Effects of bioenergetics, temperature and cadmium on liver mitochondria reactive oxygen species production and...

A by-product of mitochondrial substrate oxidation and electron transfer to generate cellular energy (ATP) is reactive oxygen species (ROS). Superoxide anion radical and hydrogen peroxide (H₂O₂) are the proximal ROS produced by the mitochondria. Because low levels of ROS serve critical regulatory roles in cell physiology while excessive levels or inappropriately localized ROS result in aberrant physiological states, mitochondrial ROS need to be tightly regulated. While it is known that regulation of mitochondrial ROS involves balancing the rates of production and removal, the effects of stressors on these processes remain largely unknown. To illuminate how stressors modulate mitochondrial ROS homeostasis, we investigated the effects of temperature and cadmium (Cd) on H₂O₂ emission and consumption in rainbow trout liver mitochondria. We show that H₂O₂ emission rates increase with temperature and Cd exposure. Energizing mitochondria with malate-glutamate or succinate increased the rate of H₂O₂ emission; however, Cd exposure imposed different patterns of H₂O₂ emission depending on the concentration and substrate. Specifically, mitochondria respiring on malate-glutamate exhibited a saturable graded concentration-response curve that plateaued at 5 μM while mitochondria respiring on succinate had a biphasic concentration-response curve characterized by a spike in the emission rate at 1 μM Cd followed by gradual diminution at higher Cd concentrations. To explain the observed substrate- and concentration-dependent effects of Cd, we sequestered specific mitochondrial ROS-emitting sites using blockers of electron transfer and then tested the effect of the metal. The results indicate that the biphasic H₂O₂ emission response imposed by succinate is due to site II_F but is further modified at sites I_Q and III_Qo. Moreover, the saturable graded H₂O₂ emission response in mitochondria energized with malate-glutamate is consistent with effect of Cd on site I_F. Additionally, Cd and temperature acted cooperatively to increase mitochondrial H₂O₂ emission suggesting that increased toxicity of Cd at high temperature may be due to increased oxidative insult. Surprisingly, despite their clear stimulatory effect on H₂O₂ emission, Cd, temperature and bioenergetic status did not affect the kinetics of mitochondrial H₂O₂ consumption; the rate constants and half-lives for all the conditions tested were similar. Overall, our study indicates that the production processes of rainbow trout liver mitochondrial H₂O₂ metabolism are highly responsive to stressors and bioenergetics while the consumption processes are recalcitrant. The latter denotes the presence of a robust H₂O₂ scavenging system in liver mitochondria that would maintain H₂O₂ homeostasis in the face of increased production and reduced scavenging capacity.