Toxicity of fluorochloridone on BRL-3A cells

Background Fluorochloridone (FLC) is a widely used herbicide. Previous studies have found that FLC has potential hepatotoxicity, but associated effects and mechanisms are rarely reported.

Objective This experiment explores the toxic effect and underlying mechanism of FLC on rat BRL-3A cells.

Methods BRL-3A cells were exposed to FLC (0.1, 1, 10, and 100μmol·L^-1) for 24h, then the 1, 10, and 100 μmol·L^-1 FLC showed significant cytotoxicity by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and were used for further experiments. The levels of reactive oxygen species (ROS) in hepatocytes induced by FLC were detected by immunofluorescence. The mRNA expression levels of superoxide dismutase (SOD2) and glutathione peroxidase (GSH-Px) were detected by reverse transcription-polymerase chain reaction (RT-PCR). The mortality and apoptosis rates of cells induced by FLC were detected by flow cytometry. The protein expression levels of cyclooxygenase-3 (Cox-3), as well as apoptosis-related proteins Bax and B-cell lymphoma-2 (Bcl-2) were detected by Western blotting.

Results Compared with the control BRL-3A cells, the survival rates of cells exposed to 1, 10, and 100 μmol·L^-1 FLC decreased by (91.4±4.2) %, (73.5±13.1) %, and (66.7±5.5) %, respectively (P < 0.05). Exposure to 10μmol·L^-1 FLC increased the intracellular ROS level to (126.2±12.4) % (P < 0.05). After the 1-100μmol·L^-1 FLC treatment, the mRNA expression levels of SOD2 were decreased to (29.8±2.1) %, (25.5±2.2) %, and (6.6±0.4) %, and the mRNA expression levels of GSH-Px were decreased to (63.6±4.2) %, (49.9±4.1) %, and (26.5±2.1) %, respectively (P < 0.001). The FLC exposure at 10 and 100 μmol·L^-1 significantly elevated apoptosis rates to 2.2±0.1 and 12.5±0.8 times and the protein expression levels of Bax to 1.2±0.1 and 1.4±0.1 times of the control group (P < 0.001), while decreased the protein expression levels of Bcl-2 to (41.6±4.0) % and (41.9±3.2) %, the ratio of Bcl-2/Bax to (35.2±4.0) % and (30.9±3.1) %, and the protein expression levels of Cox-3 to (84.1±8.0) % and (54.2±4.2) % respectively.

Conclusion FLC has a significant toxic effect on BRL-3A cells, and the effect increases with a higher FLC concentration. The toxic mechanism of FLC is to induce oxidative damage of hepatocytes, and then promote apoptosis.