First International Congress on Science and Technology October 28, 2005

1. First International Congress on Science and TechnologyOctober 28, 2005 Environmental Endocrine Disruptors Dr. Joseph Colosi Dr. Arthur Kney DeSales University Lafayette College

2. Symantic Confusion • Endocrine disrupting chemicals (EDCs) • Hormonally-active agents • Environmental estrogens • Environmental hormones • Environmental chemicals • Environmental signaling • Xenoestrogens • Gender benders C. Corbitt

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

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

5. There are many 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).

6. Hormone Action in Cells

8. Do exogenous hormones affect people?

9. 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

10. Diethylstilbestrol (DES) • 1954 DES put in chicken and cattle feed3/4 • 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

11. 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

12. 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 ?

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

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

15. USGS Water-quality data, 1999-2000

16. Compounds known to bind steroid receptors in humans and animalsRooney, 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

17. 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.

18. Chemicals Found in the Environment Reported 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.

19. Chemicals Found in the Environment Reported 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.

20. Chemicals Found in the Environment Reported 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.

21. Every year, 5000 new chemical compounds are introduced. “Better living through chemistry.”

22. How do EDC’s get into the environment?

23. What it takes to be beautiful today.

25. Bethlehem Sewage Treatment Plant

27. Plastics R. Bolen

28. Phytoestrogens, especially from legumes

29. Sheep feedlot Cattle feedlot

30. Pesticides R. Bolen

31. Industrial chemicals R. Bolen

32. Do environmental EDC’s affect wildlife?

33. Reproductive and developmental abnormalities attributed to endocrine disruption R. Bolen from McLachlan (2001)

34. How Do Environmental EDC’s affect the endocrine system? McLachlan, J.A. 2001 Environmental Signaling: What Embryos and Evolution Teach Us About Endocrine Disrupting Chemicals, Endocrine Reviews 22(3): 319-341.

35. Do EDC’s in the environment affect humans?

36. from Sharpe and Irvine (2004) Trends in reproductive health

37. 23-month-old Puerto Rican girl with premature thelarche Colon et al (2000) 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 Plasma phthalate ester levels correlated with premature thelarche cases Phthalate esters used as plasticizers; have estrogenic or anti-androgenic activity in vitro C. Corbitt

38. 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

39. What is EPA doing about it?

40. EPA Screening protocols being tested

42. 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

43. Yeast Bioassay for Estrogenic CompoundsEstrogen-inducible expression system in yeast Lac Z operon reporter ONPG (Clear) Sensitive to 1 PPT 17 -estradiol 1 part per trillion is one second in 32 thousand years 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

44. PPB PPT

47. For further study: • 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?

48. 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.

49. Detection of estrogenic compounds in wastewater using a modified transgenic yeast assay • Five steps in detecting environmental estrogens with transgenic yeast • Obtain samples and filter to remove microorganisms. • 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 • Lyse cells, add ONPG substrate and incubate 2 hr. at 37C. • Read color development with spectrophotometer. • Compare OD405 values to those of standard curve.

50. 1 3 2 4 Sterile sample 1 Sterile sample 3 Sterile sample 2 Sterile sample 4 1. Obtain samples and filter to remove microorganisms. All samples collected October 27, 2005 Sample 1 Bottled water Sample 2 Tap water Sample 3 River water Sample 4 Raw sewage