Naphthenic acid fraction compounds disrupt bioenergetics, stimulate ROS, and induce necrosis in rainbow trout primary...

Naphthenic acid fraction compounds (NAFCs) are constituents of oil sands process-affected water (OSPW) and have been shown to impair mitochondrial bioenergetics in fishes. This study tested a model adamantane NAFC, 3,5-dimethyladamantane-1-acetic acid, and an extracted NAFC mixture from weathered OSPW for their effects on respiration, reactive oxygen species (ROS) generation, energy status, and cell death in rainbow trout (Oncorhynchus mykiss) primary hepatocytes. Hepatocytes were exposed for 24 h and assessed using high-resolution respirometry and fluorometry (Oroboros Oxygraph-2k FluoRespirometer) to quantify oxygen consumption and hydrogen peroxide (H₂O₂) emission, and complementary luminescent/fluorometric bioassays to quantify intracellular adenosine triphosphate (ATP) (CellTiter-Glo®), and apoptosis/necrosis (RealTime-Glo™ Annexin V). Both NAFC sources inhibited cellular oxygen consumption in a concentration-dependent manner, with the model NAFC being more potent (IC₅₀ 3.36 mM) than the OSPW-derived mixture (IC₅₀ 6.7 mM). Both NAFC sources increased H₂O₂ emission in hepatocytes, with similar potency in respirometry assays (EC₅₀s of 6.05 and 8.43 mM for commercial and extracted NAFCs, respectively). ATP was depleted at low millimolar concentrations (IC₅₀ 0.78 mM for the model NAFC; 1.77 mM for the extracted NAFC). Cell-death profiling indicated necrosis as the predominant outcome, with limited or inconsistent evidence of apoptosis across the NAFC exposure concentrations tested. Relative to intact hepatocytes, isolated mitochondria were markedly more sensitive to the commercial NAFC for both respiratory inhibition and H₂O₂ generation. These findings demonstrate that NAFC toxicity in hepatocytes is mediated by coupled bioenergetic suppression and oxidative disruption culminating in ATP depletion and necrotic cell death, and support adamantane carboxylic acids as potential mechanistic surrogates for selected oil sands NAFCs, while recognizing that OSPW mixtures are compositionally complex.