Aryl hydrocarbon receptor and
dioxin-like toxicity Dr. Jan Vondráček (IBP.CZ)
Denison et al., Chem. Biol. Interact. 141: 3
Thr343: Cj)
laboratory ytokinetics
VIM-I
Overview of aryl hydrocarbon receptor and dioxin-
like toxicity:
what is AhR;
evolution perspective; activation of AhR; AhR-dependent genes toxic effects associated with AhR activation; dioxin/like toxicity and TEF/TEQ concept; biomarkers of AhR activation and methods of
detection of AhR-mediated activity.
laboratory ytokinetics
PAS proteins:
R J. Kew/ey et a!./The International Journal of Biochemistry & Ceil Biology 36 (2004) 1X9-204
a. bHLH
b. bHLH/Zip
bHLH
dimerization DNA binding
T
E47, Myo D
hHLH uSL,
DHLH dimerization
T   ▼
Max, Myc
c. bHLH/PAS Class I
bHLH
dimerization DNA binding
T
Ah R
AhRR
PAS
secondary dimerization
___I___
B
Transactivation
^^
Xenobiotic response
HIF-a IPAS
B
B
xyA   Hypoxia signalling
SIM1&2
B
Repression
Neurogenesis
Class II
ARNT1&2
M
B
General partner factor
BMAL 1 & 2
B
Circadian rhythm
Fig. L Schematic representation of the domain structure of some bHLH transcription factor family members.
AhR =
• ligand-activated transcription factor; • important mediator of toxicity of POPs; regulator of xenobiotic metabolism and activation of
promutagens.
AhR domain structure:
bHLH           PAS  Domain I--------------1 I--------------------------------------------------------------------
NH2
[111      1 B]-       I @]                           [lQ richl[
COOH
^
L
J   L
J
■    *    '
Ligand & hsp90  Transactavation Binding
Transformation
Fig. 2. Domain structure of the AhR.
Denison etal., Chem. Biol. Interact. 141: 3
laboratory ytokinetics
										Ecdysozoa Lophotrochozoa Deuterostoma
								Arthropoda		
										
								Nematoda		
										
								Priapula		
										
								Mollusca |		
				\—						
										
					-i			Annelida             i Platyhelminthes  1 Brachiopoda       1 Echinodermata   1		
970 NT*		i—i	>							
										
										
								nemicnora<	ALCl           1	
								Chordata |		
										
										
								Cnidaria    ^ Porifera           ^		
										
										AHK sinologs identified |
										
Evolution of AhR:
AHR1 AHR2
AH RR
ARNT
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Hahn, Chem. Biol. Interact. 141: 131
AhR activation:
R.J. Kewley et all The International Journal of Biochemistry & Cell Biology 36 (2004) 189-204
19
► Xenobiotic response genes
AhR regulated genes:
contain xenobiotic response elements (XRE) or dioxin responsive elements (DRE) in their promoter region:
• phase I enzymes - CYP1A1, CYP1A2, CYP 1B1;
• phase II enzymes - UDP-glucuronosyltransferase, GST-Ya, NADP(H):oxidoreductase;
• other genes - Bax, p27Kiv1, Jun B, TGF-ß - regulation of cell cycle and apoptosis;
• AhRR.
laboratory ytokinetics
AhR activation:
R J, Kewley et al./The International Journal of Biochemistry ť£ Cell Biology 36 (2004) 189-204
► Xenobiotic response genes
Physiological role for AhR -AhR-deficient
mice:
• significant growth retardation;
• devective development of liver and immune system;
• retinoid accummulation in liver;
• abnormal kidney and hepatic vascular structures.
• resistant to BaP-induced carcinogenesis and TCDD-induced teratogenesis;
• no inducible expression of CYP 1A1 and 2.
laboratory ytokinetics
'TlttssicarAhR Ľi^and* and CVP1A1 Inducers
a               -a
2,3,7,8-1 eŤľíicliloťodibenzo-i>-<iioxLti       3,-U\4/5-P>iitadilorobinheiivl      2.3.7,8-letrachlorodibenzofu ran
Benzoŕ^pvreiifr
Ď-ľManlirhotlavonc
Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309
laboratory ytokinetics
„Non-classical  AhR ligands
M.S. Denison et al. í Chemico-Biological Interactions 141 (2002) 3  24
Ch

CI
CI-    —     or    —    -ci 2^,7^-TetrflchlorodÍbenzo-p-dÍQSÍn    2-fMethvlmerwmtn^iiffl.».
u3
CF,
O-^k-Q
2-f4'-Chlorqphenytibc!iizotliMzolc
■N^N—N-CN       SKF71739
CH3
CH2
Bilirubin
NH2 1 ^-THaminoBaphthiüeiie
0
0CONHCH3            i^V^'i   II               n.
c6 Jx^r&s
Carbarvl
Omeprazole
I3        J       CH3
OCH<
O Indirubin
or^
C6-Methvleiiedioivbeiizeiie
H Trvptamine
CH<
Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55
Epithelial hyperplasia
Tumor promotion
Induction of drug-metabolizing enzymes
Altered ER signaling
Porphyria
Deregulated lipid metabolism
Decreased serum thyroxine
Wasting    -
Metabolism of arachldonic acid to biologically active products
Persistent thyroid hormone receptor activation
EGF receptor down-regulation Lipid peroxidation
Immunosuppression
Inhibition of gluconeogenesis
Teratogenesis/embryotoxicity
Utilization of brown adipose tissue
Vitamin A depletion
Cardiac dysfunction
Figure I    Biological responses to TCDD, A wide variety of cellular processes have been shown to be affected by TCDD.
Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55
AHR SIGNALING PATHWAYS
79
AHR-ARNT-de p*nd ear gŕne expression
AHR.-ARNT

Repression «f ARNT-mcdiated gene expression
AAVM«F-lftWAIWT->C
Repression of other
AHH-mcdidtcdgcnt
eiprcssion
Drug mtMahiNl Í7Jn^. crizyiHíí Flä&iiiinu^n üUivaLm: inhibitor-2
Inleríeukin I "P
I^Wf-dflÍTiiiy sile regulated gem«
-*■ Secondary gfira regulation
Eiyihitipuieiirt VtühfUüh" Glytůlyiic enzym« Unknown genes
Unkjujwn genes
AHH-X
Sequestration of basal or PAS-spetific ťoacrivjtflrs by AHR-ARNT
AHPL-AftNT
UrtrílůLcdKtncs
Figure 6 Possible models for the mechanism of TCDD toxicity, which probably results from alterations in gene expression induced by AHR-ARNT activity. This may be either a direct effect of the activation of AHR-ARNT-regulated genes or an indirect effect resulting from a decrease in the availability of either the AHR or ARN T to panici pal c in different transactivation complexes.
Toxic equivalency factors (TEF)/TEQ concept:
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:
laboratory ytokinetics
Toxic equivalency factors for PCDDs, PCDFs
and PCBs:
Table 4. Toxic EquivalenL	Factors established by	the WHO (WHO-TEFs) for dioxins and dioxin-like PC3s [41			
PCDD Congener	WHO-TEF	PCDF Congener	WHO-TEF	PCB Congener	WHO-TEF I
2,3,7,8-TCDD	1	2,3,7,8-TCDF	0.1	Won-off ho	
	1	12,3,7,8-PeCDF	005	PCB#81	0.0005
123478-HxCDD	0.1	23478-PeCDF	0.5	PCB#77	0.0005
123ň78-HxCDD	0.1	123478-HxCDF	001	PCB#12ň	0.1
12,3,7,89-HxCDD	0.1	123Ŕ78-HxCDF	0.1	PCB#1Ů9	0.01
1234ň78-HpCDD	001	234f>78-HxCDF	0.1	Mono-orího	
OCDD	0.0001	12,3,7,89-HxCDF	0.1	PCB#105	0.0001
		1234670-HpCDF	0.01	PCB#114	00005
		1234789-HpCDF	0.01	PCB#118	0.0001
		OCDF	o.oom	PCB#123 PCB#156 PCB#157 PCB#1Ů7 PCB#189	0.0001 00005 0.0005 0.00001 0.0001
					
Eljarrat & Barceló, Trends Anal. Chem.22: 655
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Table  2.   Comparative  lists	of POPs selected for	environmental and
toxicolo^ical studies		
POPs selected at the	POPs with an	Emerging
Stockholm Convention	assigned TEF or	POPs
(2001)	REP	
Aldrin		
Chlordan c		
DD\		
Dieldrin		
bndrin		
Hep t ach lor		
H ex ac hlo roben ze n c		
Mi rex		
íoxaphene		
ľCBs	PCBs	
PCDDs/PCDhs	PCDDs/PCDhs ľCNs	
	PBDts	PBDEs
	PBDDs/PBDhs	PBDDs/PBDhs
	PBBs	PBBs
	PAHs	
Eljarrat & Barceló, Trends Anal. Chem.22: 655
Biomarkers/bioanalytical methods:
•in vivo biomarkers: EROD activity, CYP 1A1 and 1B1
expression;
• 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
laboratory ytokinetics
Detection of EROD activity:
140                       M. Till et aL / Chemico-Biological Interactions 111 (1999) 135-150
0            8           16          24          40          48          64         72
Incubation time (h)
Fig. 2. Time course of induction of CYPlAl-catalyzed 7-ethoxyresorufin 0-deethylase (EROD) activity in primary cultures of rat hepatocytes, after addition of 1.7 x ÍO-15 M benzo[ŕ/]pyrene (-▼-), 1.9 x 10 ~6 M benzo[A"]rluoranthene (-A-) or 9.4 x 10_5 M acenaphthylene (-O-). EROD activity was determined in cell homogenates. The data represent means + S.D. from four independent experiments.
CALUX/CAFLUX assays:
i hydrocarbon receptor-mediated activi rmined using in vitro reporter gene ass;
TCDD and Related Compounds     ,
a + Ah
Lighť
ChŠpŽ

Nuclear s Factors
"Activated"1, /
DRE-Luc
Increased Protein\    Modulation of Gene
Phosphorylation
Expression
Manbrane
Proteins
Cytosolic v Proteins/
Luciferase,
Adapted fron Blankenáiip (1994)
laboratory ytokinetics
Gel Retardation of AhR Binding (GRAB) assay:
[iNF:   +
AHR/ARNT DRE
-» measures the ability of
chemical or chemical mixture
to stimulate AhR
transformation and DNA
binding in vitro
laboratory ytokinetics