The rapid expansion of wireless technologies has raised concerns regarding the biological effects of radiofrequency radiation on reproductive health. This study evaluated the impact of radiofrequency electromagnetic radiation from mobile phones (4 G) and a signal generator operating at 1800 MHz and 2450 MHz for 15–120 min on mitochondrial function in TM3 Leydig cells. Flow cytometry with JC-1 dye revealed frequency- and time-dependent mitochondrial membrane potential (Δψm) depolarization. Corresponding adenosine triphosphate (ATP) assay indicated impaired mitochondrial bioenergetics after 30 min of irradiation across all exposure conditions. Pharmacological modulation revealed frequency-dependent variations in oxidative stress responses. DPI significantly reduced ROS under mobile phone and 2450 MHz exposure but showed no significant effect on superoxide levels, suggesting a comparatively limited contribution of NOX-derived oxidative stress. In contrast, Mito-TEMPO significantly reduced ROS levels across all exposure conditions and reduced superoxide levels under mobile phone and 2450 MHz exposure, while significant restoration of Δψm was observed only under 2450 MHz exposure, suggesting mitochondria as a predominant source of oxidative stress in RF-EMR-exposed Leydig cells. Rapamycin partially stabilized Δψm, whereas bafilomycin A1 and hydroxychloroquine aggravated mitochondrial depolarization, suggesting possible involvement of autophagy-associated mitochondrial quality control pathways. Collectively, these findings demonstrate that short-term RF-EMR exposure induces frequency-dependent mitochondrial dysfunction in TM3 Leydig cells through mechanisms involving oxidative stress and possible alterations in autophagy-associated cellular responses.
