Oxidative stress time course in the rat diaphragm after freezing–thawing cycles

Hyperthermia was shown to induce oxidative stress by uncoupling mitochondrial respiratory chain and to reduce superoxide dismutase (SOD) activity in muscles. Reactive carbonyl groups, malondialdehyde (MDA)–protein adducts, 3-nitrotyrosine immunoreactivity, Mn-SOD, and catalase were detected using immunoblotting in rat diaphragm specimens and homogenates thawed at room temperature (after previous storage at −80 °C) for 5, 15, 30, and 60 min, and 3, 6, and 24 h to be subsequently and immediately stored at −80 °C. Mn-SOD activity was also measured in all muscles. Both total protein carbonylation (reactive carbonyl groups and MDA–protein adducts) and nitration were significantly increased over time, reaching their peaks in the diaphragms of the 60- and 15-min groups, respectively. Mn-SOD expression and activity were significantly reduced over time, while catalase expression showed no significant variation. Protein oxidation was significantly increased in the rat diaphragms exposed to freezing–thawing cycles of different time lengths, while Mn-SOD was substantially reduced in all muscles.