In vitro exposure to atomoxetine hydrochloride reduces testosterone biosynthesis, induces cell death and DNA damage, as well as altering the oxidative profile and gene expression in TM3 Leydig cells

Atomoxetine, a drug used in the treatment of Attention Deficit/Hyperactivity Disorder, acts by increasing the availability of neurotransmitters, which can act on the male genital system. The effects of this substance on Leydig cells, which are responsible for testosterone production, have not been described. This study aimed to investigate the in vitro effects of atomoxetine hydrochloride (250, 1000 or 1750 ng/mL) on DNA damage (4 h), gene expression (12 h ), and after 24 h, cell viability and death, oxidative profile, cytokines and testosterone levels, and the colony-forming capacity in TM3 Leydig cells. Although cell viability was not altered, a reduction in testosterone biosynthesis, an increase in apoptosis and necrosis, a decrease in clonogenic capacity, and an increase in DNA damage were observed at higher concentrations. An alteration in the redox profile was noted, evidenced by a decrease in reduced glutathione and catalase enzyme activity, an increase in total, oxidized, and S-transferase glutathione enzymes, in addition to superoxide dismutase activity and increased lipid peroxidation. Genes involved in apoptotic pathways ( BAX , Tp53 ) were upregulated, as were those related to oxidative stress ( HO-1 and Nrf2 ). Conversely, the androgen receptor gene ( AR ), especially at the concentration of 1000 ng/mL, downregulated. Furthermore, there was a reduction in IL-33 levels. Even without directly compromising cell viability, possibly due to a limitation of the MTT test, or altering levels of TNF-α and IL-1β, atomoxetine hydrochloride caused important functional alteration in Leydig cells through oxidative stress, genotoxicity, and cytostatic effects, with potential implications for male reproductive function. • Atomoxetine hydrochloride (ATX) impaired the oxidative profile in TM3 Leydig cells. • ATX caused DNA damage and apoptosis without inflammatory cytokine activation. • ATX reduces testosterone synthesis and clonogenic potential. • Findings reveal potential reproductive risks associated with atomoxetine exposure.