Toxicology I: Principles & Mechanisms

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

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

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 = g-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 O2 • tissue-, sex-, and stage- specific expression • broad substrate specificity (endogenous and xenobiotic) • some inducible • nomenclature (family-subfamily-gene: e.g. CYP1A1)

9. Human P450 enzymes

10. Regulation of CYP gene expression by soluble receptors

11. Reactions - PAH metabolism EH CYP1A1 CYP1A1 DHD-DH

13. 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’,3,4,4’,5’-HCB 2,2’,4’,5,5’,6-HCB 2,2’,4,4’,5,5’-HCB Rob Letcher, Univ. of Windsor

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

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

16. PCB Hydroxy PCB 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

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

18. 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

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

21. 2,3’,4,4’-TCB 2,2’,5,5’-TCB

22. Relative ratios (Rrel) vs food for PCB congeners harbor seal otter 1 m,p H 2-3 o Cl (CYP2B) 0 m,p H 2 o Cl 0 m,p H 1 o Cl (CYP1A) Boon et al (1997) common dolphin harbor porpoise

23. Immunochemical characterization of hepatic microsomal cytochromes P450 in beluga antibody to CYP forms band 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: 45-57.

25. Immunochemical detection of CYPs in marine mammals

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 Pharmacol137: 127-140.

28. CYPs in marine mammals Immunochemical evidence and cDNA cloning

29. 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.

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

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. 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.

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. Soluble receptors involved in xenobiotic effects Receptor Endogenous Xenobiotic ligands Target genes ligands Aryl hydrocarbon (Ah) receptor (AHR) ? dioxins, PCBs, PAHs CYP1A,B; GST; UGT Constitutive androstane androstanes, barbiturates; PCBs CYP2 (CYP3), UGT, GST, receptor (CAR) bile acids OAT, MRP Pregnane X receptor (PXR) bile acids, organochlorine pesticides; CYP3; (CYP2); UGT pregnenolone PCBs Peroxisome-proliferator- fatty acids fibrates,phthalates CYP4 activated receptor (PPAR) and metabolites Farnesoid X Receptor (FXR)/ bile acids, CYP7, ABC-A1Liver X Receptor (LXR) oxysterols Retinoid receptors retinoids methoprene (RAR, RXR) Estrogen receptors (ER) 17--estradiol OC pesticides; CYP19, Vtg alkylphenols; others Androgen Receptors (AR) testosterone OC pesticides Glucocorticoid receptor (GR) glucocorticoids MeSO2-PCBs (CYP3)

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

40. nucleus hsp90 pRb Ara9 AHR ? E2F TCDD ARNT cell cycle XRE nuclear export proteasomal degradation Co-act mRNA BTF XRE cytoplasm TATA 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 Molecularbiology • 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: 51-88.

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-dependenteffects only)

48. TCDD toxic equivalency (TEQ) approachusing 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)