Biomarkers Indicators (measures) of exposure to a chemical or class of chemicals

1. Biomarkers Indicators (measures) of exposure to a chemical or class of chemicals Not generally indicators of effects Vitellogenin (phospholipoglycoprotein) is an egg yolk precursor produced in the liver of females during egg production. Males have the ability to produce egg yolk protein but generally never receive an internal signal to do so. The production of vitellogenin in male fish has been used as a biomarker of exposure to chemicals in the environment that produce estrogenic responses (xenoestrogens).

2. Estrogen (estradiol) and its metabolites (listed in order of their potency) estrone, and estriol have all been shown to be capable of inducing the production of vitellogenin in male fish. Ethynyl estradiol is a synthetic estrogen that is used in the Pill. It too has been shown to be capable of inducing the production of vitellogenin in male fish.

3. Should we care that male fish have elevated levels of VTG?

4. Individual Born Lives Reproduces Dies Death rate < Birth rate Population Increases Ova Population Persists Total Fertility 2.1 ? Population Decreases Sperm Death rate > Birth rate

5. Two systems control all physiological processes The nervous systemexerts point-to-point control through nerves, similar to sending conventional messages by telephone (hard wired). The endocrine systembroadcasts its hormonal messages to essentially all cells by secretion into blood and extracellular fluid. Like a radio broadcast it requires a receiver (cells must have a receptor for the hormone being broadcast). These two system often act together

6. Endocrinology is the study of hormones, their receptors and the intracellular signaling pathways they invoke. Distinctendocrine organs are scattered throughout the body. HypothalamusPituitaryThyroid glands Parathyroid glandsPancreasAdrenal glands Testes Ovaries In addition to the classical endocrine organs, many other cells secrete hormones, through what is sometimes called the “diffuse” endocrine system.

7. All pathophysiological events are influenced by the endocrine milieu All “large” physiologic effects are mediated by multiple hormones acting in concert There are many hormones known and little doubt that others remain to be discovered.

8. What exactly are hormones and how are they different from “non-hormones”? Hormones are chemicalmessengers secreted into blood or extracellular fluid by one cell that affect the functioning of other cells. Most hormones circulate in the blood, coming into contact with essentially all cells. However, a given hormone usually affects only a limited number of cells, which are called target cells. Target cells respond to the hormones because they have receptors for the hormone. Hormone receptors are found either exposed on thesurfaceof the cell or within the cell depending on the type of hormone. In very basic terms binding of hormone to receptor triggers a cascade of reactions within the cell that affects function.

9. Two important terms are used to refer to molecules that bind to the hormone-binding sites of receptors: Agonists are molecules that bind the receptor and induce all the post-receptor events that lead to a biologic event. In other words they act like the “normal” hormone. Antagonists are molecules that bind the receptor and block binding of the agonist, but fail to trigger intracellular signaling events. (Antagonists are like certain types of bureaucrats – they don’t themselves perform useful work, but block the activities of those that do have the capacity to contribute.) Hormone antagonists are widely used as drugs (e.g. statins).

10. Atorvastatin (Lipitor) Fluvastatin (Lescol) Lovastatin (Mevacor) Pravastatin (Pravachol) Simvastatin (Zocor) Rosuvastatin (Crestor) The statin drugs inhibit HMG CoA reductase in the liver and prevent the formation of cholesterol. Lipitor, followed closely by Zocor was the most frequently prescribed drug in the United States. All have the same MOA and probably act additively

11. Normal response Abnormal Response No Response Dormant receptor Receptor Blocked turned on by mimic Normal receptor over stimulated by mimic Timing Timing Timing

12. Endocrine System regulates biological processes • Growth and function of reproductive system (androgen [testosterone], estrogen [estradiol], and related compounds from gonads) • Control of blood sugar (pancreatic insulin) • Regulation of metabolism (adrenal cortisol and thyroid thyroxin) • Development of nervous system including brain (estrogen and thyroid hormones) • Overall development from conception to old age = homeostasis

13. Hormone Classes • Steroids : derived from cholesterol • androgens and estrogens and cortisol • Amines : synthesized from amino acids • give rise to adrenaline and noradrenaline • Peptides and proteins : a.a. chains • growth hormone • Eicosanoids - 20 carbon fatty acid derivatives • prostoglandins = adenyl cyclase activator

14. Receptor Types • Cell membrane : peptide hormones • Cytoplasmic : steroids • Nuclear receptors : thyroid hormones • a cell may contain as many as 10,000 receptors for a single hormone • ~ 50-100 genes may be controlled by a single hormone

15. Specific Case Example Fish Response to Wastewater Effluent Estrogenicity

16. Estrogen Mimics • Intended disruptors • DES (diethylstilbestrol) • ethynylestradiol • phytoestrogens (soy protein, soy milk)

17. Estrogen Mimics • Unintended disruptors • nonyl/octylphenol • commercial and domestic detergents • phthalate & Bisphenol A • plastics : bottles and liners • DDT (DDE) • PCBs

18. Estrogen Mimics : promiscuous receptor Estradiol Intended disruptors Ethynylestradiol DES

19. Estradiol Unintended disruptors -C9H19 Bisphenol A Polycarbonate plastic DDT DEHP Di(2-ethylhexyl) phthalate Nonylphenol Estrogen Mimics : promiscuous receptor

20. Gonad Vitellogenin Biomarker : fish model Endogenous / Exogenous Estrogens Vitellogenin Liver Vitellogenin – egg yolk precursor produced in the liver

21. Pilot Study Methods • Adult male fathead minnows (n=5/exposure) • 1-3 Weeks of exposure to 100% WWTP • RHW control in lab • Plasma VTG content via ELISA • Likelihood Ratio Test • ND left censored at 3,000 ng/ml

22. Pilot Study Results Number of male fish with detectable VTG levels / site with 5 fish Non-detects were left censored at 3,000 ng/ml = DL.

23. Pilot Study ResultsMean plasma vitellogenin concentrations (ng/ml) * * *Statistical significance from the controls (=0.05)

24. Comparison to Similar StudiesMean plasma vitellogenin concentrations (ng/ml) in fish exposed to wastewater effluent receiving systems.

25. Conclusions • Induction of vitellogenesis in male fish indicates the presence of estrogenic components in the wastewater effluent tested. • Vitellogenin concentrations increased and were more frequent with increased exposure duration. • Vitellogenin concentrations in male fish decreased when UNT and TWU were not in session.

26. Additional Research • Assessment of the efficacy of a constructed wetland to reduce or remove wastewater effluent estrogenicity using the vitellogenin biomarker in fathead minnows (Pimephales promelas Rafinesque, 1820). • wetland vegetative coverage and degradation activity • suspect chemical constituents • estradiol and ethynylestradiol vs • DEHP, DDT, Bisphenol A, Nonylphenol

27. 2 1 4 3 In Out Replicate minnow traps (seven male fish per trap) Wetland Exposure Design

28. Fish Measurements • Vitellogenin Content of Plasma • GSI – Gonado Somatic Index testes wt/somatic wt x 100 • HSI – Hepato Somatic Index liver wt/total body wt x 100 • Hematocrit (%packed blood cells) • Secondary Sex Characters (turbercles, fat pad, stripes) • Length • Weight • Gonadal Differentiation

29. Estradiol Chemical Analysis Ethynylestradiol -C9H19 Bisphenol A DDT DEHP Nonylphenol

30. Wetland Characterization • vegetation types and density • depth and width of channels • retention time as estimated by input flow • relative to chemical constituents & fish response

31. Fish health as measured by condition factor (K) and hematocrit values were significantly reduced at wetland sites 1 and 2. A negative trend was observed between VTG concentration and condition factor among wetland sites. A positive trend was observed between condition factor and hematocrit value. Gonadosomatic index was significantly reduced at site 1 in the wetland while the hepatosomatic index was significantly increased. Tubercle number, fatpad thickness, and stripe density were all reduced at site 1.

32. Vitellogenin concentrations in fish from site 1 were significantly elevated compared to control and other wetland sites.The constructed wetland significantly reduced VTG levels in fish and increased measures of fish well-being. As population continues to increase and pressures on available freshwater in our area increased use of “reclaimed water” will become more and more important. One of the ways to reclaim the water is to run Trinity River water through a constructed wet land and then reintroduce it into our reservoirs.

33. Vitellogenin Gene Expression in Fathead Minnows Exposed to EE2 in a Whole Lake Dosing Experiment Greg Toth *Lazorchak, JM1, Flick, R1, Lattier, D.L.1, 1 U.S. EPA, National Exposure Research Laboratory, Cincinnati, OH Kidd, K2, Palace, V2, Evans, B2 , Blanchfield, P2, Mills, K2 , Hodge, T2,. 2 Canadian Division of Fisheries and Oceans, Freshwater Institute, Winnipeg, Manitoba, Smith, ME3, Wiechman, B3,, 3 Sobran Inc., c/o U.S. EPA, Cincinnati, OH.,

34. Why look at 17a-ethynylestradiol (EE2) • effective component of birth control pills • potent estrogen mimic • 70-80 % degraded in sewage treatment • found at significant and effective concentrations downstream of municipal wastewater treatment plants K. Kidd

35. Why Work with the Canadian DFO Fathead Endpoints • Vitellogenin (spring, mid-summer, fall) V. Palace • Liver, kidney and gonad development, GSI and LSI (spring & fall) B. Evans • Secondary sex characteristics (mid-summer) P. Blanchfield • Male reproductive behaviour (mid-summer) P. Blanchfield • Nest size and egg development (mid-summer) P. Blanchfield • Population size structure, growth, abundance (spring and fall) K. Mills K. Kidd

36. Located in northwestern Ontario approximately 250 km east of Winnipeg and 50 km east-southeast of Kenora. K. Kidd

40. Lake 260 – Ethynylestradiol (EE2) Addition Lake • 34 ha in surface area • Max. depth 14 m • Outflow into lake that is • 30 times larger • Contains well-defined • populations of lake • trout, white sucker, • fathead minnow and • pearl dace • Long-term records on • plankton and water quality

41. Study Design recovery? effects on individuals & populations ethynylestradiol additions baseline data 2004 1999 2000 2001 2002 2003 2005 2006 reference lake data

42. Additions of EE2 to Lake 260, 2001-2003 • EE2 added 3 times a week for 5 months • 100-450 mg added/day to maintain • constant concentration (4.5% loss/day) • Season mean of 6.1 (2.9) and 5.0 (1.8) • ng/L in surface waters (SPE and RIA) water sampling K. Kidd

44. Epilimnion EE2 = 6.1+/- 1.3 ng/L Metalimnion 1.9 +/- 0.93 ng/L Hypolimnion 1.7 +/- 0.61 ng/L Concentrations of EE2 in Stratified Lake 260

45. U.S. EPA MERB 2001 Objectives • Evaluate exposure of indigenous male fathead minnows in Lake 260 to ethynylestradiol using vitellogenin gene expression • Evaluate short-term exposure of male fathead minnows from Lake 114 deployed in Lake 260 • Evaluate exposure of Cincinnati male fathead minnows to Lake 260 water at the US EPA facility in Cincinnati • Evaluate exposure of Cincinnati fathead minnow fry to sediment from Lake 260

46. Approach - Indigenous Male Fathead Minnows • Male Fathead Minnows were collected from Lake 260 after 7 weeks, 9 weeks and 12 weeks of dosing. • Male minnows were collected from Lake 114 at the same timepoints. • Livers were collected. • RT-PCR was performed on samples and vitellogenin expression quantified relative to 18s ribosomal RNA expression.

47. 2001 Semi-quantitative PCRGel Based

48. 2001 Results of Indigenous Male Fathead Minnows

49. EPA MERB Approach - Deployment Study • Fish were collected in Lake 114 using minnow traps two days before deployment. • On day of deployment males were separated from females and placed in cages in Lakes 114 and 260. • Minnows were retrieved from cages on days 1, 3, 7 and 13. • Livers were collected and RT-PCR performed.

50. 2001 Results of 13-day Deployment Study