Regulation of antioxidant systems in response to anoxia and reoxygenation in Rana sylvatica.

Abstract

The wood frog (Rana sylvatica) is a remarkable species. These frogs can endure prolonged oxygen deprivation as well as dehydration to ~60% of total body water lost and, combining these two abilities, they survive whole body freezing for weeks at a time during the winter. Episodes of anoxia/reoxygenation or freeze/thaw can trigger elevated production of reactive oxygen species (ROS) causing cellular damage, especially when oxygen is reintroduced during reoxygenation or thawing. To mitigate ROS damage, stress-responsive transcription factors such as the Octamer Binding Transcription factor (OCT4) and Nuclear factor (erythroid-derived 2)-like 2 transcription factor (Nrf2) were postulated to be involved in enhancing pro-survival pathways and antioxidant defenses. The present study used immunoblotting to analyze OCT4 and Nrf2 responses (and downstream factors under their control) to 24h anoxia and 4h reoxygenation in liver and skeletal muscle of wood frogs, with an emphasis on antioxidant systems. Surprisingly, no change was observed in relative total protein expression of either of the two transcription factors in liver. Furthermore, a significant decrease in total protein levels of OCT4 and Nrf2 occurred in skeletal muscle after 4h recovery. However, essential cofactors of OCT4 and Nrf2 were significantly upregulated during anoxia and/or recovery. Downstream targets of the Nrf2-ARE pathway were evaluated, including glutathione-S-transferases (GSTs) and aldo-keto reductases (AKRs). Significant increases in GSTT1 and GSTP1 were observed in liver and muscle whereas AKRs showed a tissue specific response to both anoxia and recovery from anoxia. This study demonstrates activation of antioxidants as a cell protective mechanism against generation of reactive oxygen species during anoxia in wood frogs.