L. Camus & B. Gulliksen

1. L. Camus & B. Gulliksen Antioxidant defense properties of Arctic amphipods: comparison betweendeep-, sublittoral and surface-water species Presented by Lara Jarvis

2. ROS What are Reactive Oxygen Species? • Reactive molecules that contain oxygen atoms • Reactive because of presence of unpaired valence shell electrons • Formed through partial reduction of molecular oxygen during aerobic metabolism • Examples: 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.

3. Dual Role for ROS • Play a major role in cellular damage and disease BUT also play an important role in normal cellular function • Apoptosis • Defense against pathogens in higher plants • Mediation of morphogenic events (Lesser 2006)

4. Mediation of morphogenic events • With onset of mutualistic symbiotic associations • Symbiosis of the serpiolid squid (Euprymnascolopes) light organ and the bioluminescent bacterium Vibriofisheri (Lesser 2006)

5. Prooxidant Forces vs. Antioxidant Defences • In living cells ROS production from natural cell activity must be kept in check • Defenses • low-molecular-weight free-radical scavengers • glutathione • a number of specific enzymes • superoxide dismutase and catalase

6. ROS/Antioxidant Pathways Electron transfer from NADPH to molecular O2 O2.- 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-) www.bioscience.org

7. Temperate vs. Polar Species Antioxidant Levels • Oxidative stress processes have been well studied in temperate species • Interest in these processes in cold adapted marine animals is growing • Low metabolic rate and internal ROS production with high antioxidant defenses • What 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 Sources • ROS in water • Formed by photoreactions of dissolved organic carbon and oxygen in seawater • Ozone depletion could be speeding up this process • 24 hour illumination periods

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

10. Methods and Materials Testing for antioxidant defense levels

11. 3 Amphipod Species Gammaruswilkitzkii • 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 eol.org oceanexplorer.noaa.gov

12. 3 Amphipod Species Anonyxnugax • 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 Species Eurythenesgryllus • 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 Species G.wilkitzkii Surface, under ice nugax 800 m E. gryllus 2000 m

15. TOSC Assay • Total oxyradical scavenging capacity • based 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 gas • More ethylene = less antioxidants • Less ethylene = more antioxidants

16. TOSC Assay • Examining antioxidant response to 3 ROS • Peroxyl assay • Highly reactive oxygen radical • Hydroxyl assay • Most reactive oxygen radical • Attacks all biological molecules in a diffusion controlled fashion • Peroxynitrite assay • Can diffuse across membranes 400X faster than superoxide • Highly reactive, especially with lipids (Lesser 2006)

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

18. Exposure to H2O2 • Exposed G. wilkitzkii and A. nugax only • G. wilkitzkii • 2 groups of 5 individuals • Control Group: placed in 2L of seawater • Experimental Group: placed in 2L of seawater + 5mM H2O2 • Exposed for 7 days • Extracted hemolymph and froze appendageless bodies

19. Exposure to H2O2 • A. nugax • Same procedure used with the following modifications • Used seawater + 2.5mM H2O2 concentration • Exposed for 5 days • Extracted 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 peroxynitrite • Low Hydroxyl susceptibility suggested due to low TOSC values • G. wilkitzkii has lower values than A. nugax: ?

22. Discussion • G. wilkitzkii has lower TOSC values for peroxyl and peroxynitrite, compared to A. nugax • Contradictory? • Could be caused by: • Dietary differences • Omnivorous/Carnivorous vs. Scavenger • Metabolic rates • G. wilkitzkii is among the lowest for Arctic or sub-Arctic species • Habitat differences • G. wilkitzkii live in a very unstable environment = salinity, temperature change

23. TOSC Assay: Hemolymph • TOSC for peroxyl was significantly different from the peroxynitrite • G. 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. Discussion • Indicates 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 hydroxyl • Possible formation of a biological adaptive mechanism to prevent hydroxyl formation • Removal of superoxide by higher activity SOD?

25. TOSC Assay: Digestive Tract • Indicating the relative importance of low-molecular-weight scavengers compared to larger antioxidant proteins • The high percentages for hydroxyl indicate the low-molecular-weight scavengers are most important in keeping these radicals in check • Percent 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. Exposure to H2O2: Digestive Gland A. 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

27. Exposure to H2O2: Hemolymph A. nugax G. wilkiztkii • In A. nugax TOSC values increased significantly toward peroxyl, but not hydroxyl or peroxynitrite

28. Discussion • Results again indicate G. wilkitzkiipossess a mechanism of resistance for exogenous ROS • Mechanism that either prevents the diffusion of external H2O2 through the gills OR • Helps excrete internal H2O2 (based on Wilhelm et al. 1994) • A. nugax appears highly susceptible • This coupled with higher basal TOSC support the observations of limited environmental antioxidant forces in benthic Arctic habitats • Hypothesized that polar filter-feeding bivalves require a high TOSC because of low turnover

29. Conclusions • First 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. wilkitzkiidemonstrated an adaptive mechanism for living in highly prooxidant Arctic surface waters • Exclusion or secretion of ROS?

30. Critique • Exposure to H2O2 not well executed • Was 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.

31. 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?

32. References Camus 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 RadicBiol 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.