1. We are working in Tier I with mechanisms Are mechanisms similar or dissimilar across phyla, species, classes, etc.? In other words – What is the phylogeny of the various mechanisms? Attempt at ‘Problem Statement’

2. In particular, what is the phylogeny of: 1.Nuclear receptors – as they bind the the ligands and activate particular genes and pathways 2.P450s – as they synthesize and metabolize most NR ligands Regrettably, will emphasis more of what we don’t know, than what we know

3. Nuclear Receptors Metazoan signaling pathways 5 Basic evolutionary groups: - based on DNA element structure (a repeated sequence, some serial repeats, some mirrored, and with n nucleotides spacer) - based on gene structure – intron/extron point in DNA binding region of protein - based on amino acid sequence homology At this time, steroid family (E, A, G, P, M) appears to be unique to vertebrates.

4. Echinoderms Hemichordates Urochordates Cephalochordates Vertebrates PoriferansCnidariansCtenophorans Gastrotrichs Nematodes Priapulids Kinorhynchs Onychophorans Tardigrades Arthropods Bryozoans Entoprocts Platyhelminthes Pogonophorans Brachiopods Phoronids Nemerteans Annelids Echiurans Molluscs Sipunculans Gnathostomulids Rotifers Metazoans Bilateria DeuterostomesProtostomes LophotrochozoansEcdysozoans

5. Gastrotrichs Nematodes Priapulids Kinorhynchs Onychophorans Tardigrades Arthropods Bryozoans Entoprocts Platyhelminthes Pogonophorans Brachiopods Phoronids Nemerteans Annelids Echiurans Molluscs Sipunculans Gnathostomulids Rotifers Metazoans & Nuclear Receptors ER , AR, GR, MR, & PR  TR , ERR , VDR, RAR , PPAR  Note: Greek letters indicate separate (multiple) forms of the receptor exist - action, ligand specificity, cell distribution may vary Vertebrates only: Steroid Family (??) “Others” also restricted to a few phyla Echinoderms Hemichordates Urochordates Cephalochordates Vertebrates

6. Implications 1. Is there a scientific basis or rationale to extrapolate an EAT based screen to other phyla? At this time, probably not! 2. This means we have no screens for the other phyla. They are unprotected! We have serious scientific gaps that need to be filled!!!

7. Metazoans & Nuclear Receptors Gastrotrichs Nematodes Priapulids Kinorhynchs Onychophorans Tardigrades Arthropods Bryozoans Entoprocts Platyhelminthes Pogonophorans Brachiopods Phoronids Nemerteans Annelids Echiurans Molluscs Sipunculans Gnathostomulids Rotifers ?? ?? ?? RXR  COUP  EcdR, KNIRPS  For invertebrates, outside Arthropods far less known about NucRec’s and their transcriptional roles – some are identified only as genes Echinoderms Hemichordates Urochordates Cephalochordates Vertebrates

8. Briefly, the P450s Far more ancient Far more diverse But a parallel way to analyze the problem

9. Lipid synthetic tree of ligands DNA element Several core structures intron-exon break in DNA region organization abcd abcd dcba nucleotide(s) spacing

10. Suggested analysis What are the nuclear receptors that are potential ED targets? What are the ligands (‘mimic targets’)? What are the synthetic steps (P450scc, aromatase, 5-alpha reductase, …)? What then is the appropriate phyla for which a screen can and cannot be used? (And have a rationale basis to validate)

11. N-terminusC-terminus DBD – DNA Binding Domain Zinc finger interaction Often repeated Several configurations distinguish receptor subclasses LBD – Ligand Binding Domain Large, 3 sided cavity Envelopes ligand Largely hydrophobic amino acids lining the cavity Nuclear Hormone Receptor Structure

12. Nuclear Hormone Receptor Structure N-terminusC-terminus AF-1 Activation Factor 1 (site for activation by several kinase or phosphorylation pathways) AF-2 Activation Factor 2 (interaction with other protein families – one represses and one activates) Essential for transcription

13. DNA Response Element Nuclear Hormone Receptor Ligand Triggering Action #1 NH – receptor Ligand Bound Heat Shock Protein (HSP) chaperone complex CoRepressor Dissociation Receptor Dimer Formation This ‘free’ dimer appears susceptible to proteolysis (ubiquitin) Binding DNA Element Sites L NH – receptor Ligand Bound L NH – receptor Ligand Bound L NH – receptor Ligand Bound L NH – receptor Ligand Bound L

14. Transcribing Genes Into mRNA RNA polymerase II or pol II complex actually transcribes, binds DNA at TATA box DNA site TATA Box Gene Start (AUG codon) 1. Pol II Binds TATA Box 2. Pol II Moves Down DNA 3. Pol II Initiates mRNA transcription Pol II transcribing Base 0 for geneBase “X” # bases upstream for gene

15. Nuclear Hormone Receptor- Ligand Complex Action #2 DNA Response Element TATA Box RNA polymerase II or pol II complex actually transcribes, starting at TATA box DNA site Response element for receptor is upstream of TATA box RNA pol II normally cannot bind to TATA box alone, transcription blocked Upstream of target gene by X bases

16. Nuclear Hormone Receptor- Ligand Complex Action #2 DNA Response Element TATA Box RNA polymerase II or pol II complex must bind TATA box DNA site to transcribe target gene Receptor-ligand (holo-receptor) does not interact directly with the Pol II complex NH – receptor Ligand Bound L NH – receptor Ligand Bound L Upstream of target gene by X bases Upstream of the TATA box Y bases or X + Y of target gene

17. Nuclear Hormone Receptor- Ligand Complex Action 2 DNA Response Element TATA Box RNA polymerase II or pol II complex actually transcribes, starting at TATA box DNA site NH – receptor Ligand Bound L NH – receptor Ligand Bound L Recruitment of Coactivators is Necessary They are an Obligate ‘Bridge’ to Pol II complex* * Receptor-ligand can bind DNA and NOT recruit pol II = Antagonist

18. Chemical Numbers vs Chemical ‘Doses’ Working Examples Perfume Raw Materials Salvito et al. Env. Toxicol. Chem. 21(6): 1301-1308 Prediction of Environmental Concentrations (PECs) for > 2,100 chemicals Prediction of PNEC – PEC/PNEC ratio - prioritization Simonich et al. Env. Sci. Tech. 36(13): 2839-2847. Measurement and Validation of PECs

19. Thyroid Toxicological History Adverse Effects and Mechanisms Thyroid – goiter/developmental toxicity Mechanisms elucidated 1940-1990: - Iodide deficiency or uptake blockade to thyroid - Inhibition of thyroid peroxidase - Blockage of thyroid T3/T4 release - Increased T3/T4 metabolism - Inhibition of 5’-deiodinase - Life stage sensitivity vs consequences Clear endpoints: thyroid histopathology, circulating T3/T4 and TSH