Antioxidant defense properties of Arctic amphipods: comparison between - PowerPoint Presentation

1. Antioxidant defense properties of Arctic amphipods: comparison betweendeep-, sublittoral and surface-water speciesL. Camus & B. GulliksenPresented by Lara Jarvis

2. ROSReactive molecules that contain oxygen atomsReactive because of presence of unpaired valence shell electronsFormed through partial reduction of molecular oxygen during aerobic metabolismExamples: superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl radicals ( OH), peroxyl radicals (ROO ), alkoxyl radicals (RO ) and peroxynitrite (HOONO)Can cause cell damage, leading to oxidative stress and ultimately cell death.What are Reactive Oxygen Species?

3. Dual Role for ROSPlay a major role in cellular damage and disease BUT also play an important role in normal cellular functionApoptosisDefense against pathogens in higher plantsMediation of morphogenic events (Lesser 2006)

4. Mediation of morphogenic eventsWith onset of mutualistic symbiotic associationsSymbiosis of the serpiolid squid (Euprymna scolopes) light organ and the bioluminescent bacterium Vibrio fisheri(Lesser 2006)

5. Prooxidant Forces vs. Antioxidant DefencesIn living cells ROS production from natural cell activity must be kept in checkDefenseslow-molecular-weight free-radical scavengers glutathionea number of specific enzymes superoxide dismutase and catalase

6. ROS/Antioxidant Pathwayswww.bioscience.orgElectron transfer from NADPH to molecular O2O2.- is dismutated to hydrogen peroxide (H2O2) by superoxide dismutases (SOD) In the presence of Fe, H2O2 may form the highly reactive hydroxyl (OH.) species through the Fenton and Haber-Weiss reactions O2.- also reacts with nitric oxide (NO) to form peroxynitrite (ONOO-)

7. Temperate vs. Polar Species Antioxidant LevelsOxidative stress processes have been well studied in temperate speciesInterest in these processes in cold adapted marine animals is growingLow metabolic rate and internal ROS production with high antioxidant defensesWhat is going on?Responding to external ROS?Lack of research examining the link between external prooxidant sources and the antioxidant defences of species in the cold polar environment

8. Possible External SourcesROS in waterFormed by photoreactions of dissolved organic carbon and oxygen in seawaterOzone depletion could be speeding up this process24 hour illumination periods

9. PurposeCamus and Gulliksen proposed to: Aquire a preliminary understanding of the antioxidant capabilities of three species of amphipod from different ocean depth regionsCompare the antioxidant response of two of those species following laboratory exposure to a ROS (H2O2)

10. Methods and MaterialsTesting for antioxidant defense levels

11. 3 Amphipod SpeciesGammarus wilkitzkiieol.orgoceanexplorer.noaa.gov Collected at surface, under-ice using a SCUBA –operated suction sampler Body length ca. 3 cm n= 5 Extracted hemolymph and removed appendages from body, frozen in liquid nitrogen

12. 3 Amphipod SpeciesAnonyx nugax Collected at 800m depth using trawl Body length ca. 4 cm n= 5 Extracted hemolymph and digestive tract, frozen in liquid nitrogen

13. 3 Amphipod SpeciesEurythenes gryllus Collected at 2000 m depth using baited traps Body length ca. 6 cm n= 10 Extracted hemolymph and digestive tract, frozen in liquid nitrogen

14. Spatial Location of Amphipod SpeciesG. wilkitzkiiSurface, under icenugax800 mE. gryllus2000 m

15. Total oxyradical scavenging capacitybased on the oxidation of KMBA to ethylene upon reaction with certain oxyradicals and on the ability of various antioxidants to inhibit this reaction ( Regoli and Winston 1999, Winston et al. 1998)Measurements are relative rates of production of ethylene gasMore ethylene = less antioxidantsLess ethylene = more antioxidantsTOSC Assay

16. TOSC Assay Examining antioxidant response to 3 ROSPeroxyl assayHighly reactive oxygen radicalHydroxyl assay Most reactive oxygen radicalAttacks all biological molecules in a diffusion controlled fashionPeroxynitrite assayCan diffuse across membranes 400X faster than superoxideHighly reactive, especially with lipids(Lesser 2006)

17. TOSC AssayData expressed as TOSC unit per milligram protein (digestive tract) and TOSC unit per microliter (hemolymph) TOSC unit/mg =  oxyradical scavenging capacity

18. Exposure to H2O2Exposed G. wilkitzkii and A. nugax onlyG. wilkitzkii 2 groups of 5 individualsControl Group: placed in 2L of seawaterExperimental Group: placed in 2L of seawater + 5mM H2O2Exposed for 7 daysExtracted hemolymph and froze appendageless bodies

19. Exposure to H2O2A. nugax Same procedure used with the following modificationsUsed seawater + 2.5mM H2O2 concentrationExposed for 5 daysExtracted hemolymph and digestive tract

20. Results & Discussion

21. TOSC Assay: Digestive Tract Indicates digestive gland is more susceptible to exposure to peroxyl and peroxynitrite A. nugax had significantly higher TOSC values toward peroxyl and peroxynitriteLow Hydroxyl susceptibility suggested due to low TOSC valuesG. wilkitzkii has lower values than A. nugax: ?

22. DiscussionG. wilkitzkii has lower TOSC values for peroxyl and peroxynitrite, compared to A. nugaxContradictory? Could be caused by: Dietary differencesOmnivorous/Carnivorous vs. ScavengerMetabolic ratesG. wilkitzkii is among the lowest for Arctic or sub-Arctic speciesHabitat differencesG. wilkitzkii live in a very unstable environment = salinity, temperature change

23. TOSC Assay: HemolymphTOSC for peroxyl was significantly different from the peroxynitriteG. wilkitzkii TOSC profile similar to the digestive tract TOSC profiles G. wilkitzkii had significantly lower and higher TOSC values for hydroxyl and peroxynitrite, respectively

24. DiscussionIndicates presence of active scavengers of ROS in the cell-free hemolymph of amphipod crustaceans Important as first line of defense!The lower hydroxyl scavenging capacity seen in G. wilkitzkii suggests a lower formation of hydroxylPossible formation of a biological adaptive mechanism to prevent hydroxyl formationRemoval of superoxide by higher activity SOD?

25. TOSC Assay: Digestive TractIndicating the relative importance of low-molecular-weight scavengers compared to larger antioxidant proteinsThe high percentages for hydroxyl indicate the low-molecular-weight scavengers are most important in keeping these radicals in checkPercent contribution of soluble fraction to the TOSC value of the total cytosolic fraction reached 94%, 100%, and 89% for hydroxyl radicals for E. gryllus, A. nugax, and G. wilkitzkii, respectively.

26. DiscussionSignificantly different TOSC values within each species indicates the endogenous generation rates of the three ROS examined are different.Low TOSC toward hydroxyl indicates a low susceptibility to hydroxylBeing removed some other way?Lower TOSC toward peroxynitrite and peroxyl in E. gryllus compared to A. nugas is explained by lower SOD activityLower metabolic rate

27. Exposure to H2O2: Digestive GlandA. Nugax G. wilkitzkii *In both digestive gland and hemolymph observed a significant TOSC response in A. nugax, but not in G. wilkitzkii* In A. nugax TOSC decreased significantly toward peroxyl and peroxynitrite, decreased toward hydroxyl but not significantly

28. Exposure to H2O2: HemolymphA. nugaxG. wilkiztkii In A. nugax TOSC values increased significantly toward peroxyl, but not hydroxyl or peroxynitrite

29. DiscussionResults again indicate G. wilkitzkii possess a mechanism of resistance for exogenous ROSMechanism that either prevents the diffusion of external H2O2 through the gills ORHelps excrete internal H2O2 (based on Wilhelm et al. 1994) A. nugax appears highly susceptibleThis coupled with higher basal TOSC support the observations of limited environmental antioxidant forces in benthic Arctic habitatsHypothesized that polar filter-feeding bivalves require a high TOSC because of low turnover

30. ConclusionsFirst baseline datasets for the TOSC in the digestive system and cell-free hemolymph with respect to different oxidants in cold-adapted amphipods from surface, sublittoral, and deep-sea habitats.G. wilkitzkii demonstrated an adaptive mechanism for living in highly prooxidant Arctic surface watersExclusion or secretion of ROS?

31. CritiqueExposure to H2O2 not well executedWas this appropriate to publish?Discussion was not well organized Figure 2 is not well described, and it’s significance to the paper is not explained well.

32. Questions?What do you think their results really showed?Were their methods rigorous enough?What could explain the variation seen in the TOSC values for each oxidant?Were the ROS they chose appropriate?Are claims made in discussion supported?

33. ReferencesCamus L, Gulliksen B (2005) Antioxidant defense properties of Arctic amphipods: comparison between deep-, sublittoral and surface-water species. Marine Biology 146: 355-362.Winston GW, Regoli F, Dugas AJ, Fong JH, Blanchard KA (1998) A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fluids. Free Radic Biol Med 24: 480-493.Lesser MP (2006) Oxidative stress in marine environments: biochemistry and physiological ecology. Annu Rev Physiol 68: 253-278.Halliwell B (2005) Free radicals and other reactive species in disease. ELS www.els.net.