L. Camus & B. Gulliksen Presented by Lara Jarvis

 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. What are Reactive Oxygen Species?

 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)

 With onset of mutualistic symbiotic associations  Symbiosis of the serpiolid squid (Euprymna scolopes) light organ and the bioluminescent bacterium Vibrio fisheri (Lesser 2006)

 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

Electron transfer from NADPH to molecular O 2 O 2.- is dismutated to hydrogen peroxide (H 2 O 2 ) by superoxide dismutases (SOD) In the presence of Fe, H 2 O 2 may form the highly reactive hydroxyl (OH. ) species through the Fenton and Haber-Weiss reactions O 2.- also reacts with nitric oxide (NO) to form peroxynitrite (ONOO - )

 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

 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

Camus and Gulliksen proposed to: 1. Aquire a preliminary understanding of the antioxidant capabilities of three species of amphipod from different ocean depth regions 2. Compare the antioxidant response of two of those species following laboratory exposure to a ROS (H 2 O 2 )

Testing for antioxidant defense levels

Gammarus wilkitzkii eol.org oceanexplorer.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

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

Eurythenes 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

G. wilkitzkii Surface, under ice A.nugax 800 m E. gryllus 2000 m

 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

 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)

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

 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 H 2 O 2  Exposed for 7 days  Extracted hemolymph and froze appendageless bodies

 A. nugax  Same procedure used with the following modifications ▪ Used seawater + 2.5mM H2O2 concentration ▪ Exposed for 5 days ▪ Extracted hemolymph and 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: ?

 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

 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

 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?

 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.

 Significantly 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 hydroxyl  Being removed some other way?  Lower TOSC toward peroxynitrite and peroxyl in E. gryllus compared to A. nugas is explained by  lower SOD activity  Lower metabolic rate

A. NUGAXG. 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

A. NUGAXG. WILKIZTKII  In A. nugax TOSC values increased significantly toward peroxyl, but not hydroxyl or peroxynitrite

 Results again indicate G. wilkitzkii possess 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

 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. wilkitzkii demonstrated an adaptive mechanism for living in highly prooxidant Arctic surface waters  Exclusion or secretion of ROS?

 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.

 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?

Camus L, Gulliksen B (2005) Antioxidant defense properties of Arctic amphipods: comparison between deep-, sublittoral and surface-water species. Marine Biology 146: 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: Lesser MP (2006) Oxidative stress in marine environments: biochemistry and physiological ecology. Annu Rev Physiol 68: Halliwell B (2005) Free radicals and other reactive species in disease. ELS