Shen Tong, Ma Tai, Ye Liang-ping, Wang Li-jie, Zhu Qi-xing
Department of Toxicology, School of Public Health, Anhui Medical University, Hefei 230032, China.
Zhonghua Yi Xue Za Zhi. 2009 Dec 1;89(44):3101-5.
To explore the potential mechanism of trichloroethylene (TCE)-induced apoptosis in normal human epidermis keratinocyte (NHEK) by assaying the Caspase activities, mitochondrial membrane potential (DeltaPsim) and apoptosis in vitro.
NHEK was exposed to TCE and Caspases-3, 8 and 9 activities were determined using a commercial assay kit. Apoptosis and DeltaPsim were detected by flow cytometry (FCM) after double-stained with annexin-V and PI, Rh123 and PI respectively. NHEK was pretreated with inhibitor of Caspase-3 or 9 to verify the activation of Caspases by TCE treatment.
Various dose of TCE exposure could increase the Caspases-3 and 9 activities in dose- and time-dependent way. There was marked difference between TCE-treated group and control at 12 or 24 h. But no significant influence of Caspase-8 activity was evoked. 0.125, 0.25, 0.5, 1.0 and 2.0 mmol/L TCE treated NHEK 4 h then cultured for 12 h. Annexin-V(+)/PI(-) proportion were (20.1 +/- 4.1)%, (30.0 +/- 7.5)%, (42.1 +/- 8.2)%, (56.0 +/- 6.1)% and (79.1 +/- 4.3)% respectively. There was marked difference between TCE-treated group except for 0.125 mmol/L and control (9.4 +/- 3.0)% (all P < 0.05). FITC(+)/PI(-) proportion were marked positive correlation with Caspase-3 and 9 activities, r = 0.786, 0.736 (both P < 0.05). Caspase-3 activities had also a marked positive correlation with Caspase-9 activities, r = 0.845 (P < 0.05). There was no correlation with Caspase-8 activities. Pretreatment for 1 h with 100 micromol/L Z-DEVD-FMK decreased the Caspase-3 activities from (0.963 +/- 0.043) to (0.349 +/- 0.045) nmol pNA * min(-1) * ml(-1), annexin-V(+)/PI(-) proportion decreased from (80.0 +/- 5.5)% to (16.3 +/- 3.2)% in 2.0 mmol/L TCE treated NHEK with a significant difference (P < 0.01), but there was no change of Caspase-9 activities. 100 micromol/L Z-LEHD-FMK pretreatment decreased the Caspase-3 activities to (0.338 +/- 0.011) nmol pNA * min(-1) * ml(-1), annexin-V(+)/PI(-) proportion decreased to (16.1 +/- 1.7)% in 2.0 mmol/L TCE treated NHEK. And the Caspase-9 activities decreased from (0.821 +/- 0.031) to (0.240 +/- 0.043) nmol pNA * min(-1) * ml(-1) with a significant difference (P < 0.01). NHEK was cultured for 4, 8, 12, 24 h after a 4-hour treatment with 2.0 mmol/L TCE. Rhod123 fluorescence intensity (FI) were respectively with a marked decrease as compared with 0 h (18.7 +/- 0.5, all P < 0.01). At 0.125, 0.5 and 2.0 mmol/L TCE treated NHEK for 4 h then cultured 8 h, Rh123 FI were 16.1 +/- 0.5, 12.1 +/- 0.6 and 8.1 +/- 0.6 with a marked decrease as compared with control (18.1 +/- 0.5, all P < 0.01).
TCE-induced NHEK apoptosis is mediated intrinsically through the mitochondrial pathway of the decrease of DeltaPsim and the Caspase-9 dependent activation of Caspase-3.
通过检测半胱天冬酶活性、线粒体膜电位（ΔΨm）及体外细胞凋亡情况，探讨三氯乙烯（TCE）诱导正常人表皮角质形成细胞（NHEK）凋亡的潜在机制。
将NHEK暴露于TCE中，使用商业检测试剂盒测定半胱天冬酶-3、8和9的活性。分别用膜联蛋白-V和碘化丙啶（PI）、罗丹明123和PI进行双重染色后，通过流式细胞术（FCM）检测细胞凋亡和ΔΨm。用半胱天冬酶-3或9抑制剂预处理NHEK，以验证TCE处理对半胱天冬酶的激活作用。
不同剂量的TCE暴露可使半胱天冬酶-3和9的活性呈剂量和时间依赖性增加。在12或24小时时，TCE处理组与对照组之间存在显著差异。但未引起半胱天冬酶-8活性的显著影响。0.125、0.25、0.5、1.0和2.0 mmol/L TCE处理NHEK 4小时，然后培养12小时。膜联蛋白-V(+)/PI(-)比例分别为(20.1±4.1)%、(30.0±7.5)%、(42.1±8.2)%、(56.0±6.1)%和(79.1±4.3)%。除0.125 mmol/L外，TCE处理组与对照组(9.4±3.0)%之间存在显著差异（均P<0.05）。异硫氰酸荧光素(+)/PI(-)比例与半胱天冬酶-3和9的活性呈显著正相关，r=0.786，0.736（均P<0.05）。半胱天冬酶-3的活性与半胱天冬酶-9的活性也呈显著正相关，r=0.845（P<0.05）。与半胱天冬酶-8的活性无相关性。用100 μmol/L Z-DEVD-FMK预处理1小时，可使2.0 mmol/L TCE处理的NHEK中半胱天冬酶-3的活性从(0.963±0.043)降至(0.349±0.045) nmol pNA·min(-1)·ml(-1)，膜联蛋白-V(+)/PI(-)比例从(80.0±5.5)%降至(16.3±3.2)%，差异有统计学意义（P<0.01），但半胱天冬酶-9的活性无变化。用100 μmol/L Z-LEHD-FMK预处理，可使2.0 mmol/L TCE处理的NHEK中半胱天冬酶-3活性降至(0.338±0.011) nmol pNA·min(-1)·ml(-1)，膜联蛋白-V(+)/PI(-)比例降至(16.1±1.7)%。半胱天冬酶-9的活性从(0.821±0.031)降至(0.240±0.043) nmol pNA·min(-1)·ml(-1)，差异有统计学意义（P<0.01）。用2.0 mmol/L TCE处理NHEK 4小时后，再培养4、8、12、24小时。罗丹明123荧光强度（FI）与0小时相比均显著降低（18.7±0.5，均P<0.01）。在0.125、0.5和2.0 mmol/L TCE处理NHEK 4小时后再培养8小时，罗丹明123 FI分别为16.1±0.5、12.1±0.6和8.1±0.6，与对照组(18.1±0.5)相比均显著降低（均P<0.01）。
TCE诱导的NHEK凋亡通过线粒体途径内在介导，其机制为ΔΨm降低及半胱天冬酶-9依赖性激活半胱天冬酶-3。
