l. camus & b. gulliksen

Antioxidant defense properties of Arctic amphipods: comparison betweendeep-, sublittoral and surface-water species

L. Camus & B. Gulliksen

Presented by Lara Jarvis

ROS 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?

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)

Mediation of morphogenic events With onset of mutualistic symbiotic

associations Symbiosis of the serpiolid squid

(Euprymna scolopes) light organ and the bioluminescent bacterium Vibrio fisheri

(Lesser 2006)

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

ROS/Antioxidant Pathways

www.bioscience.org

Electron 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-)

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

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

Purpose

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 (H2O2)

Methods and MaterialsTesting for antioxidant defense levels

3 Amphipod Species

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

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

3 Amphipod Species

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

Spatial Location of Amphipod Species

G. wilkitzkiiSurface, under iceA. nugax

800 m

E. gryllus2000 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

TOSC Assay

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)

TOSC Assay

Data expressed as TOSC unit per milligram protein (digestive tract) and TOSC unit per microliter (hemolymph)

TOSC unit/mg = oxyradical scavenging capacity

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

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

Results & Discussion

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: ?

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

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

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?

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.

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

Exposure to H2O2: HemolymphA. NUGAX G. WILKIZTKII

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

Discussion 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

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

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.

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?

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.