The Aryl hydrocarbon Receptor-Comparative Toxicology

Toxicology I: Principles & Mechanisms Marine Mammal Toxicology Spring 2004 Mark Hahn Woods Hole Oceanographic Institution Exposure 1. Absorption/route of entry Dose 1. Distribution/toxicokinetics 2. Biotransformation 3. Excretion Tissue concentration 1. Molecular mechanism 2. Pathogenesis Effect (individual) Approaches to studying toxicological mechanisms in marine mammals • Direct exposure? • Semi-field studies (feeding studies) • Extrapolation • Biomarkers of exposure, effect, susceptibility • Field associations (chemicals and effects) • in vitro studies - tissues and subcellular fractions - cloned, in vitro expressed proteins - tissue/cell culture Dose-Response • shapes of curves; thresholds • timing of exposure and effects (acute vs chronic) (algal toxins versus POPs) (exposure and effects separated in time) • low-dose extrapolation Distribution/toxicokinetics • hydrophobicity and lipid content • protein binding • effect of physiological condition (fasting, pregnancy) • compartmental analysis • physiologically based pharmacokinetic models Biotransformation (Metabolism) • Phase I (add functional group) - cytochrome P-450s (CYP) (hydroxylation) - flavin monooxygenases (N-, S-oxidation) - esterases,hydrolases, dehydrogenases… • Phase II (conjugation) - glutathione transferases (GSH = g-glu-cys-gly) - sulfotransferases - UDP-glucuronosyl transferases - acetylases; methylases Cytochrome P450 (CYP) • multiple forms (57 in humans) • mostly in endoplasmic reticulum (microsomal) • hemoproteins • require NADPH and O2 • tissue-, sex-, and stage- specific expression • broad substrate specificity (endogenous and xenobiotic) • some inducible • nomenclature (family-subfamily-gene: e.g. CYP1A1) Human P450 enzymes Famil y 1 2 3 4 5 7 8 11 17 19 20 21 24 26 27 39 46 51 TOTAL # subfamili es 2 11 1 6 1 2 2 2 1 1 1 1 1 3 3 1 1 1 # genes 3 16 4 12 1 2 2 3 1 1 1 1 1 3 3 1 1 1 57 ste roids (progesterone 21) vit. D retinoids vit. D OH-cholesterol bile acids lanosterol cholesterol, steroid 11 ste roids (pregnenolone 17) test osterone estrogens cholesterol substrates (examples) PAH, non-ortho-PCB, E2, xenobiotics ortho -PCB, b arbiturates, ste roids, ethan ol, xenobiotics ste roids, xenobiotics fat ty acids inducers (examples) PAH, non-ortho-PCB, dioxins pheno barbital, ort ho-PCB, DDT, ethano l glucocort icoids, ( PCBs) phtha late esters, ( PCBs) Regulation of CYP gene expression by soluble receptors Transc ription fact or AHR Dimerizat ion partne r ARNT Exam ples of ligands Dioxins, non- ortho PCBs, some PAHs, bilirubin, etc . Phen obarbital (PB), TCPOBOP, chlorinate d pesticides, ortho -PCBs, androst anol/ androst enol (inhibits) PB, ortho -PCBs, organochlorine pesticides, dexamet hasone, pregnenalone, corticoste rone, bile ac ids (lithoc holic ac id) Fibrate drugs, ph thalate esters, linoleic acid, arachidonic acid Cholestero l; (24 S)- hydroxych olesterol Bile acids, chenod eoxycho lic ac id Struct urally d iverse x enoest rogens Genes Regulated CYP1A, CYP1B GST, UGT, NQO CYP2B, CYP3A GST, ABC trans porters CYP3A, CYP2B, CYP7A (repression) GST, ABC trans porters CAR RXR PXR (SXR) RXR PPAR RXR CYP4A, CYP7A (re press ion), CYP8B, LXR CYP7A, ABC tra nsporters, LXR Represses CYP 7A, CYP8B, CYP27A CYP19 LXR FXR ER RXR RXR ER Reactions - PAH metabolism EH CYP1A1 CYP1A1 DHD-DH Reactions - PCB metabolism Differential susceptibility to biotransformation: Preferential loss of 3,4-unsubstituted congeners [CB ] to [CB-138] Ratio x 1.5 1.0 0.5 0 0.5 1.0 1.5 2,2’,5,5’-TCB CB-52 CB-70 2,2’,4,5,5’-PCB CB-92 CB-101 CB-99 CB-110 CB-118 CB-128 CB-138 *** CB-138 *** CB-153 CB-156 CB-156 CB-99 CB-105 CB-118 2,2’,3,4,4’,5’-HCB 2,2’,4,4’,5,5’-HCB 2,2’,4’,5,5’,6-HCB CB-149 CB-153 CB-180 Technical PCB mixture Clophen A50 PCB congeners in mink muscle Rob Letcher, Univ. of Windsor Reactions - PCB metabolism OH-PCB Formation Pathways Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl O n O Cl Cl HO Cl Cl Cl n OH Cl Cl Cl Cl Epoxide opening Cl Cl + Cl Cl Direct insertion Cl Cl 1,2-shift Cl Cl Epoxide opening Cl Cl Cl OH CB-101 + Cl Cl Cl OH Formation Pathway for Persistent MeSO2-PCBs Cl Cl Cl Cl Cl SG Cl Cl Cl SH -G Cl Cl Cl Cl Cl SCysNAC Cl Cl Cl Cl Cl Cl Cl Cl SH SCH3 Cl Cl Cl SO2CH3 (-SO2 Me) Cl Cl + Cl Cl OH Cl Cl Cl Cl Cl Cl SCys SG OH -H2O Cl Cl Cl Cl Cl SG MAP Cl Rob Letcher, Univ. of Windsor Reactions - PCB metabolism OH-PCB Formation Pathways Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl O n O Cl Cl HO Cl Cl Cl n OH Cl Cl Cl Cl Epoxide opening Cl Cl Cl CYP2B Cl Cl + Cl Direct insertion Cl Cl 1,2-shift Cl Cl Epoxide opening Cl Cl Cl OH CB-101 + Cl Cl Cl OH Formation Pathway for Persistent MeSO2-PCBs GST Cl Cl Cl Cl Cl SG Cl Cl Cl OH Cl Cl Cl Cl Cl SG Cl SG Cl Cl SH -G Cl Cl Cl Cl Cl SCysNAC Cl Cl Cl Cl Cl Cl SH SCH3 Cl Cl CYP Cl Cl Cl SO2CH3 (-SO2 Me) + OH -H2O Cl Cl Cl NAT Cl MAP -lyase Cl Cl MeT FMO Cl SCys Cl Rob Letcher, Univ. of Windsor OH-PCBs Cln OH Clm • Formed by CYP1A and CYP2B • Less hydrophobic than parent PCBs • Most readily excreted; some persist in blood (m- and p-hydroxy w/ o-Cl) • Poor substrates for conjugation (glucuronidation and sulfation) • Multiple effects - displace T4 from transthyretin - inhibit sulfotransferase (T4, E2, 3-OH-BaP) - inhibit glucuronosyl transferase (3-OH-BaP) - agonists for estrogen receptors log P 8 6 4 2 1 2 3 4 5 6 7 8 Number of Chlorine Atoms PCB Hydroxy PCB OH-PCBs as inhibitors of T4 transport by transthyretin (TTR) Brouwer et al 1998 Methylsulfonyl-PCBs • Formed by sequential enzymatic reactions • Less hydrophobic than parent PCBs but still persistent • Bioaccumulate and persist in tissues (m- and p-MeSO2 w/ 2,5,(6)-Cl) (liver, lung > fat) - likely role for CYP2B epoxidation as initial step • adipose [MeSO2-PCB]/[PCB] = .01-.25 (highest in Baltic ringed and grey seal) • Protein interactions - uteroglobin (progesterone-binding protein) - glucocorticoid receptor antagonist - estrogen receptor antagonist? • Induce CYP2B,C and CYP3A enzymes Biotransformation in marine mammals • What is the capacity for xenobiotic metabolism in MM? Are there species differences in xenobiotic-metabolizing enzymes? - diversity - expression - inducibility - catalytic function (rates and specificity) • Direct measurement of metabolites • Inferences from contaminant patterns in MM tissues • Direct assessment in vitro - immunochemical detection - in vitro catalytic assay (model substrates; correlations; ± inhibitors) - cloning, expression, characterization Biotransformation capacity inferred from patterns of PCB congeners (Dall’s porpoise vs human) m-p unsub (CYP2B) o-m unsub o-m unsub (CYP1A) m-p unsub Tanabe et al (1988) Capacity and mode of PCB metabolism in marine mammals 2,2’,5,5’-TCB 2,3’,4,4’-TCB Relative ratios (Rrel) vs food for PCB congeners harbor seal otter 0 m,p H 2 o Cl 0 m,p H 1 o Cl (CYP1A) 1 m,p H 2-3 o Cl (CYP2B) Boon et al (1997) harbor porpoise common dolphin Immunochemical characterization of hepatic microsomal cytochromes P450 in beluga antibody to CYP forms band in beluga hepatic microsomes + +(1) + + MAb fish 1A1 PAb rodent 1A1/2 PAb fish “2B” PAb rat 2B1 MAb rat 2B1 PAb rabbit 2B4 PAb dog 2B11 PAb rat 2E1 PAb rat 2E1 + +(2) White, et al. (1994) Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whales (Delphinapterus leucas). Toxicol. Appl. Pharmacol. 126: 45-57. Immunochemical detection of CYPs in marine mammals Letcher, et al (1996) Immunoquantitation and microsomal monooxygenase activities of hepatic cytochromes P4501A and P4502B and chlorinated hydrocarbon contaminant levels in polar bear (Ursus maritimus). Toxicol Appl Pharmacol 137: 127-140. CYPs in marine mammals Immunochemical evidence and cDNA cloning CYP1 CYP2 CYP3 CYP4 Cetacea – odontocetes Cetacea – mysticetes Pinnipeds Mustelids Sirenians Ursids ++ (1A1, 1B) ++(1A) ++(1A1, 1A2) + (+/-) + + + + + + ++(1A) ++(2B) Catalytic characterization of hepatic microsomal cytochromes P450 in beluga White, et al. (1994) Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whales (Delphinapterus leucas). Toxicol. Appl. Pharmacol. 126: 45-57. Rates of PCB metabolism by hepatic microsomes (pmol/min/mg protein) beluga (male) PCB-77 (3,3’,4,4’-TCB) PCB-52 (2,2’,5,5’-TCB) 22 1.1 rat (con) (low) 0-10 rat (3MC) 18-50 (low) rat (PB) (low) 66-1450 White et al. (2000) Compar. Biochem Physiol. 126, 267 Fig. 9. (White et al. (2000)) Proposed pathways for the metabolism of 3,3',4,4'-TCB in beluga whale liver microsomes. The thickness of the arrows reflects the significance of an indicated pathway. The 4-hydroxy3,3',4',5-TCB reflects a positional shift of a Cl. StL HB R.J. Letcher, et al. (2000). Methylsulfone PCB and DDE metabolites in beluga whale (Delphinapterus leucas) from the St. Lawrence river estuary and western Hudson Bay, Canada. Environ. Toxicol. Chem. 19(5), 1378-1388. Molecular mechanisms of toxicity • covalent binding to protein or DNA • oxidative stress (e.g. via Reactive Oxygen Species) - lipid peroxidation - oxidative DNA damage - oxidative damage to proteins (-SH) • enzyme inhibition (e.g. OP pesticides & AChE) • interference with ion channels - e.g. saxitoxin, brevetoxin • interference with receptor-dependent signaling - membrane bound receptors (neurotransmitter) - intracellular receptors (hormone) Soluble receptors involved in xenobiotic effects Receptor Endogenous ligands Xenobiotic ligands Target genes Aryl hydrocarbon (Ah) receptor (AHR) ? Constitutive androstane receptor (CAR) Pregnane X receptor (PXR) Peroxisome-proliferatoractivated receptor (PPAR) Farnesoid X Receptor (FXR)/ Liver X Receptor (LXR) Retinoid receptors (RAR, RXR) Estrogen receptors (ER) Androgen Receptors (AR) Glucocorticoid receptor (GR) dioxins, PCBs, PAHs barbiturates; PCBs OAT, MRP CYP1A,B; GST; UGT CYP2 (CYP3), UGT, GST, androstanes, bile acids bile acids, pregnenolone fatty acids bile acids, oxysterols retinoids 17--estradiol testosterone glucocorticoids organochlorine pesticides; CYP3; (CYP2); UGT PCBs fibrates,phthalates and metabolites CYP4 CYP7, ABC-A1 methoprene OC pesticides; alkylphenols; others OC pesticides MeSO2-PCBs (CYP3) CYP19, Vtg Definitions • Receptor (P. Erlich, 1913; J.N. Langley, 1906) A macromolecule with which a hormone, drug, or other chemical interacts to produce a characteristic effect. Two essential features: – chemical recognition – signal transduction • Ligand: A chemical that exhibits specific binding to a receptor. Definitions • Specific binding (SB): High-affinity, low capacity binding of ligand to receptor • Non-specific binding (NSB): Low-affinity, high capacity binding of ligand to other proteins • Agonist: A ligand that binds to a receptor, increasing the proportion of receptors that are in an active form and thereby causing a biological response. • Antagonist: A ligand that binds to a receptor without producing a biological response, but rather inhibits the action of an agonist. • Partial agonist: An agonist that produces less than the maximal response in a tissue, even when all receptors occupied. Partial agonists have properties both of agonists and of antagonists. Definitions • Potency: The concentration or amount of a chemical required to produce a defined effect. Location along the dose axis of dose-response curve (property of ligand and tissue). • Efficacy: The degree to which a ligand can produce a response approaching the maximal response for that tissue (property of ligand and tissue). • Affinity: The tenacity with which a ligand binds to its receptor (property of ligand). • Intrinsic Efficacy: Biological effectiveness of the ligand when bound to the receptor; e.g. ability to “activate” receptor once bound (property of ligand). Affinity, Efficacy, and Potency Ligand + Receptor I AFFINITY Kd LigandReceptor I INTRINSIC EFFICACY LigandReceptor A TISSUE COUPLING RESPONSE EFFICACY KE POTENCY EC50 Hestermann et al. 2000 nucleus hsp90 pRb AHR Ara9 ? E2F TCDD ARNT cell cycle nuclear export XRE proteasomal degradation Co-act BTF cytoplasm XRE TATA mRNA e.g. CYP1A1 Evidence for role of Ah receptor in effects of dioxins / planar PCBs Genetics • inbred strains of mice (responsive and “non-responsive”) Pharmacology • Structure-activity relationships for AHR binding and toxicity Cell Biology • Mouse hepatoma cell mutants Molecular biology • AHR-null mice Structure-activity relationships log ED50 for Thymic Atrophy in Rats 4 3 2 1 0 (1) y = 1.119x + 8.374 r 2 = 0.642 (2) (9) (8) (7) (6) (5) (4) log Kd for AHR binding The toxic potencies of many halogenated aromatic hydrocarbons are related to their AHR-binding affinities. Data from Safe, S. (1990) CRC Crit. Rev. Toxicol. 21: 51-88. 3D Structure of PCBs: Calculated Dihedral Angle 100 Cl Dihedral Angle [°] 80 60 40 Cl Cl Cl Cl Cl Cl PCB 118 Cl Cl Cl Cl Cl PCB 95 Cl Cl Cl Cl PCB 153 Cl Cl 20 0 Cl Cl Cl PCB 126 0 1 2 3 4 Number of ortho-Chlorine Atoms Hans-Joachim Lehmler, Univ. of Iowa post-AHR mechanisms of dioxin/PCB toxicity • induction of CYP1A (metabolism of endogenous compound; release of ROS) • altered expression of other target genes (cell proliferation/differentiation) • recruitment of AHR away from endogenous function • competition for factors required for other signaling pathways (ARNT, coactivators; HIF, SIM) • cross-talk with other signaling pathways (estrogen, progesterone) PAH vs PCB as agonists for the AHR PAH aff inity timing of activat ion clearance of ligand nature of m etabolites biomarkers variable (high) transient rapid reactive (electrophiles) PCB variable (high) sust ained some s low stable but bioactive CYP1A (early) CYP1A protein or DNA adducts parent comp ounds and me tabolites Mechanisms of toxicity of PCBs and their metabolites Congener/metabolite non-o rtho and mono ortho -PCBs di-orth o PCBs (PCB-95: 2,2’,3,5,6-PCB) Molecular Target aryl hydr ocarb on r eceptor (AHR) ryanod ine recepto r Action altered gene expr ession (CYP 1A and others); ox idative stress ? altered c alcium h omeostas is, neurot oxicity? altered neurotra nsmitte r metabolism (dopamine & seroton in) induction of CYP2B di-orth o PCBs ortho PCBs (PCB-164: 2,3,3’,4’,5’,6-HCB) ?? constitut ive androst ane recepto r (CAR) rodent PXR (agonists ) human SXR (antagonists) transthy retin (TTR) highly ch lorinated PCBs (PCB-184, -196, -153) induction of CYP3A; va ries by s pecies OH-PCB inhibition of thyro id hormon e tra nsport an d retinoid homeostas is (rodents > hum ans; TTR vs TBG) OH-PCB PCB and O H-PCB methy lSO2-PCB methy lSO2-PCB sulfotrans ferase, glucuronosyl tra nsferase estrogen recepto r (ER) uteroglobin glucocort icoid recepto r inhibition of sulfotrans ferase (E2 a nd T4 , 3-OH-BaP) ER ago nist or anta gonist displaceme nt of progesterone? ? GR antagonist Toxic equivalency (TEQ) approach using toxic equivalency factors (TEFs) (AHR-dependent effects only) TCDD toxic equivalency (TEQ) approach using toxic equivalency factors (TEFs) chemical type conc (ng/kg lw) 45 983 2,351 119,000 376,000 5,320,000 7,630,000 13,448,334 Total PCB (ng/kg lw) TEF (mammals) 1 0.1 0.0001 0.0001 0.0001 0 0 TEQ (ng/kg lw) 45.00 98.30 0.24 11.90 37.60 0.00 0.00 % of TEQ % of [PCB] 2,3,7,8-TCDD PCB-126 PCB-77 PCB-105 PCB-118 PCB-153 other PCB non-ortho non-ortho mono-ortho mono-ortho di-ortho 23.31 50.92 0.12 6.16 19.48 0.00 0.00 0.007 0.017 0.885 2.796 39.559 56.736 193.04 Total TEQ (ng TCDD-Eq/kg lw ) • Calculated TEQs versus Bioassay-derived TEQs TEQ approach: Assumptions • compounds act via common mechanism • additivity (no synergism, antagonism) • no differences in intrinsic efficacy (all full agonists) • similar structure-activity relationships for endpoints of concern and endpoints used to generate TEF values • similar structure-activity relationships for species of concern and species used to generate TEF values Ross et al (2000) Receptor-dependent mechanisms of toxicity in marine mammals • Species differences in receptor characteristics? - diversity - expression - function (affinity, SAR, target genes) Differential Sensitivity to Dioxin (2,3,7,8-TCDD) Mammals - laboratory species: 5000-fold variability (lethality) - humans: ? - marine mammals: ? Birds: up to 1000-fold variability among species Reptiles: ? Amphibians - anurans: 1000-fold less sensitive than fish - other amphibians: ? Bony fishes: 40-fold variability among species Ligand-binding assays • High affinity, low capacity binding (Specific Binding) Total [3H]-TCDD Free (loosely bound) Bound (Total) Non-specific Specific binding binding Analysis of AHR specific binding on sucrose density gradients AHR + [3H]TCDD AHR + [3H]TCDD + TCDF (100x) 10% sucrose Total binding Non-specific binding 30% sucrose • Incubate • Spin for 2 hours • Fractionate • Count Fractions Sucrose gradient analysis of in vitro-expressed and tissue-derived AHR proteins cloned, in vitro expressed 1600 1200 tissue-derived Beluga Liver Cytosol Beluga AHR 1600 dpm TB 1200 800 800 400 0 0 10 20 NSB 30 40 400 0 0 10 20 30 40 2500 Mouse AHR 2500 2000 1500 1000 500 0 10 20 30 40 0 Mouse Liver Cytosol 2000 dpm 1500 1000 500 0 0 10 20 30 40 fraction number fraction number Jensen & Hahn (2001) Saturation binding analysis of in vitro-expressed AHR proteins beluga AHR 1500 mouse AHR 2000 human AHR 1000 pSP64belAHR Kd = 0.34 nM pSPORTmoAHR Kd = 0.75 nM pSPORThuAHR K d = 1.23 nM TB DPM 1000 DPM 1500 750 DPM SB 500 1000 500 500 250 NSB 0 0 1 2 3 4 5 6 Fre e TCDD (nM ) 0 0 1 2 3 4 5 Fre e TCDD (nM ) 0 0 1 2 3 4 5 Fre e TCDD (nM ) B [35S]methioninelabeled proteins M H UPL Equilibrium Dissociation Constants (Kd) for in vitro-expressed AHR proteins mean Kd (n=4) beluga AHR mouse AHR 0.43 ± 0.16 nM ** 0.68 ± 0.23 nM * human AHR 1.63 ± 0.64 nM *p<0.05 versus human AHR **p<0.01 versus human AHR Beluga express a high-affinity (low Kd) AHR In vitro binding affinity vs. In vivo tissue burdens KD for TCDD: 0.43 nM in vitro TCDD-Eqs in liver of St. Lawrence beluga: 0.13 nM (adult male) (Muir et al. 1996 Environ. Pollut.) Result: 23% AHR occupancy (% Maximum response depends on receptor concentration) Jensen & Hahn (2001) Relative Potencies or Toxic Equivalency Factors (TEFs) for dioxin-like compounds in wildlife TEF values congener PCDD/PCDF IUPAC # rodent 1 0.1 2,3,7,8-TCDD 2,3,7,8-TCDF non-ortho PCB 3,3’,4,4’,5-PeCB 3,3’,4,4’,5,5’-HCB 3,4,4’,5-TCB 3,3,’4,4’-TCB mono-ortho PCB 2,3,3’,4,4’-PeCB 2,3’4,4’,5-PeCB 2,3,3’,4,4’,5-HCB 126 169 81 77 marine mammals 1 ? 0.1 0.01 0.0001 0.0001 ? ? ? ? 105 118 156 0.0001 0.0001 0.0005 ? ? ? Source: van den Berg, et al. (1998) Environ. Health Persp. 106: 775-792. Competitive binding of PCB congeners using in vitro expressed AHRs and [3H]TCDD Beluga AHR 1.0 0.8 0.6 0.4 0.2 TCDD TCDF 126 169 77 81 105 118 156 128 0.0 IC50: One-site competition model (Prism) -5 -4 -3 -2 -1 0 1 2 3 4 5 6 log[HAH] nM Mouse AHR KI: From IC50, [3H]TCDD (Cheng and Prusoff) 1.0 0.8 0.6 0.4 0.2 0.0 TCDD TCDF 126 169 77 81 105 118 156 128 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 log[HAH] nM Jensen & Hahn (2001) Correlation between beluga and mouse AHR binding affinities 105 104 x=y beluga KI (nM) 103 118 156 128 Mono-ortho PCBs 105 Di-ortho PCB 102 101 100 10-1 10-2 81 77 169 126 TCDF TCDD Non-ortho PCBs PCDD/F 10-1 100 101 102 103 104 105 mouse KI (nM) Harbor seal versus mouse AHR dpm/fraction 2000 A Harbor seal 1500 1000 500 0 0 2000 5 10 15 20 25 30 35 B Mouse dpm/fraction 1500 [35S]methioninelabeled proteins 1000 500 [3H]TCDDbinding 0 5 10 15 20 25 30 35 0 2000 C UPL dpm/fraction 1500 1000 500 0 0 5 10 15 20 25 30 35 Fraction Kim & Hahn (2002) Bound 3H-TCDD (fmol) Mouse AHR 100 TB SB 50 mouse AHR KD = 1.70 ± 0.26 nM NSB 0 0 2 4 6 8 10 [free 3H-TCDD] (nM) 100 Bound 3H-TCDD (fmol) Seal AHR 75 TB SB NSB 0 2 4 6 8 10 50 seal AHR KD = 0.93 ± 0.19 nM 25 0 [free 3H-TCDD] (nM) Kim & Hahn (2002) Trainer & Baden (1999) High affinity binding of red tide neurotoxins to marine mammal brain. Aquat Toxicol. 46: 139-148. Weight of evidence approach for assessing impact of contaminants on marine mammals Epidemiological and observational studies in wildlife species Comparative mechanistic studies Mechanistic studies in laboratory animals