Activation of antioxidant defenses in response to freezing in freeze-tolerant painted turtle hatchlings

Background: Hatchlings of the painted turtle. Chrysemys picta marginata can endure long-term freezing of their extracellular body fluids. We hypothesized that freezing survival would include adaptive up-regulation of antioxidant defenses to deal with ischemia-reperfusion injuries associated with the freeze-thaw cycle. A number of antioxidant enzymes are under the control of the NF-E2 related factor 2 (Nrf2) transcription factor including members of the glutathione S-transferase (GST) and aldo-keto reductase (AKR) families. Methods: RT-PCR and Western immunoblotting were used to measure changes in transcript and protein levels in response to 5-h freezing exposure of hatchlings. Results: Transcript levels of Nrf2 increased in turtle brain, liver, and muscle by 1.5- to 2-fold, and protein levels increased in the brain and muscle by 1.6- to 2.3-fold in response to freezing. GSTs responded strongly to freezing in turtle brain with amounts of GSTP1, M1, K1, and A3 elevated by 1.5- to 2.4-fold. GSTM3 and T1 rose by 1.8- to 2.3-fold in gut, whereas reduced levels of GSTP1, M1, M3, and K1 were found in livers of frozen animals. Levels of the AKR1B4 isozyme rose 2.1-fold during freezing in brain. Conclusions: Freezing triggered tissue-specific changes in the antioxidant defenses in C picta marginata organs. General significance: These data indicate that activation of an antioxidant response is an important aspect of natural freeze tolerance in turtles. (C) 2010 Elsevier B.V. All rights reserved.