Document rpz1MnZjw26jKqr0557QNxQx0

Cee Bative report Hedin T0225 Be f seamed +o SRPT T0245 -35 Xenochemical Receptor trans-Activation by Perfluorooctane-based Chemicals T-29s" 35 AR226- 1723 oF or T-6375.357 E36 9 en. 3 piE-BY T-1563:) Contributing Scientists at Boston University: Jonathan Shipley Megon Walker DaJ.vWaixmdan dated12/08/2000 between 3M Medical Department, Corporate Toxicology 3M Center 220-2E-02 Aun: Andrew M. Seacat, Ph.D. Tel. 651-575-3161; Fax. 651-733-1773 and Boston University Laboratory of Dr. David J. Waxman Department of Biology 5 Cummington St. Boston MA 02215 Tel. 617-353-7401; Fax. 617-353-7404; Email: djw@bu.edu Report date: September 12, 2001 3 &Eon = 83 ~ Sn THE & TE 2194 3M-BU Report.doc - 9/12/01 ~ Page | `Summary - Peroxisome proliferator-activated receptors (PPAR) are ligandactivated transcription factors that activate target genes involved in lipid metabolism, energy homeostasis and cell differentiation in response to diverse compounds, including environmental chemicals. The liver-expressed receptor PPAR. mediates peroxisome proliferative responses associated with rodent hepatocarcinogenesis, while PPARY plays a central role in various physiological and pathophysiological processes in multiple tissues, including adipogenesis, angiogenesis, and macrophage foam cell differentiation. Previous studies have established that certain perfluorooctanesulfonamide-based chemicals (PFOSAs) alter lipid metabolism and are weak hepatic peroxisome proliferators in rodents, suggesting that they may activate PPARy or PPAR, respectively. The present study investigates this question and characterizes the activation of PPAR. and PPAR by PFOSAS using a luciferase reporter gene frans-activation assay. Perfluorooctanesulfonate (PFOS), an end-stage metabolite common to several PFOSASs, was found to activate both PPARc: and PPAR. Dose-response studies suggested that ~2-4-fold higher concentrations of PFOS may be required to activate human PPARe: compared to mouse PPAR; however, additional data is required to validate this finding. Whereas PFOS treatment led to maximal activation of PPARc:, PPARY was only partially activated at saturating concentrations of the fluorochemical. Perfluorooctanesulfonamide (FOSA) displayed activity towards both PPARc: and PPARy, however, cellular toxicity associated with this compound resulted in dose-response profiles that varied between experiments. Studies of 2-N-ethylperfluorooctanesulfonamidoethyl alcohol (N-EFOSE) were less informative due to its insolubility, although weak PPAR0 and PPARY activation was seen in some experiments. More severe solubility problems were encountered with two amido acetate derivatives of 3M-BU Report.doc - 9/12/01 - Page 2 PFOS, designated N-EtFOSAA and FOSAA, precluding an evaluation of PPAR activation by these compounds. The present finding that PFOS and FOSA activate both PPARc: and PPARYhelps explain some of the diverse biological responses associated with these fluorochemicals. Further investigation of the interaction of PFOSAs with PPARs, with particular reference to possible species differences in receptor responsiveness and the identification of downstream target genes, may aid in the evaluationof human and environmental risks associated with exposure to this class of perfluorochemicals. 3M-BU Report.doc - 9/12/01 - Page 3 Introduction Perfluorooctanesulfonate (PFOS; C,F;,SOy) and perfluorooctanesulfonamide derivatives (PFOSAs) constitute a class of fluorochemicals (FCs) which have been used in many industrial and consumer applications as powerful surfactants or components of `products which provide oil and water-resistant properties to paper and fabrics. In May 2000, 3M Company announced that it would voluntarily cease producing PFOS due to concerns about its biopersistence and its widespread exposure to human populations and environmental distribution (1-3). A recent epidemiological study, that documents human occupational exposure to PFOS, revealed no associations of PFOS with hepatic: enzyme and lipid clinical chemistry parameters (4). The 3M Company is presently engaged in an extensive research effort to characterize the metabolic actions and potential toxicities associated with these FCs, with the goal of evaluating the risk associated with exposures at both occupational and environmental levels. PFOS and related chemicals can have a variety of metabolic and other effects. PFOS and N-ethyl perfluorooctanesulfonamidoethanol (N-ECFOSE) stimulate hepatic peroxisome proliferation when administered to rodents. Perfluorooctanesulfonamide (FOSA) is a suspect peroxisome proliferator, as well as a potent uncoupler of mitochondrial oxidative phosphorylation (5). PFOS is the ultimate metabolite of these FCs in mammalian systems, where it tends to accumulate in the liver (6,7). The peroxisome proliferative response of PFOSAs in rodent species is weaker than the peroxisome proliferation response of perfluorooctanoate (PFOA), a chemically distinct EC (8). PFOSAS can also increase liver triglycerides and free cholesterol levels, lower serum cholesterol, cause hypolipidemia, increase liver weight and cause anorexia (9-13). These diverse biological effects of PFOSAs are similar to those caused by long-chain 3- 3M-BU Report.doc - 9/12/01 - Page 4 thia fatty acids, non-B-oxidizable fatty acid analogs that induce peroxisome proliferation a4). Peroxisome proliferation and certain other metabolic effects of PFOSAs may be a consequence of the activation of the nuclear receptors PPARc: and/or PPARY. PPARs are members of the nuclear receptor superfamily and mediate a broad range of biological responses to fatty acids, their synthetic analogs and structurally diverse lipophilic chemicals containing an acidic group (15-17). Peroxisomes are organelles that are involved in fatty acid oxidation, cholesterol metabolism, and hydrogen peroxidelinked respiration (18). Activation of PPARG: and PPARY leads to altered regulation of distinct sets of genes involved in lipid metabolism and homeostasis, peroxisome proliferation and cell growth (19). Long-term exposure of rodents to peroxisome proliferator compounds causes an increased incidence of liver tumors (20), raising concerns about the safety of these compounds for humans and other species (21,22). In contrast to rats and mice, guinea pigs and other species, including cynomolgus monkeys (13), show little or no evidence for peroxisome proliferation when treated with FCs or other peroxisome proliferating compounds. Studies with human hepatocytes indicate that humans are also likely to be poorly responsive to hepatic peroxisome proliferators. `This species-dependence reflects several factors, one of which is the expression of a substantially higher level of PPARct in rat and mouse liver compared to humans and other unresponsive species (23). A second important factor contributing to the species-specificity of peroxisome proliferative responses is the lower intrinsic responsiveness of human PPARs compared to rodent PPARs to some, but not all, peroxisome proliferator chemicals (24, 25). Using a reporter gene assay involving transfection into COS-1 monkey kidney cells of 3M-BUReport.doc - 9/12/01 - Page 5 PPARG: together with a PPARo-activated reporter gene, PFOA has been shown to trans-activate both mouse and human PPAR (10). PFOA maximally activated mouse PPARG: at 5-10 uM, whereas human PPARa: required somewhat higher concentrations (10-20 jM PFOA) for maximal stimulation. Other studies confirm the differential responsiveness of human and mouse PPAR to a subsetof PPAR: activators. For example, 5 to 6-fold higher concentrationsofthe classical peroxisome proliferator Wy14,643 are required to maximally activate human PPARc: compared to mouse PPAR, `whereas no major species difference in PPAR responsiveness is seen with the plasticizer metabolite mono[2-ethylhexyl]-phthalate (MEHP) (25). Important differences in PPAR ligand specificity are also apparent in the comparison of PPARo: and PPARY (26). For example, PPARG: but not PPARY is readily activated by classic rodent peroxisome proliferators, such as Wy-14,643 and fibrate drugs (e.g., clofibrate, nafenopin), whereas PPARY is preferentially activated by a distinct set of chemicals, including anti-diabetes type II drugs of the thiazolidinedione class (e.g, troglitazone). PPAR can, however, be efficiently activated by certain activators of PPAR, as shown in recent studies of the phthalate mono-ester and environmental pollutant MEHP (25). Presently, it is not known whether PFOSAs activate PPARo. or PPARY. Itis also uncertain whether species-dependent differences in receptor responsiveness, which could be important for human risk assessment, characterize this class of FCs. `Thespecificgoalsofthepresentstudyare: 1) to ascertain whether, and with what `potency, PFOSAS activate PPAR, the major mediator of hepatic peroxisome proliferation responses, as determined using an intact cell-based rans-activation assay; 2) to ascertain whether PFOSAs activate PPARY, which plays a major role in adipocyte differentiation and could contribute to the observed effects of PFOSAs on fat `metabolism and lipid homeostasis; and 3) to ascertain whether human PPARs (PPAR. 3M-BUReport.doc - 9/12/01 - Page 6 or PPARY) exhibit altered sensitivity to PFOSAs compared to the corresponding mouse PPARs, a finding that could be important in extrapolating to human toxicology and risk assessment data obtained in rodent model studies. MatearndiMeathlodss Chemicals - Troglitazone was a gift from Hidekuni Takahagi (Sankyo Co. Ltd, Tokyo, Japan). Wy-14,643 and DMSO were purchased from Sigma Chemical Co (St. Louis, MO). PFOS, N-EtFOSE, FOSA, N-EtFOSAA and FOSAA were obtained from Dr. Andrew Seacat (3M Corpn.). Plasmids - The Firefly luciferase reporter pHD(x3)Luc, obtained from Dr. J. Capone (McMaster University, Toronto, ON, Canada), contains three tandem copies of a PPARactivated DNA response element (PPRE) from the rat enoyl-CoA hydratase/3- hydroxyacyl-CoA dehydrogenase gene linked directly to a minimalor core promoter (27), which provides binding sites for assemblyofthe RNA polymerase II-containing transcription preinitiation complexandcan respond to adjacent transcriptional activators, such as PPAR. Mouse PPAR cloned into the expression plasmid pCMV5 was obtained from Dr. E. Johnson (Seripps Research Institute, La Jolla, CA) (28). The PPARY] expression plasmid pSV-Sport-mPPARy] was obtained from Dr. J. Reddy (Northwestern University, Chicago) (29). The human PPAR0. expression plasmid PSGS-PPAR0 (30) was obtained from Dr. F. Gonzalez (National Cancer Institute, Bethesda, MD). A human PPARY] expression plasmid, pSG5-PPARY1 (31) was provided by Dr. S. Kliewer (Glaxo Wellcome Inc., Research Triangle Park, NC). wD. ' Renilla luciferase reporter plasmid, pRL-CMV, was purchased from Promega (Madison 3IM-BU Report.doc - 9/12/01 ~ Page 7 Cellcultureandtransienttransfectio-n COS-1 cells, an SV40-transformed African green monkey cell line (ATCC #CRL-1650) were grown in Dulbecco's modified Eagles `medium (DMEM) with 10% fetal calf serum. COS-1 cells were transfected ata density of 3x10* cells/well ofa48-well tissue culture plate. Cells were plated in 500 ul of culture medium. Transfection of COS-1 cells was carried out using 0.3 sl FuGENE 6 transfection reagent (Roche) per well. Twenty four hr after additionofthe DNAFuGENE mixture to the cells, the medium was changed to serum-free DMEM containing FCs at the specified concentrations. Cells were lysed 24hrlater, and Firefly luciferase and Renilla luciferase activity were measured using a dual luciferase assay kit (Promega). Transfections were performed using the following amounts of plasmid DNA/well unless indicated otherwise: 90 ng of PPAR reporter plasmid pHD(X3)Luc, 5 ng of PPAR expression plasmid (mPPARct, mPPARY1, hPPARe: or hPPAR1), and 1 ngof pRL-CMV. Salmon sperm DNA was added to the plasmid DNA mix to give 250 ng total DNA. In later experiments, the amount of Salmon sperm DNA added to the plasmid DNA mix was decreased to give a totalof200 ng. Celltreatment - FCs were dissolved in DMSO solvent to give a 1 M stock solution. Serial dilutions were prepared in DMSO, followed by a final 1000-fold dilution into serum-free DMEM for cell treatment. For the experiments shown in Figs. 5-12, 144, C, D and F, stock solutions of each FC in DMSO were prepared in triplicate, followed by dilution of each stock solution into DMEM in duplicate, to give 6 replicates (tested in 6 parallel wells) for each FC at each concentration. Each experiment included two negative controls, each in triplicate: no treatment (indicated by *-*; first bar in each panel of Figs. 1, 3-14) and 0.19% DMSO solvent control, as marked on each figure. In addition, a positive control for activation of PPARe: (5 tM Wy-14,643) or PPARY (3 3M-BU Report.doc - 9/12/01 - Page 8 WM troglitazone) was included in triplicate for each experiment. Data presented are mean values based on n =3 independent dilutions of the FC, with each value calculated from the average of the duplicate determinations. All other experiments were based on asingle serial dilution of the FC, followed by asingle dilution into DMEM and treatment of 3 replicate wells at each FC concentration. Dataanalysis - Luciferase activity values were normalized for transfection efficiency by dividing the measured Firefly luciferase activity values by the Renilla luciferase activity obtained for the same cell extract, i.c., (Firefly/Renilla) x 1000. Data presented are mean + SD values for n = 3 independent FC dilutions, where each n = the mean of a duplicate, as indicated above. Normalized activity values and the corresponding raw Firefly luciferase data are presented alongside in cach figure. EvaluationofdirectFC effectsonluciferaseactivity -- For the experiment shown in Fig. 2, cells were transfected with Firefly luciferase and Renilla luciferase expression plasmids for 24hr followed by cell Iysis. Cell lysate was incubated at 37 C for 1 hr with each FC at the concentrations indicated followed by measurement of Firefly and Renilla luciferase activity. Results 1. Effect of PFOSAs on cellular expression of transfected Firefly and Renilla luciferase reporter activity We first sought to determine whether treatment of COS-1 cells with PFOSAs interferes with cellular expression of either Firefly or Renilla luciferase enzyme activity. This experiment was carried out to establish the range of FC concentrations over which 3M-BU Report.doc - 9/12/01 - Page 9 trans-activation assays could be carried out without significant toxicity to cellular expression of the luciferase reporter. The plasmids pGL2-Luc and pRL-CMV direct the expression of Firefly luciferase and Renilla luciferase, respectively, in aconstitutive `manner, i.., in the absence of PPAR and in the absenceof a PPAR ligand. COS-1 cells were transiently transfected with pGL2-Luc and pRL-CMV for 24 hr followed by treatment with each FC, or with solvent control (0.1% DMSO in DMEM), for a further 24hrat concentrations ranging from 15.5 iM to 1000 iM (n=3 ateach concentration). Cells were lysed and luciferase activities determined. EOS, C,F,;SO; - PFOS had little or no effect on the expression of Firefly or Renilla luciferase activity up to 250 tM. At 500 uM PFOS there was a notable decrease in luciferase activity, and at 1 mM PFOS there wasnoexpression of either luciferase (Fig. 1A; left (Firefly) and right (Renilla). N-E{FOSE, CyF;;SO,N(C;H,)CH,CH,0H. N-EtFOSE had no significant effect on Renilla luciferase activity at all concentrations tested, but Firefly activities were more. variable (Fig. 1B). Later studies suggest that this lack of effect of N-E(FOSE may be due its poor solubility (See Table 1, below). FOSA, C;F,;SO,NH,. Firefly and Renilla activities were both decreased to undetectable levels at FOSA concentrations > 62.5 jtM. This decrease in activity was abrupt, and `was not characterized by a smooth dose-dependence (Fig. 1C). N-EIFOSAA, CyF,,SO,N(C2Hs)-CH,COO'. Luciferase values were substantially reduced, in a dose-dependent manner, in cells treated with N-EXFOSAA. This inhibitory effect was more prominent on Firefly luciferase than Renilla luciferase (Fig. 1D). 3M-BU Report.doc - 9/12/01 - Page 10 EOSAA, CF,SO;NHCH,COO'. Firefly luciferase activity was essentially constant at all concentrations of FOSAA. The apparent increase in Renilla activity seen at the two highest concentrationsof FOSAA is not readily explained (Fig. 1E). 2. Solubility of PFOSAs Initial experiments revealed that some of the FCs became insoluble upon dilution from DMSO into DMEM culture medium. Attempts were made to maximize the solubility of the FCs by using different solvents, by heating and by shaking the samples. All of the FCs were found to be soluble in DMSO at 1 M. PFOS, FOSA and N-EtFOSE, but not N-EtFOSAA or FOSAA, remained in solution when the DMSO stock was diluted 1000fold to give a nominal 1 mM solution in DMEM. In viewofthe insolubilityofthe acetates, N-EtFOSAA and FOSAA, subsequent experiments focused on PFOS and FOSA. A limited number of studies included N-EFOSE, which was found to be insoluble in a series of analytical solubility tests carried out at the 3M Company by Fred L. DeRoos (Table 1). 3. Test for direct inhibitory effectsof PFOSAs on Firefly and Renilla Luciferase activity `We next investigated whether the decrease in Firefly and Renilla luciferase activity seen in cells treated with some of the FCs (Fig. 1) is the result ofa direct inhibition of enzyme activity, rather than cell toxicity leading to FC inhibitionofFirefly or Renilla luciferase gene expression. COS-1 cell lysate prepared from cells transfected with constitutively expressed Firefly and Renilla luciferase plasmids was incubated for 60 min at 37 C in the presence of FOSA, N-EtFOSAA, FOSAA or 0.1% DMSO solvent control, followed by analysis of luciferase enzyme activity. PFOS and N-E(FOSE were 3M-BU Report.doc - /12001 - Page 11 excluded from this experiment because minimal inhibitory effects were observed with these FCs in Fig. 1. FOSA at 62.5 11M had no effect on either enzyme activity in this in vitro experiment (Fig. 2A), whereas it reduced Firefly and Renilla values to background upon treatment of COS-1 cells (Fig. 1C). Thus, FOSA is not directly inhibitory to either luciferase enzyme. Rather, FOSA exhibits cell toxicity that is manifest as inhibition of expression of both luciferase plasmids at concentrations above 62.5 UM (Fig. 1C). No inhibitory effects were seen with N-EIFOSAA or FOSAA (Fig. 2A, 2B). However, difficulties were encountered with the solubility of these FCs, so results from those `experiments may not be informative. 4. trans-Activation assay for PPAR activity: time course study and serum dependence To investigate the potential ability of the FCs to activate the nuclear receptor PPAR, COS-1 cells were transiently transfected with a PPAR expression plasmid, together with the reporter plasmid pHD(x3)luc, which contains three PPAR binding sites (PPRESs) linked to a minimal promoter controling the gene for Firefly luciferase. Cells were cotransfected with an expression plasmid encoding Renilla luciferase, which serves both as an internal control to normalize individual samples for variation in transfection efficiency and as a general monitor of cellular toxicity of the FC under the conditions of the experiment. The PPAR receptor forms included in these experiments are the and y isoforms, cloned from mouse (m) and human (h) sources: mPPARG, hPPARG, mPPARy and hPPARY. In each experiment, the peroxisome proliferator chemical Wy14,643 (5 WM) was used as a positive control for activation of PPARc and the thiazolidinedione anti-diabetic drug troglitazone (3 WM) was used as a positive control for activation of PPARY. 3M-BU Report.doc - 9/12/01 - Page 12 Time-coursestudy - In a typical trans-activation assay, cells transfected with receptor (PPAR) and reporter (pHDx3-Luc) plasmids are stimulated with the test chemical for 24 h, followed by assayofcell extracts for luciferase reporter activity. In view of the cell toxicity seen with someofthe FCs (Fig. 1), we investigated 1) whether PPARdependent reporter gene activity could be assayed reliably using a shorter time period of FC stimulation, and 2) whether the use oaf shorter time period improves the results, i.c., decreases cellular toxicityofthe FC, as monitored by expression of reporter gene activity. Fig. 3 shows a time course for FC activation ofmPPARY, assayed 6, 16 and 25 hr after stimulationofthe transfected cells with either PFOS or FOSA. At each time point, PFOS induced a dose-dependent increase in mPPAR-activated reporter gene activity. As anticipated, the 6 hr treatment time gave a smaller fold-increase in activity relative to the no solvent control, or the DMSO control, compared to either the 16 hr or 25 hr time. points. This smaller activity increase reflects the shorter time allowed for gene expression (Fig. 3). In the case ofFOSA, reporter activity was stimulated at the lower concentrations (10-40 WM), but toxicity was apparent at the higher concentrations tested (60-120 1M). This toxicity is indicated by the substantial inhibitionofRenilla. luciferase expression (also see FOSA inhibitionof luciferase expression shown in Fig. 1C). Unfortunately, no significant reductionofthis toxicity was apparent in the 6 hr treatment samples. Given this lackof improvement in toxicity, and given the decreased responsivenessofthe reporter activity seen at 6 hr (c.f, lower Firefly activity values and lower fold-stimulation by PFOS compared to the DMSO control), all subsequent experiments were carried out using a standard 24 hr period of FC treatment, 3M-BU Report.doc - 9/12/01 -Page 13 Effects of Fetal Bovine Serum (EBS) - The standard PPAR transactivation assay is carried out in the absence ofFBS. We investigated whether the inclusionofFBS might increase the solubility and thereby enhance the stimulatory activityofthe PFOSAs. Figure 4 shows that the fold-activation ofmPPAR was unchanged when FC treatment was carried out in the presence of FBS. Subsequent experiments were therefore carried out in the absence of FBS. 5. Activation of PPAR and PPARy by PFOS (Figs. 5-8; Table 2) PFOS activated all four PPAR forms, although a greater response was observed with PPARG than with PPARY. In general, the pattemofresponse seen in the normalized reporter activity data (Firefly/Renilla luciferase; upper setofgraphs) paralleled that of the raw Firefly luciferase data (lower sets ofgraphs), indicating no major toxic effect on the Renilla luciferase internal control. PPAR0 -- PPAR: was activated 4-6-fold by PFOS. This is compared to the maximal activation observed in parallel wells using the established PPAR. ligand Wy-14,643. Based on the data obtained, maximal activation ofhPPARc: compared to mPPARG may require up to a 2-4-fold higher concentrationofPFOS. PPARy~ PFOS activated both mouse and human PPARYy, although the extent of activation seen in transfection experiment #108 (T-108) was lower than that achieved in experiment T-92 or that shown in the time-course study presented in Fig. 3. PFOS activation ofhPPARY was also seen, but may be weaker than that ofmPPARY. The maximal activation of PPARY by PFOS was consistently lower than the maximal activation seen in cells stimulated with the established PPARY ligand troglitazone. Some inconsistency in the dose-response data between replicate experiments was 3M-BU Report.doc - 9/12/01 -- Page 14 apparent. This may be due, in part, to the low fold-activation of PPARy induced by PFOS. 6. Activation of PPARc and PPARy by FOSA (Figs. 9-12; Table 2) PPAR activation data obtained with FOSA were more variable, both in termsofthe. fold-activation and the consistency of the dose-response values when compared to PFOS. Strong activation of PPARata single concentration was sometimes observed, but this was not consistently seen (c.f, Fig. 9). Interpretation of these findings is complicated by the atypical variation in extent of PPAR activation by the positive control obtained in someofthe transfection experiments (e.g., poor response of mPPARG: to Wy-14,643 in the normalized luciferase data for experiment T-102 (Fig. N). PPARa - FOSA activated PPAR, but to a variable extent. For example, a 3.3-fold activation was seen with mPPARc: in experiment T-102, albeit with significant inter sample variation, compared with a more modest 1.6-fold activation observed in T-107 (Fig. 9). In the case ofhPPARG, substantial activation by FOSA (4.3-fold) was seen in one experiment (T-107) but not in a second experiment (T-102; Fig. 10).Ofnote, the `Wy-14,643 positive control responseofhPPAR. was also particularly strong in experiment T-107. PPARY-FOSA also had variable effects on PPARy activation. Overall, there appeared to be little activation of mPPARoyrhPPARyby FOSA in one experiment (T-116), but in a second experiment (T-102) a 4-5-fold activation of both mPPARyand hPPARY was seen at the highest non-toxic concentration of FOSA examined (45-60 uM) (Fig. 12). 3M-BU Report.doc - 9/12/01 - Page 15 e ee -------- ere-- s -- ---- -- -- 7. Effects of N-EtFOSE on PPAR activity PPAR - Treatment of the transfected cells with N-EFOSE did not induce Firefly luciferase activity in the case of mouse PPARG: or human PPAR.. Although Figs. 13A and 13B (normalized luciferase activity) suggest an apparent activationof mouse PPAR, examinationofthe corresponding raw Firefly luciferase data (Figs. 13C, 13D) revealed this apparent receptor `activation' is largely due to a dose-dependent decrease in Renilla activity values (data not shown). PPARy- Mouse and human PPARY were activated ~2-fold by N-EFOSE in experiments where the Renilla luciferase intemal control remained constant (compare Fig. 14B and 14E). Its unclear why the activation ofhPPARYseen in T-108 was not reproducible (see T-116; Fig. 14C). Discussion PFOS and FOSA were both shown to activate PPAR and PPARY when assayed in a COS-1 cell-based trans-activation assay. Examinationofthe dose-response curves for PPAR activation suggests that mPPARc may be maximally activated by PFOS at a somewhat lower concentration than hPPARG: (32 uM for mPPAR. vs, 64-128 uM for hPPARG; Table 1). This latter conclusion should be viewed as preliminary, as further experimentation is needed for confirmation and validation. The findingthathPPARo: can be activated by PFOS is consistent with the hepatic peroxisome proliferative activity seen when PFOS is administered to rodents (8). PFOS is an end-stage `metabolite of N-E{FOSE and related FCs, and accumulates in liver when administered to rats (7). PFOS was found to activate mPPAR: and hPPARG: to nearly the same maximum extent as the potent peroxisome proliferator Wy-14,643, albeit at much 3M-BU Report.doc - 9/12/01 - Page 16 higher concentrations (c.f, Figs. 5 and 6). This finding is consistent with the report that PFOS is almost as potent a rodent peroxisome proliferataosr the established peroxisome proliferator PFOA (8). By contrast, the maximal activation of PPARyobserved in cells treated with a saturating concentration of PFOS was consistently lowerthan that obtained in parallel samples stimulated with the strong PPARy agonist troglitazone. `The reason for this difference in the degreeof maximal activation of PPARG. vs, PPARY following PFOS stimulation is unclear, but it may suggest that PFOS acts as apartial agonist in the caseofthe latter receptor. Further study is required to clarify this point. A somewhat higher concentration of PFOS than FOSA was required to activate PPAR. `The actual difference is reduced somewhat when the lower aqueous solubility and availability of PFOS (Table 1)is taken into account. Accordingly, PFOS and related FCs may be somewhat more potent activators of PPAR: or PPARYthan would otherwise be apparent by examining the nominal concentration data presented here. Some inconsistencies and variations in the experimental data have been noted. This variability is not a general characteristic of the cell-based trans-activation assay used in the present study. Moreover, this variability cannot be explained by the use of frozen FC stock solutions in some experimentsys,freshly prepared stocks in others (data not shown). These inconsistencies make it more difficult to establish precise dose-response curves for receptor activation by FCs. The present studies are nevertheless informative, as they establish the capabilityofthis class ofFCs to activate PPAR, which based on rodent data may be associated with hepatocarcinogenesis (17), and PPARY, a PPAR isoform expressed at high levels in multiple human tissues. Further investigation is warranted to better establish the PPAR-activation capability of these perfluorochemicals using cell-based assays employing endogenous PPAR receptors and endogenous, 3M-BU Report.doc - 9/12/01 - Page 17 J ---- chromatin-associated target genes. Extension of these studies to include PPARS would also beof interest, in viewofthe near ubiquitous tissue expressionofthis PPAR form, its roleinreverse cholesterol transport (32), and its importance for developmental processes (33, 34). Peroxisome proliferators and other chemicals may activate PPARs by two types of mechanisms: by binding directly to the receptor (i.c., as ligands), orby perturbing lipid `metabolism and transport in a manner that stimulates the synthesis and/or release of endogenous PPAR ligands. We cannot, at present, distinguish between these two mechanisms in the case of the activation of PPAR by PFOS and FOSA reported in the present study. Previous investigations carried out by scientists at 3M have shown, however, that PROSAs can displace endogenous ligands from liver fatty acid binding protein (L-FABP) (35, 36, an intracellular lipid carrier that binds and transports fatty acids, acyl-CoA, and a variety of other hydrophobic molecules within hepatocytes. Functions of L-FABP include stimulation of phospholipid synthesis, regulation of lipid `metabolism and protection of the cell by maintaining the concentration of free fatty acids at sub-toxic levels. PFOSAs may thus induce their hypolipidemic and peroxisome proliferating effects, in part, by interfering with the capacity of L-FABP to bind fatty acids, cholesterol and other lipids. Displacement of fatty acids could, in turn, stimulate the activationof PPARG: or PPARY by endogenous ligands. Further investigation, including a more direct examination of the ability of PFOSAS to bind directly PPARc. or PPARy should help resolve this question. In contrast to PPARG, which is expressed at comparatively low levels in human tissues (23), PPARY is expressed at relatively high levels in a broad range of human tissues. These include adipose tissue, where many lipophilic foreign 3M-BU Report.doc - 9/12/01 - Page 18 chemicals tend to accumulate, as well as colon, heart, liver, testis, spleen and hematopoietic cells (37, 38). Most interestingly, studies of adipocyte differentiation have revealed that PPARY serves as a master transcription factor which regulates adipogenesis in response to PPARY ligands. Further studies are planned to characterize the adipogenic response of the PPARy-activating FCs identified in the present report using established cellular models for adipogenesis. Other biological responses linked to PPARy ligands and activators include the inhibitionof human vascular endothelial cell differentiation and angiogenesis (39), which could impact on developmental processes, and colon tumorigenesis (40). PPARyactivation also leads to foam cell macrophage differentiation, although itis uncertain whether this latter PPAR-dependent response is likely to contribute to generation ofan atherosclerotic plague (41). The extent to which PFOSAS may alter these physiological or pathophysiological responses to endogenous or other foreign chemical PPARy ligands is uncertain, and is an important area for further research. Abbreviations used: FCs, fluorochemicals Perfluorooctanesulfonate, PFOS PFOSAS, PFOS and perfluorooctanesulfonamide-based chemicals Perfluorooctanoic acid, PFOA Perfluorooctanesulfonamide, FOSA N-ethyl-perfluorooctanesulfonamido acetate, N-E(FOSAA Perfluorooctanesulfonamidoacetate, FOSAA 2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EFOSE mPPARG, mouse PPAR. PPAR, human PPAR. mPPARy, mouse PPARY hPPARy, human PPARY 3M-BUReportdoc - 9/12/01 - Page 19 References 1. Hansen, K.J., Clemen, L. A., Ellefson, M. E., and Johnson, H. O. Compoundspecific, quantitative characterization of organic fluorochemicals in biological `matrices. Environ Sci Technol, 35: 766-770., 2001. 2. Giesy, J. P. and Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ Sci Technol, 35: 1339-1342., 2001. 3. Kannan, K., Koistinen, J, Beckmen, K., Evans, T., Gorzelany, J. F., Hansen, K. 1., Jones, P. D., Helle, E., Nyman, M., and Giesy, J. P. Accumulation of perfluorooctane sulfonate in marine mammals. Environ Sci Technol, 35: 15931598., 2001. 4. Olsen, G. W., Burris, J. M., Mandel, J. H., and Zobel, L. R. Serum perfluorooctane sulfonate and hepatic and lipid clinical chemistry tests in fluorochemical production employees. J Occup Environ Med, 41: 799-806., 1999. 5. Schnellmann, R. G. and Manning, R. O. Perfluorooctane sulfonamide: a structurally novel uncoupler of oxidative phosphorylation. Biochim Biophys Acta, 1016: 344-348., 1990. 6. Gibson, S. 1. Johnson, J. D., and Ober, R. E. (eds.) Absorption and biotransformation of N-ethyl FOSE and tissue distribution and elimination of carbon-14 after administration of N-ethyl FOSE-14 in feed. St. Paul, MN: Riker Laboratories Ind, 1983. 7. Butenhoff, J. L. and Seacat, A. M. 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Zhu, Y., Alvares, K., Huang, Q, Rao, M. ., and Reddy, J. K. Cloning ofa new `member of the peroxisome proliferator-activated receptor gene family from mouse liver. J Biol Chem, 268: 26817-26820, 1993. 30. Sher, T., Yi, H. F., McBride, O. W., and Gonzalez, F. J. cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry, 32: 5598-5604, 1993. 31. Kliewer, S. A, Sundseth, S. S., Jones, S. A., Brown, P. J, Wisely, G. B., Koble, C.S., Devchand, P., Wahli, W., Willson, T. M., Lenhard, J. M., and Lehmann, J. M. Proc. Natl. Acad. Sci. USA, 94: 4318-4323, 1997. 32. Oliver, W.R,, Jr., Shenk, J. L., Snaith, M. R., Russell, C. S., Plunket, K. D., Bodkin, N. L., Lewis, M. C., Winegar, D. A., Sznaidman, M. L., Lambert, M. H., Xu, H. E., Stembach, D. D., Kliewer, S. A., Hansen, B. C., and Willson, T. M. A 3M-BU Report.doc - 9/12/01 - Page 23 selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport. Proc Natl Acad Sci US A, 98: 5306-5311., 2001. 33. Peters, ].M, Lee, S. S., Li, W., Ward, J. M., Gavilova, O., Everett, C., Reitman, M. L., Hudson, L. D., and Gonzalez, F. J. Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta). Mol Cell Biol, 20: 5119-5128., 2000. 34. Lim, H. Gupta, R. A, Ma, W. G., Paria, B. C., Moller, D. E., Morrow, J. D., DuBois, R. N., Trzaskos, J. M., and Dey, S. K. Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta. Genes Dev, 13: 1561-1574., 1999. 35. Nabbefeld, D., Butenhoff, I, Bass, N., and Seacat, A. Displacement ofa fluorescently labeled fatty acid analogue from fatty acid carrier proteins by. Wyeth-14,643, ammonium perfluorooctanoate, potassium perfluorooctane sulfonate and other known peroxisome proliferators. Toxicologist Abstract, 1998. 36. Nabbefeld, D. An investigation of the effects of flurochemicals on liver fatty acid-binding protein. University of Minnesota, 1998. 37. Greene, M. E., Blumberg, B., McBride, O. W., Yi, H. F., Kronquist, K., Kwan, K., Hsieh, L., Greene, G., and Nimer, S. D. Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping. Gene Expr, 4: 281-299, 1995. 38. Vidal-Puig, A. J., Considine, R. V., Jimenez-Linan, M., Werman, A., Pores, W. 3,, Caro, J. F., and Flier, J. S. Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids. J Clin Invest, 99: 2416-2422, 1997. 39. Xin, X., Yang, S., Kowalski, I, and Gerritsen, M. E. Peroxisome Proliferatoractivated Receptor gamma Ligands Are Potent Inhibitors of Angiogenesis in Vitro and in Vivo. J Biol Chem, 274: 9116-9121, 1999. 3M-BU Report.doc - 9/12/01 -- Page 24 40. Saez, E., Tontonoz, P., Nelson, M. C., Alvarez, J. G., Ming, U. T., Baird, S. M., `Thomazy, V. A., and Evans, R. M. Activators of the nuclear receptor PPARgamma enhance colon polyp formation. Nat Med, 4: 1058-1061, 1998. 41. Rosen, E. D. and Spiegelman, B. M. Peroxisome proliferator-activated receptor gamma ligands and atherosclerosis: ending the heartache. J Clin Invest, 106: 629631.,2000. 3M-BUReport.doc - 9/12/01 - Page 25 Signature page [52 / QD DDre.paDatvmiedntJ. oWfaBximolaongy BSotstyonDiUrneacitvoerrsity Yen Date. ATonxdicoouOloMng.yiSSehpaceatc,siPAD.1D RoniadTM 3SpMonCsoorprosrRaetpereTsoexnitcaotliovgey lof:ud[or 3M-BUReport.doc - 9/12/01 Page 26 Table 1. Solubility of PFOS, FOSA and NEF in MEM culture medium Theoretical |Measured Percent of|Measured Percent of|Measured Percent of| f[oEomww J[ [owr[JwJf fw wr w nmJss w | e]] ff[emEww JJxooJw f mw ww s w JsJ|s]) foorw J J&a [fewwwJJss |] ] Data provided byFred L. DeRoos; March 29, 2001 PFOS and FOSA anion concentrations were analyzed by HPLC coupled to electrospray `mass spectrometry (ESI-MS). N-EtFOSE concentrations were determined using capillary GC with flame ionization detection (GC-FID). 3M-BU Report.doc - 9/12/01 - Page 27 Table 2. Activation of PPARsby PFOSAS -- Shown is a summaryofthe transfection studies presented in Figs. 5-14. Data shown for PFOS and FOSA are from two independent experiments (marked i and i, with the transfection experiment number as marked); N-EtFOSE data are based on a single experiment with each receptor, with the exceptionofhPPARY. ald Pros mPPAR mPPARY [i Ti [i Ta [i Ta [i [i | number [Fold [a [62 [35 [42[42[25[17 |28| [Concentration (uM)[32 [32 [125 [64 [125 [32 [32[32| Fold of +ve control[44 [65[6 [25|75[123145 [6 | [Fosa Ti Ta [i Ta [i [a [i [i | `Transfection 107 [102 [10[ 7 2[1s] 102 [116 number [Fold [33 [16 [21 [43[52[18 [41 [15| [ConcentrationGM)[45 [8 [45 [45 |60 Jeo [45 [14| Fold of +ve conwol[14 [41[18 [7.1 [33 [89 [27 [61| [Eos Ti | Transfection number Fold sec text Concentration M)[ - | Foldof +vecontrol [49| Ti[Ti T |-| 2| 2 [250 | [87 [| Ti Ti | 26 [1 [250[| [89 [46| Fold - the maximum fold activation for the normalized data in that particular experiment relative to vehicle (DMSO)-treated samples; Concentration concentration at which maximal or near maximal activation of PPAR was observed;Foldof+ve(positive)control - 5 uM Wy-14,643 for experiments with PPAR: and 3 uM Troglitazone for experiments PPARY. 3M-BU Report doc - 9/12/01 - Page 28 FigureLegends FIG. 1. Eff ofe PFc OSAts on cellular expressionof transfected Firefly and Renilla luciferase activity. COS-1 cells co-transfected with Firefly luciferaseexpression plasmid, pGL2Luc, and Renilla liciferase expression plasmid, pRL-CMV, were treated for 24 hr with PFOS (A), N-EtFOSE (B), FOSA (C), N-EtFOSAA (D)or FOSAA (E) at the indicated concentrations. Firefly and Renilla luciferase were assayed as described +-SD,n=3. under Materials and Methods. Data shown are mean luciferase activities (light units) FIG. 2. FOSA, N-EtFOSAA and FOSAA are not direct-acting inhibitorsofFirefly and Renilla luciferase activity. COS-1 cell lysate containing Firefly and Renilla Iuciferase activity was incubated for 60 min at 37C with FOSA, N-EtFOSAA and FOSAA at the concentrations indicated. Shown are mean luciferase activities -/+ SD, n =3. Asterisks -- no luciferase activity was detected, indicating strong inhibition of luciferase gene expression. FIG. 3. Time-course for stimulation of mPPARy by PFOS are FOSA. COS-1 cells were co-transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding mPPARy. Transfected cells were treated with Troglitazone, PFOS or FOSA at the concentrations indicated. Cells were lysed either 6, 16 or 25 hr after additionofthe PPAR activator under study then assayed for Firefly and Renilla luciferase activity. Shown are mean Firefly activities (D-F), and the Firefly activity normalized to the Renilla internal control (A-C), n= 3. Asterisk --Renilla luciferase activity not detectable. 3M-BU Report.doc - 9/12/01 - Page 29 FIG. 4. Activation of mPPARG. by PFOS in the presence of Fetal Bovine Serum (FBS). COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding mPPARG.. Transfected cells were treated for 24 hr with 125 UM PFOS in the absence or presence of 10% FBS, as indicated. FIG. 5. Effect of PFOS on mPPAR activity. COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding mPPAR. Transfected cells were treated for 24 hr with DMSO (vehicle control), 5 uM Wy14,643, or PFOS at the concentrations indicated. Mean + SD Firefly activity (C+D), and Firefly activity normalized to the Renilla internal control (A+B) are shown for two Separate experiments (transfections T-96 and T-97), n = 3, where eachofthe 3 values is the mean of duplicate determinations (see Materials and Methods). FIG. 6. Effect of PFOS on hPPAR activity. Details as for Fig. 5, substituting PPAR: for mPPARG. FIG. 7. Effect of PFOS on mPPARY activity. Details as for Fig. 5, substituting mPPARY for mPPARG, and using 3 iM Troglitazone instead of Wy-14,643 as a positive control. FIG. 8. Effect of PFOS onhPPARy activity. Details as for Fig. 5, substituting hPPARy for mPPAR, and using 3 tM Troglitazone instead ofWy-14,643 as apositive control. FIG. 9. Effect of FOSA on mPPARc: activity. COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding mPPARG. 3M-BU Report.doc - 9/12/01 - Page 30 `Transfected cells were treated for 24 hr with DMSO (vehicle control), 5 uM Wy14,643,orFOSA at the concentrations indicated. Mean + SD Firefly activity (C+D), and Firefly activity normalized to the Renilla internal control (A+B) are shown for two separate experiments, n= 3, where eachofthe 3 values is the mean of duplicate determinations. FIG. 10. Effectof FOSA on hPPARG activity. Details as for Fig. 9, substituting hPPARG for mPPARc:. FIG. 11. Effect of FOSA on mPPARy activity. Details as for Fig. 9, substituting mPPARY for mPPARG, and using 3 iM Troglitazone insteadof Wy-14,643 as a positive control. FIG. 12. Effectof FOSA on hPPARY activity. Details as for Fig. 9, substituting hPPARY for mPPAR, and using 3 11M Troglitazone insteadofWy-14,643 as a positive control. FIG. 13. Effect of N-E(FOSE on PPAR: activity. COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding `mPPARG: (A+C) or hPPARa: (B+D). Transfected cells were treated for 24 hr with DMSO (vehicle control), 5 tM Wy-14,643 or N-EtFOSE at the concentrations indicated. Mean + SD Firefly activity (C+D), and Firefly activity normalized to the Renilla internal control (A+B) are shown; mea4nn =3. 3M-BU Report.doc - 9/12/01 - Page 31 FIG. 14. Effect of N-EtFOSE on PPARY activity. COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding `mPPARY (A+D) or hPPARY (B, C, E and F). Transfected cells were treated for 24 hr with DMSO (vehicle control), 3 tM Troglitazone or N-EtFOSE at the concentrations indicated. Mean + SD Firefly activity (C+D), and the Firefly activity normalized to the Renilla intemal control (A+B). n = 3. In Fig. 14A, C, E and F, n = 3, where each of those 3 is the meanof a duplicate. In Fig. 14B and E, n=3. Library: PPAR.ms.refs/Can Res 3M-BU Report.doc - 9/12/01 - Page 32 tenes crnros E seEgeREg i" oes8g eegg "Mid, : mm IEEE IEEE N-EtFOSE (uM) N-EtFOSE (uM) EEREEE EEREEE wn D. Firefly Luciferase + PFOSAA am Renilla luciferas+e PFOSAA - Lo. rosa - ---- f "i EEl EELi EE "three. Ay tetas55s i oe325wo 8A88 WFiignurSe 2MLucfEioferrs1ashe incubated ooA Firefly luciferase + PFOSA i.oo oE PFOSA (uM) B.Firefly luciferase + PFOSAA Renilla luciferase + PFOSA " I. 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