Copper, cadmium, and zinc trigger multifaceted effects and interactions on cardiac mitochondrial bioenergetics and reactive oxygen species production in rainbow trout.

Mitochondria are key intracellular targets and mediators of metals toxicity. We probed the effects of copper (Cu), cadmium (Cd), zinc (Zn), and their binary mixtures on mitochondrial bioenergetics and reactive oxygen species (ROS: hydrogen peroxide, H₂O₂) emission in permeabilized rainbow trout (Oncorhynchus mykiss) cardiac fibers. Mitochondria were fueled with glutamate-malate in state 2 (LEAK) and state 3 (OXPHOS) conditions and exposed to the metals singly and in binary combinations. Respiration and H₂O₂ emission were measured simultaneously using an Oroboros O2k fluorespirometer. Zn demonstrated greater potency in inhibiting cardiac mitochondrial OXPHOS than Cu or Cd (IC₅₀ values, μM: Zn 13.4; Cd 114; Cu 178). Both Cu and Cd stimulated LEAK respiration but inhibited OXPHOS, while Zn suppressed both respiratory states. Cd antagonized Cu-induced OXPHOS inhibition and LEAK stimulation caused by Cu or Zn. Cu elicited biphasic H₂O₂ emission patterns in LEAK and OXPHOS states. Cu and Cd interacted antagonistically on H₂O₂ emission, while Cu and Zn acted synergistically to enhance LEAK H₂O₂ emission. Cd and Zn showed partial additivity on LEAK H₂O₂ emission but no interaction during OXPHOS. H₂O₂ emission was consistently higher in LEAK state than OXPHOS. Metals exposure expanded the dynamic range of respiration and H₂O₂ emission by up to 9-fold and 19-fold, respectively, with a weak negative correlation (R² = 0.11) between the two. These findings demonstrate that the effects of Cu, Cd, Zn, and their binary mixtures on mitochondrial function depend on mitochondrial metabolic state, metal identity, concentration, and inter-metal interactions.