Oxidative stress, autophagy, and apoptosis induced by doxycycline in loach fin cells in vitro

Cytotoxicity, molecular function disorder, mitophagy, and apoptosis were studied in loach fin cells in vitro after exposure to doxycycline (DOX). The semi-lethal concentration of DOX in loach cells was calculated as 668.96 +/- 2.83 mol/L. Loss of cell viability and increases in vacuoles and autolysosomes were evident in cells exposed to DOX at 200 and 400 mu mol/L, and apoptotic bodies occurred at 600 mu mol/L. In addition, Superoxide Dismutase (SOD), catalase (CAT), Na+-K+-ATPase, and Ca2+-ATPase activities increased significantly in cells exposed to 200 mu mol/L DOX, and dose-dependent inhibitory effects on activities were observed in cells exposed to 400 and 600 mu mol/L DOX. Quantitative gene expression showed that 400 and 600 mu mol/L DOX could induce caspase-3- and caspase-8-mediated apoptosis as well as caspase-activated DNase in loach cells. Transcriptome sequencing in DOX vs. control groups found 16,288 differentially expressed genes, among which protein binding (2633, 31.91%) was the most significant in Gene Ontology terms. Furthermore, 11,930 genes were enriched in 298 Kyoto Encyclopedia of Genes and Genomes (KEGG)pathways. The top three upregulated pathways included lysosome , protein processing in endoplasmic reticulum , and proteasome . FPKM analysis indicated that most genes associated with autophagy and in protein processing in the endoplasmic reticulum , TNF signaling pathway , and NF-kappa B signaling pathway were upregulated. This suggests that at lower concentrations, DOX induces reactive oxidative species (ROS) in loach fin cells to reduce cell proliferation. ROS in turn stimulate oxidant stress, ion excretion capability and mitophagy to maintain cell homeostasis. Apoptosis was induced in cells subjected to higher concentrations of DOX. The transcriptome data and pathways determined in this study will provide a foundation for the analysis of DOX toxicity in loach cells, which must be examined thoroughly to further understand the cytotoxic mechanism of antibiotics in fish cells.

Automated summary

This study investigated the cytotoxicity, molecular function disorder, mitophagy, and apoptosis in loach fin cells exposed to doxycycline (DOX) in vitro. The results showed that DOX could induce the production of reactive oxidative species (ROS) at lower concentrations, leading to reduced cell proliferation, while higher concentrations of DOX induced cell apoptosis.