"From field to lab" and "from lab to field

Transcription

1 "From field to lab" and "from lab to field chemicals, in vitro, in vivo vs. in situ Luděk Bláha 1, Klára Hilscherová 1, Edita Mazurová 1, John P. Giesy 2, Rita Triebskorn 3, Ivan Holoubek 1 RECETOX Research Center for Environmental Chemistry and EcoTOXicology Masaryk University, Brno, CR 2 U of Saskatchewan,, Canada; ; City C University of Hong-Kong, China 3 University of Tübingen, Germany

2 The in situ problem Pilnok reservoir Ostrava-Karvina region Pontastacus leptodactylus INTERSEX: Females w/ male gonopods Males carrying eggs?

3 Major questions / Objectives What is the cause of intersex occurence? Can ED-chemical chemicals be identified? Can the mechanism be understood? Can we induce ED experimentally?

4 Integrated assessment Sediments collected Pilnok Reference localities ies Karvina pond, Steinlach creek Organic extractione In n vivo effects Chemical analyses In vitro effect testing

5 Chemical contamination?

6 Organics (ng/g d.w.) Pilnok Karvina Steinlach Dřevnice Morava 16 PAHs Σ PCBs Σ DDTs High concentrations of PAHs in PILNOK Other POPs (PCBs, OCPs) ~ low-average

7 Metals (μg/g d.w.) Pilnok Karvina Steinlach Pb Cu Zn

8 Bio-TEQs (H4IIE.luc bioassay) 120 AhR-mediated activity % TCDD max induction TCDD calibration Steinlach Pilnok 0 1.0E-2 1.0E-1 1.0E+0 1.0E+1 1.0E+2 ng TCDD/well 1.0E-4 1.0E-3 1.0E-2 1.0E-1 1.0E+0 1.0E+1 Sediment extracts (mg/well)

9 Bio-TEQs (H4IIE.luc bioassay)

10 TEQs + Analytical data (ng( ng/g d.w.) Pilnok Karvina Steinlach Morava 16 PAHs Σ PCBs Σ DDTs Chem-TEQs TEQs* Bio-TEQs TEQs crude Bio-TEQs H 2SO crude <0.005 <0.005 <0.005 * WHO TEFs for PCBs & IEFs for PAHs (Machala et al. 2001)

11 Effects in vitro?

12 Assessment of in vitro effects OH HO ACTION OF STEROID HORMONES P!! ER AhR P hsp90 hsp90 hsp90 hsp90 AhR HO ER RAR OH ER AhR P ER AhR RAR

13 Assessment of in vitro effects Cholesterol SYNTHESIS OF STEROID HORMONES! CYP11A Pregnenolone 3β-HSD Progesterone CYP21 11-Deoxycorticosterone CYP11B2 Corticosterone CYP11B2 Aldosterone Zona glomerulosa CYP17 17α-OH- Pregnenolone 3β-HSD 17α-OH- Progesterone CYP17 CYP21 11-Deoxycortisol CYP11B1 Cortisol Zona fasciculata CYP17 DHEA CYP17 3β-HSD Androstene -dione 17β-HSD Testosterone CYP19 17β-Estradiol Zona reticularis HO OH OH HO ACTION OF STEROID HORMONES P!! ER AhR P hsp90 hsp90 hsp90 hsp90 AhR HO ER RAR OH ER AhR P ER AhR RAR

14 MVLN bioassay 120 Estrogenity (ER-mediated activity) % E2 max induction E2 calibration 0 1.0E-5 1.0E-4 1.0E-3 1.0E-2 1.0E-1 ng E2/well Steinlach Pilnok 1.0E-2 1.0E-1 1.0E+0 1.0E+1 Sediment extracts (mg/well) PILNOK estrogenic Reference sediments - no effects

15 AR-yeast bioassay 120 Antiandrogenity (AR-mediated activity) 100 % T max induction T calibration Steinlach Pilnok E-5 1.0E-3 1.0E-1 1.0E+1 1.0E+3 ng testosterone (T)/well 1.0E-4 1.0E-3 1.0E-2 1.0E-1 1.0E+0 Sediment extracts (mg/well) PILNOK anti-androgenic androgenic Reference sediments - no effects

16 Effects on steroidogenesis Cholesterol CYP11A Pregnenolone 3β-HSD Progesterone CYP21 11-Deoxycorticosterone CYP11B2 Corticosterone CYP11B2 Aldosterone Zona glomerulosa CYP17 CYP17 17α-OH- Pregnenolone 3β-HSD 17α-OH- Progesterone CYP21 11-Deoxycortisol CYP11B1 Cortisol Zona fasciculata CYP17 CYP17 DHEA 3β-HSD Androstene -dione 17β-HSD Testosterone CYP19 17β-Estradiol Zona reticularis - gene modulation (DNA->mRNA mrna) Real Time PCR - protein levels - enzyme activities - hormones produced HO OH H295R cell line bioassay development supported by US EPA grant

17 Pilnok sediment extracts modulate steroidogenesis in H295R cells CYP11A 3βHSD Cells DMSO Crude Acid- Treated Crude Acid- Treated Cells DMSO Crude Acid- Treated Crude Acid- Treated Sed.No.2 Pilnok Sed.No.3 Ref.Sed. Sed.No.2 Pilnok Sed.No.3 Ref.Sed. CYP21 CYP11B Cells DMSO Crude Acid- Treated Crude Acid- Treated Cells DMSO Crude Acid- Treated Crude Acid- Treated Sed.No.2 Pilnok Sed.No.3 Ref.Sed. Pilnok Sed.No.2 Ref.Sed. Sed.No.3

18 Effects on steroidogenesis Cholesterol CYP11A Pregnenolone 3β-HSD Progesterone CYP21 11-Deoxycorticosterone CYP11B2 Corticosterone CYP11B2 Aldosterone Zona glomerulosa CYP17 CYP17 17α-OH- Pregnenolone 3β-HSD 17α-OH- Progesterone CYP21 11-Deoxycortisol CYP11B1 Cortisol Zona fasciculata CYP17 CYP17 DHEA 3β-HSD Androstene -dione 17β-HSD Testosterone CYP19 17β-Estradiol Zona reticularis HO OH

19 In vivo effects?

20 Assesment of in vivo effects Control 25% Pilnok 75% Pilnok 100% Pilnok 50% Pilnok Solventcontrol 25% Pilnok 50% Pilnok 75% Pilnok Potamopyrgus antipodarum 2,5,8 weeks: mortality, embryos, hsp70 Gammarus fossarum 12 weeks: mortality, juveniles, histopathology

21 Sex-dependent differences in effects I! sensitivity of males 60% A females males 8 hsp70, Männchen Hsp70 levels 40% 6 75% Pilnok 75% Pilnok 20% 0% B C 50% Pilňok * * 100% Pilňok C 50% Pilňok 100% Pilňok hsp70-level Males hsp70-level Females hsp70, Weibchen T0 K 25% 50% 75% 100% LK 25% 50% 75% T0 K 25% 50% 75% 100% LK 25% 50% 75%

22 Sex-dependent differences in effects II variable effects in hepatopankreas A male B male C control 50μm 50μm 50μm D E 50μm female 50μm female

23 Pilnok sediments shift reproduction cycle in females (towards maturity) Anteil (ohne Atresie) Relative ratio 2,0 1,5 1,0 0,5 0,0 OOCYTE Ovarienreifenzustand STAGE (vitellogenesis) PVO FVO SVO RO Pre- Early- Later- Mature Oozytenreifenstadien Kontrolle Control Lockwitzbach Steinlach 50% Pilnok 100% Pilnok

24 Pilnok sediments induce production and size of F1 juveniles Number of specimens adults juveniles * * C 50%Pilňok 100%Pilňok

25 Pilnok sediments (and organic extracts) stimulate production of embryos No. Embryos / female (% % of control) * * 75% Pilnok 75% Pilnok week Woche Pilnok-Sediment Pilnok-Extrakt

26 SUMMARY 1 ) Routine (PAHs, PCBs, OCPs) analytical data did not clearly indicate excessive EDs

27 SUMMARY 1 ) Routine (PAHs, PCBs, OCPs) analytical data did not clearly indicate excessive EDs ) Biological experiments complement ment chemical analyses and suggest elevated levels of unknown EDs in PILNOK pond (?? PAH derivatives)

28 SUMMARY 1 ) Routine (PAHs, PCBs, OCPs) analytical data did not clearly indicate excessive EDs ) Biological experiments complement ment chemical analyses and suggest elevated levels of unknown EDs in PILNOK pond (?? PAH derivatives) ) In vitro assessment indicate feminization - estrogenicity and anti-androgenicity androgenicity

29 SUMMARY 1 ) Routine (PAHs, PCBs, OCPs) analytical data did not clearly indicate excessive EDs ) Biological experiments complement ment chemical analyses and suggest elevated levels of unknown EDs in PILNOK pond (?? PAH derivatives) ) In vitro assessment indicate feminization - estrogenicity and anti-androgenicity androgenicity ) New mechanism? ( CYP11B2 / steroidogenesis)

30 SUMMARY 2 ) In vivo experiments with two invertebrate species revealed EDCs in Pilnok sediments stimulated reproduction

31 SUMMARY 2 ) In vivo experiments with two invertebrate species revealed EDCs in Pilnok sediments stimulated reproduction (?) ) Differences - vertebrate in vitro: feminization - invertebrate in vivo: reproduction

32 Answers to our questions? What is the cause of intersex occurence? - contaminants associated with sediments Can ED-chemical chemicals be identified? - partially yes: organic (labile) contaminants Can the mechanism be understood? - (partially) yes: feminization / speeding up reproduction Can we induce ED experimentally? - Yes!

33 Acknowledgements J.P. Giesy,, P.D. Jones, M. Hecker R. Triebskorn D. Jungmann J. Ohlmann Z. Ďuriš,, I. Horká K. Hilscherová,, I. Holoubek, E.Mazurová recetox.czcz FINANCIAL: GAČR, Ministry of Education CR

34 Acknowledgements J.P. Giesy,, P.D. Jones, M. Hecker R. Triebskorn D. Jungmann J. Ohlmann Z. Ďuriš,, I. Horká K. Hilscherová,, I. Holoubek, E.Mazurová recetox.czcz FINANCIAL: GAČR, Ministry of Education CR