氟咯草酮对肝细胞BRL-3A的毒性作用
Toxicity of fluorochloridone on BRL-3A cells
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摘要:
背景 氟咯草酮（FLC）是一种广泛使用的除草剂。动物毒理研究发现FLC具有潜在肝毒性，但对FLC暴露肝细胞的毒性作用和机制的研究十分有限。
目的 探索FLC对大鼠肝细胞BRL-3A的毒性作用和潜在毒性机制。
方法 采用四甲基偶氮唑盐微量酶反应比色法（MTT法）考察0.1、1、10、100 μmol·L-1 FLC染毒24 h后对BRL-3A肝细胞活性的影响，确定后续试验染毒浓度为1、10、100 μmol·L-1。应用免疫荧光技术检测肝细胞内的活性氧（ROS）水平的变化，用逆转录聚合酶链反应（RT-PCR）方法检测锰超氧化物歧化酶（SOD2）和谷胱甘肽过氧化物酶（GSH-Px）的mRNA表达水平，采用流式细胞术检测肝细胞的死亡率及凋亡率，采用Western blotting法检测环氧合酶-3（Cox3）、凋亡促进因子Bax和B-淋巴细胞瘤-2（Bcl-2）蛋白的表达水平。
结果 相对于对照组：1、10、100 μmol·L-1浓度FLC染毒肝细胞BRL-3A后，细胞存活率分别为（91.4±4.2）%、（73.5±13.1）%和（66.7±5.5）%（均
P < 0.05）；10 μmol·L-1染毒细胞内ROS水平增高至（126.2±12.4）%（P < 0.05）；1~100 μmol·L-1的FLC染毒后SOD2 的mRNA表达降低至（29.8±2.1）%、（25.5±2.2）%和（6.6±0.4）%，而GSH-Px 的mRNA表达降低至（63.6±4.2）%，（49.9±4.1）%和（26.5±2.1）%（P < 0.001）；10、100 μmol·L-1浓度FLC分别促进细胞凋亡率达（2.2±0.1）、（12.5±0.8）倍，Bax的蛋白表达增高至（1.2±0.1）、（1.4±0.1）倍（P < 0.001），Bcl-2的蛋白表达降低至（41.6±4.0）%和（41.9±3.2）%（P < 0.001），Bcl-2/Bax值降低至（35.2±4.0）%和（30.9±3.1）%（P < 0.001），同时Cox-3的蛋白表达下调至（84.1±8.0）%和（54.2±4.2）%。结论 FLC对肝细胞BRL-3A具有明显的毒性效应，随浓度的升高而增强，其毒性机制可能是通过诱发肝细胞氧化损伤，进而促使细胞凋亡。
Abstract: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 ofSOD2 were decreased to (29.8±2.1) %, (25.5±2.2) %, and (6.6±0.4) %, and the mRNA expression levels ofGSH-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.
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