Copper Alters the Effect of Temperature on Mitochondrial Bioenergetics in Rainbow Trout, Oncorhynchus mykiss

Abstract

We investigated the interaction of temperature and copper (Cu) on mitochondrial bioenergetics to gain insight into how temperature fluctuations imposed by natural phenomena or anthropogenic activities would modulate the effects of Cu on cellular energy homeostasis. Mitochondria were isolated from rainbow trout livers and, in the first set of experiments, exposed to Cu (0-2.5 mM) at 5, 11, and 25 °C with measurement of mitochondrial complex II (mtCII)-driven respiration. In the second set of experiments, unenergized mitochondria were incubated for 30 or 60 min with lower concentrations (0-160 μM) of Cu to measure the effects on mtCII enzyme activity. Whereas maximal (state 3) respiration was inhibited by high Cu exposure, low Cu doses stimulated and high Cu doses inhibited resting (state 4) and 4ol (proton leak) respirations. High temperature alone increased mitochondrial respiration in all states. The Q10 values for state 3, state 4, and proton leak respirations suggested active processes with state 4 respiration and proton leak exhibiting greater thermal sensitivity than state 3 respiration. The differential thermal sensitivity of resting relative to phosphorylating mitochondrial state led to uncoupling and limitation of mitochondrial oxidative capacity at both high temperature and doses of Cu. Moreover, exposure to high Cu caused loss of thermal dependence of the mitochondrial bioenergetics culminating in Q10 values well below unity and decreased activation energies (E a) for both maximal and resting respiration rates. In addition, mtCII activity was increased by low and decreased by high doses of Cu indicating that direct effects on this enzyme contribute to Cu-induced mitochondrial dysfunction. Taken together, it appears that the substrate oxidation (electron transport chain and tricarboxylic acid cycle) and proton leak subsystems are targets of the deleterious effects of Cu and increased temperature on mitochondrial bioenergetics. However, mitochondrial effects of Cu and temperature may not be easily distinguished because they are generally qualitatively similar.

Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss

“…All of the latter studies reported that beyond a species-specific critical temperature the RCR decreased, thus lending support to the notion that the highest temperature (25 • C) tested in the present study was well-tolerated by rainbow trout liver mitochondria. Copper exposure on the other hand induced varying effects ranging from absence of effect to decreased or increased RCR depending on the temperature-Cu concentration combination, which is consistent with our recent study on complex II-driven respiratory flux (Sappal et al, 2014). These dissimilar changes in coupling can be explained by the inhibitory effect of high Cu and stimulatory effect of high temperature on maximal respiration, the stimulatory effect of low concentrations of Cu and high temperature on basal respiration, and the inhibitory effect of high Cu concentrations on basal respiration.…”

“…Because we did not observe inhibition of maximal respiration at 25 • C as would be expected at unfavorably high temperature (Zukiene et al, 2010;Guderley, 2011;Iftikar and Hickey, 2013;Blier et al, 2014) we concluded that under the experimental conditions employed, this temperature level alone did not constitute sufficient stress to impair mtCI-driven respiration in rainbow trout liver isolated mitochondria. Copper exposure on the other hand inhibited the maximal respiration in agreement with previous studies in rats (Saris and Skulskii, 1991;Belyaeva et al, 2004Belyaeva et al, , 2011, rainbow trout (Sappal et al, 2014) and bivalves (Collins et al, 2010;Ivanina and Sokolova, 2013). The inhibition of maximal respiration was consistently greater at higher temperature and interestingly, lower Cu concentrations caused greater inhibition at 5 • C compared with the putative control (15 • C).…”

“…Although high Q 10 values at lower temperatures are frequently reported for maximal mitochondrial respiration (Guderley and Johnston, 1996;Abele et al, 2002;Lemieux et al, 2010), a consistent theme remains elusive because the converse has also been reported (Chamberlin, 2004). Overall, regarding interactive effects, the present findings and those from other studies (Sokolova, 2004;Cherkasov et al, 2006;Sappal et al, 2014) suggest that temperature sensitizes mitochondrial maximal respiration to metals.…”

“…Livers were immediately dissected out, rinsed with mitochondrial isolation buffer [MIB: 250 mM sucrose, 10 mM Tris-HCl, 10 mM KH 2 PO 4 , 0.5 mM EGTA, 1 mg/ml BSA (fatty acid free), 2 g/ml aprotonin, pH 7.3], blotted dry and weighed. Thereafter mitochondrial isolation was done according to our routine procedure (Adiele et al, 2010;Onukwufor et al, 2014;Sappal et al, 2014). Briefly, 2 or 3 livers were diced into smaller pieces, pooled and homogenized on ice in 3 volumes of MIB using Potter-Elvehjem homogenizer (Cole Parmer, Anjou, QC) with a loosely fitting Teflon pestle.…”

“…Because the mitochondria are the primary sites of ROS generation in cells (Chen et al, 2003;Kowaltowski et al, 2009;Murphy, 2009), it is feasible that Cu can increase mitochondrial ROS generation and exacerbate damage to these organelles. Indeed, several studies have shown that mitochondria are sensitive Cu targets in endotherms and ectotherms (Saris and Skulskii, 1991;Sokol et al, 1993;Belyaeva et al, 2011;Sappal et al, 2014) with a key effect being impairment of oxidative phosphorylation (OXPHOS).…”