EstrogensYou can’t live without ‘em:Reproductive failureBone lossVasomotor disturbances (hot flashes)Some cardiovascular system vulnerabilitiesSome cognitive declines, mood disordersSkin changes

You can’t live with ‘em:Cancer (breast, uterus, colon, pituitary)Blood clotsNausea or eating disorders

If you have the wrong ones (xenoestrogens) you’re in big trouble: endocrine disruption of many types

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Xenoestrogens (XEs) are threats to both sexes

Physiologic E levels vs. sex, age, cycles, & pregnancy

Watson, STKE Dec 1999

How do estrogens and xenoestrogens signal in the cell?

Both:From the nucleus (classical genomic mechanism)

From the membrane(novel, nonclassical nongenomic mechanism)

ERER

GPR30/(ERα36)

Nataliya Bulayeva

Confocal views of E2-peroxidase binding in GH3/B6/F10 pituitary tumor cells

Visualizing Membrane Estrogen Receptors (Binding)

Detection of PROTEIN•PROTEIN epitope interactions – the proximity

ligation assay (PLA)

R Ab

mAb (AB-N09)

mAb

mAb +R Ab -

Either two ERα Abs or Abs for two differenent proteins

Slice 3D

epitope proximity for ER

Two epitopes from within ERα

Guangzhen Hu

ERα partnering with either Gαi or Caveolin

3DSlice

Guangzhen Hu

15 min

Estrogens (either physiologic or environmental) cause rapid disappearance of mERα Ab recognition (H151 hinge region epitope) while other epitope signals are not affected – rapid conformational change.

veh3 min DES(Similar for:E2, dieldrin, endosulfan, nonylphenol)

Such responses are both rapid and potentCeleste Campbell

GH3/B6/F10 pituitary tumor cells

Ever since then we have been examining mechanisms of rapid E and XE actions, related to - functional outcomes – looking at ways XEs can disrupt normal function.

All small molecules ~270-400 MW

We picked these 16 originally to compare because of interests our interests in:

■ life-stage prevalence of physiological estrogens

■ xenoestrogen toxicities and disease associations

■ structural features/groupings

■ therapeutic uses

So many estrogens… so little time!

… and we are adding more compounds all the time to enhance our stucture-activity analyses

Some typical results using these assays:

have taught us important concepts about how these

compounds act via nongenomic mechanisms

Andrea Norfleet/Jennifer Jeng

We see non-monotonic dose-responses for E2-induced PRL release from GH3/B6/F10 pituitary cells at 6 min

(also true for other estrogens)P

RL

sec

reti

on

Prolactin (PRL) secretion

Xenoestrogens also cause PRL release in a nonmonotonic pattern

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PR

L/C

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Ann Wozniak

* *

*

Bisphenol A

Big news for the toxicology world -- Nonmonotonic responses make it impossible to extrapolate back from a high dose to see what the lowest effective dose would be.

PR

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10-12M 10-11M 10-10M 10-9M 10-8M

D9 Cell Time Dependent PRL Release at 10-8M E2

D9 Cell Dose Dependent PRL Release at 3 Minutes

Ann Wozniak

E2 Concentration (M)

ERα is necessary for many of these responsesCells selected for very low mERα levels (D9 subclone) ….can’t elicit the PRL

response to estrogens.

To efficiently identify specific receptors involved, we have also used

--siRNA knockdowns of NRs ER primarily responsible, , and GPR30 sometimes modulatory

--receptor-selective agonists --receptor-selective antagonists

To prove action from the membrane we have used:--various impeded ligands (E2-BSA, E2-peroxidase, E2-dendrimers)

--ERα Ab triggering of responses

What signaling mechanisms may be provoked by xenoEs acting on mERs

Ca++

mitogen-activated protein kinases (MAPKs) cAMP and PKA G protein activations other kinases/phosphatases caspases downstream transcription factor post-translational modifications

and how they relate to functional endpoints

Cell signaling is complicated - not just a linear signaling cascade. Many things must be coordinated. It is a web.

Signaling is like a Rube Goldberg Machine http://blueballfixed.ytmnd.com/

created by the Web site Something Awful

Mitogen-activated protein kinases (MAPKs) like ERK:

Are signal integrators. …..many signaling pathways funnel into the ERK “integrator”.

Are associated with major cellular destinies, like cell proliferation

- non-nuclear feeder signaling streams we have tested & found to be involved in E-induced downstream ERK signaling

Signaling starting at the membrane funnels into the downstream kinases ……

and then fans out again to coordinate major complex downstream functions.

The MAP kinases are the integrators that separate these two phases

MAPKsERKsJNKsp38

cell cycle

DNA and proteinsynthesis for replication

Proliferation

new proteins

Differentiationcaspase cascades

Death

2nd messengers and signaling enzymes (kinases, and phosphatases, lipases, NT-cyclases) and scaffolds

receptors

SecretionMigration

One of our most useful techniques is a fixed-cell 96-well plate immunoassay which:

--is optimized for each cell type, epitope-Ab, and cell compartments (based on permiabilization); makes use of the many Abs now available to the activated (usu. p’ated) form of proteins

-- is relatively high throughput allowing us to compare multiple xenoestrogens at a wide range of concentrations acting on multiple antigens

-- allows us to do physiologic Es + XEs in incremental mixtures, the real-life scenarios of XE toxic exposures

E2

DES Endosulfan

Nataliya Bulayeva

0 3' 6' 10' 15' 30'80

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Dieldrin

Coumestrol

E2-Peroxidase

96-well plate immunoassays for phospho-ERK ligands all at 1nM

Responses oscillate with time; XEs cause different phasing than E2

control -14 -13 -12 -11 -10 -9 -8

E2 (3 min)*

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control-14 -13 -12 -11 -10 -9 -8

Nonylphenol (30 min)

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control -14 -13 -12 -11 -10 -9 -880

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control -14 -13 -12 -11 -10 -9 -880

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Concentration Nataliya Bulayeva

Responses are often non-monotonic: U-, V-, or M-shaped dose-responses

Why do MAPK responses oscillate oscillate with time

and concentration.

The sum of pathways A & B is an oscillating curve over time or concentration

Here is a new example of one of the most rapid -- Gi activation (charged with GTP and assayable with an Ab to the charged form).

Because it is so proximal to receptor-binding it is much more rapid (peaks at 15 seconds) than downstream signal integrator responses like for MAPKs (several minutes). Again see differences between E2 and XEs

Guangzhen Hu

Some responses are very rapid – seconds

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GTP-bound G

i -- %

of vehicle

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160E2 NP BPA

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Seconds

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Jennifer Jeng

log [M]

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BPAEPPPOPNP

Con -15 -14 -13 -12 -11 -10 -9 -8 -7 -6

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Both physiologic estrogens and xenoestrogens cause pituitary cells to proliferate (just as pituitaries and their tumors grow in response to estrogens)…

Proliferation function downstream of MAPKs are affected by XEs

Rene Vinas

Mechanisms that affect cell number – apoptosis – are elicited by XEs

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Jennifer Jeng

Examples: Elk and ATF2, with some differences in response pattern

…Es and XEs (in this case phytoEs) can also rapidly activate (phosphorylate) transcription factors downstream of ERKs

altered timing (phasing)

altered dose-response patterns and levels

imperfect mimicry or inhibition of multiple estrogenic actions activated via the nongenomic pathways

To summarize the 1st part…endocrine disruption by xenoestrogens includes:

Can a structure/chemical characteristic of an estrogen predict its activity in a nongenomic

response?

Here again are the ones we have studied

They can all be described by their physical characteristic of lipophilicity – the partition coefficient between octanol and water

Short chain alkylphenols are less lipohillic than long chain ones

13 estrogens – their activities in various functional and signaling responses compared to their hydrophobicity

(Chlorinated compounds were left out as they significantly reduced the correlations)

Jennifer JengMikhail Kochukov

The most complicated - both genomic and nongenomic pathway control

Best correlations are for structures that vary in simple ways (side-chain length)

Chemists can use such information to design less estrogenic versions of these compounds

But in real life these estrogens are rarely present by themselves

You have physiologic estrogens on board already – and then you get exposed to xenoestrogens on top of that.

Do xenoestrogens interfere with physiologic estrogens? Do they disturb the normal signaling patterns? How?

In different ways - examples for ERK signaling follow that demonstrate some

principles of estrogenic endocrine disruption.

Temporal phasing of response….XenoEs can delay the response after causing an initial dephosphorylation

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160EP EP+E3

E3

Jennifer JengTime in Minutes

pE

RK

XEs can enhance an estrogenic response at concentrations where it is less effective by itself; inhibit at higher concentrations – this is the

most typical effect of combinations

Jennifer Jeng

(physiologic estrogens all at 1 nM for these studies; 5 min responses)

p-E

RK

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of c

on

tro

l)

Log [Concentration (M)]

Have assessed for 3 physiologic estrogens challenged by 5 different XEs

Phospho-activation of ERKE2[10-9M]+ BPA[10-14M] + NP[10-8M]

Time (min)

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Rene´ Viñas

Extreme Disruption by Multiple XEs - can completely wipe out a physicologic E response – we have seen this now with multiple

combinations of XEs.

Any of these alterations “disrupts” the normal pattern

or extent of physiologic estrogen signaling.

In summary we have learned that Es/XEs acting via nongenomic pathways: ► signal more potently via the membrane versions of estrogen receptors

► activate multiple signaling pathways

► responses often oscillate with time and are non-monotonic (U- V- or even M-shaped dose-responses and XEs alter this to disrupt signaling – a difficult target for regulation

► mediated primarily via ER (depending on tissue); modulated by other ER subtypes

► XEs elicit tissue-specific disfunctions (secretion, differentiation, cell proliferation, enzymatic cascades, components of inflammatory responses, behavior, ROS) in different nontransfected cell types: pituitary, breast, prostate, neuronal, cells of the immune system……

► have particular developmental window / stage-specific vulnerabilities to XEs

► chemical structures of Es/XEs can predict strength/potency of nongenomic responses

► XE mixtures (which is what we are exposed to) dramatically disrupt physiologic E responses

How we would like to use our newly determined principles learned via high throughput signaling assays: Green Chemistry Design

We are currently collaborating with labs who view endocrine disruption at different (increasingly more complicated) levels.

remediation of existing contamin-

ants

animal develop-

ment - later assigned to

a NR

nongenomic cell signaling

- later assigned to

a NR

NR binding and NR-based

genomic response

in silico chemical- receptor dockingREDESIGN

+ -+++

AcknowledgementsWatson lab – over the years working on nongenomic E signaling projects

Todd PappasCeleste Campbell (Finnerty)Andrea NorfleetAdrian JakubasBridget HawkinsGaga ZivadinovicNataliya BulayevaTeresa ReedLeanne LashAnn WozniakRebecca AlyeaYow-Jiun Jennifer JengMikhail KochukovStephanie LaurenceAnannya BangaLuke KoongJune GuptarakGuangzhen HuRene VinasManish Saraf

Acknowledgements

FundingNIEHS (R01s and training grant)NIDA (P20 Center and training grant)Am. Inst. for Cancer ResearchUTMB Center for Addiction ResearchSealy Center for Environ. Health & MedicinePassport Fndn. Innovator Award

Collaborators: Bahiru Gametchu, Mary Thomas, Kathryn Cunningham, Randy Goldblum & Terumi Midoro-Horiuti

Green Chemistry Collaborators: Ruben Abyuan & Fiona McRobb, Bruce Blumberg, Susan Jobling, Terry Collins, John Warner, Pete Myers & Karen O’Brien

Fix cells: various methods to match epitope and cell type -- unpermeabilized measures membrane proteins-- permeabilized (usu. with detergent) measures intracellular proteins

(We have used it for various ERs, DAT, pMAPKs, and p-transcription factors, and GTP-G proteins in many different cell types)

Incubate with 1°Ab; biotinylated 2° Ab; avidin-conjugated alk. phos.

Incubate with pNpp → pNp at 37° in dark, & read at 405 nm Ag quantitation

Wash off reagents

Stain with 0.1% crystal violet, wash, extract, read at 562 nM cell number (normalization for each well)

inside

outsideImmuno-assay for quantitation of membrane vs. intracellular

proteins, their trafficking, and their activation state

molecules- receptors -- cell signaling - organisms- remediation genomics nongenomics

In silico – Ruben Abyuan and Fiona …..Receptor binding and genomic activation – Bruce BlumbergNongenomic cell-type specific signaling – Cheryl WatsonDeveloping organisms – Susan JoblingRemediation – Terry Collins, John WarnerCementing it all together – Pete Myers and Karen O’Brien

In silico cell-free cell dependent development chemical capture of amphib & fishes and degradation

Do TAML activators activate ERK – NO!!!!!

www.secondlife.comGenome Island, Genome (139,145, 37)

CW searching for receptors, wherever they are in the cell

Crucial difference between our studies and past studies on xenoestrogens:

● we look at rapid nongenomic effects

●we use very low (fMnM) concentrations (very relevant to those found commonly in the environment) within very wide concentration ranges ….and sensitive, quantitative cellular response assay systems

● we use non-transfected cell systems to avoid overexpression and heteroexpression artifacts

The assays we plan to do in GH3/B6/F10 pituitary cells for

the Green Chem Center Project

• Signaling assays--G protein activation (proximal signaling)

--ERK (+JNK & p38) kinase activations (downstream integrated signaling)

• Function assays--PRL release (nongenomic functional endpoint)

--Cell proliferation (complex functional endpoint)

If the money got bigger I could add:

• Other cell types in which we have already monitored nongenomic signaling effects (breast cancer, prostate cancer, neuronal, immune system)

• Other myriad signaling pathways (IPs, ions, other kinases, activation of transcription factors)

• Other functional endpoints