Mitochondrial oxidative stress and ion-channel suppression reveal a distinct neurotoxic profile of fentanyl analogues in a human neuronal model

Fentanyl analogues, including butyrylfentanyl (BUF) and 4-fluorobutyrylfentanyl (4F-BUF), are increasingly implicated in severe toxicity and fatal overdoses. While their pharmacological activity at µ-opioid receptors is well established, the cellular mechanisms underlying their neurotoxic effects remain incompletely understood, particularly with respect to mitochondrial dysfunction and its relationship with neuronal functional impairment. In this study, differentiated SH-SY5Y cells were used as a human neuronal model to evaluate the toxicodynamic effects of morphine, fentanyl, BUF, and 4F-BUF. Cell viability assays were performed to define sub-toxic concentrations, while oxidative stress was assessed by measuring cytoplasmic reactive oxygen species (ROS) and mitochondrial superoxide at early (25 min) and prolonged (24 h) time points. Functional alterations were examined using whole-cell patch-clamp recordings of voltage-gated inward and outward membrane currents. All compounds induced concentration-dependent cytotoxicity, with BUF and 4F-BUF exhibiting the lowest IC₂₅ values (8.39 ± 0.55 and 8.36 ± 0.67 µM, respectively), compared with fentanyl (37.87 ± 4.23 µM) and morphine (59.09 ± 4.72 µM). Cytoplasmic ROS levels increased similarly across all treatments, whereas mitochondrial superoxide generation displayed a time-dependent divergence, with 4F-BUF producing the highest mitochondrial superoxide levels after prolonged exposure (334.7 ± 26.1% of control), significantly exceeding fentanyl (281.6 ± 21.6% of control). Electrophysiological analysis revealed that fentanyl preferentially reduced inward currents, whereas BUF and 4F-BUF induced a broader suppression of both inward and outward membrane conductance. Importantly, mitochondrial ROS scavenging with mitoTEMPO markedly attenuated 4F-BUF-induced mitochondrial superoxide accumulation (278.6 ± 24.8% vs. 152.5 ± 7.8% of control) and prevented the associated suppression of voltage-gated membrane currents. These findings indicate that fentanyl analogues exert distinct cellular effects characterized by enhanced mitochondrial oxidative stress and associated alterations in membrane conductance. Notably, mitochondrial superoxide emerges as an indicator of fentanyl analogue toxicity, revealing differences not captured by cytoplasmic ROS measurements and highlighting mitochondrial dysfunction as a relevant target for toxicological evaluation.