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