1. Marta Ferreira CIIMAR/CIMAR Oxidative stress biomarkers in aquatic species, and applications in environmental monitoring July 2010 Reis-Henriques, M.A. and Moradas-Ferreira, P. CIIMAR/CIMAR, IBMC/UP, ICBAS/UP

2. Contaminants In the last decades a increasing number of different chemicals has been released to the environment. The aquatic environment, being a final reservoir for these chemicals, is particularly sensitive. Acute Exposition→ easily recognizable. Chronic Exposition→ will have a negative effect, not only in the environment but also in the food chain.Introduction

3. The presence of pollutants in the aquatic environment can lead to the formation of Reative Oxidant Species (ROS) that will induce oxidative stress in the species exposed to them Oxidative Stress Biomarkers Antioxidant Enzyme activities Oxidative Damages Superoxide anion radical – O 2 - Hydrogen peroxide–H 2 O 2 Hydroxide radical- OH Strong oxidants with the ability to react with macromolecules like, lipids, proteins and DNAIntroduction

4. Pollutant O2-O2- SOD H2O2H2O2 CAT ½ O 2 + H 2 O H2OH2O GPx GSH GSSG GR NADPH NADP + ROS formation  Antioxidant Enzymes: Detoxify ROS generated by the presence of contaminants  Oxidative damages: ROS can damage macromolecules (lipids, proteins nucleic acids)  Assessed by the activity of different enzymes: SOD, CAT, GPx e GRIntroduction Oxidative Stress

5. Case Study

6. Contaminants:  PCBs DDTs  PAHs Biomarkers:  Oxidative Stress Group I – After capture Group II – 1 month depuration Group IV – 4 months depuration Group VIII – 8 months depuration Mullet Flounder Experimental Approach

7. CAT in Mullet SpringSummerAutumnWinter 0 5 10 15 20 25 CAT(mmol/min/mg protein) Group I * p<0,05 CAT in Flounder SpringSummerAutumnWinter 0 4 8 12 16 CAT (mmol/min/mg protein) Group I Group II *  The presence of contaminants in the estuary increases CAT activity in mullet that decreases after one month depuration  In flounder the responses are not consistent, that could be related to the fact that flounder did not ate during the depuration period. 13% 5% 3% 35% 18%Catalase

8. tSOD in Mullet SpringSummerAutumnWinter 0 4 8 12 16 SOD (U/mg protein) Group I * p<0,05 tSOD in Flounder SpringSummerAutumnWinter 0 4 8 12 16 SOD (U/mg protein) Group I Group II * 21% 25% 2% Total SOD  The depuration period lead to a decresae in tSOD activity (higher than CAT).  The lack of food seems to induces this enzyme in flounder.

9. LP in Mullet SpringSummerAutumnWinter 0 10 20 30 MDA (nmol/mg protein) Group I * p<0,05 LP in Flounder SpringSummerAutumnWinter 0 10 20 30 40 MDA (nmol/mg protein) Group I Group II * * 29 % 32 % Lipid Peroxidation  An increase in LPO was observed in mullet that was related to an increase in temperature in these periods in the laboratory.  The lack of food induces an increase in LPO in flounder

10. I I II II Flounder I II I Mullet I – Group I II – Group II Oxidised Proteins  In both species an increase in OP was observed after one month depuration.  The decrease in antioxidant enzymes activities could be responsible for the increase in OP in mullet liver

11. Case Study

12. To analyse changes in the levels of antioxidant defences and oxidative damages after long-term depuration. RECOVER ??? PCBs DDTs Metals PAHs xenoestrogens Oxidative stress biomarkersGoal

13. After long term depuration a decrease in oxidative stress biomarkers was observed Oxidative Stress

14. 1 2 3 4 5 6 Lanes 1 and 3 – Group I Lanes 2 and 4 – Group II Lane 5 – Group IV Lane 6 – Group VIII Detection of liver protein carbonyls Oxidised Proteins  First month depuration leads to an increase and the extent of time in unpolluted water is associated with a decrease in the levels of oxidised proteins.

15. RECOVER PCBs DDTs Metals PAHs xenoestrogens Oxidative stress biomarkers YESConclusions Antioxidant enzymes activities levels and oxidative damages can be considered as suitable biomarkers in mullets to monitor pollution.

16. SEAQUA Project: POCI/MAR/59094/2004 Case Study

17. METHODOLOGIES Class I: 10g Class II: 60g Class III: 150g Class IV: 300g Class V: 450g Wild: 375g Metal content in tissues, and food pellets Catalase (CAT) and Superoxide Dismutase (SOD) were assessed as antioxidant defences, and LP and OP as oxidative damages, in liver and muscle.

18. RESULTS Metals (  g/g dw) in food pellets ClassCuAsCdPb I and II8.92.30.0740.089 III8.81.50.860.38 IV and V8.81.70.480.26 Class III food pellets showed higher Cd and Pb content. Cu levels were the same in all food pellets feed to white seabream.

19. RESULTS In general, wild white seabream presented higher levels of metals in liver and muscle Metals (  g/g dw) in tissues

20. RESULTS Antioxidant Enzymes in Liver Significant Higher levels for CAT were observed in wild white seabream. SOD activity was higher in Class V and wild animals. a a a b b c b,c a,b a a,c dd Cu: 0.77 As: 0.55 Cd: 0.74 Pb:0.57 Cu: 0.39 As: 0.71 Cd: 0.56

21. RESULTS Oxidative Damages in Liver Higher levels of LPO were observed in Class III and Class IV. No differences were registered for levels of OP in liver. a a b b b,c c

22. RESULTS Oxidative Damages in Muscle Lower levels of LPO were observed in higher size classes and wild. No differences were registered for levels of OP in muscle. 0.72

23. SEAQUA Project: POCI/MAR/59094/2004 Case Study

24. Class I: 30g Class II: 120g Class III: 220g Class IV: 450g Wild: 740gApproach Metal content in tissues, and food pellets. Catalase (CAT) and Superoxide Dismutase (SOD) were assessed as antioxidant defences, and LP and OP as oxidative damages, in liver and muscle.

25. Results In general, wild seabass presented higher levels of metals in liver and muscle Metals (  g/g dw) in tissues

26. a a a a b a a b b bResults As: 0.71 Cd: 0.69 Pb:0.58 Pb: 0.41 As: 0.59 Cd: 0.49 Wild seabass presented higher levels of antioxidant enzymes activity in agreement with higher metal content in tissues Antioxidant Enzymes in Liver

27. Results a a a b b b b a a a Lower levels of LPO and OP were observed in wild seabass. Oxidative Damages in Liver

28. Results a,b a c a,b,c b,c a a a,b b b No differences between wild and cultured seabass were observed in muscle. Oxidative Damages in Muscle

29. Conclusions These studies showed that fish are facing oxidative stress due to environmental pollutants. Oxidative stress adaptations are species dependent, and abiotic factors can change the responses. Oxidative stress parameters are good biomarkers to apply in environmental monitoring studies. Oxidative damages, can also be a useful tool to evaluate fish quality.

30. Thank you for the attention