BI 242 Environmental Science

Environmental Endocrine Disruptors (EDC’s)

Dr. Joseph ColosiDeSales University

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

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Insulin, a protein hormone

Steroid hormones

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

Types of hormone receptors

R

Enzyme effector

R G-protein

Enzyme effector

R Ion channel

HORMONE

NUCLEUS

R

R

DNA binding sites

Nuclear membrane

CELL MEMBRANE

Protein hormones

Intracellular Receptors

Plasma Membrane Receptors

Steroid hormones

C. Corbitt

Protein product

HYPOTHALAMUS

PITUITARY

TESTIS

Testosterone

The H-P-G Axis

-

-

- = negative feedback

GnRH-

- LH

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It’s actually much more complicated

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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 DES miscarriage benefit

Diethylstilbestrol (DES)

• 1954 DES put in chicken and cattle feed• 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 (CDC DES page http://www.cdc.gov/des/consumers/)

DES Daughters CDC DES daughter page

http://www.cdc.gov/des/consumers/about/effects_daughters.html

• One third have reproductive tract abnormality • 100X greater risk for clear cell adenocarcinoma, from 0 to 1/1000• 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

Flame retardant

Insect repellant

Fungicide

antibacterial

surfactant

surfactant

surfactantplasticizer

Survey study 2002 http://pubs.acs.org/hotartcl/est/es011055j_rev.html

New study on pesticides 2006 http://pubs.usgs.gov/fs/2006/3028/

US

GS

Wa

ter-

qu

alit

y d

ata,

19

99-

200

0

PPB

PPT

Detection limits for USGS study

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)

Every year, 5000 new chemical compounds are introduced.

“Better living through chemistry.”

How do these chemicals get into the environment?

What it takes to be beautiful today.

Where do these chemicals go when we shower and pee?Why pee?

Bethlehem Sewage Treatment Plant

Plastics

R. Bolen

Cattle feedlot Sheep feedlot

Pesticides

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Industrial chemicals

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Do these chemicals in the environment affect wildlife?

There are hundreds of research articles relating EDC’s to effects on wildlife:• feminization of male alligators, tadpoles

• masculinization of female gastropods; female fish

• impaired immune and decreased reproductive function in Baltic seals

• eggshell thinning in water birds, eagles• all have resulted in population decline.

R. Bolen

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 Work?

Do these chemicals in the environment affect humans?

Trends in human reproductive health• testicular dysgenesis syndrome

– decreased semen quality (reduced numbers, motility, altered morphology of sperm)

– increased frequency of male reproductive tract abnormalities• hypospadias (urinary tract opening located not at the tip of the penis)• cryptorchidism (testis fails to descend by age 12 months)

• altered sex ratio (fewer male babies)• premature breast development (thelarche)• premature onset of menstruation (menarche)• increase in rates of breast cancer, testicular cancer

Are these trends In humanscaused by EDC’S?

R. Bolen from Sharpe and Irvine (2004)

What evidence links 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• Premature thelache in Puerto Rico associated with high blood

phthalate levels.• Prenatal blood phthlate associated with feminization of male babies.

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Premature Thelarche

23-month-old Puerto Rican girl with premature thelarche

Thelarche: breast development, timing 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, I, D Caro, CJ Bourdony, and O.Rosario. 2000. Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development. Environ. Health Perspect. 108:895-900

Plasma phthalate ester levels correlated with premature thelarche cases

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

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But, McKee, RH. 2004. Phthalate exposure and early thelarche. Environ. Health Perspect. 112:A541-A543. Questions the results because the blood levels are too high and phthalates do not act like estrogens in female rodents

penis

Anogenital distanceAGD

AGI = AGD/weight

. August 2005

anus

Environmental Health Perspectives 113: 1056-1061

What is EPA doing about it?

EPA Screening protocols being testedTier 1 Screening will include a battery of screening assays that would identify substances that have the potential to interact with the estrogen, androgen, or thyroid hormone systems.

Tier 2 Testing AssaysWill determine whether a substance may cause endocrine-mediated effects through or involving estrogen, androgen, or thyroid hormone systems,

Will determine the consequences to the organism of the activities observed in Tier 1

Will establish the relationship between doses of an endocrine-active substance administered in the test and the effects observed.

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 Lifecycle

Invertebrate Lifecycle (Whole mysid

shrimp)

Mammalian 2-Generation (Whole rats)

Tier (to be determined)

In Utero through Lactation (Whole rats)

http://www.epa.gov/scipoly/oscpendo/edspoverview/chronology.htm 1996-2005

EPA will test for pesticide concentration in the the 20 most consumed foods for pesticides

http://www.epa.gov/fedrgstr/EPA-TOX/2005/September/Day-27/t19260.pdf

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.

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 10 PPT 17 -estradiol

1 part per trillion is one second in 32 thousand years.

A playing card on a football field is one part per million. Cut the card into 1 million pieces. One of these pieces on a football field is one part per trillion.

ONPG

(Clear)

Yellow

PPB

PPT

Witters et al. (2001) Method for Extraction of Estrogenic Compounds from Wastewater

2.5 Liters of wastewater passed through 3 sets of filters (final pore size 0.45μm), then solid phase extraction disk. Estrogenic compounds eluted with acetone, dried with nitrogen, and dissolved in 100μl DMSO.

Revised method -0.2μm sterile filter

Standard Curve for Quito 10/27/2005

y = -2E+30x3 + 3E+20x2 + 6E+09x - 0.0092

R2 = 0.9999

0

0.5

1

1.5

2

2.5

0.00E+00 2.00E-11 4.00E-11 6.00E-11 8.00E-11 1.00E-10 1.20E-10 1.40E-10 1.60E-10

Estradiol Molar Concentration

OD

405

Estrogenic compounds stimulate the yeast to convert the ONPG substrate into a yellow pigment. More yellow signifies more estrogenic compounds in the water.

Estradiol Equivalents in Six Lehigh Valley, PA Wastewater Treatment Plants

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

Pberg Easton Usaucon Nazareth Bath Allentown

Wastwater source

Est

rad

iol

Eq

uiv

alen

t,

Par

ts p

er T

rill

ion 7/20/04 Influent

7/20/04 Influent

10/8/04 Influent

10/8/04 Effluent

3/14/05 Influent

3/14/05 Effluent

6/28/05 Influent

6/28/05 Effluent

•What is the daily pattern of estrogenic chemicals in influent wastewater?•What is the seasonal pattern?•Do these vary with source of wastewater?•How much is the concentration reduced by the 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 devolpmental 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.