天然除虫菊素诱导人肝细胞DNA氧化损伤研究
DNA oxidative damage induced by natural pyrethrins in human liver cells
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摘要:
背景 天然除虫菊素被广泛应用于环境和家庭卫生领域。研究显示，其具有潜在的肝脏毒性，但具体机制尚不明确。
目的 探讨天然除虫菊素对人肝细胞DNA损伤的影响。
方法 本研究以人肝细胞QSG7701为体外测试模型，以未处理细胞为对照组，以经系列质量浓度（后称浓度）（5、10、20和40 μg·mL−1）天然除虫菊素暴露6 h或24 h后作为处理组。分别采用荧光探针法经荧光显微镜检测细胞活性氧（ROS）的荧光强度；采用比色法经酶标仪检测细胞内硫代巴比妥酸反应物质（TBARS）的水平；采用彗星电泳试验镜下观察DNA断片迁移以检测DNA损伤程度；采用免疫荧光试验通过激光共聚焦对细胞磷酸化H2AX（γH2AX）及8-氧鸟嘌呤（8-oxoG）水平等指标进行检测。
结果 随着天然除虫菊素浓度的升高，ROS荧光强度呈剂量依赖性升高，与对照组相比，10 μg·mL−1及以上浓度组差异均有统计学意义（
P <0.01），其中20 μg·mL−1和40 μg·mL−1处理组ROS水平是对照组的2.17和3.05倍；TBARS水平随着天然除虫菊素浓度升高而升高（P <0.01），20 μg·mL−1和40 μg·mL−1天然除虫菊素处理组的TBARS水平是对照组2.46和3.01倍；彗星试验结果显示，各浓度组细胞DNA呈现不同程度的拖尾，随着天然除虫菊素暴露浓度的增高，尾部DNA含量（TDNA%）、尾长（TL）、尾矩（TM）和Olive尾矩（OTM）等指标呈剂量依赖性增高，与对照组相比，20 μg·mL−1及以上浓度组差异均有统计学意义（P <0.01），其中40 μg·mL−1浓度组TDNA%、TL、TM及OTM指标分别为（46.92 ± 3.52）%、（64.67 ± 4.16）μm、30.96 ± 2.94和22.64 ± 3.89。细胞免疫荧光结果显示天然除虫菊素可诱导γ H2AX和8-oxoG的形成，且荧光强度呈剂量依赖性增高，与对照组相比，在10 μg·mL−1及以上浓度组相对荧光强度的增高均有统计学意义（P <0.01）。结论 天然除虫菊素可诱导人肝细胞DNA损伤，且ROS介导的氧化应激可能在其诱导的肝细胞遗传毒性中发挥了重要作用。
Abstract:Background Natural pyrethrins have long been widely used in the fields of environmental and household hygiene. Studies have reported that natural pyrethrins have potential liver toxicity, but their specific mechanisms are still unclear yet.
Objective To explore the effect of natural pyrethrins on DNA damage in human liver cells.
Methods This study used human liver cell QSG7701 as an
in vitro testing model. After exposure to DMSO and a series of concentrations of natural pyrethrins (5, 10, 20, and 40 μg·mL−1) for 6 and 24 h, reactive oxygen species (ROS) was detected by fluorescence microscopy using a fluorescence probe, thiobarbituric acid reactive substance (TBARS) by colorimetric method using a microplate reader, DNA damage by comet assay through observing DNA fragment migration under microscope, and phospho H2AX (γ H2AX) and 8-oxoguanine (8-oxoG) by immunofluorescence assay using a laser confocal microscope.Results As the exposure concentration of natural pyrethrins increased, the fluorescence intensity of ROS significantly increased in a concentration-dependent manner. The differences in ROS between the 10 μg·mL−1 and above groups and the control group were statistically significant (
P <0.01), and the ROS levels in the 20 μg·mL−1 and 40 μg·mL−1 treatment groups were 2.17 and 3.05 times higher than that in the control group respectively. The TBARS level increased in a concentration-dependent manner in natural pyrethrins treated cells (P <0.01), and the levels in the 20 μg·mL−1 and 40 μg·mL−1 treatment groups were 2.46 and 3.01 times higher than that in the control group respectively. The results of comet assay showed trailing formation of cellular DNA in each dose group; as the exposure concentration of natural pyrethrins increased, indicators such as tail DNA content (TDNA%), tail length (TL), tail moment (TM), and Olive tail moment (OTM) increased in a concentration-dependent manner. Compared with the control group, the differences in the indicators between the 20 μg·mL−1 and above groups and the control group were statistically significant (P <0.01), especially in the 40 μg·mL−1 treatment groups, where TDNA%, TL, TM, and OTM were (46.92 ± 3.52) %, (64.67± 4.16) μm, 30.96 ± 2.94, and 22.64 ± 3.89, respectively. The cellular immunofluorescence results showed that natural pyrethrins induced the formation of γH2AX and 8-oxoG, the fluorescence intensities of γH2AX and 8-oxoG increased in a concentration-dependent manner, and the differences between the 10 μg·mL−1 and above groups and the control group were statistically significant (P <0.01).Conclusion Natural pyrethrins could induce DNA damage in human liver cells, and ROS-mediated oxidative stress may play an important role in its liver cell genotoxicity.
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