HERRAMIENTAS MOLECULARES Y CELULARES EN ORGANISMOS CENTINELA PARA DETERMINAR EL ESTADO DE SALUD DE ECOSISTEMAS COSTEROS Y ESTUARINOS

Manu Soto 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 5 AGUIÑO APRIL 04/05/06 6 CALDEBARCOS 16 ARRIGUNAGA 7 CAMELLE 17 GORLIZ JULY 03/04/05 8 SEGAÑO 18 SEPTEMBER 03 9 ESTACA 19 20 OCTOBER 04/05 21 ORIO 22 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

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 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