HERRAMIENTAS MOLECULARES Y CELULARES EN ORGANISMOS CENTINELA PARA DETERMINAR EL ESTADO DE SALUD DE ECOSISTEMAS COSTEROS Y ESTUARINOS Manu Soto Plentzia Marine Station PiE- UPV/EHU Human Impact on Oceans’ health The health of the world’s oceans and marine life is degrading rapidly as a result of excess human activities Over exploitation of resources Fishing activities Pollution (Persistent, Spills,…) Population increase Over-exploitation of resources Fuel extraction & accidental oil spills & other accidents Mutagenic, highly carcinogenic Great Pacific Garbage Patch (Pacific Trash Vortex ) Charles Moore (1997) Lost of biodiversity Persistant, chronic & long-term pollution Synthetic Bisphenol A Hormones Pharmaceuticals & cosmetics Phtalates Reproductive problems & cancer Pesticides Peripheric and central nervous systems, liver DDT Long Island, 1945 Biomagnification Bioaccumulation DDT Methylmercury DDTconcentrations in living organisms tissues Tissue concentrations rise along the trophic chain rise with age Biological effects: Human Health Minamata / Hg Biological effects: Human Health Itai-itai / Cd Origin and fate of contaminants ECOSYSTEM HEALTH ASSESSMENT Ecosystem health protection for Human health prevention HEALTH PROGNOSIS DIAGNOSIS THERAPY SENTINEL SPECIES Anchovy Engraulis encrasicolus (pelagic) Hake Merluccius merluccius (demersal) Mussel Mytilus galloprovincialis Thicklip grey mullet Chelon labrosus Cod fish Gadus morhua Slug Arion ater Turbot Earthworm Scophthalmus maximus Eisenia fetida LOW ECOLOGICAL Biochemical RELEVANCE BIOMARKER parameters (early-warning signals) Detoxification Measurements at single level of biological enzymes complexity that indicate the presence of pollutants (exposure biomarkers) or the magnitude of organism responses (effects Immunological biomarkers) (MacCarthy and Shugart, 1990) Physiological parameters parameters Histopathological parameters SHORT-TERM Condition LONG-TERM RESPONSE bioenergetics RESPONSE Population & community Reproductive Short-term indicators of competence long-term biological effects HIGH ECOLOGICAL RELEVANCE Biological effects Intersex Hermaphroditism (Urdaibai) Hermaphrodite mussel gonad Organometallic compounds (TBT) Antifouling agent Imposex Forbidden 2003 (Regulation 782/2003 EC ) 1979: Oysters Arcachon (France) Cantabria Tissue-level Biomarkers Structural Changes in Digestive Alveoli Normal Stressed Digestive cells loss and basophilic cells hypertrophy measure as relative proportion of VvBAS 100 µm 100 µm Thinning of the digestive tubule wall Digestive cell Basophilic cell Histopathological lesions 100 µm 100 µm Parasites Development of inflammatory and degenerative lesions 100 µm 100 µm SCREENING Mussel Watch: effects of POS 10 LLANES 1 SÃO BARTOLOMEU 11 SAN VICENTE 2 ONS 12 SUANCES 3 CIES 13 PEDREÑA 4 OIA 14 LAREDO 15 MUSKIZ 5 AGUIÑO APRIL 04/05/06 6 CALDEBARCOS 16 ARRIGUNAGA 7 CAMELLE 17 GORLIZ JULY 03/04/05 8 SEGAÑO 18 BAKIO SEPTEMBER 03 9 ESTACA 19 MUNDAKA 20 MUTRIKU OCTOBER 04/05 21 ORIO 22 HONDARRIBIA Mussel Watch: effects of POS Camelle Arrigunaga General condition of the c digestive gland ITC tissue 50 μm 50 μm Extremely low histological integrity c ICT a a ICT Disorganized ICT, c hemocytic infiltration, 100 μm 100 μm disrupted VCTC and April-03 April-03 ACTC Dilated digestive c ducts, atrophy of 50 μm digestive alveoli ICT ICT c Signs of recovery a ICT after July-05 a 100 μm 100 μm April-06 April-06 LYSOSOMAL MEMBRANE Mussel Watch: effects of POS STABILITY HEALTHY STRESSED Galician coast Cantabrian coast Basque coast 10-Llanes 1-São Bartolomeu 6-Caldebarcos 15-Muskiz 19-Mundaka 11-San Vicente 2-Ons 7-Camelle 16-Arrigunaga 20-Mutriku 12-Suances 3-Cies 8-Segaño 17-Gorliz 21-Orio 13-Pedreña 4-Oia 9-Estaca 18-Bakio 22-Hondarribia 5-Aguiño 14-Laredo 25 HEALTHY (LP > 20 min) 20 15 LP (min) LP 10 5 STRESSED (LP < 10min) J S A J O A J O A J S A J O A J O A J S A J O A J O A 2003 2004 2005 2006 2003 2004 2005 2006 2003 2004 2005 2006 Viarengo et al. (2000) & Izagirre (2002) Mussel Watch: effects of POS 3 10-Llanes Lysosomal Enlargement 15-Muskiz 11-San Vicente 16-Arrigunaga 12-Suances 17-Gorliz Lysosomal 13-Pedreña 2 18-Bakio 14-Laredo 19-Mundaka 20-Mutriku Response Index 21-Orio 1 22-Hondarribia (LRI) Lysosomal Destabilisation 0 0.5 1 1.5 2 2.5 3 LP Component (B) Highly stressed (LRI >√2) July-03 July-03 Moderately stressed (1<LRI<√2) Sept-03 -1 Sept-03 April-04 April-04 Distress signal (0.5 <LRI<1) July-04 July-04 Oct-04 Oct-04 Unstressed (LRI<0.5) April-05 -2 April-05 July-05 July-05 Oct-05 Oct-05 April-06 Lysosomal Size Reduction April-06 -3 July 2003 Sep 2003 April 2004 July 2004 Oct 2004 April 2005 July 2005 Oct 2005 April 2006 Biscay Bay IDENTIFICATION BIOLOGY ANALYTICAL CHEMISTRY Mussel Watch: battery of biomarkers Correlations between 2004-2006 biological and chemical Pearson tPAH Naphthalene variables AOXexp -0,006 0,031 p 0,939 0,71 n 154 143 Spearman tPAH Naphthalene AOXeff 0,032 -0,07 LP -0,326 -0,432 p 0,697 0,405 p <0,0001 <0,0001 n 154 143 n 154 143 VvL 0,146 0,183 GI 0,229 0,221 p 0,083 0,037 p 0,004 0,008 n 141 130 n 154 143 S/VL -0,176 -0,265 CIIR -0,146 -0,215 p 0,037 0,002 p 0,071 0,01 n 141 130 n 154 143 VvNL 0,044 0,137 CIPI 0,107 -0,045 p 0,589 0,104 p 0,186 0,595 n 154 143 n 154 143 VvBAS -0,067 -0,068 p 0,409 0,423 n 154 143 MLR/MET 0,233 0,338 p 0,004 <0,0001 AOX activity from Orbea et al. (in prep) tPAHs and Naphthalene from Bartolomé (2007) n 154 143 Monitoring Campaign POS Direct (Nov 2002) impact cessation < DISEASE Oia < TISSUE Mundaka BIOLOGICAL RESPONSE BIOLOGICAL DAMAGE Chemical Direct Biol. Secondary Biol. < LYSOSOMAL Exposure Impact Impact RESPONSE 2002 2003 2004 2005 2006 2007 2011 YEAR 3,5-4,5 cm (3 yr) shell length Affected by POS at different Affected adults stage juvenile stage Presumably developmental By POS never affected stages Omics: genomics, transcriptomics….” Mugil: 8300 genes!! Aquatic Organisms Aquatic Organisms… one example PASAIA HARBOUR, BASQUE COUNTRY Pasaia exterior (PE) 22-23ºC 21-22ºC Pasaia interior (PI) Juveniles C. labrosus de Plentzia enjaulados 21 días en Pasaia LOW DENSITY MULLET MICROARRAY Pasaia Inner vs Outer TBTbp1 TBT PPARγ CYP3; CYP7; ApoE Liver obesogen Lipid metabolism Chelon labrosus 5S rRNA b 30 30 b c b b b b b b c 25 b 25 a b b a b a a b 20 a a CT 20 CT a a a a a 15 15 10 (Pasaia) 10 Jul Sep Nov Dic Ene Mar May * Hembra n=5 Intersex U test, * * Macho p<0.05 * Diaz de Cerio et al., (2012). ES&T 46: 7763-71 Ecological Health Condition Chart (EHCC) Ecosystem health indices 13 10 11 12 14 EHCC 15 17 19 20 21 22 16 18 Biscay Bay AOX AOX exp eff April-03 July-03 LP VvL MLR Sep-03 VvBAS MET April-04 CIIR CIPI July-04 10. LLANES 11. SAN VICENTE Oct-04 12. SUANCES No problematic zone 13. PEDREÑA 14. LAREDO April-05 Zone with potential 15. MUSKIZ environmental problem 16. ARRIGUNAGA July-05 17. GORLIZ Zone with 18. BAKIO environmental risk 19. MUNDAKA Oct-05 20. MUTRIKU Deteriorated 21. ORIO environmental zone 22. HONDARRIBIA April-06 After RBU-Rep (1994) Comprehensive integration Tissues LYSOSOMES OXIDATIVE STRESS GENOTOXICITY HISTOPATHOLOGY Stakeholders Science Decission makers based Public administrations diagnosis PIE PLENTZIAKO ITSAS-ESTAZIOA (PIE) ITSAS-BIOLOGIA ETA -BIOTEKNOLOGIARAKO ESTAZIO ESPERIMENTALA ESTACION EXPERIMENTAL DE BIOLOGIA Y BIOTECNOLOGIA MARINAS U EXPERIMENTAL STATION FOR MARINE BIOLOGY AND BIOTECHNOLOGY /EH UPV MARKERS ASSAYS TOXINS BiO DRUGS SENSORS REMEDIATION ENVIRONMENTAL BIOTECHNOLOGY FOR ECOSYSTEM HEALTH Eskerrik asko!!! Thanks!!! Cell Biology in Environmental Toxicology Res Grp www.ehu.es/GrupoBCTA Cell Biology and Histology Lab - Plentzia Marine Station Zoology and Animal Cell Biology Dept University of the Basque Country .