铅对肾上腺皮质细胞线粒体的氧化损伤

目的】 研究铅对肾上腺皮质细胞氧化应激和线粒体功能的 影响，为了解其肾上腺皮质毒作用机制提供依据。【方法】 原代分离培养豚鼠肾上腺皮质 细胞，以0、6.25、12.5、25、50、100 μmol/L醋酸铅(PbAc)处理细胞，观察PbAc诱导肾 上腺皮质细胞活性氧(ROS)产生和线粒体损伤作用。ROS检测采用荧光分光光度法，线粒体膜 电位(MMP)和细胞存活状态采用Rh123和PI双标记、流式细胞术检测，细胞ATP水平采用化学 发光法测定。【结果】 PbAc染毒后肾上腺皮质细胞ROS形成水平随剂量增加而增加,具有剂 量效应关系[y=4.16+10.21×1g(x+1)，P<0.01，R2=0.64 1］；线 粒体膜电位呈剂量依赖性降低，Rh123的平均荧光强度(MFI)在6.25～100 μmol/L各剂量组 依次为1.01,0.94,0.96,0.95和0.91，与对照组(1.35)比较具有显著性差异(P<0 .01)；染毒后细胞死亡率轻度增加，与对照组(1.02%)比较，50 μmol/L和100 μmol/L的 PbAc组分别为3.16%和3.40%，差异具有显著性(P<0.05)；ATP水平降低与PbAc剂量之 间存在剂量效应关系[y=212965.7-51592.5×1g(x+1)，P<0.01,R2= 0.568］，剂量和时间对ATP水平的影响呈协同抑制作用(P<0.01)。【结论】 线粒体 氧化应激介导肾上腺皮质细胞毒性可能是PbAc毒作用机制之一，线粒体损伤是铅致肾上腺皮 质毒作用的早期细胞和分子事件。

Mitochondria Oxidative Damage of Adrenocortical Cells Induced by Lead Acetate in vitro

【Objective】 To study the effect of lead acetate on m itochondria oxidative stress and functional alteration of adrenocortical cells. 【Methods】 Male guinea pig fasciculata-glomerulosa (FG) cells were dispersed and primarily cultured as the testing system. The changes in the formation of re active oxygen species (ROS) and the mitochondria damage were observed when the c ells were incubated with lead acetate at different dosages of 0, 6.25, 12.5, 2 5, 50 and 100 μmol/L. The indices and their corresponding detecting methods inc luded: ① ROS (fluorescence spectrometry); ② Mitochondria membrane potential (M MP) and dead cells rate ［flow cytometry with dual-labeling of rhodamine 123(Rh 123) and propidium iodide(PI)］; ③ ATP level (chemoluminescence assay). 【Resul ts】 The formation of ROS in adrenocortical cells was increased in dose-depen dent manner after lead acetate incubation ［y=4.16+10.21×1g(x +1)，P<0.01，R2=0.641］；MMP decreased as the dose of PbAc increased . The mean fluorescent intensity(MFI) of Rh123 dropped from 1.01 to 0.91 as th e dosage increased from 6.25 μmol/L to 100 μmol/L, which were significantly lower than that of the control(1.35, P<0.01)； Dead cells rate appeared to be slightly increased. Dead cells rates in the grou ps of 50 μmol/L and 100 μm ol/L PbAc were 3.16% and 3.40% respectively when compared with the control (1 .02%, P<0.05)；ATP levels tended to decrease, resulting from the synergist ic inhibition by the dose and incubation period of PbAc. The regression analysis showed that dose-effect relationship existed between the decreased ATP and the increased dosage of PbAc ［y=212965.7-51592.5×1g(x+1)，R 2=0.568，P <0.01］. 【Conclusion】 This study demonstrated that cytotoxicity of adrenoco rtical cells induced by lead acetate is probably attributed to mitochondria oxid ative stress. Mitochondria damage might be the early cellular and molecular even t in toxic effect of lead on adrenal cortex.