Document 2qpJxYBboqweaJeoZKVeNK0K6

P-1 "McCrea, Deborah" < m ccrea@ taftlaw .com > 1 2 /28/20 09 01 :55 PM To N C IC O P P T @ E P A cc "Bilott, R obert A." < bilott@ taftlaw .com > bcc Subject 1 2 /2 8 /2 0 0 9 Letter To EPA D ocket C enter Taft/ Deborah McCrea / Legal Assistant Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1800 Cincinnati, Ohio 45202-3957 Tel: 513.381.2838 Fax: 513.381.0205 www.taftlaw.com / mccrea@taftlaw.com ovO c? f J.3 --O :.a .. co cn CO Internal Revenue Service Circular 230 Disclosure: As provided for in Treasury regulations, advice (if any) relating to federal taxes that is contained in this communication (including attachments) is not intended or written to be used, and cannot be used, for the purpose of (1) avoiding penalties under the Internal Revenue Code or (2) promoting, marketing or recommending to another party any transaction or matter addressed herein. This message may contain information that is attorney-client privileged, attorney work product or otherwise confidential. If you are not an intended recipient, use and disclosure of this message are prohibited. If you received this transmission in error, please notify the sender by reply e-mail and delete the message and any attachments. 2612_001.pdf *5 CONTAINS NO CBI P-2 Taft/ Taft Stettmkis & Holster UP 42S W alnut Street, Suite 1800 / Cincinnati. OH 45202-3957 / Tel: 513.381.2838 / Fax: 513.381.0205 / www.taftlaw.com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix / Beijing R08OtTA.Bn.0TT 513-357-9638 bflotl8 tafllavr.com December 28, 2009 FEDERAL EXPRESS EPA Docket Center, MC 2822T U.S. Environmental Protection Agency EPA W est, Room 3334 1301 Constitution Avenue, NW Washington, D.C. 20004 Re: Submission to IRIS and AR-226 Database For PFOA/PFOS: EPA-HQQ R D -20 0 3 -00 1 6 ________________________________________________ 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. Un, C.-Y., et, et. a i, "Investigation of the Associations Between Low-Dose Serum Perfluorinated Chemicals and Liver Enzymes in US Adults," Am. J. Gastroenterology (doi: 10.1038/ajg.2009.707)(Dec. 15,2009); and 2. Guruge, K. S., et a i, "Effect of Perfluorooctane Sulfonate (PFO S) on Influenza A Virus-Induced Mortality in Female B6C3F1 Mice," 34 J. Toxicol. Sci. 687-91 (2009). RABimdm Enclosures Very truly yours, w\ f b Robert A. Bilott 11587425.1 December 28,2009 Page 2 cc: Gloria Post (NJDEP)(w/ enei.) (via U.S. Mail) Helen Goeden (MDHXw/ end.) (via U.S. Mail) Lora Werner (ATSDR)(w/ end.) (via U.S. Mail) {W1405808.1} P-4 1 P-5 American Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 1 o f 11 h I I /./ / U \\ V Tiie American journal of i GASTROF.NTF.ROl. Liver and Biliary Tract V Subject Category: L iv e r a n d B ilia ry T r a c t A m J G astroenterol advance online publication 15 December 2009; doi: 10.1038/ ^ . 2009.707 Investigation o f the Associations Between Low-Dose Serum Perfluorinated Chemicals and Liver Enzymes in US Adults Chien-Yu lin MD, MPH1*3*3*2, Lian-Yu Lin MD, PhD4*2, Chih-Kang Chiang MD, PhD4, WeiJie Wang MD5, Yi-Ning Su MD, PhD6, Kuan-Yu Hung MD, PhD4*6 and Pau-Chung Chen MD, PhD3 'D epartm ent of Internal Medicine, En Chu Kong Hospital, Taipei County, Taiwan i n s titu te of Occupational Medicine an d Industrial Hygiene, National Taiwan University College o f Public Health, Thipei, Taiwan 3School o f Medicine, Fu J e n Catholic University, Taipei County, Taiwan 4Departm ent of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan 5D epartm ent o f In te rn a l Medicine, Taoyuan G eneral Hospital, Taoyuan, Taiwan 6Departm ent of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan TThese authors contribute equally to this work 8Co-correspondence . Correspondence: Pau-Chung Chen, MD, PhD, Institute of Occupational M ediane and Industrial Hygiene, National Taiwan University rvmpg. 0 f public H ealth, #17 Syujhou Road, T aipei 10055. Taiwan. E-mail: p c h e n @ n tu - e d u .tw Received 16 May 2009: Accepted 17 Novem ber 2009; Published online 15 December 2009. A b stract O BJECTIVES: P erflu orin ated ch em icals (PFC s) have b een larg ely u sed fo r years in a v a rie ty o f p ro d u cts w orld w id e. H ow ever, th e to x ic e ffe ct o f PFCs o n exp osu re to th e liv e r in th e g en eral p o p u latio n h as n o t y e t b een determ in ed. M ETH OD S: In th is stud y, 2 ,2 16 a d u lts (18 y ea rs o f age o r o ld er) w e re re cru ite d in a N a tio n a l H ealth an d N u tritio n E xam in ation S u rvey (N H AN ES) in 19 9 9 -2 0 0 0 an d 2003 200 4 to d eterm in e th e relatio n sh ip betw een serum le v e l o f PFCs an d th e levels o f liv er en zym es. T h e d ata w ere a d ju sted fo r a ll o th er con foun din g varian ts. R ESU LTS: A fte r p erfo rm in g m ath em atical a n alysis, w e d eterm in ed w h en seru m logp erflu o ro o cta n o ic a d d (PFO A) in creases in on e u n it, th e serum alan in e am in otran sferase (A L T ) co n ce n tra tio n (U A ) in c re a se s b y 1.8 6 u n its (95*> co n fid en ce in te rv a l (C l), 1.2 4 -2 .4 8 ; p= o.oc> 5), "I th e seru m lo g-Y -glu tam yitran sferase (G G T ) co n cen tra tio n (U /l) is 0.0 8 u n it h ig h e r (95X C l, 0 .0 5 -0 .11; P = o .o i9 ). T h e a sso d a tio n b etw een PFO A an d liv e r enzym es w as m o re evid en t in obese su b jects, as w e ll as su b jects w ith in su lin resistan ce an d /or m etab o lic syn d rom es. W hen d ivid in g th e seru m PFO A in to q u a rtiles in th e fu lly adjusted http://www.nature.com/ai g/\ oumal/vaop/ncurrent/full/ai g2009707a.html 12/22/2009 P-6 American Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 2 o f 11 models in subjects with a body mass index > 3okg/m 2, the ALT level trend across the serum PFOA quardles was significant (P=*o.oo3). CONCLUSIONS: (hi die basis o f these data, we conclude that a higher serum concentration o f PFOA m ay cause liver enzymes to increase abnormally in the general population, particularly in obese individuals. Further studies are warranted to clarify the casual relationship between PFCs and these liver enzymes. INTRODUCTION Perfluorinated chemicals (PFCs) consist o f a 4 -14 carbon backbone and a charged functional moiety (prim arily carboxyiate, sulfonate, or phosphorate). PFCs are man-made contemporary ehTM! that have only been used in the last half-century. Until recently, PFCs have been considered to be biologically inactive. Human and wildlife monitoring studies have identified PFCs worldwide. This finHmg led to efforts to better understand the hazards that may be inherent in these compounds, as w ell as die elobal distribution of PFCs GO- The two most w idely known PFCs are perfluorooctanoic ad d (PFOA) and perfluorooctane m ifato (PFOS), which belong to the 8-carbon backbone subgroup (l)- PFOA (primarily ammonium salt) can be used as a ' surfactant and an em ulsifier in the production o f polytetrafhioroethylene as well as other fluoropolymers and fluorpelastom ers. N-alkyl substituted derivatives o f PFOS have been used in a wide variety of industrial and consumer products including protective coatings for carpets and apparel, paper m atin g insecticide form ulations, and surfactants. Other PFCs, like perfluorononanoic ad d (PFNA), are used as' surfactants in the production o f fluoropolym er polyvinylidene fluoride. In contrast, perfluorohexane sulfonic add (PFHxS) is mostly used in carpet-treatment applications (g). Although the major manufacturer o f PFOS, 3M, has phased out o f production since 2002, the potential risk o f PFCs to humans still needs to be continually evaluated (3). The possible routes o f human exposure to PFCs are currently being investigated. Potential routes indude contaminated drinking water, dust, food, food packaging, and cookware. Animal studies have shown that PFCs are well absorbed orally but are poorly eliminated. PFCs mainly distribute extracellulariy. PFCs have a binding affinity for 0-Kpoproteins, as well as albumin and liver fatty add-binding protein. PFCs are not m etabolized and distributed through enterohepatic circulation to fixe serum and the kidney. However, PFCs are mainly distributed to the liver with concentrations being several timg high- thn serum concentrations (3). The half-life o f serum elimination o f PFCs in humans seems to be lmg The longer the carbon chain length, the longer PFCs persist in the body. For example, perfiuorobutane sulfonate (a 4-carbon PFC) is eliminated, on average, in a little over 1 month in humans, whereas PFOA and PFOS (8-carbon compounds, referred to as C8 compounds) are eliminated in 3.8 and 5.4 years, respectively. However, PFHxS (a 6-carbon compound) is an exception to the rule as it is elim inated'in 8.5 years (4). Exposure to PFCs at relatively high concentrations is associated w ife damage to liver function in animal m odels (5,6,2). The hepatotoxidty of PFOS and PFOA has been linked to the functions of these compounds as peroxisome proliferator-activated receptor oc (PPARa) agonist and thus, their ability to alter the expression o f genes involved in peroxisome proliferation, cell cycle control, and apoptosis (8,2,10)- In addition, other PFCs have also been shown to act as strong peroxisomal 0-oxidation inducers Ib human beings, the causal biochemical mechanisms o f hepatic toxicity after exposure to PFCs are not clearly defined. In occupational population, several studies have failed to establish a definite association between exposure to PFCs and adverse health effects (13,14,15,16). A few cross-sectional and longitudinal occupational studies have proposed a positive correlation among PFOA, serum lipid, and hver enzymes levels GUMS). In a non-worker population, examination of PFOA exposure through contaminated drinking water showed an insignificant correlation between abnormal nllmrai markers and th e serum PFOA concentration fio ) . The relationship between the serum PFC levels and liver enzymes in a nationally representative survey o f ariults has never been performed. We hypothesized that PFCs m ight have adverse effects on liver chemistry in th e general US population according to the large scale data set o f PFCs (20) and liver e n g in e profiles released by US National Health and Nutrition Examination Survey (NHANES) performed between 1999-2000 and 2003-2004. httD://w w w .nature.com /aie/ioum al/vaoD /ncurrent/full/ai22009707a.htm l 12/2212009 p.7 American Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 3 of 11 M ETHODS T h e te ^ e ^ a d o p te d fr o m 1999-2000 and 2003-2004 NHANES (PFCs were not measured in 2001 2002 NHANES). NHANES is a population-based survey designed to collect information on m e neaitn and nutrition o f the US household population and to obtain a representative sample of the noninstitutionalized civilian US population. The survey data are published biannually. Detailed contents of the NHANES 1999-2000 and 2003-2004 are available at the NHANES website (21)- In the 1999-2000 and 2003-2004 NHANES, the participants were older than 18 years of age, were not pregnant or nursing at the tim e of the examination, and were randomly assigned to receive examinations (n=10,224). Individuals were excluded based on the following criteria: individuals who had fasted <6h at the tim e of the examination (>1=1,802); individuals that were hepatitis B virus or hepatitis C virus carriers by serology (n=168); individuals in which data were not available for body weight, body height, ediFPitiffl! attainment, or smoking habits (71=113); and individuals without serum tests for PFCs, liver function or the five components of metabolic syndrome (11=5,925). A total of 2,216 participants were left for final analysis. A flow chart o f algorithm is shown in F ig u re i . In NHANES, a subset of the participants who received a morning fasting examination (71=1,114) had blood fasting and insulin levels measured. Insulin resistance status was determined for this subset o f participants. F igu re 1. Flow chart algorithm. HBV, hepatitis B virus; HCV, hepatitis C virus; PFCs, perfluorinated chemicals. Full figure and legend f125IQ Potential causes o f elevated liver enzymes In accordance with earlier studies (22,23), in addition to chronic hepatitis viral infection excluded from this study), we considered excessive alcohol consumption, smoking, and increased serum m arkers of iron stores as potential causes of elevated fiver enzymes. As obesity, insulin resistance, and m etabolic syndrome are strong predictors of increased fiver enzyme activity (M ), we also considered body m index (BMI), insulin resistance, and metabolic syndrome as potential confounders m hver function tests. The data were collected at all study sites by trained personnel using standardized procedures. Sociodemographic information such as age, gender, race/ethnicity, history of medication, and education level was recorded during the household interview. The education level was categorized as either above high school a diploma or high school diploma and below. The degree of alcohol intake was determined by a m estioim ^re^ind categorized into the following four categories: < i2drinte, <6odnnks, and >240 drinks a year). Smoking status was subdivided into an active smoker, a former sm o k er.o rh ^ never smoked by serum cotinine levels and as described on the questionnaire (25)- Serum iron and tota iron binding capacity were measured by the Beckman/Coulter LX20 analyzer. The transferrin saturation value was calculated using the following equation: (iron/total iron binding capacity) x 100%. Weight and height were measured by standard methods and digitally recorded. The BMI was calculated as weight (in kg) divided by the square of height (in m). The homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) index (tiie product o f basal glucose and insulin levels divided by 22.5) is regarded as a simple, inexpensive, and reliable surrogate http://www.nature.com/aig/ioumal/vaop/ncurTent/full/aig2009707a.html 12/22/2009 p.8 American'Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 4 o f 11 ' measure o f insulin resistance The serum glucose level and the plasma insulin concentration were determined by die hcxokinase enzymatical method and the immunoenzymometric assay, respectively. Search: j The National CholesterolEducatioirProgram ThiitiAuurrTreatm ent Panel fa v l has established guidelines for m etabolic syndrome with modifications for the different sexes. People who were 18 years old and above were defined as victim s o f metabolic syndrome if they meet at least three o f the following criteria: a w aist measurement >88 cm for women and >102 cm for men; serum triglyceride > 1.69 mM; serum high-density lipoprotein cholesterol <1.03 mM in men and <1.29 mM in women; systolic blood pressure > 130 mmHg or diastolic blood pressure > 85 mmHg or self-report o f anti-hypertensive medications; and fasting glucose level > 6.10 mM or self-report o f anti-hyperglycemic medications. Three, sometimes four, blood pressure determinations were taken with a mercury sphygmomanometer by a physician using the right arm unless otherwise specified. The averaged systolic and diastolic blood pressure was obtained The level o f triglycerides was measured enzymatically. Levels o f high-density lipoprotein cholesterol were measured after precipitation o f other lipoproteins using a Hitachi model 704 analyzer (Roche Diagnostics, Indianapolis, IN). Assessment o f liver enzyme parameters Total bilirubin, alanine aminotransferase (ALT'), aspartate aminotransferase, Y-glutamyltransferase (GGT), and alkaline phosphatase were the liver enzymes parameters available from NHANES. Bilirubin is m ostly derived from the metabolism o f hemoglobin. Increases in bilirubin are highly specific for diseases o f the liver or bile ducts fa8 ). Aspartate aminotransferase and ALT are enzymes presented in liver parenchymal cells. Both o f these enzymes are elevated during acute liver damage. Increased ALT activity has been used as a surrogate measure for the presence o f liver disease in earlier population-based studies (a g .2 3 .aA). GGT is found on the cell surface o f all cells. Particularly high concentrations o f GGT are found in the liver, the bile ducts, and the kidney. GGT increases occur earlier and persist longer than alkaline phosphatase in cholestatic disorders fa o ). In the study, w e used total bilirubin, ALT, and GGT as m arkers o f liver enzymes. Serum total bilirubin, GGT, and ALT levels were measured by enzymatic m ethods through automated biochemical profiling Beckman Synchron LX20). Total bilirubin was calculated in micromolar (pM) and GGT and ALT were calculated as units/litre (U/l). Assessm ent o f PFCs concentration Thirteen kinds o f PFCs are available in NHANES. However, in nine, over 70% of the PFCs are below the lim it o f detection. Therefore, w e used serum samples o f PFOA, PFOS, PFHxS, and PFNA for analysis in th is study. A b rief summary o f the PFCs assessment (30) is as follows: the serum was diluted with 0.1 M form ic add w ithout protein predpitation and a 100 pi aliquot of the serum was injected into a commercial colum n switching system for the determination of the concentration o f the analytes on a C18 solid-phase extraction column. This column was placed autom atically in front of a C8 analytical high-performance liquid chromatography column for chromatographic separation of the analytes. Detection and quantification were done by negative-ion TurbolonSpray ionization tandem m ass spectrometry and isotope-labeled internal standards. The lim it o f detection for PFOA was 0.2 ng/m l in the NHANES 1999 2000 and 0.1ng/m l in the NHANES 2003-2004 data sets. For PFOS, the lim it o f detection was 0.2 and 0.4 ng/ml in th e NHANES 1999-2000 and 2003-2004, respectively. The lim it o f detection for PFHxS w as 0.1 and 0.3 ng/ml in 1999-2000 and 2003-2004, respectively. For PFNA, the lim it of detection was 0 .2 ng/ml in 1999-2000 and o.in g/m l in 2003-2004. For concentrations below the detection limits (0.4% for PFOA, 0.1% for PFOS, 2.3% for PFHxS, and 1.796for PFNA), a value equal to the detection limit divided by the square root o f 2 was used (20). S tatistics PFCs concentration was expressed as the geometric mean with a 95% confidence interval (Cl). Log transform ation was performed for HOMA-IR, serum GGT, and PFCs levels with significant deviation from the norm al distribution before further analyses. A ll the log-transformed data in the study had a norm al distribution and no significant outliers were found. For linear regression models, we used an extended model approach for covariates adjustment o f potential confounders. Model 1 adjusted for age, gender, and race/ethnicity. Model 2 adjusted for age, gender, race/ethnicity, life style (smoking status, drinking status, and education level), and measurement data (BMI, HOMA-IR, metabolic syndrome, and iron saturation status). To avoid ` model-dependent association," the association was considered http://www.nature.com/aig/ioumal/vaop/ncunent/fiill/ajg2009707fl hfml 12/22/2009 P-9 American Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 5 o f 11 significant only when it remained statistically significant in all models. Each PFC was modeled separately. Sampling weights that accounted for unequal probabilities of selection, over-sampling, and non-response and variance estimation accounting for complex survey design were applied to all analyses by the Complex Sample Survey module of SPSS 13.0 for Windows XP (SPSS Inc. Chicago, IL). A mobile . examination center weight variable was created by assigning halfo f the 2-year weight for 1999-2000 and a s s i g n i n g half o f the 2-year weight for 2003-2004. RESULTS The basic demographic of the sample population is outlined in T a b le 1 . The study sample consisted of 1,076 men and 1,140 women. In accordance with an earlier NHANES study (20). the results indicate that males have a higher average concentration o f PFOS, PFOA, and PFHxS than females. Hispanic Americans have low er mean serum concentrations (ng/ml) for these three compounds than non-Hispanic whites and non-Hispanic blacks. In addition, the concentration o f PFOS and PFNA was higher in the more highly educated cohort The four PFCs were moderately correlated with one another. PFOA and PFOS were m ost strongly correlated, with a Spearman correlation coefficient of 0.68; PFHxS and PFNA were the least correlated at 0.24. T a b le 1 - B asic d em ograp h ics o f th e sam p le su b jects in clu d in g geom etric m ean s fe.e.) o f PFC con cen tration s. Full table a:WOtiMotimW--Wr,nBWv rWnH"*itn'I'm rurniw* wsati Unadjusted mean liver enzymes across quartiles of PFCs (ng/ml) are shown in T a b le 2. The serum ALT levels (U/l) increased across quartiles of PFOA and PFOS (Pvalue <0.001 and 0.030, respectively). sim ilar to ALT, the serum level of GOT (U /l) also increased across quartiles of PFOA and PFOS (Pvalue 0.012 and 0.010, respectively). The serum total bilirubin level QiM) increased across quartiles of PFHxS and PFNA (Pvalue <0.001 and 0.014, respectively). T a b le 2 - U n ad ju sted liv e r w iw m e s fs .e .l a cro ss q u a rtiles o f PFCs, P u ll ta h ie A summary of the association between serum concentration of log-PFCs (ng/ml) and liver enzymes after th e adjustment for other potential covariates is listed in T a b le s . When the four PFCs were entered into th e full regression models separately, one unit increase in serum log-PFOA concentration was associated w ith a 1.86 unit (95% C l, 1.24-2.48; P = o.oos) increase in serum ALT concentration (U/l), a 0.08 unit (9 5 & C l, 0.05-0.11; P = o.oi9 ) increase in serum log-GGT concentration (U/l). PFOS associated with ALT, w hereas PFNA was associated with total bilirubin with borderline statistical significance. The PFHxS http://w w w .nature.com /aisi/ioum al/vaoD /ncurrent/full/aic2009707a.htm l 12/22/2009 American 'Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 6 o f 11 concentration was not associated with liver function tests. When the four PFCs were entered into the full regression models at the same time, one unit increase in serum log-PFOA concentration was associated with a 2.19 unit (95% C l, 1.4-2.98; P " 0.009) increase in serum ALT concentration (U/l), a 0.15unit (95X C l, 0.11--0.19; P = o .o o i) increase in serum log-GGT concentration (U/l). One unit increase in serum logPFOS concentration was negatively associated with a 1.06 unit (95X C l, -1.3 3 to -0 .79; P= 0.001) decrease in serum total bilirubin concentration (pM) and was negatively associated with log-GGT concentration with borderline statistical significance. One unit increase in serum log-PFNA and logPFHxS concentration were associated with a 0.75 unit (P--0.004 n i 0.001, separately) increase in serum total bilirubin concentration (pM). T a b le n - lin e a r re g re s s io n c o e ffic ien ts f s .e .l o f b lo o d analyte with a u n it increase 111 Full table i lin e a r regression coefficients (s.e.) o f blood analytes (ALT and GGT) with a unit increase in log-PFOA in the different subpopulations o f the sample subjects are shown in T a b le A. Subjects with iron saturation above 50X were excluded because o f the small sample size (AT=19). The association between ALT and PFOA was significant n the following subgroups o f non-Hispanic Caucasians, individuals with a lower education level, higher BMI, non-smoking, lower alcohol consumption, higher HOMA-IR, and subjects diagnosed as having metabolic syndromes. On the other hand, the association between GGT and PFOA w as significant in subgroups o f non-Hispanic white, higher BMI, lower alcohol consumption, and higher HOMA-IR. Thi*. A - lin e a r re g re ssio n co e fficie n ts (s .e .) o f M ood an aivte with a unit in c re a s e in log-PFOA c o n c e n tra tio n s (n g /m l) in su b p o p u la tio n s o f th e sa m p le su b je cts. W hen dividing serum PFOA into quartiles in the fully adjusted models in subjects with BMI> 30 kg/m2, th e adjusted levels of ALT are shown in F ig u re a . The trend in ALT levels across quartiles o f serum PFOA was significant (P=o.oo3), whereas the trend in log-GGT was not significant. F ig u re a- The adjusted geometric m eans o fALT across qoartfles of the serum PFOA concentrations. The data are from the fully adjusted model (age. gender, race/ethnicity, smoking status, drinking status, education level, metabolic syndrom e, and iron saturation status) in subjects w ith a BMI &30 kg/ ms. The trends in ALT level across the quartiles of the scrum PFOA were significant ( P - 0.003). ALT, alanine am inotransferase; BMI, body m ass index; PFOA, perfluorooctanoic acid. h ttn ://w w w .nature.com /aie/ioum al/vaoD /ncurrent/full/aig2009707a.htm l 12/22/2009 American Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 7 o f 11 F ull figu re and legend fso lO OMWtoWWOMeWU D ISC U SSIO N To our knowledge, our report is the first to link serum PFC levels to liver enzymes in a nationally representative survey. In this study, we showed that increased serum PFOA concentrations are associated with elevated serum liver markers. PFOS and PFOA show liver toxicity in rodents and non-human primates fs .6.7). A number o f short-term studies focusing on rats and mice have shown that PFOS and PFOA are capable o f inducing peroxisome proliferation via activation o f PPAR<x (8,9,10 ,11). There is strong evidence to support the idea that PFOA-induced liver toxicity occurs through a PPARot agonistic mode o f action in rodents (8). However, adverse hepatic effects o f PFOA stQl exist in PPAR null mice f a il. These findings imply that PFOA may exert its toxic effects through both PPAR as w ell as other alternative pathways. As the key events of hepatic toxicity induced by PFOS are not consistent with a PPARa-agonistic model, the relevance of PPARcx-induced toxicity dependent on dose-response in humans is a current scientific debate (6,32). Some studies have used other PFCs that have been conducted in mice fix.12 V All of the compounds tested induced hepatomegaly and peroxisomal (1-oxidase activity. The potency of PFCs to cause hepatomegaly and peroxisomal p-oxidase activity is shown as follows: PFNA>PFOA>perfluoroheptanoic acid>perfluorohexanoic arid. These results indicate that the longer the perfluoroalkyl chain of the PFC, the higher the accumulation o f the compound in the mouse liver. In some occupational epidemiology studies, the association between PFCs exposure and abnormal liver function tests could not be established f ia .iA .is .i61. Only one cross-sectional occupational study fiv l observed a positive relationship between serum PFOA and GGT. In the meantime, one longitudinal occupational study (18) revealed that serum PFOA is related to total bilirubin (0.008 mg/dl decline/1,000ppb) and serum aspartate aminotransferase (0.35 units increase/1,000 ppb). A study was conducted to testify whether PFOA affected the non-working in a community located near a fluoropolymer production facility using either hematologic or biochemical clinical markers f io l. In this non-occupational population, the median serum PFOA was 354 ng/ml (an interquartile range, 184-571 ng/m l). This serum concentration of PFOA is higher than the level in the general US population (median o f 4.4 ng/ml). However, there was no significant correlation between abnormal clinical markers and serum PFOA concentration in that study. In our study, serum PFOA was associated with a change in both ALT and GGT, but not other PFCs. However, the potential biological significance between PFOA and liver enzymes is sm all and subdinical in this general population. As PFOA are metabolically inert, it is difficult to detect the sam e metabolic effect in the low exposure group of the general population and in the occupational studies presented with a high concentration level. One possible explanation is the bias of die "healthy worker effects," which states that severely ill or disabled people are more susceptible to PFOA exposure and excluded from employment. Another possible explanation is that the dose-response effects o f PFOA on liver enzymes may not be a linear relationship in humans. PFOA exerts the maximal effects at a low dose already and no further consistent or potentially relevant clinical changes occur at an even higher level. In our study, th e association between PFOA and liver enzymes was more evident in subjects suffering from obesity, insulin resistance, and the metabolic syndromes. However, there was no significant difference in regards to serum PFOA concentration between these groups. Studies o f gene expression profiles in rat livers treated with PFOA showed that the largest category of induced genes are those involved in metabolism and transport of lipids, particularly fatty acids (33,34)- An increase in lipid httD://www.nature.com/aie/ioumal/vaoo/ncurrent/full/aic2009707a.html 17/7.7/2009 American'Joumal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 8 o f 11 droplets due to alteration in lipoprotein metabolism after exposure to PFOA have also been observed (35). In obese individuals, a liver loaded with fat w ill increase liver enzymes and become insulin resistant fa61. Therefore, it is possible that PFOA m ay further increase the free Catty add accumulation in subjects with hepatic steatosis. Furthermore, there m ight be some synergistic effects on hepatotocridty between PFOA and obesity. PFOA is prim arily distributed to the liver in rats (3). In the liver, m ultiple proteins from th e cytosol, nuclei, and mitochondria fractions are capable o f specifically landing PFOA (3 2 )Another possible explanation is that in obese subjects with hepatic steatosis, intrahepatic PFOA accumulation, m ight be higher than non-obese subjects despite the sam e serum level. W ith the higher accumulation o f PFOA in the liver, the effect o f PFOA on liver is more evident. We found that the association between PFOA and fiver enzymes was m ore evident among non-smokers and those with lower alcohol consumption. One possible explanation is that the effect o f PFOA on liver enzymes is much weaker than the effect o f alcohol and tobacco smoke. When considering the hepatotoxic o f PFOA in the smoking or higher alcohol consumption population, the trend is too sm all to become statistic significant. Alternatively, it is also possible that the association between PFOA and smoking tobacco or drinking alcohol is opposite to the possible synergistic effect between PFOA and obesity mentioned above. There are several limitations o f our study. First, the cross-sectional design does not perm it any causal inference. Second, we did not indude other environmental chemicals, which may be important covariates or explanatory variables for the outcomes o f our study. Third, we did not take into account any rredfratiTM that may cause elevated ALT or GGT. Fourth, a common physiology could influence both serum PFCs and liver enzymes rather than exposure affecting outcome. Finally, the status of the liver tissue was not available to determine hepatic steatosis, inflammation, or fibrosis. In conclusion, ning the NHANES data from the US adult population, w e found that a higher serum concentration o f PFOA was associated with elevated liver enzymes. These findings provide dues to the adverse effects o f low-dose PFOA in humans. Although the potential biological significance between PFOA and liver enzymes is small and subdinical in the general US population, our data suggest that it ^ would be prudent to monitor the liver enzymes of people with low level exposure of PFOA, particularly in subjects who are obese. Further studies are needed to confirm these findings and to clarify whether these associations are causal. C O N FL IC T O F IN TE R E ST G u a ra n to r o f th e a rtic le : Pau-Chung Chen, MD, PhD. S p e c ific a u th o r co n trib u tio n s: Designed the study, drafted the artide, and interpretated the data: Chien-Yu Lin; designed the study's analytic strategy and helped conduct the literature review: Lian-Yu Lin; helped conduct the literature review: Chih-Kang Chiang, W ei-Jie Wang, and Yi-Ning Su; helped conduct the literature review, approved the analytic strategy, and approval o f the final version: Kuan-Yu Hung; contributed to the study's conception, reviewed the study, and final approval of the version to be published: Pau-Chung Chen. F in a n d a l su p p o rt: This was an investigator-initiated unfunded study. A ll authors had access to the data and the statistical analysis report. Each author approved the final article and attested to the validity o f the results. P o te n tia l co m p e tin g in te re s ts : None. STU D Y H IG H LIG H TS partner ofAGORA, HINAR1, PARK, INASP, C rouR ef and COUNTER http://w w w .nature.com /aig/iouraal/vaop/ncurrcnt/full/aie2009707a.htm l 12/22/2009 American Journal o f Gastroenterology - Investigation o f the Associations Between Low-Dose Se... Page 9 o f! 1 R eferences 1. Houde M , Martin JW, Letcher RJ et al. Biological monitoring o f polyfluoroalkyl substances: a review. Environ S d Technol 2006;40:3463-3473.1A rtic le | P iib M ed | C h em P o rt | 2. Prevedouros K, Cousins IT, Buck RC et al. Sources, fate and transport of perfluorocarboxylates Environ S d Technol 2006;40:32-44. | A rtic le | P ubM ed \C h em P o rt | 3. Lau C, Anitole K, Hodes C et al. Perfluoroalkyl acids: a review of monitoring and toxicological findings. Toxicol S d 2007;99:366-394.1A rtic le | P ubM ed | C h em P o rt | 4. Olsen GW, Burris JM, Ehresman DJ et al. 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Biochem Pharmacol 2002;63:1893-1900. i Article IPubMed I ChemPort | http://w w w .nature.com /ajg/joum al/vaop/ncurrent/ftill/ajg2009707a.htm l 12/22/2009 American Journal o f Gastroenterology - Investigation o f the Associations Between Low -D ose... Page 11 o f 11 ' 32. Shipley JM, Hurst CH, Tanaka SS et al. Trans-activation o f PPAR and induction o f PPAR target genes by perfluorooctane-based chemicals. Toxicol Sci 2004:80:151 160.1 Article ] PubMed | CbemPort | 33. Guruge KS, Yeung LWY, Yamanaka N et a l Gene expression profiles in rat liver treated with perfluorooctaonic acid (PFOA). Toxicol Sci 2006;89:93-107. | A rtic le | P ubM ed 34. Rosen MB, Thibodeaux JR, Wood CR et al. Gene expression profiling in the lung and liver of PFOA exposed mouse fetuses. Toxicology 2007;239:15-33. | A rtic le | P ubM ed | C h em P o rt | 35. Peters JM , Hennuyer N, Staels B et al. Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha-deficient mice. J Biol Chem 19975272:27307 27312. { A r tic le I P u b M ed { IS I j C h em P o rt | 36. Tilg H, Moschen AR. Insulin resistance, inflammation, and non-alcoholic fatty liver disease. Trends Endocrinol Metab 2008;19:371-379. | A rtic le ] P ub M ed | C h em P o rt | 37. Han X, Kemper RA, Jepson GW. Subcellular distribution and protein binding o f perfluorooctanoic acid in rat liver and kidney. Drug Chem Toxicol 2005;28:197-209. | P ubM ed 1C h em P o rt | Acknow ledgm ents W e thank the many people who have contributed to the National Health and Nutrition Examination Survey data w e have examined, including all o f the anonymous participants in the study. We are particularly grateful to Antonia M Calafot, who carried out the laboratory assays of PFC concentration at the Division o f Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention. http://w w w .nature.com /aig/ioum al/vaop/ncurrent/fiill/aiB 2009707a.him l 17/79/7000 p. 16 2 The Journal o f Toxicological Sciences (J. Toxicol. Sci.l Vol.34, N o.6,687-691, 2009 687 Letter Effect of perfluorooctane sulfonate (PFOS) on influenza A virus-induced mortality in female B6C3F1 mice Keerthi S. Guruge1, Hirokazu Hikono3, Nobuaki Shimada1, Kenji Murakami, Jun Hasegawa1, Leo W.Y. Yeung4, Noriko Yamanaka1and Nobuyoshi Yamashita4 `Safety Research Team. `Research Teamfo r Advanced Biologicals. `Research Teamfo r Viral Diseases, National Institute o fAnimal Health, Kannondai 3-1-5 Tsukuba, Ibaraki 305-0856, Japan 4National Institute o fAdvanced Industrial Science and Technology. Onogawa 16-1, Tsukuba. Ibaraki 305-8569, Japan {Received July 23, 2009; Accepted August 16,2009) ABSTRACT -- Recent studies showed that perfluorooctane sulfonate (PFOS) affects the mammalian immune system at levels reportedly found in the general human population. It has been demonstrated that exposure to immunotoxic chemicals may diminish the host resistance o f animals to various pathogenic challenges and enhance mortality. Therefore, the current study was carried out to characterize the effect of a 21 day pre-administration of zero, 5, or 25 pg PFOS/kg bw/day in female B6C3F1 mice on host resist ance to influenza A virus infection. At the end of PFOS exposure, body/orgati weights did not significant ly change whereas PFOS distribution in blood plasma, spleen, thymus and lung was dose-dependentiy increased. PFOS exposure in mice resulted a significant increase in emaciation and mortality in response to influenza A virus. The effective plasma concentrations in female mice were at least several fold lower than reported mean blood PFOS levels from occupationally exposed humans, and fell in the upper range of blood concentrations of PFOS in the normal human population and in a wide range of wild animals. Hence, it should be important to clarify the precise mechanism(s) for excess mortality observed in the high dose group. Key words: PFOS, Bioaccumulation, Immunotoxic, Host resistance, Mortality INTRODUCTION Perfluorooctane sulfonate (PFOS) is one of the new ly listed Persistent Organic Pollutants (POPs) under the Stockholm Convention. Recent studies have demonstrat ed that exposure to perfluorinated alkyl substances can modulate rodent humoral and cellular immune functions (Dewitt el al, 2008; Peden-Adams et al., 2008). Suppres sion o f the primary antibody response was reported in mice exposed to PFOS, at serum concentrations 14 times lower than the average concentrations of occupationally exposed workers, and in the upper range of levels report ed for the general population (Peden-Adams et at., 2008). Exposure to xenobiotics and the resultant alteration of immune function may effect in a modification o f an organ ism 's ability to resist infectious disease. Several studies have shown that persistent pollutants enhance susceptibil ity to viral, bacterial, parasitic and neoplastic challenges (Burleson et al., 1996). However, no studies have been done to determine the level of susceptibility to pathogens that altered due to PFOS exposure. The rodent influenza virus model has been widely used to determine the immu notoxic effects of persistent chemicals such as dioxins on viral host resistance (Burleson et al., 1996; Nohara et al., 2002). This study represents the first preliminary investi gation of increase mortality caused by influenza A virus infection in mice pre-exposed to PFOS. MATERIALS AND METHODS Animals Female B6C3FI mice were obtained from Japan SLC Inc. (Shizuoka, Japan). They were acclimatized to the environment for at least I week before both the prelim inary virus test (9- to 10-weeks old) and PFOS/virus administration (6- to 7-weeks old). Mice were housed in HEPA filtered disposable cages (Inocage; Oriental giken, Tokyo, Japan) in a light- (12 hr light-dark cycle), tempera- Correspondence: Keerthi S. Guruge (E-mail; guruge@affrc.go.jp) 688 K.S. Guruge et at. turc- (22 2C) and humidity- (SO 10%) controlled Bio Safety Level-2 facility at the National Institute o f Animal Health (NIAH), Japan. They received food and water ad libitum. Bedding, food, and water were changed weekly. All procedures used in this experiment were reviewed and approved by the biosafety, animal care and ethical com mittees o f NIAH, Japan. Influenza virus infection Mouse adapted influenza virus, A/PR/8/34 (HIND, (obtained from Dr. Hideki Hasegawa of National Insti tute o f Infectious Diseases, Japan) was used as the infec tious agent. Aliquots (0.1 ml) o f 1 x JO7plaque forming units (pfu) o f virus were prepared and stored in -80*C. Virus dilution with phosphate buffer saline (PBS) was carried out immediately prior to use. Mice were anes thetized by intraperitoneal injection (i.p.) of Avertin and intranasally infected with 30 pi o f virus suspension. As a preliminary test, susceptibility to the current passage of influenza A virus was examined using 9- to 10-week old female B6C3F1 mice in order to achieve a known lethal dose in control animals. Virus diluted at eight concentra tions between 6.25 and 800 pfu was tested in 7 mice for each concentration. The general appearance and weight of infected mice was evaluated for twenty days. The prelim inary test showed that between 100 and 200 pfu o f virus inoculation caused approximately 40% mortality (data not showing). No mortality was observed using lower virus regimes tested on female mice. Therefore, the effect of PFOS on the mortality was assessed using 21 days of PFOS exposure followed by 100 pfu o f virus infection. PFOS exposure The potassium salt o f PFOS (CAS number 2795-39-3) was acquired from Fluka Chemical (Steinlieim, Switzer land). Stock solution was prepared in Milli-Q water con taining 0.5% Tween 20 at a concentration o f 0.2 mg/ml. Dosing solutions were prepared weekly by serial dilu tion. Control mice received Milli-Q water containing only 0.5% Tween 20. Mice were dosed by gavage for 21 days with either vehicle. 5. or 25 pg PFOS/kg body weight/day prior to virus inoculation. Experimental design for measuring PFOS effect on host resistance Mice (6- to 7-weeks old) were randomly divided into 30 animals/dose group and 6 animals/cage. They were weighed weekly during the PFOS dosing period. At the end o f a 21 day PFOS exposure, 3 animals from each group were randomly sacrificed by pentobarbital over dose, and blood samples were collected via cardiac punc ture using heparinized syringes. Blood plasma was sepa rated by centrifugation at 3,000 rpm for 10 min and kept at -20C until PFOS residue analysis. Liver, kidney, lung, spleen and thymus were collected and weighed. Organs were kept at -20C until PFOS residue analysis. The remaining mice were anesthetized by i.p. injection o f Avertin and intranasally infected with 100 pfu (in 30 pi o f PBS) influenza A virus suspension. Following virus inoculation, mice were observed for health conditions and mortality twice a day and weight was measured daily for twenty days. PFOS residue analysis The blood plasma samples were thawed at room tem perature, fortified with l,C-PFOS (Wellington Laborato ries, Guelph, Canada), and extracted. Analysis o f PFOS in piasma (0.2 ml) was carried out using the ion pairing method (Guruge et a/., 2005: Yamashita et al., 2004). The individual or pooled organ portions (approximately 0.1 g) were thawed, fortified with uC-PFOS and then 1 ml of Milli-Q water was added followed by homogenization with a Micro Homogenizing System (Tomy Seiko Inc., Tokyo, Japan) at 3,000 rpm for 3 min. The entire homoge nate was used for the extraction similar to the procedure used with plasma. Matrix recoveries o f l3C-PFOS in plas ma and organs were 93 8 (mean S.D.) and 96 8%. respectively. The limit o f quantifications for plasma and organs was 0.2 ng/tnl and 0.25 ng/g wet weight. The con centrations o f PFOS in the samples were not corrected for recoveries. RESULTS At foe end o f the PFOS exposure, foe measured organ masses were not significantly different among PFOStreated and control groups (Table 1).The distribution o f PFOS in foe body increased with repeated gavage (Table 2). At the end o f PFOS exposure, mean plasma concentra tion was significantly higher in 25 pg/kg exposure group compared to that in the 5 pg/kg exposure group. PFOSdistribution in the various samples ranked as follows: lung ~ plasma > spleen = thymus. Fig. 1 shows the change in body weight during PFOS gavage and virus infection. There was no significant weight change due to PFOS exposure alone. The mean body weights of both PFOS-treated groups have great er decreasing tendency compared with the control group from post-infection days 4 to 11, however, statistically significant differences were observed only at day 9 (P = 0.04 for 5 pg/kg group and P = 0.02 for 25 pg/kg group, Dunnett's test). Vol. 34 No. 6 689 PFOS effect on virus-induced mortality in mice Table 1. Mean body (g) and organ mass* o f adult B6C3F1 female mice dosed with PFOS for 21 days1 Dose Body weight Spleen` Thymus ` Liver` K idney` Lung` Control 20.4 0.95 0.34 0.04 0.36 0.07 3.7 0.24 1.1 0.04 0.79 0.13 5 pg/kg'bw /day 20.1 0 .4 5 0.31 0 .0 3 0.32 0.05 3.7 0.13 1.1 0 .0 3 0.67 a o s 25 pg/kg/bw/day 20.2 0.33 0.31 0 .0 2 0.26 0.03 3.9 0.27 12 0 .0 3 0.67 0.03 O rg an m ass = organ w eight (gV body w eight (g) x 100. ` D ata are reported as m ean S.E. (n = 3). The survival rate at each time point of female B6C3FI mice exposed to PFOS followed by infection with virus is shown in Fig. 2. The mean survival time based on 20 days observation period was 14.1,13.2, and 11.4 days in the control, 5 and 25 pg PFOS/kg bw/day groups, respec tively. There was no significant difference in survival time among the three groups (P >0.05, Kaplan-Meier log-rank test). The survival rate o f the mice on day 20 alter virus infection was 46%, 30% and 17% in the control, 5 and 25 pg PFOS/kg bw/day groups, respectively. A signifi cantly dose-dependent increase in mortality was observed with PFOS exposure {P - 0.014, Cochran-Armitage trend test). Additionally, the eventual survival rate differed sig nificantly between 25 pg PFOS/kg bw/day group and control group (P = 0.035. logistic regression Wald test). There was no statistically significant difference between 5 pg PFOS/kg bw/day group and control group (P = 0.28, logistic regression Wald test). DISCUSSION Exposure to higher levels of PFOS often produces sig nificant reduction in rodent body weight and this may influence their immunological outcome. Therefore, we have selected only two PFOS doses (5 and 25 pg PFOS/ kg bw/day) for this experiment to try to reproduce envi ronmentally existing blood levels in humans and wildlife. In our exposure regime, there was no significant change in body weight observed among all three mice groups at the end o f PFOS exposure. Likewise, no significant change in organ mass was found for immune-responsive organs. The results for body and organ weight gain were similar in both male and female mice exposed to PFOS at compa rable exposure levels (Peden-Adams et al., 2008). Never theless, the body weight reduction during virus infection was clearly greater in PFOS dosed mice compared to the controls. Therefore. PFOS accumulation is likely super imposed on viral illness in mice to increase mortality. The mean plasma levels of PFOS at the beginning of virus infection were 189 14 and 670 47 ng/ml wet wt. in 5 and 25 pg PFOS/kg bw/day exposed groups, respec- T able 2. M ean S.D . co n cen tratio n (w et w eight) in . blo o d plasm a an d o rg an s in ad u lt B 6C 3F1 fem ale m ice exposed to PFO S Sample type Dose Concentration* Plasma Control 2.1 0.3 5 pg/kg bw/day 189 14 25 pg/kg bw/day 670 47 Spleen ` Control <0.25 5 pg/kg bw/day 84 25 pg/kg bw/day 260 Thymus Control <0.25 5 pg/kg bw/day 60 5 2 25 pg/kg bw/day 260 68 Lung Control 1.2 0.2 5 pg/kg bw/day 190 6 5 25 pg/kg bw/day 970 145 1Concentrations in plasma are given in ng/ml, in other organs arc given in ng/g. Three mice for each dose were analyzed. ` Pooled samples. tively (Table 1). Our data are similar to recently pub lished senim levels in female B6C3F1 mice exposed to a comparable PFOS exposure regime (Peden-Adams et al., 2008). The PFOS concentrations found in mice plasma were within the range o f those reported in sera for occu pationally exposed workers in Decatur, Iowa, USA (range 145 - 3,490 ng/ml wet wt.), Antwerp, Belgium (mean: 1,480 ng/ml), adult donors (range 4 - 1,656 ng/ml wet wt.) in the USA and the upper level found in the normal population in China (Olsen et al., 1999, 2003 and 2007; Jin et al., 2007). Several studies reported that concentra tions o f PFOS in the blood of wildlife were similar to our data for mice treated with 5 and 25 pg PFOS/kg bw/day. In the late I990's, mean PFOS concentrations found in blood o f ringed seals and bottlenose dolphins were 242 and 143 ng/ml wet wt. in European waters (Kannan et al., Voi. 34 No. 6 690 K.S. Guruge et al Fig. 1. Effect o f PFOS on mean body w eight in mice challenged with influenza A vims. Adult fem ale B6C3F1 m ice dosed with 0, 5 and 25 p g PFO S/kg bw /day for 21 days and infected intranasally. N um ber o f anim als a t day 0 infection: control = 24, 5 l>g PFOS/kg bw/day - 2 3 .2 5 fig PFOS/kg bw/day - 2 4 . ' Significantly different (P < 0.05, D unnett's test) from the control group. Standard deviation o f body weight during whole experiment was 0.30-3.8,0.25-5.01 and 0.37-479 for mice dosed with 0 ,5 and 25 fig PFOS/kg bw/day, respectively. 0 2 4 6 8 10 12 14 16 18 20 Day after infection F ig. 2. Effect o f PFOS on host resistance to influenza A vims. Adult female B6C3FI mice dosed with 0, 5 and 25 fig PFOS/kg bw/ day for 21 days and infected intranasally. Number o f animals at day 0 infection: control = 2 4 ,5 fig PFOS/kg bw/day = 23, 25 fig PFOS/kg bw/day = 24. ' Significantly different (P < 0.05, logistic regression Wald test) from the control group. 2001, 2002).The PFOS concentrations in plasma o f bottlenose dolphins from the Gulf of Mexico and die Atlan tic Ocean were ranged from 46 to 3,073 ng/g wet wt (Houde et ai., 2005). Interestingly, a significant associa tion between infectious disease and elevated PFOS con centration in the livers o f sea otters has been described (Kannan et a!., 2006). As shown in Fig. 2, we observed that PFOS administration resulted in a dose-dependent enhanced mortality. In particular, 25 fig PFOS/kg bw/day exposed group exhibited a significantly reduced survival rate compared to the control group. It is important to note that female mice treated with similar PFOS levels to this study had suppressed plas ma IgM antibody production (Peden-Adams et a!., 2008). Numerous components o f the immune defense system such as cytotoxic T cells, NK cells and other humoral responses are activated and play an important role dur ing a viral infection. Hence, any alteration of this mcch- Vbl. 34No.6 691 PFOS effect on virus-induced mortality in mice anism may contribute to suppression o f viral clearance caused by exposure to PFOS. It has been suggested that slight alterations of several immunological functions may together result in significant immunosuppression that can be detected as an increased susceptibility to infectious dis eases and measured by host resistance models (Burleson et al., 1996). Therefore, the mechanism(s) o f enhanced mortality may be related to several important immunolog ical functions which response to viral clearance. In conclusion, this is the first study that shows the effect of PFOS on host resistance to a pathogen in labora tory animals. Our results suggest that PFOS accumulation may associated with mortality in influenza virus-infect ed female mice. It will be essential to examine numerous immunological endpoints before concluding that PFOS accumulation directly leads to an alteration in host resist ance to pathogens in animals. ACKNOWLEDGMENTS This study was supported in part by a Grant-in-aid from the Global Environment Conservation Research Fund to Dr. K.SG by the Environmental Ministry o f Japan (Year 2004-2008). The authors thank Dr. Hideki Hasegawa of National Institute o f Infectious Diseases, Japan for pro viding mouse-adapted influenza virus. Mr. Hitoshi Ohashi and Mr. Akira lino of NIAH, Japan are thanked for their invaluable assistance during the experiment. Prof. Paul Lam o f City University of Hong Kong is acknowledged for providing postgraduate studentship to LWYY. REFERENCES Burleson, G.R., Lebrec. H., Yung, Y.G., Ibanes, J.D., Pennington, K..N. and Bimbaum, L.S. (1996): Effect of2,3.7.8-tetrachlorodibenzo-p-dioxin (TCDD) on influenza virus host resistance in mice. Fundam.AppL Toxicol., 29,40-47. D ew itt, J.C ., Copeland, C.B., Sirynar, M.J. and Luebke, R.W. (2008): Perfluorooctanoic acid-induced immunomodularion in adult CS7BL/6J or C57BL/6N female mice. Environ. 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