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Mechanisms of Feminization

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Mechanisms of Feminization Presentation Transcript

  • 1. Evaluation of Endocrine Disruption in Benthic Flatfish of Southern California Collected Near A Publicly Owned Treatment Works Outfall Daniel Schlenk, Ph.D.University of California Riverside, Department of Environmental Sciences
  • 2. Thanks • EDWG-Southern California,• Xin Deng Coastal Water Research• Mary Ann Irwin-Rempel Project (SCCWRP) – City of LA• Jeff Armstrong – City of San Diego• Michael Coronado – LACSD• Bruce Brownawell – OCSD• Sharanya Reddy – CSULB• David Sedlak – UCSD• Ed Kolodziej • Orange County Sanitation• Shane Snyder District • CEQI-UC-Marine Council
  • 3. Environmental EstrogensNatural 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. Pharmacodynamic Dispositional Processes Exposure Elimination AbsorptionStorage Distribution Target Biotransformation
  • 6. Hiramatsu et al. 2005
  • 7. Potential Spatial and TemporalImpacts of Environmental Stressors Adams et al. 2002
  • 8. Vitellogenin Expression in Demersal Flatfish of So. Cal Bight Vitellogenin Expression (ODU) Hornyhead Turbot 0.42 ng/g 14 English Sole Bigmouth Sole 12 10 8 0.048 ng/g 6 4 2 0 T11 T11 T1 T0 T6 T14 (female) (outfall) Sampling Site Roy et al. 2003
  • 9. Proportion of plasma vitellogenin levels at given concentrations in hornyhead turbot 100% 90% 80% 70% >0.45 ng/ug Percentage 60% 0.31-0.45 ng/ug 50% 0.15-0.30 ng/ug 40% <0.15 ng/ug 30% 20% 10% 0% Outfall Outfall Outfall Reference July 03 n=5 Jan 04 n=7 Aug 04 Jan 04 n=10 n=54 Sam pling Site and Period Rempel et al. 2006
  • 10. Comparison of male hornyhead turbot plasma vitellogenin levels to average vitellogenic female levels 9 8 ng Vitellogenin / ug Protein 7 6 Outfall 5 Reference 4 Female 3 2 1 0 Vitellogenic July 03 January 04 August 04 Female Rempel et al. 2006
  • 11. Plasma estradiol concentrations (pg/ml) ofhornyhead turbot and English sole collected from the outfall and reference sites Sampling Outfall Reference Species Period Male Female Male Female January 201.5±3.7 206.6±6.4 208.0±8.2 212.9 Hornyhead 2003 (n=5) (n=4) (n=5) (n=1) Turbot January 329.9±255.6 302.6±165.2 388.6±215.0 245.0±143.3 2004 (n=7) (n=5) (n=17) (n=2) January 191.4±2.8 184.0±1.1 205.2±10.3 200.5 English 2003 (n=2) (n=3) (n=4) (n=1) Sole January 142.1±206.3 209.4±282.3 169.4±92.0 108.9±94.5 2004 (n=8) (n=11) (n=12) (n=6) Rempel et al. 2006
  • 12. Regression analysis of sperm DNA damage(expressed as tail moment) versus estradiolplasma levels in male hornyhead turbot from the outfall 800 700 R2 = 0.712 600 Estradiol (pg/ml) 500 400 300 200 100 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Tail Moment Rempel et al. 2006 p < 0.05
  • 13. Males with detected Vtg levels Number of Number of Month Site Males with Vtg % Males induction DP 19 0 0 Jan O1 18 3 15 O11 20 6 33 DP 31 5 16 May O1 24 6 25 O11 5 0 0 DP 20 2 10 August O1 10 9 90 O11 20 14 70 DP 23 0 0November O1 18 0 0 O11 23 0 0 DP 74 7 9.5 Total O1 52 15 29 O11 48 14 29
  • 14. Vitellogenin levels--Males 0.24 0.20 a a a 0.16 a Vtg (ng/u g pro tein) a a ab* 0.12 a 0.08 b* 0.04 b b b 0.00 -0.04 Feb -DP May-DP Au g-DP Nov-DP Feb-O11 Feb-O1 M ay-O1 May-O11 Aug-O11 Nov-O11 Aug-O1 Nov-O1 Mean Mean¡ S E À Mean¡ S D À Mo nth & SiteWilcoxon / Kruskal-Wallis Tests P < 0.0001, different letters denote significantlyseasonal difference. * indicates significant site difference of the same season.
  • 15. Gonadosomatic Index--Male 0.14 a 0.12 a1/2 GSI (% bo dy weight) 0.10 a b 0.08 0.06 0.04 0.02 0.00 -0.02 Feb -DP May-DP Au g-DP Nov-DP Feb-O11 Feb-O1 M ay-O1 May-O11 Aug-O11 Nov-O11 Aug-O1 Nov-O1 Mean Mean¡ S E À Mean¡ S D À Mo nth & Site ANOVA, P< 0.0001, different letters denote significantly seasonal difference.
  • 16. Developmental Stages of Males1 3 4 Ova-testis5 6
  • 17. Developmental Stages-Male(1) 19 18 20 31 24 5 20 19 20 23 18 23100% 90% 80% 70% 60% St age 3 50% St age 2 40% St age 1 30% 20% 10% 0% Feb- DP ay- DP Aug- DP Nov- DP Feb- O11 ay- O11 Aug- O11 Nov- O11 Feb- O1 ay- O1 Aug- O1 Nov- O1 M M MChi-square test, P<0.0001
  • 18. Ova-testisSite Feb May Aug NovDP 0 0 0 0O1 0 0 0 1O 11 0 0 1 2
  • 19. Sex Ratio (M:F) in 2006-07 c 14 DP O1 O 11Gender Ratio (M:F) 12 4 a a 3 a a a a b 2 b ab b b 1 0 N ovem ber F ebruary M ay A ugust N ovem ber January A pril M o n th
  • 20. Sex Ratio (M:F) at O1 and O11(2003-2006) 20 * 20 15 15Sea Ratio (M:F) 10 10 O1 5 * 5 O11 2 2 1 1 0 0 Jan/Feb May July/Aug Nov Month
  • 21. Proportion of hornyhead turbot and English solemales collected at the outfall and reference site 1988 to 2004 100 Hornyhead Turbot From 90 Outfall Percentage of Males 80 Hornyhead 70 Turbot From 60 Reference 50 English Sole 40 From Outfall 30 20 English Sole 10 From 0 Reference 1985 1990 1995 2000 2005 Expected Year of Sampling
  • 22. Abundance of 3 Flatfish Species Collected Near the outfall of OCSD (T-1) 1986-2001 100 90 80 70 60 hornyhead 50 bigmouthCPU 40 English 30 20 10 0
  • 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 SCBin 2002. 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< cal= detected by instrument, but below calibration curve.
  • 26. Bioassay guided fractionation of sediment extracts from LAC and OC 18 16 14 12 LAC Vtg Expression ng/mg 10 8 6 4 2 0 10% 25% 50% 75% 100% Crude Extract E2 NPs OCs 18 16 14 OC 12 10 8 6 4 2 0 10% 25% 50% 75% 100% Crude Extract Schlenk et al. 2005
  • 27. 0.20 Detector A (254nm) 75%fr 081704 EtOH081704fr.dat 0.15 0.10 Volts 254 nm 0.05 0.00 0 5 10 15 20 25 30 35 40 Minutes OCs E2, NPsEEQ Schlenk et al. 2005
  • 28. Oxybenzone• Historical Background• New York City wastewater – Bioassay guided fractionation for estrogenic activity – 19 ng/L oxybenzone OH O O• LA County Sanitation District (LACSD) Sediment extracts – Bioassay guided fractionation for estrogenic activity – 8 ng/g oxybenzone
  • 29. Effect of Oxybenzone on the Expression of Rainbow Trout Vitellogenin Results Exposure. after 14 day Aqueous• Results showed significant difference in vitellogenin level in the 2 oxybenzone exposed fish at 1000ppb. * 1.8 Acetone Control Vtg ng/µg plasma protein Oxybenzone 10ppb• P-value < .01 Oxybenzone 100ppb 1.6 Oxybenzone 1000ppb 1.4 Positive Control 1.2 PPB = µg/L 1 0.8 0.6 0.4 0.2 0 Vitellogenin Concentration
  • 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??