赭曲霉毒素A对BHK细胞毒害作用的研究

Effect of ochratoxin A on viability of BHK cell

以叙利亚仓鼠肾细胞(BHKcell)为模型,用噻唑兰(MTT)法检测赭曲霉毒素(ochratoxin A,OTA)对细胞活力的影响,用单细胞凝胶电泳法和DNA梯形条带法检测OTA对细胞DNA的损伤,用黄嘌呤氧化酶法检测细胞中SOD活力的变化,用硫代巴比妥酸(TBA)法测定细胞培养液中MDA的含量。结果显示:OTA对BHK细胞活力的影响呈现时间和剂量依赖性的抑制作用。染毒12h后,2.5、5、10、20和40μg·mL-1OTA剂量组BHK细胞的活力均显著降低(P<0.05),至染毒24h,40μg·mL-1OTA染毒剂量组细胞活力下降达90%。OTA对BHK细胞DNA的损伤作用同样存在明显的剂量效应关系,与对照组相比,除2.5μg·mL-1剂量组外,其余各剂量组的彗尾DNA含量和拖尾率差异极显著(P<0.01),40μg·mL-1剂量组的细胞拖尾率是对照组的80倍。40μg·mL-1OTA处理BHK细胞24h后检测到特征性的凋亡梯形条带,而对照组则未检测到。OTA作用于BHK细胞使细胞内SOD活力显著降低(P<0.05),细胞培养液中MDA含量则显著升高(P<0.05)。OTA染毒24h后,40μg·mL-1OTA剂量组细胞内SOD活力降低至对照组的25%,培养液中MDA含量较对照组增高了500%。上述结果表明:OTA对BHK细胞活力的影响与细胞内过氧化物的大量生成和抗氧化酶活力下降有关,且对细胞DNA造成损伤,引起细胞凋亡。

Effect of ochratoxin A （OTA） on the viability and its action mechanisms were studied using BHK cells as empirical model.Cell viability was test by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay,DNA damage by single cell gel electrophoresis assay,cell apoptosis by DNA ladder method,SOD activity by xanthine oxidase assay and MDA value in cell cultured solution by 2-thiobarbituric acid assay.The results showed that,a dose-and time-dependent inhibitory effect of OTA on cell viability were observed on BHK cells.Cell viability significantly decreased after 12 h exposure to 2.5,5,10,20 and 40 μg·mL-1 OTA （P 0.05）.24 hours after treated with 40 μg·mL-1OTA,nearly 90% of the cell viability were lose.DNA damage increased as compared with that observed in untreated control.Average tail length and the tail rate in each treated group were significantly enhanced compared with that in the control （P 0.01）,except 2.5 μg·mL-1 OTA treated group.40 μg·mL-1 OTA led to a 80-fold increase of the tail rate vs.control.The activity of SOD decreased significantly （P 0.05）,on the contrary MDA concentration increased greatly （P 0.05） in each toxin group.The activity of SOD of the high dose group （40 μg·mL-1 OTA） have only one fourth,though MDA concentration increased 5 times that of the control at 24 h after exposure.DNA ladder detected in the experiment treated with 40 μg·mL-1OTA for 24 h.These results demonstrate that the increasing concentration of lipid peroxidation and decreasing activity of SOD induced by OTA effect a change in cell viability.Moreover,OTA led to DNA damage,finally it induced cell apoptosis.