Jean E. Grundy, Ph.D. Chemistry, 1996

Antioxidant defenses during estivation in the spadefoot toad, Scaphiopus couchii



Antioxidant enzymes and lipid peroxidation damage were analyzed in an estivating species, to determine whether estivation (a dormancy induced by hot, dry environmental conditions) represented an oxidative stress and to determine whether changes in organ antioxidant systems were made in response to estivation. The model animal used was Scaphiopus couchii, the spadefoot toad, that estivates for about 10 months per year in the Arizona desert during which it loses about 50% of its body water and builds up high levels of electrolytes and osmolytes in body fluids. Activities of six antioxidant enzymes (AOE) and xanthine oxidase/xanthine dehydrogenase as well as the small molecular weight free radical scavenger glutathione were measured in liver, lung, heart, kidney, gut and leg muscle of control and dormant toads. Damage to membranes was assessed by measurement of extent of lipid peroxidation by three different methods. The glutathione ratio, (GSSG) /(GSH), rose significantly in several tissues during estivation. In addition, activities of key AOE were reduced in several tissues, perhaps indicating that the metabolic cost of keeping antioxidant defenses high during estivation was greater than the cost of maintaining AOE at their previous level. Three antioxidant enzymes were further examined: Catalase (CAT) and glutathione reductase (GR) were purified and characterized from liver and glutathione S-transferase (GST) from liver and muscle of awake and estivated toads. CAT activity was lower in liver during estivation but the kinetic properties of catalase were the same in estivated and awake toads. Catalase from toad liver was an active dimer, which has not been demonstrated previously in a eukaryote, to our knowledge. GR and GST of toad liver had altered kinetic properties during estivation, indicating pre- or post-translational modification of the enzyme in the dormant animal. However, GST in muscle was the same for awake and estivated toads. GST is a multiclass family of enzymes which generally render lipid-soluble material soluble in the cytosol, via conjugation to glutathione. Three class of GST, Mu Pi and Alpha, have different substrate specificities; these parameters were used to classify frog and toad GSTs. GST, GR and CAT were relatively insensitive to physiological urea concentrations but were strongly modulated by KCl, suggesting that urea buildup during estivation may serve to modulate passive KCl accumulation as a result of dehydration.