BioSci 2B - Environment and Human Health

1. 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

2. Rules for freshman seminars • Attendance • at least 8/10 lectures • Participation • Make a presentation • Participate in discussions • Be involved in your own education

3. 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)

4. 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 • 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?

5. 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

6. 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

7. DNA-binding AGGTCA n n n D I E Nuclear Hormone Receptors - A Large Family of Ligand Modulated Transcription Factors ligand-bindingtranscriptional activationdimerization half-site recognitionhalf-site spacingdimerization

8. 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

9. 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 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,γDHR38ROR α,β,γ DHR3Rev-erb α,β E75, E78SF-1 α,β FTZ-F1 α,βCOUP α,β,γsvpHNF-4 α, β HNF-4Tlx α,βtll 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

10. 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

11. Environment and Development • Deformed frogs in Minnesota and throughout north America • Many indications that these frogshave altered retinoid signaling • Purifying compound(s) from highlyaffected 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

12. 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

13. 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

14. 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?

15. SXR Responds to Many Steroids

16. The mammalian xenobiotic response • SXR ligands stimulate theexpression of genesinvolved in xenobiotic metabolism. • Phase I - oxidation • CYP3A4, CYP2B6CYP2B9, 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

17. Pharmacology of Mouse and Human SXR 30 20 10 30 20 10 control Mouse PXR Human SXR RIF nifedipine tamoxifen spironolactone PCN dexamethasone corticosterone cortisone testosterone estradiol DES coumestrol control Human ERα Rat ERα RIF nifedipine tamoxifen spironolactone PCN dexamethasone corticosterone cortisone testosterone estradiol DES coumestrol

18. 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

19. 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

20. C l C l C l C l C l C l C l C l C l C l C l O H H O C l C l C l C l b i s p h e n o l A P C B 1 8 4 P C B 1 9 6 C l C l C l C l C l C l S C H O 2 5 P O C l C H O C l C l 2 5 C l C l D D E O D D T C h l o r p y r i f o s C l O H O O n o n y l p h e n o l O b i s - p h t h a l a t e EDCs Can Activate SXR