Daniel Schlenk, Ph.D.
University of California Riverside, Department
of Environmental Sciences
Evaluation of Endocrine Dis...
Thanks
• EDWG-Southern California,
Coastal Water Research
Project (SCCWRP)
– City of LA
– City of San Diego
– LACSD
– OCSD...
Environmental Estrogens
Natural Products POPs Pharmaceuticals Industrial
Compounds
Genistein DDT Ethinyl estradiol Bisphen...
Mechanisms of Feminization
• Direct Acting
– Receptor Based (ER)
• Indirect Acting
– Estradiol synthesis
• Central-HPG axi...
Exposure
Absorption
Storage Distribution Target
Biotransformation
Elimination
Pharmacodynamic Dispositional Processes
Hiramatsu et al. 2005
Potential Spatial and Temporal
Impacts of Environmental Stressors
Adams et al. 2002
0
2
4
6
8
10
12
14
T11
(female)
T11 T1
(outfall)
T0 T6 T14
Sampling Site
VitellogeninExpression(ODU)
Hornyhead Turbot
Engl...
Proportion of plasma vitellogenin levels at given
concentrations in hornyhead turbot
0%
10%
20%
30%
40%
50%
60%
70%
80%
90...
Comparison of male hornyhead turbot plasma
vitellogenin levels to average vitellogenic
female levels
0
1
2
3
4
5
6
7
8
9
V...
Plasma estradiol concentrations (pg/ml) of
hornyhead turbot and English sole collected
from the outfall and reference site...
Regression analysis of sperm DNA damage
(expressed as tail moment) versus estradiol
plasma levels in male hornyhead turbot...
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
...
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-...
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-D...
Developmental Stages of Males
1 3 4
5 6
Ova-testis
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-D...
Ova-testis
Site Feb May Aug Nov
DP 0
0
0
0 0 0
O 1 0 0 1
O 11 0 1 2
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 D...
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...
Proportion of hornyhead turbot and English sole
males collected at the outfall and reference site
1988 to 2004
0
10
20
30
...
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
hornyh...
Summary and Conclusions
• Estrogenic activity is present in demersal flatfish
throughout the Southern California Bight.
• ...
Unknowns??
Table 2. E2, E1, AP and APE concentrations (ng/g) of sediments collected from the SCB
in 2002.
< cal= detected by instrume...
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 Ext...
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,...
Oxybenzone
• Historical Background
• New York City wastewater
– Bioassay guided
fractionation for estrogenic
activity
– 19...
Results
• Results showed significant difference in vitellogenin level in the
oxybenzone exposed fish at 1000ppb.
• P-value...
Summary and Conclusion-Cont.
• In vivo bioassays can be used for the determination of matrix estrogenic
activity and ident...
Questions??
Mechanisms of Feminization
Mechanisms of Feminization
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Mechanisms of Feminization

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Mechanisms of Feminization

  1. 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. 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. 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. 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. 5. Exposure Absorption Storage Distribution Target Biotransformation Elimination Pharmacodynamic Dispositional Processes
  6. 6. Hiramatsu et al. 2005
  7. 7. Potential Spatial and Temporal Impacts of Environmental Stressors Adams et al. 2002
  8. 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. 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. 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. 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. 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. 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. 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. 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. 16. Developmental Stages of Males 1 3 4 5 6 Ova-testis
  17. 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. 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. 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. 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. 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. 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. 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. 24. Unknowns??
  25. 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. 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. 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. 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. 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. 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. 31. Questions??
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