BioSci 2B - Environment and Human Health

• Bruce Blumberg– 2113E McGaugh Hall – open office hours– phone 824-8573– blumberg@uci.edu

• check e-mail and noteboard daily for announcements, etc..– If you do not have ready access to e-mail or the web

speak with me ASAP– Please use the course noteboard for discussions of the

material

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)

• 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– Bring light snacks for everyone – I will pay up to $20/class

• I would like to post these on course web site – is that OK?

Requirements for the oral presentation - presenter

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 Laboratoryhttp://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

half-site recognitionhalf-site spacingdimerization

ligand-bindingtranscriptional activationdimerization

Nuclear Hormone Receptors - A Large Family of Ligand Modulated Transcription Factors

BXRRXR

NUCLEUS

CYTOPLASM

LIGAND

APO

INTRA

DNA LIGANDA/B C D E F

DNA-binding

AGGTCA

n

n

n

D

I

E

Nuclear Hormone Receptors

• 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

DNA LIGANDA/B C D E F

The Nuclear Hormone Receptor Superfamily

Known Receptors

Classical receptors (from biochemistry)GR cortisolMR aldosteroneAR α,β testosteronePR α,β progesteroneER α,β estradiolVDR 1,25-(OH)2 vit D3TR α,β triiodothyronineEcR 20-OH ecdysone

Orphan Receptors

Vertebrate DrosophilaTR-2 α,β DHR78NGFI-B α,β,γ DHR38ROR α,β,γ DHR3Rev-erb α,β E75, E78SF-1 α,β FTZ-F1 α,β COUP α,β,γ svpHNF-4 α, β HNF-4Tlx α,β tll

No known homologsERR α,β,γ knirpsDAX-1 knirps-relatedSHP egonGCNF DHR96

C. elegans ~250 nuclear receptorsD. melanogaster ~20 nuclear receptorsH. sapiens ~48 genesArabidopsis no family members

DNA LIGANDA/B C D E F

EX-OrphansRAR α,β,γ all-trans retinoic acidRXR α,β,γ 9-cis retinoic acidPPAR α,β,γ fatty acids, eicosanoidsLXR α,β oxy-sterolsFXR α,β bile acidsBXR α,β benzoates

Nearly EX-orphans (natural ligands?)CAR androstanes, xenobioticsSXR/PXR steroids, xenobiotics

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 EupatorMithridates VI Eupator The Royal Toxicologist The Royal Toxicologist

King of Pontus (120-63 BC)King of Pontus (120-63 BC)aka Mithridates the Great aka Mithridates the Great

Mithridates Mithridates by A.E. Housmanby A.E. Housman

They put arsenic in his meatThey put arsenic in his meat

And stared aghast to watch him eat;And stared aghast to watch him eat;

They poured strychnine in his cupThey poured strychnine in his cup

And shook to see him drink it up:And shook to see him drink it up:

They shook, they stared as white’s their They shook, they stared as white’s their shirt:shirt:

Them it was their poison hurt.Them it was their poison hurt.

--I tell the tale that I heard told.--I tell the tale that I heard told.

Mithridates, he died oldMithridates, he died old

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

0

2

4

6

8

10

12

14

solve

nt

corti

coste

rone

preg

neno

lone

dehy

droe

piandr

oste

rone

dihyd

rote

stoste

rone

prog

este

rone

estra

diol

corti

sol

corti

sone

dexa

met

haso

ne

Fo

ldIn

du

ctio

n

The mammalian xenobiotic response• SXR ligands stimulate

the expression of genesinvolved in xenobiotic metabolism.

• Phase I - oxidation– CYP3A4, CYP2B6

CYP2B9, CYP2C8CYP2C9, 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

controlRIF

nifedipinetamoxifen

spironolactonePCN

dexamethasonecorticosterone

cortisonetestosterone

estradiolDES

coumestrol

controlRIF

nifedipinetamoxifen

spironolactonePCN

corticosteronecortisone

testosteroneestradiol

DEScoumestrol

dexamethasone

30 20 10 10 20 30

HumanSXR

MousePXR

RatERα

HumanERα

Pharmacology of Mouse and Human SXR

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

OH OH

bisphenol A

Cl

Cl

Cl

Cl

Cl

Cl Cl

PCB 184

Cl

Cl Cl

Cl

Cl

Cl Cl

Cl

PCB 196

Cl Cl

ClCl Cl

DDTCl Cl

Cl Cl

DDE

Cl

Cl

Cl

OP

SC2H5O

C2H5O

Chlorpyrifos

OH

nonylphenol

O

O

O

O

bis-phthalate