1. PRESTIGE’S FUEL SOLUBLE PHASE ACTIVATES THE ARYL HYDROCARBON RECEPTOR Susana CASADO, Mar BABÍN, José V. TARAZONA, José M. NAVAS. Department of the Environment, Laboratory for Ecotoxicology, Spanish National Institute for Agriculture and Food Research and Technology (INIA), A-6 Km 7, 28040 Madrid, Spain.

2. EROD INDUCTION AFTER ARYL HIDROCARBON RECEPTOR (AhR) ACTIVATION CYP1A mRNA CYP1A EROD F RECEPTOR AhR F XRE AhR F

3. AhR MODEL LIGANDS Model ligands exhibit coplanar, polyaromatic and polycyclic molecules. This is the case of dioxins, furans, polychlorinated biphenyls and polyaromatic hydrocarbons (PAHs).

4. ¿WHY TO STUDY AHR INDUCTION? After Prestige spillage much organisms were affected by direct contact with solid fuel But, ¿What about the fraction of the fuel disolving in water (WSF)? Inespecific toxicity of the WSF Navas et al. also in this symposium ¿Specific toxicity? PAHs are an important fraction of the fuel. Much of them are considered model ligands of the aryl hidrocarbon receptor (AhR)

5. OBJECTIVE To characterize the activation of the AhR by the WSF obtained from the solid fuel of the Prestige. The AhR activation route was monitored at several levels: -LEVEL 1: Ethoxyresorufin-O-deethylase induction (EROD) -LEVEL 2: XRE activation -LEVEL 3: AhR- ligand interaction

6. WSF PREPARATION Sonication 15 min F FiltrationAgitation 48 hours WSF ready for use

7. Celular lines RTL-W1 and RTG-2 were used in order to determine EROD induction Dose response curves were obtained by serial dilutions of the WSF. FD ½, beginning by dilution 1/1. EROD activity was measured by fluorescence (em 530/ex 590) At least, three independent experiments were done LEVEL 1: EROD INDUCTION CYP1A mRNA CYP1A EROD F RECEPTO R AhR F XRE AhR F

8. EROD INDUCTION ASSAY 24 hours FLUORESCENCE Resorrufin Ethoxyresorufin 48 hours P450 1A1 WSF in culture medium (L15, EMEM) RTLW1 RTG2

9. EROD DOSE RESPONSE CURVE IN RTL-W1 p<0.05 ED50= 1/100 NOED= 1/128 * * * * * EROD(pMol/mg/min) CTRL dilution

10. EROD DOSE RESPONSE CURVE IN RTG-2 ED50= 1/227 NOED= 1/1024 EROD(pMol/mg/min) P<0.05 * * * * * * * * Log dilución * * * * * * * CTRL dilution

11. Probably a saturation of the enzymatic activity occurs due to high concentration of the toxics. In this case, the transcription of the CYP1A should not be altered at those concentrations RT-PCR IN RTL-W1 Dose response curves were obtained by serial dilutions of the WSF. CYP1A and ß-actin (housekeeping gene) expression were studied WHY DO HIGH CONCENTRATIONS OF WSF PRODUCE EROD INHIBITION?

12. High concentrations of the WSF produce higher gene transcription. CYP 1A1 ½, 1/8, 1/32, 1/128, 1/512, CM ßACTIN ½, 1/8, 1/32, 1/128, 1/512, CM RT-PCR IN RTL-W1

13. DR-CALUX cell line Permanently transfected cell line. XRE activation produces the expression of the reporter gene (luciferase). Dose response curves were obtained by serial dilutions of the WSF. At least, three independent experiments were performed LEVEL 2: XRE ACTIVATION CYP1A mRNA CYP1A EROD F RECEPTO R AhR F XRE AhR F

14. XRE ACTIVATION ASSAY 48 hours 24 hours LUMINISCENCE substrate XRE+luciferase gene WSF in  -MEM DR-CALUX

15. DOSE RESPONSE CURVE IN DR-CALUX ED50= 1/47 NOED<1/512 * * * ** * * * * UL/µg protein p<0.05 dilution

16. LEVEL 3: DOES WSF INTERACT WITH AHR BY LIGAND- BINDING MECHANISM? AhR inmunoassay® CYP1A mRNA CYP1A EROD F RECEPTO R AhR F XRE AhR F Several dilution of WSF were added to the AhR inmunoassay® plate. ßnaphthoflavone was used as a model ligand Three independent experiments were done

17. R F XRE are fixed in the wells X100 ABSORBANCE λ =405 F AhR XRE Antibody 1 Antibody 2 AhR INMUNOASSAY ®

18. AhR INMUNOASSAY ® RESULTS

19. Water soluble fraction of Prestige´s fuel induces CYP1A at enzymatic and also transcriptional levels, after its interaction with AhR by a ligand-binding mechanism. CONCLUSION FUTURE The activation of the AhR has been associated by several authors with disruption of the estrogen receptor (ER) mechanisms of action. Lead to reductions of the reproductive performance. Several experiments are being performed in vivo (medaka) to study the disruption caused by WSF AhR activation on ER pathways

20. THANKS FOR YOUR ATTENTION