Document gXQNkr2ypKmmMQQjapbaMb0L

fl v'epork f i lg-Cl- 1M T* C SRPT T-c.2i^ ^^^W-ecl-fo '5 5 Xenochemical Receptor ftwis-Activation by Perfluorooctane-based Chemicals r ?$~ (p'vp lo ll -3 iv sa .s T-156SM Contributing Scientists at Boston University: Jonathan Shipley Megon Walker David J. Waxman Final Report for University Research Agreement dated 12/08/2000 between 3M Medical Department, Corporate Toxicology 3M Center 220-2E-02 Attn: 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 T\F a m 3M-BU Report.doc - 9/12/01 - Page 1 Summary - Peroxisome proliferator-activated receptors (PPARs) 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 PPARa 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 PPARa, respectively. The present study investigates this question and characterizes the activation of PPARa and PPARy by PFOSAs using a luciferase reporter gene trans-activation assay. Perfluorooctanesulfonate (PFOS), an end-stage metabolite common to several PFOSAs, was found to activate both PPARa and PPARy. Dose-response studies suggested that ~2-4-fold higher concentrations of PFOS may be required to activate human PPARa compared to mouse PPARa; however, additional data is required to validate this finding. Whereas PFOS treatment led to maximal activation of PPARa, PPARy was only partially activated at saturating concentrations of the fluorochemical. Perfluorooctanesulfonamide (FOSA) displayed activity towards both PPARa and PPARy, however, cellular toxicity associated with this compound resulted in dose-response profiles that varied between experiments. Studies of 2-N-ethylperfluorooctanesulfonamido)ethyl alcohol (N-EtFOSE) were less informative due to its insolubility, although weak PPARa 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 PPARa and PPARy helps 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 evaluation of human and environmental risks associated with exposure to this class of perfluorochemicals. 3M-BU Report.doc - 9/12/01 - Page 3 Introduction Perfluorooctanesulfonate (PFOS; C8F17S 0 3') 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-EtFOSE) 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 FC (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-(3-oxidizable fatty acid analogs that induce peroxisome proliferation (14). Peroxisome proliferation and certain other metabolic effects of PFOSAs may be a consequence of the activation of the nuclear receptors PPARa 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 PPARa 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 PPARa 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-BU Report.doc - 9/12/0] - Page 5 PPARa together with a PPARa-activated reporter gene, PFOA has been shown to trans-activate both mouse and human PPARa (10). PFOA maximally activated mouse PPARa at 5-10 pM, whereas human PPARa required somewhat higher concentrations (10-20 pM PFOA) for maximal stimulation. Other studies confirm the differential responsiveness of human and mouse PPARa to a subset of PPARa activators. For example, 5 to 6-fold higher concentrations of the classical peroxisome proliferator Wy14,643 are required to maximally activate human PPARa compared to mouse PPARa, whereas no major species difference in PPARa 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 PPARa and PPARy (26). For example, PPARa 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). PPARy can, however, be efficiently activated by certain activators of PPARa, as shown in recent studies of the phthalate mono-ester and environmental pollutant MEHP (25). Presently, it is not known whether PFOSAs activate PPARa or PPARy. It is also uncertain whether species-dependent differences in receptor responsiveness, which could be important for human risk assessment, characterize this class of FCs. The specific goals of the present study are: 1) to ascertain whether, and with what potency, PFOSAs activate PPARa, the major mediator of hepatic peroxisome proliferation responses, as determined using an intact cell-based ira/w-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 (PPARa 3M-BU Report.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. Materials and Methods 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/3hydroxyacyl-CoA dehydrogenase gene linked directly to a minimal or core promoter (27), which provides binding sites for assembly of the RNA polymerase Il-containing transcription preinitiation complex and can respond to adjacent transcriptional activators, such as PPAR. Mouse PPARa cloned into the expression plasmid pCMV5 was obtained from Dr. E. Johnson (Scripps Research Institute, La Jolla, CA) (28). The PPARyl expression plasmid pSV-Sport-mPPARyl was obtained from Dr. J. Reddy (Northwestern University, Chicago) (29). The human PPARa expression plasmid pSG5-PPARa (30) was obtained from Dr. F. Gonzalez (National Cancer Institute, Bethesda, MD). A human PPARyl expression plasmid, pSG5-PPARyl (31) was provided by Dr. S. Kliewer (Glaxo Wellcome Inc., Research Triangle Park, NC). Renilla luciferase reporter plasmid, pRL-CMV, was purchased from Promega (Madison W I). 3M-BU Report.doc - 9/12/01 - Page 7 Cell culture and transient transfection - 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 at a density of 3xl04cells/well of a 48-well tissue culture plate. Cells were plated in 500 (il of culture medium. Transfection of COS-1 cells was carried out using 0.3 pi FuGENE 6 transfection reagent (Roche) per well. Twenty four hr after addition of the DNAFuGENE mixture to the cells, the medium was changed to serum-free DMEM containing FCs at the specified concentrations. Cells were lysed 24 hr later, 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 (mPPARa, mPPARyl, hPPARa or hPPARyl), and 1 ng of 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 total of 200 ng. Cell treatment - 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. 5-12,14A, 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 0.1% DMSO solvent control, as marked on each figure. In addition, a positive control for activation of PPARa (5 |lM Wy-14,643) or PPARy (3 3M-BU Report.doc - 9/12/01 - Page 8 |iM 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 a single serial dilution of the FC, followed by a single dilution into DMEM and treatment of 3 replicate wells at each FC concentration. Data analysis - 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.e., (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 each figure. Evaluation n f direct FC effects on luciferase activity - For the experiment shown in Fig. 2, cells were transfected with Firefly luciferase and Renilla luciferase expression plasmids for 24 hr followed by cell lysis. 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 frans-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 a constitutive manner, i.e., in the absence of PPAR and in the absence of 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 24 hr at concentrations ranging from 15.5 pM to 1000 pM (n=3 at each concentration). Cells were lysed and luciferase activities determined. PFOS. C8F17S 0 3' - PFOS had little or no effect on the expression of Firefly or Renilla luciferase activity up to 250 pM. At 500 pM PFOS there was a notable decrease in luciferase activity, and at 1 mM PFOS there was no expression of either luciferase (Fig. 1A; left (Firefly) and right (Renilla)). N-EtFOSE. C8F 17S 02N(C2H5)CH2CH20H. N-EtFOSE had no significant effect on Renilla luciferase activity at all concentrations tested, but Firefly activities were more variable (Fig. IB). Later studies suggest that this lack of effect of N-EtFOSE may be due its poor solubility (See Table 1, below). FOSA. C8F17S 0 2NH2. Firefly and Renilla activities were both decreased to undetectable levels at FOSA concentrations ^ 62.5 pM. This decrease in activity was abrupt, and was not characterized by a smooth dose-dependence (Fig. 1C). N-EtFOSAA. C8F17S 0 2N(C2H5)-CH2C 0 0 . Luciferase values were substantially reduced, in a dose-dependent manner, in cells treated with N-EtFOSAA. This inhibitory effect was more prominent on Firefly luciferase than Renilla luciferase (Fig. ID). 3M-BU Report.doc - 9/12/01 - Page 10 FOSAA. C8F17S 0 2NHCH2C 0 0 . Firefly luciferase activity was essentially constant at all concentrations of FOSAA. The apparent increase in Renilla activity seen at the two highest concentrations of FOSAA is not readily explained (Fig. IE). 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 1000 fold to give a nominal 1 mM solution in DMEM. In view of the insolubility of the acetates, N-EtFOSAA and FOSAA, subsequent experiments focused on PFOS and FOSA. A limited number of studies included N-EtFOSE, 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 effects of 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 of a direct inhibition of enzyme activity, rather than cell toxicity leading to FC inhibition of Firefly 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-EtFOSE were 3M-BU Report.doc - 9/12/01 - Page 11 . excluded from this experiment because minimal inhibitory effects were observed with these FCs in Fig. 1. FOSA at 62.5 pM 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 pM (Fig. 1C). No inhibitory effects were seen with N-EtFOSAA 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. rans-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 (PPREs) 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 a and y isoforms, cloned from mouse (m) and human (h) sources: mPPARa, hPPARa, mPPARy and hPPARy. In each experiment, the peroxisome proliferator chemical Wy14,643 (5 pM) was used as a positive control for activation of PPARa and the thiazolidinedione anti-diabetic drug troglitazone (3 pM) was used as a positive control for activation of PPARy. 3M-BU Report.doc - 9/12/01 - Page 12 I Time-course study - 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 assay of cell extracts for luciferase reporter activity. In view of the cell toxicity seen with some of the 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 of a shorter time period improves the results, i.e., decreases cellular toxicity of the FC, as monitored by expression of reporter gene activity. Fig. 3 shows a time course for FC activation of mPPARy, assayed 6, 16 and 25 hr after stimulation of the transfected cells with either PFOS or FOSA. At each time point, PFOS induced a dose-dependent increase in mPPARy-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 of FOSA, reporter activity was stimulated at the lower concentrations (10-40 fiM), but toxicity was apparent at the higher concentrations tested (60-120 jj.M). This toxicity is indicated by the substantial inhibition of Renilla luciferase expression (also see FOSA inhibition of luciferase expression shown in Fig. 1C). Unfortunately, no significant reduction of this toxicity was apparent in the 6 hr treatment samples. Given this lack of improvement in toxicity, and given the decreased responsiveness of the 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 (FBS) - The standard PPAR transactivation assay is carried out in the absence of FBS. We investigated whether the inclusion of FBS might increase the solubility and thereby enhance the stimulatory activity of the PFOSAs. Figure 4 shows that the fold-activation of mPPARa 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 PPARa and PPARyby PFOS (Figs. 5-8; Table 2) PFOS activated all four PPAR forms, although a greater response was observed with PPARa than with PPARy. In general, the pattern of response seen in the normalized reporter activity data (Firefly/Renilla luciferase; upper set of graphs) paralleled that of the raw Firefly luciferase data (lower sets of graphs), indicating no major toxic effect on the Renilla luciferase internal control. PPARa - PPARa was activated 4-6-fold by PFOS. This is compared to the maximal activation observed in parallel wells using the established PPARa ligand Wy-14,643. Based on the data obtained, maximal activation of hPPARa compared to mPPARa may require up to a 2-4-fold higher concentration of PFOS. PPARy-PFO S activated both mouse and human PPARy, 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 of hPPARy was also seen, but may be weaker than that of mPPARy. 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 PPA Ra and PPARy by FOSA (Figs. 9-12; Table 2) PPAR activation data obtained with FOSA were more variable, both in terms of the fold-activation and the consistency of the dose-response values when compared to PFOS. Strong activation of PPAR at a 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 some of the transfection experiments (e.g., poor response of mPPARa to Wy-14,643 in the normalized luciferase data for experiment T-102 (Fig. 9)). PPARa - FOSA activated PPARa, but to a variable extent. For example, a 3.3-fold activation was seen with mPPARa 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 of hPPARa, substantial activation by FOSA (4.3-fold) was seen in one experiment (T-107) but not in a second experiment (T-102; Fig. 10). Of note, the Wy-14,643 positive control response of hPPARa 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 mPPARy or hPPARy by FOSA in one experiment (T-l 16), but in a second experiment (T-102) a 4-5-fold activation of both mPPARy and hPPARy was seen at the highest non-toxic concentration of FOSA examined (45-60 fiM) (Fig. 12). 3M-BU Report.doc - 9/12/01 - Page 15 7. Effects of N-EtFOSE on PPAR activity PPARa - Treatment of the transfected cells with N-EtFOSE did not induce Firefly luciferase activity in the case of mouse PPARa or human PPARa. Although Figs. 13A and 13B (normalized luciferase activity) suggest an apparent activation of mouse PPARa, examination of the 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-EtFOSE in experiments where the Renilla luciferase internal control remained constant (compare Fig. 14B and 14E). It is unclear why the activation of hPPARy seen in T-108 was not reproducible (see T-l 16; Fig. 14C). Discussion PFOS and FOSA were both shown to activate PPARa and PPARy when assayed in a COS-1 cell-based ira/w-activation assay. Examination of the dose-response curves for PPAR activation suggests that mPPARa may be maximally activated by PFOS at a somewhat lower concentration than hPPARa (32 pM for mPPARa vs* 64-128 pM for hPPARa; Table 1). This latter conclusion should be viewed as preliminary, as further experimentation is needed for confirmation and validation. The finding that hPPARa 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-EtFOSE and related FCs, and accumulates in liver when administered to rats (7). PFOS was found to activate mPPARa and hPPARa 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 proliferator as the established peroxisome proliferator PFOA (8). By contrast, the maximal activation of PPARy observed in cells treated with a saturating concentration of PFOS was consistently lower than that obtained in parallel samples stimulated with the strong PPARy agonist troglitazone. The reason for this difference in the degree of maximal activation of PPARa vs. PPARy following PFOS stimulation is unclear, but it may suggest that PFOS acts as a partial agonist in the case of the latter receptor. Further study is required to clarify this point. A somewhat higher concentration of PFOS than FOSA was required to activate PPARa. 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 PPARa or PPARy than 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 experiments vs* 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 capability of this class of FCs to activate PPARa, 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 - 9/12A)l - Page 17 chromatin-associated target genes. Extension of these studies to include PPARS would also be of interest, in view of the near ubiquitous tissue expression of this PPAR form, its role in reverse 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.e., as ligands), or by 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 PFOSAs 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 activation of PPARa or PPARyby endogenous ligands. Further investigation, including a more direct examination of the ability of PFOSAs to bind directly PPARa or PPARy should help resolve this question. In contrast to PPARa, which is expressed at comparatively low levels in human tissues (23), PPARyis 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 inhibition of human vascular endothelial cell differentiation and angiogenesis (39) , which could impact on developmental processes, and colon tumorigenesis (40) . PPARy activation also leads to foam cell macrophage differentiation, although it is uncertain whether this latter PPARy-dependent response is likely to contribute to generation of an atherosclerotic plaque (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-EtFOSAA Perfluorooctanesulfonamidoacetate, FOSAA 2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE mPPARa, mouse PPARa hPPARa, human PPARa mPPARy, mouse PPARy hPPARy, human PPARy 3M-BU Report.doc - 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 Sei Technol, 35: 766-770., 2001. 2. Giesy, J. P. and Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ Sei Technol, 35: 1339-1342., 2001. 3. Kannan, K., Koistinen, J., Beckmen, K., Evans, T., Gorzelany, J. F., Hansen, K. J., Jones, P. D., Helle, E., Nyman, M., and Giesy, J. P. Accumulation of perfluorooctane sulfonate in marine mammals. Environ Sei Technol, 35: 1593 1598., 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. J., 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. S., and Reddy, J. K. Cloning of a 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 U S A , 98: 5306-5311., 2001. 33. Peters, J. M., Lee, S. S., Li, W., Ward, J. M., Gavrilova, 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, J., Bass, N., and Seacat, A. Displacement of a 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., Pories, W. J., 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, J., 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: 629 631., 2000. 3M-BU Report.doc - 9/12/01 - Page 25 Signature page L>?Jt Dr. David J. Waxman Department o f Biology Boston Unaiversity Study Director L/ Q^ 9f)^ /d\jQ ) Date /yl'V ^ ^ X t^ O Andrew M. Seacat, Ph.D. Toxicology Specialist 3M Corporate Toxicology Sponsor's Representative ______ / / ! /& / Date 3M-BU Report.doc - 9/12/01 - Page 26 Table 1. Solubility of PFOS, FOSA and NEF in MEM culture medium Theoretical Measurec Percent of Measurec Percent of Measurec Percent of Concentratior [PFOS] Theoretica [FOSA] Theoretica [N-EtFO! Theoretica 1000 pM 190 19 120 12 <5 -- 500 pM 130 26 57 11 <5 -- 250 pM 70 28 97 39 <5 -- 125 pM 48 38 87 70 <5 -- 64 pM 28 44 81 130 <5 -- 32 pM 17 50 28 88 <5 -- 16 pM 8.7 54 18 110 <5 -- 8 pM 3.8 48 8.8 110 <5 -- Data provided by Fred 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 PPARs by PFOSAs - Shown is a summary o f the transfection studies presented in Figs. 5-14. Data shown for PFOS and FOSA are from two independent experiments (marked i and ii, with the transfection experiment number as marked); N-EtFOSE data are based on a single experiment with each receptor, with the exception of hPPARy. PFC mPPARa PFOS i ii Transfection 96 97 number Fold 4 6.2 Concentration (pM) 32 32 Fold of +ve control 4.4 6.5 hPPARa mPPARy i ii i ii 96 108 92 108 hPPARy i ii 96 97 3.5 4.2 4.2 2.5 1.7 2.8 125 64 125 32 32 32 6 2.5 7.5 12.3 4.5 6 FOSA i ii i Transfection 102 107 102 number Fold 3.3 1.6 2.1 Concentration (pM) 45 8 45 Fold of +ve control 1.4 4.1 1.8 ii i ii i ii 107 102 116 102 116 4.3 5.2 1.8 4.1 1.5 45 60 60 45 14 7.1 3.3 8.9 2.7 6.1 N-EtFOSE i i i i ii Transfection 108 108 116 108 116b number Fold see see 2 2.6 1 text text Concentration (pM) - - 250 250 - Fold of +ve control 4.9 2 8.7 8.9 4.6 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; Fold of+ve (positive) control - 5 pM Wy-14,643 for experiments with PPARa and 3 pM Troglitazone for experiments PPARy. 3M-BU Report.doc - 9/12/01 - Page 28 Figure Legends FIG. 1. Effect of PFOSAs on cellular expression of transfected Firefly and Renilla luciferase activity. COS-1 cells co-transfected with Firefly luciferase expression 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 under Materials and Methods. Data shown are mean luciferase activities (light units) +/- SD, n = 3. FIG. 2. FOSA, N-EtFOSAA and FOSAA are not direct-acting inhibitors of Firefly and Renilla luciferase activity. COS-1 cell lysate containing Firefly and Renilla luciferase 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 addition of the 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 mPPARa 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 mPPARa. Transfected cells were treated for 24 hr with 125 (xM PFOS in the absence or presence of 10% FBS, as indicated. FIG. 5. Effect of PFOS on mPPARa activity. COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding mPPARa. Transfected cells were treated for 24 hr with DMSO (vehicle control), 5 pM 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 each of the 3 values is the mean of duplicate determinations (see Materials and Methods). FIG. 6. Effect of PFOS on hPPARa activity. Details as for Fig. 5, substituting hPPARa for mPPARa. FIG. 7. Effect of PFOS on mPPARy activity. Details as for Fig. 5, substituting mPPARy for mPPARa, and using 3 pM Troglitazone instead of Wy-14,643 as a positive control. FIG. 8. Effect of PFOS on hPPARy activity. Details as for Fig. 5, substituting hPPARy for mPPARa, and using 3 pM Troglitazone instead of Wy-14,643 as a positive control. FIG. 9. Effect of FOSA on mPPARa activity. COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding mPPARa. 3M-BU Report.doc - 9/12/01 - Page 30 Transfected cells were treated for 24 hr with DMSO (vehicle control), 5 (iM Wy14,643, or FOSA 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 each of the 3 values is the mean of duplicate determinations. FIG. 10. Effect of FOSA on hPPA Ra activity. Details as for Fig. 9, substituting hPPARa for mPPARa. FIG. 11. Effect of FOSA on mPPARy activity. Details as for Fig. 9, substituting mPPARy for mPPARa, and using 3 (xM Troglitazone instead of Wy-14,643 as a positive control. FIG. 12. Effect of FOSA on hPPARy activity. Details as for Fig. 9, substituting hPPARy for mPPARa, and using 3 |i.M Troglitazone instead of Wy-14,643 as a positive control. FIG. 13. Effect of N-EtFOSE on PPA Ra activity. COS-1 cells were transfected with the PPAR reporter plasmid pHD(x3)Luc and an expression plasmid encoding mPPARa (A+C) or hPPARa (B+D). Transfected cells were treated for 24 hr with DMSO (vehicle control), 5 (iM 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; mean n = 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 pM Troglitazone or N-EtFOSE at the concentrations indicated. Mean SD Firefly activity (C+D), and the Firefly activity normalized to the Renilla internal control (A+B). n = 3. In Fig. 14A, C, E and F, n = 3, where each of those 3 is the mean of a duplicate. In Fig. 14B and E, n=3. Library: PPAR.ms.refs/Can Res 3M-BU Report.doc - 9/12/01 - Page 32 A Firefly Firefly FIGURE 1. A. Firefly Luciferase + PFOS 300000 200000- 100000 min inw m; <<Nn o in omo ooo co T" PFOS (hM) B. Firefly Luciferase + N-EtFOSE 3000001 200000* 100000 m ocn<M tf1) O O N-EtFOSE (>iM) C. Firefly Luciferase + PFOSA 300000 200000' 100000- in m in in s eg oo s ^m a S 8 PFOSA (mM) Renilla luciferase + PFOS 200000 = 100000- g in in in in o o o 2 8 - S PFOS (mM) Renilla luciferase + N-EtFOSE 2000001 = 100000- <n m O N-EtFOSE (nM) Renilla luciferase + PFOSA 200000 S 100000- m in ai m ID mfSi o <nI D Qo *On oO PFOSA (|iM ) Firefly D. Firefly Luciferase + PFOSAA Renilla luciferase + PFOSAA 400000-1 Firefly PFOSAA (nM) E. Firefly Luciferase + M556 300000*1 M556 (nM) to in ooo x*i- *^- <gt>j 2 8 8 8r- PFOSAA (nM) Renilla luciferase + M556 500000 400000- f M556 ((iM) Firefly Figure 2. Luciferase incubateci with 3M-FCs for 1 h A. Firefly luciferase + PFOSA 15000 10000 5000 PFOSA (nM) B. Firefly luciferase + PFOSAA PFOSAA (nM) C. Firefly luciferase + M556 M556 (|iM) M556 (nM) (F lre fly/R e n illa )*1000 IS O 8 16 32 64 125 250 FIGURE 3. Time course A. mPPARy - 6 h 7.51 5.0- 2.5- 0.0 1 ' a0t 1-- 2n CO filili P F O S (nM) ili n0 Jg 00 CM <0000 0 CM 2 P F O S A (ixM) B. mPPARy - 1 6 h 301 1 o> 0 C O C D C M ^ O Q $ P F O S (jiM) CO OJg O i- OO <N OO O CD CD CM P F O S A (nM) C. mPPARy - 25 h 50- 40- S i 30- 20- J I I n-I h>O0--ft 1 CO a lii! O co co cm m 0 eS2n " CO (D CM to cm P F O S (nM) 0Jg 0 0 CM 0^ 00 0 CO CO CM P F O S A (jiM) Firefly D. mPPARy - 6 h E. mPPARy-16 h 75001 r F. mPPARy - 2 5 h 150001 5000- s u. 2500- J I l ill l.ll. aot trM- CinM O or - oCM oTt o<DoCDoCM 2O_--_--_-_--_-_--_--Z. PFOS (ftM) PFOSA (nM) 10000- 8 5000- j a l l l i i l * iil 'a ntocj^o r > o o o o o o O Wis CO CD C--M- JCOM W r - CM CD CO CM*-- 2Q Q * PFOS (nM) PFOSA(|iM) Figure 4. A. mPPARa + PFOS + FBS B. 10000- 7500- >i | 5000- (2OO0 8 $ FIGURE 5. mPPARct + PFOS A. (T-96> 75"| C 3000-1 i 2 --------------- m PFOS (pM) mPPARa + PFOS (T-96) 2000- f z 1000- s a go 2 S 53 (0z z _______ O ----------------------- ID PFOS OiM) mPPARa + PFOS B. (T-97) 75 250 PFOS (|iM) D. 2000-1 mPPARa + PFOS (T-96) PFOS OiM) Firefly (Firefly/RenHla)*1000 P F O S (mM) Firefly (Flreflymenllla)MOOO PFOSCiM) FIGURE 7. mPPARy + PFOS 2 11 PFOS (nM) c. 1500- mPPARy + PFOS (T - , 11000- 1A 1 -r I 500- la1i i. l .l H. l Ml l l O-' ? 8" *83g Q r> PFOS (pM) D 20000 f* 10000- I mPPARy + PFOS (T-108) mPPARy + PFOS (T-108) o * s m s s g<M 3 n PFOS (nM) FIGURE 8. hPPARy + PFOS (T-96) C. 1500-1 hPPARy + PFOS (T-96) 1000- hPPARy + PFOS B. (T-97) 501 PFOS (jiM) mPPARa + PFOSA mPPARa + PFOSA (Flrefly/Renina)*1000 Firefly PFOSA (mM) PFOSA (jM) FIGURE 10. hPPARa + PFOSA A. (T-102) 401 30- 20io- PFOSA (jiM) hPPARa + PFOSA (T-102) 3 PFOSA <^M) hPPARa + PFOSA B. (T-107) " ? 838 PFOSA iiM ) D. 7500T hPPARa + PFOSA (T-107) 5000- >> 1 2500 O 553 tn o tn 8 S 'Q 8 PFOSA tM) FIGURE 11. mPPARy + PFOSA A. (T-102) 150n 100 PFO SA (mM ) mPPARy + PFOSA C. 750 T (T-102) Q? 8 S $ 8 8 a PFOSA (iM ) mPPARy + PFOSA B. (T-116) D. 15000- mPPARy + PFOSA (T-116) 10000- > I c 5000- o?? 8 S9 g 23 D CO PFOSA 4iM) FIGURE 12. hPPARy + PFOSA A. (T-102) C. 15001 hPPARy + PFOSA (T-102) 8 ? fi 5 9 8 8 O 3 PFOSA (jjM) CO hPPARy + PFOSA (T-116) PFOSA OiM) hPPARy + PFOSA (T-116) 83 ; ? s s $ 8? I- ______ , ... ----------------------------------- o PFOSA (nM) to FIGURE 13. mPPARa + N-EtFOSE A. (T-108) C. 2000 m PPARa + N-EtFOSE (T-108) r ` * s 8 8 N-EtFOSE (jiM) hPPARa + N-EtFOSE B. (T-108) io n N-EtFOSE (jiM) hPPARa + N-EtFOSE (T-108) 23n N-EtFOSE (nM) FIGURE 14. mPPARy + N-EtFOSE A. (T-116) hPPARy + N-EtFOSE B. (T-108) hPPARy + N-EtFOSE (T-116) (Ftrefty/RenlBa)*1000 Firefly mPPARy + N-EtFOSE D. (T-116) hPPARy + N-EtFOSE E. (T-108) hPPARy + N-EtFOSE F. 150001 (T-116) 10000- >* 5000 N-EtFOSE (nM)