Metals exposure dissociates mitochondrial NAD(P)H redox state from H₂O₂ emission

Mitochondria integrate energy metabolism and redox signaling through NAD(P)H-dependent processes that regulate reactive oxygen species (ROS) production. While the thermodynamic relationship between NAD(P)H redox state and hydrogen peroxide (H₂O₂) emission is well-established under physiological conditions, how environmental stressors disrupt this coupling remains poorly understood. This study investigated how exposure to copper (Cu), cadmium (Cd), and zinc (Zn), individually and in binary mixtures, affects the coupling between NAD(P)H redox state and H₂O₂ emission in rainbow trout (Oncorhynchus mykiss) heart mitochondria. Using substrate-specific assays and electron transport system inhibitors, we quantified NAD(P)H autofluorescence and H₂O₂ production at five redox sites associated with 2-oxoacid dehydrogenases and complex I. Results revealed that effects of metals were highly context-dependent, varying with substrate type, redox site, metal concentration, and co-exposure conditions, with antagonism predominating in binary mixtures. Under control conditions, NAD(P)H redox state and H₂O₂ emission were moderately correlated (R² = 0.53 for all sites and R² = 0.41 for whole pathways), with the correlation being stronger for site-specific measurements. Cu and Cd predominantly oxidized NAD(P)H pool, decreasing the degree of reduction by up to 94% whereas Zn frequently increased NAD(P)H reduction. Effects on H₂O₂ emission were complex, with metals exhibiting biphasic patterns at several sites. Critically, metals exposure weakened the positive correlation between NAD(P)H redox state and H₂O₂ emission, lowering the overall R² to 0.36 and site-specific values to 0.00-0.28. These findings demonstrate that metals do not simply impose uniform oxidative stress but rather alter ROS production through mechanisms that decouple it from NAD(P)H redox status.