Naphthenic Acid-Induced ROS Emissions by Rainbow Trout Mitochondria

Naphthenic acid fraction compounds (NAFC) are prevalent in petrochemical wastewater, including from oil sands processing, and induce reactive oxygen species (ROS) emissions in isolated mitochondria. The purpose of this study was to verify if a primary carboxylic acid, the moderately hydrophobic NAFC 3,5-dimethyladamantane-1-acetic acid, would cause the mitochondrial ROS (hydrogen peroxide; H₂O₂) production and affect its consumption by mitochondria in multiple bioenergetic states. Intact mitochondria isolated from rainbow trout (Oncorhynchus mykiss) liver were exposed to commercially available 3,5-dimethyladamantane-1-acetic acid. The emission of ROS during States 3 and 4 respiration was quantified using fluorometry with an Oroboros fluorespirometer. Subsequently, select ROS emission sites in the mitochondrial complexes were isolated using inhibitors, and the ROS emission of each site was measured using the Amplex UltraRed-horseradish peroxidase (AUR-HRP) system. The compound 3,5-dimethyladamantane-1-acetic acid was equally potent in causing ROS emission in State 3 and State 4 ROS sites. The baseline (no NAFC) proportion of ROS emission by site was II_F > I_Q > III_Qo > I_F. The 3,5-dimethyladamantane-1-acetic acid compound increased ROS emission in a dose-dependent manner at II_F with an EC₅₀ of 0.2 mM, which was not significantly different than the State 3 and 4 Oroboros response. In contrast, there was no consistent concentration-effect response at the other three ROS sites (I_Q, III_Qo, and I_F). Malonate, an inhibitor of succinate dehydrogenase, eliminated ROS production in Oroboros experiments. These findings identify site II_F as the predominant source of NAFC-stimulated ROS and provide mechanistic insight into how adamantane-type NAFCs impair mitochondrial redox balance in fishes.