Document 2O6jodrJw5dJqZEXkMykp9Qp

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 This area has become increasingly important as new epidemiological studies are released. Plausibility Coherence Experiment Analogy 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. Individual Publication by Year Tim es Cited 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 - Do not enter into the biochemical reactions that use fatty acids as substrate. However, PFAs may present as FFAs. - Transporters - Receptors - Carrier proteins 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., PPARa) 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 Male 0.75 mg/kg 184 d 1Seacat 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 - PPARa activation - Other processes (e.g., CAR and PXR) - Adaptation vs. pathological change Diversion #1 - Molecular Biology Xenobiotics, Hormones, Cytokines, Growth Receptors & Transcription Factors PPARa CAR Gene Expression, Transcription CYP4A mRNA CYP2B mRNA Protein Synthesis Enzymes Other Compounds Metabolism Metabolic Products Based on: Waxman (1999) Arch. Biochem. Biophys. 369, 11-23. Some Common Nuclear Receptors Controlling CYP Induction Receptor PPARa PPARy car PXR LXRa FXR RXR TR Ah1 Typical Activator Fatty acids, Fibrates Rosiglitazone Phenobarbital Steroids, Dexamethasone Cholesterol Bile acids Retinoic acid Triiodothyronine Polycyclic aromatics, Dioxin 1 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 PPARa Humans less responsive than rodents - Lower human levels of PPARa - Human PPARa not associated with hyperplasia Use of genetically-modified mice1'2'34 - Using specific activators of PPARa mPPARa (natural) - hypertrophy and hyperplasia hPPARa - hypertrophy but NO hyperplasia No PPARa - 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. Relative Lytifflm sn Attivi Differential Activation of PPARa in an Engineered System PFOA is a weak activator of PPARa 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 PPARa - Less potent than clofibrate and endogenous long chain FFA PFOS and PFOA are weak agonists for PPARy - Much less potent than rosiglitazone No activation of RXRa or LXRp PFOA and PFOS more specific and less potent than endogenous long-chain FFAs. 1Vanden Heuvel et al. (2006) Toxicol Sci 92, 476-489. Human vs. Rat Liver Cells in Primary Culture and PPARa Activation by PFAs > All PFAs at 25 pM in cell culture media. > C < 4 PFAs have little or no effect. CKeonudrtaelsl yWoafllDacr.e, 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) 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 PPARa 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-a-hydroxylase - Increased liver size - Increased fatty acid oxidation - Suggests a PPARa 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 - ^ G ro u p 1: control - - G roup 4: 0.006 % PFHS --^ G ro u p 2: 0 .0 3 % F e n o fib ra te - * - G ro u p 3: 0 .0 3 % P F B S - - G roup 5: 0.003 % PFOS C h o le ste ro l (m m o l/L ) 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 pg/mL PFBS PFHS PFOS Albumin 93.5 > 99.9 99.8 y-Globulin < 0.1 26.1 24.1 a-Globulin < 0.1 13.7 59.4 Fibrinogen < 0.1 < 0.1 < 0.1 a-2-Macro- globulin < 0.1 < 0.1 < 0.1 Transferrin < 0.1 6.4 <0.1 |3-Lipo-protein thern Research Institute, unpublished data PFOA H 99.7 3.0 11.0 < 0.1 < 0.1 2.1 39.6 Percent Binding to Isolated Human Serum Protein Fractions at 10 pg/mL Albumin y-Globulin a-Globulin Fibrinogen a-2-Macro- globulin Transferrin 3-Lipo-protein PFBS 93.5< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 PFHS > 99.926.1 13.7 < 0.1 < 0.1 (M 64.1 PFOS PFOA H 99.8- 99.7- 24.1 3.0 59.4 11.0 < 0.1 < 0.1 < 0.1 < 0.1 CO CO 95.6 39.6 Additional Experimental Approaches Binding interaction studies - Exploit 35S-PFOS made at Stockholm University in Ake 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 Mean %Change in Body Weight Maternal Dose (mg PFOS/kg/day) Mean Pup Weight Gain/Liller (grams) Body Weight Hypotheses - Increased burning of fat Uncoupling of oxidative phosphorylation (mitochondria) Inc- reOanslyedwimthitcoecrthaoinndsurilafolnbaomdidieess ((PNFOOTAP)FOS or PFOA) PP- AERvaideancctievafrtoiomn rat and monkey studies - 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 PPARa 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 PPARa activation - Oxidative stress - Potential for contribution of CAR activation Testicular Leydig cell adenoma - Consequences of PPARa activation - Induction of aromatase enzyme leading to increased estrogen Pancreatic acinar cell adenoma - Consequences of PPARa 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. BrDU staining showing proliferation of acinar cells and not ductal or islet cells in rat pancreas stimulated with T3. W. f ^ / a ? .1 f "2 y ' n A il fSsSm&L,*a#$ r 9 t \' /* A"tO p^ 7 ,D*S&n? Thyroid hormone (T3) Peroxisome proiferator Retinoid Tumorigenesis - PFOS Was PPARa activation responsible? PFOS - CXR investigation results - Liver PFOS is a mixed agonist in the rat PPARa, 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 PFHxS PFOS PFBA PFOA 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 PPARa 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) 0 5 10 Air Space (%) 63.9 1.5 56.7 2.1 55.2 2.2* Septal Space (%) 31.6 1.3 41.2 2.0* 43.6 1.9* Alveolar Structure Surfactant prevents lungs from collapsing during end-expiration by reducing the surface tension at the air-liquid interface Alveolar olar type I cell Lamellar body Tubular Alveolar flui Air space PFOS? Alveolar macrophag 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 Endpoint PFA Apelberg Fei Monroy Gestational PFOS NS NS NS Age PFOA NS NS NS Birth Weight PFOS (g) PFOA NS (-69a,Tb) NS NS (-104a,T) -10.6 NS NS Birth Length (cm) PFOS PFOA NS NS NS -0.69 N/A N/A Head Circum. PFOS (cm) PFOA NS N/A NS N/A Abdominal PFOS Circum. (cm) 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 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 PPARa 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 Oatpl 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 Oatpl may be reabsorption transporters A Schematic for a Physiologically-Motivated Renal Resorption Pharmacokinetic Model1 1Andersen e t al. (2006) Toxicology 226, 156-164. Uptake transporters in renal proximal tubule cells Lumen Blood 5 Oatplal Oat2 Oatpl a3 Oatp4c1 Oat1 Oat3 Apical Membrane Basolateral Membrane 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 serum12 3 - Saturable1 - Competition with endogenous substrates Steroid hormones1 Thyroid hormones4 - Carbon number (size) and solubility Jones 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 v/c (M-1) Binding o f P F O S to H S A 35000 y = -4 0 2 2 7 + 22376 R2 = 0.7454 30000 25000 i 20000 * 15000 i 10000 5000 0 0 234 v (molPFOS/molHSA) 5 6 [ b o u n d O A - H S A ) (|j M) Binding of PFOS to TTR Binding of PFOS to TTR Determination of the Ka for binding of PFOS to TTR [total PFOS] (mM) Saturable Competition between PFOS and thyroxine for binding to TTR 700 i Q. 400 300 T3 00 > * w 5 10 15 20 25 30 [thyroxine] (mM) 5 uM PFOS fixed concentration 1 -2 binding sites 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