Cytotoxic, genotoxic, and apoptotic effects of manganese nitrate on RAW264.7 macrophages via reactive oxygen species accumulation and mitochondrial injury

BACKGROUND: Manganese (Mn) is an essential trace element involved in multiple physiological processes. However, excessive Mn exposure is well known to induce neurotoxicity and has been extensively studied in neuronal and glial models. In contrast, the effects of manganese nitrate (Mn(NO₃)₂) on innate immune cells, particularly macrophages, remain poorly understood. Given the critical role of macrophages in immune regulation and inflammation, this study investigated the cytotoxic, genotoxic, and apoptotic effects of Mn(NO₃)₂ in RAW264.7 macrophages, with emphasis on oxidative stress, mitochondrial injury, and apoptotic signalling pathways. METHODS: Macrophages were treated with various concentrations of Mn(NO₃)₂. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and genotoxic effects were analysed using micronucleus and comet assays. Apoptosis was quantified using sub-G1 DNA analysis, Annexin V/PI staining, and the TUNEL assay. Furthermore, intracellular reactive oxygen species (ROS) generation, mitochondrial membrane potential, caspase activities, and apoptosis-related protein (BCL2, BAD, Fas, and TNFR1) expression were evaluated. FINDINGS: Mn(NO₃)₂ exposure was associated with concentration-dependent reductions in cell viability, along with increases in micronucleus formation, DNA strand breaks, and nuclear fragmentation. Elevated ROS generation and mitochondrial depolarisation levels were also observed. Moreover, BCL2 downregulation, BAD upregulation, and caspase-3, caspase-8, and caspase-9 activation were observed. Fas and TNFR1 upregulation confirmed the involvement of both intrinsic and extrinsic apoptotic pathways. CONCLUSION: In conclusion, the findings demonstrate that Mn(NO₃)₂ induces cytotoxicity, genotoxicity, and apoptosis in RAW264.7 macrophages through ROS-mediated mitochondrial dysfunction. Mn(NO₃)₂ activates both intrinsic and extrinsic apoptotic pathways, as evidenced by caspase-3/caspase-9 and caspase-8 activation along with BCL2 downregulation, BAD upregulation, and Fas and TNFR1 expression alterations. These findings provide new mechanistic insights into Mn-induced immunotoxicity.