蒋伟,
朱洪坤,
清江^,
上海出入境检验检疫局工业品与原材料检测技术中心, 上海 200135

作者简介: 蒋伟(1969-),男,研究员,研究方向为危险化学品鉴定,E-mail:jiangw@shciq.gov.cn.

通讯作者: 清江,qingj@shciq.gov.cn

基金项目: 质检总局科技计划项目(2017IK216)


中图分类号: X171.5

Cytotoxicity Evaluation of Phenylamines to L929 Cell Lines in Vitro

Jiang Wei,
Zhu Hongkun,
Qing Jiang^,
Technical Center for Industrial Products and Raw Materials Inspection and Testing, Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200135, China

Corresponding author: Qing Jiang,qingj@shciq.gov.cn

CLC number: X171.5

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摘要:探讨不同浓度的苯胺化合物对小鼠成纤维细胞L929的体外毒性作用,为满足危险化学品分类鉴定和风险评估需求、更好地预防环境化合物对人体健康的损害提供理论依据。体外培养L929细胞,分别将不同浓度的苯胺化合物(0、0.001、0.01、0.1、1、10 μg·mL^-1)加入培养基中处理细胞,在不同时间点分别取材,MTT检测细胞增殖率,AO/PI染色检测细胞存活率,Annexin V-PI染色后流式细胞仪检测细胞凋亡,相关检测试剂盒测定细胞内活性氧(ROS)的含量及总谷胱甘肽的含量。结果表明,高浓度(1、10 μg·mL^-1)的苯胺化合物对L929细胞的生长和增殖具有显著的抑制效应。即使浓度降低至0.1 μg·mL^-1,在处理48 h后,细胞的存活数量显著下降,说明其对细胞增殖具有浓度和时间的累积效应和依赖性。同时,对各组细胞采用流式细胞仪测定细胞凋亡,发现低浓度的苯胺对细胞凋亡影响并不明显,但高浓度的苯胺处理后,细胞凋亡和坏死数量显著增加。ROS含量和GSH含量检测结果显示,较低浓度的苯胺对细胞氧化应激反应并无显著影响,但浓度至0.1 μg·mL^-1时,细胞较对照组出现明显的氧化应激反应,说明细胞已受损,再继续培养将会导致细胞死亡。上述研究结果提示,高浓度苯胺化合物可显著影响L929细胞存活率,且具有时间和浓度累积效应。其毒性机制可能与细胞内活性氧增加、谷胱甘肽耗竭有关。
关键词: 苯胺/
L929细胞/
细胞毒性/
细胞凋亡

Abstract:The cytotoxicity of phenylamines to L929 cell lines was investigated in vitro to meet the needs of hazardous chemicals classification and risk assessment. The different concentration of phenylamines solutions were added to the culture mediums to treat the cells for 24 h, 48 h and 72 h, respectively. Then MTT method was used to measure the survival rate of the cells. After the preliminary experiment, we selected the suitable doses for AO/PI staining to observe the live or dead cells and for flow cytometry with Annexin V-PI staining to detect apoptosis. Finally, the ROS detecting Kit and GSH detecting Kit were used to analyze the ROS and GSH level in the treated cells. After treating with 1 and 10 μg·mL^-1 phenylamines, the growth and proliferation of L929 cell were significantly inhibited. When the concentration reduced to 0.1 μg·mL^-1, apoptosis and necrosis were still observed after treating for 48 h, indicating that the inhibiting effect of phenylamines was related to the treating time and concentration. The cell apoptosis was not obvious in the low concentration of phenylamines with 0, 0.001, 0.01 μg·mL^-1. The ROS level and GSH level detection showed that the cells generated obvious oxidative stress response after 0.1 μg·mL^-1 phenylamines treating. After treated with different concentrations of phenylamines for 24 h, the survival rate of the cells was significantly lower than that of the cells in control group, and the apoptosis and oxidative stress response was obviously enhanced. These results showed that high concentrations of phenylamines would cause cell damage, which may be related to the increase of ROS and the depletion of GSH.
Key words:phenylamines/
L929 cell line/
cytotoxicity/
apoptosis.