Neurotoxic effects of synthetic phenolic antioxidants on dopaminergic, serotoninergic, and GABAergic signaling in larval zebrafish (Danio rerio)

Synthetic phenolic antioxidants (SPAs) are an environmental concern because they are widely detected in aquatic ecosystems and can pose potential threats to organisms. Studies have reported developmental deficits and behavioral changes in response to SPAs, indicating possible neurotoxic effects. However, their neuroactive potency as well as their mode of action (MoA) remain unclear. As such, this study evaluated the potential neurotoxicity of three SPAs [butylated hydroxytoluene (BHT), 2,4-di-tert-butylphenol (2,4-DTBP), and 4-tert-octylphenol (4-t-OP)] at three concentrations (0.01, 0.1 and 1 μM) to zebrafish larvae. Both 2,4-DTBP and BHT decreased spontaneous tail coiling (STC) at 28 hpf (hours post fertilization) whereas 4-t-OP increased STC. Locomotor activity, based on the velocity and distance of larvae (144 hpf) travelled, was promoted by 2,4-DTBP while it decreased in larvae with exposure to 4-t-OP and BHT. In the light-dark preference assay, exposure to either 2,4-DTBP or BHT resulted in variability in the visiting frequency to the dark zone, and larvae (144 hpf) spent less time in the dark, suggesting anxiety-like behavior. Conversely, zebrafish exposed to 4-t-OP, especially at 1 μM concentration, were hypoactive and spent more time in dark, suggestive of anxiolytic-like responses. RNA-seq was conducted to discern mechanisms underlying behavioral responses. Transcriptomic analysis revealed that gene networks related to neuroactive ligand-receptor interaction as well as neurotransmitter-related pathways were altered by all three SPAs based on gene set and subnetwork enrichment analysis. Modulation of dopaminergic, serotoninergic, and/or GABAergic signaling at the transcript level was noted for each of the three SPAs, but different expression patterns were observed, indicating SPA- and dose-specific responses of the transcriptome. The present study provides novel insight into potential mechanisms associated with neurotoxicity of SPAs congeners.