We are working in Tier I with mechanisms

Are mechanisms similar ordissimilar across phyla, species,

classes, etc.?

In other words – What is the phylogeny ofthe various mechanisms?

Attempt at ‘Problem Statement’

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 whatwe don’t know, than what we know

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) appearsto be unique to vertebrates.

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MetazoansBilateria

DeuterostomesProtostomes

Lophotrochozoans Ecdysozoans

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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 distributionmay vary

Vertebratesonly:

SteroidFamily (??)

“Others”also restrictedto a few phyla

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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 thatneed to be filled!!!

Metazoans & Nuclear Receptors

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EcdR, KNIRPSFor invertebrates, outside Arthropodsfar less known about NucRec’s and their transcriptional roles – some are identified only as genes

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Briefly, the P450s

Far more ancient

Far more diverse

But a parallel way to analyze the problem

Lipid synthetictree of ligands

DNA elementSeveral corestructures

intron-exonbreak in

DNA region

organizationabcd abcdabcd dcba

nucleotide(s)spacing

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)

N-terminus C-terminus

DBD –DNA Binding DomainZinc finger interactionOften repeatedSeveral configurations distinguish receptor subclasses

LBD –Ligand Binding DomainLarge, 3 sided cavityEnvelopes ligandLargely hydrophobic amino acids lining the cavity

Nuclear HormoneReceptor Structure

Nuclear HormoneReceptor Structure

N-terminus C-terminus

AF-1Activation Factor 1(site for activationby several kinase orphosphorylationpathways)

AF-2Activation Factor 2(interaction with otherprotein families – one represses and one activates)Essential for transcription

DN

A R

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Nuclear Hormone ReceptorLigand Triggering Action #1

NH – receptorLigand Bound

Heat Shock Protein(HSP) chaperone

complex

CoRepressor

DissociationReceptor Dimer

Formation

This ‘free’dimer appearssusceptible to

proteolysis(ubiquitin)

Binding DNAElement Sites

L NH – receptorLigand Bound

LNH – receptorLigand Bound

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Transcribing GenesInto mRNA

RNA polymerase IIor 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 IItranscribing

Base 0 for geneBase “X” # bases upstream for gene

Nuclear Hormone Receptor-Ligand Complex Action #2

DNA Response Element TATA Box

RNA polymerase IIor 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

Nuclear Hormone Receptor-Ligand Complex Action #2

DNA Response Element TATA Box

RNA polymerase IIor pol II complex

must bind TATA boxDNA site to transcribe

target gene

Receptor-ligand (holo-receptor) doesnot interact directly

with the Pol II complex

NH – receptorLigand Bound

LNH – receptorLigand Bound

L

Upstream of target gene by X bases

Upstream of the TATA box Y basesor X + Y of target gene

Nuclear Hormone Receptor-Ligand Complex Action 2

DNA Response Element TATA Box

RNA polymerase IIor pol II complex

actually transcribes,starting at TATA box

DNA site

NH – receptorLigand Bound

LNH – receptorLigand Bound

L

Recruitment of Coactivators is NecessaryThey are an Obligate ‘Bridge’ to Pol II complex*

* Receptor-ligand can bind DNA and NOT recruit pol II = Antagonist

Chemical Numbers vs Chemical ‘Doses’

Working ExamplesPerfume 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

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