BIF-induced ROS-mediated cytotoxicity and genotoxicity in embryonic cell culture of Daphnia magna

Bifenthrin (BIF) is a widely used synthetic pyrethroid insecticide that poses significant risks to the environment, particularly to aquatic ecosystems. In the present study, the cytotoxic and genotoxic effects of BIF on Daphnia magna cells were evaluated using in vitro methods. To achieve this, we developed a novel embryonic cell culture system from D.magna using Modified Schneider's Insect Medium (MSIM), which demonstrated remarkable viability for over two months. The lethal concentration 50 (LC₅₀) values of BIF were determined using this cell culture system through XTT (2,3-bis-(2‑methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide)assays, yielding values of 7.4 µg/mL and 4.3 µg/mL for 24 h and 48 h exposures, respectively. A fluorometric intracellular reactive oxygen species (ROS) assay was employed to measure ROS production, revealing that BIF exposure induced oxidative stress in a dose-dependent manner. The activities of Glutathione peroxidase (GPx), glutathione (GSH), and glutathione-S-transferase (GST) were significantly reduced, indicating oxidative damage. Co-treatment with N-acetylcysteine(NAC) mitigated these effects, restoring antioxidant enzyme activity and reducing (ROS) levels. Gene expression analysis via quantitative real-time PCR (qPCR) showed upregulation of stress-related genes (hsp70, hsp90) and antioxidant genes (Mn/ZnSod, cat) following exposure to LC₅₀ concentrations of BIF. However, prolonged exposure led to a downregulation of these genes, suggesting cumulative effects over time. The comet assay confirmed that BIF caused genotoxicity, as evidenced by significant increases in comet and tail lengths. Co-treatment with NAC effectively mitigated these genotoxic effects. This study highlighted the cytotoxic and genotoxic potential of BIF in aquatic organisms and suggested the need for environmentally friendly pest control strategies. Also, the findings confirmed the reliability of D. magna embryonic cell cultures for assessing the toxicological effects of environmental pollutants, offering new possibilities for in vitro toxicity testing at cellular and molecular levels.