Effect of environmental stress on reactive oxygen species metabolism and oxidative stress response in rainbow trout (Oncorhynchus mykiss)

Aquatic organisms are exposed to different environmental stressors including temperature variations, low dissolved oxygen and metals pollution that may cause physiological perturbation attributed to altered mitochondrial function and dysregulation of reactive oxygen species (ROS) metabolism. Therefore, it is important to understand how these stressors act and interact to disrupt ROS homeostasis leading to oxidative stress. The study investigated the effects of temperature, anoxia-reoxygenation and cadmium (Cd) on rainbow trout (O. mykiss) liver mitochondrial ROS (as hydrogen peroxide, H2O2) production and consumption under different bioenergetic states. The overall hypothesis was that temperature, anoxia-reoxygenation and Cd will stimulate ROS emission and that the combined effect of Cd and temperature or anoxia-reoxygenation will heighten the individual stressor effects. The first study investigated the effect of temperature and Cd on H2O2 emission and consumption in liver mitochondria and showed that temperature and Cd acted cooperatively to increase the rate of H2O2 emission. In addition, Cd imposed different H2O2 emission response patterns depending on the concentration and substrate. Cd evoked a graded H2O2 emission response that plateaued at 5 μM for mitochondria oxidizing glutamate-malate but a biphasic concentration-response with a spike in H2O2 emission at 1 μM Cd followed by a gradual diminution at higher Cd concentration during succinate oxidation. The biphasic H2O2 emission response observed in mitochondria oxidizing succinate was due primarily to site IIF while the saturable graded H2O2 emission in mitochondria oxidizing glutamate-malate was consistent with the effect of Cd on site IF. However, Cd and temperature did not affect the kinetics of mitochondrial H2O2 consumption irrespective of the bioenergetic state. The second study investigated the biotic and abiotic factors that affect mitochondrial H2O2 emission and showed that optimization of assay conditions is critical for quantitation of maximal H2O2 emission. Moreover, the study showed that NADPH oxidase 4 is a major source of H2O2 emission in unenergized mitochondria. The third study showed that anoxia-reoxygenation attenuated H2O2 emission while Cd evoked monotonic responses for glutamate-malate and palmitoylcarnitine-malate, but evoked a biphasic response for succinate oxidizing mitochondria. Anoxia-reoxygenation more severely inhibited mitochondrial respiration with palmitoylcarnitine-malate compared with succinate or glutamate-malate. The effect of anoxia-reoxygenation and Cd on site-specific H2O2 emission depended on substrate and site. Contrary to the expectation, anoxia-reoxygenation blunted the effect of Cd on mitochondria site-specific H2O2 emission. The fourth study investigated the effect of Cd and anoxia-reoxygenation on mitochondria H2O2 emission during glycerol 3-phosphate (G3P) oxidation and showed that Cd evoked a low concentration stimulatory-high concentration inhibitory pattern of H2O2 emission that was blunted by anoxia-reoxygenation. The study showed that mitochondrial glycerol phosphate dehydrogenase activity was very low and that state 2 and state 3 respiration during oxidation of G3P was also low. Overall, the research unveiled the mechanisms by which temperature, anoxia-reoxygenation and Cd disrupt mitochondrial ROS metabolism, and increased the understanding of how these stressors cause oxidative stress