E i t l b Environmental obesogens:

Is the environment making us fat?

Bruce Blumberg, Ph.D.
Department of Developmental and Cell Biology
Developmental Biology Center
Institute for Genomics and Bioinformatics
University of California, Irvine y ,
The Worldwide Obesity Epidemic
60 million people in the US are clinically obese
>30% above ideal body weight
Obesity accounts for 8% of healthcare costs in Western countries
$75 billion annually in US $75 billion annually in US
Obesity is associated with metabolic syndrome -> type 2 diabetes
and cardiovascular disease and cardiovascular disease
Central (abdominal obesity)
Atherogenic dyslipidemia (high triglycerides, high LDL, low HDL)
Hypertension
Insulin resistance
Prothrombotic state
Pro-inflammatory state (elevated CRP)
Hormonal control of weight
Hormonal control of appetite and
metabolism
Leptin, ghrelin, resistin Leptin, ghrelin, resistin
adiponectin are key players
Hormonal control of fat cell
development and lipid balance
Regulated through nuclear Regulated through nuclear
hormone receptors RXR-PPAR
PPAR master regulator of
fat cell development fat cell development
increased fat cell differentiation
Increased fat storage in existing cells
Increased insulin sensitivity Increased insulin sensitivity
From Nature Medicine 10, 355 - 361 (2004)
How does obesity occur?
P ili i d h t t d Prevailing wisdom couch potato syndrome
Positive energy balance from too much food and increasingly
sedentary lifestyles
Other factors?
Stress
Inadequate sleep Inadequate sleep
Thrifty genes which evolved to make the most of scarce calories
What about role of prenatal nutrition or in utero experience? What about role of prenatal nutrition or in utero experience?
Maternal smoking decreases birth weight and increases obesity in
exposed offspring
What about the role of industrial chemicals?
Baillie-Hamilton (2002) postulated a role for chemical toxins in
the etiology of obesity
Noted that the obesity epidemic coincides with the marked
increase of chemical use in the environment
Endocrine Disrupting Chemicals (EDCs)
Endocrine disrupter - a compound that mimics or blocks the
action of endocrine hormones, either directly or indirectly
Disturb development, physiology and homeostasis
Persistent pollutants or dietary components
Frequently act through nuclear hormone receptors Frequently act through nuclear hormone receptors
environmental estrogens
Anti-androgens
h d Anti-thyroid
Nuclear Receptors - A Large Family of Ligand
Modulated Transcription Factors p
DNA LIGAND
A/B C D E F
NUCLEUS LIGAND
Bind to specific DNA targets - Hormone Response Elements
Most are activators
Some constitutive
NUCLEUS LIGAND
Ligands are small lipophilic
l l th t f l t ll
Few inactivate
BXR RXR
APO
molecules that freely enter cells
Diffuse from source
& penetrate to a target
CYTOPLASM
INTRA
Respond to low levels of hormone
Parts per billion levels
Regulation of levels is important g p
Can be disrupted by environmental
contaminants
The Nuclear Hormone Receptor Superfamily
A/B C D E F
Known Receptors Orphan Receptors
DNA LIGAND
A/B C D E F
Known Receptors
Classical receptors (from biochemistry)
GR cortisol
MR aldosterone
AR testosterone
Orphan Receptors
Vertebrate Drosophila
TR-2 , DHR78
NGFI-B ,, DHR38
ROR DHR3 AR testosterone
PR progesterone
ER , estradiol
VDR 1,25-(OH)
2
vit D3
TR , triiodothyronine
ROR ,, DHR3
Rev-erb , E75, E78
SF-1 , FTZ-F1 ,
COUP ,, svp
HNF-4 , HNF-4
EcR 20-OH ecdysone Tlx , tll
No known homologs
ERR ,, knirps
DAX-1 knirps-related
EX-Orphans
RAR ,, all-trans retinoic acid
RXR ,, 9-cis retinoic acid
SHP egon
GCNF DHR96
C. elegans ~250 nuclear receptors
PPAR ,, fatty acids, eicosanoids
LXR , oxy-sterols
FXR , bile acids
BXR , benzoates
D. melanogaster ~20 nuclear receptors
H. sapiens ~48 genes
Arabidopsis no family members
Nearly EX-orphans (natural ligands?)
CAR androstanes, xenobiotics
SXR/PXR steroids, xenobiotics
The Nuclear Hormone Receptor Superfamily
A/B C D E F
Known Receptors Orphan Receptors
DNA LIGAND
A/B C D E F
Known Receptors
Classical receptors (from biochemistry)
GR cortisol
MR aldosterone
AR testosterone
Orphan Receptors
Vertebrate Drosophila
TR-2 , DHR78
NGFI-B ,, DHR38
ROR DHR3 AR testosterone
PR progesterone
ER , estradiol
VDR 1,25-(OH)
2
vit D3
TR , triiodothyronine
ROR ,, DHR3
Rev-erb , E75, E78
SF-1 , FTZ-F1 ,
COUP ,, svp
HNF-4 , HNF-4
EcR 20-OH ecdysone Tlx , tll
No known homologs
ERR ,, knirps
DAX-1 knirps-related
EX-Orphans
RAR ,, all-trans retinoic acid
RXR ,, 9-cis retinoic acid
SHP egon
GCNF DHR96
C. elegans ~250 nuclear receptors
PPAR ,, fatty acids, eicosanoids
LXR , oxy-sterols
FXR , bile acids
BXR , benzoates
D. melanogaster ~20 nuclear receptors
H. sapiens ~48 genes
Arabidopsis no family members
Nearly EX-orphans (natural ligands?)
CAR androstanes, xenobiotics
SXR/PXR steroids, xenobiotics
Do obesogens exist?
Obesogens - chemicals that inappropriately stimulate
adipogenesis and fat storage
Early postnatal exposure to environmental estrogens (ER)
increases weight
Bisphenol A, alkylphenols, DES, genistein
Prenatal exposure reduces weight
Thiazolidinedione anti-diabetic drugs (PPAR)
Increase fat storage and fat cell size at all ages
Phthalates cause fat cell differentiation in vitro (PPAR)
Organotins cause fat cell differentiation (PPAR)
Endocrine disruption by organotins invertebrates
Endocrine disrupter - a compound that mimics or blocks the action of
Organotins ->imposex in molluscs
Endocrine disrupter - a compound that mimics or blocks the action of
endocrine hormones, either directly or indirectly
Direct inhibitory effect on aromatase
(CYP19) enzymatic activity
Also blocks testosterone esterification
S
Cl
Alters shell development in bivalve molluscs
Tributyltin-Cl
Sn
Routes of exposure to organotins
Various modes of toxicity in vertebrates
Sperm, liver, immune system, neural, sex reverses some fishes
Marine ship paints Marine ship paints
Trialkytins are potent biocidal antifouling agents for molluscs
Widely used 1960-1970s, regulated but incompletely phased out
Contaminates seafood Contaminates seafood
Other uses and routes of exposure
Fungicide on high value food crops
( t t i l )
Cl
(potatoes, rice, celery, pecans)
Wood preservative
Catalysts for organic synthesis
H bili i f f
Tributyltin-Cl
Sn
Cl
Heat stabilizers in manufacture of
polyolefin plastics (PVC)
Bioaccumulative
BCF of ~2-11K (oysters), 17K-350K (mussels), ~2.5K-12K (fishes)
BCF of ~30K in algae
Organotins and endocrine disruption
TBT alters sex determination typically female -> male
S d t i ti i t id t iti l ti i Sex determination requires sex steroids at critical times in
development
Sex steroids act through nuclear receptors
TBT effects are seen at nM doses and below
Alter activity of transcription factors? y p
Possible ligand for nuclear receptors?
Hypothesis TBT alters the activity of one or more nuclear receptors Hypothesis TBT alters the activity of one or more nuclear receptors,
thereby causing endocrine disruption
Test nuclear receptors for activation or inhibition by organotins
E t ff t t id t ti it Expect effect on steroid receptor activity
Nuclear receptor activation by TBT
F ld ti ti
Construct
Fold activation
(60 nM TBT) Permissive RXR heterodimer?
RXR (human) 60 Yes
RXR ( ) 25 Y RXR (xenopus) 25 Yes
RXR (xenopus) 7.0 Yes
PPAR (mouse) 0.7 Yes
PPAR (human) 5.3 Yes
PPAR (human) 1.7 Yes PPAR (human) 1.7 Yes
RAR (human) 0.7 No
TR (h ) 0 4 N TR (human) 0.4 No
VDR (human) 0.5 No
SXR (human) 1.0 No
RXR is a key partner for many pathways
RAR RXR RAR RXR
Known ligands
RAR ,, all-trans RA
RAR RXR RAR RXR
TR , thyroid hormone
VDR 1,25-(OH)
2
-VD3
PPAR ,/, fatty acids, eicosanoids
LXR oxysterols LXR , oxysterols
FXR , bile acids
BXR , benzoates
EcR ecdysteroids
OR RXR OR RXR
EcR ecdysteroids
Activatable Orphans
SXR/PXR steroids xenobiotics SXR/PXR steroids, xenobiotics
CAR xenobiotics, androstanes
Inappropriate RXR activation may be expected to cause
wide ranging disturbances in the bodys homeostatic hormonal controls
What is the effect of TBT treatment, in vivo?
Newborn Liver TBT (in utero)
TBT Vehicle (corn oil)
Pregnant C57BL6 dams dosed with TBT (0.5 mg/kg i.p.) from E12-E18. 20 m tissue cryosections
were prepared from newborn pups and stained with Oil Red O (lipid stain) and counterstained with
eosin/hematoxylin.
Growth following prenatal TBT exposure
25
TBT Male
Control Male
p =.58
20
g
m
s
)
Control Male
TBT Female
Control Female
p =.05
15
W
e
i
g
h
t
(
5
10
B
o
d
y
W
0
5
0 10 20 30 40 50 60
70
Time (days post birth)
C57BL6 Weight Gain: 6-9 months
120
%
)
Cont ol
Males
120
%
)
Control
Females
110
115
h
a
n
g
e

(
%
Control
TBT
110
115
h
a
n
g
e

(
%
Control
TBT
100
105
W
e
i
g
h
t

C
100
105
W
e
i
g
h
t

C
90
95
m
a
l
i
z
e
d

W
90
95
m
a
l
i
z
e
d

W
80
85
6 9
N
o
r
m
80
85
6
9
N
o
r
m
Time (months)
6 9
Time (months)
6
9
Conclusions organotins and nuclear receptors
Organotins are very potent regulators of RXR and PPAR that drive
fat cell development in vivo.
Is organotin exposure a contributing factor for obesity? Is organotin exposure a contributing factor for obesity?
Acute neonatal exposure permanently alters adult phenotype
Acute adult exposure rapidly induces adipogenic genes
Are humans exposed to sufficient levels of TBT for concern?
PVC is up to 3% w/w (0.1 M) organotins
Prevalent contaminants in dietary sources
ATSDR (2003) average of 15-100 nM in seafood tested ATSDR (2003) average of 15 100 nM in seafood tested
ATSDR (2003) average blood level of 27 nM in 32 random people
Human exposure to organotins may reach levels sufficient to
activate high affinity receptors activate high affinity receptors
Is the environment making us fat?
Organotins?
Environmental estrogens?
Collaborators Collaborators
UCI - Blumberg Lab UCI NINS Okazaki, Japan UCI Blumberg Lab
Dejoie Blumberg
Emily Grossman
Felix Grn
UCI
Dave Gardiner
John Greaves
Edward Nelson
NINS Okazaki, Japan
Taisen Iguchi
Hajime Watanabe
Felix Grn
Shirley Guan
Lan Li
Monica McCallum
Edward Nelson
NIHS - Tokyo, Japan
Jun Kanno
City of Hope
Barry Forman
Monica McCallum
Jun-ichi Nishikawa
Win Phyu
Amy Shah Amy Shah
Jason Shiotsugu
Suman Verma
John Ycaza John Ycaza
Chetman Yu
Lauren Maeda Lauren Maeda
Zamaneh Zamanian