BioSci 2B - Environment and Human Health Bruce Blumberg –2113E McGaugh Hall – open office hours –phone check and noteboard daily for announcements, etc.. –If you do not have ready access to 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 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 Laboratory 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 recognition half-site spacing dimerization ligand-binding transcriptional activation dimerization Nuclear Hormone Receptors - A Large Family of Ligand Modulated Transcription Factors DNA-binding AGGTCA n DIEDIE

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

The Nuclear Hormone Receptor Superfamily Known Receptors Classical receptors (from biochemistry) GRcortisol MRaldosterone AR α,βtestosterone PR α,β progesterone ER α,β estradiol VDR1,25-(OH) 2 vit D3 TR α,βtriiodothyronine EcR20-OH ecdysone Orphan Receptors VertebrateDrosophila TR-2 α,βDHR78 NGFI-B α,β,γ DHR38 ROR α,β,γ DHR3 Rev-erb α,βE75, E78 SF-1 α,βFTZ-F1 α,β COUP α,β,γ svp HNF-4 α, βHNF-4 Tlx α,βtll No known homologs ERR α,β,γknirps DAX-1 knirps-related SHP egon GCNFDHR96 C. elegans~250 nuclear receptors D. melanogaster~20 nuclear receptors H. sapiens~48 genes Arabidopsisno family members EX-Orphans RAR α,β,γall-trans retinoic acid RXR α,β,γ 9-cis retinoic acid PPAR α,β,γ fatty acids, eicosanoids LXR α,βoxy-sterols FXR α,β bile acids BXR α,βbenzoates Nearly EX-orphans (natural ligands?) CARandrostanes, xenobiotics SXR/PXRsteroids, 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 Eupator The Royal Toxicologist King of Pontus ( BC) aka Mithridates the Great 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

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

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

control RIF nifedipine tamoxifen spironolactone PCN dexamethasone corticosterone cortisone testosterone estradiol DES coumestrol control RIF nifedipine tamoxifen spironolactone PCN corticosterone cortisone testosterone estradiol DES coumestrol dexamethasone Human SXR Mouse PXR Rat ERα Human ERα 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 O H bisphenolA Cl Cl Cl Cl Cl Cl Cl PCB184 Cl Cl Cl Cl Cl Cl Cl Cl PCB196 Cl Cl Cl Cl Cl DDT ClCl ClCl DDE Cl Cl Cl O P S C 2 H 5 O C 2 H 5 O Chlorpyrifos OH nonylphenol O O O O bis-phthalate