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BioSci 2B - Environment and Human Health
• Bruce Blumberg
– 2113E McGaugh Hall – open office hours
– phone 824-8573
– [email protected]
• check e-mail daily for announcements, etc..
– If you do not have ready access to e-mail or the web speak with
me ASAP
Rules for freshman seminars
• Attendance
– at least 8/10 lectures
• Participation
– Make a presentation
– Participate in discussions
• Be involved in your own education
Requirements for the oral presentation - presenter
• Powerpoint presentation on a topic related to the environment and
human health
– Please approve it with me in advance
– Send out links to the articles, web sites, etc in advance
• 30 minutes with time allowed for discussion (max of ~20 slides)
– What is the problem of interest – the big picture question
– Present background – what is known, what is unknown
– Present the story, research, etc
– Present an analysis of the story or article
• Which parts are credible
• Which are suspect
• How would you study the problem
– Point out a few papers for further reading (reviews, followups,
etc)
Requirements for the oral presentation - presenter
• Logistics
– Prepare presentation and either e-mail to me or bring it on a
• CD-ROM
• Floppy disk
• USB flash memory drive
– Or bring your own laptop
• I would like to post these on course web site – is that OK?
Requirements for the oral presentation listener
• READ THE PAPER or article
– Think about it – make notes on parts you have questions about
– Listen to the speaker
• If presentation is unclear, ask the speaker to elaborate
• Always feel free to ask questions – we want an open
discussion
Blumberg Laboratory
http://blumberg-serv.bio.uci.edu
• The role of nuclear hormone receptors in:
– development
– physiology
– endocrine disruption
– cancer
• Functional genomic approaches to:
– identify cellular partners for known genes
– define new members of signaling pathways
– find downstream target genes
– develop new drug targets
Nuclear Hormone Receptors - A Large Family of
Ligand Modulated Transcription Factors
A/B
C
D
E
DNA
F
LIGAND
ligand-binding
transcriptional activation
dimerization
half-site recognition
half-site spacing
dimerization
DNA-binding
AGGTCA
D
n
I
n
E
n
NUCLEUS
LIGAND
APO
RXR BXR
INTRA
CYTOPLASM
Nuclear Hormone Receptors
A/B
C
DNA
D
E
LIGAND
• Bind to specific DNA targets - hormone response elements
• Most activate transcription upon ligand binding
– Some are constitutive
– A few are deactivated by ligand binding
• Ligands are small lipophilic molecules that freely enter cells
– Diffuse from source
– Penetrate to a target
• Typically respond to low levels of hormone ~3 ppb (10-8 M)
– Regulation of levels
– Environmental agents
F
The Nuclear Hormone Receptor Superfamily
A/B
C
D
DNA
E
F
LIGAND
Known Receptors
Orphan Receptors
Classical receptors (from biochemistry)
Vertebrate
Drosophila
GR
MR
AR α,β
PR α,β
ER α,β
VDR
TR α,β
EcR
TR-2 α,β
NGFI-B α,β,γ
ROR α,β,γ
Rev-erb α,β
SF-1 α,β
COUP α,β,γ
HNF-4 α, β
Tlx α,β
DHR78
DHR38
DHR3
E75, E78
FTZ-F1 α,β
svp
HNF-4
tll
cortisol
aldosterone
testosterone
progesterone
estradiol
1,25-(OH)2 vit D3
triiodothyronine
20-OH ecdysone
EX-Orphans
RAR α,β,γ
RXR α,β,γ
PPAR α,β,γ
LXR α,β
FXR α,β
BXR α,β
No known homologs
all-trans retinoic acid
9-cis retinoic acid
fatty acids, eicosanoids
oxy-sterols
bile acids
benzoates
Nearly EX-orphans (natural ligands?)
CAR
SXR/PXR
androstanes, xenobiotics
steroids, xenobiotics
ERR α,β,γ
DAX-1
SHP
GCNF
knirps
knirps-related
egon
DHR96
C. elegans
D. melanogaster
H. sapiens
Arabidopsis
~250 nuclear receptors
~20 nuclear receptors
~48 genes
no family members
Developmental Functions of Nuclear Receptors
• What is the role of RARs in anterior patterning?
– RAR-mediated repression is required for head development
• RAR signaling in posterior patterning
– How do RAR and growth factor signaling pathways interact?
• Identification of RAR target genes
– macroarray
– microarray
• Function of BXR during development
– gain of function
– loss-of-function (morpholino, DN)
– endogenous ligand identification
incubate until
mid neurula
stage
Environment and Development
• Deformed frogs in Minnesota and throughout north America
– Many indications that these frogs
have altered retinoid signaling
– Purifying compound(s) from highly
affected sites that activate RAR
– Several candidate compounds
• Activate RAR
• Found in multiple affected sites
– Scale-up purification is underway
• Structure elucidation
• Animal testing
• Thyroid axis disruption in Montana
– Axolotls metamorphose in city water
– Thyroid problems on Indian reservation
Solving an ancient mystery
• Sometimes science leads us in unexpected directions
• We identified a very strangely behaving hormone receptor that is
very important in our response to chemicals in foods
Mithridates VI Eupator
The Royal Toxicologist
Mithridates
by A.E. Housman
They put arsenic in his meat
And stared aghast to watch him eat;
They poured strychnine in his cup
And shook to see him drink it up:
They shook, they stared as white’s their shirt:
Them it was their poison hurt.
--I tell the tale that I heard told.
Mithridates, he died old
King of Pontus (120-63 BC)
aka Mithridates the Great
Long Standing Questions
• Mithridatum - generalized tolerance to poison
• Adaptive hepatic response (Hans Selye)
– Exposure to certain “catatoxic”
chemicals elicits protection against
later exposure to others
– chemical immunity
– Apparently mediated via large
increase in hepatic microsomes
• What is the mechanism?
SXR Responds to Many Steroids
14
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Fold Induction
10
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The mammalian xenobiotic response
• SXR ligands stimulate
the expression of genes
involved in
xenobiotic metabolism.
• Phase I - oxidation
– CYP3A4, CYP2B6
CYP2B9, CYP2C8
CYP2C9, CYP2C19
• Phase II - conjugation
– glutathione-S-transferase (GST), sulfotransferase (SULT),
and UDP-glucoronosyltransferase (UGT) families.
• Phase III genes - transport
– MDR1, MRP2 and Oatp2
• SXR is a master regulator of xenobiotic metabolism
Pharmacology of Mouse and Human SXR
30
20
10
10
Human
SXR
control
RIF
nifedipine
tamoxifen
spironolactone
PCN
dexamethasone
corticosterone
cortisone
testosterone
estradiol
DES
coumestrol
Human
ERα
control
RIF
nifedipine
tamoxifen
spironolactone
PCN
dexamethasone
corticosterone
cortisone
testosterone
estradiol
DES
coumestrol
30
20
Mouse
PXR
Rat
ERα
Model Systems
• Effects on animals predict effects on humans
– Fundamental assumption: biochemistry, endocrinology and
metabolism are the same
– Nuclear receptors behave virtually identically across species
• Different pharmacology of SXR and PXR suggests that there are
important differences in metabolism
• These differences may be highly relevant for toxicology (including
developmental effects), drug interactions and endocrine disruption
• Cross-species extrapolation must account for differences in response
of xenobiotic sensors
– SXR
– CAR
SXR and Endocrine Disruption
• SXR regulates the oxidation, conjugation and clearance of ingested
steroids and xenobiotics
• Activation of SXR may predict effects of suspected EDC
– SXR activators may be detoxified by CYP action and not a human risk
– But activators may also be toxified by CYP action, increasing the risk.
– EDC may have no effect on SXR and therefore more likely to act on other
receptors, e.g. ER
• Metabolism will play important role in shape of dose response curves
– A compound could have effects at low doses but induce its own
metabolism at high doses, masking the low-dose effect
• SXR is a molecular assay for potential activity of EDCs
• Different pharmacology of SXR and PXR suggests that differences in
metabolism may exist and be relevant for risk assessment
EDCs Can Activate SXR
Cl
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OH
HO
bisphenol A
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PCB 184
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PCB 196
Cl
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DDT
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DDE
Cl
O
OH
O
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nonylphenol
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bis-phthalate
S
C2H5O
P O
C2H5O
Chlorpyrifos
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