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Mechanisms of Feminization

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  • 1. Daniel Schlenk, Ph.D. University of California Riverside, Department of Environmental Sciences Evaluation of Endocrine Disruption in Benthic Flatfish of Southern California Collected Near A Publicly Owned Treatment Works Outfall
  • 2. Thanks • EDWG-Southern California, Coastal Water Research Project (SCCWRP) – City of LA – City of San Diego – LACSD – OCSD – CSULB – UCSD • Orange County Sanitation District • CEQI-UC-Marine Council • Xin Deng • Mary Ann Irwin-Rempel • Jeff Armstrong • Michael Coronado • Bruce Brownawell • Sharanya Reddy • David Sedlak • Ed Kolodziej • Shane Snyder
  • 3. Environmental Estrogens Natural Products POPs Pharmaceuticals Industrial Compounds Genistein DDT Ethinyl estradiol Bisphenol A Naringenin Kepone DES Phthalate esters Coumestrol PAHs Alkylphenols Zearalenone PCBs/OH-PCBs Endosulphan B-sitosterol Dioxins Triazines
  • 4. Mechanisms of Feminization • Direct Acting – Receptor Based (ER) • Indirect Acting – Estradiol synthesis • Central-HPG axis (gonadotropin, aromatase) – Anti-androgens – Estradiol elimination • CYP or Phase II inhibition
  • 5. Exposure Absorption Storage Distribution Target Biotransformation Elimination Pharmacodynamic Dispositional Processes
  • 6. Hiramatsu et al. 2005
  • 7. Potential Spatial and Temporal Impacts of Environmental Stressors Adams et al. 2002
  • 8. 0 2 4 6 8 10 12 14 T11 (female) T11 T1 (outfall) T0 T6 T14 Sampling Site VitellogeninExpression(ODU) Hornyhead Turbot English Sole Bigmouth Sole Vitellogenin Expression in Demersal Flatfish of So. Cal Bight 0.42 ng/g 0.048 ng/g Roy et al. 2003
  • 9. Proportion of plasma vitellogenin levels at given concentrations in hornyhead turbot 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Outfall July '03 n=5 Outfall Jan '04 n=7 Outfall Aug '04 n=10 Reference Jan '04 n=54 Sampling Site and Period Percentage >0.45 ng/ug 0.31-0.45 ng/ug 0.15-0.30 ng/ug <0.15 ng/ug Rempel et al. 2006
  • 10. Comparison of male hornyhead turbot plasma vitellogenin levels to average vitellogenic female levels 0 1 2 3 4 5 6 7 8 9 Vitellogenic Female July '03 January '04 August '04 ngVitellogenin/ugProtein Outfall Reference Female Rempel et al. 2006
  • 11. Plasma estradiol concentrations (pg/ml) of hornyhead turbot and English sole collected from the outfall and reference sites Outfall Reference Male Female Male Female January 2003 201.5±3.7 (n=5) 206.6±6.4 (n=4) 208.0±8.2 (n=5) 212.9 (n=1) January 2004 329.9±255.6 (n=7) 302.6±165.2 (n=5) 388.6±215.0 (n=17) 245.0±143.3 (n=2) January 2003 191.4±2.8 (n=2) 184.0±1.1 (n=3) 205.2±10.3 (n=4) 200.5 (n=1) January 2004 142.1±206.3 (n=8) 209.4±282.3 (n=11) 169.4±92.0 (n=12) 108.9±94.5 (n=6) English Sole Hornyhead Turbot Species Sampling Period Rempel et al. 2006
  • 12. Regression analysis of sperm DNA damage (expressed as tail moment) versus estradiol plasma levels in male hornyhead turbot from the outfall R2 = 0.712 0 100 200 300 400 500 600 700 800 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Tail Moment Estradiol(pg/ml) Rempel et al. 2006 p < 0.05
  • 13. Males with detected Vtg levels Month Site Number of Males Number of Males with Vtg induction % Jan DP O1 O11 19 18 20 0 3 6 0 15 33 May DP O1 O11 31 24 5 5 6 0 16 25 0 August DP O1 O11 20 10 20 2 9 14 10 90 70 November DP O1 O11 23 18 23 0 0 0 0 0 0 Total DP O1 O11 74 52 48 7 15 14 9.5 29 29
  • 14. Vitellogenin levels--Males Mean Mean¡ ÀSE Mean¡ ÀSD Feb-DP Feb-O11 Feb-O1 May-DP May-O1 May-O11 Aug-DP Aug-O1 Aug-O11 Nov-DP Nov-O1 Nov-O11 Month & Site -0.04 0.00 0.04 0.08 0.12 0.16 0.20 0.24 Vtg(ng/ugprotein) a a a a a a ab* b b b b* a Wilcoxon / Kruskal-Wallis Tests P < 0.0001, different letters denote significantly seasonal difference. * indicates significant site difference of the same season.
  • 15. Gonadosomatic Index--Male Mean Mean¡ ÀSE Mean¡ ÀSD Feb-DP Feb-O11 Feb-O1 May-DP May-O1 May-O11 Aug-DP Aug-O1 Aug-O11 Nov-DP Nov-O1 Nov-O11 Month & Site -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 1/2GSI(%bodyweight) a a ba ANOVA, P< 0.0001, different letters denote significantly seasonal difference.
  • 16. Developmental Stages of Males 1 3 4 5 6 Ova-testis
  • 17. Developmental Stages-Male(1) 2024 1920 185 2331 23 0 % 1 0 % 2 0 % 3 0 % 4 0 % 5 0 % 6 0 % 7 0 % 8 0 % 9 0 % 1 0 0 % Feb-DP Feb-O1 Feb-O11 May-DP May-O1 May-O11 Aug-DP Aug-O1 Aug-O11 Nov-DP Nov-O1 Nov-O11 St age 3 St age 2 St age 1 19 18 20 Chi-square test, P<0.0001
  • 18. Ova-testis Site Feb May Aug Nov DP 0 0 0 0 0 0 O 1 0 0 1 O 11 0 1 2
  • 19. Sex Ratio (M:F) in 2006-07 M onth Novem ber February May August Novem ber January April GenderRatio(M:F) 0 1 2 3 4 12 14 DP O1 O11 a aa ab b a b a b a b c
  • 20. Sex Ratio (M:F) at O1 and O11(2003-2006) Month Jan/Feb May July/Aug Nov SeaRatio(M:F) 0 1 2 5 10 15 20 0 1 2 5 10 15 20 O1 O11* *
  • 21. Proportion of hornyhead turbot and English sole males collected at the outfall and reference site 1988 to 2004 0 10 20 30 40 50 60 70 80 90 100 1985 1990 1995 2000 2005 Year ofSampling PercentageofMales Hornyhead Turbot From Outfall Hornyhead Turbot From Reference EnglishSole FromOutfall EnglishSole From Reference Expected
  • 22. Abundance of 3 Flatfish Species Collected Near the outfall of OCSD (T-1) 1986-2001 0 10 20 30 40 50 60 70 80 90 100 hornyhead bigmouth English CPU
  • 23. Summary and Conclusions • Estrogenic activity is present in demersal flatfish throughout the Southern California Bight. • Three histological lesions indicative of feminization in 2006-2007 samples • No significant effects on developmental stage • Populations tend to display greater than 50% males (seasonal) (2:1) with no relationship to Vtg, histopath endpoints, or overall abundance
  • 24. Unknowns??
  • 25. Table 2. E2, E1, AP and APE concentrations (ng/g) of sediments collected from the SCB in 2002. < cal= detected by instrument, but below calibration curve. LAC SD OC Reference E1 0.6 < 0.03 < 0.03 < 0.03 E2 0.3 0.3 0.45 0.16 NP 198 122 3200 130 NPCl <DL <DL 100 <DL NPBr <DL <DL <DL <DL NP 1EO 36 11.6 330 76 NP 2EO 19.2 3.2 600 92 NP 3EO 15.6 1.9 3900 92 OP 8.2 1.9 <DL <DL OP 1EO <DL <DL <cal 21 OP 2EO <DL <DL <cal 8 OP 3EO <DL <DL 42 5.8 Schlenk et al. 2005
  • 26. Bioassay guided fractionation of sediment extracts from LAC and OC 0 2 4 6 8 10 12 14 16 18 10% 25% 50% 75% 100% Crude Extract 0 2 4 6 8 10 12 14 16 18 10% 25% 50% 75% 100% Crude Extract LAC OC VtgExpressionng/mg E2 NPs OCs Schlenk et al. 2005
  • 27. Minutes 0 5 10 15 20 25 30 35 40 Volts 0.000.050.100.150.20 Detector A (254nm) 75%fr 081704 EtOH081704fr.dat 254nm OCs E2, NPs EEQ Schlenk et al. 2005
  • 28. Oxybenzone • Historical Background • New York City wastewater – Bioassay guided fractionation for estrogenic activity – 19 ng/L oxybenzone • LA County Sanitation District (LACSD) Sediment extracts – Bioassay guided fractionation for estrogenic activity – 8 ng/g oxybenzone O OH O
  • 29. Results • Results showed significant difference in vitellogenin level in the oxybenzone exposed fish at 1000ppb. • P-value < .01 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Vitellogenin Concentration Acetone Control Oxybenzone 10ppb Oxybenzone 100ppb Oxybenzone 1000ppb Positive Control * Vtgng/µgplasmaprotein Effect of Oxybenzone on the Expression of Rainbow Trout Vitellogenin after 14 day Aqueous Exposure. PPB = µg/L
  • 30. Summary and Conclusion-Cont. • In vivo bioassays can be used for the determination of matrix estrogenic activity and identification of estrogenic compounds – In vitro responses tend to underestimate “total” estrogenic activity (Industrial input) – Should be used in concert with cell-line screening and AC if possible • Estrogenic compounds (known and unknown) are present in sediments near SC municipal facilities at concentrations that elicit feminizing effects in laboratory fish – Acute responses do not appear to be due to steroidal exposures but likely due to exposures to multiple compounds at < ppb concentrations – Chronic responses??
  • 31. Questions??