The Aryl hydrocarbon (Ah) Receptor: Comparative Toxicology and Possible Role as a Biomarker of Dioxin Susceptibility

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Toxicology I Principles Mechanisms
Marine Mammal Toxicology Spring 2004 Mark
Hahn Woods Hole Oceanographic Institution
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Exposure
1. Absorption/route of entry
Dose
1. Distribution/toxicokinetics
2. Biotransformation
3. Excretion
Tissue concentration
1. Molecular mechanism
2. Pathogenesis
Effect (individual)
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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

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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

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Distribution/toxicokinetics

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

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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

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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)

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Human P450 enzymes
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Regulation of CYP gene expression by soluble
receptors
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Reactions - PAH metabolism
EH
CYP1A1
CYP1A1
DHD-DH
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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
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Reactions - PCB metabolism
Rob Letcher, Univ. of Windsor
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Reactions - PCB metabolism
CYP2B
GST
NAT
CYP FMO
MeT
b-lyase
Rob Letcher, Univ. of Windsor
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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

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

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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

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Biotransformation capacity inferred from patterns
of PCB congeners(Dalls porpoise vs human)
Tanabe et al (1988) Capacity and mode of PCB
metabolism in marine mammals
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2,3,4,4-TCB
2,2,5,5-TCB
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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
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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.
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Immunochemical detection of CYPs in marine mammals
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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.
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CYPs in marine mammals Immunochemical evidence
and cDNA cloning
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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.
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Rates of PCB metabolism by hepatic
microsomes (pmol/min/mg protein)
White et al. (2000) Compar. Biochem Physiol. 126,
267
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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.
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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.
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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)

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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, barbitur
ates PCBs CYP2 (CYP3), UGT, GST, receptor (CAR)
bile acids OAT, MRP Pregnane X receptor
(PXR) bile acids, organochlorine
pesticides CYP3 (CYP2) UGT pregnenolone PCBs P
eroxisome-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 Glucocor
ticoid receptor (GR) glucocorticoids MeSO2-PCBs (
CYP3)
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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.

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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.

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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).

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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
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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
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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

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

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PAH vs PCB as agonists for the AHR
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Mechanisms of toxicity of PCBs and their
metabolites
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Toxic equivalency (TEQ) approach using toxic
equivalency factors (TEFs) (AHR-dependenteffects
only)
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TCDD toxic equivalency (TEQ) approachusing toxic
equivalency factors (TEFs)
Calculated TEQs versus Bioassay-derived TEQs
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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

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Ross et al (2000)
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Receptor-dependent mechanisms of toxicity in
marine mammals

• Species differences in receptor
  characteristics? - diversity - expression -
  function (affinity, SAR, target genes)

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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

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Ligand-binding assays
High affinity, low capacity binding(Specific
Binding)
Total 3H-TCDD
Free (loosely bound)
Bound (Total)
Specific binding
Non-specific binding
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Analysis of AHR specific binding on sucrose
density gradients
AHR 3HTCDD
AHR 3HTCDD TCDF (100x)
10 sucrose
Total binding
Non-specific binding
30 sucrose
Fractions

• Incubate
• Spin for 2 hours
• Fractionate
• Count

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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)
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Saturation binding analysis of in vitro-expressed
AHR proteins
beluga AHR
mouse AHR
human AHR
TB
SB
NSB
35Smethionine- labeled proteins
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Equilibrium Dissociation Constants (Kd) for in
vitro-expressed AHR proteins
plt0.05 versus human AHR plt0.01 versus human
AHR
Beluga express a high-affinity (low Kd) AHR
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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)
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Relative Potencies or Toxic Equivalency Factors
(TEFs) for dioxin-like compounds in wildlife
TEF values
congener IUPAC rodent marine PCDD/PCDF mammal
s 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-TC
B 81 0.0001 ? 3,3,4,4-TCB 77
0.0001 ? mono-ortho PCB 2,3,3,4,4-PeCB 105
0.0001 ? 2,34,4,5-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.
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Competitive binding of PCB congeners using in
vitro expressed AHRs and 3HTCDD
IC50 One-site competition model (Prism) KI
From IC50, 3HTCDD (Cheng and Prusoff)
Jensen Hahn (2001)
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Correlation between beluga and mouse AHR binding
affinities
105
xy
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
102
101
103
104
105
mouse KI (nM)
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Harbor seal versus mouse AHR
35Smethionine-labeled proteins
3HTCDD-binding
Kim Hahn (2002)
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TB
SB
mouse AHRKD 1.70 0.26 nM
NSB
TB
seal AHRKD 0.93 0.19 nM
SB
NSB
Kim Hahn (2002)
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Trainer Baden (1999) High affinity binding of
red tide neurotoxins to marine mammal brain.
Aquat Toxicol. 46 139-148.
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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