Aryl-hydrocarbon Receptor: Signaling, Toxicity, and UDP-

GlucuronosyltransferasesAnna Radominska-Pandya

Department of Biochemistry and Molecular Biology

University of Arkansas for Medical Sciences

Little Rock, Arkansas, US

October 2010; Gdansk University of Technology

Aryl-hydrocarbon Receptor (AhR)

• What is an ‘Aryl-hydrocarbon’?

– Aromatic hydrocarbon or arene

– “Cyclic molecule with characteristic electron density that is highly unsaturated and unexpectedly stable” (B&F, 1998)

• AhR is also know as the Dioxin Receptor

– Dioxin is the popular name for the family of chlorinated organic compounds consisting of:• Polychlorinated dibenzofurans (PCDFs)

• Polychlorinated dibenzodioxins (PCDDs)

– Dioxins have been shown to bioaccumulate in humans and wildlife due to their lipophilic properties.

-Aryl

Aryl-hydrocarbon Receptor (AhR)

• AhR was isolated in 1991

• Ligand-dependent transcription factor regulating gene expression

• Mediates toxicity of environmental contaminants exerting broad toxicological effects– Belongs to the basic helix-loop-helix/Per-Arnt-sim family

(bHLH/PAS) of chemosensors

– Highly conserved throughout evolution

– Expressed in:• Liver

• Central nervous system

• Reproductive organs

• Cardiovascular system

Aryl-hydrocarbon Receptor (AhR)

• Activated by a variety of environmental pollutants – Polycyclic aromatic hydrocarbons (PAHs)

• benzo[a]pyrene (BaP),

– Halogenated aromatic hydrocarbons (HAHs)• 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD)

– Polychlorinated biphenyls (PCBs)

• Natural endogenous ligands have not been identified– Potential candidates are:

• 7-keto-cholesterol

• Oxidative products of arachidonic acid

• Indols (products of the degradation of tryptophan),

• Retinoic acid

• Bilirubin

Exogenous ligand Structure example

Halogenated aromatic

hydrocarbons (HAH)TCDD

Polycyclic aromatic hydrocarbons

(PAH)

3-MC B[a]P

Flavonoids β-Naphthoflavone

Ligands for AhR

(Denison & Nagy, 2003)

Cl

ClO

OCl

Cl

CH3

O

O

Endogenous ligand Structure

2-(1’H-indole-3’-carbonyl)-thiazole-4-

carboxylic acid methyl ester

(Song et al., 2002) ITE

Tryptophan metabolites(?)

(Adachi et al., 2001)Indirubin

Bilirubin

Endogenous Ligands for AhR

NH O

CH3

HN

S

N O

O

O

AhR: Mechanisms of Toxicity

• Ligand biotransformation

• Induction/repression of genes

• Carcinogenesis

• Endocrine disruption

Ligand Biotransformation

• PAHs undergo oxygenation to generate diol epoxides or radical cations

B[a]P

(Cavalieri & Rogan, 1993)

PeroxidasesOxidants CYP1A1

B[a]P radical cation+

e-

OH

CYP1A1 and

epoxide hydroxylase

HO

O

B[a]P diol epoxide

DNA Adduct Formation

• Reactive electrophiles bind covalently to DNA

B[a]P radical cation

+

Guanine

HN

N

N

NH

O

H3C

..HN

N

N

NH

O

H3C

B[a]P-6-N7Gua

(Cavalieri & Rogan, 1993)

AhR and Carcinogenesis

• DNA adduct provides carcinogenesis initiation

• Activate protooncogenes genes

• Inactivate tumor suppressor genes

• DNA adduct formation suppressed in AhR-KO mice• non-AhR mediated DNA adduct formation

(Kondraganti, 2003)

2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD)

• Most persistent and bioaccumulative environmental contaminant

• The most biologically active environmental contaminant

• The highest acute toxicity of any environmental contaminant

• Environmentally and biologically stable

• Mutagenic in vivo and classified as a tumor promoter

• Endocrine disruptor

Cl

ClO

OCl

Cl

HAH Mediated-Toxicity and Carcinogenesis

Dioxin exposure

Free Dioxin in tissues

Dioxin binding to AhR in tissues

AhR-Dioxin complex binding with DNA

Gene regulation

mRNA regulation

Protein synthesis

Biochemical alterations

Early cellular responses (cell growth stimulation)

Delayed tissue response (cancer)

Ligand-Activated AhR

• The biological activity of AhR ligands is mediated by AhR and may contribute to:– Malignant transformation – Carcinogenesis

– Arteriosclerosis – Immunosuppression

– Endocrine responses – Wasting

– Neurotoxicity – Reproductive toxicity

– Diminished viral resistance – Chloracne

Physiological Role of AhR

• Observations in mice lacking WT AhR

– Hepatic growth and development stunted (Schmidt et al. 1996)

– Cardiac hypertrophy in adult AhR null mice (Lund et al. 2003)

– Cell proliferation: • Angiotensin II and ET-1 implicated (Lund et al. 2003)

– Lack of teratogenic response to TCDD (Mimura et al. 1997)

Role of Chemoprotective Phytochemicals

Can be both AhR agonists and antagonists:

Resveratrol and Flavonoids

Resveratrol

• Structure

• Occurrence

– Grapes

– Wine

– Grape juice

– Peanuts

– Berries of Vaccinum species (blueberries, cranberries, etc.)

Flavonoids

• Basic Structure

• Structure of Isoflavonoid, Daidzein

• Occurrence– Soy Beans

– Red wine

– Berries of Vaccinum species (blueberries, cranberries, etc.)

• Biological Activities– Lower cholesterol

– Reduction of cardiac risk

– Decrease cancer incidence• Breast

• Prostate

– Increased longevity

O

O

O

OH

HO

Endocrine Disruption

• Crosstalk between AhR and ER

– Ligand-activated AhR-Arnt complex can activate unliganded ER to mimic estrogens

• Disruption of thyroid hormone

• Crosstalk between AhR and GR

Brosens & Parker (2003). Nature 423: 487.

AhR signaling

AhR Pathway Participants

90

X XAP2 (hepatitis B virus X-associated protein)

HSP90 x 2 (90kDa heat shock protein)

L

L

Exogenous ligand

Endogenous ligand

AhR Arnt

XRE promoter gene

IC Initiation complex

Target DNA

AhR Signaling Pathway

Cytoplasm Nucleus

9090

X

AhR

L

L

9090

X

L

9090

X

L

L

9090

X

L

or Arnt

AhR Signaling Pathway

XRE promoter gene

Translation

Increased expression CYP1A1 protein

Increased expression of other gene products

+

AhR/Arnt heterodimer

mRNA

IC

+

Xenobiotic Response Element (XRE)

• Dioxin response element

• AhR and Arnt binding to different parts of XRE

• XRE in enhancer in upstream regulatory sequence• Phase I enzymes – CYP 1A1 (eight XREs in enhancer)

• Phase II enzymes – UGTs

• AhRR

XRE gene

5’TNGC GTG3’

AhR

Arnt

(Whitlock, 1999)

AhR Target Genes

Drug Metabolizing Enzymes:

P450s and UGTs

Aryl hydrocarbon Receptor (AhR)

• Target Genes: Drug Metabolizing Enzymes

– CYP450s• Ex) 1A1, 1A2, and 1B1

– UGTs

– GSTs

• Induces the expression of growth factors and proto-oncogenes

• Many, many more…

Examples of AhR Target Genes in the Cardiovascular Domain

• Genes– iNOS– Endothelial NOS– Cyclooxygenase 2– PG H Synthase– PG endoperoxide synthase– 5-lipoxygenase– 15-lipoxygenase– CD23 p45– NF-kB p65– NF-kB p50– I kappa B alpha– Meloperoxidase

• Interlukines, Chemokines– IL7– RANTES– IL-1β

• Viral Receptors– CD4– CXCR4– Coxsackle-adenovirus receptor

• Membrane Receptors– Thromboxane A2 receptor– IL-2 receptor a – IL-2 receptor b– IL-8 receptor a – IL-8 receptor b– TNF receptor p75/80– Interferon gamma receptor II– Connexin 43– CD36– ICAM (CD54)– LDL receptor

• Viruses– Cytomegalovirus IE-1– HIV 1 isolate BRU (LAV-1)– HIV 2– Herpes virus HSV1– Hepatitis A virus– Adenovirus type 2– Adenovirus type 12– Adenovirus type 19A

Sequential metabolism of BaP and 2,3,7-triCDD by P450 and UGT in human liver

microsomes.

Putative Interactions Between P450s and UGTs

• Evidence points to proteomic coupling between P450s (phase I oxidative enzymes) and UGTs (phase II conjugative enzymes).

• This coupling can be:A. Tight resulting in effective

detoxification

B. Loose resulting in toxicity.

C. Unbalanced resulting in toxicity.

• Figure modified from Nebert et al. JBC 2004