Rotenone‐Induced Cytotoxicity in Human Umbilical Vein Endothelial Cells (HUVECs): Involvement of Oxidative Stress, Mitochondrial Dysfunction, and Intrinsic Apoptosis

ABSTRACT Rotenone is a well‐defined inhibitor of Mitochondrial Complex I, which induces oxidative stress and apoptosis in various cell types. However, its cytotoxicity and molecular mechanisms in human umbilical vein endothelial cells (HUVECs) remain unexplored. Therefore, the purpose of this study was to examine the effects of rotenone on HUVECs. Cells were exposed to rotenone at concentrations of 5–100 μM for 24 h. Cell viability and cytotoxicity were assessed using MTT and LDH assays, respectively. Morphological alterations were examined using light microscopy. Further, using cytotoxic doses of rotenone, glutathione (GSH) levels, lipid peroxidation (LPO), catalase activity, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Apoptosis was assessed by measuring caspase‐3 and caspase‐9 activities, and the mRNA expression of apoptosis‐related genes (caspase‐3, caspase‐9, Bax, Bcl‐2, p53, and p21) was analyzed using quantitative real‐time PCR. Our results showed that rotenone induced concentration‐dependent cytotoxicity in HUVECs. Morphological studies showed cell shrinkage, membrane damage, and cell detachment after rotenone treatment. Rotenone exposure caused oxidative stress in HUVECs, as evidenced by a significant increase in ROS and LPO levels and depletion of GSH and catalase activity. The loss of MMP observed upon rotenone treatment suggests mitochondrial dysfunction. Moreover, rotenone significantly increased the activities of caspase‐3 and caspase‐9; enhanced the mRNA expression of caspase‐3, caspase‐9, Bax, p53, and p21; and reduced the expression of Bcl‐2, which further confirmed the induction of apoptosis through mitochondrial signaling. Overall, rotenone caused substantial cytotoxicity in HUVECs through oxidative stress, mitochondrial dysfunction, and activation of the intrinsic apoptotic pathway.