giesy keynote edcs

8/3/2019 Giesy Keynote EDCs

1/50

Zoology Dept. & National Food Safety and Toxicology Center,

Center for Integrative Toxicology, Michigan State University

Society of ToxicologyRegional Meeting

November 4, 2005East Lansing, MI


2/50



Endocrine Disruptor Mechanisms:

Beyond Receptor BindingJ.P. Giesy

M. Hecker, K. Hilscherova, X. Zhang,J. Newsted, P.D. Jones, E. Higley

Michigan State University, Dept. Zoology

National Food Safety and Toxicology CenterInstitute of Environmental Toxicology

R. Wu, M. Murphy,R. Yu

Dept. Biology and ChemistryCity University of Hong Kong

Kowloon, Hong Kong, SAR, PRC


3/50



Thanks To

This work was facilitated through a grant from the U.S.Environmental Protection Agency.

Gary Timm (US-EPA)Ralph Cooper (US-EPA)

Anne-Marie Vinggaard (Danish Institute for Food and

Veterinary Research, DK)


4/50



ENDOCRINE DISRUPTION (ED)

...an exogenous agent that interferes withthe synthesis, secretion, transport,

binding, action, or elimination of naturalhormones in the body that are responsiblefor the maintenance of homeostasis,reproduction, development, and/orbehavior.

Kavlock et al., 1996. Research needs for the assessment ofenvironmental effects of endocrine disruptors: a report of the USEPA-sponsored workshop


5/50



Endocrine Disruption

Major Functions of the Endocrine System:

1. Coordinate homeostasis in the body

2. Allow communication among organs


6/50



Maintaining Homeostasis

is a Complex Process

Involves many signal transduction pathways

Many alternative pathways

Many enzymes involved

Rate limiting steps unknown

Cybernetic feedback loops


7/50



Levels of EDC Organism Interaction

Hormone Synthesis

Hormone Action

Hormone Transport

Hormone Metabolism

Stimulation

Suppression



8/50



ENDOCRINE DISRUPTION

Direct - (Mimics)Agonists

AntagonistsPartial Agonists

Environmental estrogens are only one mimic

Indirect

Induction of Enzymes that Directly orIndirectly Affect Hormone Concentrations

Alteration of signal transduction pathways


9/50




In the United States current attention is focuseprimarily on compounds that can affect steroidhormones

Reauthorization of the Clean Water Act

Food Safety Protection Act

Endocrine Disrupter Screening and TestingCommittee (EDSTAC)

Estrogen receptor (ER)Androgen Receptor (AR)

Thyroid Receptor (TR)


10/50



Endocrine Disruption & Food Safety

(USA)

When setting new or reassessing existingtolerances and tolerance exemptions, EPA

must also evaluate the potential for endocrinedisruption. The law directs the Agency to use

its authority to require specific tests andinformation on estrogenic effects for allpesticide chemical residues.


11/50



Endocrine Disruption & Food Safety

(EU) Council Directive 96/22/EC of 29 April 1996 concernin

the prohibition on the use of stock-farming of certainsubstances having a hormonal or thyrostatic actionand of-agonists (amended by Dir. 2004/73/EC)

Council Directive 91/414/EEC of 15 July 1991concerning the placing of plant protection products othe market: Endocrine disrupting activity: waiting for test methodology

to be endorsed by OECD. In the meantime in case ofsuspicion of ED potential fish full life cycle test.


12/50



Endocrine Disruptor Screening Program

Universe of chemicals - 87,000 chemicals:

- 900 pesticide active ingredients- 2,500 other pesticide formulate ingredients- 75,500 industrial chemicals- 8,000 cosmetics, food additives and nutritional

supplements

Initial focus on:

- pesticide actives- high production volume inerts


13/50



Proposed Tier 1 Screening Battery (EDSTAC

In VitroScreens ER Binding / Reporter Gene AssayER Binding / Reporter Gene Assay

AR Binding / Reporter Gene AssayAR Binding / Reporter Gene Assay

Steroidogenesis Assay with minced testisSteroidogenesis Assay with minced testis

In VivoScreens

Rodent 3Rodent 3--day Uterotrophic Assay (sc)day Uterotrophic Assay (sc) Rodent 20Rodent 20--day Pubertal Female Assay with Thyroidday Pubertal Female Assay with Thyroid

Rodent 5Rodent 5--7 day Hershberger Assay7 day Hershberger Assay

Frog Metamorphosis Assay (FETAX)Frog Metamorphosis Assay (FETAX) Fish Reproduction Screening AssayFish Reproduction Screening Assay

Alternate assays have also been proposed


14/50



1. Exposure - outcome linkages- are effects occurring in humans?

2. Comparative toxicology

- extrapolations between species?

3. Multiple mechanisms of action

4. Cumulative exposure/latency between exposureand outcome

Uncertainties and Concerns


15/50



5. Cost - $200,000 per substance for Tier 1

6. Animal welfare

- Estimate that 0.6 - 1 million animals would beused per 1,000 substances (1999, Toxicol. Sci. 52, 141-

7. Do endocrine disruptors require specialconsideration in risk assessment?

8. Assay and test validation (Interagency CoordinatingCommittee for the Validation of Alternative Methods -ICCVAM)

Uncertainties and Concerns contd


16/50



Mechanism of Action for ER-Activation

ProteinPhosphorylationof ER

Ligand- Independent Activation

DNA Binding

Estrogen

or xenoestrogen

mRNA

ER

ER-Responsive Genes

ER

NuclearFactors

+

ER

P P

Estrogenic Effects


17/50



Limitations of Screening Methods

If used in a sequential decision processFalse negatives

If negative in the binding assay, may still be

positive as an endocrine disruptingcompound


18/50



Binds to EstrogenReceptor or is

predicted to do sofrom QSAR

YesNo

Further Testing because compound maybe an endocrine disruptor

The proposed sequential testing provides useful information

for designing additional testing, but does not allow for a sortingof compounds and does not assist in prioritizing compounds

for additional testing


19/50



Example: Triazine Herbicides

Do not bind to ER!

Results in estrogenic effect in vitro

Sanderson. J.T., W. Seinen, J.P. Giesy and M. van den Berg. 200

2-chloro-S-Triazine Herbicides Induce Aromatase (CYP-19) ActivityH295R Human Adrenocortical Carcinoma Cells: A Novel Mechanis

for Estrogenicity. Toxicol. Sci. 54:121-127.

I d ti f CYP19 RNA


20/50



Induction of CYP19 mRNAby triazines

0

50

100

150

200

250

300

Control30 M

S

imazine

100 M

8Br-cA

MP

A

mplifica

tionres

ponsera

tioof

CYP19/beta-actin

(%o

fcontr

olratio)

A

trazine

P

ropazine

DMS

O


21/50



Effects of Other Compounds on

Aromatase activity H295R cells Imidazole-type fungicides decrease aromatase

activity. Competitive inhibitors

imazalilprochlorazdifenoconazol

penconazolePropiconazoleDiclobutrazoleTricyclazolePaclobutrazoleNuarimol

Sanderson et al. 2001. Organohalogen Compounds. 53:10-13.

the structurally similar

fungicide Vinclozolinincreases cAMP 150%,whereas forskolin increasescAMP 300%


22/50



Proposed Mechanisms of

Action for Triazines Aromatase induction / promotion

via protein kinase A pathwayvia steroidogenic pathways

Inhibition of phosphodiesterase

Results in less conversion of c-AMP to AMP so that AMPincreases

c-AMP increases signal transduction of Protein Kinase A

Protein kinase A increases CREB and SF-1

Aromatase m-RNA is up-regulated such that morearomatase is formed and aromatase activity increases

P t i Ki A Si li P th


23/50



Protein Kinase A Signaling Pathway

Ligands (ACTH, LH, FSH, GnTH) Membrane-boundreceptors

Adenylyl cyclase

cAMP AMP

G-protein

Protein Kinase A

SF-1

aromatase

CREB

STAR

phosphodiesterase

MIS

Cholesterol-

esteresterase

cholesterol

AtrazinInhibit


24/50



In vitroModel System

Needed a flexible assay system that wouldallow for rapid studies of mechanisms ofaction

Needed to express major enzyme systems

Needed to be stable so results are

reproducible


25/50



H295R cell line

Human female adrenocortical carcinoma

Produces many steroid progestins,androgens & estrogens hormones

glucocorticoids, mineralocorticoids

Express most of the important

steroidogenic enzymesCYP11A, CYP11B, CYP17, CYP19, CYP21


26/50



Derived from the NCI-H295 pluripotentadrenocortical carcinoma cell line (Gazdar

et al. 1990) from a carcinoma of theadrenal cortex that arose in a 48 y.o. black

female.Modified cells retain the ability to produce

aldosterone, cortisol and C19 steroids(adrenal androgens).

H295R cell line


27/50



H295R Cells

The cells maintain the capacity to synthesize

most of the steroid hormones characteristicof three phenotypically distinct zones of theadult adrenal cortex

Zona glomerulosa

Zona fasciculata

Zona reticularis


28/50



Pregnenolone

Progesterone

11-Deoxycorticosterone

Corticosterone

Aldosterone

17-OH-Pregnenolone

17-OH-Progesterone

CYP17

CYP17

CYP11A

3-HSD

CYP21

CYP11B2

CYP21

11-Deoxycortisol

3-HSD

CYP11B1

CortisolCYP11B2

Zona glomerulosa

Zona fasciculata

DHEA

Androstene-dione

Testosterone

CYP17

Zona reticularis

3-HSD

17-HS

CYP19

17-Estradiol

CYP17

Cholesterol


29/50



Effects on steroidogenesis

At level of expression

measure mRNA levels: RT-PCR

measure amount of enzyme present

Effects on enzyme concentrations

measure catalytic activities: selectivesubstrates

Effects on metabolism of steroidhormones

measure steroid hormone concentrations


30/50



Objectives

Develop and optimize a rapid screening test todetermine effects of chemicals on sex steroidsynthesis:

ProgesteroneTestosterone

17-estradiol Demonstrate the performance of the assay with

known inhibitors and inducers of steroidogenic

pathways

Develop methods to screen for effects onsteroidogenic enzymes


31/50



Objectives (Cont.)

Establish an assay that will integrate possible effectson multiple parts of the steroidogenic pathway:

1. Steroidogenic signal transduction

2. Regulation of cholesterol transport by theSTAR-Protein

3. Conversion of cholesterol to testosterone by: P450SCC

3HSD & 17-HSD P450C17

4. Androgen conversion to estrogen by CYP19aromatase


32/50



Objectives (Cont)

Assess and quantify sources of variability inthe assay to:

Establish performance criteria for large scalescreening of chemicals

Demonstrate flexibility and transferability ofthe protocol to other laboratories prior toconducting ring tests

Develop optimized protocol for inter-laboratoryvalidation phase.

OVERALL APPROACH


33/50



The responses profiles will beused in infomatic analyses ofthe effects of the compoundsscreened.

Cytotoxicity assay

Infomatic analysis

Responses profiles

H295R Cell exposure

RT-qPCR

(11 genes)

Model chemicals Environmentaltoxicants

OVERALL APPROACH

Model chemicals: inhibitors &Inducers

Incubation time: 48 h

Gene will be monitored indose range without causingcell death


34/50



Real-time Reporters

Non-specific DNA binding dyes1) Ethidium Bromide (first reported by Higuchi, 1993)

2) SYBR Green ISpecific Hybridization Probes

1) TaqMan probes

2) Molecular beacons *3) others (dual-oligo FRET pairs)

To indicate the accumulation of PCR products

M l l B


35/50



Molecular Beacons

MolecularBeacon

RQ

BHQ-2ROX

BHQ-1TET

BHQ-0TAMRA

DABCYLFAM

QuenchersReporters

Fluorescence Resonance Energy Transfer (FRET)


36/50



Molecular Beacons

MolecularBeacon

R Q

l

Taq

Denaturation

RQ

Taq

R

Q

Extension

Detection

Primers

RQ

Annealing

Prim

Taq


37/50



HMGR + -actin:HMGR:Diluted in a 5-fold manner-actin:Fixed at a concentrationTriplicate analyses

Amplification plot for HMGR

Standard Curve for HMGR

Example of duplex PCR


38/50



Benefits of RT PCR (1)

Due to: Inhibitors of the polymerase Reagent limitation (primers)

Eventually, Plateau Phase

PCR cycle #

Lo

gPCRproductamount

Plateau Phase

Theo

retically

Exp

onen

tial

To provide a more accurate andprecise measurement, it isnecessary to collect

quantitative data in theexponential phase ofamplification


39/50



Benefits of RT PCR (2)

PCR cycle #

Fluorescence

Signal

Baseline

Threshold

0 10 20 30 40

0

0.1

1

10

17 19 CT = 19-17 = 2

By the reliabledetection of products

generated duringeach cycle of PCR,Real-Time PCRallows us to analyze

reactions duringexponential phase

More specifically, sensitively, and reproducibly


40/50



Proteasome inhibitorTajima et al., 2001MG132StAR

Not specific (CYP19, StAR) (H295Feltus et al., 2002dexamethasone3 -HSD

Not specific ( CYP11A, CYP17,

STAR, CYP11B1/ B2, 3HSDCell testing

Valle et al., 1995forskolinCYP21

Cell testingSanderson et al., 20008Br-cAMP, Vinclozolin

Cell testing; increases intracellulcAMP levels

Zhao et al., 1997Prostaglandin (PG) E2CYP19

Not specific (CYP11A, STAR,CYP21, 3bHSD) (H295R)

Holland et al., 1993Angiotensin IICYP11B2

Not specific (CYP11B1, CYP 17)Cell testing

Valle et al., 1995ACTHCYP11B1

Not specific (3-bHSD,CYP17);Cell testing

Payne et al., 1992cAMP (cyclicadenosinemonophosphate)

CYP11A

NotesReferenceInducerGene

Inducers of Steroidogenic Genes


41/50



Exposure Results

H295R Methods to Measure Effects


42/50



H295R Methods to Measure Effectson Hormone Production

Incubated for24 hours

Cells cultured in FlaskRenew media 2-3 x weeklySplit cell when ~90% confluent

Trypsinize

Seed Plate

Dose Cells

Replace

Media

IncubateFor 48 hrs

Extract Medium

with ether

Analyze for Hormone

ELISA, RIA, LC/MS

Collect CellsFreeze in Liquid N2

(gene expression, enzyme analysis)

Model Chemical Exposure


43/50



Model Chemical ExposureProchloraz

-6.00

-5.00

-4.00

-3.00

-2.00

-1.00

0.00

1.00

2.00

3.004.00

5.00

6.00

0.001 0.01 0.1 1 10

Prochloraz [M]

relativ

echange

* = sign. P

+ = sign. T@ = sign. E2

***

******

***

@@@

@

@@@

@@@

+++

+++ +++

Progesterone Testosterone Estradiol

@@@


44/50



Inter-laboratory Comparison

Participating Laboratories:

US Environmental Protection AgencyEndocrinology Laboratory, U.S.A.

Chemicals Assessment Center

Chemical Evaluation and Research Institute, Japan

Danish Institute for Food and Veterinary Research

Department of Toxicology and Risk Assessment,Denmark


45/50



Inter-laboratory Comparison

Performance based comparison. Use of:

Same cells

Different cell culture protocols/conditions

Same seeding density Same acclimation and exposure

protocols/conditions

Different hormone detection methods

P li i I t L b C i


46/50



Preliminary Inter Lab Comparison

Prochloraz (Progesterone)

-2

-1

0

1

2

3

4

5

6

0.001 0.01 0.1 1 10 100

uM Prochloraz

ChangerelativetoSC(

=0)

MSU (USA)

AMV (DK)


47/50



3

4

5

6

7

8

Preliminary Inter Lab Comparison

Prochloraz (Progesterone)

MSU AMV

y 1.4886x + 7.109 1.4696x + 6.6189

R2 0.9398 0.9006

EC25 0.228 mM 0.109 mM

EC50 0.079 mM 0.038 mM-4 -3 -2 -1 0 1 2

log uM Prochloraz

pro

bit


48/50



Future Directions & Upcoming Events

Present results during 2nd Meeting of the OECD

Validation Management Group for Non-AnimalTesting: 14-15 December 2005, Paris

Good chance that this test system will be adoptedinto the OECD Chemical Testing Guidelines forEndocrine Disrupter Testing and Assessment

ICCVAM Validation By NICETUM (NTP-NIH)


49/50



Questions ???????

SO

T

Th k Y


50/50

Zoology Dept & National Food Safety and Toxicology Center

Thank You

John P. Giesy

Dept. Zoology

Michigan State University

East Lansing, Michigan, 48824, USA

Tel: (517) 353-2000

Fax: (517) 432-1984

Email: [email protected]

Web Site: http://www.msu.edu/user/giesy

giesy keynote edcs

Documents