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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.

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slide1

Toxicology I: Principles & Mechanisms

Marine Mammal Toxicology

Spring 2004

Mark Hahn

Woods Hole Oceanographic Institution

slide2

Exposure

1. Absorption/route of entry

Dose

1. Distribution/toxicokinetics

2. Biotransformation

3. Excretion

Tissue concentration

1. Molecular mechanism

2. Pathogenesis

Effect (individual)

slide3

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
slide4

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
slide5

Distribution/toxicokinetics

  • hydrophobicity and lipid content
  • protein binding
  • effect of physiological condition (fasting, pregnancy)
  • compartmental analysis
  • physiologically based pharmacokinetic models
slide6

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
slide7

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)
slide11

Reactions - PAH metabolism

EH

CYP1A1

CYP1A1

DHD-DH

slide13

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

slide14

Reactions - PCB metabolism

Rob Letcher, Univ. of Windsor

slide15

Reactions - PCB metabolism

CYP2B

GST

NAT

CYP

FMO

MeT

b-lyase

Rob Letcher, Univ. of Windsor

oh pcbs

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
slide17

OH-PCBs as inhibitors of T4

transport by transthyretin (TTR)

Brouwer et al 1998

methylsulfonyl pcbs
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
slide19

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
slide20

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

slide21

2,3’,4,4’-TCB

2,2’,5,5’-TCB

slide22

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

slide23

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.

slide27

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.

slide28

CYPs in marine mammals

Immunochemical evidence and cDNA cloning

slide29

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.

slide30

Rates of PCB metabolism by hepatic microsomes

(pmol/min/mg protein)

White et al. (2000) Compar. Biochem Physiol. 126, 267

slide31

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.

slide32

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.

slide34

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)
slide35

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)

definitions

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.

definitions1

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.

definitions2

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
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

slide40

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

evidence for role of ah receptor in effects of dioxins planar pcbs
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

slide42

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.

3d structure of pcbs calculated dihedral angle
3D Structure of PCBs: Calculated Dihedral Angle

Hans-Joachim Lehmler, Univ. of Iowa

slide44

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)
slide47

Toxic equivalency (TEQ) approach using

toxic equivalency factors (TEFs)

(AHR-dependenteffects only)

slide48

TCDD toxic equivalency (TEQ) approachusing toxic equivalency factors (TEFs)

• Calculated TEQs versus Bioassay-derived TEQs

slide49

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
slide52

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
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
Ligand-binding assays

• High affinity, low capacity binding(Specific Binding)

Total [3H]-TCDD

Free (loosely bound)

Bound (Total)

Specific

binding

Non-specific

binding

slide55

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

Fractions

  • • Incubate
  • Spin for 2 hours
  • • Fractionate
  • • Count
slide56

Sucrose gradient analysis of

in vitro-expressed and tissue-derived AHR proteins

cloned, in vitro expressed

tissue-derived

Beluga Liver Cytosol

Beluga AHR

1600

1600

1200

TB

1200

dpm

800

800

NSB

400

400

0

0

0

10

20

30

40

0

10

20

30

40

Mouse AHR

Mouse Liver Cytosol

2500

2500

2000

2000

1500

dpm

1500

1000

1000

500

500

0

0

0

10

20

30

40

0

10

20

30

40

fraction number

fraction number

Jensen & Hahn (2001)

slide57

B

M

H

UPL

Saturation binding analysis of in vitro-expressed AHR proteins

beluga AHR

mouse AHR

human AHR

TB

SB

NSB

[35S]methionine-

labeled proteins

slide58

mean Kd (n=4)

beluga AHR 0.43 ± 0.16 nM **

mouse AHR 0.68 ± 0.23 nM *

human AHR 1.63 ± 0.64 nM

Equilibrium Dissociation Constants (Kd)

for in vitro-expressed AHR proteins

*p<0.05 versus human AHR

**p<0.01 versus human AHR

Beluga express a high-affinity (low Kd) AHR

slide59

In vitro binding affinity vs. In vivo tissue burdens

KD for TCDD: 0.43 nMin 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)

slide60

Relative Potencies or Toxic Equivalency Factors (TEFs) for dioxin-like compounds in wildlife

TEF values

congener IUPAC rodentmarine

PCDD/PCDF#mammals

2,3,7,8-TCDD 1 1

2,3,7,8-TCDF 0.1 ?

non-ortho PCB

3,3’,4,4’,5-PeCB 126 0.1 ?

3,3’,4,4’,5,5’-HCB 169 0.01 ?

3,4,4’,5-TCB 81 0.0001 ?

3,3,’4,4’-TCB 77 0.0001 ?

mono-ortho PCB

2,3,3’,4,4’-PeCB 105 0.0001 ?

2,3’4,4’,5-PeCB 118 0.0001 ?

2,3,3’,4,4’,5-HCB 156 0.0005 ?

Source: van den Berg, et al. (1998) Environ. Health Persp. 106: 775-792.

slide61

Beluga AHR

TCDD

TCDF

1.0

126

169

0.8

77

81

0.6

105

118

0.4

156

128

0.2

0.0

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

log[HAH] nM

Mouse AHR

TCDD

1.0

TCDF

126

0.8

169

77

0.6

81

105

0.4

118

156

0.2

128

0.0

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

log[HAH] nM

Competitive binding of PCB congeners using in vitro expressed AHRs

and [3H]TCDD

IC50: One-site competition model

(Prism)

KI: From IC50, [3H]TCDD

(Cheng and Prusoff)

Jensen & Hahn (2001)

slide62

Correlation between beluga and mouse AHR binding affinities

105

x=y

104

118

Mono-ortho PCBs

103

156

105

Di-ortho PCB

128

102

81

beluga KI (nM)

77

101

169

Non-ortho PCBs

100

126

TCDF

10-1

PCDD/F

TCDD

10-2

10-1

100

101

102

103

104

105

mouse KI (nM)

slide63

Harbor seal versus mouse AHR

[35S]methionine-labeled proteins

[3H]TCDD-binding

Kim & Hahn (2002)

slide64

TB

SB

mouse AHRKD = 1.70 ± 0.26 nM

NSB

TB

seal AHRKD = 0.93 ± 0.19 nM

SB

NSB

Kim & Hahn (2002)

slide65

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
Weight of evidence approachfor assessing impact of contaminants on marine mammals

Epidemiological and observational studies in wildlife species

Comparative mechanistic studies

Mechanistic studies in laboratory animals