Antioxidant defenses in the tolerance of freezing and anoxia by garter snakes

The garter snake Thamnophis sirtalis parietalis can readily tolerate several hours of freezing or anoxia exposure. Both stresses halt oxygen availability to tissues and to endure these stresses snakes must cope with potential oxidative stress arising as a result of the ischemic/anoxic condition followed by reperfusion of aerated blood during recovery. To determine whether antioxidant defenses are important for freezing and anoxia survival, we monitored the activities of antioxidant enzymes and the levels of glutathione (GSH and GSSG) during freezing (5 h at -2.5 degrees C) and anoxia (10 h under N2 gas at 5 degrees C) exposures in three organs (muscle, liver, and lung) of snakes. Freezing resulted in a significant rise in the activity of muscle and lung catalase (by 183 and 63%) and in muscle glutathione peroxidase (52%). Anoxia enhanced muscle and liver superoxide dismutase activities (by 59 and 118%) and also caused a 57% increase in muscle GSH levels. The increase in muscle GSH concentration in anoxia (from 0.45 to 0.71 mM) could also stimulate muscle glutathione peroxidase activity in vivo by 1.5-fold because of its low affinity for GSH (Km = 11 mM). The ratio of GSSG/GSH was not affected by experimental state in any tissue, suggesting that oxidative stress did not occur during the freezing or anoxic exposure. Rather, H2O2- and O2(-)-detoxification systems may be activated in preparation for possible oxygen free radical overgeneration during thawing or reoxygenation. Antioxidant defenses appear to be part of the adaptive machinery for reptilian tolerance of freezing and anoxia.