Abstract: OBJECTIVE: The objective of this study was to investigate the hepatotoxicity of ceftriaxone and its impurities in zebrafish. METHODS: Wild-type strain zebrafish at 72 h post-fertilization(hpf) and transgenic zebrafish Tg(fabp10a:Ds Red) with liver-specific fluorescence labeling were chosen as experimental animals.Zebrafish larvae were treated with different concentrations of ceftriaxone(at concentrations of 1, 2, 5 mmol/L) and its impurities A, B, C, D and impurities E(at concentrations of 0.1, 0.5, 1 mmol/L) for 2 days. The liver morphology of wild-type zebrafish larvae and the fluorescence intensity of transgenic zebrafish larvae were observed, and the hepatotoxicity of each group was assessed by comparison with the control group. Whole-body Oil Red O staining was employed to observe changes in liver fat content. Additionally, transcriptomic sequencing was performed to detect the gene expression profiles of zebrafish in each treatment group. Differential expression genes were screened(735 differential genes in ceftriaxone group, 237 in impurity A group and 237 in impurity C group), and Gene and Genomes(KEGG) pathway enrichment analysis was conducted. RESULTS: Ceftriaxone and impurities A and C caused an enlargement of the zebrafish liver region or an increase in fluorescence intensity compared to the control group(P<0.05 or 0.01). Impurities B, D, and E primarily resulted in a reduction of the liver region or a decrease in fluorescence intensity compared to the control group(P<0.05 or 0.01). Overall Oil Red O stain assays indicated that both ceftriaxone and its impurities could cause an increase in liver fat. Differential gene expression was observed in each treatment group through transcriptome sequencing.KEGG pathway analysis revealed different pathway enrichments in each group. Genes related to ceftriaxone were mainly enriched in 10 pathways, including metabolism. Genes related to impurity A were mainly enriched in signaling pathways such as tryptophan metabolism. Genes related to impurity C were mainly enriched in signaling pathways, including calcium signaling. CONCLUSION: The study provides insights into the hepatotoxic effects of ceftriaxone and its impurities in zebrafish, demonstrating varied impacts on liver morphology, fluorescence intensity, and gene expression profiles. The findings highlight potential pathways through which these substances may induce hepatotoxicity.

Key words: ceftriaxone, hepatotoxicity, zebrafish, differentially expressed genes, impurity