TOXICOLOGICAL RESEARCH PROGRAM IN PERFLUORINATED CHEMISTRIES John L. Butenhoff, Ph.D. Medical Department 3M Company Value of Legacy Fluorochemical Toxicological Research • • • • Association of Chemistry with 3M Reduced Uncertainty in Risk Assessment Credibility in the Health Science Field Causal Perspective for: – Employee medical surveillance – Epidemiological investigation • Defensive Barriers to Litigation • Application to Current and New Products Causal Perspective for Epidemiology The Environment and Disease: Association or Causation?1 1Hill (1965) Proc Royal Soc Med 58, 295-300. Bradford-Hill Criteria • Strength • Consistency • Specificity • Temporality • Biological Gradient • Plausibility • Coherence • Experiment • Analogy This area has become increasingly important as new epidemiological studies are released. Flood of New Science • Frequency of new scientific papers has • • increased. Appreciation of the whole field by the newer authors is obviously limited. Increasing attempts to associate effects with general population exposures. 3M Publication Impact • 54 3M-authored, peer-reviewed fluorochemical papers cited 1804 times in scientific literature. 160 140 100 80 60 40 20 Individual Publication by Year 20 09 20 08 20 07 20 06 20 05 20 04 20 03 0 19 9 19 8 9 20 9 0 20 0 02 Times Cited 120 Two Broad Areas of Research • Pharmacodynamics – Biochemical interactions – Biochemical and physiological responses • Adaptive or pathological • Pharmacokinetics – Absorption, distribution, metabolism, excretion Current Research Strategies • Internal 3M research – Pharmacodynamics and pharmacokinetics • Collaborative research – E.g., USEPA NHEERL, Universities • Contract research – E.g., TNO • 3M-sponsored university research – U of MN, Stockholm U, UKMC, U of Houston, Penn State Chemical and Physical Properties • Perfluorinated alkyls (PFAs) – – – – – – – Exceptionally stable Non-reactive Solubility varies Amphiphilic, “organic” acids with low pKa Essentially dissociated under most conditions Surface active Low Van der Waal’s forces in carbon chain Physical/Chemical Determinants • Resemble free fatty acids (FFAs); although… – Non-reactive – Not metabolized PFOA – Do not enter into the biochemical reactions that use fatty acids as substrate. Octanoic acid • However, PFAs may present as FFAs. – Transporters – Receptors – Carrier proteins Oleic acid 18:1 (9) Biological Interactions of PFAAs • Expected interactions – Biological membranes – Organic anion transport processes • Induction, competition – Protein ionic binding sites • Competition with endogenous substrates (e.g., FFA, hormones) – Activation of biochemical processes • Nuclear receptor activation (e.g., PPARα) Pharmacodynamics Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • • • • • • • • Liver function and health Serum lipid chemistry Body-weight change Tumorigenesis Reproduction/Development Immune system Nervous system Endocrine system (hormones) Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • Liver function and health • • • • • • • Serum lipid chemistry Body-weight change Tumorigenesis Reproduction/Development Immune system Nervous system Endocrine system (hormones) Monkey Liver at 0.75 mg/kg/d K+PFOS (human equivalent dose = 53 mg/d) Electron micrographs of liver cells from six-month monkey study with K+PFOS1 Male control 184 d 1Seacat Male 0.75 mg/kg 184 d et al. (2002) Toxicol Sci 68, 249-264. Male 0.75 mg/kg after 211 d recovery Liver Effects • Increased liver weight – Enlarged cells (hypertrophy) • Adaptation or pathological change? – Increased numbers of cells (hyperplasia) • Pathological change (hyperplasia → tumor → cancer) • Metabolic and biochemical changes – e.g., increased burning of fat • Human relevance – PPARα activation – Other processes (e.g., CAR and PXR) – Adaptation vs. pathological change Diversion #1 - Molecular Biology Xenobiotics, Hormones, Cytokines, Growth Factors Fatty Acid Phenobarb. PFOA PFOA Receptors & Transcription Factors Gene Expression, Transcription CYP4A mRNA Protein Synthesis Metabolism PPARα CAR CYP2B mRNA Enzymes Other Compounds Metabolic Products Based on: Waxman (1999) Arch. Biochem. Biophys. 369, 11-23. Some Common Nuclear Receptors Controlling CYP Induction Receptor • PPARα • PPARγ • CAR • PXR • LXRα • FXR • RXR • TR • Ah1 1 Typical Activator Fatty acids, Fibrates Rosiglitazone Phenobarbital Steroids, Dexamethasone Cholesterol Bile acids Retinoic acid Triiodothyronine Polycyclic aromatics, Dioxin PAS transcription family member, not a nuclear receptor Experimental Approaches • Engineered nuclear receptor domains • Primary cell culture • In-life exposure followed by biochemical • and molecular biological methods Transgenic mouse studies – Remove or repress receptor – Insert human form of receptor Species Differences in PPARα • Humans less responsive than rodents – Lower human levels of PPARα – Human PPARα not associated with hyperplasia • Use of genetically-modified mice1,2,3,4 – Using specific activators of PPARα • mPPARα (natural) – hypertrophy and hyperplasia • hPPARα – hypertrophy but NO hyperplasia • No PPARα – NO hypertrophy and NO hyperplasia 1Cheung et al. (2004) Cancer Res 64, 3849-3854. 2Morimura et al. (2006)Carcinogenesis 27, 1074-1080. 3Shah et al. (2007) Mol Cell Biol 27, 4238-4247. 4Yang et al. (2008) Toxicol Sci 101, 132-139. Differential Activation of PPARα in an Engineered System PFOA is a weak activator of PPARα compared to ciprofibrate and natural fatty acids. Nuclear Receptor Activation by PFOA and PFOS in an Engineered System • Mouse, rat, human receptor forms • PFOA and PFOS activate PPARα – Less potent than clofibrate and endogenous long chain FFA • PFOS and PFOA are weak agonists for PPARγ – Much less potent than rosiglitazone • No activation of RXRα or LXRβ • PFOA and PFOS more specific and less potent than endogenous long-chain FFAs. 1 Vanden Heuvel et al. (2006) Toxicol Sci 92, 476-489. Human vs. Rat Liver Cells in Primary Culture and PPARα Activation by PFAs Cyp4A1 mRNA  All PFAs at 25 μM in cell culture media.  C ≤ 4 PFAs have little or no effect. Courtesy of Dr. Kendall Wallace, U of MN. Carboxylates Sulfonates Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • Liver function and health • Serum lipid chemistry • • • • • • Body-weight change Tumorigenesis Reproduction/Development Immune system Nervous system Endocrine system (hormones) Serum Lipids • Hypolipidemia – Reduced serum total cholesterol with • PFOS; PFHxS; PFBA; PFOA (not consistently) • Early onset clinical observation in lab animals – Apparent reduction in HDL (female monkeys) • PFOS • A basis for MDH HRL for PFOS – Mode(s) of action • PPARα activation (evidence strong) • HMG CoA reductase inhibition (evidence weak) Serum Lipids • Hyperlipidemia – Inconsistent epidemiological association of serum PFOS and PFOA with increased serum cholesterol in humans • C8 Science Panel Report – “In multivariate models adjusting for other factors … all lipid outcomes except HDL were higher when serum PFOA and PFOS levels were higher. The positive trends were statistically significant in all cases, again with the exception of HDL.” A Case of Reverse Causation? • Do higher serum lipids increase serum • • binding capacities for PFOA and PFOS? Is there a experimental basis for causation? Continuing areas of research – Serum lipid biochemical studies – Binding of PFOS and PFOA to serum lipoproteins – Pharmacokinetic distribution studies Serum Lipids Experimental model: – – – – – “Humanized” lipoprotein-profile transgenic mice Developed by TNO in The Netherlands Studying PFBS, PFHxS, PFOS Western-style diet (high fat) PFOS, PFHxS, PFBS at ~ 3, 6 and 30 mg/kg body weight/d in diet, respectively. APOE*3Leiden Mouse Study • PFOS and PFHxS – – – – – Reduced total cholesterol and triglycerides Decreased cholesterol 7-α-hydroxylase Increased liver size Increased fatty acid oxidation Suggests a PPARα agonist mode of action • PFBS had no effect. APOE*3Leiden.CETP Mouse Studies • Incorporate cholesterol ester transfer protein • PFOS and PFHxS – reduced total cholesterol and triglycerides via • decreased VLDL production • increased VLDL lipolysis and clearance • increased HDL clearance • PFBS – reduced total cholesterol and triglycerides • to a lesser extent and via • reduced VLDL production and • increased VLDL clearance • no effect on HDL PFBS, PFHS, PFOS & Hypolipidemia APOE*3Leiden.CETP Mouse Group 1: control Group 4: 0.006 % PFHS Group 2: 0.03 % Fenofibrate Group 5: 0.003 % PFOS Group 3: 0.03 % PFBS Cholesterol (mmol/L) 2.5 VLDL 2.0 1.5 HDL 1.0 IDL 0.5 LDL 0.0 0 5 10 15 Fraction 20 25 Association of PFOS and PFOA with Hyperlipidemia in Epi Studies • APOE*3Leiden mouse model argues • • against causation. Serum binding studies show affinity of PFOS and PFOA for lipoproteins. Additional serum binding work may help prove reverse causation. Percent Binding to Isolated Human Serum Protein Fractions at 10 µg/mL PFBS PFHS PFOS PFOA Albumin 93.5 > 99.9 99.8 99.7 γ-Globulin < 0.1 26.1 24.1 3.0 α-Globulin < 0.1 13.7 59.4 11.0 Fibrinogen < 0.1 < 0.1 < 0.1 < 0.1 α-2-Macro- globulin < 0.1 < 0.1 < 0.1 < 0.1 Transferrin < 0.1 6.4 <0.1 2.1 β-Lipo-protein < 0.1 64.1 95.6 3M Company and Southern Research Institute, unpublished data 39.6 Percent Binding to Isolated Human Serum Protein Fractions at 10 µg/mL PFBS PFHS PFOS PFOA Albumin 93.5 > 99.9 99.8 99.7 γ-Globulin < 0.1 26.1 24.1 3.0 α-Globulin < 0.1 13.7 59.4 11.0 Fibrinogen < 0.1 < 0.1 < 0.1 < 0.1 α-2-Macro- globulin < 0.1 < 0.1 < 0.1 < 0.1 Transferrin < 0.1 6.4 <0.1 2.1 β-Lipo-protein < 0.1 64.1 95.6 39.6 Additional Experimental Approaches • Binding interaction studies – Exploit • 35S-PFOS made at Stockholm University in Åke • Bergman’s lab. Biochemical expertise of Joe DePierre’s research group. • In-life experiments under consideration – Exploit APOE*3Leiden.CETP mice • Dietary manipulation of lipoprotein profile Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • Liver function and health • Serum lipid chemistry • Body-weight change • • • • • Tumorigenesis Reproduction/Development Immune system Nervous system Endocrine system (hormones) Body Weight • Decreased weight gain in growing animals Rat pups • Weight loss at sufficient dose Male monkeys Body Weight • Hypotheses – Increased burning of fat • Uncoupling of oxidative phosphorylation (mitochondria) – Only with certain sulfonamides (NOT PFOS or PFOA) • Increased mitochondrial bodies (PFOA) – Evidence from rat and monkey studies • PPARα activation – Strong evidence from mouse studies – Decreased appetite • Some evidence – Malabsorption of nutrients • Not fully investigated Biological Interactions - Mitochondria • 3M sponsored – Starkov and Wallace (2002) Toxicol Sci 66, 244-252. – O’Brien et al. (2008) Toxicol Appl Pharmacol 227, 184-195. – Berthiaume and Wallace (2002) Toxicology Lett 129, 23-32. – Butenhoff et al. (2002) Toxicol Sci 69, 244-257. – Mitochondrial proliferation mode of action (current) • NTP sponsored (i.e., they think its important) – Mitochondrial interactions of PFCs in vitro (Wallace) Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • Liver function and health • Serum lipid chemistry • Body-weight change • Tumorigenesis • • • • Reproduction/Development Immune system Nervous system Endocrine system (hormones) Tumorigenicity in SD Rats • PFOA – At 300 ppm in diet (~15 mg/kg body weight) • Hepatocellular adenoma (males) • Pancreatic acinar-cell adenoma (males) • Testicular Leydig-cell adenoma (“Tumor triad” pattern seen with other PPARα agonists) • No increased tumor incidence in females • PFOS – At 20 ppm in diet (~1 mg/kg body weight) • Hepatocellular adenoma (males and females) • Thyroid follicular cell adenoma (20 ppm stop-dose males) Tumorigenesis - PFOA • Hepatocellular – Consequences of PPARα activation – Oxidative stress – Potential for contribution of CAR activation • Testicular Leydig cell adenoma – Consequences of PPARα activation – Induction of aromatase enzyme leading to increased estrogen • Pancreatic acinar cell adenoma – Consequences of PPARα activation – Increased cholecystokinin hormone (evidence weak) – Mitogenic activity of thyroid hormone, retinoids (not tested). Pancreatic acinar cell proliferation From: Ohmura et al. (1997) Can Res 57, 795-798. Thyroid hormone (T3) is a strong mitogen for rat pancreatic acinar cells, as are BR931 and 9-cisRA. Thyroid hormone (T3) BrDU staining showing proliferation of acinar cells and not ductal or islet cells in rat pancreas stimulated with T3. Peroxisome proiferator Retinoid Tumorigenesis - PFOS • Was PPARα activation responsible? • PFOS - CXR investigation results – Liver • PFOS is a mixed agonist in the rat • PPARα, CAR, PXR – Thyroid • No effect of PFOS Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • • • • Liver function and health Serum lipid chemistry Body-weight change Tumorigenesis • Reproduction/Development • Immune system • Nervous system • Endocrine system (hormones) PFAs Studied for Reproduction and Developmental Effects - PFBS - - PFBA Results of Major Laboratory Studies • No effect on functional aspects of • • • • reproduction Structural anomalies associated with dosing causing maternal stress Developmental delays noted in some cases Birth weight and weight gain affected in some cases Neonatal mortality with PFOS and PFOA Modes of Action - Current Thoughts • Although in utero exposure of both PFOS and PFOA caused neonatal mortality, the adverse effects may be mediated by separate mechanisms • PFOA likely acts through the PPARα signaling pathway that regulates intermediary metabolism • PFOS likely interacts with phospholipids of lung surfactant and interferes with lung inflation and pulmonary function Lung Histology and Morphometry Control PFOS Dose (mg/kg) Air Space (%) Septal Space (%) 0 63.9 ± 1.5 31.6 ± 1.3 5 56.7 ± 2.1 41.2 ± 2.0 * 10 55.2 ± 2.2* 43.6 ± 1.9 * Alveolar Structure Surfactant prevents lungs from collapsing during end-expiration by reducing the surface tension at the air-liquid interface PFOS? Modified from Hawgood & Clements, 1990. PFOS and Pulmonary Surfactant • PFOS was detected in amniotic fluid that bathed the fetal lung • Oral gavage of newborn rats failed to cause mortality – chemical has to reach within the lung • PFOS interacts with phospholipids (Xie et al., 2007) – Dipalmitoylphosphatidylcholine (DPPC) is a major component of lung surfactant – In vitro study: PFOS had strong tendency to partition into and disrupt DPPC bilayers – PFOS > PFOA >>OS • Definitive evidence is needed Non-Occupational Human Studies - Summary a Endpoint PFA Apelberg Fei Monroy Gestational Age PFOS PFOA NS NS NS NS NS NS Birth Weight (g) PFOS PFOA NS (-69a,Tb) NS (-104a,T) NS -10.6 NS NS Birth Length (cm) PFOS PFOA NS NS NS -0.69 N/A N/A Head Circum. (cm) PFOS PFOA -0.32 (T) -0.27 (T) NS NS N/A N/A Abdominal Circum. (cm) PFOS PFOA N/A N/A NS -0.059 N/A N/A Ponderal Index PFOS PFOA -0.074 (T) -0.074 (T) NS NS N/A N/A Placental Weight PFOS PFOA N/A N/A NS NS N/A N/A Stat. sign. when adjusted for gest. age but not sign. in fully-adjusted analysis. b Log transformed (change for 2.7-fold change in PFA concentration). Birth Weight - Another Case of Reverse Causation? • Plasma volume expansion positively • • associated with increased birth weight. Concentrations of plasma constituents may decrease during pregnancy. Research approach: – Modeling of pharmacokinetics in pregnancy – Contract with The Hamner Instutues Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • • • • • Liver function and health Serum lipid chemistry Body-weight change Tumorigenesis Reproduction/Development • Immune system • Nervous system • Endocrine system (hormones) PFOS and PFOA & Immune System • Suppression of adaptive immunity in mice • • • • • – Thymic and splenic atrophy Enhancement of innate immunity in mice Attenuated by knocking out PPARα Appears to be a high-dose effect (DePierre) However, Peden-Adams report on PFOS effect at 91 ppb PFOS in serum. Epi studies? Immune System and PFOS - Mice • Dr. DePierre’s research group at Stockholm University – Carefully repeated Peden-Adams et al. work. – Not able to reproduce observed effects. – Likely due to methodological issues with Peden-Adams et al. study. • Human data would be helpful Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • • • • • • Liver function and health Serum lipid chemistry Body-weight change Tumorigenesis Reproduction/Development Immune system • Nervous system • Endocrine system (hormones) Nervous System • Decreased habituation consistently observed with PFOS in developing male rats and mice (transient) – Publishing DNT study • Delayed pupillary reflex in male rats given PFOA and PFBA – Grant to Dr. Donald Fox, U of Houston • Brain uptake studies – Collaborative with USEPA – Grant to Dr. Grant Anderson, U of MN Responses of Laboratory Animals To Perfluorinated Alkyls (PFAs) • • • • • • • Liver function and health Serum lipid chemistry Body-weight change Tumorigenesis Reproduction/Development Immune system Nervous system • Endocrine system (hormones) Endocrine System • PFAs can interfere with free hormone • measurement Current focus on thyroid hormones – Publication of remaining PFOS work – Publication of PFBA work • Human thyroid hormone displacement • studies planned Follow-up to PFBA planned using ultrafiltration and LC-MS/MS T4 method Pharmacokinetics Key Questions • What are the mechanisms of PFAA transport and elimination? • What are the determinants of interspecies elimination differences? • How can interspecies dose-response extrapolations best be accomplished? 3M-Sponsored Research • Joe DePierre’s lab at Stockholm U – Distribution and binding • Hagenbuch’s lab at KUMC – Renal and liver tgransport • Anderson’s lab at Univ of MN – Thyroid hormone transport interactions – Brain uptake • The Hamner Institute – Pharmacokinetic modeling Pharmacokinetics – Tissue Distribution of Radiolabelled PFCs • Recent synthesis of 35S-PFOS at Stockholms Universitet: – Initial distribution study in mice completed. – Whole-body distribution in progress – Fetal, age effects, intracellular investigations planned – Protein binding studies to be addressed Role of Organic Anion Transport • Active renal proximal tubular reabsorption • First suggested by Kudo et al. (2002) – Based on increased mRNA for Oatp1 in male rats • First modeled by Andersen et al. (2006) – Cynomolgus monkey PK data for PFOA and PFOS fit resorption model • Evidence in rat by Katakura et al. (2007) – Oat3 and Oatp1 may be reabsorption transporters A Schematic for a Physiologically-Motivated Renal Resorption Pharmacokinetic Model1 1 Andersen et al. (2006) Toxicology 226, 156-164. Uptake transporters in renal proximal tubule cells Based on subcellular localization, Oat1 and Oat3 may be responsible for active renal secretion of PFHA, PFOA and PFNA while Oatp1a1 may be responsible for reabsorption of PFDA, PFNA and PFOA. (From poster by Weaver and Hagenbuch, 2008). Pharmacokinetics – PBPK Models • The Hamner Institutes (3M funding) • Andersen et al. (2006) Toxicology 227, 156-164. • Tan et al. (2008) Toxicol Lett 177, 38-47. • EPA • Wambaugh et al. (2008) J Pharmacokinet Pharmacodyn 35, 683-713. • Harris and Barton (2008) Toxicol Lett 181, 148-156. • Lou et al. (2009) Toxicol Sci 107, 331-341. Protein Ionic Binding • Albumin – Major carrier protein in serum1,2,3 – Saturable1 – Competition with endogenous substrates • Steroid hormones1 • Thyroid hormones4 – Carbon number (size) and solubility 1Jones et al. (2003) Environ Toxicol Chem 22, 2639-2649. 2Han et al. (2003) Chem Res Toxicol 16, 775-781. 33M and Southern Research Institute, unpublished report, USEPA Docket AR-226. 4Chang et al (2008) Toxicology 243, 330-339. Binding of PFOS to HSA Binding of PFOS to HSA y = -4022.7x + 22376 R2 = 0.7454 35000 30000 v/c (M-1) 25000 20000 15000 10000 5000 0 0 1 2 3 4 5 6 v (molPFOS/molHSA) 50 uM PFOS fixed concentration Competition between PFOS and OA for binding to HSA [bound OA-HSA) (µM) 4000 3500 3000 2500 2000 1500 1000 500 0 0 500 1000 1500 2000 [PFOS] (µM) 10 uM OA fixed concentration 2500 Binding of PFOS to TTR Determination of the Ka for binding of PFOS to TTR Binding of PFOS to TTR 60000 y = -31575x + 57114 14 50000 12 40000 v/c (M-1) [bound PFOS] (µM) 16 10 8 20000 4 10000 0 0 0 100 200 300 [total PFOS] (µM) 400 500 Saturable 700 600 500 400 300 200 100 0 0 5 10 15 20 25 [thyroxine] (µM) 5 uM PFOS fixed concentration 0.0 0.5 1.0 30 1.5 v (mol PFOS / mol TTR) 1 -2 binding sites Competition between PFOS and thyroxine for binding to TTR [bound PFOS-TTR] (dpm) 30000 6 2 2 R = 0.7217 2.0 Summary – Key Research Areas • Differential effects: human vs. lab animals • Mechanism of effects on serum lipids • Immune effects – human relevance • • • • Transporters – species differences Pharmacokinetic models; e.g, pregnancy Distribution studies Binding studies