# High Doses of Norfloxacin Nicotinate Induce Apoptosis, Developmental Neurotoxicity, and Aberrant DNA Methylation in Zebrafish (Danio rerio) Larvae

## TL;DR

High doses of norfloxacin nicotinate harm zebrafish larvae by causing cell death, brain development issues, and DNA changes.

## Contribution

This study identifies new biological mechanisms of toxicity in zebrafish larvae exposed to high doses of NOR-N.

## Key findings

- High-dose NOR-N induces apoptosis in zebrafish larvae with increased Cas3 and Cas9 activity.
- Exposure to NOR-N disrupts DNA methylation and neurodevelopment-related gene expression in larvae.
- NOR-N exposure leads to developmental neurotoxicity and altered DNA methylation patterns.

## Abstract

Norfloxacin nicotinate (NOR-N), a derivative and effective substitute of norfloxacin (NOR), has been widely used in livestock production and aquaculture sectors. However, the adverse impacts of NOR-N on non-target animals and their associated biological response mechanisms are still not completely elucidated. This study revealed that exposure to the high doses of NOR-N induced apoptosis, developmental neurotoxicity and aberrant DNA methylation in zebrafish (Danio rerio) larvae. These findings contribute to uncovering the response mechanisms of fish to NOR-N exposure and enhancing understanding of the potential hazards that NOR-N may pose to aquaculture ecosystems.

This study aimed to evaluate the response mechanisms of zebrafish larvae to Norfloxacin nicotinate (NOR-N) exposure. Embryos were exposed to NOR-N from 4 h post-fertilization (hpf) until 96 hpf. The exposure concentrations included 0.002, 0.2, 1, and 5 mg/L (simulating both normal and exceptionally high environmentally relevant levels of NOR), as well as a high dose of 25 mg/L. Subsequent analyses focused on apoptosis, neurodevelopment, and DNA methylation in the resulting zebrafish larvae. The results showed that high-dose NOR-N (≥5 mg/L) induced obvious apoptotic cell death in zebrafish larvae, accompanied by increased activities of Cas3 and Cas9, up-regulated P53, Bax, Puma, Apaf1, Cas3 and Cas9 genes expression, and reduced Mdm2 levels and Bcl2/Bax ratio. Moreover, exposure to 5 and/or 25 mg/L NOR-N resulted in a significant up-regulation of neurodevelopment-related genes (Sox2, Sox3 and Sox19a), concomitantly with a marked decline in the transcription of DNA methylation genes, including Dnmt1, Dnmt3a1, Dnmt3b1, Dnmt3b2 and Dnmt3b4. Overall, our findings demonstrated that NOR-N exposure could induce apoptosis, developmental neurotoxicity and aberrant DNA methylation in zebrafish larvae. These findings provide insights to guide the safe application of NOR-N in aquaculture and support the assessment of its potential ecological risks to aquatic ecosystems.