First International Congress on Science and Technology

October 28, 2005

Environmental Endocrine Disruptors

Dr. Joseph Colosi Dr. Arthur KneyDeSales University Lafayette College

Symantic Confusion• Endocrine disrupting chemicals (EDCs)

• Hormonally-active agents

• Environmental estrogens

• Environmental hormones

• Environmental chemicals

• Environmental signaling

• Xenoestrogens

• Gender benders

C. Corbitt

The Endocrine system

GlandTransport

Target Cell

Hormone

General Features of the endocrine system:

ductless

rich blood supply

secreted into the blood

can reach virtually every cell in the body

hormone receptors are very specific

C. Corbitt

Endocrine Glands

don’t forget the heart, placenta, fat

All of these glands produce hormones and are also targets for hormones

C. Coebitt

Link to diagram showinglocations of the endocrine glandsThyroid-stimulating hormone (TSH)protein (201)Anterior lobe of pituitaryFollicle-stimulating hormone (FSH)protein (204)Luteinizing hormone (LH)protein (204)Prolactin (PRL)protein (198)Growth hormone (GH)protein (191)Adrenocorticotropic hormone (ACTH)peptide (39)Antidiuretic hormone (ADH)(vasopressin)peptide (9)Posterior lobe of pituitaryOxytocinpeptide (9)Thyrotropin-releasing hormone (TRH)peptide (3)HypothalamusGonadotropin-releasing hormone (GnRH)peptide (10)Growth hormone-releasing hormone (GHRH)peptides (40)Corticotropin-releasing hormone (CRH)peptide (41)Somatostatinpeptides (14, 28)DopamineTyrosine derivativeMelatoninTryptophan derivativePineal glandThyroxine (T4)Tyrosine derivativeThyroid GlandCalcitoninpeptide (32)Parathyroid hormone (PTH)protein (84)Parathyroid glandsGlucocorticoids (e.g., cortisol)steroidsAdrenal cortexMineralocorticoids (e.g., aldosterone)steroidsAndrogens (e.g., testosterone)steroidsAdrenaline (epinephrine)Tyrosine derivativeAdrenal medullaNoradrenaline (norepinephrine)Tyrosine derivativeEstrogens (e.g., estradiol)steroidOvarian follicleProgesteronesteroidCorpus luteum and placentaHuman chorionic gonadotropin (HCG)protein (237)Trophoblast and placentaAndrogens (e.g., testosterone)steroidTestesInsulinprotein (51)Pancreas (Islets of Langerhans)Glucagonpeptide (29)Somatostatinpeptides (14, 28)Amylinpeptide (37)Erythropoietin (EPO)protein (166)KidneyCalcitriolsteroid derivativeCalciferol (vitamin D3)steroid derivativeSkinAtrial-natriuretic peptide (ANP)peptides (28,32)HeartGastrinpeptides (14)Stomach and intestineSecretinpeptide (27)Cholecystokinin (CCK)peptides (8)Somatostatinpeptides (14,28)Neuropeptide Ypeptide (36)Ghrelinpeptide (28)PYY3-36peptide (34)Insulin-like growth factor-1 (IGF-1)protein (70)LiverAngiotensinogenproteinThrombopoietinprotein (332)LeptinproteinFat cellsNote (1): Numbers within parentheses indicate the number of amino acids in the protein or peptide(s).

There are many hormones

Steroid Hormone Receptors (inactive)

Pol II Complex

SRE

Nucleus

DNA

Protein Product

Responsive Cell

Hormone

mRNA

Steroid Hormone Receptor Action

Hormone Action in

Cells

Do exogenous hormones affect people?

Diethylstilbestrol (DES)

• 1938 Charles Dodds synthesized DES

• 1941 Harvard: DES enhances pregnancy and prevents miscarriage

• 1947 FDA Approved DES for pregnancy

• 1947-1971 DES prescribed for 5 million US pregnant women, dose = 700 birth control pills

• 1953 U of Chicago study: no prevention of miscarriage benefit DES

Diethylstilbestrol (DES)

• 1954 DES put in chicken and cattle feed3/4

• 1959 DES banned for chickens and lambs

• 1964 Charles Dodds Knighted

• 1971 Surgeon General warned against DES for pregnant women

• 1979 USDA banned DES for cattle feed

• 1970’s DES prescribed to many thousands of pregnant women throughout the world

DES Outcomes (http://www.cdc.gov/des/consumers/)

DES Daughters• One third have reproductive tract abnormality

• 100X greater risk for clear cell adenocarcinoma

• 2.5X greater chance of miscarriage

• 2 to 3X greater chance of ectopic pregnancy

• 33% increase in infertility

• 25% increase in premature delivery

DES Outcomes (http://www.cdc.gov/des/consumers/)

DES Sons• 4X increase in non-cancerous epidymal cysts

• 3 to 4X increase in genital deformity?

DES Grandchildren

?

So avoid high doses of steroid drugs and you’ll be safe.

First nationwide survey to detect 95 pharmaceuticals, hormones, and other organics in 139 urban and agricultural streams in 30 states. Two chemicals found in 80% of the samples, and 82 chemicals occurred in at least one sample.

USGS Water-quality survey, 1999-2000

http://jeq.scijournals.org/cgi/content/full/32/2/466

US

GS

Wat

er-q

ual

ity

dat

a, 1

999-

2000

Compounds known to bind steroid receptors in humans and animals

Rooney, AA, and LJ Gillette, Jr. Contaminant interactions with steroid receptors: evidence for receptor binding. In Guillette, LJ, Jr., and DA Crain. 2000 Environmental Endocrine Disrupters: An Evolutionary Perspective. Taylor and Francis.

• Diethylstilbestrol (DES) Food contaminant• Butylated hydroxyanisole Food additive• Vinclozolin fungicide• Alachlor Herbicide• DDT Insecticide• P-Nonylphenol Industrial chemical• Genistein Phytoestrogen

Compounds known to bind steroid receptorsRooney, AA, and LJ Gillette, Jr. Contaminant interactions with steroid receptors: evidence for receptor binding. In Guillette, LJ, Jr., and DA Crain.

2000 Environmental Endocrine Disrupters: An Evolutionary Perspective. Taylor and Francis.

Pesticides Industrial chemicals PhytoestrogensVinclozolin

Alachlor

Atrazine

Cyanazine

Desiopropyl

Simazine

Prometryne

o,p’-DDD

p,p’-DDD

p,p’-DDE

o,p’-DDE

o,p’-DDT

p,p’-DDT

DDOH

2,4-Dichlorophenol

Chlordecone (Kepone)

Dicofol

Endosolfan sulfate

-Endosolfan

Methoxychlor

cis-Nonachlor

trans-Nonachlor

Toxaphene

Tamoxifen (drug)

Aroclor 1242 (PCB

mixture)

p-sec Amyl phenol

p-sec-Amyl phenol

p-Isoamyl phenol

p-tert Amyl phenol

o-sec-amyl phenol

Bensophenone

Bisphenol-A

bis(2-Ethylyexyl) phthalate

bis(2-Ethylyexyl) adipate

6-Bromonaphthol-2

4-sec-Butylphenol

n-butyl-benzene

Di-n-Butylphthalate

Butylbenzyl phthalate

4,4’-Dihydroxy

2’chlorobiphenyl

4,4’-Dihydroxybiphenyl

4-Hydroxy, 2’,4’,6’-

trichloro biphenyl

4-Hydroxy, 2’,3’,4’,5’-

tetrachloro biphenyl

1-Naphthol

2-Naphthol

4-nitrotoluene

p-Nonylphenol

Nonylphenol

4-Nonylphenoxy carboxylic acid

4-tert-Octylphenol

-Phenylcresol

Tetrahydronaphthol-2

3,4,3’,4’-tetrachloro biphenyl

Anglolensin

Apigenin

Biochanin-A

Chrysin

Coumestrol

Daidzein

o-Desmethylangolensin

4,4’Dihydroxychalcone

4,7’-Dihydroxyflavanone

Equol

Genistein

Isoliquiritigenin

Kaempferide

Luteolin

Miroestrol

Naringenin

Phloretin

-Sitosterol

Zearalenone (Mycoestrogen)

Zearalenone (Mycoestrogen)

Zearalanol (Mycoestrogen)

Chemicals Found in the EnvironmentReported to be Estrogenic

McLachlan, J.A. 2001 Environmental Signaling: What Embryos and Evolution Teach Us About Endocrine Disrupting Chemicals, Endocrine Reviews 22(3): 319-341.

McLachlan, J.A. 2001 Environmental Signaling: What Embryos and Evolution Teach Us About Endocrine Disrupting Chemicals, Endocrine Reviews 22(3): 319-341.

Chemicals Found in the EnvironmentReported to be Estrogenic

McLachlan, J.A. 2001 Environmental Signaling: What Embryos and Evolution Teach Us About Endocrine Disrupting Chemicals, Endocrine Reviews 22(3): 319-341.

Chemicals Found in the EnvironmentReported to be Estrogenic

Every year, 5000 new chemical compounds are introduced.

“Better living through chemistry.”

How do EDC’s get into the environment?

What it takes to be beautiful today.

Bethlehem Sewage Treatment Plant

Plastics

R. Bolen

Phytoestrogens, especially from legumes

Cattle feedlot Sheep feedlot

Pesticides

R. Bolen

Industrial chemicals

R. Bolen

Do environmental EDC’s affect wildlife?

Reproductive and developmental abnormalities attributed to endocrine disruption

from McLachlan (2001)

Species Observation Contaminant

alligators abnormal gonads, decreased phallus size, altered sex hormone levels

DDT, DDE, dicofol

snails masculinization,, imposex, formation of additional female organs, malformed oviducts, increased oocyte production

tributylin, bisphenol A, octylphenol

seals impaired reproductive function coumestrol

water birds egg shell thinning, mortality, developmental abnormalities, growth retardation

DDE, PCBs, AhR agonists

mosquito fish abnormal expression of secondary sex characteristics, masculinization

androstenedione

R. Bolen

McLachlan, J.A. 2001 Environmental Signaling: What Embryos and Evolution Teach Us About Endocrine Disrupting Chemicals, Endocrine Reviews 22(3): 319-341.

How Do Environmental EDC’s affect the endocrine system?

Do EDC’s in the environment affect humans?

from Sharpe and Irvine (2004)

Trends in reproductive health

Premature Thelarche

23-month-old Puerto Rican girl with premature thelarche

Thelarche: breast developmenttiming depends on estrogen/androgen ratio

Premature thelarche “epidemic” in Puerto Rico

Premature Thelarche and Early Sexual Development Registry tracked 4,674 cases in P.R. since 1969

Several causes hypothesized: diet, EDCs

Colon et al (2000)

Plasma phthalate ester levels correlated with premature thelarche cases

Phthalate esters used as plasticizers;have estrogenic or anti-androgenic activity in vitro

C. Corbitt

Evidence linking human reproductive problems to EDCs

• girls exposed to higher levels of PBCs and DDE in utero entered puberty an average of 11 months earlier than controls

• higher levels of organochlorine chemicals found in mothers of men with testicular cancer

• exposure of boys to endosulfan associated with delayed puberty

R. Bolen

What is EPA doing about it?

EPA Screening protocols being testedTier 1 Screening Assays

Tier 2 Testing Assays

Amphibian Metamorphosis

(Tadpoles)

Androgen Receptor (AR)

binding

Aromatase

Estrogen Receptor (ER)

binding

Fish Screen (Whole fish)

Hershberger (Whole rats)

Pubertal Female (Whole rats)

Pubertal Male (Whole rats)

Amphibian 2 (Whole frogs)

Avian 2-Generation (Whole

birds)

Fish Life Cycle

Invertebrate Lifecycle (Whole

mysid shrimp)

Mammalian 2-Generation

(Whole rats)

Tier (to be determined)

In Utero through Lactation

(Whole rats)

Current US regulations on environmental EDC’s

•Banned: aldrin, chlordane, DDT, dieldrin, endrin, kepone, mirex, PCB’s, toxaphene•Restricted: dicofol, dienochlor, endosulfan, heptachlor, lindane, methoxychlor•Not regulated: Vast majority of EDC’s.

•Last update of EDC webpage: 2002

Yeast Bioassay for Estrogenic CompoundsEstrogen-inducible expression system in yeast

Routledge, EI, JP Sumpter. 1996. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environmental Toxicology and Chemistry 15: 241-248

Sensitive to 1 PPT 17 -estradiol

1 part per trillion is one second in 32 thousand years

ONPG (Clear)

Yellow

Lac Z operon reporter

PPB

PPT

0 1 2 30.25 0.5 0.75 1.25 1.5 1.75 2.25 2.5 2.75

Optical Density

1E-012

1E-011

1E-010

1E-009

Es

tra

dio

l C

on

ce

ntr

ati

on

(M

)Standard Curve NSF Laboratory - July 20, 2004

Polynomial Fit Results

Equation Y = 1 .03E-011 + 4 .32E-010 * X - 5 .34E -010 * X ^2 + 3 .42E-010 * X^3 - 6 .51E-011 * X^4

D egree = 4N um ber o f data po in ts used = 7

Estrogenic Compounds at Lehigh Valley Wastewater Treatment Plants

0.0

10.0

20.0

30.0

40.0

50.0

60.0

Pberg10/12/04

Usaucon10/12/04

Bath10/08/04

Easton10/09/04

Nazareth10/09/04

Atown07/19/04

Wastewater Treatment Plants

Est

rad

iol

Eq

uiv

alen

t n

g/L

(p

pt)

Influent

Effluent

•What is the daily pattern of EDC’s in influent wastewater?•What is the seasonal pattern?•Do these vary with source of wastewater?•How much is EDC concentration reduced by wastewater treatment? Are some treatments more effective?•Do the levels in the effluent affect aquatic wildlife? terrestrial wildlife? Humans?

For further study:

Summary

•The endocrine system is complex and depends on extremely low concentrations of hormones.•Many chemicals are found in US surface waters.•Many of these are EDC’s.•There are disturbing trends in sexual and developmental dysfunction in wildlife and humans.•Progress on detection of EDC’s is slow.•Transgenic yeast has potential to expand our knowledge about the occurrence of EDC’s.

Detection of estrogenic compounds in wastewater using a modified transgenic

yeast assay

Five steps in detecting environmental estrogens with transgenic yeast

1. Obtain samples and filter to remove microorganisms.2. a) Add yeast and medium to filtered samples and incubate at

30oC overnight. b) Prepare duplicate set of tubes with estradiol spike for positive

control.c) Prepare set of standardized samples

3. Lyse cells, add ONPG substrate and incubate 2 hr. at 37C.4. Read color development with spectrophotometer.5. Compare OD405 values to those of standard curve.

1. Obtain samples and filter to remove microorganisms. All samples collected October 27, 2005

Sample 1Bottled water

Sample 2Tap water

Sample 3River water

Sample 4Raw sewage

Sterile sample 1

1

Sterile sample 3

3

Sterile sample 2

2

Sterile sample 4

4

Sterile sample 1

1

Sterile sample 3

3

Sterile sample 2

2

Sterile sample 4

4

2a) Add yeast and medium to filtered samples and incubate at 30 C overnight.

500µl 100µl 500µl 100µl 500µl 100µl

1/2 1/10

500µl 100µl

1/2 1/10 1/2 1/10 1/2 1/10

Two dilutions for each sample

500µl 100µl

1/2 1/10

Sterile sample 1

12X

medium and yeast

500µl500µl

Sterile DI water

2a) Detail of preparation of tubes for incubation of yeast with filtered samples. This procedure is for each sample.

400µl

Incubate each tube for 24 hours at 30 C

Two dilutions for each sample completed

500µl 100µl

1/2S 1/10S

Sterile sample 1

12X

medium and yeast

500µl500µl

Sterile DI water

2b) Detail of preparation of duplicate tubes with estradiol spike for positive control for each sample.

400µl

1.56µl E-08 Estradiol (S7)

1.0µl

Incubate each tube for 24 hours at 30 C.

2X medium

and yeast

500µl

Sterile DI water

2c) Detail of preparation of standard curve

S4 S6S5 S8S7 DMSO

500µl

1.0µl

Estradiol

1.25E-7M 6.25E-8M 3.12E-8M 1.56E-8M 7.80E-9M DMSO

BLANK

3. Lyse cells, add ONPG substrate and incubate 2 hr. at 37C

Transfer 100 ul of each sample tube and standard curve tube, in which yeast has incubated for 24

hours, to a new tube

1

1

100 µl

New tube – Mix with 900 ulof Z buffer/ONPG

Z buffer with ONPG (substrate)

900 µl

Incubate for 1 to 2 hours at 35oC

1

Stop buffer

400 µlAfter incubation periodstop reaction in

new tube

4. Read color development with spectrophotometer

Cuvette

Each tube

Pour about ½ ml into cuvette

Use DMSO as blank for each sample and standard curve tube. Read OD405 for each tube. Pour out contents from each tube and use cuvette for next tube. Read tubes with less color than tubes

with more color.

5. Compare OD405 values to those of standard curve.

0 1 2 30.25 0.5 0.75 1.25 1.5 1.75 2.25 2.5 2.75

Optical Density

1E-012

1E-011

1E-010

1E-009

Es

tra

dio

l C

on

ce

ntr

ati

on

(M

)

Standard Curve NSF Laboratory - July 20, 2004

Polynomial Fit Results

Equation Y = 1 .03E-011 + 4 .32E-010 * X - 5 .34E -010 * X ^2 + 3 .42E-010 * X^3 - 6 .51E-011 * X^4

D egree = 4N um ber o f data po in ts used = 7

Estrogenic Compounds at Lehigh Valley Wastewater Treatment Plants

0.0

10.0

20.0

30.0

40.0

50.0

60.0

Pberg10/12/04

Usaucon10/12/04

Bath10/08/04

Easton10/09/04

Nazareth10/09/04

Atown07/19/04

Wastewater Treatment Plants

Est

rad

iol

Eq

uiv

alen

t n

g/L

(p

pt)

Influent

Effluent