Haloperidol Triggers RAW264.7 Macrophages Injury through Oxidative Stress-Driven Mitochondrial Dysfunction, Genomic Instability, and Apoptotic Cell Death.

Background and Objective: Haloperidol is a widely used first-generation antipsychotic for the management of schizophrenia and acute neurocognitive disorders. However, its potential immunotoxic effects remain poorly understood. This study aimed to investigate the cytotoxic and genotoxic mechanisms of haloperidol in RAW264.7 macrophages, focusing on oxidative stress, mitochondrial dysfunction, and apoptosis. Methods: RAW264.7 macrophages were treated with haloperidol (0-150 μM). Cell viability (MTT), DNA damage (micronucleus and comet assays), ROS production, mitochondrial membrane potential, cytochrome c release, apoptosis (annexin V/PI), death receptor expression and caspase activity were evaluated. N-acetylcysteine (NAC)-based pharmacological rescue was further performed to assess the involvement of ROS in haloperidol-induced cytotoxicity, intracellular ROS generation, and caspase-3 activity. Results: Haloperidol induced significant cytotoxicity, with marked effects observed at concentrations ≥50 μM. DNA damage was evidenced by increased micronucleus formation and elevated comet assay parameters. ROS levels were significantly increased, accompanied by mitochondrial membrane depolarization and cytochrome c release. In addition, apoptotic and necrotic cell populations and sub-G1 accumulation were markedly elevated. Mechanistically, both intrinsic and extrinsic apoptotic pathways were activated, as indicated by increased caspase-9, caspase-8, and caspase-3 activities, along with upregulation of Fas and TNFR. NAC pretreatment attenuated haloperidol-induced ROS accumulation, caspase-3 activation, and loss of cell viability, suggesting that ROS accumulation contributes, at least in part, to haloperidol-induced macrophage injury. Conclusions: The present study demonstrates that haloperidol induces cytotoxicity and DNA damage in macrophages through oxidative stress-mediated mitochondrial dysfunction and activation of caspase-dependent apoptotic pathways under high-exposure in vitro conditions. These findings identify macrophages as a potential target of haloperidol toxicity and highlight the importance of considering immune-related effects in safety evaluation.