不同形态砷化合物对非致瘤性人源性肝细胞的毒性作用

Cytotoxicity in human liver cells QSG7701 induced by different speciations of arsenic

目的探讨不同形态砷化合物对非致瘤性人源性肝(QSG7701)细胞的毒性及氧化应激作用。方法将处于对数生长期的QSG7701细胞分别暴露于亚砷酸钠(砷浓度为1、5、25、100μmol/L)、砷酸钠(砷浓度为10、25、100、500μmol/L)及MMA和DMA(砷浓度均为100、500、1 000、2 000μmol/L)培养24 h,并设对照(含10%胎牛血清的RPMI-1640培养基)。采用四甲基偶氮唑盐(MTT)法测定细胞活性;检测细胞培养液中乳酸脱氢酶(LDH)释放率、谷草转氨酶(AST)活力、总超氧化物歧化酶(T-SOD)活力和丙二醛(MDA)含量。结果一定浓度的As^(Ⅲ)(≥1μmol/L)、As~Ⅴ(≥10μmol/L)、MMA(≥100μmol/L)和DMA(≥2 000μmol/L)均能显著降低QSG7701细胞的存活率,差异有统计学意义(P<0.05);且随着As^(Ⅲ)、As~Ⅴ及MMA和DMA染毒浓度的升高,QSG7701细胞的存活率呈逐渐降低的趋势。经计算,QSG7701细胞暴露于As^(Ⅲ)、As~Ⅴ、MMA和DMA 24 h的IC_(50)分别为170.89、863.73、2 235.67、4 045.31μmol/L,对QSG7701细胞生长的抑制作用依次为As^(Ⅲ)>As~Ⅴ>MMA>DMA。在当As^(Ⅲ)浓度为5~100μmol/L、As~Ⅴ浓度为10~500μmol/L及MMA中的砷浓度为1 000、2 000μmol/L和DMA中的砷浓度为2 000μmol/L时,QSG7701细胞的LDH释放率均高于对照组,差异有统计学意义(P<0.05);另外,当As^(Ⅲ)浓度为1~100μmol/L、As~Ⅴ浓度为10~500μmol/L及MMA和DMA中的砷浓度为100~2 000μmol/L时,QSG7701细胞培养液中的AST活力均高于对照组;且随着As^(Ⅲ)、As~Ⅴ及MMA和DMA染毒浓度的升高,QSG7701细胞的LDH释放率和细胞培养液中的AST活力呈上升的趋势。与对照组比较,当As^(Ⅲ)浓度为5~100μmol/L、As~Ⅴ浓度为10~500μmol/L时,QSG7701细胞中的MDA含量均较高,而T-SOD活力均较低;与对照组比较,当MMA和DMA中的砷浓度均为500~2 000μmol/L时,QSG7701细胞中的MDA含量均较高;而当MMA中的砷浓度为100~2 000μmol/L和DMA中的砷浓度为100、500μmol/L时,QSG7701细胞中的T-SOD活力均较低,差异有统计学意义(P<0.05)。结论在本实验条件下,4种砷化合物均可不同程度地损伤肝细胞膜,破坏肝细胞的氧化平衡状态,产生氧化应激并导致脂质过氧化,对人肝细胞QSG7701的毒性作用依次为As^(Ⅲ)>As~Ⅴ>MMA>DMA。

Objective To investigate the cytotoxicity and oxidative stress induced by different speciations of arsenic in human liver ceils （QSG7701）. Methods Cultured QSG7701 cells were exposed to sodium arsenite （at the dose of 0,1,5,25 and 100 μmol/L respectively） , sodium arsenate （at the dose of 0,10,25,100 and 500 μmol/L respectively） , sodium methylarsonate and cacodylic acid sodium salt trihydrate（both at the dose of 0,100,500, 1 000 and 2 000 μmol/L respectively） for 24 h. MTT assay were used to evaluate the cell viability. The leakage of lactate dehydrogenase （LDH）,the activity of the aspartate aminotransferase （AST） and superoxide dismutase （SOD）,also the malondialdehyde （MDA） content were detected respectively. Results AsⅢ（≥ 25 μm01/L）, As V（≥ 100 μmol/L）, MMA （≥ 500 μmol/L） and DMA （≥ 2 000 μmol/L） could all decrease the cell viability in a certain dose range （P〈0.05） and as their exposure concentration increased,QSG7701 cell viability was gradually decreased. The IC50 values of QSG7701 cell exposed to AsⅢ, AsV, MMA and DMA for 24 h were determined to be 170.89 μmol/L,863.73 μmol/L,2 235.67 μmo//L and 4 045.31μmol/L,respeetively. The inhibition of four different speciations of arsenic to QSG7701 cell growth were Asm〉AsV〉MMA〉DMA. Ash（5-100 μmol/L）,AsV（10-500 μmol/L）,MMA （1 000 and 2 000 μmol/L） and DMA （2 000 μmol/L） showed the higher leakage of LDH than the control group （P〈0.05）. Ash（I-100 μmol/L）,As v （10-500 μmol/L）, both MMA and DMA （100-2 000 μmol/L） could significantly increase AST vitality compared with the control group （P〈0.05）. As their exposure concentration increased,leakage of LDH and AST vitalities in the cuhure medium gradually increased. AsⅢ （5-100 μmol/L） and Asv （10-500 μmol/L） could increase the MDA contents and decreased the SOD activity at the same time （P〈0.05）. When the exposure concentration of MMA and DMA are 500-2 000 p, mol/L,the MDA contents increased（P〈0.05）, and MMA （100-2 000 μmol/L） and DMA（100 and 500 μmol/L） showed the lower SOD activity than the control group （P〈0.05）. Conclusion The four different speciations of arsenic can damage the liver cell membrane, breaking the oxidation equilibrium of liver cells,leading to the oxidative stress and the lipid peroxidation under the experimental conditions.