Whole-transcriptome sequencing (RNA-seq) analyses of the zebrafish liver cell line, ZFL, after acute exposure to Cu2+ ions

Abstract

All cells require Cu as a cofactor, but Cu²⁺ induces toxicity and oxidative damage. A strict system is thus needed to maintain Cu homeostasis. Using the ZFL zebrafish liver cell line as a model, we studied the cellular responses after exposure to Cu²⁺, using whole-transcriptome shotgun sequencing (RNA-seq) to screen nearly all transcriptomes in cell samples and identify changes in gene expression. ZFL cells were treated with 100, 200, or 400 μM CuCl₂ and harvested after 4 and 24 h. RNA was then extracted and subjected to RNA-Seq and qPCR validation. Exposure to 400 μM CuCl₂ for 4 h and 24 h led to the regulation of 5993 and 4235 genes, respectively. In a gene ontology enrichment analysis, Cu²⁺ exposure enriched the nitrogen compound metabolic process and antioxidant activity but did not significantly affect cellular copper, zinc, iron and calcium ion homeostasis. In a KEGG pathway enrichment analysis, anti-oxidative stress induced the glutathione metabolism pathway. Furthermore, Cu²⁺ also induced genes related to apoptosis and arrested the cell cycle in the G2 phase. This study was based on the full gene expression profile combined with pathway analysis details, providing a full cellular response picture for Cu.