Chronic antibiotic use exacerbates cortico-hippocampal damage induced by aflatoxin B1 and diethylnitrosamine via oxidative stress and apoptotic pathways.

We examine how antibiotic misuse may induce gut dysbiosis, which influences the neurotoxicity of co-exposure to the environmental neurotoxicants Aflatoxin B1 (AFB1) and Diethylnitrosamine (DEN) in rats. Wistar rats (n = 45; male) were assigned to Control, DEN (200 mg/kg, i.p.), or AFB1 (2 mg/kg, i.p.) groups, each further divided into saline, Ampicillin (50 mg/kg; p.o), or Ciprofloxacin (12.5 mg/kg; p.o) treatments over 21 days-14 days of antibiotics followed by 7 days of AFB1 or DEN co-treatment. Rat behaviour was assessed through the open field test, sucrose splash test, and Morris's water maze. Biochemical analyses measured neurotransmitters, oxido-nitrosative stress (ONS), inflammation, and apoptosis markers in the prefrontal cortex and hippocampus. The mechanisms and pharmacokinetics of the compounds were investigated using network pharmacology, molecular docking, and ADMET analyses. Antibiotic co-treatment reduced locomotion and exploration, increased anxiety, and impaired memory and learning across all tests. The co-treated groups demonstrated increased AChE activity, decreased antioxidant biomarkers, and heightened ONS and apoptosis in the prefrontal cortex and hippocampus. Network pharmacology identified 31 common targets among antibiotics, AFB1/DEN, and oxidative-stress-related neurotoxicity. STAT3, HSP90AB1, EGFR, MMP2, KDR, and PDGFRB emerged as core hub genes. PPI and enrichment analyses highlighted their roles in kinase signalling, cell death regulation, and cancer pathways. Molecular docking indicated strong binding of AFB1 and CPX to HSP90AB1 and EGFR, suggesting potential interference with oxidative and apoptotic pathways. ADMET assessment showed antibiotics possess favourable drug-like properties, whereas AFB1 and DEN are more toxic, lipophilic, and neurotoxic, supporting their role in cortico-hippocampal injury. Antibiotic misuse may intensify the neurotoxic effects of AFB1 and DEN by increasing oxido-inflammatory responses and neuronal damage, leading to memory impairment and heightened anxiety-like behaviours. Addressing antibiotic misuse and promoting gut microbiota could improve brain health.