# Butylated Hydroxyanisole (BHA) Disrupts Brain Signalling in Embryo–Larval Stage of Zebrafish Leading to Attention Deficit Hyperactivity Disorder (ADHD)

## TL;DR

This study shows that BHA, a common preservative, harms zebrafish embryos and larvae, causing ADHD-like symptoms through disrupted brain signaling and oxidative stress.

## Contribution

The study reveals BHA's impact on zebrafish brain development and behavior, linking it to ADHD-like effects at low concentrations.

## Key findings

- BHA exposure caused developmental deformities and reduced hatching and heart rates in zebrafish embryos.
- BHA led to increased apoptosis and oxidative stress, with reduced antioxidant enzyme activity in larvae.
- BHA decreased AChE activity, serotonin levels, and gene expression of DRD4, COMT, 5-HTR1aa, and BDNF, causing anxiety and memory impairment.

## Abstract

Background: Butylated hydroxyanisole (BHA) has been extensively used in several commercial industries as a preservative. It causes severe cellular and neurological damage affecting the developing fetus and might induce attention deficit hyperactivity disorder (ADHD). Methods: Zebrafish embryos were subjected to five distinct doses of BHA—0.5, 1, 2, 4, and 8 ppb up to 96 h post fertilization (hpf). Hatching rate, heart rate, and body malformations were assessed at 48 hpf, 72 hpf, and 48–96 hpf, respectively. After exposure, apoptotic activity, neurobehavioral evaluation, neurotransmitter assay, and antioxidant activity were assessed at 96 hpf. At 120 hpf, the expression of genes DRD4, COMT, 5-HTR1aa, and BDNF was evaluated by real-time PCR. Results: BHA exposure showed a delay in the hatching rate and a decrease in the heart rate of the embryo when compared with the control. Larvae exhibited developmental deformities such as bent spine, yolk sac, and pericardial edema. A higher density of apoptotic cells was observed in BHA-exposed larvae at 96 hpf. There was a decline in catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and superoxide dismutase (SOD) activity, indicating oxidative stress. There was a significant decrease in Acetylcholinesterase (AChE) activity and serotonin levels with an increase in concentration of BHA, leading to a dose-responsive increase in anxiety and impairment in memory. A significant decrease in gene expression was also observed for DRD4, COMT, 5-HTR1aa, and BDNF. Conclusions: Even at lower concentrations of BHA, zebrafish embryos suffered from developmental toxicity, anxiety, and impaired memory due to a decrease in AChE activity and serotonin levels and altered the expression of the mentioned genes.