Redox Imbalance and Epigenetic Reprogramming Underlie Polysubstance-Induced Neuronal Dysfunction.

Polysubstance use is a major contributor to neurotoxicity, yet the integrated redox-dependent mechanisms underlying polydrug exposure remain poorly understood. Cocaine (Coc), nicotine (Nic), and alcohol (Alc) are commonly co-abused substances associated with oxidative stress, mitochondrial dysfunction, and neuronal impairment. Here, we investigated the effects of Coc, Nic, Alc, Nic+Alc, and Coc+Nic+Alc exposure on redox homeostasis, mitochondrial function, and epigenetic regulation in mice brain and human microglial cells (HMC3). Individual and polydrug exposures increased reactive oxygen species, altered mitochondrial and antioxidant defense pathways, including GSS, SOD2, PGC-1α, TFAM, and Trx/TXNIP signaling. Polydrug exposure further reduced oxidative phosphorylation complexes, altered NRF1/NRF2-mediated mitochondrial pathways, and decreased Mfn1/2 expression in mice brain tissue, consistent with impaired mitochondrial function and fusion. In HMC3 cells, polysubstance exposure similarly increased oxidative stress, as demonstrated by MitoSOX, DHE, and DCFH-DA assays. These redox and mitochondrial alterations were accompanied by changes in DNMT and TET enzymes, consistent with altered epigenetic regulation. Behaviorally, both individual and polydrug exposures produced significant conditioned place preference. Notably, polydrug exposure was associated with reduced tyrosine hydroxylase immunoreactivity in the substantia nigra, along with altered dopamine transporter and dopamine D2 receptor expression, increased caspase-3 activation and ASC expression, and changes in microglial P2RY12 expression, together with enhanced neuroinflammatory responses. Collectively, these findings identify interconnected redox, mitochondrial, and epigenetic alterations associated with polysubstance-induced neuroadaptation and neurotoxicity. Although selected molecular pathways exhibited partial counter-regulation, persistent mitochondrial dysfunction, neuroinflammatory responses, and alterations in dopaminergic markers were observed following polysubstance exposure, highlighting potential therapeutic targets for substance use disorders.