Comparative In Vitro Assessment of Levothyroxine‐Induced Cytotoxicity, Oxidative Stress, and Genotoxicity in TM3 LEYDIG and TM4 SERTOLI Cells

ABSTRACT Levothyroxine, the synthetic form of thyroxine, is widely prescribed for hypothyroidism, including in men of reproductive age. While generally considered safe, its potential direct effects on male reproductive somatic cells remain poorly defined. This study investigated the in vitro reproductive toxicity of levothyroxine by evaluating its cytotoxic, oxidative, and genotoxic effects on murine TM3 Leydig and TM4 Sertoli cells. Cells were exposed to levothyroxine at 0.1–200 μM for 24 h. Cytotoxicity was assessed using MTT and neutral red uptake assays, intracellular ROS by DCFDA fluorescence, and DNA damage by alkaline comet assay. Levothyroxine significantly reduced cell viability in a concentration‐dependent manner. Based on MTT results, the IC 50 values were 121.22 μM for TM3 cells and 114.16 μM for TM4 cells, whereas neutral red uptake assays yielded IC 50 values of 104.16 and 104.61 μM, respectively. ROS levels increased with dose but did not reach statistical significance. Comet assay results revealed mild, nonsignificant DNA damage in TM3 cells at 100 μM and a nonmonotonic pattern in TM4 cells, peaking at 1 μM. These findings reveal subtle, cell‐type‐specific responses to supraphysiological exposure to levothyroxine. Importantly, TM4 Sertoli cells exhibited slightly greater sensitivity than TM3 Leydig cells across assays, consistent with their critical role in supporting the seminiferous epithelium. Although direct clinical extrapolation is not possible, this represents the first comparative analysis of levothyroxine's effects on Leydig and Sertoli cells in vitro. In conclusion, levothyroxine induces modest, dose‐dependent cytotoxicity and subtle oxidative/genotoxic alterations in testicular somatic cells, with Sertoli cells being more susceptible.