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Aryl hydrocarbon receptor and
dioxin-like toxicity
Dr. Jan Vondráček (IBP.CZ)
Denison et al., Chem. Biol. Interact. 141: 3
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Overview of aryl hydrocarbon receptor and dioxin-
like toxicity:
ˇ what is AhR;
ˇ evolution perspective;
ˇ activation of AhR; AhR-dependent genes
ˇ toxic effects associated with AhR activation;
ˇ dioxin/like toxicity and TEF/TEQ concept;
ˇ biomarkers of AhR activation and methods of
detection of AhR-mediated activity.
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PAS proteins:
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AhR =
ˇ ligand-activated transcription factor;
ˇ important mediator of toxicity of POPs;
ˇ regulator of xenobiotic metabolism and activation of
promutagens.
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AhR domain structure:
Denison et al., Chem. Biol. Interact. 141: 3
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Evolution of AhR:
AHR1
AHR2
AHRR
ARNT
Hahn, Chem. Biol. Interact. 141: 131
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AhR activation:
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AhR regulated genes:
contain xenobiotic response elements (XRE) or dioxin
responsive elements (DRE) in their promoter region:
ˇ phase I enzymes - CYP 1A1, CYP 1A2, CYP 1B1;
ˇ phase II enzymes - UDP-glucuronosyltransferase, GST-
Ya, NADP(H):oxidoreductase;
ˇ other genes - Bax, p27Kip1, Jun B, TGF- - regulation of
cell cycle and apoptosis;
ˇ AhRR.
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AhR activation:
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Physiological role for AhR - AhR-deficient
mice:
ˇ significant growth retardation;
ˇ devective development of liver and immune system;
ˇ retinoid accummulation in liver;
ˇ abnormal kidney and hepatic vascular structures.
ˇ resistant to BaP-induced carcinogenesis and TCDD-
induced teratogenesis;
ˇ no inducible expression of CYP 1A1 and 2.
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Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309
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,,Non-classical" AhR ligands
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Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55
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Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55
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TEFs provide a simple, single number that is indicative of overall
toxicity of a sample containing a mixture of dioxins and dioxin-
like compounds. TEFs are consensus values based on REPs
across multiple species and/or endpoints. TEFs are based upon a
number of endpoints, from chronic in vivo toxicity to in vitro
toxicity with the former having the greatest importance in
determining overall TEF.
The total potency of a mixture can be expressed in TCDD TEQ
concentration:
Toxic equivalency factors (TEF)/TEQ concept:
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Toxic equivalency factors for PCDDs, PCDFs
and PCBs:
Eljarrat & Barceló, Trends Anal. Chem.22: 655
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Biomarkers/bioanalytical methods:
ˇ in vivo biomarkers: EROD activity, CYP 1A1 and 1B1
expression;
ˇ in vitro:
EROD in H4IIE rat hepatoma cells;
CALUX/CAFLUX assays;
GRAB assay (AhR-DNA binding)
yeast bioassay;
immunoassays;
detection of CYP1A mRNA or protein
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Detection of EROD activity:
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Aryl hydrocarbon receptor-mediated activity
determined using in vitro reporter gene assay
Aryl hydrocarbon receptor-mediated activity
determined using in vitro reporter gene assay
TCDD and Related
Compounds
LightLight Luciferase
Nuclear
Factors
1
P
+ AhR
HSP90
HSP90
Src
HSP90
HSP90
Adapted from Blankenship (1994)
DRE-Luc
AhR
ARNT
Modulation of Gene
Expression
ARNT
Src
"Activated"
2
P
P
Cytosolic
Proteins
Membrane
Proteins
Increased Protein
Phosphorylation
TCDD and Related
Compounds
LightLight Luciferase
Nuclear
Factors
1
P
+ AhR
HSP90
HSP90
SrcSrc
HSP90
HSP90
Adapted from Blankenship (1994)
DRE-Luc
AhR
ARNT
Modulation of Gene
Expression
ARNT
Src
"Activated"
2
P
P
Cytosolic
Proteins
Membrane
Proteins
Increased Protein
Phosphorylation
CALUX/CAFLUX assays:
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Gel Retardation of AhR Binding (GRAB) assay:
measures the ability of
chemical or chemical mixture
to stimulate AhR
transformation and DNA
binding in vitro