BI 242 Environmental Science Environmental Endocrine Disruptors EDCs

BI 242 Environmental Science Environmental Endocrine Disruptors (EDC’s) Dr. Joseph Colosi De. Sales University

The Endocrine system General Features of the endocrine system: Transport Gland Hormone Target Cell rich blood supply secreted into the blood ductless C. Corbitt can reach virtually every cell in the body hormone receptors are very specific

Endocrine Glands don’t forget the heart, placenta, fat All of these glands produce hormones and are also targets for hormones C. Coebitt

Steroid hormones Insulin, a protein hormone

There are many hormones Link to diagram showing locations of the endocrine glands. Thyroid-stimulating hormone (TSH)protein (201)Anterior lobe of pituitary. Follicle-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 pituitary. Oxytocinpeptide (9)Thyrotropin-releasing hormone (TRH)peptide (3)Hypothalamus. Gonadotropin-releasing hormone (Gn. RH)peptide (10)Growth hormone-releasing hormone (GHRH)peptides (40)Corticotropin-releasing hormone (CRH)peptide (41)Somatostatinpeptides (14, 28)Dopamine. Tyrosine derivative. Melatonin. Tryptophan derivative. Pineal gland. Thyroxine (T 4)Tyrosine derivative. Thyroid Gland. Calcitoninpeptide (32)Parathyroid hormone (PTH)protein (84)Parathyroid glands. Glucocorticoids (e. g. , cortisol)steroids. Adrenal cortex. Mineralocorticoids (e. g. , aldosterone)steroids. Androgens (e. g. , testosterone)steroids. Adrenaline (epinephrine)Tyrosine derivative. Adrenal medulla. Noradrenaline (norepinephrine)Tyrosine derivative. Estrogens (e. g. , estradiol)steroid. Ovarian follicle. Progesteronesteroid. Corpus luteum and placenta. Human chorionic gonadotropin (HCG)protein (237)Trophoblast and placenta. Androgens (e. g. , testosterone)steroid. Testes. Insulinprotein (51)Pancreas (Islets of Langerhans)Glucagonpeptide (29)Somatostatinpeptides (14, 28)Amylinpeptide (37)Erythropoietin (EPO)protein (166)Kidney. Calcitriolsteroid derivative. Calciferol (vitamin D 3)steroid derivative. Skin. Atrial-natriuretic peptide (ANP)peptides (28, 32)Heart. Gastrinpeptides (14)Stomach and intestine. Secretinpeptide (27)Cholecystokinin (CCK)peptides (8)Somatostatinpeptides (14, 28)Neuropeptide Ypeptide (36)Ghrelinpeptide (28)PYY 3 -36 peptide (34)Insulin-like growth factor-1 (IGF-1)protein (70)Liver. Angiotensinogenprotein. Thrombopoietinprotein (332)Leptinprotein. Fat cells. Note (1): Numbers within parentheses indicate the number of amino acids in the protein or peptide(s).

Types of hormone receptors HORMONE Plasma Membrane Receptors Protein hormones R R Enzyme effector R G-protein Enzyme effector Ion channel R Nuclear membrane Protein product Intracellular Receptors Steroid hormones R DNA binding sites NUCLEUS CELL MEMBRANE C. Corbitt

The H-P-G Axis HYPOTHALAMUS - Gn. RH - PITUITARY - - LH TESTIS Testosterone C. Corbitt = negative feedback

It’s actually much more complicated C. corbitt

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 100 X greater risk for clear cell adenocarcinoma, from 0 to 1/1000 2. 5 X greater chance of miscarriage 2 to 3 X greater chance of ectopic pregnancy 33% increase in infertility 25% increase in premature delivery

DES Outcomes (http: //www. cdc. gov/des/consumers/) DES Sons • 4 X increase in non-cancerous epidymal cysts • 3 to 4 X increase in genital deformity? DES Grandchildren ?

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

USGS Water-quality survey, 1999 -2000 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. Survey study 2002 http: //pubs. acs. org/hotartcl/est/es 011055 j_rev. html New study on pesticides 2006 http: //pubs. usgs. gov/fs/2006/3028/ Insect repellant Flame retardant antibacterial surfactant Fungicide surfactant plasticizer

USGS Water-quality data, 1999 -2000

Detection limits for USGS study PPB PPT

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

Compounds known to bind steroid receptors 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. Pesticides Vinclozolin Alachlor Atrazine Cyanazine Desiopropyl Simazine Prometryne o, p’-DDD p, p’-DDE o, p’-DDT p, p’-DDT DDOH 2, 4 -Dichlorophenol Chlordecone (Kepone) Dicofol Endosolfan sulfate -Endosolfan Methoxychlor cis-Nonachlor trans-Nonachlor Toxaphene Industrial chemicals 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 4 -Nonylphenoxy carboxylic acid 4 -tert-Octylphenol -Phenylcresol Tetrahydronaphthol-2 3, 4, 3’, 4’-tetrachloro biphenyl Phytoestrogens 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) 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 R. Bolen

Industrial chemicals R. Bolen

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 • Bolen all have resulted in population decline. R.

Reproductive and developmental abnormalities attributed to endocrine disruption 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, Ah. R agonists mosquito fish abnormal expression of secondary sex characteristics, masculinization androstenedione R. Bolen from Mc. Lachlan (2001)

How Do Environmental EDC’s Work? Mc. Lachlan, J. A. 2001 Environmental Signaling: What Embryos and Evolution Teach Us About Endocrine Disrupting Chemicals, Endocrine Reviews 22(3): 319 -341.

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 humans caused 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. R. Bolen

Premature 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 23 -month-old Puerto Rican girl with premature thelarche Several causes hypothesized: diet, EDCs Plasma phthalate ester levels correlated with premature thelarche cases Phthalate esters used as plasticizers; have estrogenic or anti-androgenic activity in vitro 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 But, Mc. Kee, RH. 2004. Phthalate exposure and early thelarche. Environ. Health Perspect. 112: A 541 -A 543. Questions the results because the blood levels are too high and phthalates do not act like estrogens in female rodents C. Corbitt

. August 2005 Environmental Health Perspectives 113: 1056 -1061 penis Anogenital distance AGD anus AGI = AGD/weight

What is EPA doing about it?

EPA Screening protocols being tested Tier 1 Screening Tier 2 Testing Assays 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. Will 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 20 most consumed foods for pesticides http: //www. epa. gov/fedrgstr/EPA-TOX/2005/September/Day-27/t 19260. 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 Compounds Estrogen-inducible expression system in yeast Sensitive to 10 PPT 17 -estradiol 1 part per trillion is one second in 32 thousand years. ONPG (Clear) 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. Yellow 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

PPB PPT

Witters et al. (2001) Method for Extraction of Estrogenic Compounds from Wastewater Revised method 0. 2μm sterile filter 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.

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

For further study: • 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?

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.