Document M4qRzR49GBY8Ex4xEjwYVjV2V

Jean B. Sweeney Vice President 3M Environmental, Health and Safety Operations S36M5t.1PC7aeu3nl7,teM3r,5N6B9u5i5ld1i4n4g-O10.202004-o6-vi-03 February 16, 2010 CERTIFIED MAIL C3 NO CBI Document Processing Center EPA East - Room 6428 Attn: Section 8(e) Office of Pollution Prevention and Toxics, U.S. EPA 1200 Pennsylvania Avenue NW Washington, DC 20460-0001 Re: TSCA 8(e) Substantial Risk Notice: Sulfonate-based and Carboxylic-based Fluorochemicals, Docket 8EHQ-0598-373 - Results from a mechanistic investigation of the effect of PFBS, PFHS, and PFOS on lipid and lipoprotein metabolism in transgenic mice Dear Sir or Madam: rc':v:*: <r.; 3M is submitting this notice to supplement previous submissions on sulfonyl and carboxylic-^' based fluorochemicals (FCs), and more specifically our July 28, 2006 and January 8, 2007 cz:< submittals concerning data generated by TNO Laboratories in Leiden, Netherlands. These . .. data suggest an effect of perfluorooctanesulfonate (PFOS) and perfluorohexanesulfonate - r (PFHS) on body weight, food consumption, and serum cholesterol in 15% dietary fat fed E3V? Leiden transgenic mice. o CO Enclosed is a final report for follow up investigation to the aforementioned study. The current study was conducted at TNO Laboratories in Leiden, Netherlands using APOE*3Lieden/huCETP transgenic mice. Results help to explain the mechanism of action by which perfluorobutanesulfonate (PFBS), PFHS, and PFOS alter lipid and lipoprotein metabolism in animals treated with these chemicals. An effect noted in this study that has not been clearly demonstrated in other studies on these chemicals was a decrease in high density lipoprotein (HDL) in animals treated with PFOS and PFHS. If you have any questions, please contact Deanna Luebker at (651) 737-1374 or djluebker@mmm.com. Sincerely, Jean B. Sweeney Vice President Environmental, Health and Safety Operations Enclosure CONTAINS NO CBI 3^ 4^ 71 P-2 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 TNO-Report for the study entitled: Mechanism of the effect of different PFAS's (PFBS vs PFHS vs PFOS) on lipid and lipoprotein metabolism in APOE*3-Leiden/huCETP transgenic mice TNO Biosciences Gaubius Laboratory Zernikedreef 9, 2333 CK Leiden The Netherlands General Phone +31 71 518 1818 Specific Fax +31 71 518 1901 Drafted by: Dr. Hans M.G. Princen Elsbet Pieterman B.Sc. In assignment of: 3M, Medical department Project number: 031.12685 Study number: 3M#03 Date: 17 November 2009 Final version Previous version: Draft 2 October 2009 Number of pages: 153 CONFIDENTIAL Signature and date: J.W.A. van den Hoorn M.C.E. Maas E.J. Pieterman BSc. Principal Investigator Dr. J.R.O. Hanemaaljer Dr. H.M.G. Princen Team Manager Study Director All rights reserved. No part of this report may be reproduced and/or published In any form by print, photoprint, microfilm or any other means without the previous written permission from TNO. All Information which is classified according to Dutch regulations shall be treated by the recipient in the same way as classified Information of corresponding value in his own country. No part of this information will be disclosed to any third party. In case this report was drafted on Instructions, the rights and obligations of contracting parties are subject to either the Standard Conditions for Research Instructions given to TNO, or the relevant agreement concluded between the contracting parties. Submitting the report for inspection to parties who have a direct interest is permitted. 2009 TNO 1 p.3 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Summary Introduction and Aim Perfluorinated alkyl sulphonates are fully fluorinated amphiphilic organic molecules with strong surface-tension reducing properties. They are stable to environmental and metabolic degradation. Perfluorooctanesulfonate (PFOS) is widely dispersed in humans, fish-eating wildlife, and surface waters. Toxicological studies in rats and monkeys have shown a reduction in serum cholesterol after treatment with PFOS; however, such reductions have not been observed among exposed workers. In the present study the focus is put on a further elucidation of the mechanism responsible for the observed changes in plasma triglycerides and cholesterol levels in the atherogenic apoB-containlng (VLDL, IDL, LDL) lipoproteins and to investigate the effect of PFBS, PFHS and PFOS on HDL metabolism! The APOE*3-Leiden(E3L)/huCETP transgenic mouse model was used to study these effects. The aim of the study Is further to emphasize the differences In biological effects between PFBS, PFHS and PFOS and to publish these data. Material and methods The study, which was subdivided in three experiments, had the following design: after a run-in period of 4 weeks on a Western type diet (containing 14 % beef tallow, 1 % corn oil, 0.25 % cholesterol), mice received a Western type diet (control) or a western type diet containing 0.03 % PFBS (31.8 mg/kg body weight/day), 0.006% PFHS (6.0 mg/kg body weight/day), 0.003 % PFOS (3.1 mg/kg body weight/day) or as a positive control 0.03% fenofibrate (31.1 mg/kg body weight /day). In all experiments body weight, food intake, plasma cholesterol, HDL cholesterol and triglycerides were measured after 4 weeks of treatment. In experiment 1 lipolytic activity (LPL and HL activity) was measured from post-heparin plasma after 5 weeks of treatment and feces were collected for the determination of bile acids, neutral sterols and fatty acids. At the end of the experiment (6 weeks of treatment) VLDL-triglyceride and de novo apoB production was measured and lipid composition of VLDL was determined. In experiment 2 after 4 weeks of treatment at the end of the experiment the gall bladder was cannulated and during 45 minutes bile flow, biliary cholesterol, phospholipids and bile salt output were measured. Directly hereafter the in vivo clearance of VLDL-like triglycerides-rich particles was determined. In experiment 3, next to plasma ApoAl and CETP activity and mass determinations, the in vivo clearance of autologous labeled HDL was measured after 4 weeks of treatment. From this experiment livers were collected and a part was used for liver histology analysis, a part was used for liver lipid analysis and a part was used for microarray analysis. Mechanism of action of PFBS PFBS reduced plasma cholesterol and triglyceride levels by about 25% and 45%, respectively. The data from the physiological experiments indicate that the decreases in lipid levels are caused by increased clearance of VLDL-TG and VLDL-CE and mildly reduced VLDL-particle production. PFBS had no clear effect on HDL-cholesterol and apoAl levels. PFBS showed no effect on genes involved in HDL metabolism, in line with the unchanged HDL levels. PFBS mildly increased liver weight, but had no effect on ALT and development of hepatosteatosis. Based on mRNA signals PFBS appears to have mild PPARa-agonistic activity ((3-oxidatlon increased and liver size increased). The present data indicate that PFBS has no increased CVD risk profile. Mechanism of action of PFHS PFHS reduced plasma cholesterol and triglyceride levels by about 60% and 75%, respectively. The data from the physiological experiments supported by hepatic mRNA levels indicate that the decreases in lipid levels are caused by increased lipolysls and clearance of VLDL-TG and VLDL-CE, and strongly reduced VLDL-TG and VLDL-particle production. In line with the higher lipolytic activity in plasma, the hepatic mRNA signal for LPL (4.3-fold) was increased. PFHS strongly decreased HDL-cholesterol (about -75%) and apoAl (about -75%) levels. Based on mRNA signals we conclude that PFHS reduces HDL levels by down-regulation of apoAl synthesis and HDL maturation (ABCal, LCAT). These adverse changes are most likely the result of PXRagonistlc activity. Increased remodeling (PLTP) and decreased uptake (SR-B1) are suggested to be responsible for the formation of larger HDL particles. PFHS increased liver weight, ALT, and resulted in hepatosteatosis, as observed by biochemical and histological measures. 2 P-4 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Based on mRNA signals PFHS has strong PPARa-agonistic (lipolysis increased, (5-oxidation increased, FA uptake increased, liver size increased) and PXR-agonistic activity (FA uptake increased, FA synthesis increased and HDL synthesis and maturation decreased, liver size increased). Mechanism of action of PFOS: PFOS reduced plasma cholesterol and triglyceride levels by about 65% and 70%, respectively. A similar mechanism of action is active with PFOS as with PFHS. The data from physiological experiments supported by hepatic mRNA levels indicate that the decreases in lipid levels are caused by increased lipolysis and clearance of VLDL-TG, and strongly reduced VLDL-TG and VLDL-particle production. In line with the higher lipolytic activity in plasma, the hepatic mRNA signal for LPL (2.1-fold) was increased. PFOS increased mRNA levels of cholesterol synthesizing enzymes and cholesterol esterification genes. Moreover, the gene coding for the major rate-limiting enzyme in the bile acid synthetic pathway, and genes involved in biliairy cholesterol excretion were decreased by PFOS. Inhibition of cholesterol metabolism and excretion may form an explanation for increased hepatic cholesterol levels found with PFOS. PFOS strongly decreased HDL-cholesterol (about -65%) and apoAl (about -80%) levels. Based on mRNA signals we conclude that PFOS reduces HDL levels by down-regulation of apoAl synthesis and HDL maturation. These adverse changes are most likely the result of PXR-agonistic activity. PFOS increased liver weight, ALT, and resulted in pronounced hepatosteatosis and liver cholesterol accumulation, as observed by biochemical and histological measures. Based on mRNA signals PFOS has strong PPARa-agonistic (lipolysis increased, (5-oxidation increased, FA uptake increased, liver size increased) and PXR-agonistic activity (FA uptake increased, FA synthesis increased and HDL synthesis and maturation decreased, liver size increased). Involvement of other nuclear transcription factors in the regulation of lipid and lipoprotein metabolism by PFHS and PFOS Involvement of CAR and LXR in the changes in lipid and lipoprotein metabolism caused by PFHS and PFOS cannot be fully excluded, but is less likely. Little is know about the role of CAR in lipid metabolism. CAR has been shown to decrease p-oxidation genes as CPT1 and enoyl CoA hydratase. The latter genes were, however, increased in the present experiments. LXR increases fatty acid synthesis by induction of SREBPlc expression, which was 2-fold decreased, however. In addition, LXR induces expression of CETP mRNA, whereas in the present experiments a decrease in CETP activity was found. It cannot be excluded that this is caused by a strongly decreased acceptor pool for CE transfer. Involvement of RXR in the observed effects cannot be excluded, since RXR forms a heterodimer together with a larger number of nuclear transcription factors like PPARa, PXR and LXR. However, direct activation of RXR, for instance with bexarotene leads to opposite effects with increased levels of triglycerides and apoB-containing lipoproteins. Mechanism of action o f fenofibrate Fenofibrate reduced plasma cholesterol and triglyceride levels by about 40% and 70%, respectively. The data from the physiological experiments supported by hepatic mRNA levels indicate that the decreases in lipid levels are caused by strongly increased lipolysis and clearance of VLDL-TG and VLDL-CE, despite the increased VLDL-TG production rate. In line with the higher lipolytic activity in plasma, the hepatic mRNA signal for LPL (4.6-fold) was increased. LDLR mRNA as marker for increased uptake of VLDL remnant particles was enhanced by 1.5-fold. Fenofibrate paradoxically increases VLDL-TG production despite reducing plasma TG, which may be caused by enhanced hepatic free fatty acid uptake resulting from strongly accelerated peripheral LPLmediated lipolysis of VLDL or by increased de novo hepatic TG synthesis. Fenofibrate increased HDL-cholesterol (+50%) and formation of large HDL-1 particles, and had no effect on apoAl. Fenofibrate strongly decrease in CETP activity, which was found majorly responsible for the increased HDL levels upon treatment with fenofibrate and PPARa,y-agonists. Fenofibrate increased liver weight, without effects on ALT and hepatosteatosis, and decreased liver cholesterol content. Based on mRNA signals fenofibrate has strong PPARa-agonistic activity (lipolysis increased, FA uptake increased, |3-oxidation increased; HDL remodeling decreased). 3 p. 5 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Contents Summary Contents .............................................................................................................2 4 1 Introduction & a im ................................................................................. 7 1.1 Aim of the study...................................................................................................8 2 Materials and methods................................................................................................. 9 2.1 Test substances & reference substance................................................................... 9 2.2 Mice .................'......................................................................................... 9 2.3 Animal welfare......................................................................................................9 2.4 Diets ........................................................................................................... 9 2.5 Compound administration...................................................................................... 9 2.6 Treatment groups...............................................................................................10 2.7 Study design...................................................................................................... 10 2.8 Measurements experiment 1 ................................................................................ 13 2.9 Measurements experiment 2 ................................................................................ 14 2.10 Measurements experiment 3 ................................................................................ 14 2.11 Statistical analysis...............................................................................................16 3 Deviations from the protocol......................................................................................17 4 Results........................................................................................................................18 4.1 Results study 1 ................................................................................................... 18 4.1.1 Markers of general well-being....................................................................18 4.1.2 Body weight............................................................................................ 18 4.1.3 Liver and perigonadal fat weight............................................................... 19 4.1.4 Food intake.............................................. 21 4.1.5 Plasma A LT ............................................................................................. 22 4.1.6 Plasma cholesterol...................................................................................23 4.1.7 Plasma HDL-cholesterol.............................................................................24 4.1.8 Plasma triglycerides..................................................................................25 4.1.9 Lipoprotein profiles.................................................................................. 27 4.1.10 Plasma free glycerol.................................................................................28 4.1.11 Plasma free fatty acids............................................................................. 29 4.1.12 Post-heparin LPL and HL activity............................................................... 30 4.1.13 Fecal lipids..............................................................................................32 4.1.14 VLDL-triglycerides and de novo ApoB production.........................................38 4.2 Results study 2 ................................................... 43 4.2.1 Markers of general well-being....................................................................43 4.2.2 Body weight............................................................................................ 43 4.2.3 Liver and perigonadal fat weight............................................................... 44 4.2.4 Food intake............................................................................................. 46 4.2.5 Plasma A LT ............................................................................................. 47 4.2.6 Plasma cholesterol...................................................................................47 4.2.7 Plasma HDL-cholesterol.............................................................................49 4.2.8 Plasma triglycerides.................................................................................. 50 4.2.9 Lipoprotein profiles.................................................................................. 51 4.2.10 Biliary bile adds, cholesterol and phospholipids production........................... 52 4.2.11 In vivo clearance of VLDL-like TG-rich particles and uptake in tissues.............57 4 P-6 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3 Results study 3 ...................................................................................................64 4.3.1 Markers of general well-being.................................................................... 64 4.3.2 Body weight............................................................................................ 64 4.3.3 Liver and perlgonadal fat weight................................................................65 4.3.4 Food intake............................................................................................. 67 4.3.5 Plasma A LT ............................................................................................. 68 4.3.6 Plasma cholesterol...................................................................................68 4.3.7 Plasma HDL-cholesterol............................................................................ 69 4.3.8 Plasma triglycerides..................................................................................71 4.3.9 Lipoprotein profiles...................................................................................72 4.3.10 Plasma A p o A l....................... 73 4.3.11 Plasma CETP m ass...................................................................................74 4.3.12 Plasma CETP activity................................................................................ 75 4.3.13 In vivo clearance of autologous HDL.......................................................... 76 4.3.14 Liver microsomal DGAT activity................................................................. 79 4.3.15 Liver lipid analysis....................................................................................80 4.3.16 Liver histology.........................................................................................82 4.3.17 Liver microarray analysis.......................................................................... 85 5 Conclusions and comments............................................... 89 6 References................................................................................................................ 92 7 Appendices............. ................................................................................................... 94 Appendix I Body weight........................................................................................ 94 Appendix II Tissue weight....................................................................................... 97 Appendix III Food intake........................................................................................ 100 Appendix IV Plasma cholesterol.............................................................................. 103 Appendix V Plasma HDL-cholesterol....................................................................... 106 Appendix VI Plasma triglycerides............................................................................ 109 Appendix VII Plasma free glycerol............................................................................ 112 Appendix VIII Plasma free fatty acids........................................................................ 113 Appendix IX Plasma A p o A l.....................................................................................114 Appendix X Plasma CETP mass.............................................................................. 115 Appendix XI Plasma CETP activity........................................................................... 116 Appendix XII Post heparin LPL and HL activity........................................................... 117 Appendix XIII Fecal lipids.........................................................................................118 Appendix XIV VLDL-triglycerides and de novo ApoB production..................................... 123 Appendix XV Biliary bile acids, cholesterol and phospholipids..................................... 126 Appendix XVI In vivo clearance of VLDL-like TG-rlch particles and uptake in liv er.........130 Appendix XVII In vivo clearance of autologous H D L..................................................... 140 Appendix XVIII Liver microsomal DGAT activity.............................................................141 Appendix XIX Liver lip id s......................................................................................... 142 Appendix XX Liver microarray analysis..................................................................... 143 Appendix XXI Summary tab le.................................................................................. 153 5 p.7 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Sponsor of the study 3M, Medical Department 3M Center, Building 0220-02-E-02 St.Paul, Minnesota 55144-1000 USA Study contact John L. Butenhoff, Ph.D., CIH, DABT Corporate Scientist Medical Department 3M Center, Building 0220-02-E-02 St.Paul, Minnesota 55144-1000 USA Phone: +1.651.733.1962 Fax: +1.651.733.1773 E-mail: jlbutenhoff@mmm.com Testing facility TNO, Business Unit Biosciences Mail address: P.O.Box 2215, 2301 CE Leiden Delivery address: Zernikedreef 9, 2333 CK Leiden The Netherlands Responsible personnel Study director Dr. Hans M.G. Princen Phone: +31 71 518 1471 Fax: +31 71 518 1901 E-mail: hans.princen@tno.nl Technical staff Marian Bekkers B.Sc. Simone Droog B.Sc. Annemarie Maas B.Sc. Elsbet Pieterman B.Sc. Karin Toet B.Sc. Marijke Voskuilen B.Sc. Dr. Marjan van Erk Dr. Anita van den Hoek Dr. Jos van der Hoorn Dr. Patrick Rensen Advisors Prof. Dr. Louis M. Havekes E-mail: louis.havekes@tno.nl Dr. Patrick Rensen E-mail: p.c.n.rensen@lumc.nl 6 P-8 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 1 Introduction & aim Perfluorinated alkyl sulphonates are fully fluorlnated amphiphilic organic molecules with strong surface-tension reducing properties. They are stable to environmental and metabolic degradation. Perfluorooctanesulfonate (PFOS) Is widely dispersed In humans, fish-eating wildlife, and surface waters. Toxicological studies In rats and monkeys have shown a reduction in serum cholesterol after treatment with PFOS; however, such reductions have not been observed among exposed workers. In order to obtain more insight into the possible mechanism of action the APOE*3-Leiden transgenic mouse, a well-recognized animal model for hyperlipidemia and atherosclerosis, has been used in a previous study to investigate the in vivo effects of three perfluoro-alkyl-sulphonates with different chain length, PFBS (C4), PFHS (C6) and PFOS (C8), on plasma lipids and lipoproteins and bile acid metabolism. Of the three PFAS, PFHS and PFOS have been withdrawn from the market by 3M in the beginning of 2000 because of environmental issues, whereas PFBS is marketed in various industrial applications. APOE*3Leiden transgenic mice exhibit elevated plasma cholesterol and triglyceride levels, mainly confined to the VLDL/LDL sized lipoprotein fraction (1). Extensive previous research showed that, in contrast to wild-type mice, APOE*3Leiden transgenic mice are highly responsive to fat and cholesterol feeding as far as the effects on plasma VLDL and chylomicron levels are concerned (2, 3). In addition, we have found that drugs and dietary compounds influencing either the chylomicron and VLDL production and/or the hepatic clearance of lipoproteins exert relatively strong effects on plasma cholesterol and triglyceride levels (4-11, see for review ref. 12). In contrast, in normal wild-type mice the plasma cholesterol and triglyceride levels are very low and (almost) not responsive to diet and hypolipidemic drugs. This animal model has been proven to be representative for the human situation regarding plasma lipoprotein levels, lipoprotein profiles, its responsiveness to hypolipidemic drugs (like statins, fibrates etc.) (4-8, 10) and nutrition (9, 11). In addition, depending on the level of plasma cholesterol APOE*3Leiden mice develop atherosclerotic lesions in the aorta resembling those found in humans with respect to cellular composition and morphological and immunohistochemical characteristics (3). TNO in collaboration with the Leiden University Medical Center has recently developed the APOE*3Leiden(E3L)/huCETP transgenic mouse, which has proven to be very suitable for testing the effects of drugs and nutritional factors on plasma HDL and triglyceride levels, atherosclerosis and metabolic syndrome. In the newly generated mouse, human cholesterol ester transfer protein (huCETP) under control of its natural flanking regions is introduced into the APOE*3-Leiden mouse resulting in a more human-like lipoprotein profile with transfer of cholesterol ester from HDL to the apoB-containing lipoproteins in exchange for triglycerides. As a result of this adverse lipoprotein distribution and the higher amount of atherogenic apoB-containing lipoproteins, the E3L.CETP transgenic mice develop increased atherosclerosis on a Western-type diet as compared to E3L transgenic mice (13). The E3L.CETP transgenic mice respond to treatment with (registered) drugs as fibrates (14), statins (15), niacin (16) the CETP inhibitor torcetrapib (7) at similar dosages and in a similar way to humans, with decreases in the apoB-containing lipoproteins and an increase in HDL levels. From the previous study in APOE*3-Leiden transgenic mice, it was concluded that PFHS and PFOS have strong cholesterol and triglycerides lowering effects. Lipoprotein profiles of PFHS and PFOS treated APOE*3-Leiden mice also showed the formation of a "large HDL" particle, presumably an apoE-, cholesteryl ester-rich HDL-1 particle. Moreover, PFHS and PFOS treated animals showed increased plasma ALAT levels and liver size, decreased bile acid synthesis and cholesterol-7ahydroxylase activity, increased fatty acid oxidation, and decreased body weight, epididymal fat and increased energy expenditure via burning of fat, all suggesting PPARa agonist activity for these two chemicals. PFBS differed markedly from PFHS and PFOS by having no or only minor effects on the measured parameters, whereas PFHS appeared to have intermediate effects taking into account the higher dose applied as compared to PFOS. In the present study the focus is put on a further elucidation of the mechanism responsible for the observed changes In plasma triglycerides and cholesterol levels in the atherogenic apoB-containing (VLDL, IDL, LDL) lipoproteins and to investigate the effect of PFBS, PFHS and PFOS on HDL metabolism. Therefore, in this study the more appropriate APOE*3-Leiden(E3L)/huCETP transgenic 7 P-9 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 mouse model was used. The aim of the study is further to emphasize the differences in biological effects between PFBS, PFHS and PFOS and to publish these data. 1.1 Aim of the study To elucidate the mechanism of action responsible for the observed changes in plasma triglycerides and cholesterol levels in the atherogenic apoB-containing (VLDL, IDL, LDL) lipoproteins by PFHS and PFOS and to investigate the effect of PFBS, PFHS and PFOS on HDL metabolism in E3L.CETP transgenic mice. To emphasize the differences in biological effects between PFBS, PFHS and PFOS and to publish these data. 8 p. 10 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 2 Materials and methods 2.1 Test substances & reference substance Test substances: Reference substance: PFBS (perfluorobutanesulfonate): L-7038, lot 2 (1999) PFHS (perfluorohexanesulfonate): 127498-80, lot L9051 PFOS (perfluorooctanesulfonate): FC-95, lot 217 Fenoflbrate: F6020, lot 117K1486 PFBS, PFHS and PFOS were provided by 3M Medical Department (sent to TNO on 10-Jun-04). Fenoflbrate was purchased from Sigma (St. Louis, USA). 2.2 Mice Based on the large difference In half-life between males (17h) and females (3h) in CD-I mice, we have used males in the proposed experiments. One-hundred-twenty male heterozygous APOE*3Leiden.CETP mice, 7-10 weeks of age, from the SPF breeding stock at TNO-Biosciences (Leiden) have been used, and housed during the experiment in macrolon cages (maximal 4 mice per cage), in clean-conventional animal rooms at TNO-Leiden (relative humidity 50-60%, temperature ~21C, light cycle 7 am to 7 pm). Individual animals are marked by ear punch-holes. Mice were supplied with food and acidified tap water ad libitum. 2.3 Animal welfare Experiments were performed conform to the rules and regulations set forward by the Netherlands Law on Animal Experiments. Experiments had been approved by the Animal Experiment Committee of TNO under registration number 2483. The study director was entitled to terminate experiments in case of serious unexpected animal discomfort. 2.4 Diets Mice received Western type diet (WTD; semi synthetic diet containing 14% beef tallow, 1% corn oil and 0.25% of cholesterol), purchased from ABdlets (Woerden, The Netherlands). This resulted in total cholesterol (TC) plasma levels of about 8 mmol/L and triglyceride levels of about 2 mmol/L. Diets were renewed once per week. 2.5 Compound administration The required quantities of PFBS, PFHS and PFOS were supplied by 3M (sent to TNO on 10-Jun-04). Fenoflbrate (Sigma, St. Louis) was used as a positive control. All compounds were administered orally as admix to the WTD. Preparation of all diets (with different compounds) were performed according to: Operation procedure number 14 of "Laboratorlum procedures Llpiden" entitled "Preparation of diet chunks from powdered food". The tyophilized diet chunks were stored in vacuum bags in an alarm-secured -20C room. 9 p. 11 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 2.6 Treatment groups Group 1: WTD diet Group 2: WTD + 0.03 % fenofibrate (w/w) or 31.8* mg/kg body weight/day (positive control) Group 3: WTD + 0.03 % PFBS (w/w) or 31.1* mg/kg body weight/day Group 4: WTD + 0.006 % PFHS (w/w) or 6.0* mg/kg body weight/day Group 5: WTD + 0.003 % PFOS (w/w) or 3.1* mg/kg body weight/day * Average intake of compounds based on average body weight and food intake in the three indicated experiments 2.7 Study design Design experiment 1 Weeks of treatment: 1 -4| -3| -2| -1 I 0 I 1 I 2 I 3 I 4 I 5 I ~6 Group 1: diet WTD Group 2: WTD + 0.03 % fenofibrate Group 3: WTD + 0.03 % PFBS Group 4: WTD + 0.006 % PFHS Group 5: WTD +0.003 % PFOS -* x -------------------------- + X --------------------------------------------------------- -* X --------------------------------------------------------- - X --------------------------------------------------------- - X --------------------------------------------------------- Collection of extra plasma for measurements of plasma levels of the compounds by 3M Experiment 1 was performed according to the scheme as outlined above. In short: A run-in period of 4 weeks was started with 54 male E3L.CETP mice on a semi synthetic western type diet (containing 14 % beef tallow, 1 % corn oil, 0.25 % cholesterol). In week 0 the animals were randomized on body weight, plasma cholesterol, HDL-cholesterol and triglycerides (after 4h fasting) in 5 groups of 8 animals and the 6-weeks treatment was started. Body weight, food intake, plasma cholesterol, HDL-cholesterol, triglycerides, glycerol and free fatty acids (after 4h fasting) were measured at t=0 weeks and 4 and 6 weeks after start of treatment. Plasma ALAT as a parameter for liver damage and lipoprotein profiles were measured in pooled samples per group at t=0, 4, 6 weeks (ALAT) and t=4, 6 weeks (lipoprotein profiles). Extra plasma (without radioactivity) was collected at 6 weeks for measurements of plasma levels of the compounds by 3M. After 5 weeks of treatment lipolytic activity (LPL and HL activity) was measured from postheparin plasma from fasted mice (4h fasting) and feces was collected for the determination of bile acids, neutral sterols and fatty acids. At the end of the experiment VLDL-triglyceride and de novo apoB production was measured and lipid composition of VLDL was determined. After sacrifice EDTAplasma, serum and perigonadal adipose tissue and liver was collected. Both tissues were directly frozen in liquid nitrogen and stored below - 70 C. The run-in period of study 1 started on 25 March 2008, mice were sacrificed on 6 June 2008. 10 p. 12 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Design experiment 2 Weeks of treatment: | -4| -3| -2| -1 | 0 | 1 | 2 | 3 | 4 Group 1: diet W TD Group 2: WTD + 0.03 % fenofibrate Group 3: WTD + 0.03 % PFBS Group 4: WTD + 0.006 % PFHS Group 5: WTD + 0.003 % PFOS x * x x *> x x X x X x X * Collection of extra plasma for measurements of plasma levels of the compounds by 3M Experiment 2 was performed according to the scheme as outlined above. In short: A run-in period of 4 weeks was started with 38 male E3L.CETP mice on a semi synthetic western type diet (containing 14 % beef tallow, 1 % corn oil, 0.25 % cholesterol). In week 0 the animals were randomized on body weight, plasma cholesterol, HDL-cholesterol and triglycerides (after 4h fasting) in 5 groups of 6 animals and the 4-weeks treatment was started. Body weight, food intake, plasma cholesterol, HDL-cholesterol and triglycerides (after 4h fasting) were measured at t=0 and 4 weeks. Plasma ALAT as a parameter for liver damage and lipoprotein profiles was measured in pooled samples per group at t=0 and 4 weeks (ALAT) and t=4 weeks (lipoprotein profiles). Extra plasma (without radioactivity) was collected at 4 weeks for measurements of plasma levels of the compounds by 3M. After 4 weeks of treatment at the end of the experiment the gall bladder was cannulated and during 45 minutes bile flow, biliary cholesterol, phospholipids and bile salt output were measured. Directly hereafter the in vivo clearance of VLDL-like triglycerides-rich particles was determined. After sacrifice EDTA-plasma, serum and perigonadal adipose tissue and liver was collected. The perigonadal adipose tissue and liver were directly frozen in liquid nitrogen and stored below -70 C. The run-in period of study 2 started on 16 June 2008, mice were sacrificed on 15 August 2008. The extra mice from study 3, used for bile cannulation were sacrificed on 2 October 2008. ll p. 13 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Design experiment 3 Weeks of treatment: | -4| -3| -2| -1 | 0 | 1 | 2 | 3 | 4 Group 1: diet WTD Group 2: WTD + 0.03 % fenofibrate Group 3: W TD + 0.03 % PFBS Group 4: W TD + 0.006 % PFHS Group 5: W TD + 0.003 % PFOS x --------------- x ------------------ x --------------- x ------------------ x --------------- x ------------------ x --------------- x -- -- ---- -- - x --------------- x ----------------- - Experiment 3 was performed according to the scheme as outlined above. In short: A run-in period of 4 weeks was started with 38 male E3L.CETP mice on a semi synthetic western type diet (containing 14 % beef tallow, 1 % corn oil, 0.25 % cholesterol). In week 0 the animals were randomized on body weight, plasma cholesterol, HDL-cholesterol and triglycerides (after 4h fasting) in 5 groups of 7 animals and the 4-weeks treatment was started. Body weight, food intake, plasma cholesterol, HDL cholesterol and triglycerides and plasma ApoAl, CETP activity and mass (after 4h fasting) was measured at t=0 and 4 weeks. ALAT as a parameter for liver damage and lipoprotein profiles was measured in pooled samples per group at t=0 and 4 weeks (ALAT) and t=4 weeks (lipoprotein profiles). Extra plasma (without radioactivity) was collected at 4 weeks for measurements of plasma levels of the compounds by 3M. After 3 weeks of treatment one mouse of each group was sacrificed by C 02 and serum was collected from the retro-orbital vein. A part of the liver was directly frozen in liquid nitrogen. HDL was isolated and radiolabeled with 3H-cholesteryl oleyl ether. The in vivo clearance of autologous HDL was determined in 5 mice per group for 24 hr after an injection of 3H-cholesteryl oleyl ether labeled autologous HDL. After sacrifice EDTA-plasma, serum and small intestine, perigonadal adipose tissue and liver were collected. The small intestine was directly frozen in liquid nitrogen and stored below -70 C for mRNA isolation. The liver was cut in four parts; two parts of the liver were directly frozen in liquid nitrogen and stored below - 70 C for mRNA isolation and liver lipid analysis, one part was used fixed in buffered formalin for liver histology and a part was used for the isolation of microsomes. Perigonadal adipose tissue was directly frozen in liquid nitrogen and stored below - 70 C. One mouse per group was used for an extra bile cannulation at t=5 weeks. The run-in period of study 3 started on 28 July 2008, mice were sacrificed on 25 September 2008, except the extra mice from study 3. These mice, used as extra mice for the bile cannulation experiment for study 2, were sacrificed on 2 October 2008. 12 p. 14 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 The last measurement (CETP activity) was performed on 7 September 2009. Liver histology analysis was performed in week 38, 2009. 2.8 Measurements experiment 1 1) Body weight and food intake (per cage) at 0, 4 and 6 weeks. 2) Plasma cholesterol, HDL cholesterol, triglycerides, glycerol and free fatty adds (after 4h fasting) at 0, 4 and 6 weeks. Plasma cholesterol and triglycerides were determined using kit "Cholesterol CHOD-PAP" and kit "Triglycerides GPO-PAP" both from Roche (Mannheim, Germany). Plasma glycerol was determined using "the free glycerol determination kit" from Sigma (St. Louis, USA). Plasma free fatty acids were determined using kit "NEFA C" from WAKO (Neuss, Germany). Plasma HDL-cholesterol was determined using kit "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) after the precipitation of apoB containing lipids by MnCI2 (0.2 mol/L) and heparin (500 IU/mL). 3) Lipoprotein profiles at group level at 4 and 6 weeks. Lipoproteins were separated by FPLC analysis using an AKTA apparatus. Analyses were performed In freshly obtained pooled samples per group. Cholesterol and phospholipid profiles were measured In the fractions using kit "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) and kit "Phospholipids" from Instruchemie (Delfzijl, the Netherlands), respectively. 4) Plasma ALAT at group level at 0, 4 and 6 weeks. Plasma ALAT was measured using the spectrophotometric assay of the Roche Reflotron plus system (Mannheim, Germany). 5) Lipoprotein lipase and hepatic lipase activity at 5 weeks. Lipolytic activity (lipoprotein lipase and hepatic lipase activity) was determined as described previously (18). In short: Postheparin plasma from fasted mice (4h) was collected from the tail vein at 20 minutes after intraperitoneal injection of heparin (0.5 IU/g body weight). Postheparin plasma triacylglycerol hydrolase activity was determined in the presence or absence of 1 mol/L NaCI to estimate both the lipoprotein lipase (LPL) and hepatic lipase (HL) activity. LPL activity was calculated as the portion of total lipase activity inhibited by 1 mol/L NaCI. 6) VLDL-triglyceride and VLDL-apoB production and lipid composition of nascent VLDL at 6 weeks. The rate of hepatic VLDL-triglyceride production, de novo apoB secretion and lipid composition of nascent VLDL was determined in 4 hr (or overnight) fasted mice as described previously (18, 19). In short: Mice were anesthetized with fluanisone-fentanylmidazolam intraperitoneally and injected intravenously with 0.1 ml phosphate-buffered saline containing 100 pCi Tran3SS-label (ICN Biomedicals, Irvine, USA) to measure de novo apoB synthesis. After 30 min, the animals received a Triton WR1339 injection (500 mg/kg body weight), which virtually completely inhibited VLDL clearance by blocking LPL mediated lipolysis. Blood samples were drawn 0, 15, 30, 60 and 90 min after Triton WR1339 injection and plasma triglycerides concentrations were measured. After 90 minutes mice were killed and blood was collected by heart punction for isolation of VLDL and subsequent determination of de novo apoB synthesis and VLDL composition. For that purpose VLDL particles (density < 1.019) were separated from other lipoproteins by density gradient ultracentrifugation. The protein content of the VLDL fraction was determined by a Lowry protein determination, and triglycerides and total cholesterol concentrations were determined using kit "Cholesterol CHOD-PAP" and kit "Triglycerides GPO-PAP" both from Roche (Mannheim, Germany). Phospholipid and free cholesterol concentrations were determined using kit "Phospholipids" and kit "Free cholesterol C" both from Instruchemie (Delfzijl, The Netherlands). The 3SS-apoB content of VLDL was measured after selective precipitation of apoB with isopropanol. The VLDL-TG production rate was calculated by curve fitting (trendlines with the equation: plasma TG=a*time + b, in which a is the calculated production rate). Lipid composition of VLDL was calculated per newly synthesized ApoB and expressed as pmol/dpm ApoB. 7) Fecal excretion of bile acids, neutral sterols and fatty acids at 5 weeks (n= 6 per group). Fecal bile acids, neutral sterols and fatty acids were determined in feces collected during a 48-72 h time period in 3 subgroups at 2 consecutive time points during week 5, by gas chromatographic (GC) analysis as described previously (19, 20). 8) After sacrifice at 6 weeks (end of VLDL-triglyceride and VLDL-apoB production experiment) EDTA-plasma (also needed for isolation of VLDL and subsequent determination of de novo apoB synthesis and VLDL composition), serum, perigonadal adipose tissue and liver. EDTAplasma and serum were collected from heart punction. The liver and perigonadal adipose tissue was weighed and directly frozen in liquid nitrogen and stored below -70 C. 13 p. 15 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 9) Plasma taken at 6 weeks after 4h fasting was sent to 3M for measurements of plasma levels of compounds. Samples were sent on dry Ice by Fedex courier on December 1st 2008 and arrived at the 3M center on December 2nd 2008. 2.9 Measurements experiment 2 1) Body weight and food Intake (per cage) at 0 and 4 weeks. 2) Plasma cholesterol, HDL cholesterol and triglycerides (after 4h fasting) at 0 and 4 weeks. Plasma cholesterol and triglycerides were determined using kit "Cholesterol CHOD-PAP" and kit "Triglycerides GPO-PAP" both from Roche (Mannheim, Germany). Plasma HDLcholesterol was determined using kit "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) after the precipitation 6f apoB containing lipids by MnCI2 (0.2 mol/L) and heparin (500 IU/mL). 3) Lipoprotein profiles at group level at 4 weeks. Lipoproteins were separated by FPLC analysis using an AKTA apparatus. Analyses were performed in freshly obtained pooled samples per group. Cholesterol and phospholipid profiles were measured in the fractions using kit "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) and kit "Phospholipids" from Instruchemle (Delfzijl, the Netherlands) respectively. 4) Plasma ALAT at group level at 0 and 4 weeks. Plasma ALAT was measured using the spectrophotometric assay of the Roche Reflotron plus system (Mannheim, Germany). 5) Bile cannulation, bile flow, biliary cholesterol, phospholipids, bile acids at 4 weeks. Determination of biliary lipid secretion and bile flow was determined as described previously (20). In short: The common bile duct of anesthetized mice was ligated, and the gallbladder was cannulated. Bile was collected In 15 minutes intervals for 45 minutes. Bile flow was measured. Cholesterol and phospholipid concentrations in bile were determined determined using kit "Cholesterol CHOD-PAP" and kit "Phospholipids" from Roche (Mannheim, Germany) and Instruchemie (Delfzijl, The Netherlands), respectively. Total bile acids were determined in bile using kit "total bile acids assay" from Lucron Bioproducts (Milsbeek, the Netherlands) 6) Clearance of VLDL-like particles and uptake in liver at 4 weeks. The in vivo clearance of VLDL-like trlglycerldes-rich particles was adapted from what was described previously (19). In short: Directly after bile cannulation experiment VLDL-like emulsion particles containing 200 pCi 3H-triolein and 20 pCI 14C-cholesteryl oleate were Injected Into the tail vein at a dose of 1 mg triglycerides per mouse. At 2, 5, 10, 20 and 30 min blood samples (50 pi) were taken from the tail vein. 3H and 14C activities were counted in 10 pi serum and corrected for total serum volume. At the end of the experiment liver, heart, perigonadal fat, spleen and muscle (femoralls) were collected. All tissues were weighed. Lipids were extracted from the tissues by an overnight incubation at 60 C in 500 pi Solvable (PerklnElmer, Wellesley, USA) and radioactivity was measured. The clearance rate of VLDL-like particles was calculated by curve fitting (trendlines with the equation: % of Injected dose=b*etime/a, in which a is the calculated half life of the labeled VLDL-like particles). 7) After sacrifice at 4 weeks (end of in vivo VLDL clearance experiment) collection of EDTAplasma, remainder liver and perigonadal adipose tissue. EDTA-plasma was collected from heart punction. The remainder of the livers and perigonadal fat tissues were directly frozen In liquid nitrogen and stored below -70 C. 8) 15 pi of (non-radioactlve) plasma taken at 4 weeks after 4h fasting was sent to 3M for measurements of plasma levels of compounds. Samples were sent on dry Ice by Fedex courier on December 1st 2008 and arrived at the 3M center on December 2nd 2008. 2.10 Measurements experiment 3 1) Body weight and food intake (per cage) at 0 and 4 weeks. 2) Plasma cholesterol, HDL-cholesterol and triglycerides (after 4h fasting) at 0 and 4 weeks. Plasma cholesterol, HDL-cholesterol and triglycerides were determined using kit "Cholesterol CHOD-PAP" and kit "Triglycerides GPO-PAP" both from Roche (Mannheim, Germany). Plasma HDL-cholesterol was determined using kit "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) after the precipitation of apoB containing lipids by MnCI2 (0.2 mol/L) and heparin (500 IU/mL). 3) Lipoprotein profiles at group level at 4 weeks. Lipoproteins were separated by FPLC analysis using an AKTA apparatus. Analyses were performed in freshly obtained pooled samples per group. Cholesterol and phospholipid profiles were measured in the fractions using kit 14 p. 16 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) and kit "Phospholipids" from Instruchemie (Delfzijl, the Netherlands), respectively. 4) Plasma ALAT at group level at 0 and 4 weeks. Plasma ALAT was measured using the spectrophotometric assay of the Roche Reflotron plus system (Mannheim, Germany). 5) Plasma CETP activity (17) and mass (16) at 0 and 4 weeks. CETP activity was determined using kit "Roar CETP Activity assay kit" from Roar Biomedical Inc (New York, USA). CETP mass was determined using the CETP ELISA from Dalichi Pure Chemicals Co. (Tokyo, Japan). 6) Plasma ApoAl concentration at 0 and 4 weeks. Plasma ApoAl concentrations were determined using a sandwich ELISA (16). Rabbit anti-mouse ApoAl polyclonal antibody from Abeam Pic. (Cambridge, UK) was coated overnight (3 pg/mL) onto Costar strips (New York, USA) at 4C and was incubated with diluted mouse plasma (dilution 1:400,000) for 90 min at 37C. Subsequently, goat anti-mouse ApoAl antibody from Rockland Immunochemicals Inc. (Gllbertsvllle, USA; dilution 1:3,000) was added and incubated for 90 min at 37C. Finally, HRP-conjugated rabbit anti-goat IgG antibody from Rockland Immunochemicals Inc. (Gilbertsville, USA; dilution 1:15,000) was added and incubated for 90 min at 37C. HRP was detected by incubation with tetramethylbenzidine from Organon Teknika (Boxtel, The Netherlands). Purified mouse ApoAl from Biodesign International (Saco, USA) was used as a standard. . 7) In vivo clearance of autologous HDL at 4 weeks. The radiolabeling of autologous HDL and In vivo clearance of autologous HDL was performed as described previously (14). In short: For the radiolabeling of autologous HDL, one mouse from each experimental group was euthanized by C 0 2, and blood was drawn from the retro-orbital vein. (One piece of the liver was collected and directly frozen in liquid nitrogen and stored below -70 C for mRNA isolation and liver lipids determination). Serum was collected and HDL was isolated after density ultracentrifugation. HDL (0.32 pmol HDL-cholesterol) was radiolabeled by incubation (37C, 24 h) with 3H-cholesteryl oieyl ether labeled egg yolk phosphatidylcholine vesicles (40 pCi, 0.5 mg of phospholipid) in the presence of lipoprotein-deficient serum (1 m,L) from E3Leiden.CETP mice. Subsequently, HDL was relsolated after density ultracentrifugation. For the in vivo clearance of autologous HDL, mice were Injected via the tail vein with a trace of autologous 3H-cholesteryl oieyl ether labeled HDL (0.1 pCi in PBS) at 8 am. Blood was collected at 1, 2, 4, 8 and 24 h after Injection and 3H-actlvlty was counted in plasma samples to determine the plasma decay of 3H-cholesteryl oieyl ether. HDL clearance was calculated by curve fitting timeframe 0-8 h (trendlines with the equation % of Injected dose=100*ea*tlme, In which a Is the calculated fractional catabolic rate of HDL). 8) After sacrifice at 4 weeks (end of in vivo HDL clearance experiment) collection of EDTAplasma, serum, liver, small Intestine and perigonadal adipose tissue. EDTA-plasma and serum was collected from heart punction. The liver was weighed and cut in 4 parts, two parts and the small Intestine were directly frozen in liquid nitrogen and stored below -70 C. One part was fixed in phosphate buffered formalin (10%) for liver histology. The remainder of the liver was used to isolate microsomes for DGAT activity measurements (per liver microsomes were isolated) Perigonadal adipose tissue was weighed and directly frozen in liquid nitrogen and stored below -70 C. 9) 15 pi of (non-radioactive) plasma taken at 4 weeks after 4h fasting was sent to 3M for measurements of plasma levels of compounds. Samples were sent on dry Ice by Fedex courier on December 1st 2008 and arrived at the 3M center on December 2nd 2008 10) DGAT2 activity in liver microsomes. The DGAT activity in liver microsomes was determined as described previously (7, 18) in the presence of 5 mmol/L or 100 mmol/L MgCI2. Since DGAT2 activity is inhibited at higher concentrations of MgCI2 (100 mmol/L), DGAT2 activity can be calculated by subtracting the DGAT activity in the presence of 100 mmol/L MgCI2 (only DGAT1 activity) from the DGAT activity in the presence of 5 mmol/L MgCI2 (DGAT1 and DGAT2 activity). 11) Micro array analysis livers: RNA was isolated from collected liver pieces. After quality control of the RNA, microarray analysis was carried out at Service XS B.V. (Leiden, the Netherlands) using the Affymetrix technology platform and Affmetrix GeneChip mouse genome MOE430-2.0 arrays. Data were sent to TNO Zeist for gene expression data analysis ( 21). 12) Liver lipids (FC, CE, TG and PL) in 6 samples per group and liver histology in 5 samples per group. Liver lipids were determined by a HPTLC analysis as previously described (22). 15 p. 17 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 2.11 Statistical analysis Significance of differences between the groups was calculated non-parametrically, using the computer program SPSS. A Kruskall-Wallis test for several independent samples was used, followed by a Mann-Whitney U-test for independent samples. A P-value < 0.05 was considered statistically significant. The resulting p-values were given in separate tables in the report. 16 p. 18 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 3 Deviations from the protocol Experiment 1 - Lipolytic activity determination: Post-heparin plasma was collected in week 5 instead of week 4 of treatment. Instead of 1 IU heparin/g body weight 0.5 IU heparln/g body weight heparin was injected intraperitoneally, 20 minutes before collection of post-heparin plasma. - Feces were collected during a 48 h and a consecutive 72 h time period In 3 subgroups instead of 2 consecutive time points of 48 hours. - We decided not to collect liver pieces for histology analysis, liver lipid analysis and mRNA isolation and subsequent micro array analysis in experiment 1 but in experiment 3, since injection of Triton WR-1339 (used to block VLDL clearance) could have an effect on liver gene expression and liver lipids. Intestine was collected in experiment 3 Instead of experiment 1. - At sacrifice no serum was collected, only EDTA-plasma. All of the plasma was used for VLDL isolation (via ultracentrifugation). - Lipid composition of VLDL is expressed per dpm 35S-ApoB instead of per mg protein, since the protein concentration was very low in the PFHS and PFOS treatment, and could not be measured properly. Experiment 2 - 5 extra mice from experiment 3 (one mouse per group) were used to perform extra bile cannulations for determination of bile flow, biliary cholesterol, phospholipids, bile acids. These extra mice were used since some mice from experiment 2 had to be excluded from analysis because of a very low bile production. The excluded mice cooled off too much during the cannule placement operation, and the mice were not warm enough during the bile collection, which affected bile production drastically. - Clearance of VLDL-like particles and uptake In liver at 4 weeks: We decided to only measure uptake in liver of VLDL-like particles at the end of the VLDL-clearance experiment, instead of taking liver biopts at every blood sampling point, to minimize loss of mice. Next to the liver, uptake of VLDL-like particles was measured in heart, muscle (femoralis), perigonal fat and spleen. - At sacrifice no serum was collected, only EDTA-plasma. No intestine was collected and stored In liquid nitrogen. Experiment 3 - Treatment was started with 5 groups of 7 animals Instead of 5 groups of 6 animals. The extra mouse per group was used for an extra bile cannulatlon (see also deviations in experiment 2). - Instead of 0.4 pM HDL per group, 0.32 pM HDL was used for radiolabeling. The reason for this was that less than 0.4 pM HDL was isolated from the PFHS and PFOS treatment groups. - We decided not to collect liver pieces for histology analysis, liver lipid analysis and mRNA isolation and subsequent micro array analysis in experiment 1 but in experiment 3, since injection of Triton WR-1339 (used to block VLDL clearance) could have an effect on liver gene expression and liver lipids. Intestine was collected in experiment 3 instead of experiment 1 for that same reason. - After consultation with the Sponsor (see email correspondence of 28-Jan-2009), we decided to perform micro array analysis on the livers instead of liver and intestinal mRNA analysis of some selected genes. - Microsomes were isolated per liver instead of combining 2 livers; per group 5 remainders of livers were used for microsomes isolation. 17 p. 19 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4 Results 4.1 Results study 1 4.1.1 Markers of general well-being During the study one mouse (mouse 22) was put in a separate cage because of an open wound from fighting. Another mouse (mouse 15) showed some scabs in the belly area. No other specific clinical signs were observed during the study. At sacrifice, besides the livers no macroscopic differences were observed between the groups. Livers were visibly somewhat enlarged in the fenofibrate group and strongly enlarged in the PFHS and PFOS group. 4.1.2 Body weight Values are absolute values (grams) and are means S.D. from 8 mice per group. Individual body weights are given in appendix I. Body weight (g) Control Fenofibrate PFBS PFHS PFOS 0 avg 26.3 sd 2.4 avg 26.8 sd 2.9 avg 26.1 sd 1.7 avg 27.3 sd 2.2 avg 27.2 sd 2.9 time (weeks) 14 27.5 28.6 2.5 27.6 2.3 27.2 2.6 28.0 2.3 28.0 1.8 28.5 1.8 29.1 2.2 2.1 28.0 2.6 28.2 2.4 6 29.0 2.9 28.8 2.6 28.4 1.8 28.9 2.0 28.1 1.8 Body weight: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 0.635 0.574 1.000 0.382 0.382 0.279 0.878 0.959 0.234 0.279 0.878 time (weeks) 14 0.781 0.782 0.798 1.000 0.959 0.645 0.328 0.645 0.574 0.721 0.382 0.721 0.382 0.721 0.878 0.798 0.279 0.161 0.505 1.000 0.798 0.442 6 0.888 0.959 0.959 0.878 0.721 0.505 0.798 0.382 0.505 0.721 0.382 During the study an increase in body weight was seen. Between groups no significant differences were observed. 18 p. 20 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.2 Body weight 4.1.3 Liver and perigonadal fat weight Values are absolute values (grams) and are means S.D. from 8 mice per group. Individual tissue weights are given in appendix II. Tissue weight (g) Control Fenofibrate PFBS PFHS PFOS Liver avg 1.27 sd 0.18 avg 1.83 sd 0.27 avg 1.45 sd 0.17 avg 3.32 sd 0.23 avg 3.23 sd 0.29 Perigonadal fat 0.62 0.24 0.50 0.11 0.51 0.15 0.45 0.14 0.38 0.10 Tissue weight: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS Liver <0.001 0.001 0.028 <0.001 <0.001 0.003 <0.001 <0.001 <0.001 <0.001 0.645 Perigonadal fat 0.077 0.234 0.328 0.083 0.007 0.798 0.442 0.065 0.645 0.130 0.382 19 p. 21 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Livers were significantly enlarged in all treatment groups. Fenofibrate and PFBS treatment increased liver weights by respectively +44% and +15%. The PFHS and PFOS groups showed a robust induction in liver weights, respectively by +162% and +155 %, which was also significantly higher as compared to the PFBS group. Although a slight decrease was seen in all treatment groups, only PFOS significantly decreased perigonadal fat weight (by 39%, p=0.007). Figure 4.1.3a Liver weight 5 Control Fenofibrate a PFBS 4 PFHS o> 3 o> '3 2 1 0 * p<0.05 vs. control week 6 Figure 4.1.3b 1.2 Perigonadal fat weight _1.0 O) 0) 0.8 0) 5 -S 0.6 (0 rao Oo>0.4 Q. 0.2 0.0 * p<0.05 vs. control week 6 20 Control Fenofibrate a PFBS PFHS PFOS p. 22 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.1.4 Food intake Values are absolute values (gram/mouse/day) and are means S.D. from 3-4 cages per group. Food intake values per individual cage are given in appendix III. Food Intake (g/day/mouse) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd -1-0 2.8 0.2 2.8 0.2 2.8 0.2 2.8 0.2 2.8 0.2 time (weeks) 0-1 3-4 2.7 2.8 0.2 0.4 2.7 3.3 0.2 0.2 2.9 3.0 0.3 0.1 2.8 2.9 0.4 0.3 2.8 3.0 0.2 0.4 5-6 2.9 0.5 3.3 0.3 3.4 0.6 2.9 0.3 2.9 0.4 Food intake: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0-1 .4 1.000 0.400 1.000 0.700 0.400 1.000 0.629 1.000 1.000 1.000 time (weeks) 3-4 0.253 0.114 0.700 0.700 0.400 0.114 0.114 0.400 1.000 0.700 1.000 All groups showed a similar food Intake. Figure 4.1.4 4 Food intake 5-6 0.384 0.400 0.400 1.000 1.000 1.000 0.229 0.229 0.400 0.229 1.000 >. 03 "3O5 J to 3 O E o) 2 o> to o1 0 - 1-0 * p<0.05 vs. control 0-1 3-4 Time (weeks) -" C o n tro l Fenofibrate ""P F B S - PFHS --PFO S 5-6 21 p. 23 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.1.5 Plasma ALT Values are absolute values (U/L) from measurements In pooled plasma samples per group (8 mice per group) at t=0, 4 and 6. Group 1 Plasma ALAT OJ/D 1 t=0 weeks t=4 weeks t=6 weeks 174 240 128 F e n o fib ra te 224 104 96 210 156 115 i 196 272 250 P FO S 206 726 390 Although no statistical analysis could be performed on pooled ALT levels, PFOS and to a lesser extend PFHS seem to Increase plasma ALT levels. Figure 4.1.5 Plasma ALT 22 p. 24 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.1.6 Plasma cholesterol Values are absolute values (mmol/L) and are means S.D. from 8 mice per group. Individual plasma cholesterol levels are given In appendix IV. Cholesterol (mmol/L) Control Fenofibrate PFBS PFHS PFOS time (weeks 046 avg 7.9 8.1 9.0 sd 1.5 1.9 1.1 avg 7.8 5.0 5.6 sd 1.5 0.6 1.1 avg 7.9 6.2 6.8 sd 1.5 0.9 1.4 avg 8.1 3.2 3.6 sd 1.5 0.7 0.6 avg 8.0 3.0 3.2 sd 1.6 0.7 1.1 Cholesterol: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 0.999 1.000 1.000 0.959 0.959 0.959 0.878 0.798 0.878 0.878 1.000 time (weeks 4 <0.001 0.001 0.050 <0.001 <0.001 0.028 <0.001 <0.001 <0.001 <0.001 0.574 6 <0.001 <0.001 0.010 <0.001 <0.001 0.065 <0.001 0.002 <0.001 <0.001 0.382 All treatment groups showed a significant reduction In plasma cholesterol at t=4 and 6 weeks of treatment. Fenofibrate decreased plasma cholesterol by -38% at both time points. PFBS, PFHS and PFOS treatment reduced plasma cholesterol levels respectively by -23%, 60% and -63% after 4 weeks of treatment and by -24%, -60% and -64% at 6 weeks of treatment. PFHS and PFOS decreased cholesterol levels to a larger extent than PFBS (p<0.001 at both time points). PFHS and PFOS showed a similar inhibition. 23 p. 25 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.6 Plasma cholesterol 14 C o n tro l Fenoflbrate __.12 "P F B S o E 10 E. PFHS --PFO S 22 8 C0O) oO 6 CO . E4 (C0O El _ I I ......ii.r -- -- , i , ...... .. r | -1 0 234 56 7 Time (weeks) * p<0.05 vs. control 4.1.7 Plasma HDL-cholesterol Values are absolute values (mmol/L) and are means S.D. from 8 mice per group. Individual plasma HDL-cholesterol levels are given in appendix V. HDL-Cholesterol (mmol/L) Control Fenoflbrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd 0 0.94 0.17 0.96 0.19 0.93 0.23 1.05 0.32 0.94 0.25 time (weeks 4 1.36 0.21 2.06 0.32 1.52 0.24 0.85 0.16 0.62 0.12 6 1.50 0.41 2.31 0.54 1.46 0.50 0.69 0.08 0.44 0.13 HDL-Cholesterol: P-value Between groups Control vs: Fenoflbrate vs PFBS vs PFHS vs Fenoflbrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 0.789 0.721 0.878 0.328 0.959 0.574 0.442 0.574 0.328 0.959 0.442 time (weeks 4 <0.001 0.001 0.328 0.001 <0.001 0.003 <0.001 <0.001 <0.001 <0.001 0.028 6 <0.001 0.007 0.959 <0.001 <0.001 0.010 <0.001 <0.001 0.010 <0.001 0.001 24 p. 26 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasma-HDL levels were increased at t=4 and 6 weeks in the fenofibrate group (respectively by +52% and +54%). PFBS treatment showed no significantly different plasma HDL-cholesterol levels as compared to the control group, but both PFHS and PFOS significantly decreased HDLcholesterol after 4 and 6 weeks of treatment (PFHS respectively by -38% and -54%, PFOS by -54% and -70%). Compared to PFBS treatment, PFHS and PFOS also significantly decreased HDL-cholesterol levels. When comparing PFHS and PFOS treatment, PFOS significantly decreased HDL-cholesterol to a larger extent (p=0.028 and p=0.001 at t=4 and 6 weeks respectively). Figure 4.1.7 Plasma HDL-cholesterol -1 0 1 2 3 4 5 6 7 Time (weeks) * p<0.05 vs. control 4.1.8 Plasma triglycerides Values are absolute values (mmol/L) and are means S.D. from 8 mice per group. Individual plasma triglycerides levels are given In appendix VI. Triglycerides (mmol/L) Control Fenofibrate PFBS PFHS PFOS time (weeks 0 avg 2.24 4 1.91 6 1.91 sd 0.75 avg 2.19 0.43 0.77 1.01 0.52 sd 0.76 0.47 0.29 avg 2.08 1.08 1.02 sd 0.43 avg 1.97 0.42 0.59 0.63 0.41 sd 0.81 0.18 0.13 avg 2.17 sd 0.70 0.74 0.09 0.52 0.12 25 p. 27 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Triglycerides: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 0.971 1.000 0.798 0.721 0.798 0.959 0.878 1.000 0.574 0.959 0.382 time (weeks 4 <0.001 0.002 0.003 <0.001 <0.001 0.028 0.798 0.195 0.003 0.015 0.050 6 <0.001 0.001 0.028 <0.001 <0.001 0.015 0.234 0.234 0.001 0.002 0.161 All treatment groups showed significant reductions in plasma triglycerides at t=4 and 6 weeks of treatment. Fenofibrate decreased plasma triglycerides by -60% and -73%, respectively. PFBS, PFHS and PFOS treatment reduces plasma triglycerides levels respectively by -44%, -69% and -61% at t=4 weeks of treatment and by -46%, -79% and -73% at t=6 weeks of treatment. PFHS and PFOS decreased triglycerides levels to a larger extent than PFBS (respectively p=0.003 and p=0.015 at t=4 weeks and p=0.001 and p=0.002 at t= 6 weeks). When comparing the PFHS and PFOS group, at t=4 weeks a borderline significance was seen (p=0.05) towards lower triglycerides levels in the PFHS group, but at t=6 weeks no significant differences were seen between groups. Figure 4.1.8 Plasma triglycerides 26 p. 28 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.1.9 Lipoprotein profiles Lipoproteins were separated on a superose column. Values are absolute values (mM) from cholesterol (left column) and phospholipids (right column) measurements In pooled plasma per group (with 8 mice per group) at t=4 and 6. We consider fractions 4-8 as VLDL; 9-14 as LDL; 13-16 as large-HDL (HDL1) and 17-24 as HDL. After 4 and 6 weeks of treatment, plasma cholesterol levels were decreased in the VLDLLDL peak In all groups as compared to the control group. Fenofibrate and PFHS showed the strongest and a similar reduction, PFOS seemed to decrease VLDL to a lesser extend than PFHS. PFBS decreased VLDL-LDL t<? a lesser extent than PFHS and PFOS. HDL was increased In the fenofibrate group. Whereas PFBS showed no clear effects on HDL, both PFHS and PFOS strongly decreased the HDL peak. PFHS seemed to decrease HDL to a lesser extent than PFOS and In the profile of the PFHS group HDL1 (large HDL) was Increased. The changes in the lipoproteins (apoB-contalnlng lipoproteins (VLDL-LDL) and HDL) are In line with changes in plasma cholesterol and HDL. Figure 4.1.9 Lipoprotein profiles 0 5 10 15 20 25 fraction Cholesterol (mmol/L) Cholesterol (mmol/L) 27 p. 29 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.1.10 Plasma free glycerol Values are absolute values (mmol/L) and are means S.D. from 8 mice per group. Individual plasma free glycerol levels are given in appendix VII. Free glycerol (mmol/L) Control Fenofibrate PFBS PFHS PFOS time (weeks 04 6 avg 0.23 0.26 0.19 sd 0.04 0.03 0.03 avg 0.23 0.20 0.14 sd 0.03 0.05 0.03 avg 0.25 0.21 0.16 sd 0.05 0.03 0.04 avg 0.22 0.13 0.09 sd 0.04 0.03 0.01 avg 0.23 0.15 0.09 sd 0.05 0.03 0.03 Free glycerol: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 0.724 0.878 0.442 0.721 0.959 0.382 0.442 1.000 0.234 0.505 0.574 time (weeks 4 <0.001 0.021 0.028 <0.001 <0.001 0.878 0.007 0.028 0.001 0.001 0.195 6 <0.001 0.005 0.130 <0.001 <0.001 0.195 0.001 0.015 <0.001 0.002 0.574 Fenofibrate, PFHS and PFOS showed significant reductions In free glycerol levels at 4 and 6 weeks of treatment (respectively by -23%, -51% and -43% at t=4 weeks and by -28%, 52% and -54% at t=6 weeks). PFBS showed a transient decrease at t=4 weeks (by -17%), but at t=6 weeks plasma free glycerol levels were not significantly different from the control group. PFHS and PFOS decreased free glycerol levels to a larger extent than PFBS (respectively p=0.001 and p=0.001 at t=4 weeks and pcO.OOl and p=0.002 at t=6 weeks). No significant differences were seen between the PFHS and PFOS group. 28 p. 30 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.10 Plasma free glycerol 4.1.11 Plasma free fatty acids Values are absolute values (mmol/L) and are means S.D. from 8 mice per group. Individual plasma free fatty acid levels are given in appendix VIII. Free fatty acids (mmol/L) Control Fenoflbrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd 0 0.99 0.27 0.98 0.17 0.90 0.14 1.02 0.37 1.02 0.37 time (weeks 4 0.69 0.10 0.72 0.11 0.66 0.06 0.41 0.09 0.44 0.06 6 0.74 0.07 0.68 0.10 0.67 0.12 0.39 0.06 0.29 0.03 Free fatty acids: P-value Between groups Control vs: Fenoflbrate vs PFBS vs PFHS vs Fenoflbrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 0.941 0.721 0.721 0.959 0.959 0.382 0.798 0.798 0.721 0.505 0.798 time (weeks 4 <0.001 0.721 0.574 <0.001 <0.001 0.382 <0.001 <0.001 <0.001 <0.001 0.574 6 <0.001 0.382 0.279 <0.001 <0.001 0.798 <0.001 <0.001 <0.001 <0.001 0.003 29 p. 31 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Free fatty acid levels were similar in the control, fenofibrate and PFBS groups. Both PFHS and PFOS significantly decreased free fatty acid levels after 4 and 6 weeks of treatment (PFHS respectively by -41% and -48%, PFOS respectively by -37% and -60%). After 4 weeks of treatment no significant differences were seen between the PFHS or PFOS group but after 6 weeks the PFOS group showed significantly lower FFA levels than the PFHS group (p=0.003). Figure 4.1.11 Plasma free fatty acids 1 0 1 2 3 4 5 6 7 Time (weeks) * p<0.05 vs. control 4.1.12 Post-heparin LPL and HL activity Values are absolute values (pmol FFA/hr/mL) and are means S.D. from 8 mice per group. Individual post-heparin LPL and HL activity values are given in appendix XII. Lipolytic activity (pmol FFA/hr/mL) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd HL activity 7.4 0.9 12.3 1.4 5.3 2.1 8.5 3.0 9.0 1.4 LPL activity 12.0 2.5 25.1 3.1 14.3 2.4 20.8 5.1 18.5 2.9 30 p. 32 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Lipolytic activity: P-values Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS HL activity <0.001 <0.001 0.050 0.798 0.021 <0.001 0.010 <0.001 0.050 0.003 0.442 LPL activity <0.001 <0.001 0.105 0.001 0.001 <0.001 0.050 0.001 0.007 0.010 0.328 Post-heparin lipoprotein lipase activity was strongly induced after fenofibrate treatment (by +110%). Both PFHS and PFOS increased LPL activity to a lesser extent (respectively by +74% and +54%). Hepatic lipase activity was significantly increased in the fenofibrate group and the PFOS group (respectively by +67% and +22%). In contrast PFBS significantly decreased hepatic lipase activity by -28% (p=0.05). PFBS had significantly lower levels of LPL and HL activity as compared to PFHS (0.007 and 0.05, respectively) and PFOS (0.010 and 0.003, respectively). Figure 4.1.12 Post heparin LPL and HL activity HL * p<0.05 vs. control t=5 weeks* LPL 31 p. 33 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.1.13 Fecal lipids Values are absolute values (pmol/lOO gram mouse/day) or relative values (% of total neutral sterols, phytosterols, bile acids and fatty adds, respectively) and are means S.D. from 3 (combined) cages per group at 2 consecutive time points. Values per (combined) cage per time point are given in appendix XIII. Fecal lipids (umol/100 gram mouse/day} Control avg Fenofibrate sd avg PFBS sd avg PFHS sd avg sd PFOS avg sd Total neutral sterols 36.3 10.7 44.5 9.8 38.1 3.0 39.1 5.2 34.0 12.9 Total Dhvtosterols 3.5 0.7 3.4 0.6 3.6 0.4 2.9 0.2 3.4 0.6 Total bile acids 5.5 1.6 3.4 0.8 5.0 1.4 3.3 0.9 2.8 0.3 Total fatty adds 257 222 410 121 240 79 159 128 188 86 Fecal lipids: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS Total neutral sterols 0.462 0.180 0.310 0.310 0.818 0.394 0.310 0.180 1.000 0.937 0.589 Total phytosterols 0.175 0.937 1.000 0.065 0.937 0.485 0.394 0.818 0.002 0.937 0.132 Total bile acids 0.001 0.015 0.589 0.004 0.002 0.093 0.699 0.240 0.015 0.002 0.093 Total fatty acids 0.030 0.180 0.485 0.394 0.937 0.009 0.026 0.009 0.065 0.240 0.310 Nor fecal neutral sterols (figure 4.1.13a), neither fecal phytosterols (figure 4.1.13b) were affected by fenoflbrate, PFBS, PFHS or PFOS treatment. Total bile add excretion was significantly lower in the fenofibrate, PFHS and PFOS groups (figure 4.1.13c). Fecal bile acids were decreased by -38%, -41% and -50%, respectively. Although the fenofibrate group showed a somewhat higher fatty acid excretion, none of the treatment groups were significantly different from the control group (figure 4.1.13d). 32 p. 34 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.13a Total neutral sterol excretion 70 60 c >to o 'S 50 w so X0) o E 40 E w 2 o>30 0 o o 0 |2 0 z 10 Control Fenoflbrate PFBS PFHS PFOS * p<0.05 vs. control Fecal neutral sterols (%) Control Fenofibrate PFBS PFHS PFOS coprostanol avg 0.14 sd 0.04 avg 0.12 sd 0.04 avg 0.12 sd 0.02 avg 0.12 sd 0.04 avg 0.14 sd 0.04 t=5 weeks cholesterol 93.7 0.6 94.8 0.8 94.4 0.4 93.1 0.9 92.4 1.5 cholestanol 3.0 1.2 2.0 0.4 2.6 0.5 4.1 1.2 4.5 1.6 lathosterol 3.2 0.7 3.1 0.7 2.9 0.2 2.6 0.5 3.0 0.5 Fecal neutral sterols: P-value Between groups Control vs: Fenofibrate vs PFBS vs t f vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS coprostanol 0.673 0.699 0.589 0.310 0.699 0.937 0.937 0.310 0.818 0.240 0.394 cholesterol 0.002 0.026 0.093 0.310 0.093 0.394 0.015 0.002 O.4 0.002 0 589 cholestanol ...... 0.04 0.240 0.310 0.132 0.240 0.065 0.004 0.002 0.041 0.004 0.818 lathosterol .401 0.485 0.937 0.132 0.618 0.937 0.240 0.818 0.132 0.818 0.180 The fecal neutral sterol composition of the fenofibrate group was somewhat changed, the percentage excreted cholesterol was slightly but significantly increased as compared to the control group (94.8% vs 93.7%). The other treatment groups showed the same composition as the control group. 33 p. 35 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.13b Total phytosterol excretion Control Fenoflbrate PFBS * PFHS c PFOS o ? 8 0) t=5 weeks * p<0.05 vs. control Fecal phytosterols (%) Control Fenoflbrate PFBS PFHS PFOS campesterol avg 26.1 sd 0.5 avg 26.9 sd 0.9 avg 26.1 sd 0.2 avg 26.1 sd 1.4 avg 24.2 sd 1.1 Fecal phytosterols: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenoflbrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS campesterol 0.006 0.180 0.485 0.818 0.002 0.065 0.310 0.002 1.000 0.002 0.041 stigmasterol 9.2 0.5 8.4 1.3 8.7 1.2 9.5 3.7 14.8 1.8 stigmasterol 0.011 0.310 0.485 1.000 0.002 0.818 1.000 0.002 1.000 0.002 0.026 b-sltosterol 64.7 0.7 64.7 1.1 65.2 1.1 64.4 2.5 61.0 1.3 b-sitosterol 0.009 0.818 0.485 0.937 0.002 0.699 0.818 0.002 0.818 0.002 0.009 Only PFOS treatment affected fecal phytosterol composition as compared to the control group; PFOS showed an increased stigmasterol (14.8 % vs 9.2 %) and a decreased bsitosterol and campesterol conteht (61.0% vs 64.7% and 24.2% vs 26.1%, respectively). 34 p. 36 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.13c 10 Total bile acid excretion s; 8 n c o "w0 3 o 16 X0 E o 2 'r5a O) o 4 o 00 Control Fenofibrat PFBS PFHS PFOS 0 * p<0.05 vs. control t=5 weeks Fecal bile adds (%) Control Fenofibrate PFBS PFHS PFOS a-muricholate deoxycholate avg 7.1 sd 1.1 avg 6.0 sd 1.2 avg 6.5 sd 2.4 avg 6.3 sd 2.9 avg 5.5 sd 2.4 14.2 5.1 10.6 4.3 17.9 7.8 16.7 10.5 13.5 10.5 cholate 12.7 4.2 13.4 4.5 11.3 5.2 15.1 8.3 13.5 6.1 llthocholate 10.6 1.4 16.6 3.7 17.3 7.3 11.1 5.4 6.3 3.0 b-muricholate w-murlcholate hyodeo/urso 25.5 2.8 18.4 6.6 24.5 13.4 16.6 8.8 14.1 7.9 23.5 1.7 18.5 2.0 29.2 14.6 24.8 11.9 26.3 12.4 6.6 1.0 16.4 5.3 12.5 5.5 8.8 3.9 6.7 2.1 Fecal bile acids: P-value a-murlcholate deoxycholate Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0.642 0.180 0.818 0.394 0.310 0.818 0.699 0.485 0.937 0.589 0.394 .4 0.310 0.394 0.937 0.394 0.180 0.394 0.699 0.818 0.240 0.589 cholate 0.816 0.818 0.589 0.589 0.818 0.485 1.000 0.937 0.240 0.589 0.818 llthocholate 0.003 0.004 0.065 0.937 0.026 0.937 0.093 0.002 0.132 0.004 0.180 b-murlcholate w-muricholate .44 0.093 0.589 0.065 0.041 0.699 0.937 0.310 0.485 0.132 0.699 0.002 0.485 0.589 1.000 0.394 0.132 0.394 0.485 0.818 0.937 hyodeo/urso 0.03 0.002 0.015 0.132 0.699 0.310 0.015 0.002 0.240 0.041 0.394 Fecal bile acid composition was significantly changed after treatment with fenoflbrate, PFBS and PFOS. Fenofibrate increased the percentage lithochate and hyodeoxycholate/ ursocholate (16.6% vs 10.6 % and 16.4% vs 6.6%, respectively) and decreased wmuricholate content (18.5% vs 23.5%). PFBS only Increased hyodeoxycholate/ ursocholate content significantly (12.5% vs 6.6%), while PFOS decreased the percentage of llthocholate and b-muricholate (6.3% vs 10.6% and 14.1% vs 25.5% respectively). 35 p. 37 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.13d Total fatty acid excretion 600 500 <>o> 5 1 400 o E 3 0 0 o> 1 ^200 u. o E 100 Control Fenoflbrate PFBS PFHS PFOS * p<0.05 vs. control t=5 weeks Fecal fatty acids (%) Control Fenoflbrate PFBS PFHS PFOS C14:0 avg 3 sd 0.5 avg 2.2 sd 0.3 avg 2.6 sd 0.2 avg 2.6 sd 0.8 avg 2.4 sd 0.4 C16:0 46.5 1.4 46.3 2.7 48.0 0.7 46.8 4.1 46.3 1.9 C16:l 0.68 0.07 0.62 0.05 0.65 Q.03 0.86 0.24 0.69 0.07 C O 4J 2.5 43.9 2.5 41.8 1.4 41.0 4.5 43.1 3.0 C18:l 6.7 1.1 6.2 1.2 6.3 0.7 7.9 0.6 6.8 0.8 C18:2 0.75 0.08 0.66 0.09 0.62 0.03 0.73 0.10 0.74 0.04 C18:3 0.03 0.04 0.06 0.03 0.03 0.02 0.02 0.04 0.05 0.03 Fecal fatty acids: P-value Between groups Control vs: Fenoflbrate vs PFBS vs PFHS vs Fenoflbrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS C4 0.121 0.093 0.589 0.818 0.240 0.041 0.093 0.485 .i 0.240 0.180 SS3 0.192 0.937 0.041 0.180 0.937 0.132 0.394 0.818 0.937 0.065 0.310 C16:l 0.377 0.240 0.394 0.310 0.699 0.180 0.310 0.132 0.394 0.394 0.394 C55 0.159 0.310 0.699 0.065 1.000 0.093 0.180 0.589 0.065 0.589 0.093 C18.-1 0.053 0.485 0.485 0.041 0.937 0.589 0.065 0.180 0.015 0.310 0.026 C18:2 0.034 0.093 0.002 0.937 0.818 0.818 0.310 0.065 0.180 0.002 0.818 53T3 0.145 0.310 0.937 0.485 0.589 0.065 0.041 0.589 0.132 0.310 0.132 Although changes In fatty acid composition were small, PFBS decreased the percentage of C18:2 (0.62% vs 0.75%) and PFHS Increased the percentage of C18:l (7.9% vs 6.7%) significantly. No effects of fenofibrate or PFOS were seen. 36 p. 38 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.13e Fatty acid balance 10000 Control Fenofibrate PFBS PFHS PFOS * p<0.05 vs. control t=5 weeks Fatty acid balance (umol/100 qram mouse/day) Control avg Fenofibrate PFBS sd avg sd avg sd PFHS avg sd PFOS avg sd Fatty acid input-output 5492 326 6114 436 6146 910 5565 209 5593 458 Fatty acid balance: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS Fatty add Input-output 0.096 0.015 0.240 0.589 0.589 0.589 0.015 0.240 0.180 0.310 0.937 The fatty add balance (fatty acid input - output) was significantly increased by fenofibrate treatment (by 11% p=0.015). The perfluor alkyl sulfonate compounds were not significantly changed. 37 p. 39 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.1.14 VLDL-triglycerldes and de novo ApoB production Values are absolute values (mmol/L for plasma triglycerides, pmol/h for VLDL-TG production rate, 104 dpm/ml/h for de novo ApoB synthesis, pmol/104 dpm for TG production per de novo synthesized ApoB and pmol/104 dpm for lipid composition per de novo synthesized ApoB) and are means S.D. from 7-8 mice per group. Individual values are given in appendix XIV. Plasma triglycerides (mmol/L) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd 0 1.4 0.4 0.5 0.3 0.8 0.3 0.3 0.1 0.5 0.2 Time after Triton WR1339 in ection (min) 15 30 60 2.6 4.1 6.4 0.5 0.7 1.0 2.0 4.0 7.6 0.5 0.5 1.0 2.1 3.6 6.1 0.6 0.8 1.0 0.6 1.0 1.6 0.2 0.3 0.5 0.6 0.9 1.2 0.2 0.3 0.4 90 8.7 1.4 11.1 1.5 8.9 1.5 2.2 0.7 1.6 0.5 Plasma triglycerides: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 <.01 0.002 0.021 < 0.001 < 0.001 0.065 0.195 0.878 < 0.001 0.028 0.028 Time after Triton WR1339 in ection (min) 15 30 60 < 0.001 < 0.001 < 0.001 0.072 0.094 0.955 0.336 0.029 0.694 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.798 < 0.001 0.195 < 0.001 0.021 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.959 0.505 0.130 90 < 0.001 0.006 0.955 < 0.001 < 0.001 0.028 < 0.001 < 0.001 < 0.001 < 0.001 0.105 VLDL-TG production rate (pmol/h) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd production rate 6.3 1.4 9.3 1.7 6.9 1.5 1.7 0.7 0.9 0.4 VLDL-TG production rate: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS production rate <0.001 0.006 0.536 < 0 .0 0 1 <0.001 0.021 <0.001 <0.001 <0.001 < 0 .0 0 1 0.028 At t=0 min (before Triton WR1339 injection) fenofibrate, PFHS, PFOS and to a lesser extent PFBS treatment decreased plasma triglycerides levels (in line with the plasma samples, taken at 4 weeks and 6 weeks of treatment). The increase in plasma triglycerides was more or less the same in the control group and the PFBS group. Fenofibrate increased VLDLtriglycerides to a higher extent than the control group. Both PFHS and PFOS showed a significantly reduced VLDL triglycerides production. In figure 4.1.14b the VLDL-triglycerides production rate (=slope figure 4.1.14a) is shown. Fenofibrate significantly increased the production rate by +46%. PFHS and PFOS strongly decrease TG production rate, respectively by -74% and -86%, whereas PFBS showed no effects. PFHS and PFOS decreased VLDL-TG production rate as compared to PFBS (pcO.OOl for both treatments). 38 p. 40 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.1.14a Plasma triglycerides after Triton WR1339 injection * p<0.05 vs. control Figure 4.1.14b VLDL-triglycerides production rate * p<0.05 vs. control 39 p. 41 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 De Novo ApoB synthesis (104 dpm/mL/h) Control Fenofibrate PFBS PFHS PFOS ApoB synthesis avg 6.1 sd 1.0 avg 6.4 sd 0.5 avg 5.1 sd 0.9 avg 1.5 sd 0.3 avg 0.8 sd 0.2 TG production per de novo synthesized ApoB (pmol/104 dpm) TG production per ApoB Control avg 0.80 sd 0.09 Fenofibrate avg 1.12 sd 0.19 PFBS avg 1.09 sd 0.21 PFHS avg 0.86 sd 0.24 PFOS avg 0.86 sd 0.20 De Novo ApoB synthesis: P-value Between qroups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS TG production per de novo synthesized ApoB: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS ApoB synthesis < 0.001 0.463 0.072 < 0.001 < 0.001 0.005 < 0.001 < 0.001 < 0.001 < 0.001 0.001 TG production per ApoB 6.6o7 < 0.001 0.014 0.397 0.536 0.798 0.065 0.010 0.065 0.065 0.878 Fenofibrate showed no effects on de novo ApoB synthesis rate (figure 4.1.14c). Since fenofibrate increased VLDL-triglycerides production, the newly synthesized VLDLtriglycerides per ApoB were plotted in figure 4.1.14d to examine TG content of newly synthesized VLDL. Fenofibrate showed triglycerides-rich newly synthesized ApoB particles (increase in triglycerides of +40% as compared to the control). PFBS showed a decreased ApoB synthesis (by -17%) and an increase in triglycerides-rich newly synthesized particles (increase in triglycerides of +36% as compared to the control). Both PFHS and PFOS strongly inhibited ApoB synthesis (respectively by -76% and -87%), resulting in newly synthesized VLDL particles with similar triglycerides content as the control group. PFHS and PFOS had a significantly reduced de novo ApoB synthesis rate and TG production per ApoB as compared to PFBS. 40 p. 42 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 De novo ApoB synthesis * p<0.05 vs. control Figure 4.1.14d TG production per ApoB 2.5 oaCO 2 0 <-- wa> Ea. 1 4 a. tj 1,3 ll Sa 3I 1.0 O 0.5 0.0 * p<0.05 vs. control Control Fenofibrate PFBS SPFHS PFOS 41 p. 43 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Lipid composition VLOL (pmol/lO4dpm ApoB) Control Fenofibrate PFBS PFHS PFOS Total cholesterol avg 0.65 sd 0.18 avg 0.36 sd 0.07 avg 0.75 sd 0.55 avg 0.46 sd 0.15 avg 1.61 sd 1.15 Free cholesterol 0.18 0.04 0.15 0.03 0.21 0.12 0.20 0.06 0.49 0.23 Cholesterol ester 0.47 0.14 0.21 0.05 0.54 0.43 0.25 0.11 1.12 0.92 Triglycerides Phospholipids 0.79 0.19 0.99 0.15 1.08 0.20 0.80 0.22 1.09 0.18 0.23 0.05 0.22 0.04 0.29 0.08 0.22 0.04 0.39 0.12 Lipid composition VLDL: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS Total cholesterol < 0.001 O.OOl 0.613 0.029 0.014 O.OOl 0.065 < 0.001 0.007 0.007 < 0.001 Free cholesterol < 0.001 0.152 0.955 0.613 < 0.001 0.130 0.050 < 0.001 0.574 0.003 < 0.001 Cholesterol ester < 0.001 0.001 0.536 0.006 0.094 < 0.001 0.442 < 0.001 0.010 0.065 0.001 Triglycerides Phospholipids 0.012 0.040 0.014 0.779 0.014 0.382 0.083 0.234 0.028 1.000 0.021 0.001 1.000 0.054 0.955 0.001 0.050 1.000 < 0.001 0.083 0.028 < 0.001 Lipid composition of isolated VLDL (isolated VLDL is the sum of newly synthesized VLDL and VLDL present in plasma at t=0 min), calculated as pmol lipid per newly synthesized ApoB, was significantly different from control in all treatment groups. Fenofibrate decreased cholesterolester and increased triglycerides content, respectively by -55% and +26%. PFBS significantly increased triglycerides content by 37%, PFHS showed a decreased cholesterolester composition by -46%. PFOS treatment increased free cholesterol, triglycerides and phospholipids content in VLDL, respectively by +164%, +38% and +74%. When compared to the PFHS group, the PFOS group showed an isolated VLDL with increased free cholesterol, triglycerides and phospholipids, indicative for larger particles. A compared to PFBS, PFHS induced smaller particles and PFOS larger particles.* Total cholesterol Free cholesterol Cholesterol ester Triglycerides Phospholipids * p<0.05 vs. control 42 p. 44 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.2 Results study 2 4.2.1 Markers of general well-being No specific clinical signs were observed during the study. At sacrifice, besides the livers no macroscopic differences were observed between the groups. Livers were visibly somewhat larger in the fenofibrate group and strongly enlarged in the PFHS and PFOS group. One mouse (mouse 21) showed some white spots in the liver and bile stones in the bladder. 4.2.2 Body weight Values are absolute values (g) and are means S.D. from 6 mice per group. Individual values are given in appendix I. Body weight (g) Control Fenofibrate PFBS PFHS PFOS 1 time (weeks) 0 avg 26.4 1 26.7 4 i. sd 1.3 avg 26.1 1.1 26.3 1.4 27.9 sd 1.2 avg 26.6 1.2 26.6 1.6 27.8 sd 2.3 2.3 2.3 avg 26.6 26.9 28.2 sd 2.4 2.4 2.2 avg 26.2 26.3 27.2 sd 1.8 1.6 1.6 Body weight: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS 0 0.981 0.394 1.000 1.000 0.937 0.818 0.818 0.937 1.000 0.818 0.699 time (weeks) 1 0.993 0.818 1.000 0.937 0.818 0.937 0.818 0.937 0.937 0.818 0.699 4 0.913 0.937 0.818 0.937 0.485 0.818 0.937 0.589 0.699 0.699 0.485 During the study mice gained body weight in all groups. There were no significant differences between groups. 43 p. 45 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.2.2 Body weight 40 35 O) I 30 >* o o 00 25 Control Fenofibrate PFBS * PFHS PFOS 20 0 1 Time (weeks) 4 * p<0.05 vs. control 4.2.3 Liver and perigonadal fat weight Values are absolute values (g) and are means S.D. from 6 mice per group. Individual values are given in appendix II. Tissue weight (g) Control Fenofibrate PFBS PFHS PFOS Liver Perigonadal fat avg 1.43 0.50 sd 0.14 avg 1.76 0.17 0.58 sd 0.16 avg 1.63 0.12 0.58 sd 0.28 avg 3.01 0.09 0.40 sd 0.35 0.11 avg 2.97 sd 0.26 0.41 0.12 Tissue weight: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS Liver <0.001 0.009 0.310 0.002 0.002 0.394 0.002 0.002 0.002 0.002 1.000 Perigonadal fat 0.034 0.240 0.132 0.310 0.485 0.818 0.026 0.041 0.015 0.026 0.937 44 p. 46 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Liver weights were significantly increased in the fenofibrate, PFHS and PFOS group. After 4 weeks of treatment PFBS treatment did not result in significantly increased livers. Fenofibrate increased liver weights by +23%. The PFHS and PFOS groups showed a robust induction in liver weights, respectively by +110% and +107%. Although a slight decrease in perigonadal fat weight was seen after PFOS and PFHS treatment, this was not significantly different from the control group. Liver weights in the PFHS and PFOS groups were significantly higher as compared to the PFBS group, whereas weights of perigonadal fat were significantly decreased In the PFHS and PFOS groups. Figure 4.2.3a Liver weight o> O) 1 ul week 4 * p<0.05 vs. control Figure 4.2.3b 1.2 Perigonadal fat weight CD 1.0 O) I 0.8 Trree3 0.6 c o O) 0.4 a. 0.2 0.0 * p<0.05 vs. control week 4 45 Control Fenofibrate PFBS i PFHS PFOS p. 47 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.2.4 Food intake Values are absolute values (g/day/mouse) and are means S.D. or range from 2-3 cages per group. Individual values per cage are given in appendix III. Food intake (g/day/mouse) time (weeks) -1-0 0-1 Control avg sd/range 2.8 0.1 2.8 0.1 Fenofibrate avg sd/range 2.8 0.1 2.9 0.1 PFBS avg sd/range 2.8 0.1 2.7 0.2 PFHS avg 2.8 sd 0.1 2.7 0.4 PFOS avg 2.8 sd 0.1 2.7 0.2 3-4 2.8 0.1 2.8 0.1 2.7 0.2 2.8 0.4 2.7 0.2 Food intake: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS time (weeks) 0-1 3-4 0.865 1.000 0.667 0.800 0.920 0.667 0.667 0.800 0.400 0.667 0.800 0.667 0.800 0.800 1.000 0.800 1.000 1.000 0.800 0.800 1.000 1.000 All groups showed a similar food intake during the study. Figure 4.2.4 5 Food intake 0W 3 O "E5 35n j0c n o o uo. -1-0 0-1 Time (weeks) 46 p. 48 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.2.5 Plasma ALT Values are absolute values (U/L) from measurements In pooled plasma samples per group (6 mice per group) at t=0 and 4. Group F e n o fib ra te PFOS 1 Plasma ALAT (U/LI t=0 weeks t=4 weeks 201 245 169 113 123 118 129 200 105 194 1 At t=0 weeks the control group showed a higher ALT level as compared to the other groups. After 4 weeks of treatment, the control group still had the highest plasma ALT level, but PFHS and PFOS showed, with respect to t=0 levels, a higher Increase in plasma ALT. Figure 4.2.5 Plasma ALT Control Fenofibrate PFBS Time (weeks) 4.2.6 Plasma cholesterol Values are absolute values (mmol/L) and are means S.D. from 6 mice per group. Individual values are given In appendix IV. Cholesterol (mmol/L) Control Fenofibrate PFBS PFHS PFOS time (weeks) 04 avg 8.4 7.6 sd 1.3 avg 8.4 1.2 4.9 sd 2.9 avg 8.7 0.5 6.3 sd 1.9 avg 8.5 sd 2.7 avg 8.6 sd 2.3 1.7 2.5 0.7 3.0 0.7 47 p. 49 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Cholesterol: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 0 0.985 4 <0.001 0.818 0.394 0.002 0.041 0.589 1.000 0.002 0.002 1.000 0.937 0.065 0.002 0.937 0.002 0.818 0.002 0.699 0.002 0.818 0.180 As was seen in study 1, all treatment groups showed a significant reduction in plasma cholesterol at t=4 weeks of treatment. Fenofibrate decreased plasma cholesterol by -35%. PFBS, PFHS and PFOS treatment reduced plasma cholesterol levels respectively by -16%, -67% and -60% after 4 weeks of treatment. PFHS and PFOS decreased cholesterol levels to a larger extent than PFBS (p=0.002). PFHS and PFOS showed a similar Inhibition. Figure 4.2.6 12 Plasma cholesterol * p<0.05 vs. control Time (weeks) 48 p. 50 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.2.7 Plasma HDL-cholesterol Values are absolute values (mmol/L) and are means S.D. from 6 mice per group. Individual values are given In appendix V. HDL-Cholesterol (mmol/L) Control Fenoflbrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd time (weeks) 04 0.96 1.22 0.47 1.12 0.68 0.29 2.39 .24 1.10 1.72 0.53 0.37 1.03 0.37 0.80 0.12 1.07 0.58 0.43 0.10 HDL-Cholesterol: P-value Between groups Control vs: Fenoflbrate vs PFBS vs PFHS vs Fenoflbrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 04 1.000 <0.001 0.937 0.002 1.000 0.041 0.937 0.026 0.937 0.002 0.937 0.015 1.000 0.002 0.937 0.002 0.937 0.002 1.000 0.002 0.937 0.009 HDL-cholesterol levels were Increased after 4 weeks treatment in the fenoflbrate group, by +97%. In contrast to experiment 1, PFBS treatment showed a significantly increased plasma HDL-cholesterol, by +42%. Both PFHS and PFOS significantly decreased HDL-cholesterol after 4 weeks of treatment, by -34% and -54%, respectively. When comparing PFHS and PFOS treatment, PFOS significantly decreased HDL-cholesterol to a larger extent (p=0.009). PFHS and PFOS significantly decreased HDL as compared to PFBS. 49 p. 51 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.2.7 Plasma HDL-cholesterol 4 o E E3 2 aM 0 2 O Q X m1 E RS CL 0 Control Fenofibrate PFBS PFHS 04 Time (weeks) * p<0.05 vs. control 4.2.8 Plasma triglycerides Values are absolute values (mmol/L) and are means S.D. from 6 mice per group. Individual values are given in appendix VI. Triglycerides (mmol/L) Control Fenofibrate PFBS PFHS PFOS time (weeks) 0 avg 2.26 4 1.37 sd 0.71 avg 2.27 0.21 0.48 sd 0.79 avg 2.54 0.12 0.87 sd 1.02 avg 2.25 0.25 0.56 sd 0.96 0.17 avg 2.39 0.68 sd 0.76 0.21 Triglycerides: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 04 0.991 0.001 1.000 0.818 0.002 0.004 1.000 0.002 0.699 0.004 0.`818 0.009 0.937 0.485 0.818 0.041 0.818 0.026 0.937 0.180 0.818 0.310 50 p. 52 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 All treatment groups showed significant reductions in plasma triglycerides at t=4 weeks of treatment. Fenofibrate decreased plasma triglycerides by -65%. PFBS, PFHS and PFOS treatment reduced plasma triglycerides levels respectively by -37%, -59% and -50% after 4 weeks of treatment. PFHS decreased triglycerides levels to a larger extent than PFBS (p=0.026). PFOS showed no significantly different triglycerides levels as compared with PFHS and PFBS. Figure 4.2.8 Plasma triglycerides 4 Control Fenofibrate PFBS PFHS PFOS * p<0.05 vs. control Time (weeks) 4 4.2.9 Lipoprotein profiles Lipoproteins were separated on a superose column. Values are absolute values (mM) from cholesterol (left column) and phospholipids (right column) measurements in pooled plasma per group (with 6 mice per group) at t=4. We consider fractions 4-8 as VLDL; 9-14 as LDL; 13-16 as large-HDL (HDL1) and 17-24 as HDL. The profiles are more or less the same as in experiment 1. After 4 weeks of treatment, plasma cholesterol levels were decreased In the VLDL-LDL peak in all groups as compared to the control group. Fenofibrate and PFHS showed the strongest and a similar reduction, PFOS seemed to decrease VLDL to a lesser extent than PFHS. PFBS decreased VLDL to a lesser extend than PFHS and PFOS. HDL was increased In the fenofibrate group, with the appearance of a large HDL1 particle. Whereas PFBS seemed to increase HDL somewhat, both PFHS and PFOS strongly decreased the HDL peak. PFHS seemed to decrease HDL to a lesser extent than PFOS and in the profile of the PFHS group HDL1 (large HDL) was increased. The changes in the lipoproteins (ApoB-containing proteins (VLDL-LDL) and HDL) are in line with the changes in plasma cholesterol and HDL. 51 p. 53 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.2.9 Lipoprotein profiles Cholesterol (mmol/L) 0 5 10 15 20 25 fraction 4.2.10 Biliary bile acids, cholesterol and phospholipids production Values are absolute values (pL/min/kg mouse for bile flow, nmol/min/kg mouse for biliary bile acids, phospholipids and cholesterol) and are means S.D. from 5-7 mice per group. Individual values are given in appendix XV. Bile flow (pl/mln/kg mouse) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd 15 min 43.2 7.8 50.5 5.5 43.7 3.6 63.9 13.6 51.3 15.3 time 30 min 45 min 38.7 41.3 7.1 7.1 47.4 45.2 6.6 38.8 9.5 41.2 12.2 58.0 13.8 54.9 8.9 11.0 40.8 39.9 19.7 16.6 total 40.3 6.8 47.7 4.8 41.2 9.4 58.9 10.4 44.0 17.0 Bile flow: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0-15 min 0.035 0.329 0.690 0.017 0.343 0.126 0.026 1.000 0.009 0.530 0.101 time bile collection 15-30 min 30-45 min 0.040 0.245 0.177 0.394 0.841 0.429 0.009 0.093 1.000 0.628 0.177 0.429 0.026 0.180 0.445 0.445 0.052 0.177 1.000 0.755 0.035 0.101 total 0.040 0.041 0.931 0.004 1.000 0.126 0.041 0.731 0.030 1.000 0.101 Although bile flow was somewhat increased in the fenofibrate group at all collection points, this was not significantly different from the control group. However, fenofibrate did significantly increase the resulting total bile flow (by 18%, p=0.041). 52 p. 54 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 PFHS treatment increased bile flow, this was significantly different from the control group for bile collected 0-15 min and 15-30 min after the cannula was placed (by +48 and +50%, respectively). The resulting total bile flow was also significantly increased in the PFHS group (by +46%). PFBS and PFOS showed no effects on bile flow. Figure 4.2.10a Bile flow 120 oin> 100 3 O E o> 80 JC "c 60 Control Fenofibrat PFBS f PFHS PFOS o 40 m 20 0 0-15 min * p<0.05 vs. control 15-30 min 30-45 min Time bile collection total Bile acid flow (nmol/min/kg mouse) Control Fenofibrate PFBS PFHS PFOS 15 min avg 614 sd 262 avg 468 sd 43 avg 610 sd 137 avg 625 sd 123 avg 543 sd 100 time 30 min 45 min 539 615 119 189 436 469 48 140 500 603 173 255 535 514 96 101 459 483 130 201 total 573 179 457 60 571 156 558 85 495 136 Bile acid flow: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0-15 min 0.284 0.329 1.000 0.931 0.876 0.126 0.065 0.181 0.031 0.343 0.234 time bile collection 15-30 min 30-45 min 0.489 0.498 0.126 0.690 0.180 0.931 0.931 0.432 0.394 0.181 0.537 0.180 0.945 0.931 0.639 0.234 0.429 0.485 0.945 0.931 0.343 0.534 total 0.379 0.240 0.792 0.937 0.445 0.126 0.093 0.945 0.429 0.530 0.295 53 p. 55 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Although fenofibrate and to a lesser extent PFHS and PFOS seemed to decrease bile acid flow, this was not significantly different from the control group. PFBS showed the same bile add flow as the control group. Figure 4.2.10b Bile acid flow 1200 V) 3 O 1000 E o> c 800 E o E 600 c I 5= 400 T3 200Oflj 4) 5 0 0-15 min Control Fenofibrate PFBS PFHS PFOS 15-30 min 30-45 min Time bile collection total * p<0.05 vs. control Phospholipid flow (nmol/min/kq mouse) Control Fenofibrate PFBS PFHS PFOS 15 min avg 330 sd 139 avg 451 sd 132 avg 393 sd 69 avg 620 sd 166 avg 463 sd 214 time 30 min 45 min oo 310 79 139 467 457 123 180 372 448 109 192 553 576 153 148 422 443 176 181 total 346 105 459 136 405 93 583 144 443 178 Phospholipid flow: P-value Between qroups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0-15 min 0.058 0.247 0.310 0.017 0.149 0.247 0.132 0.628 0.030 0.876 0.101 time bile collection 15-30 min 30-45 min 0.048 0.370 0.030 0.394 0.548 0.931 0.004 0.132 0.268 0.836 0.429 0.792 0.310 0.310 0.366 0.731 0.126 0.126 0.639 0.876 0.101 0.138 total 0.076 0.180 0.429 0.009 0.295 0.792 0.132 0.534 0.052 0.876 0.138 54 p. 56 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Although biliary phospholipid flow was somewhat Increased in the fenofibrate group at all collection points, this was only different from the control group at t=15-30 min (by +51% , p=0.030). The calculated total phospholipid flow, however, was not significantly changed. PFHS treatment increased phospholipid flow, this was significantly different from the control group for bile collected 0-15 min and 15-30 min after the cannula was placed (by +88 and +78% respectively. The resulting total phospholipid flow was also significantly increased in the PFHS group (by +68%). PFBS and PFOS showed no effects on biliary phospholipid flow. Figure 4.2.10c Phospholipid flow 1200 1000 O <2 3 <3 O o = I-jg> 800 600 1 400 i 200 Control Fenofibrate PFBS PFHS PFOS 0 0-15 min * p<0.05 vs. control 15-30 min 30-45 min Time bile collection total Cholesterol (nmol/min/kg mouse) Control Fenofibrate PFBS PFHS PFOS 15 min avg 33.6 sd 15.7 avg 34.3 sd 10.8 avg 32.5 sd 9.8 avg 41.8 sd 7.9 avg 44.9 sd 15.1 time 30 min 45 min 33.0 38.1 16.1 13.7 38.3 37.8 9.9 29.4 11.6 38.8 13.7 41.6 14.5 39.6 11.5 4.4 42.4 41.2 21.3 12.7 total 35.4 13.0 36.8 9.0 33.6 10.4 41.0 6.6 42.8 14.4 55 p. 57 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Cholesterol flow: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0-15 min 0.284 0.792 0.841 0.247 0.268 0.931 0.093 0.234 0.177 0.202 0.628 time bile collection 15-30 min 30-45 min 0.386 0.984 0.429 1.000 0.841 1.000 0.177 0.432 0.818 0.731 0.126 0.699 1.000 0.937 0.945 0.628 0.126 0.792 . 0.343' 0.755 0.836 0.945 total 0.398 0.589 0.662 0.240 0.295 0.429 0.310 0.295 0.247 0.268 0.945 For biliary cholesterol flow, no significant changes were seen between groups. Figure 4.2.10d Cholesterol flow 100 * o 5= o<>0 3 o E 3 it) O o o E c 80 60 40 20 Control Fenofibrate PFBS PFHS PFOS 0-15 min 15-30 min 30-45 min Time bile collection* * p<0.05 vs. control total 56 p. 58 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.2.11 In vivo clearance of VLDL-like TG-rich particles and uptake in tissues Values are relative values (% of injected dose for plasma decay and uptake In different tissues) and absolute values (min for half life) and are means S.D. from 4-6 mice per group. Individual values are given In appendix XVI. [3H]-triolein decay in plasma (% of injected dose) Control Fenofibrate PFBS PFHS PFOS rime after iniection of r3Hl-triolein labeled VLDL-like particles (min 0 2 5 10 20 avg 100.0 98.2 90.5 76.8 54.7 sd 0.0 6.6 8.8 6.0 6.8 avg 100.0 78.1 63.8 35.0 11.5 sd 0.0 13.6 13.9 14.1 7.4 avg 100.0 93.0 81.1 55.4 28.0 sd 0.0 avg 100.0 sd 0.0 7.6 80.4 8.5 6.9 62.2 9.0 10.4 37.6 12.4 11.4 16.5 6.1 avg 100.0 78.6 61.6 42.4 21.7 sd 0.0 8.5 4.0 6.2 5.5 30 jy.4 9.9 5.1 2.5 13.8 7.2 7.6 3.2 12.0 3.8 [3H]-triolein decay in plasma: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS Time aft sr Iniection of f3hl-triolein labeled VLDL-like particles (mini 2 5 10 20 30 0.019 0.004 0.004 . 0.001 0.056 0.286 0.016 0.190 0.008 0.016 0.008 0.016 0.008 0.032 0.004 0.017 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.190 0.111 0.111 0.063 0.032 1.000 0.931 0.931 0.177 0.177 0.931 0.931 0.247 0.052 0.017 0.067 0.019 0.067 0.171 0.114 0.038 0.010 0.114 0.476 0.762 0.818 0.937 0.589 0.180 0.041 [JH]-triolein half-life (min) Control Fenofibrate PFBS PFHS PFOS Half-life avg 21.0 sd 6.2 avg 6.8 sd 0.9 avg 10.2 sd 3.3 avg 8.1 sd 1.3 avg 10.4 sd 1.9 [*H3-triolein half-life: P-value Between arouns Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS Half-life 0.001 0.008 0.032 0.004 0.004 0.063 0.082 0.009 0.352 0.914 0.041 The clearance of VLDL-like [3H]-trioleln labeled particles In plasma (as a measure for VLDLTG clearance) was significantly increased In all treatment groups (figure 4.2.11a). Fenofibrate showed the strongest clearance, followed by PFHS, whereas PFOS and PFBS showed a comparable clearance. In figure 4.2.11b the [3H]-trloleln half-life (=l/slope figure 4.2.11a) is shown. Fenofibrate significantly decreased TG half life by -68%. PFBS and PFOS both reduced [3H]-trioleln halflife by -51% and PFHS showed a decreased of -61%, which was a significantly stronger decrease as the PFOS group. The half-life of VLDL-TG did not significantly differ between PFBS on the one hand and PFHS and PFOS on the other hand. 57 p. 59 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.2.11a [3H]-triolein decay in plasma Figure 4.2.11b [3H]-triolein half-life* * p<0.05 vs. control Total liver [3H]-trioleln uptake was increased in all treatment groups. Fenofibrate, PFHS and PFOS showed a significant induction of respectively +98%, +149% and +84%. PFBS showed a trend towards a higher uptake (+53%, p=0.063). 58 p. 60 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 PFHS showed a significantly decreased [3H]-triolein uptake in the heart and the spleen (by 52% and -53% respectively), PFOS increased uptake in gonadal white adipose tissue (gWAT, by 164%) and fenofibrate showed a higher uptake in muscle and gonadal fat (by 65% and 162% respectively). [JH]-triolein tissue uptake (% of injected dose) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd [JH)-triolein tissue uptake: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS liver 8.7 1.8 17.3 3.7 13.4 2.6 21.8 4.2 16.1 3.6 liver 0.002 0.008 0.063 0.004 0.004 0.190 0.126 0.792 0.010 0.257 0.132 heart 1.99 0.61 1.26 0.45 1.81 0.98 0.96 0.37 1.46 0.75 heart 0.143 0.151 0.905 0.017 0.329 0.413 0.329 0.662 0.114 0.762 0.310 spleen 0.77 0.18 0.53 0.17 0.57 0.14 0.36 0.13 0.47 0.21 spleen 0.049 0.095 0.190 0.009 0.082 0.730 0.126 0.009 0.114 0.610 0.310 muscle 4.82 1.66 7.96 1.79 6.38 2.16 6.97 1.22 6.37 1.43 muscle 0.129 0.032 0.190 0.052 0.247 0.413 0.247 0.004 0.762 0.914 0.699 gWAT 0.78 0.21 2.04 0.87 1.45 1.79 1.82 1.41 2.05 1.77 gWAT 0.043 0.008 0.730 0.082 0.004 0.286 0.429 0.004 0.476 0.257 0.485 Total liver [3H]-triolein uptake * p<0.05 vs. control 59 p. 61 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.2.11d 12 Total tissue [3H]-triolein uptake Control Fenofibrate PFBS PFHS PFOS heart spleen muscle gWAT * p<0.05 vs. control The clearance of VLDL-like [14C]-cholesteryl oleate particles (as a measure for VLDLcholesterolester clearance) was significantly Increased In the fenofibrate group and the PFBS and PFHS groups (figure 4.2.lie ) . PFOS showed a similar decay as the control group. Fenofibrate showed the strongest Increase, whereas PFBS and PFHS showed a comparable decay. In figure 4.2.I l f the [14C]-cholesteryl oleate half-life (=l/slope figure 4.2.l i e ) Is shown. Fenofibrate significantly decreased CE half life by -80%. PFBS and PFHS reduced half-life by -60% and -54% respectively. PFOS was not significantly different from the control group and the PFBS group. [14C]-cholesteryl oleate decay in plasma (% of inlected dose) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd Time after Iniection of r14Cl-cholestervl oleate labeled VLDL-like particles (min) 0 2 5 10 20 30 100. 93.9 90.9 86.0 73.4 63.5 0.0 100.0 0.0 100.0 11.6 82.6 13.4 95.0 9.3 75.8 12.9 90.5 8.6 51.3 10.5 73.7 7.6 25.6 8.2 52.4 9.1 15.2 4.4 39.7 0.0 100.0 0.0 7.5 85.0 7.6 5.1 77.7 7.3 8.7 66.6 6.9 7.8 49.2 7.2 7.0 40.0 7.4 100.0 83.5 77.4 72.9 65.1 58.2 0.0 7.6 5.0 5.7 7.0 9.8 60 p. 62 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 [`"CJ-cholesteryl oleate decay in plasma: P-value Between qroups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS Time a te r Infection of f l , Cl-CO labeled VLDL-like particles fminl 2 5 10 20 30 0.185 0.036 0.002 < 0.001 < 0.001 0.151 0.056 0.008 0.008 0.008 1.000 0.730 0.063 0.016 0.016 0.247 0.052 0.009 0.004 0.004 0.177 0.052 0.030 0.126 0.429 0.286 0.190 0.032 0.016 0.016 0.792 0.931 0.030 0.009 0.004 0.537 0.662 0.009 0.004 0.004 0.067 0.019 0.257 0.914 1.000 0.067 0.019 0.914 0.038 0.010 0.937 0.818 0.180 0.009 0.002 [I4C]-cholesteryl oleate half-life (min) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd Half-life 56.6 26.2 11.0 1.7 22.5 6.1 26.3 6.7 92.4 84.2 ["CJ-cholesteryl oleate half-life: P-value Between qrcups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS Half-life < 0.001 0.008 0.016 0.009 0.931 0.016 0.004 0.004 0.610 0.010 0.004 Figure 4.2.11e [14C]-cholesteryl oleate decay re E troe a. c rree o * p<0.05 vs. control 61 p. 63 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.2.I l f [14C]-cholesteryl oleate half-life 200 c E re 150 Control Fenofibrate PFBS PFHS PFOS rree 100 4Mr*e O 50 V o 0 * p<0.05 vs. control Total liver [14C]-cholesteryl oleate was Increased in all treatment groups, except PFOS. Fenofibrate, PFBS and PFHS showed a significant induction of respectively +142%, +74% and +79%. PFHS showed a significantly decreased [14C]-cholesteryl oleate uptake in the spleen (by 52%), other tissues were not significantly affected by any treatment. [14C]-cholesteryl oleate tissue uptake (% of inlected dose) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd liver 26.3 8.1 63.8 9.7 45.9 3.5 47.1 10.9 19.1 5.0 [14C]-cholesteryl oleate tissue uptake: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS liver < 0.001 0.008 0.016 0.017 0.126 0.016 0.030 0.004 0.352 O.OIO 0.004 heart 1.40 0.32 1.45 0.64 1.50 1.04 1.05 0.45 0.96 0.37 heart 0.381 1.000 0.556 0.329 0.082 0.905 0.329 0.177 0.476 0.476 0.818 spleen 1.04 0.23 0.79 0.20 0.88 0.27 0.50 0.19 0.63 0.30 spleen 0.045 0.310 0.730 0.009 0.126 0.556 0.030 0.537 0.067 0.257 0.394 muscle 1.02 1.42 1.95 0.59 1.70 1.56 1.91 1.20 0.55 0.70 muscle 0.124 0.421 0.413 0.429 0.792 0.413 1.000 0.017 0.610 0.114 0.026 gWAT 0.02 0.03 0.39 0.17 0.18 0.30 0.33 0.32 0.41 0.64 gWAT 0.122 0.008 0.556 0.082 0.177 0.286 0.792 0.329 0.352 0.610 0.699 62 p. 64 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.2. l l g Total liver [14C]-cholesteryl oleate uptake 100 * p<0.05 vs. control Figure 4.2. l l h Total tissue [14C]-cholesteryl oleate uptake heart p<0.05 vs. control spleen muscle gWAT 63 p. 65 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3 Results study 3 4.3.1 Markers of general well-being No specific clinical signs were observed during the study. At sacrifice, besides the livers no macroscopic differences were observed between the groups. Livers were visibly somewhat increased in the fenofibrate group and largely increased in the PFHS and PFOS group. One mouse (mouse 23) showed a granular liver. 4.3.2 Body weight Values are absolute values (g) and are means S.D. from 6-7 mice per group. Individual values are given in appendix I. Body weight (g) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd Body weight: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS 0 27.6 1.3 27.0 2.9 27.6 2.0 27.4 1.1 27.1 1.2 5 0.741 0.318 0.902 0.710 0.456 0.620 0.318 0.456 1.000 0.456 0.535 time (weeks! 1 27.8 1.3 27.2 2.9 27.9 2.3 27.3 0.8 27.4 1.3 time (weeks) 1 0.737 0.383 0.620 0.456 0.383 0.456 0.318 0.456 0.902 0.902 0.902 4 29.8 1.6 29.0 2.7 30.1 2.7 28.7 0.9 28.3 0.7 4 0.262 0.310 0.937 0.132 0.015 0.485 0.818 0.937 0.394 0.132 0.589 During the study mice gained body weight in all groups. In contrast to study 1 and 2, the PFOS group showed a small but significantly decreased body weight after 4 weeks of treatment (-5%). 64 p. 66 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.3.2 40 Body weight 35 o> O) I 30 > T3 O 00 25 Control Fenofibrate PFBS * PFHS PFOS 20 0 1 Time (weeks) 4 * p<0.05 vs. control 4.3.3 Liver and perigonadal fat weight Values are absolute values (g) and are means S.D. from 6 mice per group. Individual values are given in appendix II. Tissue weight (g) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd Tissue weight: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS Liver 1.61 0.13 1.94 0.16 1.72 0.20 2.93 0.52 2.95 0.27 Liver < 0.001 0.004 0.310 0.002 0.002 0.093 0.026 0.002 0.004 0.002 0.699 Perigonadal fat 0.56 0.16 0.50 0.14 0.52 0.18 0.36 0.05 0.39 0.13 Perigonadal fat 0.083 0.485 0.699 0.009 0.132 0.937 0.093 0.132 0.132 0.180 0.818 Livers were significantly increased In the fenofibrate, PFHS and PFOS group. Fenofibrate increased liver weights by +20%. The PFHS and PFOS groups showed a robust induction in liver weights, respectively by +82% and +83%. Liver weight in the PFHS and PFOS groups was significantly higher as compared to the PFBS group. 65 p. 67 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Although a decrease in perigonadal fat weight was seen after PFOS and PFHS treatment, this was only significantly different in the PFHS group (by -36%). Figure 4.3.3a Liver weight o> o> S *w week 4 * p<0.05 vs. control Figure 4.3.3b 1.2 Perigonadal fat weight "3 1.0 - Control Fenofibrate PFBS PFHS PFOS * p<0.05 vs. control week 4 66 p. 68 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3.4 Food intake Values are absolute values (g/day/mouse) and are means S.D. from 3 cages per group. Individual values per cage are given in appendix III. Food Intake (g/day/mouse) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd -1-0 3.1 0.3 3.1 0.3 3.1 0.3 3.1 0.3 3.1 0.3 time (weeks) 0-1 2.9 0.1 2.7 0.4 2.9 0.3 2.8 0.3 2.8 0.3 3-4 2.8 0.2 2.8 0.3 2.9 0.3 2.8 0.3 2.6 0.2 Food intake: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 0-1 0.925 3-4 0.498 1.000 1.000 1.000 1.000 0.700 0.700 1.000 0.200 0.700 1.000 1.000 1.000 1.000 0.400 0.700 0.400 0.700 0.200 1.000 0.400 All groups showed a similar food intake during the study. Figure 4.3.4 Food intake 5 a(3A> 4 O E > 5m 3 o> a> n2 T3 o o1 Control Fenofibrate PFBS PFHS PFOS 0 - 1-0 * p<0.05 vs. control 0-1 Time (weeks) 67 3-4 p. 69 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3.5 Plasma ALT Values are absolute values (U/L) from measurements in pooled plasma samples per group (6-7 mice per group) at t=0 and 4. Group F e n o fib ra te PFOS Plasma ALAT (U/L! t=0 weeks t=4 weeks 168 204 167 145 123 328 162 391 125 458 After 4 weeks from treatment PFHS and PFOS showed higher ALT levels than the control group. In contrast to studies 1 and 2 PFBS treatment also seem to increase plasma ALT. Figure 4.3.5 Plasma ALT Time (weeks) 4.3.6 Plasma cholesterol Values are absolute values (mmol/L) and are means S.D. from 6-7 mice per group. Individual values are given in appendix IV. Cholesterol (mmol/L) Control Fenofibrate PFBS PFHS PFOS time (weeks) 04 avg 7.3 7.9 sd 1.0 avg 7.7 0.8 5.1 sd 1.2 0.1 avg 7.6 6.1 sd 1.9 1.1 avg 7.5 2.4 sd 1.7 0.5 avg 7.7 2.6 sd 1.4 0.6 68 p. 70 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Cholesterol: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 04 0.941 <0.001 0.535 0.002 0.620 0.026 0.535 0.002 0.620 0.002 1.000 0.015 0.805 0.002 1.000 0.002 1.000 0.002 0.710 0.002 0.805 0.699 As was seen in experiments 1 and 2, all treatment groups showed a significant reduction in plasma cholesterol at t=4 weeks of treatment. Fenofibrate decreased plasma cholesterol by -35%. PFBS, PFHS and PFOS treatment reduced plasma cholesterol levels respectively by -22%, -69% and -67% after 4 weeks of treatment. PFHS and PFOS decreased cholesterol levels to a larger extent than PFBS (p=0.002). PFHS and PFOS showed a similar Inhibition. Plasma cholesterol Time (weeks) p<0.05 vs. control 4.3.7 Plasma HDL-cholesterol Values are absolute values (mmol/L) and are means S.D. from 6-7 mice per group. Individual values are given In appendix V. 69 p. 71 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 HDL-Cholesterol (mmol/L) Control Fenoflbrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd time (weeks) 04 1.19 1.07 0.26 1.20 0.24 1.89 0.40 1.34 0.19 1.19 0.66 1.07 0.53 0.40 0.41 0.20 1.21 0.28 0.33 0.13 HDL-Cholesterol: P-value Between groups Control vs: Fenoflbrate vs PFBS vs PFHS vs Fenoflbrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 04 0.991 <0.001 0.902 0.902 0.002 0.818 0.620 1.000 0.002 0.002 1.000 0.065 0.710 0.002 1.000 0.002 0.902 0.002 1.000 0.002 0.805 0.240 HDL-cholesterol levels were increased after 4 weeks treatment In the fenoflbrate group, by +77%. Both PFHS and PFOS significantly decreased HDL-cholesterol after 4 weeks of treatment, by -62% and -74%, respectively. PFBS treatment did not result in significantly altered HDL-cholesterol levels. When compared to PFBS, PFHS and PFOS treatment showed a decreased plasma HDLcholesterol, when comparing PFHS and PFOS treatment, no significant changes were seen. Figure 4.3.7 Plasma HDL-cholesterol 70 p. 72 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3.8 Plasma triglycerides Values are absolute values (mmol/L) and are means S.D. from 6-7 mice per group. Individual values are given In appendix VI. Triglycerides (mmol/L) Control Fenofibrate PFBS PFHS PFOS time (weeks) 4 avg 1.67 1.74 sd 0.39 avg 1.70 0.42 0.38 sd 0.67 avg 1.74 0.05 0.95 sd 0.82 avg 1.81 sd 0.61 0.32 0.54 0.29 avg 1.71 sd 0.46 0.49 0.14 Triglycerides: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 04 0.988 <0.001 0.710 0.002 0.902 0.004 0.620 0.002 1.000 0.002 0.710 0.002 0.902 0.180 0.710 0.132 1.000 0.026 0.902 0.009 0.805 1.000 All treatment groups showed significant reductions In plasma triglycerides at t=4 weeks of treatment. Fenofibrate decreased plasma triglycerides by -78%. PFBS, PFHS and PFOS treatment reduced plasma triglycerides levels respectively by -45%, -69% and -72% after 4 weeks of treatment. Both PFHS and PFOS decreased triglycerides levels to a larger extent than PFBS (p=0.002). PFOS showed no significantly different triglycerides levels as compared to PFHS. 71 p. 73 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.3.8 4 Plasma triglycerides O E3 E (0A) o c 0o 2 >. o> c m E wn 1 0. Control Fenoflbrate PFBS mPFHS PFOS 0 0 4 Time (weeks) * p<0.05 vs. control 4.3.9 Lipoprotein profiles Lipoproteins were separated on a superose column. Values are absolute values (mM) from cholesterol (left column) and phospholipids (right column) measurements in pooled plasma per group (with 6-7 mice per group) at t=4 We consider fractions 4-8 as VLDL; 9-14 as LDL; 13-16 as large-HDL (HDL1) and 17-24 as HDL. The profiles are more or less the same as In experiment 1 and 2. After 4 weeks of treatment, plasma cholesterol levels were decreased in the VLDL-LDL peak in all groups as compared to the control group. Fenoflbrate showed the strongest reduction, PFOS seemed to decrease VLDL to a lesser extent than PFHS. PFBS decreased VLDL-LDL to a lesser extend than PFHS and PFOS. HDL was Increased In the fenoflbrate group, with formation of a HDL1 particle. Whereas PFBS showed no clear effects on HDL, both PFHS and PFOS strongly decreased the HDL peak. PFHS seemed to decrease HDL to a lesser extend than PFOS. In contrary to the profiles In experiment 1 and 2 HDL1 (large HDL) was not Increased in the PFHS group. The changes in the lipoprotein profiles are in line with the changes in plasma lipids. Figure 4.3.9 Lipoprotein profiles Cholesterol (mmol/L) 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3.10 Plasma ApoAl Values are absolute values (mg/mL) and are means S.D. from 6-7 mice per group. Individual values are given in appendix IX. ApoAl (mg/mL) Control Fenofibrate PFBS PFHS PFOS time (weeks) 04 avg 1.78 2.28 sd 0.40 avg 1.73 0.50 2.65 sd 0.70 avg 1.94 0.23 2.30 sd 0.82 avg 1.54 1.00 0.54 sd 0.60 0.26 avg 1.73 sd 0.30 0.43 0.08 ApoAl: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 04 0.974 <0.001 0.710 1.000 0.180 0.818 0.620 0.805 0.002 0.002 0.902 0.902 0.180 0.002 0.710 0.002 0.805 0.002 1.000 0.002 0.535 0.394 Whereas PFHS and PFOS stongly inhibited plasma ApoAl (by -76% and -81% respectively, PFBS and fenoflbrate showed not significant effects on ApoAl levels. Figure 4.3.10 4 Plasma ApoAl 04 * p<0.05 vs. control Time (weeks) p. 75 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3.11 Plasma CETP mass Values are absolute values (pg/mL) and are means S.D. from 5-7 mice per group. Individual values are given in appendix X. CETP mass (pg/mL) Control Fenofibrate PFBS PFHS PFOS time (weeks) 0 avg 13.7 4 14.7 sd 1.6 1.4 avg 13.1 9.1 sd 2.5 0.6 avg 12.8 11.8 sd 2.7 1.1 avg 13.8 9.4 sd 3.7 1.9 avg 12.6 9.1 sd 1.8 1.3 CETP mass: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 0 0.950 4 0.001 0.710 0.004 0.535 0.009 1.000 0.004 0.383 0.002 0.902 0.004 0.902 1.000 0.805 0.662 0.802 0.052 1.000 0.015 0.805 0.931 Plasma CETP levels were significantly decreased in all treatment groups. Fenofibrate decreased plasma CETP concentration by -38%. PFBS, PFHS and PFOS treatment reduced plasma CETP levels respectively by -20%, -36% and -38% after 4 weeks of treatment. PFHS and PFOS decreased levels to a larger extent than PFBS (p=0.052 and 0.015 respectively). PFOS and PFHS showed similar CETP levels. 74 p. 76 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.3.11 20 Plasma CETP mass O) 15 3 ((00 (0 E 10 UJ O m E u> 5 0. Control Fenofibrate PFBS 0 04 Time (weeks) * p<0.05 vs. control 4.3.12 Plasma CETP activity Values are absolute values (pmol/h) and are means S.D. from 4-7 mice per group. Individual values are given in appendix XI. CETP activity (pmol/h) Control Fenofibrate PFBS PFHS PFOS time (weeks) 04 avg 227 209 sd 29 avg 227 26 97 sd 37 avg 229 14 169 sd 41 avg 228 58 172 sd 44 22 avg 229 sd 17 166 19 CETP activity: P-value Between qroups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS time (weeks) 04 0.996 0.008 0.945 0.010 0.945 0.132 0.731 0.052 0.876 0.009 1.000 0.114 0.937 0.016 0.792 0.010 0.818 0.931 0.931 0.818 0.931 0.329 75 p. 77 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 CETP activity levels were significantly decreased after fenofibrate and PFOS treatment. Fenofibrate decreased plasma CETP activity by -53%. Although PFBS, PFHS and PFOS treatment reduced plasma CETP activity, this was not significantly different in the PFBS group (p=0.132). A trend toward lowered CETP activity was seen in the PFHS group (by -18%, p=0.052). PFOS decreased CETP activity by -20%. Figure 4.3.12 Plasma CETP activity 350 Control Fenofibrate PFBS si PFHS PFOS * p<0.05 vs. control Time (weeks) 4.3.13 In vivo clearance of autologous HDL Values are relative values (% of injected dose for plasma decay of labeled autologous HDL) and absolute values (pools HDL/h for fractional catabolic rate of HDL and mM HDL/h for catabolic rate of HDL) and are means S.D. from 5 mice per group. Individual values are given in appendix XVII. [JH]-cholesteryl oleyl ether decay In plasma (% of Intected dose) Control avg sd Fenofibrate avg PFBS sd avg sd PFHS avg sd PFOS avg sd Time after iniectlon of rJHl-cholestervl olevl ether labe ed autoloaous H0L(h) 0 1 2 4 8 24 100.0 69.7 56.2 38.6 20.3 4.9 0.0 6.6 4.3 2.5 2.5 1.8 100.0 67.7 60.0 46.7 31.7 9.4 0.0 14.9 5.4 2.1 1.5 100.0 64.3 52.2 32.8 17.0 1.0 2.6 0.0 100.0 5.9 54.6 8.0 38.8 3.4 23.6 4.0 9.7 0.8 1.4 0.0 5.2 4.8 4.7 2.8 0.7 100.0 55.4 40.0 23.5 8.7 1.3 0.0 5.3 4.2 2.7 1.9 0.4 76 p. 78 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 [JH]-cholesteryl oleyl ether decay in plasma: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS Time aflter iniection of f: Hl-CO-ether lat eled autoloaous HDL fhi 1 2 4 8 24 0.018 0.004 <0.001 <0.001 <0.001 0.841 0.222 0.548 0.421 0.008 0.016 0.008 0.222 0.008 0.032 0.016 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 1.000 0.095 0.310 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.222 0.008 0.008 0.008 0.008 0.032 0.056 0.008 0.016 0.056 0.032 0.095 0.008 0.008 0.008 0.841 0.841 0.841 0.841 0.841 Fractional Catabolic Rate HDL (pools HDL-C/h) Control Fenofibrate PFBS PFHS PFOS avg sd avg sd avg sd avg sd avg sd FCR 0.21 0.02 0.16 0.01 0.24 0.03 0.32 0.04 0.33 0.03 Fractional Catabolic Rate HDL: P-value Between groups Control vs: Fenofibrate PFBS PFHS PFOS Fenofibrate vs PFBS PFHS PFOS PFBS vs PFHS PFOS PFHS vs PFOS FCR <0.001 0.008 0.151 0.008 0.008 0.008 0.008 0.008 0.008 0.008 1.000 Catabolic rate HDL (mM HDL-C/h) Control Fenofibrate PFBS PFHS PFOS CR avg 0.23 sd 0.04 avg 0.31 sd 0.05 avg 0.25 sd 0.09 avg 0.12 sd 0.06 avg 0.08 sd 0.04 Catabolic rate HDL: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS POS CR 0.001 0.056 1.000 0.008 0.008 0.222 0.008 0.008 0.032 0.008 6.4ii The clearance of HDL (measured with a trace of autologous labeled [3H]-cholesteryl oleyl ether) In plasma was significantly increased in the PFHS and the PFOS groups. Fenofibrate decreased HDL clearance. PFBS showed an increased HDL clearance at two time points (4 and 24 h, figure 4.3.13a). In figure 4.3.13b the fractional catabolic rate of HDL (=slope 0-8 h figure 4.3.13a) is shown. Fenofibrate significantly decreased the fractional catabolic rate by -25%; PFHS and PFOS showed a similarly increased fractional catabolic rate (by 50% and 54%, respectively). However, calculation of the catabolic rate (figure 4.3.13c), which takes Into account the different pool sizes of HDL-cholesterol after the various treatments, showed that the clearance of HDL was actually decreased by PFHS and PFOS treatment (by -48% and -65% respectively). The catabolic rate was somewhat Increased by fenofibrate, although this was not significantly different from the control group (+32%, p=0.056). PFBS showed no effects. 77 p. 79 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.3.13a [3H]-cholesteryl oleyl ether decay in plasma >0 & *0- 100 V x L- * p<0.05 vs. control Figure 4.3.13b Fractional catabolic rate of HDL * p<0.05 vs. control 78 p. 80 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Figure 4.3.13C Catabolic rate of HDL 0.6 0.5 3 o oX .0 E 1 O 0.4 0.3 0.2 0.1 Control Fenofibrate PFBS PFHS PFOS 0.0 * p<0.05 vs. control 4.3.14 Liver microsomal DGAT activity Values are absolute values (nmol/min/mg protein) and are means S.D. from 5 mice per group. Individual values are given in appendix XVIII. DGAT activity (nmol/min/mq protein! Control Fenofibrate PFBS PFHS PFOS DGAT-l avg 2.2 sd 0.8 avg 2.1 sd 0.5 avg 2.6 sd 0.6 avg 2.6 sd 0.9 avg 3.4 sd 0.5 DGAT-2 1.6 0.4 2.8 1.0 1.7 0.7 2.4 0.8 2.8 0.9 DGAT activity: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS DGAT-l 0.068 0.841 0.222 0.421 0.056 0.222 0.310 0.016 0.690 0.095 0.151 DGAT-2 0.070 0.056 1.000 0.151 0.016 0.151 0.841 1.000 0.222 0.056 0.841 79 p. 81 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Hepatic microsomal DGAT-2 activity (which is primarily involved in VLDL assembly in the liver) was significantly increased in the PFOS treatment group (by +75%). Fenofibrate showed a trend towards significance in a higher DGAT-2 activity (by +73%, p= 0.056). PFBS and PFHS showed no significant effects. None of the treatment groups affected DGAT-1 activity (primarily involved in TG storage in the liver) significantly, although PFOS showed a trend towards significance (+53%, p=0.056). DGAT-1 and DGAT-2 activities tended to be higher in the PFOS group as compared to the PFBS treated mice. Figure 4.3.14 Liver microsomal DGAT activity DGAT-1 DGAT-2 * p<0.05 vs. control 4.3.15 Liver lipid analysis Values are absolute values (pg/mg protein) and are means S.D. from 6 mice per group. Individual values are given in appendix XIX. Liver lipids (pg/mg protein) Control Fenofibrate PFBS PFHS PFOS FC avg 14.3 sd 1.4 avg 11.7 sd 0.8 avg 11.6 sd 2.0 avg 13.1 sd 1.6 avg 16.6 sd 1.5 CE 24.6 3.5 11.9 1.5 15.9 6.4 24.2 5.7 47.6 10.3 TC 38.9 3.9 23.6 2.3 27.5 8.3 37.3 7.0 64.1 11.5 TG 74.5 17.5 59.9 15.9 61.8 30.2 113.5 24.8 217.2 47.5 80 p. 82 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Liver lipids: P-value Between groups Control vs: Fenofibrate vs PFBS vs PFHS vs Fenofibrate PFBS PFHS PFOS PFBS PFHS PFOS PFHS PFOS PFOS FC 0.001 0.004 0.041 0.240 0.026 0.589 0.065 0.002 0.240 0.002 0.002 CE <0.001 0.002 0.041 1.000 0.002 0.394 0.002 0.002 0.065 0.002 0.002 TC <0.001 0.002 0.026 0.937 0.002 0.589 0.002 0.002 0.065 0.002 0.002 TG <0.001 0.240 0.240 0.015 0.002 0.699 0.009 0.002 0.026 0.002 0.002 Hepatic triglycerides were significantly increased after PFHS and PFOS treatment (by +52% and +192%, respectively). PFOS showed a significantly higher increase than PFHS (p=0.002). Both fenofibrate and PFBS seem to decrease hepatic triglycerides somewhat, but this was not significantly different from the control. Hepatic free cholesterol and cholesterolester levels were affected by both fenofibrate and PFBS, free cholesterol was reduced by -18% and -19% and cholesterolester was decreased by -52% and -36%, respectively. PFHS did not have an effect on hepatic cholesterol levels, but PFOS showed increases in both cholesterol and cholesterol ester (by +16% and +93%, respectively). PFOS increased hepatic triglycerides and cholesterol levels to a significantly higher extent as compared to PFBS. The same holds true for triglycerides with PFHS; there was a tendency towards increased cholesterolester levels. Figure 4.3.15 Liver lipids FC * p<0.05 vs. control CE TC 81 TG p. 83 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CBTP mice TNO project number 031.12685 4.3.16 Liver histology HPS (haematoxyllne-phloxine-saffrane) stained liver slides were examined histopathologically (based on necrosis with inflammatory cells, proteinaceous droplets, hepatocellular hypertrophy and hepatocellular mlcrovacuolation). Per group 5 livers were analyzed. Table 4.3.16 Histopathologically examination of livers Group Mouse 1 2 3 4 6 fc n o fib ra te 8 fc n o fib ra te 9 fc n o fib ra te 10 fc n o fib ra te 13 fc n o fib ra te 14 16 17 18 19 ii-'Ki :jOrfSil is1' `I, llii*ri r 20 22 23 25 26 27 Pf OS 29 pros 31 pros 32 PFOS 33 PFOS 35 VS = very slight S = slight M = moderate Necrosis with Inflammatory cells VS F VS MF VS F SF SF SF SF F = focal MF = multifocal D = diffuse Proteinaceous droplets S s s s s vs vs vs vs vs vs vs/s vs/s vs/s vs vs vs vs vs vs s s s s vs Hepatocellular hypertrophy Multifocal S S s s s s M s M M M M M M M M Hepatocellular mlcrovacuolation S MD VS F MD MD MF VS F MD VS F VS F VS F MD MD MD MD s/vs Control: Only 1 out of the 5 control ApoE3L. CETP mice, mouse 2, showed hepatocellular hypertrophy and hepatocellular mlcrovacuolation. The liver histology of the other 4 mice appeared to be quite normal (figure 4.3.16a, table 4.3.16). Mouse 1 & 3 appeared to have more glycogen In the liver than mouse 4 & 6. In all control mice, proteinaceous droplets could be detected In the cytoplasm of the hepatocytes (deposition of ApoE3Leiden proteins, ref). Figure 4.3.16a HPS stained control livers A: Control (mouse 2, lOOx) with hepatocellular hypertrophy and mlcrovacuolation B: Control (mouse 1, 100x) with more glycogen (white cytoplasm) C: Control (mouse 4, lOOx) 82 p. 84 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Fenofibrate: The addition of fenofibrate to the diet resulted in fewer proteinaceous droplets. The hepatocytes were slightly hypertrophic and had a fine granular eosinophilic cytoplasm, especially in zone B & C around the central vein (figure 4.3.16b, table 4.3.16). Mouse 10 showed moderate hepatocellular vacuolation. Figure 4.3.16b HPS stained livers, treated with fenofibrate A: Drawing of liver lobule, showing zone A, B and C (lOOx) B: Fenofibrate treatment (mouse 8, lOOx) C: Fenofibrate treatment (mouse 10, lOOx) CV= central vein PFBS: The addition of PFBS to the diet resulted in fewer proteinaceous droplets. Three out of five mice showed moderate hepatocellular microvacuolation (figure 4.3.16c, table 4.3.16). Figure 4.3.16c HPS stained livers, treated with PFBS BC AT ' # .. '4 A: PFBS treatment (mouse 16, lOOx) B: PFBS treatment (mouse 17, lOOx) C: PFBS treatment (mouse 18, lOOx) 83 p. 85 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 PFHS: The addition of PFHS to the diet resulted in fewer proteinaceous droplets. The hepatocytes were hypertrophic and had a fine granular eosinophilic cytoplasm, especially In zone B & C around the central vein (figure 4.3.16d, table 4.3.16). One mouse (M23) showed moderate diffuse hepatocellular mlcrovacuolation. The other four mice only showed focally, very slight mlcrovacuolation. Focal aggregates of inflammatory cells/necrotlc hepatocytes could be detected In the livers of 3 out of 5 mice. Figure 4.3.16d HPS stained livers, treated with PFHS A: PFHS treatment (mouse 22, lOOx) B: PFHS treatment (mouse 23, 100x) C: PFHS treatment (mouse 27, 200x). Aggregates of inflammatory cells (arrows) PFOS: The addition of PFOS to the diet resulted In hypertrophic hepatocytes with fine mlcrovacuolation of the cytoplasm (table 4.3.16). Figure 4.3.16e, in which stainings of PFOS, PFHS and control livers were put next to each other, clearly showed the Increased lipid filled vacuoles In the hepatocytes after PFOS treatment. Figure 4.3.16e PFOS treatment vs PFHS treatment vs control A: PFOS treatment (Mouse 29, lOOx) B: PFHS treatment (Mouse 22, lOOx) C: Control (Mouse 4, lOOx) 84 p. 86 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 4.3.17 Liver microarray analysis Liver microarray analysis and subsequent gene expression data analysis was performed on 6 mice per group. The total transcriptome analysis report is included in appendix XX. In this paragraph tables of gene expression data of selected pathways with transcription factors and genes involved (fold change vs control, with q-values) are shown. Q-values are pvalues corrected for multiple tested. Triglyceride metabolism Lipoiysis In line with the higher lipolytic activity in plasma, hepatic mRNA signals for LPL (fenofibrate 4.6-fold, PFHS 4.3-fold and PFOS 2.1 fold) were increased. Fatty acid/triglyceride synthesis Although a major regulator of fatty acid synthesis, SREBPlc was decreased with PFBS, PFHS and PFOS several genes in the fatty acid synthesis pathway were increased, suggesting that regulation does not take place via activation of LXR but probably via PXR. Several ACS genes and DGAT1 were increased with fenofibrate, PFHS and PFOS. SCD2 was increased by PFHS and PFOS. Table 4.3.16a Gene expression data of triglyceride metabolism pathway with transcription factors and genes involved Pathway common name TG m etabolism Averaae expression f2 lo a l 1 Fenofibrate vs control 1 Control 1 Fenofibrate 1 PFBS 1 PFHS 1 PFOS 1 Fold chanqe 1 q-value PFBS vs control PFHS vs Control PFOS vs Control Fold chanqe 1q-value Fold chanqe 1 q-value Fold chanqe 1 q-value FA7T?vntK5l LXRa FXR PPARa PPARa SREBPla/C PXR FAS DGAT1 DGAT2 SCD1 SCD2 ACLY s l4 ACS. A csll ACS. Acsl3 ACS. Acsl4 ACS. Acsl5 ACS. Acssl ACS. Acss2 ACS. Acsm l ACS. Acsm2 ACS. Acsm3 ACS. Acsm5 6.77 9.78 11.03 5.58 11.05 6.74 10.06 5.99 9.25 13.15 1.98 11.10 12.36 11.68 6.89 8.29 11.73 2.04 10.77 11.23 2.09 9.74 9.85 6.76 9.82 11.33 5.28 10.90 7.10 10.99 6.56 9.02 13.47 2.10 11.02 12.75 12.79 8.51 8.95 12.31 2.03 11.58 10.89 1.95 9.92 9.77 6.77 9.77 10.90 5.48 10.28 6.78 9.56 5.92 9.13 12.52 2.16 10.52 12.00 12.07 7.14 8.54 11.58 2.03 10.45 11.27 2.34 9.40 9.53 6.52 10.04 10.54 5.90 10.10 7.10 10.68 6.47 8.93 13.39 2.70 11.24 12.59 12.57 8.47 9.35 12.23 2.34 11.73 10.98 2.12 9.34 9.77 6.42 10.06 10.55 6.18 10.03 7.41 9.89 6.44 9.04 13.10 2.30 9.79 12.53 12.48 7.67 8.78 11.76 2.28 10.43 11.18 2.05 9.25 9.94 -1.01 1.03 1.23 -1.23 -1.11 mmm 1.90 1.24 1.09 -1.05 1.31 JIAHHmkI MililMlWU -1.01 1.76 -- -- BBttl -1.10 1.13 -1.06 0.505 0.436 0.05S 0.256 0.350 0.010 0.068 0.002 0.046 0.207 0.247 0.476 0.326 0.000 0.000 0.000 O.OOl 0.487 0.068 0.000 0.130 0.168 0.373 -1.00 -1.01 -1.10 -1.07 1.03 -1.42 -1.05 -1.09 -1.54 1.14 -1.49 -1.28 1.19 1.19 -1.10 -1.01 -1.24 1.03 1.19 -1.27 -1.24 0.692 0.676 0.619 0.025 0.622 0.403 0.581 0.360 0.109 0.282 0.292 0.516 0.008 0.482 0.170 0.437 0.671 0.498 0.568 0.057 0.080 0.116 ro i*n o Iw0w d m " 4 r * 1333 ' 0.028 85 p. 87 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Pathway common name 1 Averaoe exoreaaion f2 lo a l 1 Fenofibrate vs control PFBS va control PFHS v * Control PFOS vo Control 1 1 Control 1 Fenofibrate 1 PFBS 1 PFHS 1 PFOS 1 Fold change 1 q-value Fold change 1q-value Fold chanae 1 a-value Fold chanae I a-value r n UDiaKBi tra n s v r i i Dine una SREBP2 3.32 3.50 3.24 3.35 SREBPla/c 11.05 10.90 10.28 10.10 PPARo 5.58 5.28 5.48 5.90 PXR 6.74 7.10 6.78 7.10 FABP. FabOl 14.47 14.61 14.45 14.57 FABP. Fabo2 11.78 12.22 11.40 10.75 FABP. FabD4 9.16 10.29 9.76 10.86 FABP. Fabo6 2.32 2.40 2.36 2.16 FABP. Fabo7 7.5? 5.71 7.29 6.37 FATP. Slc27al 2.82 5.01 3.07 4.35 FATP. Slc27a2 13.86 14.06 14.00 13.98 FATP. Slc27a3 FATP. Slc27a4 4.48 measured, biut not ex Passed 6.20 1 4.47 5.99 FATP. Slc27a5 13.16 13.06 1 13.16 13.01 CD36 9.45 11.23 1 10.15 11.25 LDLR PCSK9 6.51 5.42 7.11 6.14 1 6-5? j 5.31 6.70 5.91 Fabp9 i measured, u u i n u i bamiesseu 3.09 10.03 6.18 7.41 14.55 10.64 10.31 2.22 7.07 4.04 13.96 m 1.13 -1.11 -1.23 5.53 13.06 11.22 6.21 4.86 i 0.165 0.350 0.256 0*010 0.012 0.006 0.000 0.412 0.000 0.000 0.001 9E 0000 ___ 2 J 8 ___ 0.000 0.016 0.110 107 1.03 -1.02 -1.30 1.51 1.03 -1.16 1.20 1.10 - 0.552 1 1.02 1 0.400 1 -1.17 1 0.077 0 .0 2 5 hmmmammm o.ooo rim w rirfin i'ffiH o.ooo 0.619 1.25 i o .i7 9 0.042 1 0.622 _ M B '. 1 0.0 0 6 0.000 1 0.S73 . L 0 7 *... m 0 .0 4 8 1 1706 1 0.087 1 0.069 r i u r j l M M 0 .0 0 0 HllifMWffMT'iiTIIII 0 .0 0 0 1 0.077 0.000 0.000 0.643 1 -1.12.... I 0.199 -1.08 0.303 0.546 H H m 0.004 -1.35 0.169 0.414 0.000 o.ooo 1 0.060 1 0.043 0.683 W L ilE i-fa ti 0.001 0.672 1 1.14 0.636 1 1.40 0.047 0.000 0.234 0.172 -1.08 -1.23 -1.48 0.008 0.000 0.137 0.142 IVLDL aasem blaoe/form ation ADOB 14.19 14.15 14.18 14.09 AdoBEC 6.06 6.06 6.64 6.27 MTTP 8.67 9.18 8.91 9.02 Aoobec2 measured, but not exoressed Aoobec3 measured, but not exoressed Aoobec4 measured, but not exoressed 14.04 6.10 8.96 1[ 11 -11,'0002 11 * V " p ? - '? - -." i 0.374 0.515 0 000 T V .' IpTexcratlon IMDR2 10.85 I 11.20 I 11.12 I 11.05 I 11.05 [-- j S lg g J 0.672 1___ lL 2 Z ___ 1 0.073 0*045 ____ L I S ___ I 0.174 l____ L 2 2 ___ 1 nnn 1 0.413 0 020 ; ' ' .............. 1 V E 2: I 1.20 I 0.079 I 1.14 I 0.051 I 1.14 I B-oxidation Genes involved in p-oxidation of fatty acids (CPTla and lb , ACO, enoyl CoA hydratase, thiolase (acetyl-Coenzyme A acyltransferase) and ACS were increased with fenofibrate, PFHS and PFOS. Fatty acid uptake, binding Genes involved in fatty acid uptake and binding, most prominently CD36 and FATP, and to a lesser extent FABP genes were increased with fenofibrate, PFHS and PFOS. Triglyceride uptake The major receptor involved in uptake of VLDL remnants and LDL, LDLR, was increased with fenofibrate. PFBS showed no effect on genes involved in triglyceride metabolism except for mild increases in p-oxidation genes enoyl CoA hydratase and ACS and fatty acid transporter CD36. 86 p. 88 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Cholesterol metabolism Uptake The major receptor involved in uptake of VLDL remnants and LDL, LDLR, was increased with fenofibrate, in line with decreased liver cholesterol levels. Synthesis and storage In line with decreased liver cholesterol levels also cholesterol synthetic genes HMGCoA synthase and reductase and squalene synthase were increased with fenofibrate. HMGCoA synthase and squalene synthase were also increased with PFHS. Cholesterol esterification genes ACATI and ACAT2 were increased with fenofibrate, PFHS and PFOS. Bile acid metabolism and biliairy cholesterol excretion The gene coding for the major rate-limiting enzyme in the bile acid synthetic pathway, CYP7al, was strongly decreased with PFHS and PFOS. Also genes involved in bile acid uptake, NTCP, and biliairy excretion, BSEP, were decreased. Genes involved in biliairy cholesterol excretion, ABCg5/g8 were decreased by PFHS and PFHS. Inhibition of cholesterol metabolism and excretion may form an explanation for increased hepatic cholesterol levels found with PFOS. PFBS showed no effect on genes involved in cholesterol metabolism except for a decrease in SREBPla, which is not understood well. However, regulation by SREBPla/c takes place on the protein level, which was not measured. Table 4.3.16b Gene expression data of cholesterol metabolism pathway with transcription factors and genes involved 1P ath w ay 1Icommon nam a 11------------- A veraae ex oression (2 lo o ) _________ _________________ 1Control 1 Fenofibrate 1 PFBS 11 PFHS |1 PFOS ICholesterol Icholesterol synthesis, storace, uotake and m etabolism SREBP2 3.32 3.50 3.24 3.35 3.09 LXRa 6.77 6.76 6.77 6.52 6.42 SREBPla/c 11.05 10.90 10.28 10.10 10.03 1 Fenofibrate vs control 1Fold chanoe 1 Q-value 1i 1.13 -1.01 -1.11 0.165 0.505 0.350 1 PFBS vs control 1 PFHS vs Control 1 PFOS vs Control 1 Fold chanae 1a-value 1Fold chance 11Q-value 1Fold change 1 a-value 1 -1.05 -1.00 0.552 1____ L 0 2 ___ I1 0.400 1 0.692 0.025 E tn a 10 .0 0 0 1 -1.17 i 11 0.077 1 10 .0 0 0 synthesis ACLY HMG CoA reductase HMG CoA svnthase sauateen svnthase HMGCS 11.10 7.69 12.45 8.64 storace ACATI ACAT2 11.40 9.41 uotake SREBP2 SREBPla/c LXRa lxrE LDLR PCSK9 Nn Pr wT1i .HlinUcali 1 3.32 11.05 6.77 4.48 6.51 5.42 1 11.02 10.52 11.24 8.48 7.59 8.39 12.83 12.50 12.41 9.50 8.31 9.47 not measured (not on array) 11.88 10.33 11.32 11.63 9.27 10.23 3.50 3.24 3.35 10.90 10.28 10.10 6.76 6.77 6.52 4.33 4.26 4.17 7.11 6.53 6.70 6.14 5.31 5.91 i ivk i iisogui cu vi iv i v i i o n o r i 9.79 7.33 12.53 8.53 -1.05 * * 4- 1 0.476 0.039 0.000 0.003 - - ' " J; y ` ;r 1____________ 11.54 9.37 0.000 0.004 ---------- 1 3.09 10.03 6.42 4.37 6.21 4.86 l 1.13 -1.11 -1.01 1.64 0.165 0.350 0.505 0.271 0.016 0.110 -1.49 -1.07 1.03 -1.25 3i 0.292 1 ' M l 1 0.366 0.629 0.045 0.573 1___ 0 3 ___ I 0.337 0.342 0.003 l x ;v. im m s m i ^ ^ m n j -1.28 1.05 -1.08 0.003 0.204 0.244 0.360 -1.06 -1.10 -1.00 -1.16 1.02 -1.08 0.370 ?gff3 U pMM? 0 .0 0 2 0.569 * - 0.006 0.034 0.429 ................................1 0.552 1 1.02 1 0.400 1 -1.17 1 0.077 0 .0 2 5 m w m n a m o.ooo ib m m -- m o.ooo ew aa H 'ii m 0 636 [ |g jg f m tab o llm LXRb CAR 4.48 7.46 6.76 4.33 4.26 7.63 6.52 EFFE! bsep ntcg^ ewretlwT LXRb ABCG5 5.01 11.12 10.67 5.61 11.88 3.72 10.40 11.80 10.32 11.77 11.00 0.242 ` 1.00 -1.16 87 p. 89 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 HDL metabolism Synthesis, maturation and remodeling Genes involved in HDL synthesis, apoAl the major protein of HDL, and HDL maturation, ABCal and LCAT, were decreased by PFHS and PFOS. PLTP a gene involved in remodeling of HDL, leading to larger particles was increased by PFHS and fenofibrate. Uptake The major gene involved in uptake of HDL-cholesterol esters, SR-B1, was decreased by fenofibrate, PFHS and PFOS. The latter mechanism together with the strong decrease in CETP activity is responsible for the increased HDL levels with fenofibrate. However, this mechanism cannot explain the strong decrease in HDL levels observed with PFHS and PFOS. The decreased HDL levels most likely result from decreased HDL synthesis and maturation (decreased gene expression of apoAl, ABCal and LCAT). PFBS showed no effect on genes involved in HDL metabolism, in line with unchanged HDL levels Table 4.3.16c Gene expression data of HDL metabolism pathway with transcription factors and genes involved Pathway common name HDL meta holism HDL formation LXRa LXRb PPARa PXR AooAl APOA2 Averaoe ex pression (2 lo a ) 1 Fenofibrate vs control Control 11 Fenofibrate 11 PFBS I1 PFHS |1 PFOS 1Fold chanoe 1 q-value i 6.77 4.48 11.03 6.74 14.38 14.52 6.76 4.33 11.33 7.10 14.34 14.55 6.77 4.26 10.90 6.78 14.25 14.45 6.52 4.17 10.54 7.10 13.96 14.48 i 6.42 4.37 10.55 7.41 13.85 14.50 1.01 -1.11 1.23 -1.03 1.03 0.505 0.271 0.055 O.OIO 0.365 0.310 1 PFBS vs control PFHS vs Control PFOS vs Control 1 Fold chanqe 1q-value Fold chanqe 1 q-value Fold chanqe 1 q-value 1 -1.00 -1.16 -1.10 1.03 -1.10 -1.0S 0.692 1 -1-19 - 1 0-039 1 0.008 0.304 1 - --1-24. 1 0 -0 4 6 11___ i 0 ___ I 0.277 ESSI *1 *1 n ro a 0.148 0.289 1 -1.02 I 0.270 I1 -i.o i *1 *1 1I 0.373 P a th w ay HDL maturation LXRa LXRb PPARa ABCA1 LCAT 6.77 4.48 11.03 5.14 10.96 6.76 4.33 11.33 5.06 10.82 6.77 4.26 10.90 5.16 10.99 6.52 4.17 10.54 4.95 10.46 6.42 4.37 10.55 5.01 10.55 -1.01 -1.11 1.23 -1.06 -1.12 0.505 0.271 0.055. 0.244 0.080 IHDL m odellina/destabilisation LXRa 6.77 6.76 6.77 6.52 LXRb 4.48 4.33 4.26 4.17 PLTP 7.65 9.38 7.56 8.85 endothelial lioase 2.90 3.41 2.70 2.85 HL 10.82 10.42 10.60 10.05 GPIHBP1 7.41 7.52 7.09 7.66 CETP not measured (not on array) 6.42 4,37 8.05 2.65 10.33 7.85 -1.01 ___ i l l ___ 1 1.08 i n 0.505 0.271 0.000 0.045 0.000 0.344 HDL uptake LXRa LXRb PPARa FXR PXR SRB1 ATPsvnth common name 6.77 6.76 6.77 1 6.52 6.42 -1.01 0.505 4.48 4.33 4.26 1 4.17 4.37 -m i 0.271 11.03 9.78 11.33 9.82 10.90 1 10.54 10.S5 9.77 10.04 10.06 1.23 1 nj 0.055 0.436 6.74. 7.10 6.78 1 7.10 7.41 O.OIO 9.50 13.07 8.99 13.20 9.23 8.29 8.68 12.96 I 13.13 13.05 1.10 O .O O l 0.064 Averaoe ex pression>i2 lo g ) 11 Fenofibrate vs control Control 1 Fenofibrate 1 PFBS 1 PFHS 1 PFOS I1Fold chanqe !1 q-value -1.00 -1.16 -1.10 1.01 1.01 0.692 0.304 . 0.453 0.635 0.669 I 0.039 I n rm 0.277 0.003 1 0 -0 2 4 11___ 0 9 ___ I 0.092 'T 'T 'T 'I 0.000 i -1.00 -1.16 -1.07 -1.15 -1.17 -1.25 *..........- 0.692 M 0.304 m 0.629 3 3 B B 5 0.457 -1.04 0.081 0.189 1.19 0.039 0.046 -1.08 0.002 1.31 0.398 -1.19 o.ooo H H H 0.103 9H U TM 0.008 0.277 0.171 0.185 f l 0.000 0.012 S S P T i -1.00 -1.16 -1.10 -1.01 1.03 -1.20 -1.07 0.692 0.304 0.453 0.676 0.622 0.168 0.272 1 0 .0 3 9 p-- I 0.008 1 0 0 4 6 1 -1.08 1 0.277 . l V . 0.002 0.003 1 M K S . 1 0.0 3 5 I I H a E u ! ...... 1.38 ... 0 .0 0 6 0.028 0.000 1 0 .0 0 0 p ijk ia iie lii__a-| 0 .0 0 0 1.05 1 0.203 1 -lx ii ] 0.424 1 PFBS vs control 1 PFHS vs Control 1 PFOS vs Control 1 Fold chanqe 1q-vaiue 1Fold chanqe T q-value 1 Fold chanqe 1 q-value Table 4.3.16d Safetyand tran scrip tion factors Isafetv IALAT 9.58 transcription fact ors LXRa 6.77 LXRb 4.48 PPARa 11.03 PPARa 5.S8 CAR 7.46 FXR 9.78 PXR 6.74 PGClaloha 6.40 PGClbeta 7.00 PPARd CYP3A11 13.53 9.64 6.76 4.33 11.33 5.28 7.86 9.82 7.10 6.50 6.84 13.39 Gene expression data of transcription factors and safety parameters 9.25 9.57 9.80 6.77 4.26 10.90 5.48 7.63 9.77 6.78 6.21 6.70 13.51 6.52 4.17 10.54 5.90 7.44 10.04 7.10 5.01 6.53 13.75 6.42 4.37 10.55 6.18 8.32 10.06 7.41 5.12 6.55 13.73 1.04 -1.01 -1.11 1.23 -1.23 1.32 1.03 0.11 -0.16 -1.10 88 0.368 0.505 0.271 0.055 0.256 0.172 O0..4O3IO6 0.436 0.405 0.100 0.040 -1.01 0.441 - 4 i l . 7 - . - l 0.039 -1.00 -1.16 -1.10 -1.07 1.13 -1.01 1.03 -0.18 -0.30 -1. 0.692 0.304 0.453 0.619 0.546 0.676 0.622 0.547 0.458 0 651 1.25 -1.01 -0.47 1 .1 7 ,. 0.039 0.046 0.002 0.179 0.443 0.035 0.006 0.000 0.119 0007 -1.08 0.008 0.277 0.003 0.042 0.007 00 ..00 02 08 0.000 . V-0.44 " - * 0.139 145 0.013 p. 90 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 5 Conclusions and comments In appendix XXI a table is included, in which the effects PFBS, PFHS, PFOS an fenofibrate on all measured parameters (except for the micro-array data) are summarized. Mechanism of action of PFBS PFBS reduced plasma cholesterol and triglyceride levels by about 25% and 45%, respectively. The data from physiological experiments indicate that the decreases in lipid levels are caused by increased clearance of VLDL-TG and VLDL-CE and mildly reduced VLDL-partlcle production. PFBS showed no effect on genes involved in triglyceride metabolism except for mild increases in poxldatlon genes enoyl CoA hydratase and ACS and fatty add transporter CD36. PFBS also showed no effect on genes involved In cholesterol metabolism except for a decrease in SREBPla, which Is not understood well. However, regulation by SREBPla/c takes place on the protein level, which was not measured. PFBS had no effect on HDL-cholesterol and apoAl levels. PFBS showed no effect on genes Involved In HDL metabolism, in line with the unchanged HDL levels. PFBS mildly Increased liver weight, but had no effect on ALT and development of hepatosteatosls. Based on mRNA signals PFBS appears to have mild PPARo-agonistlc activity (p-oxldatlon Increased and liver size increased) The present data indicate that PFBS has no Increased CVD risk profile. Mechanism of action of PFHS PFHS reduced plasma cholesterol and triglyceride levels by about 60% and 75%, respectively. The data from physiological experiments supported by hepatic mRNA levels Indicate that the decreases In lipid levels are caused by increased lipolysis and clearance of VLDL-TG and VLDL-CE, and strongly reduced VLDL-TG and VLDL-partlcle production. In line with the higher lipolytic activity In plasma, the hepatic mRNA signal for LPL (4.3-fold) was Increased. Although a major regulator of fatty acid synthesis, SREBPlc, was decreased with PFBS, PFHS and PFOS, several genes in the fatty add synthesis pathway were increased, suggesting that regulation does not take place via activation of LXR but probably via PXR. Several ACS genes, SCD2 and DGAT1 were increased with PFHS. Increased hepatic fatty acid synthesis together with reduced VLDL-TG secretion may form an explanation for the accumulation of triglycerides in the liver. In addition, Increased uptake and binding of fatty adds (increased mRNA levels of CD36 and FATP, and to a lesser extent FABP genes) which result from enhanced lipolysis may contribute to development of hepatosteatosls. This occurred, despite the observation that genes involved in 13oxidation of fatty adds (CPTlb, ACO, enoyl CoA hydratase, thlolase (acetyl-Coenzyme A acyltransferase) and ACS were increased with PFHS. PFHS strongly decreased HDL-cholesterol (about -75%) and apoAl (about -75%) levels. Although HDL catabolic rate and the major gene involved In uptake of HDL-cholesterol esters, SR-B1 were strongly decreased, we conclude that based on mRNA signals PFHS reduces HDL levels by downregulation of apoAl synthesis and HDL maturation (ABCal, LCAT). The latter adverse changes are most likely the result of PXR-agonlstic activity (24). Increased remodeling (PLTP) and decreased uptake (SR-B1) are suggested to be responsible for the formation of larger HDL particles. PFHS increased liver weight, ALT, and resulted In hepatosteatosls, as observed by biochemical and histological measures. Based on mRNA signals PFHS has strong PPARo-agonistlc (lipolysis Increased, p-oxidation Increased, FA uptake increased, liver size increased) and PXR-agonistlc activity (FA uptake Increased, FA synthesis Increased and HDL synthesis and maturation decreased, liver size increased). 89 p. 91 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Mechanism of action of PFOS PFOS reduced plasma cholesterol and triglyceride levels by about 65% and 70%, respectively. A similar mechanism of action Is active with PFOS as with PFHS. The data from physiological experiments supported by hepatic mRNA levels Indicate that the decreases in lipid levels are caused by Increased lipolysis and clearance of VLDL-TG, and strongly reduced VLDL-TG and VLDL-particle production. In line with the higher lipolytic activity in plasma, the hepatic mRNA signal for LPL (2.1-fold) was increased. Although a major regulator of fatty add synthesis, SREBPlc, was decreased with PFBS, PFHS and PFOS, several genes in the fatty acid synthesis pathway were Increased, suggesting that regulation does not take place via activation of LXR but probably via PXR. Several ACS genes, SCD2 and DGAT1 were increased with PFOS. Increased hepatic fatty acid synthesis together with reduced VLDL-TG secretion may form an explanation for the accumulation of triglycerides in the liver. Also Increased uptake and binding of fatty acids (increased mRNA levels of CD36 and FATP, and to a lesser extent FABP genes) which result from enhanced lipolysis may contribute to development of hepatosteatosls. This occurred, despite the observation that genes involved in p-oxidatlon of fatty acids (CPTlb, ACO, enoyl CoA hydratase, thiolase (acetyl-Coenzyme A acyltransferase) and ACS were increased with PFHS. PFOS increased mRNA levels of the cholesterol synthesizing enzymes HMGCoA synthase and squalene synthase and cholesterol esterification genes ACAT1 and ACAT2. Moreover, the gene coding for the major rate-limiting enzyme in the bile acid synthetic pathway, CYP7al, and genes involved in biliairy cholesterol excretion, ABCg5/g8 were decreased by PFOS. Inhibition of cholesterol metabolism and excretion may form an explanation for increased hepatic cholesterol levels found with PFOS. PFOS strongly decreased HDL-cholesterol (about -65%) and apoAl (about -80%) levels. Although HDL catabolic rate and the major gene Involved In uptake of HDL-cholesterol esters, SR-B1 were decreased, we conclude that based on mRNA signals PFOS reduces HDL levels by down-regulation of apoAl synthesis and HDL maturation (LCAT). The latter adverse changes are most likely the result of PXR-agonistlc activity (24). PFOS increased liver weight, ALT, and resulted In pronounced hepatosteatosls and liver cholesterol accumulation, as observed by biochemical and histological measures. Based on mRNA signals PFOS has strong PPARa-agonlstic (lipolysis increased, p-oxidation increased, FA uptake increased, liver size increased) and PXR-agonlstic activity (FA uptake increased, FA synthesis increased and HDL synthesis and maturation decreased, liver size increased). Involvement of other nuclear transcription factors in the regulation of lipid and lipoprotein metabolism by PFHS and PFOS. Involvement of CAR and LXR In the changes In lipid and lipoprotein metabolism caused by PFHS and PFOS cannot be fully excluded, but is less likely. Little is know about the role of CAR in lipid metabolism. CAR has been shown to decrease p-oxidation genes as CPT1 and enoyl CoA hydratase. The latter genes were, however, Increased in the present experiments. LXR increases fatty acid synthesis by induction of SREBPlc expression, which was 2-fold decreased, however. In addition, LXR Induces expression of CETP mRNA, whereas in the present experiments a decrease In CETP activity was found. It cannot be excluded that this Is caused by a strongly decreased acceptor pool for CE transfer. Involvement of RXR in the observed effects cannot be excluded, since RXR forms a heterodimer together with a larger number of nuclear transcription factors like PPARa, PXR and LXR. However, direct activation of RXR, for instance with bexarotene (25) leads to opposite effects with increased levels of triglycerides and apoB-containlng lipoproteins (25). Using the mlceroarray database, we suggest to study the involvement of the above and other transcription factors (ArH) involved in the metabolism of xenobiotics in more detail with respect to changes in other metabolic processes and pathways like glucose metabolism, inflammation and immuneresponse. 90 p. 92 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Mechanism of action of fenofibrate Fenofibrate reduced plasma cholesterol and triglyceride levels by about 40% and 70%, respectively. The data from physiological experiments supported by hepatic mRNA levels Indicate that the decreases in lipid levels are caused by strongly Increased lipolysls and clearance of VLDLTG and VLDL-CE, despite the increased VLDL-TG production rate. In line with the higher lipolytic activity In plasma, the hepatic mRNA signal for LPL (4.6-fold) was increased. LDLR mRNA as marker for Increased uptake of VLDL remnant particles was enhanced by 1.5-fold. Several ACS genes and DGAT1 mRNA, involved in fatty acid and triglyceride synthesis, were increased with fenofibrate, whereas DGAT2 activity primarily responsible for VLDL secretion tended to be Increased by 73% (p=0.056). In addition, genes involved In fatty acid uptake and binding, most prominently CD36 and FATP, and to a lesser extent FABP genes were increased. Several genes involved in p-oxidation of fatty acids (CPTla and lb, ACO, enoyl CoA hydratase, thiolase (acetyl-Coenzyme A acyltransferase) and ACS were strongly Increased with fenofibrate. Taken together the net effect of these changes in metabolic pathways in fatty metabolism is no effect of fenofibrate on liver triglyceride levels. In conclusion, fenofibrate paradoxically Increases VLDL-TG production despite reducing plasma TG, which may be caused by enhanced hepatic free fatty acid uptake resulting from strongly accelerated peripheral LPL-medlated lipolysls of VLDL or by Increased de novo hepatic TG synthesis. Fenofibrate reduced liver cholesterol content resulting In enhanced LDLR mRNA levels and mRNA levels of cholesterol synthetic enzymes (HMGCoA synthase and reductase and squalene synthase) and decreased mRNA levels of cholesterol metabolizing enzymes (CYP7al), the latter leading to reduced fecal bile acid excretion. Fenofibrate increased HDL-cholesterol (+50%) and formation of large HDL-1 particles, and had no effect on apoAl. Although the major gene Involved In uptake of HDL-cholesterol esters, SR-B1, was decreased, the catabolic rate was not significantly decreased. Fenofibrate strongly decrease in CETP activity, which was found majorly responsible for the increased HDL levels upon treatment with fenofibrate and PPARa,y-agonists (14,26). Fenofibrate increased liver weight, without effects on ALT and hepatosteatosis, and decreased liver cholesterol content. Based on mRNA signals fenofibrate has strong PPARa-agonistic activity (lipolysls increased, FA uptake increased, p-oxidation increased; HDL remodeling decreased). 91 p. 93 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 6 References 1. Van den Maagdenberg AMJM, Hofker MH, Krimpenfort PJA, de Bruijn IG, van Vlijmen B, van der Boom H, Havekes LM, Frants RR. Transgenic mice carrying the apolipoprotein E3Leiden gene exhibit hyperlipoproteinemia. J Biol Chem 1993; 268: 10540-10545. 2. Van Vlijmen B, van den Maagdenberg AMJM, Gijbels MJJ, van der Boom H, HogenEsch H, Frants RR, Hofker MH, Havekes LM. Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice'. J Clin Invest 1994; 93: 1403-1410. 3. Groot PHE, van Vlijmen BJM, Benson GM, Hofker MH, Schiffelers R, Vidgeon-Hart M, Havekes LM. Quantitative assessment of aortic atherosclerosis in apoE3Leiden transgenic mice and its relationship to serum cholesterol exposure. Arterioscler Thromb Vase Biol 1996;16:926-933. 4. Kleemann R, Princen HMG, Emels 33, Jukema 3W Fontijn RD, Horrevoets AJG, Kooistra T, Havekes LM. Rosuvastatin reduces atherosclerosis development beyond and independent of its plasma cholesterol-lowering effect in apoE*3Leiden transgenic mice: Evidence for anti inflammatory effects of rosuvastatin. Circulation 2003; 108: 1368-1374. 5. Delsing DJM, Jukema JW, van de Wiel MA, Emeis JJ, van der Laarse A, Havekes LM, Princen HMG. Differential effects of amlodipine and atorvastatin treatment and their combination on atherosclerosis in ApoE*3-Leiden transgenic mice. J Cardiovasc Pharmacol 2003; 42: 6370. 6. Verschuren L, Kleemann R, Offerman EH, Szalai AJ, Emeis JJ, Princen HMG, Kooistra T. Effect of low dose atorvastatin versus diet-induced cholesterol lowering on atherosclerotic lesion progression and inflammation in apolipoprotein E*3-Leiden transgenic mice.Arterioscler Thromb Vase Biol 2005; 25:1-7. 7. Delsing DJM, Post SM, Groenendijk M, Solaas K, vanderBoom H, vanDuyvenvoorde W, deWit ECM, Bloks VW, Kuipers F, Havekes LM and Princen HMG. Rosuvastatin reduces plasma lipids by inhibiting VLDL production and enhancing hepatobiliary lipid excretion in ApoE*3-Leiden mice. J Cardiovasc Pharmacol 2005; 45: 53-60. 8. Kooistra T, Verschuren L, van der Weij J, Koenig W, Toet K, Princen HMG, Kleemann R. Fenofibrate reduces atherogenesis in APOE*3Leiden mice: evidence for multiple anti atherogenic effects besides lowering plasma cholesterol Arterioscler Thromb Vase Biol 2006; 26: 2322-2330. 9. Van Vlijmen BJM, Mensink RP, v a n 't Hof HB, Offermans RFG, Hofker MH, Havekes LM. Effects of dietary fish oil on serum lipids and VLDL kinetics in hyperlipidemic apolipoprotein E*3-Leiden transgenic mice. J Lipid Res 1998; 39: 1181-1188. 10. Delsing DJM, Offerman EH, van Duyvenvoorde W, van der Boom H, de Wit ECM, Gijbels MJJ, Van der Laarse A, Jukema JW, Havekes LM, Princen HMG. The acyl-CoA:cholesterol acyltransferase (ACAT)-inhibitor avasimibe reduces atherosclerosis additionally to its cholesterol-lowering effect in apoE*3Leiden mice. Circulation 2001; 103: 1778-1786. 11. Volger OL, van der Boom H, de Wit ECM, van Duyvenvoorde W, Hornstra G, Plat J, Havekes LM, Mensink RP, Princen HMG. Dietary plant stanol esters reduce VLDL-cholesterol secretion and bile saturation in apoE*3Leiden transgenic mice. Arterioscler Thromb Vase Biol 2001; 21: 1046-1052. 12. Zadelaar ASM, Kleemann R, Verschuren L, de Vries-van der Weij J, van der Hoorn J, Princen HM, Kooistra T. Mouse models for atherosclerosis and pharmaceutical modifiers. Arterioscler Thromb Vase Biol 2007; 27: 1706-1721. 13. Westerterp M, Van der Hoogt CC, de Haan W, Offerman EH, Dallinga-Thie GM, Jukema JW, Havekes LM, Rensen PCN. Cholesteryl ester transfer protein decreases HDL and severely aggravates atherosclerosis in APOE*3Leiden mice. Arterioscler Thromb Vase Biol 2006; 26: 2552-2559. 14. Van der Hoogt CC, de Haan W, Westerterp M, Hoekstra M, Dallinga-Thie GM, Romijn JA, Princen HMG, Jukema JW, Havekes LM, Rensen PCN. Fenofibrate increases HDL cholesterol by reducing cholesteryl ester transfer protein expression. J Lipid Res 2007; 48: 1763-1771. 15. De Haan W, van der Hoogt CC, Westerterp M, Hoekstra M, Dallinga-Thie GM, Princen HMG, Romijn JA, Jukema JW, Havekes LM, Rensen PCN. Atorvastatin increases HDL cholesterol by reducing CETP expression in cholesterol-fed APOE*3-Leiden.CETP mice. Atherosclerosis 2008; 197: 57-63. 16. Van der Hoorn JWA, de Haan W, Berb^e JFP, Havekes LM, Jukema JW, Rensen PCN, Princen HMG. Niacin increases HDL by reducing hepatic expression and plasma levels of 92 p. 94 3M#Q3 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 cholesteryl ester transfer protein in APOE*3-Leiden.CETP mice. Arterioscler Thromb Vase Biol 2008; 28: 2016-2022. 17. De Haan W, de Vries-van der Weij J, van der Hoorn JWA, Gautier T, van der Hoogt CC, Westerterp M, Romijn JA, Jukema JW, Havekes LM, Princen HMG, Rensen PCN. Torcetrapib does not reduce atherosclerosis beyond atorvastatin and induces more pro-inflammatory lesions than atorvastatin. Circulation 2008; 117: 2515-2522. 18. Post SM, Groenendijk M, Solaas K, Rensen PCN, Princen HMG. Cholesterol 7a-hydroxylase deficiency in mice on an apoE*3Leiden background impairs very-low-density lipoprotein production. Arterioscler Thromb Vase Biol 2004; 23: 768-774. 19. Duivenvoorden I, Voshol PJ, Rensen PCN, Duyvenvoorde W, Romijn JA, Emeis JJ, Havekes LM, Nieuwenhuizen WF. Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent lever steatosis in apoE*3Leiden mice. Am J Clin Nutr 2006; 84: 312-321. 20. Post SM, de Crom R, van Haperen R, van Tol A, and Princen HM. Increased fecal bile acid excretion in transgenic mice with elevated expression of human phospholipid transfer protein. Arterioscler Thromb Vase Biol 2003; 23: 892-897. 21. Kleemann R, Verschuren L, van Erk MJ, Nikolsky Y, Cnubben NH, Verheij ER, Smilde AK, Hendriks HF, Zadelaar S, Smith GJ, Kaznacheev V, Nikolskaya T, Melnikov A, Hurt-Camejo E, van der Greef J, van Ommen B, Kooistra T. Atherosclerosis and liver inflammation induced by increased dietary cholesterol intake: a combined transcriptomics and metabolomics analysis. Genome Biol 2007; 8: R200. 22. Havekes LM, de Wit EC and Princen HM. Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity. Biochem J 1987; 247: 739-746. 23. Mensenkamp AR, van Luyn MJ, van Goor H, Bloks V, Apostel F, Greeve J, Hofker MH, Jong MC, van Vlijmen BJ, Havekes LM, Kuipers F. Hepatic lipid accumulation, altered very low density lipoprotein formation and apolipoprotein E deposition in apolipoprotein E3-Leiden transgenic mice. J Hepatol 2000 Aug; 33: 189-98. 24. De Haan W, de Vries-van der Weij J, Mol IM, Hoekstra M, Romijn JA, Jukema JW, Havekes LM, Princen HMG, Rensen PCN. PXR agonism decreases HDL levels in APOE*3-Leiden.CETP mice. Biochim Biophys Acta 2009; 1791: 191-197, 25. de Vries-van der Weij J, de Haan W, Hu L, Kuif M, Oei HL, van der Hoorn JW, Havekes LM, Princen HM, Romijn JA, Smit JW, Rensen PC. Bexarotene induces dyslipidemia by increased very low-density lipoprotein production and cholesteryl ester transfer protein-mediated reduction of high-density lipoprotein. Endocrinology 2009; 150: 2368-75. 26. Van der Hoorn, Jukema JW, Havekes LM, Lundholm E, Camejo G, Rensen PCN, Princen HMG. The dual PPARo/y agonist tesaglitazar blocks progression of pre-existing atherosclerosis in APOE*3-Leiden.CETP transgenic mice. Brit J Pharmacol 2009; 156: 10671075. 93 p. 95 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 7 Appendices Appendix I Body weight Study 1 Group Mouse# Cage# Eartaq Body weight (q) L R week 0 week 1 week 4 1 1 2 1 29.7 31.0 32.4 2 1 0 1 25.5 25.9 26.8 3 2 1 1 29.7 31.2 32.2 4 2 2 1 26.9 28.3 29.1 5 2 1 2 25.3 27.0 28.1 6 3 1 1 25.5 26.7 28.5 7 3 2 1 23.9 25.4 26.4 8 3 1 2 23.6 24.4 25.2 26.3 2.4 27.5 2.5 28.6 2.6 fen o fib ra te 9 4 1 0 28.5 28.0 28.9 fen o fib ra te 10 4 1 1 29.6 30.2 30.1 fen o fib ra te 11 5 1 1 26.5 26.3 26.7 fen o fib ra te 12 5 2 1 26.8 27.9 27.9 fen o fib ra te 13 6 0 2 30.0 30.0 30.0 fen o fib ra te 14 6 1 0 21.0 22.8 23.0 fen o fib ra te fen o fib ra te AVERAGE 15 16 7 7 1 0 24.6 27.3 28.2 1 2 27.4 28.2 29.4 26.8 27.6 28.0 S D 2.9 2.3 2.3 17 8 1 0 26.1 26.5 26.7 18 8 0 1 29.6 31.0 31.9 19 8 2 0 26.2 27.7 27.5 20 9 21 9 1 0 25.0 25.8 26.4 1 1 24.8 26.0 27.1 22 10(A) 0 0 24.3 26.1 27.3 23 10 0 24 10 2 1 26.8 28.6 29.3 1 25.6 26.0 27.7 26.1 1.7 27.2 1.8 28.0 1.8 PFHS-' 25 11 0 0 27.0 27.7 27.5 PFHS. 26 11 1 1 27.6 28.5 29.8 PFHS 27 11 1 2 24.9 26.2 27.4 .PFHS - 28 12 0 2 28.5 29.8 29.4 PFHS 29 12 1 2 24.4 25.6 26.8 _ _ PFHS. 30 13 1 2 30.7 31.0 31.4 PFHS 31 13 0 0 25.8 27.5 27.8 : PFHS. . 32 13 1 0 29.6 31.8 32.5 2SI1SI3 m w ^ iw w 27.3 2.2 28.5 2.2 P F O S 33 14 0 1 30.8 31.8 PF05 34 14 1 2 26.3 27.7 P F O S 35 15 1 1 25.6 25.8 P F O S 36 15 1 2 27.4 27.9 P F O S 37 15 0 0 25.4 26.5 P F O S 38 16 2 2 31.7 31.7 P F O S 39 16 0 1 23.0 24.8 P F O S 40 16 1 1 27.0 27.9 AVERAGE 27.2 28.0 S D 2.9 2.6 * Week 5: mouse 22 put in separate cage (10A), because of fighting wounds 29.1 2.1 31.8 28.2 26.5 27.6 25.7 31.2 25.4 29.0 28.2 2.4 week 6 32.2 25.9 33.2 30.6 29.9 27.7 26.6 25.6 29.0 2.9 29.4 31.0 26.6 28.8 32.2 23.7 28.9 29.6 28.8 2.6 27.5 32.0 28.4 26.7 26.7 27.4 30.2 28.1 28.4 1.8 27.1 29.1 27.0 28.8 27.4 31.1 28.2 32.6 28.9 2.0 31.6 28.0 26.7 27.5 26.8 29.6 25.9 28.3 28.1 1.8 1 94 p. 96 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 StuffY 2 Group Mouse# 1 2 3 4 5 6 Cage# 1 1 1 2 2 2 fe n o fib ra te fe n o fib ra te fe n ofib rate fe n o fib ra te fe n o fib ra te fcn o fib ra te AVERAGE SD 7 8 9 10 11 12 13 14 15 16 17 18 3 3 3 4 4 4 5 5 5 6 6 6 1W.'MHfr * ..i ' * PPOS pros PFOS PROS PFC!S PFOS AVERAGE SD 19 20 21 22 23 24 25 26 27 28 29 30 7 7 8 8 9 9 10 10 11 11 12 12 Eartaa LR 10 02 12 10 20 12 01 11 20 01 02 20 00 12 01 01 02 21 00 11 21 02 11 02 00 01 10 21 11 02 week 0 24.4 25.7 26.1 27.6 27.4 27.3 26.42 1.25 24.3 27.2 26.8 27.4 25.4 25.4 26.08 1.23 27.0 29.8 25.3 25.8 23.2 28.3 26.57 2.33 29.5 26.8 23.3 26.0 24.9 29.2 26.62 2.42 24.8 27.8 25.7 28.5 26.5 23.7 26.17 1.81 Bodv weiaht fa'1 week 1 25.1 25.6 26.8 27.5 27.6 27.4 26.67 1.07 24.6 27.7 26.9 27.4 25.6 25.7 26.32 1.21 26.9 29.9 25.2 25.7 23.4 28.4 26.58 2.33 29.6 26.7 23.6 27.0 24.8 29.6 26.88 2.45 25.1 27.7 26.1 28.3 26.6 23.9 26.28 1.63 week 4 26.3 26.7 27.5 29.3 28.6 29.5 27.98 1.35 26.3 29.9 29.1 28.6 26.0 27.6 27.92 1.56 28.4 31.4 27.1 27.0 24.4 28.5 27.80 2.30 29.3 27.7 25.1 28.4 27.1 31.7 28.22 2.22 28.7 28.5 26.8 28.0 26.4 24.5 27.15 1.59 95 p. 97 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 3 Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 fe n o f'lira te ie n o fib ra re fenonbrate fen o M x a te fenobbrate fenoEbrate fenobbrate AVERAGE SD 8 9 10 11 12 13 14 15 16 17 18 19 20 21 4 4 5 5 5 6 6 1 2 1 1 2 0 1 TWmS 22 23 24 25 26 27 28 0 2 i 0 1 2 0 p ro s p ro s PFOS p ro s PFOS p ro s p ro s AVERAGE SD 29 30 31 32 33 34 35 0 1 0 0 1 0 0 Eartaq LR 01 12 01 20 02 00 20 21 12 20 10 11 01 21 17 17 07 08 18 19 29 2 10 i 10 0 11 2 11 1 12 0 12 2 12 0 13 0 13 2 13 1 14 2 14 1 15 2 15 week 0 26.9 29.4 26.2 29.3 26.2 27.3 27.7 27.6 1.3 26.7 25.4 30.6 25.2 31.4 23.9 25.7 27.0 2.9 25.1 29.0 31.0 28.6 26.6 26.3 26.5 27.6 2.0 26.7 29.0 28.6 26.2 26.1 27.4 27.6 27.4 1.1 27.6 26.0 26.4 28.4 26.3 28.9 25.9 27.1 1.2 Body weiQht (q) 1 week 1 week 4 26.9 28.4 26.8 30.1 29.2 29.3 28.1 32.9 26.3 28.0 28.1 27.8 29.3 29.9 29.8 1.3 1.6 27.1 28.1 25.4 27.1 30.3 26.0 32.6 IM iW -- -- --1 32.1 24.1 25.6 32.1 26.2 28.1 27.2 2.9 26.2 29.0 2.7 ^ T r ^ i}r r r , i s i ! r-s p 7Wk 30.0 31.7 31.7 34.5 29.0 29.9 26.8 29.1 26.0 26.5 25.8 28.8 27.9 30.1 2.3 2.7 27.2 28.9 27.8 28.3 26.2 26.4 27.2 27.7 30.0 29.2 27.3 28.0 28.7 sacftTced 'tor HDL Isottttn 27.3 28.7 0.8 0.9 27.7 27.6 28.1 sacrificed W W f c W m * - . 26.3 27.6 28.3 29.1 25.9 27.4 29.6 29.0 26.2 28.5 27.4 28.3 1.3 0.7 96 p. 98 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix II Tissue weight Study 1 Body weight, liver weight and perigonadal fat weight at sacrifice. Group Mouse# 1 2 3 4 5 6 7 8 Cage# 1 1 2 2 2 3 3 3 Eartaq LR 21 01 11 21 12 11 21 12 fen o fih ra te fen o fib ra te fen o fib ra te fen o fih ra te fen o fib ra te fen o fib ra te fen o fib ra te fen o fib ra te AVERAGE SD 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 4 4 5 5 6 6 7 7 8 8 8 9 9 10(A) 10 10 1 1 1 2 0 1 1 1 1 0 2 1 1 0 0 2 0 1 1 1 2 0 0 2 0 1 0 0 1 0 1 1 25 11 0 26 11 1 27 11 1 i bbSs 28 12 0 (AA:>J"0 29 12 1 |g |g f.y 30 13 1 j t l l l l j a a' U 31 13 0 mif s m w m4. 32 13 1 PEGS 33 14 0 PFOS 34 14 1 PFOS 35 15 1 PFOS 36 15 1 PFOS 37 15 0 PFOS 38 16 2 PFOS 39 16 0 PFOS 40 16 1 AVtRA G F SD Mouse 15: scabs on belly (from fighting?) Mouse 22: open wound at back from fighting Mouse 32: white spot on liver Mouse 38,39,40: pale liver 0 1 2 2 2 2 0 0 1 2 1 2 0 2 1 1 Body weight (g) 32.3 25.7 32.4 30.3 29.0 26.6 27.2 25.5 28.6 2.8 29.2 30.3 26.9 28.0 31.9 23.4 30.0 29.7 28.7 2.6 27.3 31.9 28.3 27.2 27.8 26.5 30.1 26.6 28.2 1.9 26.9 28.3 27.3 28.9 27.2 31.2 28.0 32.9 28.8 2.1 31.3 28.7 26.3 27.7 25.7 29.4 25.6 27.6 27.8 2.0 Liver weight Perigonadal fat weight (g) (g) 1.62 1.08 1.10 0.59 1.36 0.42 1.35 0.44 1.24 0.36 1.16 0.70 1.23 0.79 1.06 0.61 1.27 0.62 0.18 0.24 1.92 0.61 2.22 0.60 1.55 0.52 1.61 0.59 1.82 0.56 1.47 0.41 2.09 0.31 1.93 0.39 1.83 0.50 0.27 0.11 1.37 0.54 1.77 0.80 1.53 0.49 1.31 0.52 1.40 0.58 1.55 0.33 1.44 0.33 1.24 0.52 1.45 0.51 0.17 0.15 3.10 0.53 3.31 0.66 3.45 0.41 3.14 0.35 3.49 0.34 3.30 0.49 3.02 0.26 3.73 0.58 3.32 0.45 0.23 0.14 3.37 0.41 3.65 0.20 3.08 0.41 3.32 0.39 3.03 0.33 3.43 0.56 2.70 0.36 3.25 0.37 3.23 0.38 0.29 0.10 97 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Studv.2 Body weight, liver weight and perigonadal fat weight at sacrifice. Group Mouse# 1 2 3 4 5 6 Cage# 1 1 1 2 2 2 Eartaa LR 10 02 12 10 20 12 fcn ofib ratc fcn ofib ratc fcn ofib ratc fc n o ftb ra tc fcn ofib ratc fcn ofib ratc AVERAGE SD 7 8 9 10 11 12 13 14 15 16 17 18 30 31 32 40 40 42 50 5 .1 50 60 60 62 1 1 0 1 2 0 0 2 1 1 2 1 UMmmsm ES S ilS illi fW':?7TiGVA 19 20 21 22 23 24 7 7 8 8 9 9 0 1 2 0 1 0 PFOS 25 10 PFOS 26 10 PFOS 27 11 PFOS 28 11 PF O S 29 12 PF O S 30 12 0 0 1 2 1 0 AVERAGE SD Mouse 21: White spots in verHJuestonesinbiadder 0 1 1 2 1 2 0 1 0 l 1 2 Body weight (9) 25.7 26.2 26.8 29.2 28.2 29.9 27.7 1.7 26.1 29.3 28.7 29.3 26.6 27.7 28.0 1.4 28.1 31.3 27.4 26.9 26.3 28.2 28.0 1.8 29.3 27.2 25.2 28.2 27.0 32.2 28.2 2.4 28.5 28.4 26.8 28.3 27.2 26.0 27.5 1.0 Liver weight (g) 1.35 1.37 1.23 1.54 1.49 1.61 1.43 0.14 1.72 1.95 1.83 1.85 1.69 1.49 1.76 0.16 1.52 1.79 1.52 1.32 1.50 2.10 1.63 0.28 2.89 2.72 2.78 2.83 3.17 3.64 3.01 0.35 3.17 2.93 2.72 2.88 3.37 2.73 2.97 0.26 Perigonadal fat weight (g) 0.45 0.34 0.43 0.49 0.45 0.83 0.50 0.17 0.55 0.58 0.37 0.63 0.62 0.72 0.58 0.12 0.45 0.63 0.70 0.60 0.52 0.55 0.58 0.09 0.24 0.43 0.47 0.55 0.42 0.31 0.40 0.11 0.21 0.34 0.40 0.56 0.47 0.45 0.41 0.12 p. 100 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 3 Body weight, liver weight and perigonadal fat weight at sacrifice. Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 fe-'iofiiJ' -its 'enotio? ate fe io f i >r atr.' re infu.ri'fc fe n o fn va te renol'iD ; a te f e iofi i t 1 AVE: RAGE SD 8 9 10 11 12 13 14 15 16 17 18 19 20 21 4 4 5 5 5 6 6 i 2 1 1 2 0 1 i It PFHS".-.' PFHS pfh! . PFHS JPFHS PFHS AVERAGE 22 23 24 25 26 27 28 pros pros PROS p r o cf pros PFOS pros AVERAGE SD 29 30 31 32 33 34 35 Mouse 23: granular liver 0 2 1 0 1 2 0 0 1 0 0 1 0 0 Eartaa LR 01 12 01 20 02 00 20 21 12 20 10 11 01 21 i7 17 07 08 18 19 29 2 10 1 10 0 11 2 11 1 12 0 12 2 12 0 13 0 13 2 13 1 14 2 14 i 15 2 15 Body weight (9) 28.9 28.9 26.7 30.7 28.0 29.1 28.7 1.3 27.1 26.1 31.4 32.8 25.0 26.6 28.2 3.2 29.8 32.5 28.2 28.1 25.9 28.3 28.8 2.2 27.9 29.0 27.9 26.3 26.9 28.3 27.7 1.0 26.7 26.4 27.6 26.0 28.2 26.0 26.8 0.9 Uver weight Perigonadal fat weight (g) 1.61 1.83 1.53 1.69 (9) 0.74 0.46 0.58 0.74 * j.,; 1.56 1.46 0.38 0.43 1.61 0.13 0.56 0.16 1.98 1.93 2.07 2.12 1.69 1.85 0.37 0.33 0.71 -0.56 0.46 0.59 1.94 0.16 -- -- I 0.50 0.14 2.09 1.73 1.62 1.71 1.50 1.67 0.54 0.72 0.39 0.23 0.52 0.69 1.72 0.20 0.52 0.18 3.18 1.88 3.16 3.21 2.98 3.17 , 0.28 0.38 0.40 0.33 0.33 0.41 2.93 0.52 0.36 0.05 2.89 - * lil, 0.30 2.85 3.20 2.78 3.35 2.62 0.44 0.52 0.52 0.32 0.21 2.95 0.27 0.39 0.13 99 p. 101 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix III Study 1 Food intake Cage# 24 26 27 28 29 30 31 34 36 37 38 39 40 41a 41b 42 43 AVERAGE SD week-1-0 Food intake (q/mouse/dav) 2.65 2.91 3.09 2.52 2.70 2.69 2.60 3.09 2.63 2.78 3.12 2.88 2.71 2.78 2.54 2.62 2.68 2.8 0.2 Group# Cage# 1 2 3 fen ofibrate fen ofibrate fen ofibrate fen ofibrate AVERAGE SD 4 5 6 7 8 9 10 10A * . SD PFOS PFOS PFOS AVERAGE SD 1 11 12 13 14 15 16 Number of 1 Food intake (a/mouse/dav) mice week 0-1 week 3-4 week 5-6 2 2.74 2.73 2.87 3 2.90 3.21 3.46 3 2.49 2.47 2.52 2.7 2.8 2.9 0.2 0.4 0.5 2 2.94 3.28 3.39 2 2.46 2.98 2.94 2 2.67 3.33 3.22 2 2.76 3.58 3.63 2.7 3.3 3.3 0.2 0.2 0.3 3 2 3(2)* 1 3.15 2.55 2.95 2.98 2.85 3.09 3.00 2.80 4.08 3.60 2.9 3.0 0.3 0.1 3.4 0.6 3 2.42 2.62 2.70 2 2.75 2.96 2.88 3 3.18 3.25 3.27 2.8 2.9 0.4 0.3 2.9 0.3 2 2.96 3.48 3.38 3 2.85 2.76 2.67 3 2.62 2.79 2.73 2.8 3.0 0.2 0.4 2.9 0.4 * Week 5: mouse 22 put in separate cage (10A), because of fight wounds 1 100 p. 102 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 2 Cage# 105 107 108 109 110 111 112 113 114 129 130 AVERAGE SD week-1-0 Food intake (g/mouse/day) 2.73 2.83 2.74 2.83 2.83 2.88 2.75 2.71 2.89 2.84 2.84 2.8 0.1 Group# Cage# 1 2 fe n o fib ra te fe n o fib ra te AVERAGE Range 3 4 5 6 iT 'M I "1 r' >v , PFOS PFOS PFOS AVERAGE SD 7 8 9 10 11 12 Number of 1 Food intake (a/mouse/davl mice week 0-1 week 3-4 3 2.70 2.69 3 2.98 2.92 2.8 2.8 0.1 0.1 3 2.93 2.95 3 2.77 2.69 2.9 2.8 0.1 0.1 3 2.90 2.90 3 2.54 2.50 2.7 2.7 0.2 0.2 2 2.93 3.00 2 2.19 2.35 2 2.88 2.94 2.7 2.8 0.4 0.4 2 2.96 3.01 2 2.67 2.63 2 2.51 2.55 2.7 2.7 0.2 0.2 1 101 p. 103 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 SUidYS. Cage# 149 150 151 152 153 154 155 156 157 158 159 161 AVERAGE SD week-1-0 Food intake (g/mouse/day) 2.94 3.65 3.34 2.87 3.17 3.31 3.35 2.89 2.87 3.21 2.86 2.39 3.1 0.3 Group# Cage# 1 2 3 fen ofibrate fe n ofibrate fen ofibrate AVERAGE SD 4 5 6 7 8 9 i'-r.P E H S -.-i 1111111PFOS PFOS PFOS AVERAGE SD 10 11 12 13 14 15 Number of 1 mice 2 3 2 2 3 2 3 2 2 2 2 3 3 2 2 Food intake (a/mouse/dav) week 0-1 week 3-4 2.80 2.56 2.97 2.96 2.94 2.99 2.9 2.8 0.1 0.2 2.99 2.87 2.96 3.08 2.23 2.46 2.7 2.8 0.4 0.3 2.94 2.88 3.25 2.63 3.21 2.66 2.9 2.9 0.3 0.3 3.17 2.65 2.62 3.20 2.60 2.61 2.8 2.8 0.3 0.3 2.79 2.55 3.14 2.55 2.43 2.84 2.8 2.6 0.3 0.2 1 102 p. 104 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix IV Plasma cholesterol Study 1: Group Mouse# 1 2 3 4 5 6 7 8 Cage# 1 1 2 2 2 3 3 3 Eartaq L 2 0 1 2 1 1 2 1 R 1 1 1 1 2 1 1 2 fc n o f-b ra te fe n o fib ra tc fcn ofib ra tc fcn o fib ra te fe n o fib ra tc fe n o fib ra tc fc n ofib ra tc fen o fib ra tc AVERAGE SD 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 . ^ 25 26 27 28 29 30 31 32 4 4 5 5 6 6 7 7 8 8 8 9 9 10(A) 10 10 11 11 11 12 12 13 13 13 1 1 1 2 0 1 1 1 1 0 2 1 1 0 0 2 0 1 1 0 1 1 0 1 0 1 1 1 2 0 0 2 0 1 0 0 1 0 1 1 0 1 2 2 2 2 0 0 PFOS 33 14 0 1 PFOS 34 14 1 2 PFOS 35 15 1 1 PFOS 36 15 1 2 PFOS 37 15 0 0 PFOS 38 16 2 2 PFOS 39 16 0 1 PFOS 40 16 1 1 AVERAGE SD Plasma cholesterol (mmol/L) t=0 weeks t=4 weeks t=6 weeks 8.41 8.17 8.49 10.42 10.29 10.51 7.78 6.75 6.70 5.32 8.99 8.02 5.93 9.64 6.32 10.51 9.15 10.68 7.52 6.59 8.07 9.02 7.97 8.31 7.88 1.55 8.05 1.86 9.02 1.06 10.58 5.47 7.19 9.12 4.78 6.30 7.35 4.89 4.77 7.68 6.56 6.80 8.46 4.84 4.10 4.84 6.10 4.79 4.85 5.35 6.86 6.16 7.84 5.08 5.01 4.44 5.57 1.48 0.58 1.06 7.54 6.54 7.11 8.06 6.34 5.59 4.76 5.80 5.40 6.21 6.62 7.02 6.01 6.63 5.32 9.41 7.60 9.30 8.98 10.34 6.42 5.41 8.07 7.14 7.94 1.53 9.03 9.97 6.46 7.21 6.17 0.92 3.97 3.15 2.90 3.09 6.85 1.38 4.37 4.21 3.89 3.06 7.46 6.23 4.11 2.55 4.06 3.26 8.31 9.83 2.14 3.69 2.48 3.57 8.06 1.45 3.20 0.69 3.61 0.65 7.78 2.49 2.20 6.27 2.94 2.08 6.48 2.26 2.45 6.80 9.12 10.88 7.40 9.42 8.02 2.30 3.24 4.33 2.89 3.29 2.97 2.55 3.41 5.10 3.72 4.27 3.22 1.63 0.68 1.09 1 103 p. 105 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 2: Group Mouse# 1 2 3 4 5 6 fen ofibrate fe n o fib ra te fen ofibrate fen ofibrate fe n o fib ra te fe n ofibrate AVERAGE SD 7 8 9 10 11 12 13 14 15 16 17 18 s?s t ir ili! i s s a .* 1 1 PFOS PFOS PFOS PFOS PFOS PFOS AVERAGE SD 19 20 21 22 23 24 25 26 27 28 29 30 Cage# 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 8 8 9 9 10 10 11 11 12 12 Eartaa LR 10 02 12 10 20 12 01 11 20 01 02 20 00 12 01 01 02 21 00 11 21 02 11 02 00 01 10 21 11 02 Plasma cholesterol (mmol/L t=0 weeks t=4 weeks 7.44 7.17 11.01 6.62 8.36 7.79 8.51 7.09 7.78 6.83 7.44 9.92 8.42 7.57 1.34 1.22 6.42 4.85 7.12 4.92 10.10 4.40 10.39 5.57 12.13 5.40 4.36 4.45 8.42 4.93 2.92 0.48 9.37 6.36 11.59 4.71 8.60 9.46 5.90 6.17 7.93 8.51 5.26 5.99 8.65 6.33 1.86 1.65 12.95 2.39 6.09 1.46 6.82 3.53 6.47 2.89 10.67 2.39 7.99 2.25 8.50 2.49 2.74 0.69 6.27 2.82 12.95 3.18 7.58 2.44 7.61 2.61 9.13 7.98 2.80 4.33 8.59 2.33 3.03 0.68 1 104 p. 106 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 3: Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 fon o iih ra ta fe n o h n ra tc fon of ihrate fen o fib ra te icn o fib ra te fen ofib rate fo n ofm rate AVERAGE SD 8 9 10 11 12 13 14 15 16 17 18 19 20 21 4 4 5 5 5 6 6 1 2 1 1 2 0 1 -' ' IV 22 0 23 2 24 1 25 0 26 1 27 2 28 0 PFCS RFC S PFCS PFOS PFOS PFOS PFOS AVFRAGE? SD 29 30 31 32 33 34 35 0 1 0 0 1 0 0 Eartaa LR 01 12 01 20 02 00 20 21 12 20 10 11 01 21 17 17 07 08 18 19 29 2 10 1 10 0 11 2 11 1 12 0 12 2 12 0 13 0 13 2 13 1 14 2 14 1 15 2 15 Plasma cholesterol immol/L) t=0 weeks t=4 weeks 5.90 7.33 6.70 6.90 8.50 9.16 7.53 7.94 6.80 8.64 8.31 7.02 7.49 7.30 7.85 1.00 0.80 5.57 5.24 7.31 5.05 9.03 6.76 5.06 -T' 8.59 5.03 8.60 5.27 8.21 5.11 7.73 5.13 1.24 4.87 V.fc-1 0.10 9.12 8.04 5.78 6.37 10.51 8.20 5.74 5.83 7.57 5.08 7.62 5.52 7.64 6.13 1.91 1.10 6.49 1.65 4.17 2.12 8.08 2.65 8.16 2.34 8.78 2.89 8.93 2.98 7.55 7.45 2.44 1.66 0.50 7.70 1 2.15 5.78 . - -` 8.71 9.29 1 2.79 3.01 8.49 3.38 8.16 2.04 5.62 2.17 7.68 2.59 1.44 0.55 1 1 1 1 1 1 1 _\1 1 105 p. 107 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix V Plasma HDL-cholesterol Study 1 Group Mouse# 1 2 3 4 5 6 7 8 Cage# 1 1 2 2 2 3 3 3 Eartaq L 2 0 1 2 1 1 2 1 R 1 1 1 1 2 11 2 fen o fib ra tc fen o fib ra te fen o fib ra tc fen o fib ra tc fen o fib ra tc fen o fib ra tc fen o fib ra tc fen o fib ra te AVERAGE SD PFOS PFOS PFOS PFOS PFOS PFOS PFOS PFOS AVERAGE SD 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 4 4 5 5 6 6 7 7 8 8 8 9 9 10(A) 10 10 11 11 11 12 12 13 13 13 14 14 15 15 15 16 16 16 1 1 1 2 0 1 1 1 1 0 2 1 1 0 0 2 0 1 1 0 1 1 0 1 0 1 1 1 0 2 0 1 0 1 1 1 2 0 0 2 0 1 0 0 1 0 1 1 0 1 2 2 2 2 0 0 1 2 1 2 0 2 1 1 Plasma HDL-cholesterol (mmol/L) t=0 weeks t=4 weeks 1 t=6 weeks 0.67 1.34 1.37 1.19 0.75 1.64 0.92 1.78 0.89 0.96 1.07 0.90 1.00 0.94 0.94 1.46 1.34 1.46 1.44 1.26 1.36 1.26 1.07 1.95 1.70 1.96 1.50 0.17 0.21 0.41 0.91 2.10 3.01 0.94 1.12 2.63 2.12 2.99 2.19 1.20 2.05 2.33 0.94 2.09 2.36 1.03 1.95 2.43 0.56 1.45 1.46 0.99 2.10 1.70 0.96 0.19 1.10 0.94 0.88 1.17 1.16 0.88 0.47 0.83 0.93 0.23 1.33 1.39 1.07 0.91 1.35 1.16 0.68 0.51 1.05 0.32 0.84 0.82 1.19 1.27 0.85 1.20 0.54 0.80 0.94 0.25 2.06 0.32 1.29 1.53 1.50 1.70 1.81 1.25 1.25 1.81 1.52 0.24 0.92 0.99 1.02 0.95 0.58 0.87 0.72 0.71 0.85 0.16 0.60 0.53 0.77 0.73 0.75 0.61 0.49 0.49 0.62 0.12 2.31 0.54 1.74 1.42 1.58 1.64 1.99 0.61 0.81 1.88 1.46 0.50 0.83 0.79 0.63 0.66 0.72 0.63 0.60 0.67 0.69 0.08 0.31 0.33 0.49 0.54 0.65 0.55 0.31 0.36 0.44 0.13 1 106 p. 108 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 2: Group Mouse# 1 2 3 4 5 6 fe n ofibrate fe n ofibrate fe n ofibrate fe n ofibrate fe n ofibrate fe n ofibrate AVERAGE SD 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 ? . .ft,. 23 24 PFOS PFOS PFOS PFOS PFOS PFOS AVERAGE SD 25 26 27 28 29 30 Cage# 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 8 8 9 9 10 10 11 11 12 12 Eartaa LR 10 02 12 10 20 12 01 11 20 01 02 20 00 12 01 01 02 21 00 11 21 02 11 02 00 01 10 21 11 02 Plasma HDL- cholesterol (mmol/L t=0 weeks t=4 weeks 1.46 1.63 0.22 1.18 1.20 1.22 0.77 0.90 0.73 0.90 1.37 1.46 0.96 1.22 0.47 0.29 1.18 2.25 0.32 2.44 0.75 2.25 0.79 2.60 1.35 2.09 2.30 2.74 1.12 2.39 0.68 0.24 0.51 1.48 0.66 2.01 1.34 1.57 1.25 1.60 1.08 2.10 1.06 2.25 1.10 1.72 0.53 0.37 1.08 0.93 0.75 0.64 1.35 0.70 1.51 0.93 0.96 0.78 0.53 0.80 1.03 0.80 0.37 0.12 1.10 0.45 0.96 0.49 0.60 0.64 1.20 0.61 1.84 0.72 0.75 0.54 1.07 0.43 0.58 0.10 1 107 p. 109 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 3: Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 fen ofibratc fen oiibratc fe n o f'ib ra tc fen ofibratc fen ofib ratc fen ofibratc fen ofibratc AVERAGE SD 8 9 10 11 12 13 14 15 16 17 18 19 20 21 4 4 5 5 5 6 6 1 2 1 1 2 0 1 22 23 0 2 24 1 25 0 26 1 * 27 2 28 0 PFOS PROS PFOS PFOS PFOS PFOS PFOS AVERAGE SD 29 30 31 32 33 34 35 0 1 0 0 1 0 0 Eartag LR 01 12 01 20 02 0 .0 20 21 12 20 10 11 01 21 17 17 07 08 18 19 29 2 10 1 10 0 11 2 11 1 12 0 12 2 12 0 13 0 13 2 13 1 14 2 14 1 15 2 15 Plasma HDL-cholesterol (mmol/L) t=0 weeks t=4 weeks I 1.52 1.39 1.44 1.02 1.24 0.89 0.78 1.36 1.29 iB g iia s if w ii 0.93 0.89 1.16 0.87 1.19 1.07 0.26 0.24 17 1.70 1.06 1.94 1.34 i.2 i 1.78 \ sacrificed foriH B Lisolation > | 0.53 1.73 1.12 1.96 1.89 2.22 1.20 1.89 0.40 0.19 2.74 1.17 1.26 1.11 0.74 0.67 0.84 1.26 0.66 1.01 1.80 1.44 1.83 1.34 0.66 1.1$ 0.53 1.19 0.13 0.24 0.21 0.88 0.40 1.52 0.48 1.29 0.61 1.26 0.57 1.11 sacrificed for SD L Isolation 1.07 0.41 0.20 1.29 0.31 0.86 1.21 1.45 1.76 0.83 1.08 1.21 0.33 sacrificed for HDL Isolation 0.37 0.05 0.34 0.40 0.21 0.28 0.13 108 p. 110 3M#Q3 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix VI Plasma triglycerides Study 1 Group Mouse# 1 2 3 4 5 6 7 8 Cage# 1 1 2 2 2 3 3 3 Eartaa L 2 0 1 2 1 1 2 1 R 1 1 1 1 2 1 1 2 fen o fib ra te fen o fib ra tc fen ofib rate fen o fib ra tc fen o fib ra tc fen ofib rate fen o fib ra tc fen o fib ra tc AVERAGE SD 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 4 4 5 5 6 6 7 7 8 8 8 9 9 10<A} 10 10 1 1 1 2 0 1 1 1 1 0 2 1 1 0 0 2 0 1 1 1 2 0 0 2 0 1 0 0 1 0 1 1 25 11 0 0 licS'lly ;?^ 26 11 1 1 liiiiC t t l 27 11 1 2 IBteio r l i l i 28 12 0 2 feil'Gtbib.: bv'v'j 29 12 1 2 30 13 1 2 rO/'VtJ 31 13 0 0 B S fr : : m 4 32 13 10 ISVVi'.' ,v;'s! PFOS 33 14 0 1 PFOS 34 14 1 2 PFQ^ 35 15 1 1 PFOS 36 15 1 2 PFOS 37 15 0 0 PFOS 38 16 2 2 PFOS 39 16 0 1 PFOS 40 16 1 1 AVERAGE SD Plasma trialvcerides fmimol/U t=0 weeks t=4 weeks t=6 weeks 2.90 2.61 1.61 1.99 2.09 0.91 3.59 2.10 3.58 1.99 1.38 2.28 1.67 1.81 3.19 2.69 2.23 1.43 1.68 1.42 1.04 1.38 1.60 1.23 2.24 1.91 1.91 0.75 0.43 1.01 3.43 0.70 0.40 2.77 0.57 0.44 2.00 0.46 0.30 1.60 0.47 0.34 1.72 0.54 0.43 1.27 0.57 0.43 2.96 1.87 1.18 1.78 0.96 0.64 2.19 0.77 0.52 0.76 0.47 0.29 1.62 0.75 0.63 2.37 1.01 0.95 2.30 1.07 0.78 1.60 0.71 0.61 1.74 0.81 0.57 1.94 1.96 2.47 2.20 1.39 1.26 2.85 0.91 0.92 2.08 1.08 1.02 0.43 0.42 0.63 1.40 0.35 0.42 1.76 0.40 0.33 2.04 1.96 0.49 0.59 0.33 0.36 0.90 1.91 0.60 0.73 0.29 0.59 2.11 3.72 0.65 0.92 0.32 0.63 1.97 0.59 0.41 0.81 0.18 0.13 3.48 2.33 0.76 0.80 0.54 0.31 1.54 0.67 0.52 1.21 0.67 0.44 2.66 1.97 0.71 0.78 0.56 0.59 2.19 0.89 0.71 1.96 0.61 0.47 2.17 0.74 0.52 0.70 0.09 0.12 109 p. 111 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 2 Group Mouse# 1 2 3 4 5 6 fe n o fib ra te fe n o fib ra te fen ofibrate fen ofibrate fe n o fib ra te fe n o fib ra te AVERAGE SD 7 8 9 10 11 12 13 14 15 16 17 18 l ll i il i s'aac; illf P F F fliii M E e - iiS I l PSSiSil"f;r lllllfe ;/"ri. f fli PFOS PFOS PFOS PFOS PFOS PFOS AVERAGE SD 19 20 21 22 23 24 25 26 27 28 29 30 Cage# 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 .6 6 7 7 8 8 9 9 10 10 11 11 12 12 Eartaa LR 10 02 12 10 20 12 01 11 20 01 02 20 00 12 01 01 02 21 00 11 21 02 11 02 00 01 10 21 11 02 Plasma TC fmmol/D t=0 weeks t=4 weeks 1.81 1.58 3.48 1.48 1.91 1.57 2.74 1.25 2.01 1.60 1.06 1.29 2.26 1.37 0.71 0.21 1.49 2.66 0.40 0.54 3.14 0.61 2.24 0.59 2.92 1.18 0.41 0.32 2.27 0.48 0.79 0.12 3.05 1.04 4.21 1.22 1.64 0.99 1.41 0.71 2.59 2.31 0.70 0.57 2.54 0.87 1.02 0.25 3.53 0.85 1.95 0.51 1.44 0.99 0.38 0.57 2.65 2.94 0.42 0.64 2.25 0.56 0.96 0.17 1.24 0.63 3.32 0.63 2.31 0.60 2.40 0.59 1.95 0.53 3.10 1.10 2.39 0.76 0.68 0.21 TTTTT no p. 112 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Study 3 Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 fC'iiofii: rate tV nofibratc t\..noftl:ratc fe n o til: r c-.i,iC fc n o ifb ra te fenofil; rate fo n e fjR ra te AVERAGE SC 8 9 10 11 12 13 14 15 16 17 18 19 20 21 4 4 5 5 5 6 6 1 2 1 1 2 0 1 22 0 23 2 24 1 25 0 26 1 27 2 28 0 PROS > p 1-4 <, PROS Ft OS A V L K /'G l 29 30 31 32 33 34 35 0 1 0 0 1 0 0 Eartaa LR 01 12 01 20 02 00 20 1 12 20 10 11 01 21 17 17 07 08 18 19 29 2 10 1 10 0 11 2 11 1 12 0 12 2 12 1 0 13 0 13 2 13 1 14 2 14 1 15 2 15 Plasm TG (mmol/L) t=0 weeks t=4 weeks 1.64 1.47 1.38 1.21 1.50 1.49 2.14 1.80 1.16 2.24 * .m r- ' 2.27 1.64 2.18 1.67 1.74 0.39 0.42 1.49 0.41 1.36 0.32 1.62 0.43 1.68 3.15 0.39 1.58 0.33 1.03 0.37 1.70 0.38 0.67 0.05 0.57 1.46 S K is iK ? !a its 0.87 1.63 0.87 3.30 1.21 1.55 1.45 2.04 0.63 1.63 0.66 1.74 0.95 0.82 0.32 1.46 0.41 0.89 0.44 2.48 0.40 1.55 0.31 1.52 0.57 2.36 1.11 2.40 1.81 0.54 0.61 0.29 1.36 0.35 1.99 1.63 0.53 1.78 0.42 1.27 0.73 2.56 0.38 1.38 0.51 1.71 0.49 0.46 0.14 - v*! s is 111 p. 113 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix VII Plasma free glycerol Study 1 Group Mouse# 1 2 3 4 5 6 7 8 Cage# 1 1 2 2 2 3 3 3 Eartaa L 2 0 1 2 1 1 2 1 R 1 1 1 1 2 1 1 2 fcn ofib ra tc fcn o fib ra tc fcn o fib ra tc fcn o fib ra tc fcn o fib ra tc fcn o fib ra tc fcn o fib ra tc fcn o fib ra tc 9 10 11 12 13 14 15 16 4 4 5 5 6 6 7 7 10 11 11 21 02 10 10 12 AVERAGE SD 17 8 10 18 8 01 19 8 20 20 9 10 21 9 11 M-o > 22 0 0 23 10 0 1 24 10 2 1 25 11 0 0 26 11 1 1 27 11 1 2 28 12 0 2 29 12 1 2 'JOy.,Aav M M A 30 13 1 2 31 13 0 0 32 13 1 0 PF05 33 14 0 1 PFOS 34 14 1 2 PFOS 35 15 1 1 PFOS 36 15 1 2 PFOS 37 15 0 0 PFOS 38 16 2 2 PFOS 39 16 0 1 PFOS 40 16 1 1 AVERAGE SD Plasrr a free alvcerol (mmol/LI t=0 weeks t=4 weeks t=6 weeks 0.25 0.24 0.14 0.18 0.23 0.17 0.28 0.29 0.24 0.24 0.23 0.20 0.25 0.28 0.20 0.24 0.31 0.17 0.17 0.22 0.19 0.20 0.25 0.22 0.23 0.26 0.19 0.04 0.03 0.03 0.28 0.11 0.13 0.21 0.23 0.10 0.22 0.22 0.14 0.25 0.23 0.19 0.21 0.17 0.14 0.24 0.22 0.12 0.24 0.17 0.17 0.21 0.26 0.11 0.23 0.20 0.14 0.03 0.05 0.03 0.21 0.21 0.16 0.32 0.25 0.12 0.27 0.21 0.13 0.16 0.24 0.26 0.21 0.15 0.14 0.29 0.20 0.25 0.25 0.23 0.21 0.24 0.15 0.16 0.25 0.21 0.16 0.05 0.03 0.04 0.18 0.11 0.08 0.22 0.11 0.10 0.16 0.11 0.09 0.26 0.18 0.08 0.21 0.11 0.09 0.30 0.15 0.12 0.23 0.20 0.10 0.15 0.08 0.08 0.22 0.13 0.09 0.04 0.03 0.01 0.33 0.11 0.08 0.22 0.14 0.08 0.18 0.14 0.06 0.22 0.23 0.13 0.19 0.05 0.06 0.19 0.25 0.25 0.14 0.18 0.15 0.10 0.14 0.13 0.23 0.05 0.15 0.03 0.09 0.03 112 p. 114 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix VIII Plasma free fatty acids Study 1 Group Mouse# 1 2 3 4 5 6 7 8 Cage# 1 1 2 2 2 3 3 3 fen o fib ra tc fe n o fib ra tc fe n o fib ra tc fe n o fib ra tc fe n o fib ra tc fe n o fib ra tc fe n o fib ra tc fen o fib ra tc AVERAGE SD 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 4 4 5 5 6 6 7 7 8 8 8 9 9 10(A) 10 10 25 11 26 11 27 11 28 12 29 12 30 13 31 13 32 13 PF05 PFOs PFOS PFOS PFOs PFOS PFOs PFOS AVERAGE SD 33 34 35 36 37 38 39 40 14 14 15 15 15 16 16 16 Eartaa L 2 0 1 2 1 1 2 1 R 1 1 1 1 2 1 1 2 10 11 11 21 02 10 10 12 10 01 20 10 11 00 01 21 00 11 12 02 12 12 00 10 01 12 11 12 00 22 01 11 Plasma free fattv acids (rnmol/LI t=0 weeks t=4 weeks t=6 weeks 1.40 0.78 0.60 1.34 0.67 0.71 0.78 0.63 0.75 0.92 0.80 0.82 1.11 0.84 0.80 0.70 0.66 0.70 0.88 0.56 0.74 0.75 0.61 0.76 0.99 0.69 0.74 0.27 0.10 0.07 1.27 0.59 0.64 1.17 0.82 0.80 0.93 0.63 0.55 0.96 0.59 0.72 0.74 0.72 0.61 0.97 1.02 0.80 0.72 0.57 0.78 0.81 0.86 0.76 0.98 0.72 0.68 0.17 0.11 0.10 0.96 0.59 0.75 0.98 0.66 0.55 1.08 0.74 0.77 0.89 0.65 0.69 1.00 0.91 0.74 0.58 0.64 0.45 0.68 0.70 0.69 0.65 0.74 0.76 0.90 0.14 0.66 0.06 0.67 0.12 1.00 1.81 0.62 0.98 0.39 0.25 0.39 0.47 0.39 0.29 0.32 0.38 0.90 1.22 0.34 0.50 0.40 0.50 0.90 0.44 0.36 0.70 0.50 0.44 1.02 0.41 0.39 0.37 0.09 0.06 1.82 0.37 0.29 1.10 0.92 0.39 0.52 0.31 0.28 1.06 0.52 0.27 0.66 0.80 0.48 0.42 0.26 0.35 0.70 1.10 0.41 0.39 0.31 0.29 1.02 0.37 0.44 0.06 0.29 0.03 113 p. 115 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix IX Plasma ApoAl Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 Eartao LR 01 12 01 20 02 00 20 fcn ofib ratc 8 4 21 fen ofib ratc 9 4 12 fcn ofib ratc 10 5 20 fen ofibratc 11 5 10 fcn ofib ratc 12 5 11 fen ofibratc 13 6 0 1 fen ofib ratc 14 6 2 1 AVERAGE SD 15 1 1 7 16 2 1 7 17 1 0 7 18 1 0 8 19 2 1 8 20 0 1 9 21 1 2 9 22 0 2 10 23 2 1 10 24 1 0 11 m s s k iim m 25 0 2 11 ESEirr 26 1 1 12 w;.. 27 2 0 12 28 0 2 12 A#.'0F' "O'AHP PFOS 29 0 0 13 PFOS 30 1 0 13 PEGS 31 0 2 13 PFOS 32 0 1 14 PFOS 33 1 2 14 PFOS 34 0 1 15 PFOS 35 0 2 15 AVERAGE SO Plasma AdoA I Cma/mL I t=o weeks t=4 weeks 2.40 2.70 1.43 2.66 1.96 2.69 1.26 2.32 2.05 1.55 1.70 1.79 1.62 1.78 2.28 0.40 0.50 1.84 J 1.42 2.43 1.81 2.49 1.63 1 ' s s'-f - ` 0 , .i* 0.77 2.83 1.53 2.41 3.09 2.90 1.73 .5 0.70 0.23 3.53 1.67 p^fir " ) -, $" J1 ! 1.53 - E l.f 1.55 1.35 1.13 1.85 1.80 4.07 1,37 2.30 2.50 2.72 1.94 2.30 0.82 1.00 1.81 0.41 0.31 0.16 1.79 0.71 2.08 0.45 1.47 0.60 1.93 1.36 sacrificed forHfeaoDafonp1 1.54 0.54 0.60 0.26 1.38 0.47 1.49 1.93 0.53 2.19 0.32 1.95 0.48 1.67 0.39 1.54 0.37 1.73 0.43 0.30 0.08 114 p. 116 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix X Plasma CETP mass Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 Eartaa LR 01 12 01 20 02 00 20 f\ nofibrate 4 i 1 fc nofibrate 9 4 12 fenofibratc 10 5 2 0 fenofibrate 11 5 1 -0 Ivnofi brate 12 5 11 fenefi brate 13 6 0 1 fenobbratc 14 6 21 AVERAGE SD 15 1 1 7 16 2 17 17 1 0 7 18 1 0 8 19 2 18 20 0 1 9 21 1 2 9 22 0 2 10 23 2 1 10 24 1 0 11 25 0 2 11 26 1 1 12 27 2 0 12 28 0 2 12 PFOS 29 0 0 13 Ff u , 30 1 0 13 PF OS 31 0 2 13 PF-OS 32 0 1 14 PF-OS 33 1 2 14 PF;O S 34 0 1 15 PF-OS 35 0 2 15 AVLKAGL SD Plasma CETP (UQ/mL) t= 0 weeks t= 4 weeks 11.08 15.53 14.19 14.26 12.19 16.51 14.26 15.29 15.47 i# r " 15.47 *z: * r w 13.68 13.29 12.82 13.68 1.61 9.55 11.24 16.79 12.19 m:,.`T St2Sf , `J.64 14.71 1.37 8.32 9.70 - 1 1 /; 12.61 8.99 14.32 15.23 9.61 8.71 13.13 9.06 2.48 0.59 9.26 14.93 11.00 16.45 13.29 13.68 12.95 9.08 11.00 13.09 10.61 12.95 11.98 12.78 11.81 2.74 1.12 12.05 7.49 9.26 16.11 8.80 12.89 12.40 19.63 8.22 16.28 10.12 10.04 * 3 ' z 1. ' , ,, * ' . ^ 13.75 9.41 3.75 1.93 14.32 8.61 10.04 13.75 9.61 13.29 9.44 14.38 11.39 12.19 10.37 7.81 7.91 12.62 9 .J 1.81 1.34 115 p. 117 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XI Plasma CETP activity CETP activity Group Mouse# 1 2 3 4 5 6 7 Cage# 1 1 2 2 2 3 3 Eartaa LR 01 12 01 20 02 00 20 fe n o fib ra te fe n o fib ra te fe n o fib ra te fe n o fib ra te fe n o fib ra te fe n o fib ra te fe n o fib ra te 8 9 10 11 12 13 14 4 4 5 5 5 6 6 21 12 20 10 11 01 21 AVERAGE SD 15 1 1 7 16 2 1 7 17 1 0 7 18 1 0 8 19 2 1 8 20 0 1 9 21 1 2 9 22 0 2 10 23 2 1 10 24 1 0 11 25 0 2 11 26 1 1 12 27 2 0 12 28 0 2 12 PPOb 29 0 0 13 PEOS 30 1 0 13 PF-OS 31 0 2 13 PEGS 32 0 1 14 PEGS 33 1 2 14 PFOS 34 0 1 15 PFOS 35 0 2 15 AVERAGE 5D CETP activity Dmol/hl t= 0 weeks t= 4 weeks 245 175 227 211 202 218 265 207 208 A ' 1 \ 255 253 189 192 227 209 29 SinVET' ;>?;!TVGTAFEt'7SU'ZGG ;'4< 26 214 w . , 243 107 190 288 84 231 112 193 87 227 97 37 14 1 1 11 ' rV-Lwi 218 250 299 192 230 188 229 41 185 188 242 196 265 290 _n ot_ en ou g h ^ asm ajefe 228 44 228 232 216 212 -,-r - - r > ft- #>,>-t11 a 1__________ 254__________I 229 1 17 169 250 185 202 104 103 169 58 142 166 194 165 194 172 22 sacrificed fo r 157 190 187 139 162 166 19 116 p. 118 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XII Post heparin LPL and HL activity 1 Group 1 Mouse 1 2 3 4 5 6 7 8 LPL + HL activity (umol FFA/h/mL) 16.62 24.15 19.70 21.64 16.03 14.77 21.01 20.79 frrv/ibr.itc fcnofibratc fcrofifcr.tte 'rr Vibrate tcnofibrate '<r vbrate f r',`ibr.jtc: icnohbr.ite AVERAtie SD 9 10 n 12 13 14 15 16 17 18 19 20 21 22 23 24 F !f 1, 1. VI:-...........' 25 26 27 28 29 30 31 32 38.29 36.46 35.48 34.58 41.82 41.60 38.49 32.51 20.17 19.91 20.45 21.07 14.50 21.24 18.29 21.57 25.43 26.37 31.74 34.69 24.99 24.67 28.34 37.83 d ~o s - -;os a- O S 0-;o--. ?roi ::'"0S ;r 0':, AVLR.A{;;; SD 33 34 35 36 37 38 39 40 31.98 30.83 28.65 20.98 27.94 24.04 26.84 28.61 HL activity (umol FFA/h/mL) 5.99 8.63 7.66 7.71 6.73 6.56 7.41 8.32 7.37 0.90 11.89 11.85 14.24 12.44 13.88 11.36 12.87 10.05 li. ii 1.36 6.65 7.23 5.78 4.81 3.29 8.67 3.40 2.66 5.31 2.14 6.09 5.80 14.09 5.51 11.20 6.82 8.40 8.45 3.04 10.04 8.52 9.94 5.79 8.58 9.95 9.74 9.53 9.01 1.43 LPL activity (umol FFA/h/mL) ld.63 15.51 12.04 13.93 9.31 8.21 13.61 12.48 11.97 2.46 26.40 24.60 21.24 22.14 27.94 30.25 25.63 22.46 25.08 3.10 13.52 12.69 14.66 16.26 11.22 12.57 14.89 18.91 14.34 2.43 ------------------ T51B-----------------20.28 25.94 20.60 19.48 13.47 21.52 29.44 20.81 5.12 21.94 22.31 18.71 15.19 19.36 14.09 17.10 19.06 18.47 2.92 117 p. 119 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XIII Fecal lipids Fecal neutral sterols Group fen o fib ra te fen o fib ra te fen o fib ra te fen o fib ra te fen o fib ra te fen o fib ra te AVERAGE SD PROS pros pros pros pros pros AVERAGE SD Cage# 1 2 3 1 2 3 4+5 6 7 4+5 6 7 8 9 10+10A 8 9 10+10A 11 12 13 11 12 13 14 15 16 14 15 16 total neural sterols excretion (pmol/100 gr mouse/day) 33.54 54.59 32.79 31.08 42.23 23.63 36.31 10.75 32.00 55.13 43.36 33.96 49.60 53.05 44.52 9.81 38.02 34.74 43.30 38.40 38.16 35.69 38.05 2.98 33.47 33.56 43.17 37.90 40.25 46.53 39.15 5.23 43.81 26.10 15.50 50.66 39.01 29.21 34.05 12.86 coprostanol 0.18 0.18 0.13 0.11 0.10 0.11 0.14 0.04 0.19 0.15 0.12 0.11 0.08 0.09 0.12 0.04 0.11 0.13 0.16 0.11 0.09 0.12 0.12 0.02 0.17 0.16 0.12 0.10 0.09 0.08 0.12 0.04 0.13 0.16 0.21 0.09 0.13 0.15 0.14 0.04 % of neutral sterols cholesterol cholestanol 93.21 3.93 93.87 93.20 94.06 1.73 3.64 3.21 94.69 93.08 1.30 3.92 93.68 0.63 2.96 1.15 94.08 95.75 2.59 1.64 93.96 2.00 94.86 95.64 2.45 1.89 94.52 94.80 0.76 94.02 94.29 95.16 1.57 2.02 0.42 2.80 2.81 1.69 93.99 94.32 94.40 3.02 2.86 2.22 94.36 2.57 0.42 0.51 92.34 92.03 93.50 92.84 5.19 5.12 2.90 4.78 93.41 94.60 93.12 0,93 92.50 92.60 4.20 2.51 4.12 1.15 3.47 4.44 89.74 93.93 7.26 2.88 93.71 91.93 3.45 5.20 92.40 1.51 4.45 1.61 1 lathosterol 2.68 4.21 3.03 2.62 3.91 2.89 3.22 0.67 3.13 2.46 3.92 2.58 2.40 3.81 3.05 0.69 3.07 2.77 2.99 2.88 2.72 3.26 2.95 0.20 2.31 2.69 3.47 2.28 2.30 2.81 2.64 0.46 3.91 2.80 2.78 3.10 2.71 2.73 3.00 0.47 118 p. 120 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Fecal ohvtosterols Group Cage# 1 2 3 1 2 3 fe n o fib ra te fe n o fib ra te fe n o fib ra te fe n o fib ra te fen o n b ra tc fe n o fib ra te AVERAGE SD 4+5 6 7 4+5 6 7 8 9 10+10A 8 9 10+10A , ' -_s- PFOS pros PFOS pros pros PFOS AVERAGE SD 11 12 13 11 12 13 14 15 16 14 15 16 total phytosterols excretion (umol/100 qr mouse/day) 3.56 4.53 3.39 3.44 3.73 2.43 3.51 0.67 2.80 3.99 3.00 2.73 3.66 4.04 3.37 0.60 3.27 3.20 4.24 3.52 3.66 3.71 3.60 0.38 2.86 2.95 2.56 3.16 2.99 3.04 2.93 0.21 4.14 3.01 2.35 3.71 3.73 3.65 3.43 0.64 % of Dhvtosterols campesterol stiqmasterol b-sitosterol 27.00 9.10 63.91 25.93 9.55 64.52 25.97 9.43 64.60 25.68 25.63 9.52 8.34 64.80 66.02 26.42 9.34 64.25 26.10 9.21 64.68 0.52 0.46 0.73 26.83 9.20 63.97 27.14 7.03 65.83 28.24 6.47 65.29 26.91 9.68 63.40 25.40 26.92 26.91 0.90 26.43 26.16 8.89 9.22 8.41 1.33 6.50 9.44 65.71 63.86 64.68 1.05 67.07 64.40 25.75 26.10 8.95 8.97 65.30 64.92 26.04 10.13 63.83 26.15 8.43 65.41 26.11 0.22 8.74 1.23 65.16 1.11 26.93 5.80 67.27 26.85 6.28 66.88 28.21 6.16 65.62 24.70 12.99 62.31 25.11 25.10 26.15 1.39 24.36 13.36 12.24 9.47 3.74 15.20 61.53 62.66 64.38 2.51 60.44 25.40 22.54 12.36 16.10 62.24 61.36 24.79 24.94 16.54 12.79 58.68 62.27 23.16 15.94 60.90 24.20 1.11 14.82 1.80 60.98 1.34 1 119 p. 121 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Fecal bile acids Group 'eriofibrale fcnofibraic fenofibisilo fcnofibratc fcnofihmtc fenofibrale AVERAGE SD f e. w " PFQS i'FOS pros pros pros pfos AVFRAGE SD Cage# 1 2 3 1 2 3 4+5 6 7 4+5 6 7 8 9 10+10A 8 9 10+10A 11 12 13 11 12 13 14 15 16 14 15 16 00 total bile acid excretion (umoi/100 or mouse/dav) 8.33 5.80 4.22 5.95 4.98 4.01 5.5 1.6 3.67 2.37 4.37 3.52 2.54 4.13 3.4 0.8 4.21 6.83 6.74 3.46 4.86 5.0 1.4 3.74 3.57 3.46 4.07 3.13 1.60 3.3 0.9 2.87 2.90 3.29 2.31 2.66 2.66 2.8 0.3 a-murlcholate 7.93 5.78 7.29 8.38 5.80 7.24 7.1 1.1 6.56 5.83 5.10 8.32 5.43 5.06 6.0 1.2 6.38 9.56 4.98 6.50 8.54 2.81 6.5 2.4 9.81 9.31 5.72 5.67 5.76 1.73 6.3 2.9 5.40 7.94 8.86 3.42 4.12 3.18 5.5 2.4 deoxycholate 19.10 7.67 15.90 17.35 7.92 17.33 14.2 5.1 10.23 9.99 7.58 18.88 9.82 7.11 10.6 4.3 18.03 27.82 12.73 25.12 17.42 6.50 17.9 7.8 25.45 31.19 8.61 15.48 16.94 2.75 16.7 10.5 10.53 14.67 33.87 5.60 5.99 10.28 13.5 10.5 cholate 12.16 19.16 8.58 11.45 15.99 8.60 12.7 4.2 12.02 13.10 19.89 7.28 11.03 17.15 13.4 4.5 8.92 18.10 17.49 5.88 8.27 9.32 11.3 5.2 19.87 26.55 19.43 10.32 10.67 3.67 15.1 8.3 21.12 15.40 19.26 10.72 8.69 5.67 13.5 6.1 % of bile acids lithocholate b-muricholate 10.58 21.94 8.52 26.92 12.54 24.50 11.15 23.01 9.60 10.92 29.02 27.53 10.6 25.5 1.4 2.8 18.35 16.64 19.39 10.93 12.89 25.21 15.93 18.92 21.14 11.94 12.00 26.95 16.6 18.4 3.7 6.6 16.49 16.09 29.65 47.57 11.63 32.23 20.57 11.32 16.75 23.43 8.99 16.59 17.3 24.5 7.3 13.4 14.44 19.39 25.96 25.45 8.03 19.87 8.35 11.36 12.09 13.12 4.04 3.61 11.1 16.6 5.4 8.8 10.62 8.58 15.61 16.71 1.99 28.04 5.32 8.37 4.91 7.21 6.27 8.66 6.3 14.1 3.0 7.9 w-muricholate 21.91 26.31 22.82 22.19 24.82 22.68 23.5 1.7 17.63 21.23 18.78 16.50 16.52 20.46 18.5 2.0 40.86 43.85 14.60 40.27 25.28 10.17 29.2 14.6 35.27 35.36 19.61 30.81 23.66 4.30 24.8 11.9 30.96 36.72 42.81 10.93 18.62 17.88 26.3 12.4 1 hyodeo/ureo 6.38 5.64 8.38 6.47 6.86 5.70 6.6 1.0 18.57 19.53 10.56 14.16 24.13 11.27 16.4 5.3 11.53 21.37 6.84 16.51 10.95 7.63 12.5 5.5 9.03 15.22 7.47 6.91 10.42 3.52 8.8 3.9 9.21 7.19 8.53 6.65 4.38 4.03 6.7 2.1 120 p. 122 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Fecal fatty acids Group Cage# _____________ 1 1 2 3 1 2 3 fcn ofib ratc fcn ofib ratc fcn ofib ratc fcn ofib ra tc fcn ofib ratc fcn ofib ratc AVERAGE SD 4+5 6 7 4+5 6 7 9 10+10A 8 9 10+10A _____PFHS ' m m s s i: * s a m m sm sm *V .' - . PPOS Pt Ob PPOb PF05 PPOS PFOS A V tR A G C D ii 12 13 11 12 13 14 15 16 14 15 16 total fatty add excretion (umol/100 or mouse/day) 106.3 563.7 146.4 94.4 520.7 110.1 256.9 222.1 251.4 564.9 386.9 319.7 535.8 399.7 409.7 121.5 242.8 190.7 378.4 239.0 144.1 246.0 240.2 78.5 85.4 101.8 122.3 101.1 127.2 418.7 159.4 127.9 208.7 162.3 78.8 330.1 132.4 218.7 188.5 86.3 C14:0 3.29 1.99 2.66 2.74 2.42 3.05 2.69 0.46 2.74 2.21 1.83 2.21 2.06 2.20 2.21 0.30 2.56 2.68 2.33 2.52 2.81 2.66 2.59 0.16 3.18 3.12 2.91 2.89 2.61 1.16 2.64 0.75 2.38 2.57 3.07 1.74 2.28 2.19 2.37 0.44 C16:0 48.11 44.01 47.28 45.79 47.12 46.50 46.47 1.43 49.74 47.47 41.60 46.82 45.87 46.56 46.34 2.68 48.30 48.04 46.86 48.38 47.43 48.92 47.99 0.74 49.02 47.31 50.57 47.45 47.96 38.79 46.85 4.13 47.10 47.50 48.02 42.76 46.94 45.49 46.30 1.93 C16:l . 0.59 0.78 0.73 0.61 0.69 0.68 0.07 0.70 0.60 0.61 0.64 0.55 0.63 0.62 0.05 0^67 0.68 0.63 0.66 0.62 0.66 0.65 0.03 1.02 1.05 0.51 0.98 0.98 0.59 0.86 0.24 0.70 0.73 0.80 0.61 0.69 0.61 0.69 0.07 % of fattv adds C18:0 46.97 42.34 41.96 43.67 41.36 42.66 2.48 39.35 43.73 46.64 44.06 45.43 44.17 43.90 2.48 42.54 41.06 43.88 40.90 42.27 40.08 41.79 1.37 38.52 39.47 38.64 39.69 39.50 50.06 40.98 4.47 41.76 40.94 39.52 47.92 43.08 45.10 43.05 3.05 C18:l 7.44 5.57 6.23 7.92 5.46 7.69 6.72 1.10 6.82 5.37 8.38 5.59 5.44 5.67 6.21 1.19 5.30 6.88 5.60 6.90 6.23 7.00 6.32 0.73 7.65 8.26 6.79 8.26 8.14 8.48 7.93 0.62 7.23 7.51 7.81 6.10 6.26 5.87 6.79 0.82 C18:2 0.79 0.77 0.66 0.87 0.68 0.70 0.75 0.08 0.61 0.58 0.82 0.64 0.61 0.69 0.66 0.09 0.58 0.62 0.66 0.59 0.65 0.65 0.62 0.03 0.62 0.78 0.58 0.74 0.82 0.82 0.73 0.10 0.76 0.71 0.78 0.79 0.72 0.70 0.74 0.04 C18:3 0.00 0.09 0.05 0.00 0.05 0.00 0.03 0.04 0.05 0.04 0.11 0.05 0.04 0.07 0.06 0.03 0.05 0.04 0.04 0.04 0.00 0.03 0.03 0.02 0.00 0.00 0.00 0.00 0.00 0.10 0.02 0.04 0.07 0.04 0.00 0.08 0.05 0.04 .5 0.03 1 121 p. 123 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Fatty a d d balance Group Cage# 1 2 3 1 2 3 fen o fib ra te fen o fib ra te fen o fib ra te fen o fib ra te fen o fib ra te fen o fib ra te AVERAGE SD 4+5 6 7 4+5 6 7 8 9 10+10A 8 9 10+10A 11 12 13 11 12 13 pros pros pros PF05 PFOS pros AVERAGE SD 14 15 16 14 15 16 total fatty acid excretion (umol/100 ar mouse/dav) 106 564 146 94 521 110 256.92 222.07 251 565 387 320 536 400 409.73 121.45 243 191 378 239 144 246 240.17 78.53 85 102 122 101 127 419 159.41 127.93 209 162 79 330 132 219 188.49 86.31 total fatty acid Input (umol/100 qr mouse/dav) 5800 6138 5178 5461 6488 5427 5749 492 6347 6476 6393 5998 6577 7352 6524 451 5407 5739 7801 6048 5949 7373 6386 965 5323 5604 5823 5541 5896 6159 5724 296 6276 5239 5148 6430 5865 5732 5782 523 Fatty add balance (umol/100 qr mouse/dav) 5694 5574 5032 5367 5967 5317 5492 326 6095 5911 6006 5678 6041 6952 6114 436 5164 5548 7422 5809 5804 7127 6146 910 5237 5503 5701 5439 5769 5740 5565 209 6067 5077 5069 6099 5732 5514 5593 458 122 p. 124 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XIV VLDL-triglycerides and de novo ApoB production VLDL-trialvcerides production Group kMK/ll.'l'c'iU! .i-r/ibrjv r::nri'lhrjfi" icnc/ibiuK i'.:!!0:ibr,iU ic;noAhr::(r isnoSbrutc icrnobbrur-AVlRAC-:- SE? Pr 'S inus r-VU-S i-SUS PrOS u'L-AGL Su Mouse# 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 i Body weight (9) 32.3 32.4 30.3 29.0 26.6 27.2 25.5 29.0 2.8 29.2 30.3 26.9 28.0 31.9 23.4 30.0 29.7 0.7 2.6 27.3 31.9 28.3 27.2 27.8 26.5 30.1 26.6 28.2 1.9 26.9 28.3 27.3 28.9 27.2 31.2 28.0 32.9 28.8 2.1 31.3 28.7 26.3 27.7 25.7 29.4 25.6 27.6 27.8 2.0 25.7 t=Q min 1.90 1.76 1.30 1.76 0.96 1.00 1.00 1.38 0.41 0.50 0.70 0.23 0.46 0.28 0.23 1.04 0.60 0.50 0.28 0.57 1.11 1.00 0.71 0.57 1.19 0.74 0.43 0.79 0.28 0.28 0.41 0.41 0.31 0.23 0.37 0.18 0.48 0.33 0.10 0.54 0.49 0.34 0.46 0.39 0.59 0.90 0.40 0.51 0.18 0.76 Plasma triglycerides (mmol/L) t=15 min t= 30 min t=60 min 3.22 2.84 3.20 4.755 4.515 5.157 6.681 6.468 8.373 2.33 3.975 5.589 2.03 3.498 5.433 2.30 3.591 6.159 2.15 3.324 5.748 2.58 4.12 6.35 0.50 0.70 1.00 1.84 3.618 7.002 2.42 4.323 8.223 1.40 3.564 7.332 1.69 1.79 1.44 2.52 2.60 l.M 3.876 3.792 3.531 4.302 5.025 4.00 7.494 6.843 6.321 7.779 9.654 7.58 0.48 0.52 1.02 1.97 3.456 6.087 2.66 4.629 7.017 2.88 4.932 7.854 1.30 2.529 5.019 1.64 3.612 6.192 2.30 3.45 5.214 2.18 3.702 6.537 1.52 2.79 4.866 2.05 3.64 6.10 0.56 0.82 1.04 0.44 0.696 0.969 0.65 0.921 1.338 0.67 1.002 1.626 0.80 1.362 2.202 0.47 0.82 0.852 1.404 1.518 2.286 0.30 0.84 0.555 1.089 1.089 1.782 0.62 0.99 1.60 0.20 0.30 0.48 0.70 0.99 1.26 0.86 1.48 2.13 0.45 0.63 0.80 0.56 0.79 1.19 0.56 0.89 1.55 0.62 0.74 0.98 0.92 0.98 1.10 0.41 0.62 0.93 0.63 0.89 1.24 0.18 0.28 0.42 1.61 2.478 3.714 t=90 min .470 7.965 11.532 8.715 7.26 8.817 7.875 8.66 1.38 9.474 10.419 10.065 12.102 12.036 9.837 10.722 14.028 11.09 1.53 8.895 10.965 10.806 7.803 8.388 6.993 9.795 7.497 8.89 1.50 1.467 1.755 2.253 3.195 2.235 3.261 1.311 2.349 2.23 0.72 1.80 2.46 1.12 1.49 1.94 1.23 1.57 1.04 1.58 0.48 4.356 Slope 0.073 0.070 0.113 0.077 0.070 0.087 0.077 0.081 0.015 0.102 0.111 0.114 0.131 0.128 0.107 0.109 0.151 o !ll9 0.016 0.092 0.107 0.108 0.081 0.089 0.064 0.100 0.078 0.090 0.015 0.013 0.015 0.021 0.032 0.023 0.032 0.014 0.021 0.021 0.008 0.014 0.022 0.009 0.012 0.018 0.007 0.007 0.008 0.012 0.006 0.040 VLDL-TG production (pmol/h) .ii 6.11 9.25 6.03 5.04 6.37 5.30 6.35 1.38 8.06 9.09 8.25 9.87 11.05 6.77 8.85 12.09 .5 1.71 .7 9.20 8.25 5.94 6.67 4.57 8.13 5.59 6.89 1.55 0.94 1.14 1.53 2.48 1.66 2.71 1.02 1.85 1.66 0.66 1.16 1.74 0.60 0.90 1.26 0.59 0.49 0.60 0.92 0.44 2.78 123 p. 125 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 De novo AdoB synthesis and TG production oerAooB Group Mouse# Body weight pl serum used ml VLDL 35S dpm in ApoB (9) fo r VLDL isolation after UC oer 400 ul VLDL 1 32.3 200 1.197 4756.4 3 32.4 245 1.210 6062.0 4 30.3 S 29.0 6 26.6 7 27.2 8 25.5 280 1.216 10320.5 220 1.202 6896.9 280 1.162 8837.6 200 1.183 7309.8 300 1.202 8982.9 29.0 2.8 9 29.2 240 1.178 8178.3 fc n o f'b rjlv 10 30.3 185 1.219 6455.8 teri c if ;;)r Lift' frn of-D r ;ilv 11 26.9 12 28.0 250 1.208 8164.6 270 1.201 8402.8 fi'r- o fib 'i.'tx ' 13 31.9 r ( >r. nf> * 14 23,4 280 1.208 160 1.199 7718.3 5371.3 \ t ! I ' utV 15 30.0 300 1.234 9005.4 . fl 16 29.7 300 1.216 9726.5 A V F R A C .L SD 28.7 2.6 17 27.3 185 1.218 5776.8 18 31.9 19 28.3 20 27.2 21 27.8 22 26.5 23 30.1 24 26.6 190 185 200 140 280 280 290 1.214 1.215 1.187 1.186 1.212 1.205 1.212 4434.8 4785.3 4695.1 3184,5 5217.1 6610.5 9286.6 - i t S lli- . -' r*- r - , , ,, " r "? 25 26 27 28 29 30 31 32 28.2 1.9 26.9 28.3 27.3 28.9 27.2 31.2 28.0 32.9 190 195 175 250 220 300 320 320 1.217 1.212 1.203 1.204 1.206 1.190 1.207 1.191 1514.1 1545.3 1306.0 2057.2 1523.0 2655.5 1387.7 2419.8 28.8 2.1 F'F O S PI O R f-'F k >0 Pi OR PF i >S F'F 0 ,:PF .OS PI O S 33 31.3 34 28.7 35 26.3 36 27.7 37 25.7 38 29.4 39 25.6 40 27.6 190 1 1.197 200 1.216 200 1.206 220 1.200 210 1.197 330 1.203 340 1.196 300 1.167 874.7 1059.7 651.1 846.6 1216.1 987.9 1188.7 1039.2 A V P P A S I. s i; 27.8 2.0 2 _____ 2 L 1 _____ 1 190 1 1.199 1 6243.1 de novo ApoB synthesis ( * 10* d p m /m l/h r) 4.74 4.99 7.47 6.28 6.11 7.20 6.00 6.11 1.02 6.69 7.09 6.58 6.23 5.55 6.71 6.17 6.57 6.45 0.46 6.34 4.72 5.24 4.64 4.50 3.76 4.74 6.47 5.05 0.93 1.62 1.60 1.50 1.65 1.39 1.76 0.87 1.50 1.49 0.27 0.92 1.07 0.65 0.77 1.15 0.60 0.70 0.67 0.82 0.21 6.57 TG production per ApoB (um ol/10* dpm) 0.917 0.841 0.908 0.735 0.689 0.722 0.770 0.80 0.09 0.917 0.940 1.036 1.257 1.387 0.958 1.062 1.377 1.12 0.19 0.872 1.358 1.236 1.045 1.185 1.019 1.266 0.722 1.09 0.21 0.479 0,559 0.831 1.152 0.975 1.098 0.929 0.833 0.86 0.24 0.899 1.257 0.780 0.934 0.945 0.745 0.613 0.712 0.86 0.20 0.367 124 p. 126 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Lipid m m p o sitip n jisr A bq B 'OfV;iior'citc ?f(bratr enafibrato fonof ibrtHc ienofibr ate ienafibrale fenofibrau fenofibratc AVERAGE SD Mouse# 1 3 4 5 6 7 8 4 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ii 34 35 36 37 38 39 40 2 Total cholesterol 0.98 0.71 0.43 0.74 0.64 0.51 0.57 0.65 0.18 0.38 0.37 0.27 0.35 0.34 0.29 0.50 0.38 0.36 0.07 0.51 0.62 0.49 0.57 0.48 2.10 0.77 0.49 0.75 0.55 0.25 0.48 0.42 0.41 0.45 0.45 0.43 0.79 0.46 0.15 0.62 0.88 0.81 1.22 0.90 3.96 2.42 2.06 1.61 1.15 0.55 LiDld In Isolated VLDL oer AdoB (umol/104 ApoB) Free cholesterol Cholesterol ester Triglycerides 0.26 0.71 1.14 0.22 0.49 0.90 0.15 0.28 0.83 0.18 0.17 0.57 0.47 0.68 0.61 0.14 0.36 0.64 0.16 0.41 0.74 0.18 0.47 0.79 0.04 0.14 0.19 0.15 0.23 0.87 0.12 0.25 0.88 0.12 0.15 0.93 0.13 0.22 1.00 0.17 0.17 1.17 0.12 0.18 0.82 0.20 0.30 1.06 0.19 0.19 1.23 0.15 0.21 0.99 0.03 0.05 0.15 0.13 0.37 0.86 0.19 0.43 1.36 0.17 0.32 1.17 0.17 0.40 1.02 0.16 0.33 1.09 0.51 1.58 1.16 0.22 0.55 1.27 0.15 0.34 0.75 0.21 0.54 1.08 0.12 0.43 0.20 0.12 0.13 0.41 0.14 0.34 0.60 0.17 0.25 0.84 0.21 0.20 0.20 0.25 0.94 0.77 0.24 0.21 1.11 0.25 0.18 0.76 0.31 0.48 0.99 0.20 0.06 0.25 0.11 0.80 0.22 0.32 0.30 1.23 0.33 0.55 1.35 0.37 0.40 0.44 0.82 1.09 1.02 0.28 0.97 0.62 3.00 0.76 1.21 0.67 1.75 1.10 0.55 1.52 0.95 0.49 1.12 1.09 0.23 0.92 0.18 0.14 0.41 0.53 1 Phospholipids 0.32 0.25 0.19 0.24 0.20 0.18 0.21 0.23 0.05 0.23 0.20 0.19 0.20 0.25 0.17 0.26 0.28 0.22 0.04 0.21 0.32 0.27 0.24 0.27 0.44 0.33 0.22 0.29 0.08 0.14 0.19 0.23 0.24 0.22 0.24 0.23 0.28 0.22 0.04 0.34 0.34 0.34 0.32 0.26 0.65 0.47 0.43 0.39 0.12 0.21 125 p. 127 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XV Biliary bile acids, cholesterol and phospholipids Bile flow Group Mouse 1 2 3 4 5 6 7 15 min 11.70 14.80 19.10 16.60 16.70 16.30 23.90 volume (gL) 30 min 8.10 0.50 13.10 17.30 14.60 16.30 22.10 45 min 8.00 13.40 16.50 18.40 16.30 16.80 23.80 total 27.80 28.70 . 48.70 52.30 47.60 49.40 69.80 EenoPbrate Fenobb'ate Fenofib'ate FtvioFibrate renofibrate Fenofibrate extra fenofibrate AVERAGE SD 7 9.20 2.80 3.90 8 20.50 22.30 13.20 9 24.10 19.20 18.70 10 22.10 24.10 26.70 11 18.30 19.20 20.60 12 22.30 16.10 16.70 12 19.40 17.70 23.20 13 14.30 15.30 4.90 14 19.40 13.70 15.40 15 18.50 14.40 15.40 16 15.70 13.30 15.20 17 18.00 14.40 12.90 18 IdtcXfltr ,, ,,y "X 21 20.30 25.50 27.80 15.90 56.00 62.00 72.90 58.10 55.10 60.30 34.50 48.50 48.30 44.20 45.30 73.60 19 9.60 .5 0.40 10.50 20 19.00 21.00 14.80 54.80 21 22.70 22.10 22.80 67.60 22 23.90 21.30 22.70 67.90 23 33.90 26.20 24.70 84.80 24 36.70 34.80 32.70 104.20 24 25.40 21.30 21.00 67.70 p ros PFOS p ro s p ro s p ro s p ros extra P r o s AVERAGE SD mouse l mouse 2 mouse 7 mouse 13 mouse 18 mouse 19 25 16.50 10.60 9.40 36.50 26 18.00 10.00 13.30 41.30 27 22.10 18.30 14.80 55.20 28 21.20 19.50 20.50 61.20 29 23.50 18.70 18.40 60.60 30 13.70 8.80 9.90 32.40 34 34.00 32.90 29.90 96.80 mouse not warm enough canula wrong, hardly any flow, after 30 min adjusted mouse not warm enough mouse not warm enough mouse died during anaesthesia mouse not warm enough Bile flow (uL/mln/kQ mouse) 15 min 30 min |I 45 min || total 34.10 34.10 47.51 32.59 41.04 40.38 37.90 39.50 42.01 39.80 39.48 34.52 38.53 37.51 36.34 36.34 37.46 36.71 54.75 50.63 54.52 53.30 43.20 7.76 38.71 7.13 41.28 7.07 40.30 23.50 46.64 7.15 50.74 Hit.'-* 13 30.03 42.47 55.98 44.60 43.44 48.01 50.28 45.86 53.67 54.84 48.12 38.75 60.75 51.63 40.19 55.29 48.54 44.20 39.43 35.98 47.15 40.85 m B tti50.49 47.41 5.50 6.60 iifSHIgi B esM B B iw . 45.21 5^53^ 47.70 U K ) 41.32 29.18 32.80 34.43 45.01 35.04 37.47 39.17 38.91 32.96 37.67 36.51 45.63 36.50 32.70 38.28 " 47.82 60.07 65.49 57.79 43.74 38.75 41.23 41.24 357 12.23 13.78 9.43 46.57 60.05 56.50 83.70 75.98 60.69 63.92 13.55 38.60 42.25 54.98 49.94 57.60 35.13 80.38 51.27 15.30 51.47 58.47 50.35 64.69 72.05 50.90 57.99 8.88 24.80 23.47 45.52 45.94 45.83 22.56 77.78 40.84 19.71 36.27 60.32 53.66 60.99 67.70 50.18 54.85 10.96 21.99 31.22 36.82 48.29 45.10 25.38 70.69 39.93 16.64 44.77 59.61 53.51 69.79 71.91 53.92 58.92 10.41 2.46 32.32 45.77 48.06 49.51 27.69 76.28 44.01 17.01 | | | 126 p. 128 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Bile m ini flaw Group F e n n fjb i ate F e n o fib ra te F e n o fib ra te F e n o fib ra te F e n o fib ra te F e n o fib ra te e x tra fe n o fib ra te AVERAGE SD - t'FO S PFOS PFOS pros PFOS PFOS e x tia PFO S AVERAGE SD mouse 1 mouse 2 mouse 7 mouse 13 mouse 18 mouse 19 Mouse 1 2 3 4 5 6 7 Bile acid cone in bile (mM) 15 min 30 min 45 min Si 16.88 8.71 13.09 12.60 17.64 13.78 3.61 16.23 10.52 13.46 15.34 14.35 - 13.98 2.20 12.47 18.17 10.15 14.34 16.88 16.67 14.78 3.05 Bile acid flow (nmol/min/kg mouse) 15 min 30 min I1 45 min I1 Total , .-f" >i 802 330 517 458 966 r . `' lykj, 529 415 465 558 726 425 746 427 552 632 909 425 692 391 511 549 867 614 539 615 573 262 119 189 179 | I 8 9.07 8.95 9.43 423 454 283 387 9 9.19 9.43 9.67 514 421 420 452 10 8.95 9.07 9.31 450 497 565 504 11 9.19 9.31 9.43 421 448 487 452 12 8.95 9.07 9.43 480 351 379 404 12 13.12 12.38 14.35 517 445 677 546 9.74 9.70 10.27 468 436 469 457 1.66 1.32 2.00 43 48 140 60 13 .t *. tC A k -V '.iii ' L l 1* v ,, > 4'. . ,Y* 14 9.55 8.83 9.19 395 258 301 318 15 15.72 14.34 13.21 708 502 495 568 16 16.75 19.75 22.32 652 651 841 715 17 16.11 11.37 27.42 735 415 897 682 18 iSflB . T - 1 7" _______ , ... 1_____ 1 1 21 11.76 11.26 7.34 562 676 481 573 13.98 13.11 15.90 3.15 4.20 8.65 19 f y;` .. - *. - .L*,, 610 137 500 173 /-.ir * - ' 603 255 571 156 y ;ji 20 8.95 11.25 8.83 417 579 320 439 21 10.64 7.51 8.83 639 439 532 537 22 10.15 8.71 10.10 574 438 542 518 23 9.19 9.67 9.43 769 626 575 657 24 9.43 9.07 8.95 717 653 606 659 24 10.50 9.34 10.12 637 475 508 540 9.81 9.26 9.38 625 535 514 558 0.71 1.23 0.61 123 96 101 85 25 11.98 11.37 12.84 462 ~~1 282 282 342 26 15.09 18.30 17.00 638 430 531 533 27 8.82 7.88 8.15 485 359 300 381 28 11.26 12.75 13.49 562 586 651 600 29 8.82 11.76 8.82 508 539 398 481 30 12.38 16.91 15.33 435 382 389 402 34 8.82 8.15 11.76 709 634 831 725 11.02 12.45 12.48 543 459 483 495 2.38 3.98 3.22 100 130 201 136 mouse not warm enough canula wrong, hardly any flow, after 30 min adjusted mouse not warm enough mouse not warm enough mouse died during anaesthesia mouse not warm enough 127 p. 129 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Phospholipid flow Group Mouse 15 min 30 min 45 min |I 15 min 1 2 722 3 544 582 836 333 4 379 443 430 185 5 620 684 747 316 6 557 773 811 261 7 787 635 913 556 577 623 743 330 147 123 168 139 F e n o fib ra te 7 %!'! F e n o fib ra te F e n o fib ra te 8 366 443 430 220 9 709 760 747 512 F e n o fib ra te 10 925 938 874 600 F e n o fib ra te 11 900 760 696 532 F e n o fib ra te 12 620 1039 785 429 e x tra fe n o fib ra te 12 812 913 989 413 AVERAGE 722 809 754 451 5D 209 209 ^ 1 8 9 132 13 B H H H l i H i 14 709 773 Un 823 378 in 493 646 595 286 16 938 1217 1065 471 17 722 633 1788 425 18 IS iiliiiiS II 21 660 597 395 407 704 773 933 393 159 257 539 69 19 20 696 684 747 418 21 595 608 709 461 22 811 747 874 591 23 760 709 773 821 24 811 912 925 795 24 812 736 825 636 747 733 809 620 l- V / g y - S B - ' 3 87 101 81 166 PFOS PFOS PFOS PFOS PFOS PFOS extra PFO S 25 696 836 1001 347 26 722 1065 1471 393 27 572 597 559 406 28 534 660 648 344 29 307 585 509 228 30 1557 1683 1595 705 34 787 787 812 816 AVERAGE SD 739 888 942 463 394 388 437 214 mouse 1 mouse not warm enough mouse 2 canula wrong, hardly any flow, after 30 min adjusted mouse 7 mouse not warm enough mouse 13 mouse not warm enough mouse 18 mouse died during anaesthesia mouse 19 mouse not warm enough |1 30 min I1 45 min I1 Total | 245 225 304 362 415 310 79 ,,''it, . i, *' 290 437 663 472 519 424 467 ^ 12j3 443 233 371 392 642 400 139 167 419 685 464 407 601 457 180 340 215 331 338 538 346 105 226 456 649 489 452 479 459 136 291 292 517 298 111-- 463 372 109 348 339 288 289 517 502 754 492 1S B l S i l i f S iS I lS i 334 401 448 405 192 93 454 350 407 458 552 490 485 6Q5 560 592 608 673 848 808 817 483 534 551 553 576 583 153 148 144 267 284 299 322 592 436 351 266 341 391 404 380 346 296 290 490 522 573 789 740 782 422 443 443 176 181 178 128 p. 130 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Cholesterol flow Group F e n o fib ra te F e n o fib ra te F e n o fib ra te F e n o fib ra te F e n o fib ra te F e n o fib ra te e x tra fe n o fib ra te AVERAGE SD PFOS PFOS PPOS l-FO S PFOS PFOS e x t'a PFO S AVERAGE SD mouse 1 mouse 2 mouse 7 mouse 13 mouse 18 mouse 19 Mouse Cholesterol In bile (mmol/D 11 Cholesterol flow fnmol/min/ka mouse} 15 min 30 min 45 min 15 min 30 min 45 min Total 1 2 6.51 1.11 19.1 13.2 -- E J E *\ . 37.8 37.8 3 0.75 0.81 1.08 35.5 26.3 44.3 35.4 4 0.54 0.93 1.02 20.5 36.7 42.8 33.3 5 0.79 0.54 0.55 31.3 18.6 21.3 23.8 6 0.58 0.66 0.64 21.2 24.0 24.1 23.1 7 1.08 1.17 1.07 59.2 59.3 58.1 58.9 0.75 0.82 0.91 33.6 33.0 38.1 35.4 0.21 0.24 0.25 15.7 16.1 13.7 13.0 7 . X, - *- t 8 0.60 0.64 0.70 28.0 32.7 21.1 27.3 9 0.63 0.57 0.64 35.3 25.4 28.0 29.6 10 0.60 0.66 0.63 30.2 36.2 38.3 34.9 11 0.48 0.88 0.96 22.0 42.5 49.6 38.0 12 0.69 0.99 0.99 37.0 38.4 39.8 38.4 12 1.35 1.52 1.07 53.4 54.7 50.3 52.8 0.73 0.88 0.83 34.3 38.3 37.8 36.8 0.32 0.35 0.19 10.8 9.9 11.6 9.0 13 '5-"*< -'i`~t- 14 0.58 0.67 0.66 24.1 19.7 21.6 21.8 15 1.04 0.69 0.85 46.6 24.2 32.0 34.3 16 0.63 0.85 0.88 24.4 28.1 33.3 28.6 17 18 SS0.l6l3B i 11101.610118 1.50 28.7 Vi' ` 21.9 49.0 33.2 21 0.81 0.89 0.89 38.7 53.2 58.0 50.0 0.74 0.74 0.96 32.5 29.4 38.8 33.6 0.19 0.12 0.32 13.7 14.5 10.4 19 IW SBSCTl 20 0.81 1.16 1.19 37.6 59.5 43.0 46.7 21 0.51 0.46 0.54 30.6 27.1 32.6 30.1 22 0.69 0.64 0.70 39.0 32.4 37.8 36.4 23 0.64 0.75 0.63 53.9 48.5 38.4 46.9 24 0.58 0.55 0.66 44.4 39.9 44.7 43.0 24 0.74 0.82 0.82 45.2 41.9 41.3 42.8 0.66 0.73 0.76 41.8 41.6 39.6 41.0 0.11 0.24 0.23 7.9 11.5 4.4 6.6 25 1.32 1.23 1.64 50.9 30.5 36.0 39.1 26 1.17 1.05 0.99 49.4 24.6 30.9 35.0 27 1.02 1.07 1.10 56.2 48.5 40.4 48.4 28 0.82 0.98 0.98 41.2 44.8 47.1 44.4 29 0.49 1.02 1.19 28.3 46.8 53.6 42.9 30 0.65 0.82 0.89 22.9 18.6 22.5 21.3 34 0.81 1.07 0.82 65.1 82.9 58.2 68.8 0.90 0.29 1.03 0.12 1.09 0.27 44.9 15.1 42.4 21.3 41.2 12.7 42.8 14.4 mouse not warm enough canula wrong, hardly any flow, after 30 min adjusted mouse not warm enough mouse not warm enough mouse died during anaesthesia mouse not warm enough 129 p. 131 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XVI In vivo clearance of VLDL-like TG-rich particles and uptake in liver Plasm a DECAY 3H JLOO.OOl 0.SBI 104 791 m \ '68.221 100.00 94.29 85.62 73.24 42.66 21.46 100.00 105.45 100.35 77.04 58.42 34.12 100.00 100.00 95.12 78.21 58.48 42.35 100.00 88.86 77.79 69.79 55.72 45.12 100.00 . .. m m 102.16 93.48 90.47 85.65 76.79 58.37 43.94 - ,54.1a 37.40 0.00 , lit 8.84 3g 6.85 9.90 0.00 2.95 3.95 2.67 3.06 4.43 S E R U M D E C A Y 3 H (t2 = 100<H>) control 12 21 lOttOBI__ 100.00 5 1" ' 94.SS1 90.80 10 i M l f e &a1H i 77.67 20 45.24 30 41.611 22.76 SERUM HALF-LIFE 3H k ell1 1 0.0321 t 1/2 2I . 73I 2 0.053 13.08 M ouse/ORGAN M ASS (g) control 1 mouse liver heart 25.71 1.350l O.U7I spleen 0099I 2 26.2 1.370 0.136 0.099 3 100.00 95.17 73.06 55.40 32.36 3 0.040 17.42 3 26.8 1.230 0.130 0.099 4 10 0 .0 0 95.13 78.21 58.48 42.35 4 0.031 22.29 4 29.2 1.540 0.142 0.123 5 100.00 87.54 78.54 62.70 50.78 5 0.023 29.75 5 28.2 1.490 0.144 0.110 6 average std 100.00 91.50 ri1.-rmm 'i.! 0.00 " 1* f i f 83.84 r 78.27 '3 .3 57.14 55,7 43.01 36.2:8 1 - '".85 6 0.031 22.50 0.04 21.01 0.01 6.25 6 29.9 1.610 0.120 0.103 average 28.06 ' 1.45 0.13 0.1 std 1.56 0.15 0.01 0.01 SEM 0.00 1.44 1.71 2.90 4.85 0.01 2.79 SEM 0.7 0.067 0.004 0.004 O R G A N D IS T R IB U T IO N 3 H (<H> D O S IS ) control 1 liver 12.241 heart B 2| spleen 0,651 muscle gWAT -- 5.991 0 47| 2 10.51 2.91 0.66 7,10 1.01 3 10.27 1.85 0.96 5.72 0.50 4 9.13 2.26 0.72 4.78 0.92 5 6.76 1.48 0.96 3 .5 4 0.84 6 average std 7.07 8 .75 1.76 1.43 1,99 0.61 0.55 -J. , 0.77 0.18 2.97 "" '""" 3 1 5 :.'- 1 .6 6 0.62 0 .2 1 SEM 0.79 0.27 0.08 0.74 0.10 5 5 5 5 5 5 n 5 5 5 5 5 5 5 n 5 5 5 5 n 5 5 5 5 5 130 p. 132 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a D ECAY 3H SER U M D E C A Y 3 H (t2 = 100% ) fenoflbrate 7 ________ 8 ________ 9 _______ 1 0 _______ 1 1 _______ 12 average 2 10Q.0QI 100.00 100.00 100.00 100.00 100.00 .... - m m . wliil .Il5 80.84 89.69 76.68 88.35 72.04 ' TEH10 73.SM 55.10 59.25 31.88 45.20 30.25 20 39441 30 -18.201 14.75 6.85 25.47 9.86 7.41 4.32 15.37 6.36 8.66 . . 1 4 .3 3 4.77 . 6i43 std______ SEM________ n 0.00 7.54 0.00 3.37 5 5 13.16 5.88 5 7.11 3.20 0.98 5 5 SERUM HALF-LIFE 3H k el t 1/2 7 , 0.0631 u .w | 8 0.100 6.90 9 0.084 8.22 10 0.119 5.82 11 0.102 6.78 .IMI12 0.113 0.01 6.16 " " 7 # ; - 0.92 0.01 0.41 5 5 M ouse/O RG AN M A SS (g) fenoflbrate 7 mouse Hver 26.ll 1 720| heart 0.125] spleen o.oesl 8 293 1.950 0.146 0.076 9 28.7 1.830 0.147 0.108 10 29.3 1.850 0.131 0.082 11 26.6 1.690 0.124 0.077 12 average 27.7 28.32 1.490 . ..." 1 0.124 i > ...MS 0.079 0.08 std 1.16 0.18 WM 0.01 SEM 0.52 0.08 0.01 0.01 n 5 5 5 5 ORGAN DISTRIBUTION 3H (% DOSIS) fenoflbrate 7 liver 14 UBI heart 3.661 spleen 0 6S| muscle gWAT 7.S i i 2 04| 8 16.35 1.50 0.65 9.79 2.13 9 23.07 1.87 0.73 7.64 1.17 10 18.61 1.21 0.43 7.27 2.98 11 14.14 0.69 0.51 5.48 2.78 12 average std 14.35 s 1.. 17.31 1.04 r 1" '< t m 0.32 i - 5 oes 9.61 1.12 2 .1# V . few is ? SEM 1.65 0.20 0.07 0.80 0.39 n 5 5 5 5 5 131 p. 133 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a D ECAY 3H 14 0 100.00 2 96.20 5 84.54 10 53.93 20 36.23 30 1" "" mouse excluded, coded off 15 100.00 88.73 72.32 41.34 14.41 6.40 SE/W M D E C A Y 3 H (t2 s 100%; PFDS 13 2 100.00] 5 -- 9799p 10 M.OSl 20 61.981 30 33 5 3 I 14 100.00 87.87 56.06 37.66 16.13 15 100.00 81.50 46.58 16.23 7.2 1 SERUM HA LF-U FE 3 H 13 k el 0 0J9I 1 1/2 14 0.063 10.93 15 0.097 7.18 M ouse/ORGAN M ASS (g) PFBS 13 mouse liver heart 29.31 0ll.512"0l spleen ____ 0 5 *1 14 31.3 1.790 0.142 0.127 15 27.4 1.520 0.123 0.088 16 100.00 101.91 88.14 62.68 22.77 10.26 16 100.00 86.49 61.50 22.35 10.07 16 0.085 8.15 16 26.9 1.320 0.122 0.092 17 18 100.00 "*_ 11 . H 84.97 79.42 63.71 f l * w- "2; 38.65 II: . ` * 23.11 smem mouse died average 100.00;1 92.961 81.10 55.41 28.01: 13.821K-- std o.ool 7.581 6.861 10.36 11.45 13 17 100.00 93.46 74.97 4 5 .4 9 27.20 17 0.048 14.56 18 average std 0 .0 0 87.33 :im 7 59.78 / , i PfiTi 'W4 3m.4 `v" 1545 8.85 18 /.. 70.07 <0.02 3.31 17 26.3 1.500 0.117 * 0.103 . 18 average std V "^27JI 226 77 v i l 7 7 mt O R G A N D IS T R IB U T IO N 3 H f% D O S IS ) PFBS 13 liver 12.571 heart 245 spleen 0 .9 ll muscle 74 gWAT 0.521 14 15.18 2.91 0.66 5.94 0.65 15 15.74 1.54 0 .5 7 8.52 1.08 16 12.53 2.21 0 .3 7 7.50 0.00 17 10.06 0.60 0.69 3.57 4.05 18 average std . K - 13.38 2.62 ' 4.81 ' 7 ' T A '' ' 0 4 4 .... " r ' .4.4 246 1.9 SEM 0.00 3.79 3 .4 3 5.18 5.72 3.62 SEM 0.00 2.46 5.93 6 .74 4.43 0.01 1.66 SEM 1.13 0.10 0.01 0.01 SEM 1.31 0.49 0.07 1.08 0.90 n 4 4 4 4 4 4 n 4 4 4 4 4 4 4 n 4 4 4 4 n 4 4 4 4 4 132 p. 134 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a D ECAY 3H 19 20 21 22 23 24 average std 0 100.00 100.00 100.00 100.00 100.00 100.00 100.00 .........0,00 2 75.24 69.83 73.55 88.17 88.57 87.20 W3S 8.47 5 68.94 52.84 55.16 74.06 54.76 67.72 62.25 9.05 10 48.71 38.49 26.12 55.35 24.35 32.34 J 7 jt 11 "il4 | 20 21.18 16.26 30 8.92 8.14 8.12 4.80 25.07 13.06 11.84 4.49 16.26 6.28 612 3.2 S E R U M D E C A Y 3 H (t2 m 100%; PFHS 19 2 100.00 5 91.63 10 64.74 20 28.15 30 11.85 20 100.00 75.67 55.11 23.28 11.66 SERUM HALF-LIFE 3H 19 k ei 0.078 t 1/2 8.86 20 0.077 8.99 M ouse/ORGAN M ASS (g) PFHS 19 mouse liver 29.3 2.890 heart 0.142 spleen 0.094 20 27.2 2.720 0.132 0.155 21 100.00 75.00 35.51 11.04 6.53 21 0.102 6.83 21 25.2 2.780 0.101 0.073 22 100.00 83.99 62.78 28.43 14.81 22 0.070 9.96 22 28.2 2.830 0.116 0.097 23 100.00 61.83 27.49 13.37 5.07 23 0.102 6.78 23 27.0 3.170 0.128 0.102 24 100.00 77.67 average 100.00 7753 37.09 18.65 20.49 7.20 5fe"B9isa f: std 0.0* 9.97 15.75 7.39 3.SS 24 0.093 .. . 0.09 ^ 7.48 551 1.31 24 32.2 3.640 0.157 0.101 average .... 3.01 "O S 0.10 std 2.39 0.35 0.02 0.03 ORGAN DISTRIBUTION 3H f% DOSIS) PFHS 19 liver 25.93 heart 1.09 spleen 0.38 muscle 7.14 gWAT 1.03 20 22.04 1.49 0.59 7.54 0.91 21 25.47 0.77 0.26 6.84 1.26 22 23.83 1.23 0.38 8.61 0.83 23 17.18 0.59 0.33 4.88 2.45 24 16.31 0.59 0.23 6.81 4.43 average std .21.79 ...:....... f.4 5 0.37 6.35 0.lS fa g in 122 1,41 SEM 0.00 3.46 3.69 5.08 2.50 1.30 SEM 0.00 4.07 6.43 3.02 1.55 0.01 0.53 SEM 0.98 0.14 0.01 0.01 SEM 1.69 0.15 0.05 0.50 0.58 n 6 6 6 6 6 6 n 6 6 6 6 6 6 6 n 6 6 6 6 n 6 6 6 6 6 133 p. 135 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a DECAY 3H Group 5 PFOS 25 0 100.00 2 73.11 5 63.63 10 47.09 20 27.71 30 14.99 26 100.00 70.90 57.01 34.40 20.65 13.27 27 100.00 79.23 67.75 48.18 23.32 13.83 28 100.00 94.43 62.47 46.09 26.80 14.60 29 100.00 79.87 61.35 43.84 19.28 10.74 30 average std 100.00 100.00 __ 73.93 3 78.581 8.S3 57.64 34.75 12.71 '.'l 4 2 .3 9 i--.: K..,,. .&,$& 4.83 _____! 0 4 3.84 SEM 0.00 3.48 1.63 2.54 2.26 1.57 S E R U M D E C A Y 3 H (t2 = 100<Vo) PFOS 25 2 100.00 5 87.03 10 64.42 20 37.90 30 20.51 26 1 0 0 .0 0 80.42 48.52 29.13 18.72 SERUM HALF-LIFE 3H 25 k el 0.057 1 1/2 12.25 26 0.060 11.63 M ouse/ORGAN M ASS (g) PFOS 25 mouse liver 28.5 3.170 heart 0.133 spleen 0.101 26 28.4 2.930 0.131 0.098 27 1 0 0 .0 0 85.51 60.81 29.43 17.46 27 0.064 10.81 27 26.8 2.720 0 .12 1 0.094 28 100.00 66.15 48.81 28.38 15.46 28 0.063 11.02 28 28.3 2.880 0.120 0.074 29 10 0 .0 0 76.81 54.89 24.14 13.45 29 0.072 9.59 29 27.2 3.370 0.118 0.080 30 average std 100.00 ,7o.# 77.97 ?8.9fi ;' '7.48 47.00 ;'/ 54,03 7.24 17.19 V .: 6.82 6.54 T T 1 E M T .... 4,97 30 0.098 7.05 0.02 G .1,86 30 average std 26.0 1.03 2.730 ..is i 0.117 / ' ':jh G . : iS i 0.062 M s 0,02 O R G A N D IS T R IB U T IO N 3 H (% D O S IS ) PFOS 25 liver 17.74 heart 2.63 spleen 0.43 muscle gWAT 8.17 1.03 26 19.80 1.09 0.67 7.66 1.36 27 18.52 1.90 0.43 5.07 1.50 28 11.49 1.68 0.76 6.98 1.17 29 17.43 0.71 0.36 4.73 1.60 30 average 1 1 .5 3 ........ 16.09 0.75 ' 7 1-46 0.18 0.47 5.63 .. 6.37 ! 5.65 2.05 std 3.64 0.78 0.21 L41 1,77 SEM 0.00 3.05 2.95 2.79 2.03 0.01 0.76 SEM 0.42 0.10 0 .0 0 0.01 SEM 1.48 0.31 0.09 0.58 0.72 n 6 6 6 6 6 6 n 6 6 6 6 6 6 6 n 6 6 6 6 n 6 6 6 6 6 134 p. 136 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a D ECAY 14C 01 2 5 10 20 30 1 1 100,001 ' .ra m 2 100.00 92.26 90.27 87.47 65.391 49.93| 3 100.00 103.51 96.01 85.94 82.80 63.97 lvj S E R U M D E C A Y 1 4 C (t2 = 100%; control 1 2 100.00 5 98.05 10 . 91.39 20 79.71 30 70. 2 100.00 97.84 94.81 70.87 54.12 3 100.00 92.75 83.02 79.99 61.80 SERUM HA LF-LIFE 14C 1 k el t 1/2 2 0.023 30.40 3 0.015 45.01 M O U S E/O R G A N M A S S (g) control 1 mouse 25.2 liver heart 0.1171 spleen 0.09SI 2 26.2 1.370 0.136 0.099 3 26.8 1.230 0.130 0.099 4 100.00 99.46 96.21 89.28 78.41 75.25 4 100.00 96.73 89.76 78.83 75.66 4 0.011 66.01 4 29.2 1.540 0.142 0.123 5 100.00 74.50 74.98 71.93 66.09 61.97 5 100.00 100.65 96.55 88.72 83.19 5 0.007 97.63 5 28.2 1.490 0.144 0.110 6 100.00 99.73 97.16 95.25 74.20 66.15 " average std l w ; 0.00: .4 11.58 fHNN " eas] ; f i l l 6 100.00 averaae 100.00 97.43 97.08 95.50 74.40 91.93 :.... .M ai , ^ 66.32 : 68.22 std 0.00 2.84 g5.j6-2 11.44 6 0.016 j;. .45. tetti %. vW BI 43.87 4 5 6 3 8 4 - i'2&24 6 average std 29.9 28.06 1.610 ^ I * 0.120 i f " '`f e l l mm 0.01 0.103 -- " E H _____ M l O R G A N D I S T R I B U T I O N 1 4 C (% D O S IS ) control 1 liver <27.481 heart 1 82 spleen 0.771 muscle 0*001 gWAT 0 ool 2 38.23 1.59 0.94 2.87 0.03 3 28.74 1.68 1.31 2.23 0.00 4 26.78 1.41 0.88 0.00 0.00 5 17.66 1.45 1.26 0.00 0.07 6 averaae std 20.24 26.33 8.06 0.87 1.4Q 0.32 0.81 1.04 0,23 0.00 0.00 ____ M ll.Q f ____ M 31.42 SEM 0.00 5.18 4.17 3.85 3.40 4.07 SEM 0.00 1.27 2.51 3.01 5.11 0.00 11.74 SEM 0.7 0.067 0.004 0.004 SEM 3.61 0.14 0.10 0.63 0.01 n 5 5 5 5 5 5 n 5 5 5 5 5 5 5 n 5 5 5 5 n 5 5 5 5 5 135 p. 137 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a D ECAY 14C Group 2 fenofibrate 7 0 <' 100.001 2 5 97.731 10 91 9?| 20 71 30 30 52.591 mouse exttip 8 100.00 77.43 69.87 54.53 23.96 13.27 9 100.00 96.22 92.26 66.87 39.52 22.27 r 10 100.00 97.36 86.66 50.64 20.03 13.15 11 100.00 67.90 67.08 45.74 25.03 16.15 12 100.00 74.13 63.00 38.87 19.40 11.02 average 100.00 82.61 75 77 503 25.59 15.1? std 0.00 13.40 12,88 10.47 8.16 4.37 SERUM DECAY 14C (t2 = 100% ) fenofibrate 2 7 ________ 8 ________ 9 _______ 1 0 _______ U _ _______12 100.001 100.00 100.00 100.00 100.00 100.00 average 100.00 std 5 672 10 91.021 20 70.56 90.23 70.42 30.94 95.68 69.49 41.07 89.01 52.01 20.57 98.79 67.36 36.86 --',62,3* ,.89i,1i*184.98 G G f i . ? * f!V! i l l 52.44 ' iu : 26.17 w: 31.12 t - 30 OSI 17.14 23.15 13.51 23.78 14.86 C- 'A M SERUM HALF-LIFE 14C _________7_________ 8_________ 9________ 10________ 11________ 12 k el 0.024| 0.066 0.054 0.076 0.055 0.070 t 1/2 29.371 10.53 12.88 9.10 12.70 9.97 om M OUSE/ORGAN M ASS (g) fenofibrate 7 mouse 26,11 liver heart spleen 1.72ol 0. 1 2 sl o.ossl 8 29.3 1.950 0.146 0.076 9 28.7 1.830 0.147 0.108 10 29.3 1.850 0.131 0.082 11 26.6 1.690 0.124 0.077 12 averaqe std :... :...oo.:otii27.7 - .28,32 :.... -"I'M 1.490 1.76 0.124 G " J i i 0.079 - - > * !' o il ORGAN DISTRIBUTION 14C (% DOSIS) fe n o fib ra te 7 liver 48.821 heart l.l! spleen fl.aal muscle 1.5l qWAT 0.39 8 57.57 2,29 0.97 2.68 0.58 9 69.98 1.98 0.97 1.91 0.27 10 77.98 1.10 0.62 2.38 0.48 11 56.41 0.84 0.83 1.27 0.45 12 average std 56.90 63.77 9.75 1.05 MiT-filM lS 0.64 0.55 1.49 0.15 0L.7M9 iu s : e . a i fimmimm 0.17 SEM 0.00 5.99 5.76 4.68 3.65 1.95 n 5 5 5 5 5 5 SEM________ n 0.00 5 2.47 5 4.16 3.66 2.12 5 5 5 0.00 5 0.75 5 SEM 0.52 0.08 0.01 0.01 n 5 5 5 5 SEM 4.36 0.29 0.09 0.26 0.08 n 5 5 5 5 5 136 p. 138 3M#03 Mechanism o f different PFAS's on iipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a D ECAY 14C 0 2 5 10 20 30 ___ 16 100.00 104.07 97.01 84.12 47.34 36.44 ___ 17 100.00 85.81 85.14 76.13 56.77 43.42 S E R U M D E C A Y 4 C (t2 = 100%; PFBS 13 2----- 100.001 5 '"A 10 20 30 .* -- - 1 14 100.00 95.18 65.62 63.33 49.11 SER U M H ALF-LIFE 1 4C 13 k ell1 0 0 1 5 T t 1/2 ____ I 2 L 14 0.024 28.41 M ouse/ORGAN M ASS (g) PFBS 13 14 5261mouse ____ 28.il liver 1 31.3 T790 ___ 0Sl___ SLL2Zheart spleen O.I2 0 I 0.142 15 100.00 94.00 76.13 47.41 33.83 15 0.040 17.16 15 27.4 1.520 0.123 0.088 16 1 0 0 .0 0 93.22 80.84 45.49 35.02 16 0.040 17.29 16 26.9 1.320 0.122 0.092 17 18 average std 100.00 99.22 100.00 0 .0 0 5531 i P " 2.6 88.72 a s a o s 66.16 Irw a W iS M i 55 601 IB M 50.60 , . H mm-: 17 0.026 ,*. * 27.18 18 V ' 0.03 , 22.51 0,01 17 26.3 1.500 0.117 0.103 18 . J'. "H . average std 27.98 2.26 1.53 ..'.J1M , 00.1091 ______a i l O R G A N D I S T R I B U T I O N 1 4 C f% D O S IS ) PFBS 13 liver - -33.82I heart spleen muscle - '2 351 *1 f gWAT 0 001 14 47.53 2.90 1.03 3.98 0 .0 0 15 46.84 1.33 0.92 1.46 0.09 16 48.33 1.38 0.48 0 .7 5 0.00 17 40.76 0.39 1.07 0.63 0.62 SEM 0 .0 0 1.34 4.83 5.33 4.47 0 .0 0 3.06 SEM 1.13 0.10 0.01 0.01 n 4 4 4 4 4 4 4 n 4 4 4 4 137 p. 139 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a DECAY 14C Group4 PF(fS 19 0 100.00 2 87.15 5 80.98 10 64.90 20 50.02 30 35.06 20 100.00 76.23 65.54 63.52 48.81 45.05 21 100.00 77.80 74.76 55.88 36.95 27.84 22 100.00 93.89 86.27 76.12 53.51 46.59 23 100.00 81.88 75.83 67.91 47.41 39.65 24 100.00 average 100.00 93.04 82.77 95.00 77.69 71.05 66.59 58.38 49.18 45.55 Lis__ SLE std 0.001 7 .5 7.351 6 92 7 .1 1 8 -^ fs SER U M D ECA Y 14 C (t2 = lOOVo) PFHS 19 2 100.00 5 92.92 10 7 4 . 4 7 20 57.40 30 40.23 20 100.00 85.98 83.33 64.04 59.10 21 100.00 96.09 71.83 47.50 35.78 SERUM HALF-LIFE 14C 1 9 _______ 2 0 _______ 21 k el 0.032 0.018 0.039 1 1/2 21.46 37.67 17.96 M ouse/ORGAN M ASS (g) PFHS 19 mouse 29.3 liver 2.890 heart 0.142 spleen 0.094 20 27.2 2.720 0.132 0.155 21 25.2 2.780 0.101 0.073 22 100.00 91.89 81.07 57.00 49.62 23 100.00 92.61 82.93 57.90 48.43 24 average 10 0 .0 0 111 n i l . |I|IH || 8 8.97 76.36 62.75 78.33. 57.71 48.96 47.02 ..... std 0 .0 0 118 4 .79 5.94 8.13 2 2 ______ ^23_______ 24 0,026 0,027 0.025 26.46 25.67 28.29 0 .0 3 ;; 0 .0 1 ' 7 i --'""`f a 22 28.2 2.830 0.116 0.097 23 27.0 3.170 0.128 0 .10 2 24 average std m32.2 !S !y i p 8 3.640 .. 3 .0 1 ... ;.8 5 0.157 0.13 f e m i s 0.101 0.10 .. o i l ORGAN DISTRIBUTION 14C DOSIS) PFHS 19 liver 49.59 heart 0.97 spleen 0.53 muscle 2.33 gWAT 0.27 20 27.99 1.85 0.84 3 .73 0 .0 2 21 61.71 1.08 0.40 1.67 0.28 22 4 9 .3 3 1.06 0.49 1.96 0.00 23 48.4 0.890 0.439 1.778 0.76 24 45.75 0.44 0.29 0.00 0.66 average std 47.13 10.89 rm ' 0.45 ...... 0.50 0.19 . 1.91 1.20 ' 8.33 _____ & I2 SEM 0.00 3.09 3.00 2.82 2.93 3 .0 1 SEM 0.00 1.43 1.9 5 2.38 3.32 0.00 2.75 SEM 0.98 0.14 0.01 0.01 SEM 4.45 0.19 0.08 0.49 0.13 n 6 6 6 6 6 6 n 6 6 6 6 6 6 6 n 6 6 6 6 n 6 6 6 6 6 138 p. 140 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Plasm a D ECAY 14C Group 5 PFOS 25 0 100.00 2 79.76 5 78.42 10 78.30 20 73.82 30 73.97 26 100.00 77.83 71.44 67.32 68.67 65.65 27 100.00 84.08 79.37 74.51 62.95 53.61 28 100.00 97.66 85.58 80.32 70.75 57.03 29 100.00 84.26 76.10 70.70 57.81 47.97 30 average 100.00 100.00 77.32 83.49 73.48 " t i l \ 66.49 56.80 85.131 51.16 58.23 std 0. 7m .4M 5.72 7^03 9.81 SEM 0.00 3.09 2.04 2.34 2.87 4.00 SERUM D ECAY 14C (t2 = 100% ; PFOS 25 2 100.00 5 98.32 10 98.16 20 92.55 30 92.74 26 100.00 91.79 86.50 88.22 84.35 SERUM HALF-LIFE 14C 25 k el 0.003 t 1/2 239.02 26 0.005 150.68 Mouse/ORGAN M ASS (a) PFOS 25 mouse 28.5 liver 3.170 heart 0.133 spleen 0.101 26 28.4 2.930 0.131 0.098 27 100.00 94.39 88.61 74.86 63.76 27 0.016 43.32 27 26.8 2.720 0.121 0.094 28 100.00 87.62 82.24 72.44 58.39 28 0.018 39.61 28 28.3 2.880 0.120 0.074 29 100.00 90.31 83.90 68.61 56.93 29 0.020 35.36 29 27.2 3.370 0.118 0.080 30 averaae std 100.00 100.00 0.00 95.03 .. 92.91 - \ 3.79 85.99 ; - 5.64 73.46 : .:m M . , .*.84 66.17 i s '/ 14.71 30 0.015 :' o.oi 46.21 :/'.9 3 7 0.01 . W .23 30 26.0 2.730 0.117 0.062 averaae std 27.53 1.03 0.26 ' 0.08 .........0s.0i1i? O R G A N D I S T R I B U T I O N 1 4 C f% D O S IS ) PFOS 25 liver 14.95 heart 0.84 spleen 0.51 muscle 1.39 gWAT 0.43 26 17.87 0.78 1.01 1.45 0.00 27 28.00 1.54 0.57 0.00 0.18 28 15.14 1.15 0.98 0.46 0.00 29 21.60 0.45 0.48 0.00 0.17 30 17.31 1.03 0.24 0.00 1.66 averaae .s5m3 ' . 0.55 std 4.96 0.37 0.30 0.70 o . .... . 0-6* SEM 0.00 1.55 2.30 3.94 6.00 0.00 34.39 SEM 0.42 0.10 0.00 0.01 SEM 2.03 0.15 0.12 0.29 0.26 n 6 6 6 6 6 6 n 6 6 6 6 6 6 6 n 6 6 6 6 n 6 6 6 6 6 139 p. 141 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XVII In vivo clearance of autologous HDL HDL Isolation Group# fenofibrate PFOS Mouse# 5 11 IS 28 30 Isolated HDL (m m o l/L ) 0.256 0.402 0.360 0.179 0.164 Quantity pmol HDL (ml) ,, 2 0.51 2 0.80 2 0.72 2 0.36 2.4 0.39 Needed for 0.32 pmol (m l) 1.25 0.80 0.89 1.79 1.95 NB: Groups PFHS and PFOS not enough for 0.4 pmol. Therefore all groups 0.32 pmol HDL labeled, in stead of 0.4 pmol. Group# fenofibrate PFOS Mouse# 5 11 15 28 30 3H dpm aftendjaj^ste (10 pi) vial 1 18493.9 18179.9 vial 2 18398.2 16939.5 19041.4 17957 20370.3 17930.9 19147.3 19078.8 3H dpm | per 200 pi 368921 351194 394117 358879 382261 stock 1.5 1.5 1.5 1.5 1.5 needed 200000 dpm /200 pi ml+ 1 ml PBS ml+ 1.27 ml PBS ml+ 1.13 ml PBS ml + 1.46 ml PBS ml + 1.19 ml PBS ml + 1.37 ml PBS | I Group fe n o fib ra te fe n o fib ra te fe n o fib ra te fe n o fib ra te fe n o fib ra te AVERAGE SD * ' ;PFHS- PI O S PFOS PFOS PFOS pro 5 AVERAGE SO Mouse# 1 0 1 100.0 2 100.0 3 100.0 4 100.0 6 100.0 100.0 0.0 8 100.0 9 100.0 10 100.0 13 100.0 14 100.0 100.0 0.0 16 100.0 17 100.0 18 100.0 19 100.0 20 100.0 100.0 0.0 22 100.0 23 100.0 25 100.0 26 100.0 27 100.0 100.0 0.0 29 100.0 31 100.0 32 100.0 33 100.0 35 100.0 100.0 0.0 % of injected dose In time (h) 1248 74.9 57.5 40.7 21.3 63.6 56.8 39.9 20.5 71.4 58.2 39.2 20.7 76.7 61.9 60.0 48.8 38.9 34.4 22.6 16.1 69.7 6.6 78.6 54.5 56.2 4.3 67.0 54.7 38.6 2.5 49.5 45.4 20.3 2.5 32.8 31.3 87.6 63.3 55.2 63.9 44.5 48.5 33.7 30.3 54.5 59.4 45.8 30.6 67.7 60.0 46.7 31.7 14.9 5.4 2.1 1.5 63.1 65.3 55.6 60.3 33.6 31.1 14.6 16.1 59.6 49.4 31.2 13.1 59.4 39.6 29.9 17.9 73.9 64.3 5.9 47.9 53.2 62.2 56.0 53.6 54.6 5.2 58.6 48.3 60.1 58.7 51.2 55.4 5.3 56.0 52.2 8.0 30.7 38.7 41.0 43.2 40.4 38.8 4.8 40.4 39.8 40.6 45.5 33.7 40.0 4.2 38.4 32.8 3.4 15.8 23.5 24.8 27.8 26.4 23.6 4.7 24.4 21.9 24.9 26.7 19.8 23.5 2.7 23.4 17.0 4.0 6.2 9.6 9.5 14.1 9.3 9.7 2.8 10.1 8.4 9.4 10.2 5.5 8.7 1.9 | t 1/2 24 (h) 4.3 3.39 3.6 3.28 3.7 3.32 8.0 3.48 5.1 2.83 4.9 3.26 1.8 0.25 9.5 4.61 8.7 4.13 10.8 4.45 8.1 4.27 9.9 4.14 9.4 4.32 1.0 0.21 2.5 2.75 2.1 2.84 2.1 2.58 2.2 2.81 3.9 3.48 2.6 2.89 0.8 0.35 0.9 1.80 1.1 2.17 1.1 2.21 2.5 2.56 1.5 2.20 1.4 2.19 0.7 0.27 1.1 2.24 1.6 2.07 0.9 2.20 1.8 2.30 1.0 1.81 1.3 2.12 0.4 0.19 FCR (pools HDL-C/h) 0.204 0.211 0.209 0.199 0.245 0.21 0.02 0.150 0.168 0.156 0.163 0.168 0.16 0.01 0.252 0.244 0.269 0.247 0.199 0.24 0.03 0.385 0.319 0.314 0.271 0.315 0.32 0.04 0.309 0.334 0.315 0.302 0.382 0.33 0.03 HDL-C (mM) 1.39 1.02 0.89 1.36 0.89 1.11 0.25 1.70 1.94 1.78 1.96 2.22 1.92 0.20 1.26 0.74 0.84 0.66 1.80 1.06 0.47 0.13 0.21 0.48 0.61 0.57 0.40 0.22 0.31 0.37 0.05 0.34 0.21 0.25 0.13 CR (mM HDL-C/h) 0.284 0.215 0.185 0.271 0.217 0.23 0.04 0.255 0.326 0.278 0.318 0.371 0.31 0.05 0.318 0.182 0.226 0.163 0.357 0.25 0.09 0.049 0.067 0.152 0.166 0.178 0.12 0.06 0.095 0.124 0.015 0.102 0.080 0.08 0.04 140 p. 142 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XVIII Liver microsomal DGAT activity Group Mouse# protein microsomes DGAT-1 activity (mg/ml) (nmol/min/mg protein) 4.9 1.73 7.4 3.53 6.7 1.62 7.1 1.77 sacrlficetl fgr.HDL HdWHir 7.8 jH W ftK bB frcaiW tjq 2.42 *' li'ijjil' DGAT-2 activity (nmol/min/mg protein) 1.29 1.33 1.85 2.08 * %r, 1.43 l .`. b fe n ofibrate fe no fib rate fe n ofibrate fe n ofibrate fe n ofibrate fe n ofibrate fe n ofibrate AVERAGE SD '-i-'Vy.l mouse 1 mouse 16 Total 14C TAG based on average efficiency DGAT-2 is based on 1 measurement 141 p. 143 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XIX Liver lipids Group Mouse# Cage# 11 21 32 42 52 63 Eartag LR 01 12 01 20 02 00 fe n o fib ra te fen ofibrate fen ofibrate fen ofibrate fen ofibrate fen ofibrate AVERAGE SD life 1 SSBI K P pipr ' * -J , .< pros PFOS PFOS PFOS PFOS PFOS AVERAGE SD 8 9 10 11 13 14 15 16 17 18 19 20 22 23 25 26 27 28 29 30 31 32 33 35 4 21 4 12 5 20 5 10 6 01 6 21 1 17 2 17 1 07 1 08 2 18 0 19 0 2 10 2 1 10 0 2 11 1 1 12 2 0 12 0 2 12 0 0 13 1 0 13 0 2 13 0 1 14 1 2 14 0 2 15 FC 13.0 16.2 14.2 15.3 12.5 14.7 14.3 1.4 11.6 11.7 10.7 10.8 12.5 12.7 11.7 0.8 10.7 12.7 15.0 11.4 10.4 9.5 11.6 2.0 14.8 13.5 14.0 10.3 12.8 12.8 13.1 1.6 16.0 15.1 15.3 17.8 16.3 18.8 16.6 1.5 Liver lipids (gq/mq protein) CE TC 20.4 33.5 26.0 29.9 42.3 44.1 23.9 39.3 25.9 21.4 38.4 36.0 24.6 3.5 38.9 3.9 12.1 23.7 11.9 9.9 23.6 20.6 10.5 13.0 21.3 25.5 13.9 11.9 1.5 14.1 22.2 24.0 16.9 8.5 9.4 15.9 6.4 31.0 22.9 28.6 16.6 19.1 27.2 24.2 5.7 39.9 43.4 39.9 64.3 41.5 56.4 47.6 10.3 26.6 23.6 2.3 24.7 34.9 39.0 28.3 18.9 18.9 27.5 8.3 45.8 36.4 42.6 26.9 31.9 40.0 37.3 7.0 55.9 58.5 55.2 82.1 57.9 75.1 64.1 11.5 TG 77.9 106.0 72.2 70.1 53.0 67.7 74.5 17.5 50.9 60.5 39.2 51.8 76.6 80.2 59.9 15.9 35.4 96.7 104.0 47.8 41.5 45.7 61.8 30.2 140.1 134.8 122.0 75.3 94.1 114.4 113.5 24.8 171.9 186.2 195.9 275.7 193.8 279.4 217.2 47.5 | 142 p. 144 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XX Liver microarray analysis TNO Q uality o f Life OraniWtovcor CfoKrKAlepcwSL<)iSKosoerntfAricsndtOrpnta" TNO report Transcriptome analysis of effects of 3M compounds in liver of ApoE.'L.CETP mice Utrechtseweg 48 P 0 Box 360 3700 AJ Zerst The Netherlands www.tno.nl T +31 30 6 W 4 I 44 F +3130 695 72 24 Date 5 June 2009 Author* Maqan .an Erk Copy no u-f uo No of copies no ->froptp. Numb Numb of page of appendices 10 of .ipy-eiuii <*' Customer 3M Projectaame PKiiectname Projectnumber rVcir-. mumhe Ail limits reserved No part of this publication mav be reproduced and or published by pnut photoprint, microfilm 01 any other means without the previous written consent of TNO In case this report was drafted on instructions. the rights and obligations of contracting parries are subject to either the Standard Conditions for Research Instructions given to TNO. or the relevant agreement concluded between die contracting parties. Submitting the report for inspection to parries who have a direct interest is permitted. S 2009 TNO 143 p. 145 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 TN O report 2/10 Contents 1 Introduction..................................................... 3 22.1 MRNeAthoisdosla..t.i.o..n...a..n..d...m...i.c..r.o..a..r.r..a.y...h...y..b..r.i.d...i.z.ation.................................. 44 2.2 Gene expression data analysis........................................................................................ 4 333..12 LRDiepifsifduerlmtesne.t.ti.a.a.bl.lo.y.l.ie.s.x.m.p...r...e...s...s....e...d.......g....e...n.....e...s.....................................................................................................................................................................................................556 333...232..12 PPTraretah-nswescaleryciptaetnidoanlliysfstaiosc.tf.o.g.r.e.a.n.n.e.a.s.l..y....s....i..s....o....f......g....e..n....e....s......i..n....v....o....l..v....e....d......i..n......l..i..p....i..d......m......e....t..a....b....o....l..i..s....m.................................................................................................6?? 4 Conclusions.................................................................................... 10 144 p. 146 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 T N O report 3/1# 1 Introduction YMD (Vascular and Metabolic Diseases team is conducting a study for 3M. ienffveecsttsigtaotintghetheeffeefcftesctosfoffen3ocfoibmraptoeuniFdFs).inaAPpPoAE3RLalCph'EaTPacmtivicaetora.ndStcuodmypcaorimngpotuhnedses (hPuFmBaSn.s PaFndHSm. PnaFnOirSe). aTrweopeorf-ftlhiieorsotu-adlykyclosumlfpoouuantedss. wTehreesetackoemn pooffunthdes macacrukmetu, loantee iins hsleteislasllthhinarrmiusskfeus..l.F3Murthaeimrmsortoe, itnhvisesktnigoawteledefgfeecwtsillohfetlhpetsheemcotmopdoeuvnedlospincolmigphotuonfdspothteanttaiarel Tcoramnpsocunnpdtosmoen aanaplryes-isseloecftethde sleitveorf wgeilnlesbeanpderfoonrmliepdidtoanidnvleipsotipgraoteteienffmecettsabooflisthme pfeanthowfibaryast,e and to compare these effects to the effects of PPAR-alphn activator 145 p. 147 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 T N O report 2 Methods 4/10 2.1 RNA isolation aud microarray hybridization RRNNAA. wmaicsroisaorlraatyedanfarloymsislwivaesrscaorfrieAdpoouEt3aLt.CSeErTviPcemXiSceB. .AVf.te(Lr eqidueanli,tythecoNnetrtohlerolafndthse) using the Asymetrix technology platform and Affmetrix GeneClnp mouse genome MOE430-2.0 arrays. Data were sent to TNO. 2.2 Gene expression data analysis Quality control of microarray data was performed using BioCouductor packages (a.o. sfiilmesp)lewafefrye annodrmafatlyipzelmd)u. sAinlgl stahmepGleCsRpMassAedaltghoerQithCm. R(gawc-rsnigianaslloinwt)e.nFsiotiresan(nfrootmatiConELo-f pMroNbBeIs andCDsuFm-fimlearizawtiaosn ofusseigdnals(bfarosemd proobnes reEpnrterseeznGtiennge.onevgeersnieonthe c1u1s.0to.2m) (http: brflinarrayjubm.med.umich.edu Bramarray Database CustomCDF cdfreadme.ht mGgee)n.neeTsihdiwesnetrrieefsieufrilsltte.edreidn expression values for 16331 genes, lepresented by unique Entiez for expression above 5 in 3 or more samples, resulting in a set of t(1rL1aun5ns8if7noar-gmehentdetps(:btahbsaioetin2wf).a.wsSehtuaist.eiesddtiucf.aoalrualifnmuarmltyhaseirs) awwniaathslyspcioesrrfreoGcrmteionened feuoxsrpinmreguslsttihiopenlemdtoeasdtateinrawgt.eedrCeut-tl-toeogsf-tf for statistically significant changes was set at q-vaiue 0,05 (q-value = p-value wcTSoorerafrrtneewsccsatirergiedpntifGifooimrcnambfnaHutcll.yttoiMpdrliaeufnfnteaeirclseyhtnsi.nitsigGawl).elyarmsepaxnepryrf)eo.srsmFeoderdaentnadcBhthibacthoowmsepproheuerinnedv,sooglfvtewendeasrienwvlei*pr.e2id1sm(eGleeetcantbeoodmliatshrmiax.t loIaintxpviilddoeaalvtsbietoimonos,neyenncthtoihonfeltselhiisspet,eidfroofmalllteotmytwaebiantoacglbiidsbomiloibslwimoogas,syiccndahtelhoteeplserrmisostc,eienrsoefsadlettsbby:aiosliaespcdyiinddotnhbmeiGoseisestyna,nebtcohOhleionsstlmioessl,,otlegifrpyaoi'tldtaynccnaaaottcaatiabbdtooilloiibnssemmitan,, lipoprotein metabolism, lipid transport, cholesterol transport. Imneaanddeitxiporne,ssTi-opnroifnilethreacnoalnytsroisl wgraosupp.erTfohrims eadnaulyssinisgreexsuplrteesdsioinn svcaolrueess(ct-oslcloercetse)d afnodr tsthhigeenmmifiaacjjaoonrriictteyy vooafflutthheees ggfoeernnepessatiihnnwtthahyeesppaaantthhdwwbaaiyoyloaargreeicudaplo-wprernog-cureelasgstueeldsa.;teAad.npePogasatihttiiwvveeaysssccooarrneedmmbeeioaalnnossgtithchaaatlt pArohcieesrasercshwiciathl csliugsntiefriicnagntosfctohreesse(p>a4thowra<y-'s4)anind 5bioorlo6giacuaulnparloscpeessregsroaunpd wtheerne ssceolercetsedin. ah samples was generated in GenePattern (Broad Institute. MIT. USA). 146 p. 148 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 T N O report 3 Results 5/10 3.1 Differentially expressed genes Icnomthpearsetdattiostitchael caonnatlryoslisg,rotuhpe. fFeFnorfeisburlatteed(iFnF2)9. 2P4FdBiSff.erPeFnHtiSallyanedxpPrFeOssSedggreonuep.sPwFBerSe resulted in 438 differentially expressed genes. PFHS resulted in 4230 differentially expressed genes and PFOS resulted in 3986 differentially expressed genes (figure 1). 4500 significant changes FFv*Ct PFBS v* Cl PFKSrtCl PFOSvsCt Fcoigmupreare1d toNcuomnbtreorl. of differentially expressed genes m each of the interventions Figure 2 indicates the overlap ui diffetentiallv expressed genes for FF and each of the ttohrtehee33MMccoommppoouunnddss. aOrevearlaslol.r4e3g-u5la4t%edobfythFeF.genes differentially expressed in response Fshuortwhnerminofrieg,utrheer3e. Iins ctootnasli.d2e9ra4b5legeonveesrlaarpe irnegguelnaetesdrebgyulbaotethd cboymPpFoHunSdsa,ndinPaFdOdiSti.onas. 262 of these are also regulated by PFBS. 147 p. 149 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 T N O report FBS 6 i 10 FHS Freisgpuorens2e.toVFenFnanddiaPgrFaBmSs. sPhFoHwSinagndovPeFrOlaSp rienspseectstivoeflyd,ifferentially expressed genes in Figure 3. Venn diagram showing overlap between genes regulated by PFHS, PFOS and PFBS. 3.2 Lipid metabolism 3 2.J Preselected list ofgenes A set of genes of interest was defined, with a focus on PPARa metabolism and cholesterol metabolism. The expression changes as a result of the 4 interventions aie listed m the excel file added to this report. Of the S? genes in the list. 72 were expressed amntdeivtheentimonasj.orEitxyprwesassiondioffferseenvteinallgyeneexspwreasssendotimn eraessupioends(egetnoesonweeroerno11t10presoenfttohne the microarray) and expression of 10 genes was below detection. FF intervention resulted in 31 up-regulated genes and 7 down-regulated pre-selected genes. Of the 31 genes up-regulated by FF. 25 were also significantly up-regulated by PFHS, 18 were also up-regulated by PFOS and 3 were also up-regulated by PFBS. The selected set of 148 p. 150 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 TNO report 7/10 genes was grouped into smaller sets of genes related to triglyceride metabolism u(lpiptaoklyestirsa.nspofartttbyindinagc.id YtrLigDlyLceraidsseembslyangtehefosirsm, atiobne.ta PL oxeixdcarteiotino,n), fcahttoylestearcoild ((cHhDolLesftoerrmolatsiyonnt.hHesDisL, mstaotruargaet,iounp.tHakDeL, mmeotdaeblolilnisgmd,esetxacbrielitsioanti)ona.nHdDHLDuLptmakeet)a.bolism 3.TT TInranasdcdriiptitoionntfoactfoorcuasnianlgysiosnofthgeenepsrei-nsveolelvceteddinlhisptidomf egteanbeosl,ismwe aimed to identify transcription factors relevant for regulating the expression of genes involved m hpid metabolism in response to die different compounds. Genes involved in lipid metabolism were identified from Gene Ontology annotation in at least one of the following biological processes: lipid biosynthesis, lipid catabolism, lipid biosynthesis, fatty acid biosynthesis, fatty acid metabolism, fatty acid beta olixpiodpartoioteni,n cmheotlaebsotelirsoml ,mlieptiadbtorlainsmsp,orcth, ochleoslteesrtoelroblitorsaynnstphoersti.sN, ecxht,olfeosrteeraoclh ccaotmabpooluisnmd,, die significantly differentially expressed genes involved in lipid metabolism were gseelneecstefdo:r 1P4F3BgSe.nTeshefsoer sfeetnsoofifbrgaetne,es1w72ergeesnuebsmfoitrtePdFtHoSt.ra1n6s2crgipetnioens ffoarctPoFr OanSalaynsdts2i3n tahnealBysibislioisspAhedrdeedsotfotwtahree rbeypoGrte.noTmheatirxe.suAltns efxilceellifsitlse tcroannstacirnipintigonthefacretosursltsthoaft tAhirse expected to play in role in regulating expression of the significantly differentially expressed genes involved in lipid metabolism, based on presence of a binding site in the promoter region combined with cocitation in literature, or based on co-citatiou m leiqteuraaltuforer PoFnHlyS.(fPoFrOtSranasncdriPpFtiBoSn: cPoPfAacRto-rasl)p.haT.hPePtAoRp-tgharmeemaofantrdanLsXcrRip-btieotna factors is 3.3 Pathway analysis Figure 4 gives an overview of functional gene sets (based on gene ontology annotation) rreegguullaatteedd ibnyaotnleeaostr 5maonteiminaltserovfenotnieonosr.mSeolreecttreedatfmunecnttiognroalupgse.ne sets were significantly The heatniap shows a number of groups of gene sets that show a similar profile of response. Hie first cluster consists of gene sets related to transcription, which are most sretrloantegdlytodoiwnfnla-rmegmualtaitoend. bFyFPrFeHsuSltsanidn PdFoOwSn.-rTeghuelasteicoonndofcltuhsetseer gcoennesisstestso.fHgoenweevseetrs. PFBS results in up-regulanon of some gene sets, most clearly of acute-phase response manedtabinoflliasmmmaantodryenreersgpyonmsee.taTbohleismthir(dmictloucshteorndcroionnsi)s.tsFFo fmgoesnte stsreotsngrleylautepd-retgoulhapteids lipid metabolism, the effect of PFHS and PFOS is similar but less pronounced. A small set of gene sets related to Acyl-CoA hydrolysis or metabolism are strongly up-regulated bgyenFeFs.etPsFrHelSateadndtoPgFlOutSat.hTiohneesetraenffsefcetrsasceouanldd bmeorneoloaxteydgeionaPsePAacRtiaviatyctiavreatisopne.ciLfiacsatllyly, dvoiswuanl-irzeegduliantefidgubrye P5.FHS and PFOS. The effects m these clusters of gene sets are also 149 p. 151 3M#03 Mechanism of different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 T N O report 8 MO nbMpUDtMiKn*oaHuaiMwnftryicalm t n y A owofc--<*rpres&> wawrr art C m orK lM BuWIenor M fwrt*ol n jmIiIbu otm iW wH prw > I m M rwoiMtoc*, nuoT trgnsoWton mgkWeri etwweripbon, cr t am tnpoor.[fc-rtpendere oi itm u i^ uerItW MjeytUte*. b><xe>i mauNfcan K it imNaq k (ro cm nuetoc ewlW 'Bljn} MpRytitM tMSMac proeoiS nutfe* tiucfeobtt*, nurtoowrt.nurtM* texptint*nmunemoerue tanun* cy!cftieownei*uoet* ***<*imektepnws'e*'topom e reepens ic veundoq W eeiM krv f# m * tubi* *tbaaetatar '-epkn m vi wr*-tnw crcftM uM Me m *eent|Mn>4 eetMoen, etecMC IpWttMriheBojTOceM cmi>c actanctaboKera: wenoeerbexrk eMiMCeMc rtfty eco VMtabaK precast mtachoncktan enyetow wPaumUMwMwiiliaretope sstoWtohdhatpart >tocflon*latm r t m oraaraMt Inrwc wwshara Mtocwicnwffw fH ttw tnt orowartu inuilswss fewberuaw! rtrocciMr HraMMarpart HraWar naaftrane to M MW**rsrW'beurK<crenaa w^Wyram 'Vscvttwvuaras* cvtC&P. <na(aMc procact Cna hyWcMtsaactMy acyPCo* t*rnie * aertrt1 jmMvt efta*** ewbexytertwase actMtr Molestar hydrde*e ae*iviv crtcytatm t.rt>4>tsan. |M>I mciMybgMt*Ktvy.tarurg catte" cum iy n BtewOote process entadw m Ssdok presets cwiMwiS bt*e fcnj-ohaotdtvacid-'CtA sa Mv-aoat9sactivtY O-aevfeartaMrMa aOMy tantwhariMdy actnewfcdi 'fanstarata actvty,trarctar* Sfc(NWcaatraoMar****M WadiariawraraeHty vwttanrt: nnnutKir^Bnasetct*y *axi**adurjM*adfttv arttV Figure 4. Cluster of scores for functional gene groups (based on Gene Ontology) that paroesitsiivgenisfcicoarnetl(yi.ea.ffmecatjeodritiyn oaft gleeansets 5upa-nreimguallasteodf) aantdrebaltumeenintdgicraotueps. nReegdatiivnedisccaoteres (i.e. majority of genes down-regulated) 150 p. 152 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Ftriegautmreen5.tsR. esponse profiles of clusters of gene sets that show a similar response to the 151 p. 153 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 T N O report 10/10 4 Conclusions PFBS has a more limited effect on transcriptome of liver m ApoE3L.CETP mThiceerethisana PcoFnHsSidearnadbPleFoOvSerlap in genes regulated by FF and those regulated by OaPcFvtHievrSiatlyol rgaPneFudeOliSspeitdamnaeltyasbiosliisnmdicaarteesmtohastt sftartotyngalcyidupm-reetgabuolaltiesmd .byCoFAF.hfyodlrloowlaesde bthyesPeFgHeSneasnedtsPaFreOnSo.t FsFignuipfi-craengtulylataefsfelcitpeidd bbyiotshyen3thMesicsomanpdoulnipdisd. FtrFandsopwornt-, roefgauclauttees iinnffllaammmmaattoiroyn raensdpoinmsme.uSnpeerceifsipconesffee.cPtsFBofSPrFesHuSlteadndin PuFpO-rSegaurleatuiopnrreegguullaattiioonn oofftrgalnustactrhipiotinoen.transferase and monooxygenase activity and down- 152 p. 154 3M#03 Mechanism o f different PFAS's on lipid and lipoprotein metabolism in ApoE3L-CETP mice TNO project number 031.12685 Appendix XXI Summary table 153