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SUMMARY The interplay between antioxidants, heat shock proteins and hypoxic signaling is supposed to be important for passive survival of critical temperature stress, e.g. during unfavorable conditions in hot summers. We investigated the effect of mild (18°C), critical (22°C) and severe(26°C) experimental heat stress, assumed to induce different degrees of functional hypoxia, as well as the effect of recovery following heat stress on these parameters in liver samples of the common eelpout Zoarces viviparus. Upon heat exposure to critical and higher temperatures we found an increase in oxidative damage markers such as TBARS (thiobarbituric reactive substances)and a more oxidized cellular redox potential, combined with reduced activities of the antioxidant enzyme superoxide dismutase at 26°C. Together, these point to higher oxidative stress levels during hyperthermia. In a recovery-time series, heat-induced hypoxia and subsequent reoxygenation upon return of the fishes to 12°C led to increased protein oxidation and chemiluminescence rates within the first 12 h of recovery, therein resembling ischemia/reperfusion injury in mammals. HSP70 levels were found to be only slightly elevated after recovery from sub-lethal heat stress, indicating minor importance of the heat shock response in this species. The DNA binding activity of the hypoxia-inducible transcription factor (HIF-1) was elevated only during mild heat exposure(18°C), but appeared impaired at more severe heat stress. We suppose that the more oxidized redox state during extreme heat may interfere with the hypoxic signaling response.

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0°C and after long-term warming to 5°C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0°C for the polar and 6°C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.

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SUMMARY Mitochondrial respiration, energetic coupling to phosphorylation and the production of reactive oxygen species (ROS) were studied in mitochondria isolated from the eurythermal bivalve Mya arenaria (Myoidea) from a low-shore intertidal population of the German Wadden Sea. Measurements were conducted both within the range of the habitat temperatures (5-15 °C) and when subjected to heat exposure at 20 °C and 25 °C. Experimental warming resulted in an increase in the rate of state 3 and state 4 respiration in isolated mitochondria. The highest respiratory coupling ratios (RCR) were found at 15 °C; at higher temperatures mitochondrial coupling decreased,and release of ROS doubled between 15 and 25 °C. ROS production was 2-3%of total oxygen consumption in state 3 (0.3-0.5 nmol ROS mg-1protein min-1) at the habitat temperature, reaching a maximum of 4.3 % of state 3 respiration and 7 % of oligomycin-induced state 4+respiration under heat stress. Thus, state 4 respiration, previously interpreted exclusively as a measure of proton leakage, included a significant contribution from ROS formation in this animal, especially under conditions of heat stress. Oxygen radical formation was directly dependent on temperature-controlled respiration rates in states 3 and 4 and inversely related to mitochondrial coupling (RCR+) in state 4. Mitochondrial ROS formation is therefore involved in cellular heat stress in this eurythermal marine ectotherm.