Download Nuclear receptor targets for endocrine disrupting effects

Nuclear receptor targets for endocrine disrupting effects
– mechanisms of action for emerging pollutants?
Anders Goksøyr
Prosjekt 181888 - Program Norsk miljøforskning mot 2015
onsdag 19. november 2008
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AIMS
To study the mechanisms of action of selected
environmental pollutants in a comparative manner, and
to elucidate evolutionary differences in chemico-biological
interactions of the nuclear receptors.
This knowledge may lead to new and more sensitive
technologies for biological effect monitoring of
environmental contaminants.
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NR
Responserelevance
model
Based on Adams (1991)
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Fifteen Years after ʻʻWingspreadʼʼ—Environmental Endocrine Disrupters
and Human and Wildlife Health: Where We are Today and Where We
Need to Go
Hotchkiss et al. Tox Sci 2008
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Vitellogenin induction downstream sewage
treatment works
Vitellogenin
(mg/ml(mg/ml)
plasma)
Plasma
vitellogenin
0,4
0,3
0,2
0,1
0,0
upstream
downstream
downstream
1 km
downstream
2 km
Vitellogenin (mean ± SEM; mg/ml) in plasma from rainbow trout caged immediately upstream or
downstream and one and two km downstream from a sewage treatment works.
From Parkkonen et al., 2000.
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OH
HO
Estradiol-17β or
estrogen mimic
Hepatocyte
Hsp 90
ER
Hsp 90
ER ER
Vtg and Zr protein
ERE
Vtg and Zrp genes
Secretion of
Vtg and Zr protein
Compounds shown to act via ER
Vtg and Zrp mRNA
Transport to ovary and
incorporation into oocytes
equol
ß-sitosterol
Daidzein
genistein
biochanin A
formononetin
coumestrol
zearalenone
zearalenol
o,p'-DDT
o,p'-DDE
ß-HCH, g-HCH
1-hydroxychlordene
4-tert-butylphenol
4-tert-octylphenol
4-tert-pentylphenol
4-nonylphenol
4-nonylphenol-diethoxylate
4-nonylphenoxy-carboxylic acid
3-trifluoromethyl-4-nitrophenol
di-n-butylphthalate
butylbenzylphthalate
toxaphene
methoxychlor
ethinylestradiol
diethylstilbestrol
bisphenol A
tert-butyl-hydroxyanisole
from Goksøyr et al. (2003)
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Masculinization of mosquitofish due to pulp and
paper mill effluents
Masculinized mosquitofish
(G. holbrooki) captured in
Fenholloway River, FL.
Masculinized least killifish
(H. formosa).
From Davis & Bortone (1992), in Colborn & Clement, eds. (1992).
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Nature 18 Sep 2008
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Nuclear receptors
members of a large superfamily of ligand-modulated transcription
factors
target genes involved in important and diverse biological processes such
as metabolism, development and reproduction, incl. phase I (CYP
genes), phase II (various transferases) and III (ABC transporters)
mediate transcriptional response to sex steroids (progestins, estrogens,
androgens), adrenal steroids (glucocorticoids and mineralocorticoids),
vitamin D3, thyroid and retinoid hormones, and a variety of metabolic
ligands
loss of control of nuclear receptor signaling contributes to endocrinerelated diseases. e.g. breast cancer, prostate cancer, ovarian cancer,
diabetes and obesity
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Nuclear Receptors - A Large Family of Ligand Modulated
Transcription Factors
Bind to specific DNA targets - Hormone Response Elements
Most are activators
Some constitutive
Few inactivate
Ligands are small lipophilic
molecules that freely enter cells
Diffuse from source
& penetrate to a target
Respond to low levels of hormone
Parts per billion levels
Regulation of levels is important
Can be disrupted by environmental
contaminants
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EDCs and nuclear receptors
X
PXR
CAR
PPAR
TR
VDR
ER
AR
GR
PR
NR3
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NR1
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Nuclear receptors as targets for
endocrine disrupting compounds
ER
alkylphenols, bisphenol A, lindane, DDT
AR
p,p’-DDT, sitosterol
PXR
alkylphenols, bisphenol A, chlordane, lindane
CAR
methoxychlor, dieldrin, DDE
PPAR
phtalates
RXR
methoprene
AhR
dioxins, PAHs + cross-talk
Goksøyr & Male (2006) DNVA
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Is there a connection?
Goksøyr (2006) JTEH
*a
Hepatic ERα
B
2000
6
b
*b
*bc
3
**bc
mRNA levels (% of control)
6β-OHase (pmol/min/mg protein)
9
1800
**
**
NP50
NP125
1600
1400
1200
1000
800
600
*
400
200
0
C
NP10
0
Control 1
5
25
125
E2
Dose (mg 4-nonylphenol/kg fish)
Steroid metabolism
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Estrogen receptor α
13
300
(i) E2-mimic
250
Project rationale
200
150
100
50
0
200
(ii) Hormone metabolism
The focus in endocrine disruption research on
steroid receptors is based on an
oversimplified model.
% of control
150
100
The nature of the effect of some EDCs is
determined by dose-dependent routing and
cross-talk between different classes of
nuclear receptor
50
0
1600
(iii) Receptor levels
1200
800
400
0
0
25
50
75
100
125
Important species variation in response to
different compounds makes it difficult to
propose general mechanisms for biological
consequences of exposure to different EDCs
acting through NRs
Dose of 4-nonylphenol (mg/kg)
Figure 1. Dose dependent effects of NP (i) as E2mimic, (ii) modulating hormone metabolism, and
(iii) modulating receptor levels (data from Atlantic
salmon, from Goksøyr, 2006)
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PXR/SXR
• PXR/SXR has an unusually large ligand specificity
• activated by many xenobiotics, incl. PCBs
• but some PCBs are PXR antagonists
• the most potent antagonists are non-planar,
highly chlorinated (≥6 Cl) species
• large species differences exist
• PXR is poorly conserved between species
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PBDEs activate hPXR (SXR)
hPXR
hAhR
Pacyniak et al. (2007) Tox Sci
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Species specific activation of PXR - luciferase reporter assay principle
Alkylphenols, NDL-PCBs, BPA, BFRs,
PCN, Ketoconazole, Pharmaceuticals
Cotransfect
CaPO4
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Cos-7
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Species differences in NR activation (example: SXR/PXR)
human
dog
mouse
rat
chicken
Xenopus
fathead
minnow
zebrafish
medaka
octylphenol
bisphenol A
chlordane
p,p-DDE
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✘
✓
✓
✘
✘
✘
✓
✓
✓
✓
✓
✓
✓
✓
✘
✓
✓
✓
✓
✓
✓
✘
✘
✘
toxaphene 2,4-DCP
✓
✓
✓
✓
✓
✓
✓
✓
✘
✘
✘
✘
✘
✘
✘
✘
✘
✓
endosulfan
dybutylphthalate
✓
✓
✓
✓
✓
✓
✓
✘
✘
✓
✓
✓
✓
✓
✘
✓
✘
✘
?
?
?
✓>4-fold
✘<4-fold
based on Milnes et al., Environ Health Persp (2008)
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Nuclear receptors - comparative aspects
X
X
X
X
X
X
X
comparative
protein-protein
interacions
comparative
ligand
specificities
Shulman & Mangelsdorf (2005) NEJM
ChIP-PCR
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ChIP-chip
comparative
transcriptomics
ChIP-seq
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Experimental outline of ChIP assay
Target protein
T
DNA
5. Add protein A/G
Protein A/G
1. Add 1 % formaldehyde
A/G
Formaldehyde
crosslinked cells
ab
T
6. Elute DNA-protein
complex
2. Cell lysis
Cell lysate
with crosslinked
protein-DNA
complexes
A/G
ab
3.Sonication
7.Reverse crosslinks
A/G
4. Add antibody
Antibody
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ab
8. Amplify target gene
ab
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Biphasic dose-response curves
from Calabrese & Baldwin (2003)
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Biomarker discovery in Atlantic cod fry liver after
continuous exposure to produced water
pI
control
low dose PW
high dose PW
Mw
*
*
medium dose PW
E2
Kjersem (2007), PhD thesis
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The dose-response concept revisited
100
75
50
25
0
log [dose]
The nature of the effect of some EDCs is determined by dosedependent routing and cross-talk between different modes of
toxicological action - Goksøyr (2006) J Toxicol Environ Health A vol 69
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Summary
The focus in endocrine disruption research on steroid receptors is based on
an oversimplified model.
Nuclear receptors represent an expanded range of mediators of endocrine
disrupting effects compared to previous research focus
Grün and Blumberg (2006): “The link that has been forged between
organotins and adipocyte differentiation opens an important new area of
research into environmental influences on human health with respect to
obesity and related metabolic disorders such as type 2 diabetes and
cardiovascular disease”
The nature of the effect of some EDCs is determined by dose-dependent
routing and cross-talk between different classes of nuclear receptor
Important species variation in response to different compounds makes it
difficult to propose general mechanisms for biological consequences of
exposure to different EDCs acting through NRs
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Project members
• Anders Goksøyr, project leader
• Rune Male, co-PI
• Marte Rusten, post doc (at Blumberg lab, UCI,
2008-2009)
• Roger Lille-Langøy, PhD student
• Marianne Brattås, PhD student
• Astrid Mork, Master student
• Hilde Førde, Master student
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Collaborating partners
•Jonathan Verreault, Canada
•Ian Mayer, Dept of Biology, UoB, NO
•Sonnic Meier, IMR Bergen NO
•Geir Wing Gabrielsen, Norwegian Polar Institute, Tromsø NO
•Lisa Bjørnsdatter Helgason, Norwegian Polar Institute, Tromsø NO
•Lionel Camus, Akvaplan-niva AS, Tromsø, NO
•Robert Letcher, Canada
•Heli Routti, Norwegian Polar Institute, Tromsø, NO
•Audun Nerland, Dept of Molecular Biology, UoB
•Ole Jakob Nøstbakken, NIFES
•
•
•
•
•
•
•
•
•
•
Anders Goksøyr & Rune Male, Department of Molecular Biology,
University of Bergen, NO
Marit Bakke, Department of Biomedicine, UoB, NO
Gunnar Mellgren, Dept of Medicine, UoB, NO
Augustine Arukwe, Dept of Biology, NTNU, Trondheim, NO
Malin Celander, Dept of Zoophysiology, GU, SE
Joakim Larsson, Sahlgrenska, GU, SE
Helen Håkansson, KI, SE
Bruce Blumberg, UC Irvine, CA, USA
Eric F. Johnson, Scripps, La Jolla, CA, USA
Inge Jonassen, CBU, UoB, NO
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