Freezing stress adaptations: Critical elements to activate Nrf2 related antioxidant defense in liver and skeletal muscle of the freeze tolerant wood frogs.

Wood frogs (Rana sylvatica) can survive seasonal exposure to subzero temperatures. During freeze/thaw, the frogs confront oxidative stress due to concurrent stress conditions of anoxia, ischemia and dehydration. Wood frogs also need to cope with additional oxidative stress associated with hyperglycemia due to accumulation of the cryoprotectant glucose. Here we explore the transcription factor Nrf2 (nuclear factor erythroid 2 related factor 2) and Nrf2 related antioxidant enzymes in liver and skeletal muscle of wood frogs undergoing freeze/thaw and glucose injection. Nrf2 binding activity to DNA was assessed and GSK3β, an upstream regulator of Nrf2, and gsta1, a downstream gene under Nrf2 control, were also evaluated. A multiplex protein assay was used to analyze multiple Nrf2 related antioxidant enzymes. Elevated DNA binding activity was observed in frozen frogs as compared to unfrozen controls for both liver and skeletal muscle. Interestingly, high glucose also enhanced binding to the ARE (antioxidant response element) in vitro in unfrozen frogs for both tissues. However, high blood glucose concentration failed to stimulate Nrf2 dependent gsta1 gene expression in glucose loaded frogs, although this was observed in liver of frozen frogs. A multiplex protein assay revealed that Prdx2 responded robustly in both tissues, decreasing in liver but rising in muscle. Glucose loaded frogs showed tissue specific suppression of catalase, Prdx2 (Peroxiredoxin-2) and SOD2 (superoxide dismutase 2) in liver and of Prdx2 alone in muscle. Our study further extended our understanding of the roles of Nrf2 dependent antioxidant defenses in wood frog freezing survival.