Hypoxia-cadmium interactions on rainbow trout (Oncorhynchus mykiss) mitochondrial bioenergetics: attenuation of hypoxia-induced proton leak by low doses of cadmium

Abstract

The goal of the present study was to elucidate the modulatory effects of cadmium (Cd) on hypoxia/reoxygenation-induced mitochondrial dysfunction in light of the limited understanding of the mechanisms of multiple stressor interactions in aquatic organisms. Rainbow trout (Oncorhynchus mykiss) liver mitochondria were isolated and energized with complex I substrates (malate-glutamate), and exposed to hypoxia (0>P O 2 <2 Torr) for 0-60 min followed by reoxygenation and measurement of coupled and uncoupled respiration and complex I enzyme activity. Thereafter, 5 min hypoxia was used to probe interactions with Cd (0-20 μmol l) and to test the hypothesis that deleterious effects of hypoxia/reoxygenation on mitochondria were mediated by reactive oxygen species (ROS). Hypoxia/reoxygenation inhibited state 3 and uncoupler-stimulated (state 3u) respiration while concomitantly stimulating states 4 and 4 ol (proton leak) respiration, thus reducing phosphorylation and coupling efficiencies. Low doses of Cd (≤5 μmol l −1 ) reduced, while higher doses enhanced, hypoxia-stimulated proton leak. This was in contrast to the monotonic enhancement by Cd of hypoxia/reoxygenationinduced reductions of state 3 respiration, phosphorylation efficiency and coupling. Mitochondrial complex I activity was inhibited by hypoxia/reoxygenation, hence confirming the impairment of at least one component of the electron transport chain (ETC) in rainbow trout mitochondria. Similar to the effect on state 4 and proton leak, low doses of Cd partially reversed the hypoxia/reoxygenation-induced complex I activity inhibition. The ROS scavenger and sulfhydryl group donor N-acetylcysteine, administrated immediately prior to hypoxia exposure, reduced hypoxia/reoxygenation-stimulated proton leak without rescuing the inhibited state 3 respiration, suggesting that hypoxia/reoxygenation influences distinct aspects of mitochondria via different mechanisms. Our results indicate that hypoxia/reoxygenation impairs the ETC and sensitizes mitochondria to Cd via mechanisms that involve, at least in part, ROS. Moreover, we provide, for the first time in fish, evidence for a hormetic effect of Cd on mitochondrial bioenergetics -the attenuation of hypoxia/reoxygenation-stimulated proton leak and partial rescue of complex I inhibition by low Cd doses.

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

“…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.…”

Retraction: Protective effect of SENP3-mediated SUMOylation against cadmium toxicity

“…In particular, cadmium promotes mitochondrial dysfunction (Xu et al, 2013) and can promote mitochondrial-dependent apoptosis (Adiele et al, 2011). More specifi cally, cadmium inhibits the electron transport chain complexes, resulting in mitochondrial uncoupling via mitochondrial permeability transition and increased generation of ROS (Templeton and Liu, 2010) and oxidative stress (Adiele et al, 2011;Onukwufor et al, 2014).…”

Bioenergetic and volume regulatory effects of mitoKATP channel modulators protect against hypoxia-reoxygenation induced mitochondrial dysfunction

“…Mitochondria were isolated according to the method of Onukwufor et al (2014) and re-suspended in a 1:3 (weight to volume) ratio of mitochondrial respiration buffer [MRB: 10 mmol l −1 Tris, 25 mmol l −1 KH 2 PO 4 , 100 mmol l −1 KCl, 1 mg ml −1 bovine serum albumin (BSA, fatty acid free), 2 µg ml −1 aprotinin, pH 7.3]. Mitochondrial protein concentration was measured by spectrophotometry (Spectramax Plus 384, Molecular Devices, Sunnyvale, CA, USA) according to Bradford (1976) with BSA as the standard.…”

“…The protocol used for H-R was based on the study of Onukwufor et al (2014). Briefly, mitochondrial complex-1-driven oxygen consumption was measured under normoxic conditions as described above.…”