纳米二氧化钛与醋酸铅联合诱导人胚肝细胞氧化应激作用

杜海荣; 朱晓玲; 周宜开

纳米二氧化钛与醋酸铅联合诱导人胚肝细胞氧化应激作用

Joint Oxidative Stress Induced by Nanometer Titanium Dioxide and Lead Acetate in Human Derived Fetal Hepatocytes

摘要

摘要:
目的研究纳米二氧化钛(TiO₂)与醋酸铅(PbAc)联合作用于人胚肝细胞(L-02),对细胞内活性氧(ROS)、氧化应激作用及细胞活性的影响。

方法以1.000 mg/L PbAc和10.000、1.000、0.100、0.010、0.001 mg/L TiO₂单独以及1.000 mg/L PbAc及前述浓度TiO₂混合处理L-02细胞24 h,以体积分数为0.1%二甲基亚砜为阴性对照, 30 μmol/L H₂O₂为阳性对照。用噻唑蓝法检测细胞毒性,采用流式细胞术检测胞内ROS水平,检测谷胱甘肽(GSH)与超氧化物歧化酶(SOD)以评价细胞内抗氧化物质水平。

结果与阴性对照、PbAc染毒组及其他浓度TiO₂染毒组相比, 10.000 mg/L TiO₂染毒组细胞活力明显降低(P<0.05)。与阴性对照、PbAc染毒组和其他浓度混合物染毒组相比, 10.000 mg/L混合物染毒组细胞活性明显降低(P<0.05); 1.000、0.100 mg/L混合物染毒组细胞活力也明显低于阴性对照组(P<0.05)。与阴性对照、PbAc染毒组相比,各浓度混合物染毒组细胞ROS水平均明显增加(P<0.05)。与阴性对照相比, 1.000至0.010 mg/L混合物染毒组细胞GSH水平均明显升高(P<0.05), 0.100、0.010 mg/L混合物染毒组细胞SOD活性也明显升高(P<0.05)。

结论本研究条件下,低剂量纳米TiO₂和PbAc共同作用于L-02,可增加活性氧水平,同时诱导细胞增加GSH和SOD水平以自我保护;随着染毒剂量升高, ROS水平明显增加,抗氧化能力下降,导致细胞活力下降。

Abstract:
Objective To study the joint effect on reactive oxygen species (ROS) generation, cell viability, and oxidative stress induced by nanometer titanium dioxide (TiO₂) and lead acetate (PbAc) in human derived fetal hepatocytes (L-02).

Methods The experimental groups were treated with PbAc (1.000 mg/L), TiO₂ (10.000, 1.000, 0.100, 0.010, 0.001 mg/L), or all concentration combinations of PbAc and TiO₂ for 24 h. The L-02 cells were also cultured with 0.1% dimethyl sulfoxide (negative control) and 30 μmol/L H₂O₂ (positive control). Cell toxicity and ROS generation were determined using methylthiazoltetrazolium assay and flow cytometry respectively. The levels of glutathione (GSH) and activities of superoxide dismutase (SOD) were used to determine intracellular antioxidant levels.

Results When treated with 10.000 mg/L TiO₂, the cell viability was decreased significantly compared with the negative control, the PbAc, and the other single TiO₂ dosage groups (P<0.05). When interfered with the combination of 1.000 mg/L PbAc + 10.000 mg/L TiO₂, the cell viability was reduced significantly compared with the negative control, the PbAc, and the other combination groups (P<0.05). The cytotoxicity was notably higher in the combination group of 1.000 mg/L PbAc +1.000 or 0.100 mg/L TiO₂ than in the negative controls (P<0.05). There were significant increases of the ROS levels in various combination groups in comparison with the negative controls and the PbAc group (P<0.05). The GSH levels were significantly increased after combined treatment of 1.000 mg/L PbAc + 1.000, 0.100 or 0.010 mg/L TiO₂ (P<0.05), and the SOD levels were notably raised after 1.000 mg/L PbAc + 0.100 or 0.010 mg/L TiO₂ combined treatment, in comparison with the negative controls (P<0.05).

Conclusion In these study settings, co-exposure to low doses of TiO₂ and PbAc significantly induces elevated ROS levels in L-02, and subsequently elevation of GSH and SOD levels for cell self-protection. Along with increasing exposure dose, the antioxidant capacity is declined followed by significant increase of ROS level, resulting in decrease of cell viability.

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