Multiple endocrine disrupting activities in French river sediments as assessed by the combined use of in vitro bioassays and chemical analyses

Transcription

1 Multiple endocrine disrupting activities in French river sediments as assessed by the combined use of in vitro bioassays and chemical analyses Sélim Aït-Aïssa Unit of Ecotoxicological Risk Assessment

2 Modes of action of EDCs Biosynthesis of hormones Aromatase Metabolism EDC Transport PXR (CYP3A) AhR (CYP1A) SULT... Action in target tissues SHBP Excretion EDC Steroid hormone receptors: ER AR

3 Context Environmental endocrine disrupting chemicals (EDCs) : variety of chemical classes and origins multiple modes of biological action French situation estrogens in WWTP effluents (Cargouet et al, 2004, Labadie and Budzinski 2005, Muller et al 2008) limited data on contamination levels and biological impacts in freshwater systems (rivers)

4 Field monitoring of EDCs : overall approach IMPACTS WFD Sites CONTAMINATION Fish Sediments, surface water Community assemblage Biomarkers - biochemical - histological (intersex) - somatic indexes in vitro bioassays Chemical analyses? Fish-Based Index (FBI) Biomarker responses Toxic-EQs Identification

5 Case study : small agricultural streams Pressure : Fish Index : Lézarde agriculture 2 (reference site) Réveillon Urban and agriculture 5 (bad quality) Rhonelle agriculture 3 (medium quality) Estrogenicity biomarker: VITELLOGENIN in male fish Androgenicity biomarker: Three-spined stickleback (Gasterosteus aculeatus) SPIGGING in female fish

6 Case study : small agricultural streams Pressure : Fish Index : Lézarde agriculture 2 (reference site) Réveillon Urban and agriculture 5 (bad quality) Rhonelle agriculture 3 (medium quality) Vitellogenin Spigging EROD +/ Sanchez et al, Ecotoxicology (2007), Environ. Int. (in press)

7 Endocrine disruption in fish Sediment contamination by EDCs? in vitro bioassays

8 Reporter cell-based assays Target receptor Cell lines Endpoint Reference ligands (EC 50 ) Hormonal activity Xenobiotic Metabolism ER MELN Luciferase E2 (0.02 nm) (MCF-7, ERE-LUC) AR MDA-kb2 Luciferase DHT (0.1 nm) anti-ar (MDA-MD-453, MMTV-Luc) Flutamide (0.1 µm) AhR PLHC-1 EROD 4 h BaP (5 nm) EROD 24 h TCDD (0.1 nm) PXR HGPXR Luciferase SR12,813 (0.1 µm) (GAL4RE-Luc/GAL4-hPXR) HG-PXR (Lemaire et al,2006), MELN (Balaguer et al, 1999), MDA-kb2 (Wilson et al 2002)

9 Estrogenic activities in sediment (crude organic extracts) MELN cells β-E2 EC20 Réveillon Rhonelle Lézarde 10 1.E-10 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 Relative luciferase activity (100% : E2 10 nm) g (E2 or sed d.w.) / L E-EQ = EC 20 of 17b-E2 EC 20 of sample

10 10 9 Estrogenic activities E2-Eq (ng/g) E2-EQ ng/g 1 0 Lézarde Réveillon Rhonelle Anti-androgenic activities FLU-Eq (µg/g) n.d. Flutamide-EQ µg/g 0 Androgenic activities DHT-Eq (ng/g) Lézarde Réveillon Rhonelle n.d. n.d. DHT-EQ ng/g Lézarde Réveillon Rhonelle

11 AhR PAH-like activities (BaP-Eq) BaP-Eq (µg/g) Lézarde Réveillon Rhonelle PXR-mediated activities (SR12813-Eq) SR12813-Eq (µg/g) n.d. PXR 0 Lézarde Réveillon Rhonelle Nature of PXR activators? Xeno-estrogens?

12 Endocrine disruption in fish Sediment contamination by EDCs? cell bioassays Nature of endocrine active chemicals? Target chemical analyses

13 (Xeno-)estrogens (ng/g sed. d.w.) Lézarde Réveillon Rhonelle * DES <0.1 <0.1 <0.1 17α-E2 < <0.4 17β-E2 < Estrone MeEE2 <0.3 < EE2 <1.5 <1.5 <1.5 4-NP <0.19 <0.19 < OP < <0.18 BPA < (α+β)endosulfan <24 <24 E2-EQs-chem E2-EQs-bio E2-Eq and EEF are based on EC 20 values * quantification limit

14 PAHs and PCBs (ng/g sed. d.w.) Lézarde Réveillon Rhonelle 16 PAHs PCB138 <LOQ 6.9 <LOQ PCB153 <LOQ 6.7 <LOQ BaP-EQchem BaP-EQbiol Ratio 19% 21% 27% Partial contribution of the priority PAHs BaP-EQs are based on EC 20 values IEF4h were determined for individual PAH (Louiz et al, submitted)

15 Endocrine disruption in fish Site contamination by EDCs? cell bioassays Nature of endocrine active chemicals? Target chemical analyses sample fractionation : Réveillon (prel. data) Polarity : RP-HPLC Receptor affinity : ER-columns

16 RP-HPLC fractionation : ER activities in Réveillon Relative luciferase activity% (100%=E2 at 10nM) ??? E2 BPA E1? EEQ ng/g Chem-EEQ Sum of fractions Whole extract 0 Gradient : water/acn 80%/20% ACN 100% Fraction i (i=1min)

17 RP-HPLC fractionation : AhR activities in Réveillon ??? E2 BPA E1? 16 PAHs? Fraction i (i=1min) F1 F5 F9 F13 F17 F21 F25 F29 F33 F37 F41 F45 F49 F53 F57 F61 F65 F69 F73 F77 F81 F85 F89 F93 F97 F101 F105 F109 F113 F117 Identification of active fractions : to be done!

18 Purification on herα affinity column His-ERα Produced in E. coli ckel-agarose phase 1) Receptor attachment 2) Estrogenic sample Filter Column Pillon et al., Environ. Health Persp. (2005) 113,

19 Purification on herα affinity column 1) Receptor attachment 2) Estrogenic sample 3) Ligand binding Column Pillon et al., Environ. Health Persp. (2005) 113,

20 Purification on herα affinity column 1) Receptor attachment 2) Estrogenic sample 3) Ligand binding 4) Washing step Column Washing fraction

21 Purification on herα affinity column 1) Receptor attachment 2) Estrogenic sample 3) Ligand binding 4) Washing step 5) Elution Column Washing fraction Elution fraction

22 Purification on herα affinity column 1) Receptor attachment 2) Estrogenic sample 3) Ligand binding 4) Washing step 5) Elution Column 6) Testing for ER, AhR, PXR activities in bioassays Washing fraction Elution fraction

23 Preliminary results with Réveillon sediment 100% herα column % of total sample activity 80% 60% 40% 20% Washing fraction Elution fraction 0% ER AhR PXR Activities

24 Summary Occurrence of endocrine disruption in wild stickleback agricultural sites, without influence of major WWTP Occurrence of ED activities in sediments? Profiling : ER, anti-ar, AR, PXR, AhR Nature of active compounds? E2, E1 and PAHs as main contributors... but only partially Unknown : polar ER fractions Unknown : AR and PXR agonists

25 Future work Identification of isolated fractions full scan MS, qtof, IR, UV confirmation in bioassays To extend the approach to sites with androgenic responses in sticklebacks SURVAQUA project National Research Programme on EDs (PNRPE ) INERIS (JM Porcher), CEMAGREF, Bordeaux Univ, Montpellier Univ., Le Havre Univ., UCO 13 sites

26 Thanks to collaborators Biomarkers in fish, in vitro bioassays François BRION, Wilfried SANCHEZ, colas CREUSOT, Jean-Marc PORCHER Chemical analyses Saïd KINANI, Stéphane BOUCHONNET Recombinant receptor columns, in vitro models Sonia DAGNINO, Patrick BALAGUER Field surveys Jean-Maxence DITCHE Financial support : Ministère de l'écologie et du Développement Durable (BCRD , PNRPE)