (18, 19). PFOA has ven been shown (0 be present in increasing levels in East Greenland as demonstrated by a study of a subsample of 128 subadult (3-5 years) 6 p19 polar bears (Ursus maritimus) from 19 sampling years within the period 1984-2006 in which median concentrations showed a significant annual increaseof2.3%. [20] 3) PFOA in the Environment PFOA contamination has been found in fresh water bodies of the European region. The PFOA levels concentrations were measured in samples of freshwater from several countries including Germany, Italy, Norway, and Sweden and werefoundto be in the range of <0.65-S7 ng/L with approximately 75% of the data falling between the range of non-detectable and 8 ng/L. [21-25]. Levels of PFOA contamination have been found in the surface fresh waterofthe `German Rub arca and of the river Moehne and selected contaminated tributaries where PFOA concentrations up 10 3640 and 33,900 ng, respectively were found. Saito [26], `Tanaka [22] and So [27] reported that several surface fresh water bodies in Asia, from samples collected in China, Japan and other Asian areas, hed PFOA concentrations mostly in the range of 0.1041.60 ng/L, with peaks up to 456 ng/L. In studics of North American fresh water A numberofstudies of surface water of the Great Lakes region reported PFOA concentrationsof<2-59 ng/L (28-30), Atmospheric Levels of PFOA in Europe were measured in 2005 by de Voogt [24]. The PFOA levels varied from 0.226-0.828 g/m' in March 2005 and from 0.006 0.222 ng/m in November. Atmospheric PFOA levels, measured in the towns of Oyamazaki. and Morioka located in Kyoto Japan, and were found to be inthe range of 0.00159-0.919 n/m" with a Geometric meanof 0.2627 ng/m' found in Oyamazaki and a geometric meanof 0.0020 ng/m' found in Morioka [31, 32). High levels of PFOA were 7 p.20 found in samples taken over a 10-week period by the fence of a fluoropolymer manufacturing facility in the United States where Barton [33] reported PFOA concentrations up to 900 ng/m3. PFOA is also found in the home. House dust from homes (n = 102) and dey care centers (n = 10) in Ohio and North Carolina in 2000-2001 were sampled and PFOA was measured at median concentrations of 142 ng/g of dust, with a maximum PFOA concentration of 1960 ng/g [34]. PFOA was found in dust samples that were taken from 67 houses in Canada at levels in the range of 115-1234 ng/g of dust with a mean concentration of 106 ng/g [35]. Moriwaki [36] reported detection of PFOA in the indoor dustof Japanese housesat levels of 69-3700 ng/g with a mean levelof 380 ng/g. C. Chemical and Physical Properties "The following are the chemical and physical propertiesofPFOA: Molecular Formula: C8-H-F15-02 Chemical Structure: RFR FR FQ F. Poof FF FFF Molecular Weight: 414.09 (37) Boiling Point: 189 deCg (38) Melting Point: 52-54 degC [38] 8 p21 Density/Specific Gravity: 1.792 g/ml @ 20 C [38] Dissociation Constants: pKa = 2.80 [39] VaporPressure: 0.15 mm Hg @25 C [40] 9 p22 11 TOXICITY IN ANIMALS A. Acute Toxicity Olson and Anderson [41] calculated the 30 day LDS0 ofa single IP injected dose of PFOA to be 189 mg/kg (175-208 mg/kg). All rats treated with doses of PFOA of 175 mgkg or greater died within 5 days of exposure. The LDSO for oral exposure was determined tobe 680 mg/kg for male CD rats and 430 mg/kg for female CD rats by Dean and Jessup [42]. Glaza [43] determinedthatthe LDSO for female Spraque-Dawley rats to be in the range of 250-500 mg/kg and the LD 0 for male Sprague-Dawley rats to be greater than 500 mgkg which supports the results from Dean and Jessup. The LD 50 from the acute toxicity studies performed in rodents are summarized in Table 2.1 below. TSaubmlmea2r.y1 ofPFOA LDSO values, TE eT Male Fi"scher Spray Forme Smarbnitye LD 5030dey= 189 mg/kg LD 50= LD s0= LD 50= LD 50= | asosoomats pe 1] ] i " 10 B. Subchronic Toxicity Subchronic toxicity studies were performed by Goldenthal [44] in rhesus `monkeys (2 of each sex per group) at doses of 0, 3, 10, 30 and 100 mg/kg per day by gavage in 0.5% Methocel 7 fora periodof 90days. Table 2.2 contasauimmnarsyofthe Table 2.2 Summary of the PFOA levels found in surviving monkeys at sacrifice by. prosten) gm) G9 fer [re fi Jie[Te| fo Tw (Is Ts Tr 7] Tes [3 Tso "In Fre fT o m Tm [30 [145 [Dead [30 [Dead |Dead [No [61 [Dead EERE ER 100 |Dead [Dead Dead [oor 5 17 Taso no [xo Tw Iwo [Dead [4000 |Desd |Dead [Dead [Dead 1s | [350 | [Np] 750 | [Dead | [Dead | |Dead | this group also died during weeks 12 and 13. All the animals that died during the study `animals that survived until sacrifice, one male in the 30 mg/kg per day group exhibited similar marked diffuse lipid depletion in t |he adrenal glands, slight to moderate bone p2 marrow hypoplasia and atrophy of the lymphoid follicle in the lymph nodes that was present in the animals that died during th study. The absolute and relative weightofthe Hearts and the absolute brain weight were significantly decreased in females from the 10 mek per day group. Tt was also noted that the mean weight of th pititary in males fromthe 3 mg/kgper day group was significantly increased `Two subchronic toxicity stuwerdepeirfeormsed in male cynomolgus monkeys by "Thomford in 2001 [45, 46]. The fist was a 6 month study in which male cynomolgus monkeys were administered 0,2 or 20 mg/kg per day orally via a capsule. All animals survived until sacrifice and there were no adverse effects noted in either the gross or clinical studs. This study was then followed by a 26 week study in which male cynomolgus monkeys were given 0, 3, 10 or 30 mg/kg doses per day orally via a capsule. Dosing was discontinued at day 11 because of statistically significant weight loss, decreased food consumption and decreased feces production in the 30 mg/kg per day group. Treatment ofthis group was then resumed on dey 22 at a 20 mghkg per day dose. Au sacrifice significant increases were noted in the mean absolut liver weights and the Tivetor body weight percentages in al groups receiving PFOA. Thyroid hormones were also found to be decreased beginning on day 35 in the 10 and 30120 mefkg per day groups. This study demonstrated a lowest-observed-sdverse-efiect-level of 3 mg/kg per day. A 28 day study by was conducted byChristopherand Marias 47] using CHR-CD. mice (5sex per group) administered daily dietary dose of APFO (the ammonium salt of PFOA used in fluropolymer manufacturing) ranging from 0 (0 30.000 ppm. Al animals in the 1000 ppm and higher group died within the irst 9 days of treatment, while 2 p.25 death of all the animals with the exception of one male in the 300 ppm occurred within 26 daysoftreatment. There was a dose-related reduction in the mean body weightofell the treated animals. Liver weights were increased in mice receiving a doseof30 ppm or greater. Morphological changes in the livers of all treated animals were observed including changes, enlargement and/or discoloration of 1 or more lobes of the liver, & diffuse cytoplasmic enlargement of hepatocytes throughout the liver, with a random distribution ofcytoplasmic lipid vacuoles of variable sizes. "The resultsof a second 28-day study using the same APFO doscs as Christopher `and Marias were described in a report by Metrickand Marias [48]. All animals receiving a dose that was greater than 10,000 ppm died before the end of the first weekofthe study with morphological changes noted in the livers of all the animals who received APFO. "The morphological changes in th liver noted in the study consisted of paniobular diffuse hypertrophyofthe hepatocytes with accompanying focal to multifocal cytoplasmic lipid vacuoles. A 28-day study of the toxicological effects of oral exposure to PFOA and Perfluorooctane Sulfonate (PFOS) on male Spraque-Dawiey rats by gavage has also been performed (49). Cui used groups of 10 animals per group with the control group receiving no perfluorochemicals (PFCs) and the remaining group receiving doses of 5 mg/kg per day PFOA, 20 mg/kg per day of PFOA, mg/kg per day PFOS and the final group received a dose of 20 mg/kg per day of PFOS. Histological changes in the hepatocytes were observed in all exposed groups. These changes included fatty degeneration, angiectasis in the central vein, congestion in the hepatic sinusoid, acidophilic lesions, focal hemorrhage with necrosis, cytoplasmic vaculation and hepatic 13 p.26 enlargement with inflammatory cellular infiltration. A. biodistribution assay was cpoenrcfeonrtmreadtiotnos mweearseurfeo.untdhetoPbFeOiAn tchoencoerndterraotfioKnisdnieny>tihveert>alrguentg>ohregaarntsteasntdiclteh>esptilseseune and brain. C. Mutagenicity There is conflicting evidence on the genotoxic effects of PFOA. Several studies conducted for the United States Environmental Protection Agencies by an independent laboratory concluded that PFOA and APFO did not induce mutations with or without metabolic activation in AMES tests, in human lymphocytes or in Chinese Hamster Ovary (CHO) cells [50, 51. The same laboratory also tested the in-vivo mutagenic properties of APFO using micronucleus studies on the bone marrow of mice [52, 53] with negative results. Other studies have found that APFO was able to induce both chromosomal aberrations and polyploidy with the presence of metabolic. activation in human Iymphocytes (54, 55). A recent study by Yao etal. [56] using human the hepatoma cell line HepG2 found a significant increase in the tail moment in the single cell gel electrophoresis assay in HepG2 cells exposed 0 PFOA which indicates that PFOA was able to induce DNA strand breaks in HepG2 cells. Yao also found PFOA induced a dose-dependent increase in the frequency that & micronucleus was found in binucleated HepG2 cells, which indicates that chromosome breaks occurred in HepG2 cells after PFOA treatment. Shabalina et al. [57] found DNA breaks in HepG2 cells exposed to PFOA using the TUNEL procedure and propidium iodide staining of cellular DNA. 1 p.27 Although there are conflicting evidence, PFOA has been found to produce a genotoxic effect on human cells in some studies. D. Carcinogenicity Carcinogenicity studies of PFOA in rodents demonstrated that PFOA induced tumor iad which consists of liver adenomas, Leydig cell adenomas and pancreatic acinar cll tumors in male Sprague-Dawley rats. Sibinski (58) performed a 2 year studyofSpraque-Dawley rats exposed to APFO in their diet. Groupsof 50 animes from each sex consumed feed containing 0, 30 or 300 ppm APFO; this corresponded to a mean APFO daily dose of 0.0, 1.3 and 14.2 mg/kg in the males and 0.0, 1.6 and 16.1 mg/kg per day in females. The animals were observed daily with body weights and food consumption recorded weekly for the first six months `and then bi-weekly for the remainderof the study. An interim 1 year study was also performed using groups of 15 additional rats per sex fed 0 or 300 ppm APFO with the animals sacrificed at 1 year and the weights of the Kidney, liver, testes, adrenal gland and spleen recorded. Pathological examinations which included hematology, serum chemistry and urinalysis were performed on samples from 15 rodents from cach group at 3, 6, 12, 18 and 24 months. Post mortem observations were recorded on all animals that died during the study and those that were sacrificed at the 1 year interim and 2yearstudy end. At the 2 year sacrifice, the organs were weighed for 15 randomly selected animals per sex in all remaining rats in each group. On gross examination, lesions in the iver, testis and ovary were noted. 15 p.28 At the 1 year sacrifice liver hepatomegalocytosis was present in 80% of males consuming feed containing 300 ppm APFO with no hepatomegalocytosis seen in the control group. Portal mononuclear cell infiltration was also present in 87% of the 300 ppm APO dosed male animals versus 47% of the control male animals and hepatocellular necrosis was evident in40% of the 300 ppm APFO male animals with no hepatocellular necrosis observed in the control group. Testicular masses were also discovered in 40%ofthe 300 ppm dosed group at the | year sacrifice with no testicular `masses observed in the control group. At the 2 year sacrifice megalocytosis was present in 12% of the male rats consuming feed containing APFO at a concentration of 30 ppm and 80% of the males consuming feed with a APFO concentration of 300 ppm. Megalocytosis was also observed in and 2% of the female rats consuming feed containing APFO at @ concentration of 30 ppm and in 16%ofthe females that consumed feed with a APFO concentration of 300 ppm. There was no megalocytosis observed in either the male or female control groups. It was also noted that hepatic cystoid degeneration was observed in 149% and 56%of the 30 ppm and 300 ppm male groups with only 8% observed in the control group. At the 2 year sacrifice a significant increase in the incidence of testicular Leydig cell adenomas (LCT) was reported in the high dose group which was present in 14%ofthe test animals. 4% of the low dose group also developed LCT with no LCT observed in the control group. Historically, LCT have been observed in 0.82%ofcontrol animals in 2-year old Sprague-Dawley ras used in carcinogenicity studies [9]. The effects noted in this study are summarized in Table 2.3 16 ps Table23 `Summaryofthe Effects of APFO Exposure in Male Rats FE | meee | desea] Resins. [Emp | wove [wn Tw] | L000| OppmAPEO) |GUppm ARYO) Tver 1 Year Sacrifice . MaleRats Portal mononuelear : | G00 ppm APFO) oer | vow| wwe | aw] 3 Year Sacrifice Mal Rats degeneraationa |ow | we [a] Coll Adenomas * Only rats treated with 0 and 300 PPM APFO were sacrificed at | year. Bicgel et al. [60] performed a 2-year study in which APFO was introduced into the diet of male CD rats. The study was conducted using feed contining 300 ppm APFO. Increased relativeliverweights and hepatic beta-oxidation activity were observed in the PFOA trated ats atal time points, Liver adenomas were induced in 13% of the PFOA treated group versus 3% of the control group. Leydig cell adenoma was induced in 11% of the testes of the PFOA treated group versus 0% of the control group with Leydig cell hyperplasia present in 46% of the animals exposed to PFOA in thei diet Acinar cell adenoma was induced in 9% of the PFOA treated animals versus 0% of the control group. The frequencyoftumorinduction is summarized in Table 24. 1" 5.30 Tables Summaryofthe Tumor Induction Frequencyof APFO in Rats |a BuT me | conti | E Ce UR T Ra | iveubaenee [[[ poFoiweonmo nssa n| Tatoaomw]T|sveo[sww [wm [: orws [[wwoew || gee Boas perplasia_ lossiese? [ |v0o [|wew voew [|[wo3] 5www1w6 [|ww4ie6x% || ab ie | woa [wn [we[we | poowrceisienm| m0 | Noe| ome | we] o[ rmea [oowwm [|vvoom wws[|wwa ee[[ow] we] Blood was sampled at 1, 3,6, 9, 12, 15, 18, and 21 months. Testosterone, follicle- stimulating hormone (FSH), luteinizing hormone (LH), prolactin and estradiol levels were quantified in the serum from cachofthese samples with no significant differences found in serum testosterone, FSH, prolactin, or LH concentrations in the PFOA treated rats when compared to their controls. However, significant increases in serum estradiol he contol rope concentrations were present in the PFOA treated rats at all months sampled compared to 1s p31 Il. MECHANISMS OF CARCINOGENICITY A. Liver The available published scientific literature provides data. suggesting thet induction of hepatocellular adenomas by PFOA may result from: (1) generation of reactive oxygen species, (2) increased mitochondrial biogenesis, (3) the activation of Peroxisome Proliferator-ActivatedReceptor Alpha (PPAR), (4) influence on molecular pathways such as those mediated by the constitutive androstane receptor (CAR), (5) the inhibition of gap junctional intracellular communication (GJIC), (6) increased estrogen levels and (7) modulation of the immune system. Data in support of these proposed `mechaarndiesscrmibesd in the sections that follow. 1) Reactive Oxygen Species Chemically induced carcinogenesis is though to consist ofa multistage process definable by at least three steps or stages: initiation, promotion, and progression. The oxidative stress that results from the presence of ROS impacts all three stagesofthe cancer process. During the initiation stage oxidative DNA damage may produce gene `mutations and structural akerations of the DNA, resulting in a heritable mutation. During the promotion stage ROS and oxidative stress can contribute to abnormal gene expression and inhibition of GJIC which results in an increase in cell proliferation or a decrease in apoptosis in the initiated cell population. Oxidative stress may also participate in the progression stage of the cancer process by imparting further DNA alterations to the initiated call population. These changes may result in changes in enzyme activity and 19 `make the lesions resistant to normal growth control (61). This is exemplified in figure 3.1 taken from Klaunig et al. [61]. PCrtd be rb iets sss nines' wm -- pRad | [ie | = z Ir dy by Liv ot a. (62) lord est in Orson ics) toes erscptPEOS oxFOR (tn of, 1.5 1 an 0g 20) A, by increases in activities of superoxide dismutase (SOD), catalase (CAT) and glutathione spi reductase ils(GR), heme of demonstrating cothat pePFOA in ry produced cue oxidative gnstress bps and induced ri ot a [6] messed msi yn sis (0) eon 15, 5,5 5.33 and 24 h after cultures of human HepG2 hepatoma cell lines were exposed to PFOA concentrationsof200mM and 400 mM. At 3 h, 200 mM of PFOA had elicited detectable levels ofH;03 in 91%of the total cell population compared to 12% of the vehicle-treated cells and 400 mM PFOA induced Hy0; generation in 98% of the total cell population at the same time point. Superoxide anions, which are another example of ROS species, reached detectable levels in 43% and 71%ofthe total cell population afer treatment with 200 mM and 400 mM PFOA respectively (versus 10% in DMSO-treated cells). This accumulation of ROS is thought to be the result of disruption of the mitochondrial `membrane. A study by Yao ct al. [56] investigated the genotoxic potential of PFOA in human hepatoma HepG2 cells in culture using single cell gel electrophoresis (SCGE) assay and micronucleus assay. Dichlorofluorescein diacetate was used as a fluorochrome to measure the intracellular generation of ROS with the level of oxidative DNA damage being evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8 HAG) in PFOA-treated HepG2 cells. This study found significantly increased levels of ROS and 8-OHAG in the PFOA exposed cells which indicate that human HepG2 cells exposed to PFOA do produce ROS which may play a role in all three stages of the carcinogenesis process. 2) Mitochondrial Biogenesis PFOA does produce hepatic peroxisome proliferation (PP) in rats, which is discussed later; however, PFOA has been shown to stimulate mitochondrial biogenesis, `which is not a characteristic response of PPs [64]. Berthiaume exposed male Spraque- 21 p.34 Dawley rats to 100 mgkg of the PP chemicals PFOA, PFOS and Necthyl perfluorooctanesulfonamido ethanol dissolved in dimethyl sulfoxide (DMSO) via a single: ip. injection. The animals were sacrificed 3 days post injection with the liver tissue and mitochondria collected from animals in each treatment group used to examine the effects of each compound on cytochrome oxidase activity, cytochrome content, and mitochondrial DNA copy number to determine thir effect on mitochondrial biogenesis. PFOA and PFOS were capable of inducing PP in rats following injection; however, PFOA was the only compound that caused significant decreases in mitochondrial cytochrome oxidase activity with a significant increase in mitochondrial DNA copy number which reveals that a unique mitochondrial biogenesis is occurring in PFOA `exposed rats that is not generally produced by other PPs. Starkov et al. [65] also demonstrated that PFOA and PFOS caused a slight increase in the intrinsic. proton leak of the mitochondrial inner membrane, which resembled a surfactant-ike change in membrane fluidity. Since mitochondria play a significant role in cell signaling, ROS generation and apoplosis the resulting mitochondrial biogenesis seen in rodents exposed to PFOA may play a role in carcinogenesis. 3) PPARaAgonism PPARs are steroid hormone receptors that act as ligand-activated transcription factors. Three PPAR isotypes have been identified, PPAR a, (5 and v. These isotypes play & role in lipid metabolism, energy homeostasis and inflammation [66]. PPAR agonism results in an increase in the number and volume fraction of peroxisomes in the 2 5.35 cytoplasmofcells. This proliferation can be visualized utilizing electron microscopy or can be quantitated by measuring the levels of selected peroxisome enzymes such as catalase, cytochrome P-450s (e.g, CYP4Aland 4A3), and acyl coenzyme A (CoA) oxidase. The response to exposure to PP chemicals, such as PFOA, varies drastically between species with the rat and mouse being the most sensitive. PP has been shown to be present in the hamster, to a lesser degree than the rat and mouse, with relatively no PP present in the guinea pig, monkey, and human when exposed to PPAR agonists at dose levels that produce marked response in the rat and mouse [67-70]. The liver tumors induced by PFOA exposure in rodents appears to occur as a result of PFOA binding to the PPAR, which results in PP [60, 71, 72]. PFOA is believed to produce these tumors in the liver by binding with and, as a result, activating PPARa. PPAR modulates the transcription of genes involved in PP, regulation of the lifeldeath cycle of the cell and lipid metabolism. PPAR: activation produces a proliferation of the peroxisomes, an increase in cel proliferation and a decrease in apoptosis. The decrease in cell death combined with increased stimulation in cell proliferation results in increased cell reproduction. This reproduction promoting environment coupled with the presence of ROS, and their resultant oxidative stress, promotes the reproduction of DNA-damaged cells. Asa result prencoplasti foci arise and in the courseoftime develop into tumors by wayofcontinuedclonal expansion [73]. 4) CAR Activation PFOA has also been shown to activate CAR. CAR activation is essential in liver tumor promotion by other nongenotoxic liver carcinogens such as Phenobarbital (PB) 2 5.36 (74). A DNA microsrrey analysis of hepatic gene expression inCar-/- mice showed that CAR induced or repressed 70 genes [75]. In addition to up-regulation of drugmetabolizing enzymes, CAR down-regulated genes that encode gluconeogenic enzymes, those involved in fay acid oxidation, and proteins such as angiogenin and fibronectin. Nongenotoxic carcinogens such as PB are known to stimulate cell proliferation and suppress apoptosis, leading to tumor development. While PFOA has been shown to activate PPARa [71], PFOA also produces liver enlargement in the PPARa-null knockout mice [76, 77). This PPARa-independent effect suggests other mechanism of carcinogenicity may be present other than PPAR activation. A studyofgene expression changes in PPARa-null mice exposed to PFOA revealed an up-regulation of cerain Cyp2b and Cyp2e genes, such as Cyp2b10, which are markers of CAR activation [7580). Another study revealed that two days afalsiengrle i. administration (50 mg/kg) of (PFOA) and. perfluorodecanoic acid (PFDA), mRNA expression of Cyp2bl0 was increased 20-fold [81]. It was also shown that there is a strong correlation between the transcript profile of PPAR independent PFOA genes in PPARa-null mice exposed to PFOA and thoseofactivatorsofCAR including Phenobarbital (82) which would suggest that a subset of genes are controlled by CAR in the PPARa-null mouse exposed to PFOA. 5) GIIC Inhibition `When norma cells are no longer able to control their growth by way of contact inhibition, they can become transformed into tumorigenic cells 83] which may be due to defective intercellular communication [84]. These transformed cells can be benign or 2 p.37 they can develop genetic mutations that lead to a malignant state as demonstrated in `multistage carcinogenesis. It has been implied that changes occurring in the cell-to-cell `communication by way of gapjunction does play a role in the tumorigenic process [85], A recent study by Upham ct al. [86] verified that PFOA does inhibit GJIC and induce hepatomegaly inthe livers of rats while the measured levelofliver enzymes indicated no cytotoxic response. The inhibitionofGJIC was shown to be dependent on the activation of both ERK and phosphatidylcholine-specific. phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism. This study also indicated that PFOA is able to activate the extracellular receptor kinase (ERK). In a previous study, Upham et al. [87] demonstrated that PFOA reversibly inhibited GJIC and this inhibition of GJIC was dependent on the lengthofthe fluorinated tail. Since GIIC plays an essential role in preserving tissue homeostasis, disruption of gap junction function `may play a role in the carcinogenicity of PFOA, 6) Estrogenic Signaling It is known that PFOA can produce PP in rodents, however humans and certain other species, including rainbow trout, guinea pigs, and nonhuman primates, show little 10 m0 evidence of PP [70, 88, 89]. A study by Tilton etal. [90] of rainbow trout exposed 10 PFOA (1,800 ppm or 50 mg/kg/day) and DHEA demonstrated enhanced liver tumor incidence in trout exposed to PFOA at concentrations of 50 mgke per day. This carcinogenic effect was independent of PP which was determined by a lack of peroxisomal oxidation and catalase activity. Gene profiling also demonstrated that PFOA induced an estrogenic gene signature that strongly correlated with that produced 2 p.38 by 17-estradiol. The results from this study suggestthatthe tumor-promoting activities of PFOA in trout are independent of PP and instead may be related to a mechanism involving estrogenic signaling. A study by Liu et al. [91] demonstrated that PFOA possesses estrogenic activities and additional results of an estrogen receptor inhibition assay indicated that the estrogenic effect of PFCs may be mediated by the estrogen receptor pathway in primary cultured tilapia hepatocytes. A gene expression profile study was conducting using liver tissue obtained from mele and female rare minnows (Gobiocypris rarus) exposed to PFOA using a custom cDNA microarray containing 1773 unique genes by Wei et al. [92). The minnows were treated with continuous flowthrough exposure to PFOA at concentrations of 3, 10, and 30 mg/L. for 28 days. Hepatocyte swelling and vacuolar degeneration with prominent eosinophilic. hyaline droplets were observed in the cytoplasm of male and female hepatocytes exposed to 10 mg/L. The livers from the cohort exposed to 10 mg/L, PFOA were selected for further hepatic gene expression analysis. The result revealed that a number ofgenes were cither upregulated or downregulated in the livers of the minnows exposed to PFOA with a distinct induction of estrogen-responsive genes identified using a custom cDNA microarray. The fact that ther studies 93, 94] have shown that estrogen promotes liver carcinogenesis in rats makes the estrogenic signaling properties of PFOA a viable pathway for tumor promotion in the iver. 7) Immunomodulation A healthy and fully functional immune response allows the body to identify and remove abnormal cells, including initiated cells, while a weakened or suppressed immune 2 p.39 system has been associated with increased tumor development [95-97]. A study by Yang et al. [98] demonstrated that exposure of Male CS7BI/6 mice to PFOA in the diet resulted in severe thymic and splenic atrophy with the weightofthe thymus and spleen capidly returning to normal within 10 and days, respectively, following withdrawal of PFOA from the diet. It was also noted that these changes in thymus and spleen weight paralleled the changes in total thymocyte and splenocyte counts. Yang also evalusted the effects of PFOA on the adaptive immunity in mice by measuring antigen-specific antibody responses and lymphocyte proliferation in mice immunized with horse red blood cells (77). Mice were fed a diet of 30 mg PFOA/KE for 15 days after which the immune response was quantified by employing both the plaque-forming cell assay and determination of the antibody titer by ELISA. The results of the study revealed a suppression of the IgM and IgG titers relative to the titers present in the unexposed control animals and reduced plaque formations in the exposed animals compared to the `animals in the control group. The results from another study by Yang [99] revealed that `while PFOA treatment of wild type mice led to a dramatic atrophy of the thymus and spleen reflecting losses of thymocytes and splenocytes, this effect is not as dramatic in PPARa-null mice. Although the atrophy of the thymus and loss of thymocytes was not as marked as in the wild type mice, significant thymus atrophy and loss of thymocytes were observed in PPARa-null mice exposed to PFOA. This weakened response clearly points toward the involvement of both PPARa-dependent and -independent mechanisms in the immunomodulation process. Note that although immunomodulation is only discussed in ths section the immune response is a physiological responseofthe body and modulation of the immune system affects the body as a whole. As a result the 27 p.40 immunomodulation by PFOA may play a plausible role in tumorigenesis in the liver, testis and pancreas. B. Testis Leydig cell adenomes are another type of tumor that is produced by exposure to PFOA. `The available published scientific literature provides data suggesting that induction of Leydig Cell adenomas by PFOA may result from: (1) inhibition of testosterone biosynthesis and (2) estradiol modulation. Data in support of these proposed mechanisms are described inthe sections that follow. 1) InhibitionofTestosterone Biosynthesis Inhibitionoftestosterone biosynthesis may be involved in the inductionofLeydig cell tumors by PFOA which has been shown to decrease the production oftestosterone in Leydig cells that are exposed to the compound [100]. Results from a study by Gazouli et al. [101] indicate that the inhibition of testosterone production may also be mediated by PPARa. Decreased testosterone levels can lead to increased luteinizing hormone levels through a negative feedback mechanism called the hypothalamic. pituitary testicular (HPT) axis that regulates metabolism. The disruption of the hypothalamus pituitary thyroid axis in the thyroid gland has been shown to produce follicular cell carcinoma in the thyroid [102] and may be applicable to LCT formation in the tests as a disruption of theHPTaxis. Several studies have produced sustained hypersecretionof LH in the testis, which are compromised of endocrine controled tissues, with the disruption of the HPT axis [103-109]. This mechanism was demonstrated in a 1-year study where Viguier- 28 p41 Martinez et al. [108, 109] fed Sprague-Dawley rats feed containing fiutamide, which is an androgen receptor antagonist that blocks the binding of testosterone to the testosterone. receptor and results in hypersecretion of LH duc to the disruptionofthe HPT axis, and produced Leydig cell hyperplasia in the animals fed flutamide which progressed to Leydig cell adenomabythe endofthe study. 2) Estradiol Modulation Since PP chemicals have been shown to increase estradiol levels and estradiol `modulates growth factor expression in the testes then PFOA may produce Leydig cell tumors (LCT) in the testes as a result of the increased cell hyperplasia produced by estradiol [100, 110], Administcation of estradiol to mice has produced Leydig cel tumors in several studies [111-113] which further supports the plausibility that PFOA can produce these tumors due to the compounds ability to increase estradiol levels. It is also plausible that elevated estradiol levels mayact &s a mitogen andlor enhance growth factor scoretion thereby causing Leydig cell hyperplasia and tumor formation. The transforming growth facathoars been detected in Leydig cels[114] which stimulates cell proliferation and is an example of how enhanced growth factors could be involved in tumor formation. Although peroxisomes are present in both the liver and testes the limited data on Leydig cell induction by PFOA does not support PP as a plausible MOAS for tumor production in the testes. Two separate studies exposing malerats to known PP chemicals found abundant PP in the liver using biochemical assays and visual examination with electron microscopy with no PP found in the Leydig cells [115, 116]. Currently no other 2 p42 data exists to support any other altemate MOAS for LCT formation other than the hormonal mechanisms described. C. Pancreas Pancreatic acinar cell tumors (PACT) are a third twmor type produced by exposure to PFOA. The available published scientific literature provides data suggesting that induction of pancreatic tumors by PFOA may result from cholestasis. 1) Hepatic Cholestasis Steroids (such as estradiol and testosterone discussed previously), cholesteystokinin (CCK) and other growth factors, growth factor receptor overexpression (CCK receptor) and dietary fat intake have ll been shown to produce PACTS in rats when their lvls are altered [117-119], PACT induction by PFOA and other PP appears to be seen in rats,butnot mice, which indicates that PACT wmor formation may be species dependant [67, 68]. A chronic study by Osbourn et al. [120] demonstrated increases in pancreatic weights at 3 months (6% above control) and 6 months (17% above control) with an increase in CCK plasma levels in 2 year bioassayofras exposed o the PP chemical WY. The results of the study indicate that chronic exposure to WY causes liver alterations such as cholestasis, which may increase plasma concentrations of CCK. PP chemicals such as PFOA may thus induce PACT as a resultof a sustained mild increase in CCK levels produced by hepatic cholestasis. Results from a second study by Osbourn et al. supported the validity of the role of increased CCK levels in PACT 30 pas formation [121]. The diagram below (Figure 3.2) depicts the proposed mode of action (MOA) with key eventsfor pancreatic acinar cel tumors (PACTS) induced by PFOA. Figure 32 lustration detailing the proposed MOA for PFOA pancreatic acinar cell tumorigenesis | cme tedcomposition i LIVER i PFJOA | Decrease transcription of fl cholesterol 7a-hydroxylase ii (Decrease bleaacid flow andlor i Cia J-- i PANCREAS 1i (Acinar Cell') i `Tumor Formation i T 1fl IncreasedacinarTcell proiferation cell CCK receptors { Gut || LUMEMNUDCUOOSDAENAL 3| inRcreedausceesdCtCrKyprseilneacatsievfirtyom {the duodenal mucosa As described above, the experimental evidence suggests that PPARa agonists such as PFOA induce PACTS in rats by increasing CCK levels secondary to reduced bile acid synthesis andlor alterations in bile acid composition (120, 121]. PPAR agonists have been shown to decrease transcription of cholesterol Ta-hydroxylase, the ratelimiting step of bile acid synthesis, via the PPARW/RXRa heterodimer reducing hepatocyte nuclear factor-4 (HNF-4) binding to the DR-1 sequence, which regulates this `gene [122]. The inhibition of cholesterol 7a-hydroxylase results in reduced bile flow and 3 ptt hie th pon oti st SHAS 0 2 NE increases the CCK release from the duodenal mucosa. This increased release of CCK masA t niAgs3 Ares -- results in increased activation of the CCKa receptors which can increase acinar cell F`iSguumrmear3y3of Possible Mechanisms for PFOA Carcinogenicity in Rodents. = in = 3 ; i1 | 1 1 BT aE x p45 IV. HUMAN RELEVANCE As described in the MECHANISMS OF CARCINOGENICITY section, PFOA has been shown to produce cancer in laboratory animals. This section previews studies that have used human sources in an attempt to determineifthe possible mechanisms of carcinogenicity hypothesized in animal moadreaeppllicasble to human risk. A. Liver "The induction of hepatocellular adenomas by PFOA in rodents may involve any of several pathways including ROS, CAR, GIIC, increased estrogen levels, decreased immune system response and/or PPARa agonism. Many of these pathways are plausible in human carcinogenesis. 1) ROS Generation Results of a recent study established that PFOA produces genotoxic effects on human HepG2 els [56]. This DNA damage is thought to be the resultofoxidative stress induced by intracellular ROS, particularly hydrogen peroxide. In this study the fluorochrome 2,7~dichlorofluorescin was used to measure intracellular generation of ROS and 8-0HAG was used to determine the level of oxidative DNA damage. A separate study by Hu etal. [123] that exposed human Hep G2cells to PFOA and PFOS at concentrations of 50-200 pmol/l (50-200 pmol) resulted in the generation of ROS. Panaretakas et al. [63] also demonstrated thet human Hep G2 cells treated with 200 and 400 umol PFOA exhibited a dramatic increase in the cellular content of superoxide 3 p.d6 anions and hydrogen peroxide after 3 h. Hypolipidemic drugs, which are known PP, were also shown to induce an overproduction of ROS in human hepatocytes [124]. Many other studies have implicated ROS and oxidative stress in human liver tumors [125-129] which indicates that ROS generation by PFOA may play a rol in human carcinogenesis and warrants further study. 2) CAR Activation As previously discussed, PPARa-null mice exposed to PFOA developed liver enlargement and a measurable up-regulation of Cyp2b and Cyp2e genes, which are `markers of CAR activation. PB is known to stimulate cell proliferation and suppress apoptosis, leading to tumor development via the CAR activation pathway but has typically not been shown to produce liver tumors in humans [130]. Although litle evidence suggests that CAR activation by PFOA could result in tumor formation in the liver, CAR expressionshows a large amount of genetic polymorphism as demonstrated in a study by Change etal. [131] that revealed a 240-fold variability in hepatic CAR mRNA levels in humans. `This highly variable expression among individuals may explain why there are reported cases of liver tumors in humans that have been on long term PB therapy (132, 133] and could represent plausible tumorigenic pathway for a subset of the human population that highly express CAR and are exposed to hgh levelsofPFOA. 3) GIIC Inhibition Since carcinogenesis involves a disturbanceofhomeostasis and cancer cells show uncontrolled growth, it is conceivable that altered GJIC plays an important role in 3 p47 carcinogenesis. PFOA has been shown to inhibit GJIC and induced hepatomegaly in rat livers [86]. As previously discussed the inhibition of GJIC in rats appears to depend on the activation of the ERK pathway. In studies of human cancer cell lines GJIC was absent or decreased in all human cancer cell lines that were analyzed by Mesnil et al. [134] and wes found to be decreased in human hepatocellular carcinoma tissues and cell lines by other investigators as well (135-137), The inhibition of GJIC via ERK activation has been shown to bea key pathway of in human carcinogenesis [138]. The potential for cross-species effects of PFOA on GJIC was established in a study by Hu et al. where PFOS was shown to inhibit GJIC in rat liver tissue as well as dolphin kidney cells [139]. "The ability of perfluorinated chemicals to inhibit GJIC appears to be neither species- nor tissue-specific and can occur both invitro and in vivo. Currently studies evaluating GJIC inhibition by PFOA in human cell lines are lacking. Future experiments, particularly with human cell lines, on the inhibitory effect of perfluorinated chemicals on GJIC are needed as this may be a plausible MOA for carcinogenesis in humans. 4) Estrogenic Signaling Although it was discussed previously that PFOA has been shown to promate liver tumors in ish that lack PPAR through disruptions in estrogenic signaling, more studies are needed to assess the potential for PFOA to mediate carcinogenesis through the estrogenic pathway in other species such as humans. A previous study in primates [140] did not find any evidence of PFOA increasing estrogen levels which would infer that the estrogenic. pathway would not be likely involved in human hepatocercinogenesis, 35 p48 however studies involving human cel lines are needed to exclude estrogenic signaling as a plausible MOA for PFOA carcinogenesis in humans. 5) Immunomodulation As discussed previously PFOA and other PP chemicals do exert an cffect on the immune system of study animals. The mechanisms are not clear as to why these chemicals are able to modulate the immune system and more research is needed to determine the impact on the human immunc system as there docs appear to be considerable species variability in the degree of response. The fact that the immunomodulatory effects of PFOA can be present independent of PPAR activation would imply that it would be possible for PFOA to induce liver carcinogenesis in humans Via these effects as well as in other tissues and warrants further investigation. 6) PPARG Agonism PFOA has been shown to act& a strong tumor promoter in rodent livers (141] as `well a other PP chemicals [142]. The PP produced by PFOA, which is thoughttobe the result of PFOA binding to the PPAR, does not appear to be occur in human livers, as humans show litle [140] to no evidence of PP. This may be due to the lower PPARa `expression found in humans compared to rodents as reported by Palmer et al. [143] who found a >10-fold lower PPARa DNA binding activity in human liver lysates than the levels found in mouse liver lysates. There is also evidence that activationofthe human PPARreceptorelicits a different response than activationof the mouse PPARG receplor. "This has been demonstrated in studies using a PPARa humanized mouse in which 36 pds PPARa-null mice have been genetically manipulated to express human PPAR at levels comparable to the mouse PPARa found in wild-type mice. In these studies fibrates altered the expression of genes associated with lipid metabolism in both wild-type and PPARa-humanized mice, however these compounds did not induce the hepatocyte proliferation in the PPARa-humanized mouse that is observed in the wild-type mice nor id the compounds induce the liver tumor formation in the PPARa-humanized that is observed in wild-type mice. This information reinforces the theory that the effects of peroxisome proliferators are species specific and the inductionofliver tumors by PFOA duc to PPAR activation is highly unlikely [73]. B. Testis It has been shown that PFOA does exert an effect on human testicular function as evident in astudyby Jocnsen ct al. [144] that analyzed serum samples for levels of 10 different PFAAs and reproductive hormones and assessed semen quality in 105 Danish men from the general population (median ag, 19 years). They discovered the presence of measurable quantitiesof PFOS and PFOA in all subjects with a median of 24.5 and 4.9 ng/mL respectively. A significant decrease in semen production was found in men with high combined levels of PFOS and PFOA compared to those with low levels. Men `exposed to high combined levels of PFOS and PFOA were found to produce 6.2 million normal spermatozoa in their cjaculae in contrast to 15.5 million among men with low levels of these PP chemicals in their bloodstream. Not only is it apparent that PFOA exerts an effect on testicular function but it is also plausible that the estradiol modulation 3 p.50 MOA for LCT formation seen in the rat exposed to PFOA may also be relevant in humans. 1) Estradiol Modulation "The ability of PFOA to induce human LCTs via the MOA associated with increased serum estradiol and testicular growth factors discussed previously in rodents may be of relevance to humans. Several authors have reported that estrogen receptor (ER) expression is present in the normal LCs of human adult testis (145-147) which indicates that these cells are a target for estrogens. It has also been shown that the cytochrome P450 aromatase, an enzyme that catalyzes the androgen aromatization into estrogens, has been detected in Leydig cells of normal human testis [148], which further suggests that locally produced estrogens plays 2 role in steroid production and spermatogenesis (149). A recent human study by Carpino ct al. [150] investigated the expression of aromatase and estrogen receptors (ER, ERB, ERB2) in testes from two patients with LCT. A strong immunoreactivity for aromatase, ERBI, and ERB2, together with a dtectable ER immunostaining, was revealed in tumoral tissues. These findings were confirmed by Westem blot analysisoftumor extracts. The patern ofER expression inthe LCT calls was also demonstrated to be different from that of control Leydig cells `which exhibited only ERB and ERB? isoforms. The results of this study reveals thet it is plausible that high estrogen production could play a ole in the neoplastic transformation Of Leydigcellsand the exclusive presence ofER in tumoral cells could amplify estradiol 17 signaling contributing to the tumor cell growth and progression produced by PFOA. 3 p51 C. Pancreas The tumorigenic pathways of PACT induction by PFOA in rodents that was discussed previously in this review may not be relevant in humans as the expression of CCK receptors in humans are much lower than rodents with data indicating that human pancreatic acinar cells do not respond to CCK receptor activation due to an insufficient evel of receptor expression [151]. A studyof cynomolgus monkeys exposed to PFOA for 6-months did not produce any effects on CCK levelso evidence of cholestasis [140] and human epidemiological studies ofCCK levels in employees exposed to hgh levels of PFOA did not reveal any increases of CCK levels in workers (152, 153]. These studies also imply a lack of CCK receptor expression in humans. It is also important to note that the majority of human pancreatic cancers areofthe ductal type and are not derived from the acinar cells [154] as found in the PACT that i produced in rodentsexposedto PFOA. With the incidence of pancreatic cancer in humans increasing every year, it has now become the fourth leading cause of cancer deaths [155], the ability of PFOA 10 induce pancreatic tumors in humans may warrant further investigation as the exact mechanism of PFOA carcinogenesis is not known, however the PACT induced in the rat would probably not correspond to &significant cancer risk in humans. 39 p.52 V. EPIDEMIOLOGICAL STUDIES The current epidemiological studies of the carcinogenic effects of human exposure to PFOA are inconclusive. The current data is based on occupational studies most of which was conducted by the 3M company on male workers from theirplants. In 2 3M mortality study, prostate cancer mortality was the only statistically significant association found for workers exposed to high levels of PFOA in the manufacturing process; however, this was not observed in follow-up studies that utilized more specific exposure measures (156, 157). A DuPont study did reveal an increase in the incidence of Kidney and bladder cancer [158], but this study included very little data on other varisbles such as exposure to other chemicals in the plant. An epidemiological study by Olsen et al. [153] looking at hormone levels in exposed workers found estradiol to be the only hormone level increased in workers exposed to PFOA and this increase was found only in workers with the highest levels of PFOA intheirserum. As discussed in the pancreatic section previously, an epidemiological study was done on CCK levels in employees exposed to PFOA with no increase in pancreatic tumor formation found (152, 153], There is a pertinent need for collection and analysisofmore human data regarding PFOA exposure as there is currently litle epidemiological data available, most of which has been collected and analyzed by the industry that manufactures PFOA. FY) p.53 VI. DISCUSSION AND SUMMARY As previously discussed PFOA, can be detected in the entire populationof the United States. The EPA Science Advisory Board has reviewed the information that was available at the time, and suggested that the PFOA cancer data are consistent with the EPA Guidelines for Carcinogen Risk Assessment descriptor "likely to be carcinogenic to humans." On January 9, 2009, EPA's Office of Water developed Provisional Health Advisories (PHA) for PFOA and PFOS to protect against potential risk from exposure to these chemicals through drinking water. Provisional Health Advisors serve as informal technical guidance to assist federal, state and local officials in response to an urgent or rapidly developing drinking water contamination. They reflect reasonsble, health-based hazard concentrations above which action should be taken to reduce exposure to these `contaminants in drinking water. The PHA values are 0.6 ug/L for PFOA and 0.2 pg/L for PFOS. Iti striking to note that the PHA value for PFOA in drinking water is 0.4 pg/L `when the mean serum concentration in the US population was found to be 3.9 pg/L in 2003-2004 [7] and 3.4 ug/L in 2006 [7, 159]. The EPA and the cight major companies in the industry launched the PFOA Stewardship Program in 2006, in which companies committed to reduce global facility emissions and product content of PFOA and related chemicals by 95 percent by 2010 and to work toward eliminating emissions and product content by 2015. Its evident that the EPA and manufacturers ofPFOA are in agreement that PFOA is a persistent environmental contaminant that poses a potential health risk to the human population. `The pathways by which the tumor triad is produced by PFOA exposure in rodents is still not clear. It is evident that PPAR agonis plays a role in liver adenomas yet the a p54 MOA for tumor production in the rodent testes and pancreas is still unknown. Although currentdata supports that the PPAR agonistic properties ofPFOA may not be applicable to human risk, since humans show litle PP, several additional rodent tumorigenic pathways presented in this thesis may be applicable to humans. Current research supports that exposure to PFOA results in the generation ofROS in humans and ROS and oxidative stress have been shown to play a role in tumor production in humans. PFOA has been shown to interfere with GIIC by inhibiting GJIC via the ERK pathway which has been shown to be a key pathwayofcarcinogenesis in humans. The alteration of the immune system by PFOA appears to result in a weakened immune system. A weakened or suppressed immune system has been associated with increased tumor development. Increased estradiol levels were found in an epidemiologic study by Olsen described previously in this thesis and PP chemicals such as PFOA have been shown to increase estradiol levels. This modulationof estradiol has been demonstrated to play a role in LC mor formation in humans. The activationofthe CAR pathway by PFOA has also been discussed in this thesis and a subsct of the human population that highly express CAR may be susceptible to tumor formation as a result of CAR activation by PFOA. It is conceivable that eachofthese pathways may represent pathwaysof PFOA carcinogenesis in humans. "The global presence and environmental persistence of PFOA, combined with the ong halflife ofPFOA in humans (4.37 years [1601), warrants careful consideration as to the proper handling, use and disposal of this chemical. With, ile epidemiological studies conducted, most of which have been performed by industries that manuficture PFOA, and given that PFOA is accumulating in not only the occupationally exposed, but 2 p55 also in the general population is of concer. The ubiquitous presence of PFOA in the environment compounded with the possibility that viable carcinogenic pathways exist in experimental animals that are mirrored in human carcinogenesis reflect a need to further evaluate the carcinogenic potential of PFOA in humans and the need for continued epidemiological studiesofthe human population. a3 p56 VII. REFERENCES 1. LPearyf,luCor,oaAlnkiytloalceidsK:,aHroedveisew Co,fmoLnaiitoDr.i,ngPalnabdlteosx-iHcuotlcohgeincsalAfi,ndianngds. STeoexdico1l, Sci,2007. 992):p.36-94. 2. rHienldaetrilointsehri,p PMb.etweDeenLorrempeeaMtPed.,ianhnadlaKteionnnedeyxpoG.sLu.r,esPerafnldu.orpoloacsimaan.oicPaFcOidA: concentration in the ra. Toxicology, 2006. 222(1-2): p. 80-5. 3. pSeirnfcllauiorr,oaEi,tyKlimsurSfXa.c,taAntksinlaenydefLluBo.rotaenldomKearnnaalncoKh.o,lsQuraenlteiatsaetdiofnorofmgasno-npshtaiscek cookware and microwave popcorn bags. Environ Sci Technol, 2007. 41(4): p. 1180-5. 4. MAOlrasmneidnte,algGe1..WH1..B,,.aCnhHdueZrrocrbhoenTlRRL..,MR..,,MiPMleelrdfehlduJioPzr.ao,odBceuthrakrnaiessshuJilfMoZ,.n,atHNeaobnaisnledentoitKh.1e1.r,8f,lLuuOonNrdeobicelhrlegmiEJcMKa..l,,s 2i0n0t3h.e s1e1r1u1m6o):fpA.me1r8i92c-a9n0R1ed Cross adult blood donors. Environ Health Perspect, 5. OaOltnshdeenZr,obFGe.ilWu.o,LrRoC.ch,hueQrmuicachnatliTstRati.invtHehaeEnvsSaeelnurauKt.miJo,o,nofBfCuhPrierlrdifrslenuJ.oM.,roBJooucuttranenanelhsuoolffffoCnh.iaLlt.,derMe(anPn'FsdOeHSle)aLlaHtn.hd,,. 2004.2(1):p. 53-76 6. Oalssseens,smeGnWt .ofBurrwiosrkJeMr, sBeurruemw pMerMfl,uoraonodctaMnaesnudleflonaLtHe, (EpPiOdSem)ioloagnicd peexrafmilnuaotrioooncsi.anJoaOtcecup(EPnFvOiAr)on Mecdo,nc2e0n0t3r.at4i5o3n)s: p.an26d0-70m.edical surveillance 7. aPCoanlldyaffNlauu,roirtoAiaMlokn,ylExcaWhmoeimnnigactailLosYni,nSutrhKveuekUyiLSe(.nNypHioAkpNulEZaS,t)ioR2ne0:i0dd3ya-i2aJ0f0Ar4,oamnatndhdecoNNmaeptcairdoinhsaelomnHseLaw.liLtt.hh, `NHANES 1999-2000, Environ Health Perspect, 2007. 115(11): p. 1596-602. 8. pOKelJrsf,elnu,oBruGororWcits,anJeCsMhuu,lrfcohnMaatTneRdea,lndLJaortHhs,eornfalEnudBor,oZcovkbaeenmlicBaeLllRlsie,nGa,S,neerLluudnmerdlbcyeorpngocpeJnuXtlr.aa,ttiioHonanfnsrseoonmf Seattle, Washington. Chemosphere, 2004. S4(1 1); p. 1599-611. 9. PEreircfslouno,riIn,aGteodmcehzemMic,aNlasdianlblMo.o,dovfanreBsaivdeelntBs., inLiCnadtsatlroonmiaGi(,Spaanidn)DionmrielnagtioonJ.tLo, ageandgender: apilotstudy.Environ Int, 2007. 33(5):p. 616-23. " p57 10. hJiun,man., sSearituom N,l,evHelasraodfa pKerHfl,uoIrnoooucetaKn.o,ataendanKodipzeurmfiluAo.r,ooHcitsatnoericsaullftorneantdes in in Shenyang, China. Tohoku J Exp Med, 2007.212(1):p. 63-70. 11. BPKeaGrnf,nlaunoM,roooKhc.d,taCnMoesrAusl,ofloinOnalti.ev,earFonadlJa,RnedlWyasotzuedwJ,eFlFuNiol.rl.om,cahnenYmaiGnc.ga,lsKJuHimn,arHaunKmd.aS.nA,ldLBoolugosaondaKftMrh.oa,mn Several Countries. Environmental Sci&Teecnhnoclogey, 2004. 38(17): p. 44894495. 12. GK.a,rmLeavne,lsA.o,f M1u2cplelrefrluJoFr.i,navtaend BcahveemlicBa.l,sHianrdpeonolFe.d, aTuosmtrsalLi.aMn.,searnudm,Liconldlsetcrtoemd 2002-2003, in relation o age, gender, and region. Environ Sci Technol, 2006. 40012): p. 3742.8, 13. pMeirdfalsucohr,cociOa,nesulSfcohreatttegenandT._perafnlduoroAoncgieanroearte Je,xpoPsiulroet osftutdhye oGnermtahne generalpopulation. Int J Hyg Environ Health, 2006. 209(6): p. 489-96. 14. YY.e,uLnig,J,LWGi.csyMJi.yP.a,keYaYm.a,shTiatnaiyNa.s,uaSn.d, LWaamngP.YK..,, PYeurfHl.,uSoroinaMteKd,coJmipaonugnGd.s, aWnud tEontvailroanndScieTxtercahcntoalb,le20o0r8g.an4i2c3f1lu)op:r.in8e14i0n-5h.uman blood samples from China. 15. ALic,cuXm.u,lYaetiuonng oLf.Wp.e,rfTluaonriiynaastuedS.c,oLmapomunPdXs.,inYacampatsivheiBteangN.,aXlutiMge,rsan(dPanDtahierJ.a, tigris tgris)and African lions (Panthera leo Linnaeus) in China. Chemosphere, 2008. 16. PKearnfnlawnr,ooKc.,taCnoersstofloinniatSe, aFnadl.andryelsazteJd., fOlcuhormienaGt.e,d FhocyadrrdoicaSr.b,oannsd iGniesmyarJi.n.e. `EmnavmimraolnsS,fiisTheecsh,naoln,d2b0i0r2d.sf36r(o1m5)c:oaps.ts3o21f0t-h6.e Baltic and the Mediterranean Seas. 17. SKeelglaerrs, AJML.,, KeanndnaKnuckKl.,icTkanJiRya.suPeSr.f,luYoarmiansahtietdacNo.m,poDuanydsRDin,thAerepnldtasMm.aD.o,f UnloigtgeedrShteaatdes.anEdnvKiermopn'SscirTiedclhenyols,ea20t0u5rt.le3s9(f2r3o)m: pt.h9e10s1o-u8t,heastern coast of the 18. pDeari,ilJu,orLoiocMt,anJoianteY., inSarietdo N., XuM., and Wei F., Perfluoroocanesulfonate panda and giant panda from China. Environ aSncid Technol, 2006. 40(18): p. 5647-52. 19. KFaalnannadynszK,.,JP,erTfalnuioyrainsauteSd,,coYmapmoasuhnidtsainN.s,omRoesttkerorwesstkriialP.a,nZdalaeqwusatkiic Kw.i,ldlainfde s2p0e0c7.ies42f(6r)o:mp.Po7l1a5n-d9.. J Environ Sci Health A Tox Hazard Subst Environ Eng, as p58 20. pDeirefwl,iorRoa,lkyBloscsointaRm,inaRniigsetin eFaFst,grSeoenmleandCp,olaarndbeaBros m(UrEsuWs.marIintcirmesa)s:inag ewtoxic threatto the Arctic bears. Environ Sci Technol, 2008. 42(7): p. 2701-7. 21. dSkruitnlkairnegk,waDte,rsE.xnEnevriMr.o, naSncdi PFoalrlbuetrRHe.s, IPnetrf2l0u0o6r.in1a3t(e5d)s:urp.fa2c9t9a-n3t0s7.in surface and 22. ATanntaokna,A,S., `aFnudjiLiinSd.,stLrioemn GN.P, HA.,simNpolzeoepreM-,treFautmkeangtawparocHe.,duWrieroinjaPnaFgOuSd aWn.d,. CPoFmOpAouwnadtse,r2a0n0al6ys68i;s pa.n5d27i-ts53a0p.plication in several countries. Organohalogen 23. pWerefrleumoiruikn,ateAdMsu,rfaGcetarnsttsmainnnwaSt.e,rabnydLCF-rEaSnIk-HMS,/MQSu.antJiSteatpivScei,d2e0t0e6r.mi2n9a(t1i4o)n:po.f 2515 24. vdaenVoLgete,uwPe.,nBeS.r,gearndU,vadne RCooenn W.A,.,dePeWrfolluforWi.n,atHeedimosrtgaadniEc.,cMocmLpaocuhnldasn iMnSt.h,e AEmusrtoepredaanm:enAvmisrtoenrmdenatm,(NPeLr.fopr.ce1)-.126in. Report to the EU. 2006, University of 25. pLrooodsu,ctRs.,, WopelrlfglausotroJo.ctHaunbeesurlTfo,naained H(aPFnOkSe),G.,peProfllaurohreorobciicaindoeast,eph(aPrFmOaAce)u,tiacnald `naormoyulnpdheLnaokle aMnadggiisorcearibnoNxoylrattheesrnalnidaly.ethAonxayllaBtiesoanianlsCurhfeamc,e 2a0n07d. ia38p7(w4a):terp.s 1469-78 26. PSaeirtfol,uoNro,ocHtaarncaadtae K.a,ndInpoeureflKu.o,roSoacsiaaknie Ks.u,lfoYnoastheincaognaceTn.i,raatniodnsKoiinzusmuirfaAc.e, waitnJeaparn. JOccup Health, 2004. 46(1): p. 49-59. 27. YSo,amMKa,sNh.M,iiyZathkoaeu YB.',S,YeSuhinXg.JW,.,YW.a,ngHo1.XY.,MGi,esTyanJi.yP.a,suYuS.H,, aRonsdtkLoawsmkiP.P,., CPehrefmlousoprhienartee,d20c0o7m.po68u(n1d1s): ip.n 2t0h8e5-P9e5a.rl River and Yangtze River of China. 28. PKearnfnlauno,rinKa.t,edTCaoompLo,unSdisncilnaiArguEa,icPaOsrtgvaaniSsDm.s,atJuVdareioDu.sJT,roapnhdicGLieevseylsJiPn.a, GTorxeiactoloLgayk,es200F5o.o4d8(4C)h:aipn..559A-r5c6h6i.ves of Environmental Contamination and 29. aSinndclaiPre,rfE.l,uoTraonoicytaasnue S.S,ulYfaomnaastheitainN.,MiacnhdigKaanmaanndK.,NPeewrfluYoorrokoctWaantoeircs,Aciidn Organohalogen Compounds. 2004. p. 4019-4023 30. oSfipncelrafilr,uoEr,oalMkayylascukrfDa.cTt.a,niRsobinleweatKe.r,, iYsahm,asahnitdabiNr.d,safnrdomKaNnenwanYoK.r,kOSactceu.reAnrcceh Environ Contam Toxicol, 2006. S0(3): p. 398-410. 4 p59 31. Harada, K., Nakanishi S., Saito N, Tsutsui T., and 2005. KA, Airborne pJearpafnl.uorBouolclieatninoaotfe EmnavyirobnemeantsuablstCaonnttiaamlisnoautricoen caonndtaTmoixniactoiloongyi,n 2K0y05o.lo7a4r:eap,. 64-69. 32. Harada, K., Nakanishi S, Sasaki K., Furuyama K, Nakayama S., Saito N., Yamakawa K., and Koizumi A., Particle size distribution and respiratory deposition estimatesofairborne perfluoroocianoateandperfluoroocianesulfonaie in Kyoto area, Japan. Bull Environ Contam Toxicol, 2006. 76: p. 306-310. 33. Barton, CA, Characterizing pBeurlflleuroroLoEci,anoZaatrezeicnkiamCbiJe,nt Faliarhenrteyar Jthe afnednceKaliisneer ofM,a manufacturingfacility: comparing modeled and monitored values. J Air Waste Manag Assoc, 2006. S6(1): p. 48-55. 34. Strynar, M.J and Lindstrom A.B., Perfluorinated compounds in house dustfrom `OhioandNorth Carolina, USA. EnvironSci Technol, 2008. 42(10): p. 3751-6. 35. Kubwabo, C, Stewat B, Zhu J, and Maro L. Occurence of perfluorosulfonates andother perfluorochemicals in dustfrom selected homes in the city ofOtiawa, Canada. J Environ Monit, 2005. 7(11):p. 1074-8. 36. Moriwaki, H., Takatah Y., and Arakawa R., Concentrationsofperfluorooctane sculfoonalteiln(JPaeFpOaScn)estaenhdeopmedersf.luJoErnovocitraonndiMconaicti,d20(0P3.FO(A5)):inp.v7a5c3u-1u.m cleaner dust 37. L1e9w9i6s,,NReJw,YSoarxk',sNDYa:ngVearonuNsosPtrroapenrdtRieeisonhfolIdn.dustrial Materials.9th ed. Vol. 1-3. 38. NKYi.r:k-JOothhnmeWri,leEyncayncdloSpoendsi.aof Chemical Technology. Vol. 1. 1994, New York, 39. Goss, K.U., The pKa values of PFOA and other highly fluorinated carboxylic acids. Environ Sci Technol, 2008, 42(2): p. 456-8. 40. LG.ymVaoln., 1.W1,98E5n,viBroocnamReanttoanl,EFxLp:osCuRreCFPrreossm. C3h1e.micals, ed. Neely, W. and Blau 41. Olson, C.T. and Andersen ME. The acute toxicity ofperfluorooctanoic and p`eAprpflluPohraordmeaccaonlo,ic19a8c3.ids700i3n):map.le36a2i-s72.and effects on tissue fatty acids. Toxicol 42. Dean, WP. International and Jessup Research and DC, Acute Development oral toxicity Corporation, (LDS) Study No. study in 137-09], rats. May Sth, 1978. 1978, US EPA AR226-0419. 43. SGtluazday,NoS.MC.HAWcu6t1e00o1r7al60t,oxJiacintuyasrtyud10y,o1f9T97-.66169997i,nUraSts.EPCAoArRn2i2n6g-H0a4z2e0l.ton Inc. a p.60 44. IGnotledrennatthiaoln,alE.LR,esFeianraclhrepaonrdt, nDienveetlyodpamyenstubCaocrupeorrahteisoun,s mSotnukdeyy Ntoo.xici1t3y7s-t0u9d0y., November 10, 1978. US EPA AR226-0447. 1978. 45. T(hAoPmFfOo)rdi,n cP.yJn.,om4o-lWgeueskcmaopnskuelyest.oxiSctiutydyspteurdfyowmirtehdabmymoCnoivaunmepeLrafblourdartooorciteasnoInact.e MGaroduips.on SWtiusdcyonNsoi.n C5o3v7a0n4c-e2596323f9o-2r30A,PMDEeceadm-bheorc APFO Toxicology 18, 2001, 159 pp. Working. US EPA AR226-1052a. 20012. 46. pTehrofmlfuoorrdo,octanPoJa,te (2P6-FWOe)ek in ccaypnsuolmeolgulsosimcoitnykeyss.tudyStudwyi.thperjaommmroendiubmy ACoPvFaOncTeoxLiacboolroagtyorWioerskIinnc.g,GMraoudpi.sonStuWdisycNoon.siCnov5a3n7c0e4-62352992-2f3o1r, ADPecMeEmbaedr-h1o8c, 2001,463pp. US EPA AR226-1052a. 2001b. 47. mCihcrei,stFoipnhaerl,ReBp.oratn.d 1M9a7r1i,asInAd.uJs.t,ri2a6l-Bdiaoy-Toersatl tLoaxbiocriattyosrtieusd,y Iwnict.h FC-143 in albino 48. RMaettsr,icFki,naMl.Reapnodrt.Ma1r9i7a7s, AI.ndJu,st2ri8a-ldaByioo-rTaelsttoLsaibcoirtaytosrtiuedsy. with FC-143 in albino 49. tCouxii,coLlo,gicZahloeuffeQctFs,ofPLiFsOoACaYn.d PFFuOSLio,n raanids uJsiianngghiGstBol.ogiSctauldioebsserovnatitohne andchemical analysis. Arch Environ Contam Toxicol, 2009. 6(2): . 338-49. 50. CLaowllieirm,amTmaEl,ianMumtiacgernaiscoimtey rteevsetrsweitmhutaTt-i6o3n42assinaytwhiethSaalmcoonneflilram-aEtsocrhyeraiscshaiy.a CLaobmopraantyo.rySt.nuPamuble,r:MN.170U7S3A-0E-4P0A9.AR22C6o-r0n4i3n2g.H1a9z9e5.lton Inc., Vienna, VA. 3M. SI. CLaowlluemr,ammTaEl,ianMumtiacgernoiscoimtey rteevsetrsweitmhutaTt-i6o5n64assinaytwhiethSaalmcoonneflilram-aEtsocrhyeraiscsahyi.a ACRo2r2n6i-n0g4H3a2z.el1t9o9n6.Inc., Finalreport. CHVSTudyNo. 17750-0-409R. USA EPA 52. aHsaszalye.tonC,or1n,iMnugtaHgaeznliectiont,y Itnecs.t wFiitnhalT-Re6p3o4r2t iCn HaVn SintuvdiyvoNummobueser m17i0c7r3o-n0u-c4l5e5u.s December 14, 1995. US EPA AR226-0435. 1995. 53. CMourlnii,ngH.,HaMzulteatgoenniIcnict.,y tVeisetnonna,T-V6A5.64Stiundyanruinmbveivro 1m7o7u5s0e-0m4i5c5ro,nuNcolveeumsbaesrsay1.. 1996. USEPA AR226-0430. 1996. 54. hJMauarrmvleais,nts.H.w,hoCMlouertangibenlngoiocHdiatzlylyetmtepsohtnoocInyntceT.s-6V3wi4ie2tnhnam,eaasVcAu.ornifSnitgrumcdayhtronorumymobsaeosrmsaayl17a0wb7iet3rh-r0a-mt4ai4lol9nisCp0li,en November I, 1996. USEPA AR226-0433. 1996s. 8 p61 $5. NOTOX, Evaluationof the ability of T-7524 to induce chromosomal aberrations Hinertocugletnubroesdch,petrhiepNheetrhaelrlalnydmsp.ho2c0y0t0e.s. NOTOX project number 292062. 56. eYxepoo,sXe.datondpeZrfhlounogroLo.c,iGaennooitcoaxciidc.riMsuktaantidReosx,id2a0t0iv5e. D58N7A(1-d2a)m:apg.e38i-n4H4.epG? cells 57. Shabalina, 1.G., Panaretakis T, Bergstrand A. and DePierre 1.W., Effectsofthe rodentperoxisome proliferator and hepatocarcinogen, perfluoroocianoic acid, on a2p2o3p7t-o4s6i.s in human hepatoma HepG2 cells. Carcinogenesis, 1999. 20(12): p. 58. Sibinski, LJ, Final reportof a two year oral (diet) toxicity and carcinogenicity sVtouldy1o-f4,fl3uMoroCcohmepmiincya/lRFiCke-r14e3xp(.peNrof.lu0o2r1oo8cCtRa0n0e1a2m;mo8nEHiQu-m10c8ar7b-o0x3y9l4a,te)Oicntorabtesr 16, 19871981. 59. Chandra, M., Riley M.G., Sprague-Dawley rats. Arch TaonxdicJolo,hn1s9o9n2.D6E6(.7):Spp.on4t96a-n5e0o2u.s neoplasms in aged 60. Boifcgeexlt,raLhBe.p,atiHcurtttumMoEr,indFurcatmieonSRb.y,peOr'oCxoinsnoomreJ.pCr.o,liafnedraCtooroskiJn.Cm.,alMeecChaDnirsatms.s Toxicol Sci, 2001. 60(1): p. 44-55. 61. KSltaeuvneings,onJDE.,E.,XaundYW,alIbsoerngbeEr.gF.,1SJr., Bachowski S., Kolaja KL. The roleofoxidative stress in Jiang J., chemical carcinogenesis. Environ Health Perspect, 1998. 106 Suppl 1p:. 289-95, 62. Liu, C, Yuk, Shi X,, oxidative stress and Wang J, apoptosis Lam by P., PFOS Wu R.S., and and PFOA Zhou B., Induction of in primary cultured h8e2p0at)op:c.yt1e3so5-f4f3r.eshwater tilapia (Oreochromis niloticus). Aquat Toxicol, 2007. 63. `PRaenaacrtcitvaekiosx,ygTe,n Sshpaebcaileisnaan1d.Gm.i,toGcrhaonndderriaD.m,eSdhiaotsehatnheMi.nCd,uctainodnoDfePaipeorprteosJiWs.in Human hepatoma hepatocarcinogen, HepG2 cells by perfluorooctanoic. thaecidr.odeTnotxicpoelroxAipspolmePhparrolmiafceorla,tor20a0n1d 1730)p:. 56-64. 64. Berthiaume, and N-ethyl Jp.erafnlduoWraololcatcaeneKsulB.f,onPaemrifdlouoertohoacntoaln;oapteer,opxeirsfolmoeuroporcoltiafneersautlifoonnaaten,d mitochondrialbiogenesis. ToxicolLett, 2002. 1291-2): p. 23-32. 65. Sinudrukcoevd,miAtAoc.honadnrdialWadlylsfaucnectKi.oBn..,ToSxtriuccotlurSacil,d2e0t0e2r.m6i6n(a2n):tsp.of24f4l-u5o2.rochemical- 66. V1a7n7d1e8n)pH:.eu1v1e0l8,-1J2..P, The PPAR resource page. Biochim Biophys Acta, 2007. a p.62 67. aAIssDsh,ebsys,KmeeJtn.ttloBerfapdSey,rAo.xa,insdEolmcPeoumrpbcrehoalCsfeeRr,aLtoFErl.l-iiontMdteucBcheMad,nihsetpIiascthaomlcalaeyrl-cbiJan,osgeOeddneuhsmiusmJ..a,nHTuuhgmawzoEaxorpdd. Toxicol, 1994.13 Suppl 2: p. SI-117. 68. HBeenptalteiyc, pPe.,roCxailsdoemre Ip,roEllicfeormabteionC,inGrroadsesnotsP.,anSdtriitnsgesrigDn.i,fiacnadnceWifeograhnudmaHnJs.., Food Chem Toxicol, 1993. 31(11): p. 857-907. 69. CDSa,ttleyP,asRtoCo,r Wahii W., Do TDAeL,ucPaopJp, peroxisome LElAc.omRboebiCn.s,onFenDn.eE.r,-CrSicshpwePt.z, proliferating compounds pose aLBa.,hkeeTpauBtg.oGwc.oa,orcdMianJro.,gsemananindc hazardto humans? Regul Toxicol Pharmacol, 1998. 27(1Pt2): p. 47-60. 70. LStaukdei,esBoGn.nafEevnaonpsin1-.iG.n,duGcreadyheTpaJt,icKpoerroosxiisSo.Am.e,praonldifNeorarttihonC.inJ,thCeormapi,arSaytriivaen h1a4m8s-t6e0r., guinea pig, and marmoset. Toxicol Appl Pharmacol, 1989. 99(1): p. 71. PMaPlAoRnegya,mmEaK.by asntrductWuraaxllmyandiverDsJe'. enwvriarnosn-mAcetnitvaalticohnemiocfalsP.PATRoaxlipcohla Apapnld Pharmacol, 1999. 16102): p. 209-13. 72. mPaosrtpohoorl,ogTicPa,l stLuedeiesKoPf,amPmeorniiuMmAp,erflaunodrooGcitlalnieosateP-Ji.n,ducBeidochheepmaitcoamlegaanldy andperosisome proliferation. Exp Mol Pathol, 1987. 47(1): p. 98-109. 73. KPD.leAaL.u,unciPga,PJALRGEa,,lLpBahiaabDi.acYgh.o,nMiM.sctA-K,ieneBdauccReHt.dc,kreoPdKeetPnet,rstCJuomMoo.r,ks:RIoCmb,eordtCesosrRot.foAn.a,cJta.Cin.od,nFDeaannvniedrd-hCRurimMsa.pn. relevance. Crit RevToxi20c03o. 3l3(,6):p. 655-750. 74. YThaemaomropthoa,n nYu.,clMeoarorreeceRp.,loGroclodnsswtiotrutthivye Ta.cLt.i,veN/eagnidsshoistMa.n,e raencdepMtaorroinspoestseRnRti.a,l f7o1r97l-iv2e0r0t.umorpromotion byphenobarbital in mice. Cancer Res, 2004. 64(20): p. 75. LUeedham,aAn.n, JHMa.m,adaenhdHN.eKg.i,shWiebMb.,H.DKi.v,erYsaemraomleostoofY.t,heSuneuycolsehair To.r,pAhfasnharreicCe.pAt.o,r 2C0A0R.6i1n01r)e:gulp.at1i-n6g. hepatic genes in response to phenobarbital. Mol Pharmacol, 76. AS.b,boLtitn,dBs.tDr.o,m WAoBlf.C.SJ.t,tySncahrmMidL,LE.andDaLsaKu.PC..,,ZePehrrflRuDor.ooHcetlafnaonitcL.a,cNiadkianyduacmeda. pdreovleilfoepramteonrtaalctitovxaitceidtyreicneptlhoer-malopuhsae. TisoxdiecpoelnSdcein,t20o0n7.ex9p8r(e2s):sipo.n5o7f1-p8e1r.oxisome 50 p.63 77. YDeaPnigc,trQe,J.AWb.e,diP-oVtaelnutgseurpdpireMs,sioXnioefYt.h,eZahdaaoptXi.vYe.i,mMmoulnleerreG.s,poNneslesionnmBi.cDe.,upaonnd dInitetIamrmyuenxoppohsaurrmeac10olt,he20p0o2t.en2t(2p-e3r)o:xpi.s3o8m9e-9p7r.oliferator, perfluoroocianoic acid. 78. Honkakoski, P, Zelko receptor CAR-retinoid I., X Sueyoshi receptor T., and Negishi M., The heierodimer activates the muclear orphan phenobarbital- responsive 5652-8, enhancer moduleofthe CYP2B gene. Mol Cell Biol, 1998. 18(10): p. 79. PKhaewnaombaortboi,taTl,.reSsupeoynossihvieTn.u,cZleelakrotrIa,nsMlooocarteioRn.o,fWtahsehrbeucrenpiKo.r,CaAnRd NinegiinsdhuictMi.o,n ofthe CYP2Bgene. Mol Cel Biol, 1999. 19(9): p. 6318-22. 80. Wreecie,ptPo.r CZAhRanmgedJi.,atEegsasnp-eHcaiffilcexyenMo.b,ioLtiiacnigndSu.c,tiaonndofMdorourgemeDt.aDb,oliTshme. nNuactlueraer, 2000. 407(6806): p. 920-3. 81. eCnhzeynmge,sX.inamnoduKsleaalsivseerntCh.rDou,gPhearcftliuvoartoicoanorbfoxPyPliAcRaaclipdshainadnudceCcAyRtotcrharnosmceriPpt4i5o0n Sactors, Toxicol Sci, 2008. 106(1): . 29-36. 8. LRaosuenC.,,ManBd,CoLreteonJSJ.,C.,ReTnoxHi,cogVaelnloamniact dBi.s,seLcituioJn,ofWtahaelkpeesrfMl.uPo.r,ooAcbtbaontoticBaDc.i,d rtreacnespctroirpstPpPrAofRilaelpihna amnodusCeAR.livTero:xiceovlidSecni,ce20f0o8r. 1t0h3e(1)i:sopi.v4e6m-e5n6t of mclear 83. Bboertewke,enC.NoarndmaSlacChesllLs.,aTnhdeCDeilflfserTernacensifnorComneldacbty IDnihfifbeirteinotnoCfarCceilnloRgeepnlsi.caPtrioocn Natl Acad Sci US A, 1966. 56(6): p. 1705-1711. 84. Tcraorsckion,ogeJnE. and risk aUspshesasmmenBtL. . igTnhoereedmpceornocrepwtesarisn ncoanccleotrhersisikn tahsesefsisemlednto.f Mutagenesis, 2005. 2002): p. 81-92. 85. `Tarnodsckhoe,mJoEt.hearnadpyR.uCcuhrrR.DJ.r,uGgaTparjgeutsn, 2c00ta2.sit3ao(6r)gn:etsp.sf4o6r5-c8a2n.cer chemoprevention 86. Upham, Hirose A.B,LL.HasPaergkaw1aS,R.BabKiacnanoP.,J,SoavnaddinSoaiva KI.,, RSutrmumcteulre-AaMc.ti,viTtry-odsekpoenLdEe,nt rinegvuiltartomioondeocfleslylstecmosm,muEnnivciartoinoHneablythpePrefrlsupoercitn,a2t0e0d9f.at1t1y7(adc)i:dsp.us5i4n5-g51i.n vivo and 87. jUupnchtaimo,nalBLiLnt,erDceeloluclaarmpcoommNu.nDi,caWtuiroln bBy.,perafnlduoTrrionastkeodfLaEtt.y aIcnihdisbiitsidonepoefngdeanpt `on the chain lengthofthefluorinated tail. Int] Cancer, 1998. 78(4): p. 491.5. si p64 88. BpuKetJre,fnlhuooGfrofor,omcJta.aLnn.,oaGKtSee.n,innecdNyyonkGoe.mLro.l,gJPurEs,.mHoinnakdneedyrslT.theTroomxPfi-ocM,orldLSicePid,Je,2r00PP4Hh..a,8rJ2m(ua2cn)o:gkpiR.n.e3,t9Hi4ac-n4ss06eo.nf 89. COstomrmeuprcat,urriGaslAon,anoDafolnotoghheoe1e6nRhaManl,cpihnaHg-efnldeufrofierccokt-ssS-JoanfDd,rdoesChtuyredtnri-os1e7pL-ioRann,edraoonsndt.erWoianfllelaitaowmxisitnhDEt.Bh,Ie hepatocarcinogenesis in rainbow trout. Fundam Appl Toxicol, 1996. 34(1): p. 13240, 9. TPmieelrcteohinra,aniSsCCmB,.f,oOrmahenedrpatGWi.icAl.tl,uamBmoesrnnpDirEno.gmh,ootfiGfoennAoDbm.yi,pceCrapfrrlpouefoinrltoieonrcgiHarnMeovi.ecalasHceindadnriincarkltaseirnJn.baDot.ew, trout. Environ Health Perspect, 2008. 116(8): p. 1047-55. 91. Laicut,ioCn,oDfupYer.flaunodriZnhaotuedB,c,hEevmailcuaaltsiondoetfeersmtirnoegdenibcyacvtiitveitlileosgeannind miencdhuactniiosnm oinf primaryculturedtilapia hepatocytes. Aquat Toxicol, 2007. 85(4): . 267-71. 92. hWeepia,tiYc.,efLfeicutsYo.f,peWrafnlguorJo,ocTiaaonoYi.c, aacnidd DoaniraJr,eTmoixnincoogwesno(mGiobcioancaylpyrsiissorafrust)h.e Toxicol Appl Pharmacol, 2008. 226(3): p. 285-97. 93. Cbialmeearcoind,s Ro.nG.h,eIpmaatoicdaarKc.i,noTgseundeasiHs. ainnidtiIatteodNb.,y Pdrieotmhoytlinvitereofsfaemcitnse.ofsCtaenrcoiedrsRaensd, 1982. 42(6):p. 2426.5. 94. Yhaegpaetro,caIrDci,noJgre. neasnids iYnafgeemralRe,SpOrraagluec-oDnatwrlaceeyptraitvse. CsatenrcoeirdsReass, p1r9o8m0.ot4e0r(s10)o:f p. 3680-5 95. Hanatyin-ecsa,ncNerMc.hemvoatnhedrerapMyosast aRnG,e,merLgaiknegRcAon,cepatn.d CSumryrtOhpiMn.J.I,mmiumnmouln,og2e0n08i.c 20(5): p. 545-57. 96. 3Sc,h2u0s0t6e.r,12M).: pN.ec1h3a8n4s7k.y A., and Kircheis R., Cancer immunotherapy. Biotechnol 97. TSecshnlicermem,erA.F,.,AZpiettvoohgelL.L,,GhainrdinKgrheolelmierF,G.,JoIzmamuNn.,ogPeanniacrcctaaknicserT.c,ellKdeepatph:O.a, keylockparadigm. Curr Opin Immunol, 2008. 20(S): p. S04-11 98. eYtvhaiyndmgei,nccQe.faonXditehesYpi.ln,evnoiElcrviekmasetsnrotnoopfhAiynMhi,biintNdieuoclnesodofncebBly.lDp.r,tohleiafnedrpaetDrieooPxniieasrnordmeedJe.vW_e.pl,rooplFimuferenrttahteoirrn perfluorocianoic acid in mice. Biochem Pharmacol, 2001. 62(8):p. 1133-40. E p.65 99. tYhaengp,erQo,xiXsioemeY.,proAlliefxersaotnorS-.aEc,tiNveailesdonreBce.pDl,oranadlpDheaPiicnrrteheJ.Wi.m,mIunnvoomlovdeumleanttioonf c1a8u9s3e-9d0b0y. peroxisomeprollferators in mice. Biochem Pharmacol, 2002. 63(10): p. 100. pBeirefglelu,oroLoBc.t,anoLaitue oRnCL,eydHiugrctellMfuEn,ctiaonn:d inCoviotkro,1i.nC.v,ivEof,feacntds eoxfviavmomsotundiieus.m Toxicol Appl Pharmacol, 1995. 134(1): p. 18-25. 101. GEfafzeocutlio,fpMe,r,oYxaisooZmeXp,roBliofuejrraatdorNs.,onCoLretyodnigJ.cCe.l,lCpuelrtiyphMe.r,ala-ntdypPeabpeandzoopdoiualzoespiVn.e, rsteecreopitdoorgengeesinse: erxoplreesosfiotnh,e pheorromxoinseo-msetipmruollaltfeerdatocrh-oalcetsitveartoolr transport, and receptor alpha. Endocrinology, 2002. 143(7): . 2571-83. 102. HWiilllk,inRsNon,CFE.xd,reTihcyhroLiSd.f,ollPiacyunltaerrceOll.c,arcRionboegretnsesiPs.A,FuRnodsaemnthAaplplSTLo.xicaonld, 1989. 12(4):p. 629-97. 103: aCdhurlitstreantssent,reAaKte.d achnrdonPiecaaclolcykwiKt.hC.a,nInecxrceeassseofinhLuemyadnigccheolrlinonuimcbegornaindottersotepsino.f Biol Reprod, 1980. 2202): p. 383-91. 104. teFsatiimcaunl,arfC.emiannidzaWtiinotne.rJJC.lSi.,n TEnhdeoccroinntorollMoeftgaobn,ad19o7t4r.op39i(n4)s:ecpr.e6t3i1o-n8.in complete 105. HLeelyldeirg,-cCe.lGl.siaznedfoLlelaocwhinDg.Ra.d,miQnuiasnttriaftiicoantioonfohfuLmeayndicghocrelilosniacndgomneaadsoutrroepmheinnt toof normal men. JReprod Fertil, 1971. 25(2): p. 185-92. 106. Icommppelreastiot-eMscoGifnmlaelye,phJ.enaontdypiPcedteevresolnopmReEnt.. MA aleJMm pesdeu,do19h7e6r.ma61p(h2r):odpi.t2s5m1:-72t.he 107. JTorneemsblaHyW,.R,R.,JrF,olaenydT.PM.i,gJero,nCoCr.vJo,l P., Park Plasma LJc,onKceonwtarrastkiionA.,ofBlitzesztaorsdteRrMon.e,, dihydrotestosterone, `goradotrophins in the tseysntodsrtoemroenoef-omeastlreadpisoeludboi-nhdeirnmgapghlroobudliitns,m and. with pinitary testicular Jeminization. Acta Endocrinol (Copenh), 1972. 702): p. 331-41. 108. VCi.guiEeffre-cMtartoifneaz,noMn.-Cs.t,erHooicdahleraenatuiadnedrRoegevne,rsflMu.aTm.i,deB,areonntotnheB.,hyapnodthPaelrarmeoa-u phiitsutiotlaorgyicaaxlisd,atgae.niAtcatlatrEancdtocarnidntoels(tsCoinpger)o,wi1n9g83m.a1e02(r2at)s:: pe.n2d9o9c-r3i0n6o.logical and 109. VC.i,guEienr-dMaortcinrezi, nM.oaCn.l,dohHiosgcthoileocrgeiaacualldcehRaenvgieesrsinMd.uTc.e,d bBayrfelnuttoanmiBd.e, tarnedaPtemrreeonatnu tihnecidyepnoctehsaolnafmeort-ihliytpy.opAhcytsaeaEnldotcersitnicoulla(rCoapxeinsh)o,f the 1983. adult male 104(2): p. 2r4a6t-5a2.nd their 53 p.66 110. Caodoekn,omJa.Cs., bMyurraamymoSnMi,umFrpaemrfeluSoRr.o,octaanndoaHtuer:tt M.Ep.o,ssIinbdluectieonndoocfrLineey-drielgatceedll. `mechanism. Toxicol Appl Pharmacol, 1992. 113(2): p. 209-17. 111. mBoincseert,reGa.teMd.,viMtahltirginpahnentylteutmhoyulernseo.fTthhee iJnotuernsatiltioalf cPealtlhoolftohgyeatnedstBsacitnesrtiroolonggya, 1942. 5402): p. 149-154. 112. iAnnddeurcveodnti,nteHrBs.it,alS-hcielmktienstiMc.uBl.ar, taunmdorCsanitnerBAHL.BY/.c, mTihcee.grJoNwatthl oCfanecsetrroIgnesnt-, 1960.24: p. 1219-37. 113. tHouomkoerrs,inC.miWc.eoafndtPhfAeeifsfterraiCn.Ar.e,ceTihveingmoerstprhooglenosg.y.aCnadncdeevreRleosp.,me1n9t42o.f2t:espt.ic1u5l9a-r 769. 114. Tdeeteercdtsi,on KoJf, trRaonmsmfoerrmtisng EgFr,owtahndfacDtoorm-ianlgpthoan inLH.L,eydIimgmucneolhlsistdoucrhienmgictahle evelopment of the ra ests. Mol Cell Endocrinol, 1990. 69(1: . R1-6. 115. JB.iC.c,gelC,omLpBa.risHounrtofMtEhe,efFfrecatmseofSRW.y,etAhp-p1l4e,g6a4t3e iMn.,CrOi':CCoDnnBoRr aL.nCd.,FainsdherC-o3o4k4 ats. Fundam Appl Toxicol, 1992. 19(3)p:. 590-7. 116. HInudrutc,tiMonEo,fcoElalgiuotl:atGiSon.eCffoeoctks Jb.yC.W,yOebtho-u1r4n,6J4.3D,inFCrralm:eCDSRB.R raantsd. BDiregueglCLhBe.m, Toxicol, 1997. 2001-2): p. 1-10. 117. L1o0n7g)ne:skp.er5,4-D8.S, Carcinogenesis in the pancreas. Arch Pathol Lab Med, 1983. 118. pLaonncgrneeacsk.erG,ut,DS19.87.In2t8erSfuapcpel:bep.tw2e5e3-n8.adaptive and neoplastic growth in the 119. Lcaonncgenreciknetrh,e rDa.tS.. natnJdPSanucmrieatCo.l,,E1f9f9e0c.tso7(f1-3s)e:xps.t1e5r9o-i6d5.hormones on pancreatic 120. OJ.bC.o,umM,ecJhDa,niFsrmasmefoSr.R.t,heBelplanRcHre,atiJcr., Loonncgongeecnkiecr eDf.fSe.c,tsElloioftttGh.eS.p,earnodxiCsooomke proliferatorWyeth.14,643. Toxicol Appl Pharmacol, 1997. 145(2): p. 425-36. 121. O48b9o4u7m,en1h.a0n,ceFsrapmaencSrXea,sCghriouwtT.h, vSioaloamornypTs.iEn.,ianhnidbiCtoorokmeJc.Ch.a,nEivsimd.enTcoexitchoalt `Appl Pharmacol, 1997. 146(1): p. 116-26. 122. M(aPrPmAaRpaoldpih,aM).aennddaCghoniiasntgi1n.hY.i,biPecrhoolxeisstoemroelpr7oallipfhear-ahtyodrr-oaxcyilvaasteedgreenceep(iCoYrPa7lApLh)a transcription. JLipidRes,2000. 41(4):p. 514-20. 54 p.67 123. sHuul,fonXaZte.exapnodsurHeuonD.hCep,atEofmfeactHsepofG2pecreflllu.orAorocchtaTnooxaitceol,an20d09p.er8f3l(u9)o:ropo.c8t5a1n-e 61. 124. Bdreucguswev,iaPr.eaacntdivDeaocxyageMn.,spCeocimepsarinishounmoafncyatnodtorxoidceintty liinvdeucceeldlsb. yIhntypJoMlioplidMeemdi,c 2005. 163): p. 483-92. 125. bClhaednd,eCr.aJ,ndChKeindnCey.Wd.u,eWtuo MinMge,steadndinKourgoanT.iLc.,arCsaenniccerinpodtrenitnikailnginwlaitveerr,. lBurng,J Cancer, 1992. 6(5)p:.888-92. 126. IakneddaK,aKw.a,niSsahitiohM.S,,, AKomiudlatiIv,arAiraateseaYn.a,lyTssiusbootfariAs.k,fCahctaoyrasmfaorK.h,epKautmocaedlalulH.a,r cciarrrchionsoisg.eneHseipsa:toalopgryo,sp1e9c9t3i.ve18o(b1s)e:rvp.at4i7o-5no3.f795patients with viral and alcoholic 127. YKa.to,FuJj,ikKaowbaunKe.,M,T,aNkaahkasahimuMrT..a, KTuarkoaiywaamaG.,T,TakIakdeadaK.,T,TakainmdotNoiiRt.s,u Sato Y., NheopramtailtiiszaCtipoantioeefnltesvbaytpehdlheebpoattoimcy&-ahnyddrlooxwy-ir2o'ndedoiexty.guCaannacseirnReesl,eve2l0s01i.n c6h1r0o2n4)i:c p. 8697-702. 128. Saito, 1, `Watanabe Miyamura Y., Koi S., T., Ohbayashi Onji M., Ohta A., Y. Harada H., Katayame and et al., Hepatitis C T., virus Kikuchi S., infection is aUsSsocAi,at1e9d90w.i8t7h(t1h7e):dpe.ve6l5o4p7m-e9n.tofhepatocellular carcinoma. Proc Natl Acad Sci 129. Waalkes, M.P., 4):p.2414. Cadmium carcinogenesis in review. J Inorg Biochem, 2000. 79(1- 130. OJloshenns,onJHF.,B.S,cahnuldgMencGG.e,eBJo.i0c,e, AJn.tDi.epJirl,epWthicytsrneeatrmeJ,nTtraavnidsrLi.sBk.f,oWrihleplaitaobmisliGa.Mr.y, caanndcmaleignrant lymphoma. CancerRes, 1995. S5(2):p. 294-1. 131. pCrheagnnga,neTKX.reBcaenpdtioerrgaeSnMe.e,xparnedssCiohnenin.,huCmonasntiltiuvteir:veianntedrrionsdtiavnideuarlecveapriioarbialintdy `7a1n0d,correlation with CYP2BS mRNA levels. Drug Metab Dispos, 2003. 31(1): p. 132. Flievrekroc,aAu.s,edBebdymlaonJg,-taenrdm uNsoezoifckpaheJ.n, obParibgimtealn)t.edRohzehplatCocheil,lu2l0a0r3.ad8e2(n4o)m:ap.of19t2h-e . 133. `Vbaazrbqiuteurza,tes1.). and Histol MHairsitgoiplathMo.lA,.,198L9i.ve4r(-3c)e:llp. a3d01e-n3o.ma in an epileptic man on 55 p.68 134. jMuenscntiilo,naMl,intKerructeollvuslkairkchoVm.m,unOimcoartiioYn.,inannodn-YgaemnaostaokxiicH,carcRionloegeonfebsilso.ckTeodxigcaopl Lett, 1995. 82-83: p. 701-6. 135. hMceoapn,anteXax.ciDan.rd,c2inMoagaenXnd.e,siSscu.oinWnYoe.rxFli,nddaJ3nGdasmWtRraoNneAngtseWr.oLal.,n,d20E0xp2pr.roet8(se1is)ni:so,npo.f6a4ng-da8.pjthuenicrtiornolgeeneisn 136. gMaap, jXu.nDc.t,ioMnaalXg.e,neSsu,i Yc.oFn.n,exWiannd2g, Wc.oLn,neaxninddW3ainnghCuMm.a,n Shiegpnaatloctarracnisndougcetnieosniosf World J Gastroenterol, 2003. (5): p. 946-50. 137. WExiplrgeesnsbiuosn, oKf.C,x26Ki,rkCpaxt3r2ickanCd.JC, 4K3nuegcahpeljuRn.c,tiWoinllpercokteeiKn.s, ianndnoTrrmaaulb aOn,d neoplastic human tissues. Int JCancer, 1992. S14): p. 522-9. 138. pRroobteeritns,kiPnJa.se acnadscaDdeerfoCr.lt,he Ttarregaemteinngt otfhecaRncaefr.MEOnKc-oEgRenKe,mi2t0o0g7e.n-2a6c(t22i)v:aiep.d 3291310. 139. oHlfiugv,earWpa.jn,udnJcodtnoielopsnhaPil.nDi.kn,itdeUnrpceeyhllaeupmliatBrh.eLcl.o,imalmTucrneoilscklaoitniJeosEni.nbyLviapteurroCf.al,unodarniSdnparGtaiegedsucyeo-mJDpPao,wulnIendyhsirbaiitntsiroaintn vivo, ToxicolSci, 2002. 68(2): p- 429-36. 140. pBKeuertnfenlneuhodorfyofo,Gc.tJ,a,nJoC.aotsLetiaeindGe.mr,alPEe.l,ccOoylnmsobemenolGCg..,usFaanmrdornaTkrheoDy.ms,faoHfratdenrsPeo.r,nalTKo.dx,oicsIiiwtnaygiofoHfra,m6mJmuoonnngtihuRs.m., Toxicol Sci, 2002. 69(1):p. 244-57. 141. lAibvdeerllactairfc,iAn.oGg.e,nePsriesat V., by. pTearpfelruoHr.oSo.c,iaannodicR.o_baecridf,roiad. Mp.e,rTohiesommoedulpartoiliofnoerfatroar.t "Toxicol Appl Pharmacol, 1991. 1113): p. 530-7. 142. rCoaltetloefya,lRt.eCr.e,dMhielplaetrocRy-tTe.,graonwdthCoarntodndiJf.fCe.r,enPteiratoixoinsionmetupmroolrifdeervateolrosp:mepno.tenPtrioagl, Clin Biol Res, 1995. 391: p. 295-303. 143. pPraollmiefre,raCtNor.,acHtisvuatMe.dHr,e,ceGprliofrfi-nalKp.h1a,,eRxparueesysiJo.nL.,inahnudmJaonhnlsiovenr.E.MFo.,lPPehraorxmiascoomle, 1998. 53(1)p:. 14-22. 144. JJoocrngseenns,en UNN.,, DBoosspierfRlu.oroLaelfkfyelrscoHm,poJuenndssenimApAai,r hSukamkaknebsaecmkenNEqu,aliatnyd? Environ Health Perspect, 2009. 117(6): p. 923-7. 56 p.69 145. Pelletier, receptors G. and ELAIfy M., alpha and beta in the Ihmummuannocryeiporcohduecmtiicvaelorlgoacnasl.izaJtiColninoEfndeosctrriongoeln Metab, 2000. 85(12): p. 4835-40. 146. Saunders, D.S., and P.T., Sharpe Millar MR., DRiMff.e,renWtiilallieaxmpsreKs.s,ioMnaocfpoheesrisroonge.n, rUercqeupalorrt aHl,,phIarvainnde pbreitmaaptreost.eiMnoslinHtuhmetReesptersoadn,d2m0a01l.e7r(3e)p:ropd.u2c2t7i-v3e6.systemofhuman and non-human 147. Thauymlaorn,tAisHsu.esa.nJdMAoll-AEznzdoacwriinFo.,l,Im2m0u00n.ol2o4c(a1l)i:s.ati1o4n5-o5f5o,estrogen receptor beta in 148. Brodie, A. Inkster S., and Mol Cell Endocrinol, 2001. 1Y7u8e1-2W).:, pA.r2o3m-a8t.ase expression in the human male. 149. sOp'eDromnanteolgle,neLs.,is.RoEbnedrotcsornReKv,M2,001J.on2e2s03M):Ep;. 28a9n-d31S8.impson ER. Estrogen and 150. aCanrdpiensot,roA.g,enRargeocepVt.o,rPsez(zEiRaV.l,phCaa,raEniRbCe.t,ala,nd Ando S., ERbeta2) Detectionofaromatase in human Leydig cell tumor, EurY Endocrinol, 2007. 17(2): p. 239-44. 151. aJic,iBn.a,rBicYe.l,lsSimlaecoknefDun,ctMioorntaelnsreensRpoMns,esandtoLogchsodloencyCs.tDo.k,inHiunmaanndpangcarseiartiinc Gastroenterology, 2001. 121(6):p. 1380-90. 152. Ochlosleenc,ystGokWin.in aBnudmhiespatJicM,enzyBmeus,ewcholeMstMer,ol and Mandel and lipoproteins JH, Plasma in ammonium perfluorooctanoate production 2. workers. Drug Chem Toxicol, 2000. 23(4): p. 603- 153. 1OHls.e,n,AGn.We.p,idGeimliloilloagnidc.F.Di.n,veBsutriglaetwioMn .oMf,rBeuprrroidsucJtMi.ve Mandel JS. and Mandel hormones in men with o40c7c)u:patp.io6n1a4l-2e2x.posure to perfluorooctanoic acid. J Occup Environ Med, 1998. 154. Cpaontcrraena,s.R1S98,9,KTuhmearpanV.c,reaasn,dppR.ob9b8i1-n1s01S0..L.,W.PBa.thSoaluongdiecrsB,asPihisolafdeDlipsheiaas.e. The 155. Hua, Y.P, carcinoma: LcliiannigcalLaJn,alyPseinsgofBG18,9 cLaisesS..QH,epaatnodbiHluiaarnygPJaFnc.r,eaPtanDcirseaItnit,c 2h00e9a.d 8(1):p. 79-84. 156. Geinlzlyimleasnd,, liFp.oDp.rotaenidns,MaannddelchoLlSe.st,erSole:ruamspteurdfylouofroococcutpaantoiiocnaalcliydexapnodsehdepmaetni.c AmJ Ind Med, 1996. 29(5): p. 560-8. 57 p70 157. Alexander, facility. B.H., Final Mortality Report. studyofworkers employed at April 26, 2001. Division the of E3nMviCrootntmaegnetaGlroavne `Occupational Health, School ofPublic Health, University of Minnesota. US EPA AR226-1030a018. 2001. 158. wDuoproknsts,ieEp1i9d5e5m-i20o0l1o.gyUSsuErvPeAilAlRan2c2e6.r1e3p0o7rt-:6.C2a0n0c5er incidence for Washington Zobel LR, Decline in perfluarooctanesulfonaie and other pobfioroclbyl 159. Olsen, G.W., Mair D.C., Church TR. Ellefson M.E., Reagen WXK., Boyd TM., Herron R.M., Medhdizadehkashi Z., Nobiletti J.B., Rios J.A., Butenhoff J.L., and c`Theecmhincoall,s20i0n8.Am4e2r(1i3c)a:np.Re49d89C-r9o5s.s adult blood donors, 2000-2006. Environ Sci : 160. Kudo, N. and Kawashima Y., Toxicity and toxicokineticsofperfluorooctanoic acid in humans andanimals.J Toxicol Sci, 2003. 28(2): p. 49-57. CONTAINS NO CB\ ss