The Aryl Hydrocarbon (Dioxin) Receptor: Promiscuity in Activation and Diversity in Response
M.S. Denison1, J.E. Bohonowych1, Bin Zhao1, Dal-Ho Han1,A. Pandini2 and L. Bonati2
1Department of Environmental Toxicology, University of California, Davis, CA 2Departmento di Scienze dell’Ambiente e del Territorio, Universita degli Studi
di Milano-Bicocca, Milano Italy
Halogenated Aromatic Hydrocarbons (HAHs)
O
O Cl
ClCl
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin
O Cl
ClCl
Cl
2,3,7,8-Tetrachlorodibenzofuran
Cl
Cl Cl
Cl
Cl
3,4,3',4,'5-Pentachlorobiphenyl
Sources of Dioxins and Related HAHs?
Herbicide Spraying(i.e. Agent Orange)
Food Combustion
OH
Cl
Cl
Cl
Cl
Cl
ChlorophenolsTransformers (PCBs)
Spectrum of Toxic and Biological Effects Produced by TCDD in Different Species and Tissues
• Immunotoxicity• Hepatotoxicity• Wasting Syndrome• Dermal Toxicity• Teratogenicity• Lethality• Endocrine Disruption• Tumor Promotion• Porphyria• Induction of Gene Expression (Also Repression)
• Cytochrome P4501A1/2 and 1B1• Glutathione S-Transferase• UDP-Glucuronosyl Transferase 1*6• Quinone Reductase
AhR Signal Transduction Pathway
TranslationNew Polypeptides
Increased Cytochrome P-4501A1
ARNT
Exogenous and Endogenous Ligands
Endogenous Ligands
Other Gene Products
mRNA
DREs CYP1A1
DREs Other Genes
Other Factors?Translocation
Proteosome
Degradation
AhR hsp90
XAP2 hsp90p23
Toxicity
QUESTIONS REGARDING AhR SIGNALING
1. Do all high affinity Ah receptor (AhR) ligands produce the same spectrum of toxic and/or biological effects?HAHs vs PAHs?
• What accounts for the differential responsivenessof a cell and animal to AhR ligands?
• What is diversity in AhR ligand structure?
• What is the significance of AhR ligand promiscuity?
Halogenated Aromatic Hydrocarbon AhR Ligands
PCDDsO
O
12
34 6
7
89
Congeners TEF2,3,7,8-TCDD 1.01,2,3,7,8-PeCDD 1.01,2,3,4,7,8-HxCDD 0.11,2,3,6,7,8-HxCDD 0.11,2,3,7,8,9-HxCDD 0.11,2,3,4,6,7,8-HpCDD 0.01OCDD 0.0001
PCDFs1
2
34 6
7
89
O
Congeners TEF2,3,7,8-TCDF 0.11,2,3,7,8-PeCDF 0.052,3,4,7,8-PeCDF 0.51,2,3,4,7,8-HxCDF 0.11,2,3,6,7,8-HxCDF 0.11,2,3,7,8,9-HxCDF 0.12,3,4,6,7,8-HxCDF 0.1 1,2,3,4,6,7,8-HpCDF 0.011,2,3,4,7,8,9-HpCDF 0.01OCDF 0.0001
PCBs23
4
5 6 2' 3'
4'
5'6'
Congeners TEF3,3’,4,4’-TCB 0.00013,4,4’,5-TCB 0.00013,3’,4,4’,5-PeCB 0.13,3’,4,4’,5,5’-HxCB 0.01
TEF = Toxic equivalency factor (relative to 2,3,7,8-TCDD)
Comparison of the Ligand Binding Affinity and the Relative Toxic Potency of Selected HAHs
TCDD
10 -510 -610 -710 -8.0001
.001
.01
.1
1
10
Ligand Binding Affinity (Kd)
Rel
ativ
e To
xic
Pote
ncy
(WH
O T
EF)
The Mechanism of AhR Action is Dependent Upon a Series of Equilibrium Events
AhR
L:AhR L:nAhR
L:nAhR* L:nAhR:Arnt
L:nAhR:Arnt: DRE
Gene Expression
Arnt
Arnthsp90 hsp90
DRE
LL
Degradation
hsp90
Ligand Dissociation Experiments (Equilibrium Kd ~1nM)
[3H]TCDD (2nM) + cytosolic AhR - 2 hours at 20oC
[3H]TCDD:AhR + unbound [3H]TCDD
Dextran-Coated Charcoal to remove free [3H]TCDD
[3H]TCDD:AhR
Add unlabeled 2,3,7,8-TCDF (200nM)measure specific binding at various times
[3H]TCDD:AhR + TCDF:AhR(Decreased specific binding indicates loss of original ligand)
2502001501005000
20
40
60
80
100
120
Time (hours)
[3H
]TC
DD
Spe
cific
Bin
ding
(per
cent
of T
=0)
TCDD Binding to the Guinea Pig Hepatic Cytosolic AhR is Essentially Irreversible
Typical “Equilibrium Dissociation Experiment
Similar dissociation results were also obtained with other species.
Reporter Gene Induction to Identify and Characterize AhR Agonists and Antagonists
TranslationNew Polypeptides
Increased Cytochrome P-4501A1
ARNT
Exogenous and Endogenous Ligands
Endogenous Ligands
Other Gene Products
mRNA
DREs CYP1A1
DREs Other Genes
Other Factors?Translocation
Proteosome
Degradation
AhR hsp90
XAP2 hsp90p23
Firefly Luciferase
Green FluorescentProtein (GFP)
DMSO
TCDD
8765432100
1000
2000
3000
4000
5000
6000
Time (days)
GFP
Act
ivity
(RFU
)
TCDD Induces Persistent Activation of Gene Expression
O
O
CH3
3-Methylcholanthrene β-Naphthoflavone Benzo(a)pyrene
Polycyclic Aromatic Hydrocarbons
10 -410 -510 -610 -710 -810 -910 -1010 -1110 -1210 -1310 -140
1000
2000
3000
TCDDBNF
Chemical Concentration (M)
GFP
Act
ivity
(RFU
s)
Induction of AhR-Dependent GFP Reporter Gene Induction by TCDD and BNF
Proposed that theLower Potency ofPAHs was Due to their Lower AhRBinding Affinity that Allows LigandDissociation andAhR Inactivation.PAHs are also Metabolically Labile.
While PAHs have Relative High AhR Ligand Binding Affinity they Produce Little AhR-Dependent Toxicity
10-5
10-6
10-7
10-8
10-9
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
HAHsPAHs
AhR Ligand Binding Affinity (Kd)
Toxi
c Eq
uiva
lenc
y Fa
ctor
(rel
ativ
e to
TC
DD
)
BNF
TCDD
TCDD and BNF are Essentially Irreversibly Bound to the AhR
2502001501005000
20
40
60
80
100
120
140Percent of TCDDPercent of BNF
Time (hours)
Spec
ific
Bin
ding
(Per
cent
of T
ime=
0)
8765432100
1000
2000
3000
4000
5000
6000
TCDDBNF
Time (days)
GFP
Act
ivity
(RFU
)
BNF Induces Transient Activation of Gene Expression
Ah Receptor (AhR) Signal Transduction Pathway
TranslationNew PolypeptidesIncreased Cytochrome P-4501A1 and Other
Gene Products
ARNT
AhR Ligands
mRNA
DREs CYP1A1
DREs Other Genes
Translocation
AhR hsp90
XAP2 hsp90
AhR GeneAhR
HAHs
PAHs
HO-PAHs
While AhR Binding Affinity is Important, Toxicity of AhR Agonists Appears to be More Dependent upon the Metabolic Stability of the Agonist
Which can Lead to the Chronic Activation of Nascent AhR.
Differential Responses to AhR Ligands
AhR Ligands
MetabolicallyStable andHigh AffinityLigands
Toxicological andBiological Effects
MetabolicallyLabile and/or Lower Potency Ligands
Biological Effects
Are AhR Ligands Structurally Similar?
Structural Diversity of AhR Agonists and/or Antagonists“Nonclassical” Synthetic AhR Ligands and/or Agonists
O
O
Cl
Cl
NN NH2
O
H
Guanabenz
NH2
SCH3
2(Methylmercapto)aniline
NH2
NH2
1,5-Diaminonaphthalene
N
NS
NO
CH2
OCH3
CH3CH3
Omeprazole
CH3O
N
CH3
H
5-Methyl-2-phenylindole
NH2
OH
(1S,2R)-(-)-cis-1-Amino-2-indanol
NH
NH
NH2
NO2O2N
OCH3
SRN-P2:109,NH2
NNH2
CH3
1-Methyl-1-phenylhydrazine
NN NH
CN
CF3
SKF71739
S
NCH3
NH2
2-(4-Amino-3-methylphenyl) benzothiazole
N
S
S O
N
O
O
S
Cl
ClCl
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin
CH3YH439
CH3
CH3
H
NN
NH
N
Bilirubin
CH3
CH2
CH2
CH3
CH3
COOH COOHCH3
OH HO
O
CH3
CH3 CH3
CH3CH3
CH3
CH3CH3
CH3
O
CH3Canthaxanthin
COOH
OOH
OO
Prostaglandin G2
OH
OHCOOH
OH
Lipoxin A4
OCH3
OO
OH
OOH
OOCH2
OH
O
OH
OH
OH
CH3
Diosmin
O O
Dibenzoylmethane
OHOH
OCH3H3COO O
Curcumin
O
O
OCH3
H3CO
OCH3
H3CO
OCH3Tangeritin
OOH
OH
OOH
OH
OCH3
Tamarixetin
OH O
CH3
CH3
CH3 CH3
CH3
7-Ketocholesterol
Natural and Endogenous AhR Ligands,Agonists and AntagonistsStructural Diversity of AhR Agonists and/or Antagonists
Naturally-Occurring and Endogenous Indole-Containing AhR Ligands
N
O
OHNH2
Tryptophan
N
N
O
Trypthantrin
NH
NH
CHO
Malassezin
NN
3,3'-Diindolylmethane
N
N
Indolo[3,2-b]carbazole
NHNH
O
OIndirubin
NH
ONH
O
Indigo
N
N
O
O6,12-Diformylindolo[3,2-b]carbazole
N
N
O
6-Formylindolo[3,2-b]carbazole
NH
OH
Indole 3-Carbinol
NH2
O NH2
O
OH
L-Kynurenine
NO
O
OH
Indole Pyruvic Acid
NH
OH
O
Indoleacetic Acid
NH
NH2
Tryptamine
NH
Br
Br
NH
Br
Br
O
O
Brominated IndolesNH
NH
NH
N
O
O
β-Carboline-3-Ethylester
1,2,3,4-Tetrahydro- β-Carboline
β-Carbolines
Unknown Tryptophan-Derived Products
Plants
Acidic Conditions
Enteric Bacteria
Plants and Animals
Yeast and Fungi
UV Irradiation
Mammals and Plants
Marine Organisms
Plants and Marine Organisms
Activation of the Ah Receptor By Diverse Classes of Chemicals
EnvironmentalXenobiotics
(HAHs & PAHs)
Natural & DietaryXenobiotics
(I3C, ICZ etc)Endogenous Regulators
"Endobiotics"
SyntheticChemicals Pharmaceutical
Agents
Biological and/orToxicological Responses
hsp90
hsp90
XAP2
AhR
p23
OtherChemicals
*
In Vitro DNA Binding Assay for Ah Receptor Agonist Screening
TCDD, pure AhR agonist or aextract containing AhR agonists
Gel Retardation Assay
Liver Cytosol
(AhR & Arnt)
Amount of AhR:DNA complexis directly proportional to the amountof TCDD or other agonist in sample
ArntAhR
32P
32P
32P
32P
TCDD - +
Simple Extraction of AhR Agonists from Products
1. Sample is weighed and added to a tube.
2. DMSO is added at the following ratio:Absorbent products: 1 mg product : 10 µl DMSONonabsorbent products: 1 mg product : 1.5 µl DMSO
3. Tubes are left overnight at room temperature.
4. DMSO collected from tubes.
5. Aliquots (1.25 µl) are tested for their ability stimulateAhR-dependent DNA binding by gel retardation analysis.
6. Inducible AhR:DRE complex formation quantitated byPhosphorimager analysis.
DMSO Extracts of Commercial and Consumer Products Contain Chemicals that Stimulate AhR DNA Binding
DMSO, Ethanol and Water Extracts of Selected Products Stimulate AhR DNA Binding
There is a Good Correlation Between the Ability of an Extractto Stimulate Guinea Pig AhR DNA Binding and Reporter Gene Expression
1201008060402000
20
40
60
80
100
120
140
Gel Retardation Analysis (% TCDD)
Luci
fera
se G
ene
Indu
ctio
n (%
TC
DD
)
AhR Agonists in Commercial Products are Metabolically Labile
50100 50150
% TCDD00
Red rubber-band
Black stopper
Black 0-ring
Cell scraper
Yellow pad
Blue paper-Towel
Business card
Newspaper
H1L1.1c2 (4 h) H1L6.1c2 (24 h)
DMSOEthanolWater
Activation of the Ah Receptor By Diverse Classes of Chemicals
Increased Expression of Metabolic Enzymes with Diverse Substrate Specificity
Metabolism of the Inducing Chemical(s)
EnvironmentalXenobiotics
(HAHs & PAHs)
Natural & DietaryXenobiotics
(I3C, ICZ etc)Endogenous Regulators
"Endobiotics"
SyntheticChemicals Pharmaceutical
Agents
Biological and/orToxicological Responses
hsp90
hsp90
XAP2
AhR
p23 OtherChemicals
What are the Structural and Functional
Determinants of the AhR Ligand Binding Domain?
Ligand & hsp90BindingbHLH
NH2 COOHQ richA B
417288
mAhR modeled region: PAS B Domain (aa 277 - 380)
Homology Model of the AhR Ligand Binding Domain
Hypothetical Ligand-Induced Conformational Change in the Ah Receptor Ligand Binding Domain
Ligand L
hsp90 hsp90
hsp90hsp90hsp90
Conclusions I
The AhR can bind and be activated by a structurally diverse array of ligands and it plays a key role as an “environmental sensor” leading to enhanced metabolismand degradation of exogenous and endogenous chemicals.
Not all AhR agonists are created equal - while all can induce gene expression, notall can produce toxic effects. The toxic potency of an AhR agonist is primarily dependent on its metabolic stability and persistence in the cell.
The binding of ligand to the AhR (TCDD and β-naphthoflavone) is essentiallyirreversible, which invalidates current ligand binding affinity estimates and which assume equilibrium binding conditions. The measured binding affinities are actually ligand association rates.
Conclusions II
The structural diversity in AhR ligand binding is not surprising given the role ofthe AhR as an activator of expression of metabolic enzymes each of which can bind and metabolize a structurally diverse array of chemicals.
This diversity also implies that the AhR has a very promiscuous ligand binding pocket and suggests the existence of multiple endogenous physiological ligands.
QSAR modeling of structurally diverse AhR ligands and the AhR ligand binding domain will provide information with regards to the key structural and physiochemical characteristics important for AhR ligands and ligand binding.
AcknowledgementsUniversity of California• Elaine Khan• Yujuan Song• Michael Ziccardi• Michael Nantz
Universita’ degli Studi di Milano-Bicocca, Italy
• Laura Bonati• Alessandro Pandini• Davide Lacchini• Vera Meazzi• Prof. Demetrio Pitea
Supported by a NIEHS Superfund Basic Research Grant (ES04699), the NIEHS (ES07685, ES5707) and the American Taxpayers.
Woods Hole Oceanographic Institute• Mark Hahn
Imperial Cancer Research Fund.uk• Peter Parker
Rockefeller University• Laurent Meijer• Paul Greengard
University of Queensland.au• Elizabeth Gillam