Cyproconazole‐Induced Systemic Neuro‐Immunotoxicity: An Integrated In Vivo and In Silico Study of Oxidative Stress, NLRP3‐Related Inflammatory Signaling, and Cholinergic Disruption

Cyproconazole (CYPRO), a widely used triazole fungicide, is detected across food and environmental matrices, raising concerns about chronic low-dose human exposure. Its potential to disrupt the neuro-immune interface, particularly the cross-talk between cholinergic signaling and the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, remains poorly characterized. The objective of this study is to establish an integrated neuro-immunotoxicological profile of CYPRO in rats and explore molecular associations linking oxidative stress, inflammasome-related signaling, and cholinergic disruption. Male Wistar rats received oral CYPRO (4, 8, 16 or 32 mg/kg body weight (bw)/day) for 28 days in a subacute exposure design. Endpoints comprised hematology, plasma lipid profile, tissue redox status, acetylcholinesterase (AChE) activity, NLRP3/nuclear factor-κB (NF-κB)-related gene expression, p53/Caspase-3 immunohistochemistry, alkaline comet assay, brain and lymphoid histopathology, and in silico docking against eight inflammatory and apoptotic protein targets. CYPRO induced dose-dependent dyslipidemia, complex leukocyte dysregulation (lymphocytosis, monocytosis, and neutropenia), and oxidative damage in the brain, spleen, and thymus. Systemic AChE inhibition extended to the lymphoid organs. Nlrp3, Casp1, Nfkb1, Tnfα, Il1β, and Il18 mRNA were dose-dependently upregulated, accompanied by p53 and Caspase-3 induction in lymphoid tissues and severe cortical and cerebellar lesions. DNA damage in the spleen and brain reached significance from the lowest dose tested (4 mg/kg/day), below the current No Observed Adverse Effect Level (NOAEL). Docking predicted favorable binding of CYPRO to inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and NLRP3, with engagement of COX-2 (Tyr385 and Ser530) and Caspase-1 (Cys285) catalytic residues. Together, these findings suggest a plausible neuro-immunotoxic profile for CYPRO, in which oxidative stress and AChE inhibition are associated with NLRP3-related transcriptional responses and downstream genotoxic and apoptotic signals. A putative cholinergic-inflammasome cross-talk is proposed as a working hypothesis warranting direct mechanistic validation.