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Toxicology I: Principles & Mechanisms Marine Mammal Toxicology Spring 2004 Mark Hahn Woods Hole Oceanographic Institution.

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Presentation on theme: "Toxicology I: Principles & Mechanisms Marine Mammal Toxicology Spring 2004 Mark Hahn Woods Hole Oceanographic Institution."— Presentation transcript:

1 Toxicology I: Principles & Mechanisms Marine Mammal Toxicology Spring 2004 Mark Hahn Woods Hole Oceanographic Institution

2 Exposure Tissue concentration Effect (individual) Dose 1. Absorption/route of entry 1. Distribution/toxicokinetics 2. Biotransformation 3. Excretion 1. Molecular mechanism 2. Pathogenesis

3 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

4 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

5 Distribution/toxicokinetics hydrophobicity and lipid content protein binding effect of physiological condition (fasting, pregnancy) compartmental analysis physiologically based pharmacokinetic models

6 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 =  -glu-cys-gly) - sulfotransferases - UDP-glucuronosyl transferases - acetylases; methylases

7 Cytochrome P450 (CYP) multiple forms (57 in humans) mostly in endoplasmic reticulum (microsomal) hemoproteins require NADPH and O 2 tissue-, sex-, and stage- specific expression broad substrate specificity (endogenous and xenobiotic) some inducible nomenclature (family-subfamily-gene: e.g. CYP1A1)

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9 Human P450 enzymes

10 Regulation of CYP gene expression by soluble receptors

11 Reactions - PAH metabolism CYP1A1 EH DHD-DH

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13 Rob Letcher, Univ. of Windsor Reactions - PCB metabolism Differential susceptibility to biotransformation: Preferential loss of 3,4-unsubstituted congeners 2,2’,5,5’-TCB 2,2’,4,5,5’-PCB 2,2’,4’,5,5’,6-HCB 2,2’,3,4,4’,5’-HCB 2,2’,4,4’,5,5’-HCB

14 Rob Letcher, Univ. of Windsor Reactions - PCB metabolism

15 Rob Letcher, Univ. of Windsor Reactions - PCB metabolism CYP FMO NAT GST CYP2B MeT  -lyase

16 OH-PCBs 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 PCB Hydroxy PCB

17 Brouwer et al 1998 OH-PCBs as inhibitors of T4 transport by transthyretin (TTR)

18 Methylsulfonyl-PCBs Formed by sequential enzymatic reactions Less hydrophobic than parent PCBs but still persistent Bioaccumulate and persist in tissues (m- and p-MeSO 2 w/ 2,5,(6)-Cl) (liver, lung > fat) - likely role for CYP2B epoxidation as initial step adipose [MeSO 2 -PCB]/[PCB] = (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

19 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

20 Tanabe et al (1988) Capacity and mode of PCB metabolism in marine mammals Biotransformation capacity inferred from patterns of PCB congeners (Dall’s porpoise vs human) o-m unsub (CYP1A) m-p unsub (CYP2B) o-m unsub m-p unsub

21 2,2’,5,5’-TCB 2,3’,4,4’-TCB

22 Boon et al (1997) Relative ratios (R rel ) vs food for PCB congeners harbor seal common dolphin harbor porpoise 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)

23 Immunochemical characterization of hepatic microsomal cytochromes P450 in beluga antibody to CYP formsband in beluga hepatic microsomes MAb fish 1A1+ PAb rodent 1A1/2+(1) 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:

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25 Immunochemical detection of CYPs in marine mammals

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27 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:

28 CYPs in marine mammals Immunochemical evidence and cDNA cloning

29 White, et al. (1994) Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whales (Delphinapterus leucas). Toxicol. Appl. Pharmacol. 126: Catalytic characterization of hepatic microsomal cytochromes P450 in beluga

30 White et al. (2000) Compar. Biochem Physiol. 126, 267 Rates of PCB metabolism by hepatic microsomes (pmol/min/mg protein)

31 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-hydroxy- 3,3',4',5-TCB reflects a positional shift of a Cl.

32 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), StL HB

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34 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)

35 ReceptorEndogenous Xenobiotic ligandsTarget genes ligands Aryl hydrocarbon (Ah) receptor (AHR)?dioxins, PCBs, PAHsCYP1A,B; GST; UGT Constitutive androstane androstanes,barbiturates; PCBsCYP2 (CYP3), UGT, GST, receptor (CAR) bile acidsOAT, MRP Pregnane X receptor (PXR)bile acids,organochlorine pesticides;CYP3; (CYP2); UGT pregnenolonePCBs Peroxisome-proliferator-fatty acids fibrates,phthalatesCYP4 activated receptor (PPAR) and metabolites Farnesoid X Receptor (FXR)/bile acids,CYP7, ABC-A1 Liver X Receptor (LXR)oxysterols Retinoid receptors retinoidsmethoprene (RAR, RXR) Estrogen receptors (ER)17-  -estradiolOC pesticides; CYP19, Vtg alkylphenols; others Androgen Receptors (AR)testosteroneOC pesticides Glucocorticoid receptor (GR)glucocorticoidsMeSO 2 -PCBs(CYP3) Soluble receptors involved in xenobiotic effects

36 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.

37 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.

38 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).

39 INTRINSIC EFFICACY TISSUE COUPLING AFFINITY K d EFFICACY K E Ligand- Receptor I Ligand- Receptor A Ligand + Receptor I RESPONSE POTENCY EC50 Affinity, Efficacy, and Potency Hestermann et al. 2000

40 XRE TATA TCDD AHR ARNT Co-act BTF mRNA XRE nucleus cytoplasm hsp90 Ara9 pRb ? E2F cell cycle proteasomal degradation nuclear export e.g. CYP1A1

41 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

42 Structure-activity relationships 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:

43 3D Structure of PCBs: Calculated Dihedral Angle Hans-Joachim Lehmler, Univ. of Iowa

44 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)

45 PAH vs PCB as agonists for the AHR

46 Mechanisms of toxicity of PCBs and their metabolites

47 Toxic equivalency (TEQ) approach using toxic equivalency factors (TEFs) (AHR-dependent effects only)

48 TCDD toxic equivalency (TEQ) approach using toxic equivalency factors (TEFs) Calculated TEQs versus Bioassay-derived TEQs

49 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

50 Ross et al (2000)

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52 Receptor-dependent mechanisms of toxicity in marine mammals Species differences in receptor characteristics? - diversity - expression - function (affinity, SAR, target genes)

53 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

54 Ligand-binding assays High affinity, low capacity binding (Specific Binding) Total [ 3 H]-TCDD Free (loosely bound)Bound (Total) Non-specific binding Specific binding

55 AHR + [ 3 H]TCDDAHR + [ 3 H]TCDD + TCDF (100x) Fractions Incubate Spin for 2 hours Fractionate Count 30% sucrose 10% sucrose Analysis of AHR specific binding on sucrose density gradients Total bindingNon-specific binding

56 Sucrose gradient analysis of in vitro-expressed and tissue-derived AHR proteins cloned, in vitro expressedtissue-derived dpm fraction number dpm Jensen & Hahn (2001) Beluga AHR Beluga Liver Cytosol Mouse AHR Mouse Liver Cytosol TB NSB

57 beluga AHRmouse AHRhuman AHR TB NSB SB Saturation binding analysis of in vitro-expressed AHR proteins BMHUPL [ 35 S]methionine- labeled proteins

58 Equilibrium Dissociation Constants (K d ) for in vitro-expressed AHR proteins *p<0.05 versus human AHR **p<0.01 versus human AHR Beluga express a high-affinity (low K d ) AHR mean K d (n=4) beluga AHR0.43 ± 0.16 nM ** mouse AHR0.68 ± 0.23 nM * human AHR1.63 ± 0.64 nM

59 In vitro binding affinity vs. In vivo tissue burdens K D for TCDD: 0.43 nM in vitro TCDD-Eqs in liver of St. Lawrence beluga: 0.13 nM (adult male) (Muir et al Environ. Pollut.) Result: 23% AHR occupancy (% Maximum response depends on receptor concentration) Jensen & Hahn (2001)

60 Relative Potencies or Toxic Equivalency Factors (TEFs) for dioxin-like compounds in wildlife Source: van den Berg, et al. (1998) Environ. Health Persp. 106: congenerIUPACrodentmarine PCDD/PCDF#mammals 2,3,7,8-TCDD 11 2,3,7,8-TCDF 0.1? 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 ? TEF values

61 IC 50 : One-site competition model (Prism) K I : From IC 50, [ 3 H]TCDD (Cheng and Prusoff) Competitive binding of PCB congeners using in vitro expressed AHRs and [ 3 H]TCDD Jensen & Hahn (2001)

62 TCDD TCDF Non-ortho PCBs Di-ortho PCB Mono-ortho PCBs PCDD/F mouse K I (nM) beluga K I (nM) Correlation between beluga and mouse AHR binding affinities x=y

63 [ 35 S]methionine- labeled proteins [ 3 H]TCDD- binding Kim & Hahn (2002) Harbor seal versus mouse AHR

64 Kim & Hahn (2002) mouse AHR K D = 1.70 ± 0.26 nM seal AHR K D = 0.93 ± 0.19 nM TB SB NSB TB SB NSB

65 Trainer & Baden (1999) High affinity binding of red tide neurotoxins to marine mammal brain. Aquat Toxicol. 46:

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68 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


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