Neurotransmitter-Related Alterations and Vascular-Associated Brain Injury in 2,4-Di-tert-butylphenol-Exposed Adult Zebrafish: A Putative AOP-Informed Framework.

Abstract

Although previous zebrafish studies have reported developmental, immune, intestinal, and reproductive effects of 2,4-di-tert-butylphenol (2,4-DTBP), evidence linking adult neurobehavioral outcomes with brain neurotransmitter-related changes and histopathological responses remains limited. Herein, we employed a zebrafish model subjected to a 21-day exposure across an environmentally informed and sublethal concentration range (0.01-1 μM), integrating a multi-tiered assessment that combined behavioral assays, biochemistry, molecular biology, histopathology, and computational toxicology. Our results showed reduced upper-zone exploration in the Novel Tank Test (p < 0.05) and endpoint-specific variation in social approach-related measures in the social preference test. These behavioral changes were accompanied by vascular-adjacent and parenchymal histopathological alterations in the brain. At the molecular level, 2,4-DTBP exposure altered cholinergic and serotonergic endpoints, including ACh accumulation, 5-HT depletion, and selective transcriptional changes involving slc5a7a and tph2. Notably, bdnf expression was markedly upregulated (p < 0.001), consistent with a stress-related or injury-associated transcriptional response. Molecular docking predicted a pose of 2,4-DTBP within the acetylcholinesterase (AChE) aromatic gorge, with a favorable Vina score (≈ -7.57 kcal/mol) and predicted π-π/hydrophobic interactions with aromatic residues. Collectively, these findings support the use of a putative AOP-informed framework to organize neurotransmitter-related alterations, vascular-associated and parenchymal histopathological changes, and anxiety-like behavioral responses in adult zebrafish exposed to 2,4-DTBP.