三氯乙烯对角质形成细胞线粒体功能和形态的影响

Cytotoxicity of trichloroethylene in kerafinocytes involving alterations of mitochondrial function and ultrastructure

目的探讨三氯乙烯(TCE)对皮肤细胞的毒性机制。方法以不同浓度(0.125、0.500和2.000 mmol/L)TCE处理体外分离培养的正常人角质形成细胞(KC),同时设培养基对照组和体积分数为1%的丙酮对照组,然后:(1)分别进行四甲基偶氮噻唑蓝(MTF)试验和ATP酶活力测定来检测细胞毒性和线粒体的代谢变化;(2)采用罗丹明123染色方法,借助流式细胞仪检测线粒体膜电位变化情况;(3)通过透射电子显微镜观察线粒体形态学的改变。结果TCE染毒后,细胞活力随着时间延长和剂量的增加而减小,线粒体酶活力抑制率增加,ATP酶活力减小;线粒体膜电位水平从染毒开始到8h迅速下降2.000 mmol/L TCE染毒8h后Rh123荧光强度(8.20±0.66)与对照组(18.73±0.45)相比,差异有统计学意义(P<0.01);8h以后则变化不大,12和24h Rh123荧光强度与8h组比较,差异无统计学意义(P>0.05),线粒体膜电位随染毒剂量的增加呈明显的剂量一效应关系。电镜下可见,TCE处理组线粒体出现肿胀,空泡变性,基质减少,部分嵴消失,对照组线粒体结构完整,基质分布均匀,可见线粒体嵴。结论TCE可以导致KC线粒体功能和形态发生明显改变,这些变化在TCE诱导的KC毒性中具有重要的意义。

Objective To explore mechanism of dermal toxicity of trichloroethylene（TCE）. Methods Normal human keratinocytes （KC） were isolated from foreskins of healthy donors undergoing circumcision by two-step trypsin digestion and cultured in serum-free medium. Cells were treated with medium, 1% acetone （volume fraction）, 0.125, 0.500 or 2.000 mmol/L TCE for different time （4, 8, 12 or 24 hours）. After treatment, MTT assay and ATPase activity detected, inhibition ratio of mitochondrial enzyme was calculated according to optical density （A） value of MTT assay. Mitochondrial membrane potential （MMP） was detected by flow cytometry （FCM） after being stained with Rhodamine123 （Rh123）. Morphological changes were also observed through transmission electron microscope （TEM）. Results Cellular viability and ATPase activity declined with dose of TCE, while inhibition ratio of mitochondrial enzyme increased with dose of TCE. FCM results showed that after treatment with 2.000 mmol/L TCE, fluorescence density of Rh123 decreased quickly from 18.73±0.45（0 h） to 8.20±0.66（8 h） （P〈0.01）. After 8 h, fluorescence density maintained at the level equal to that of 8 h （fluorescence density of Rh123 were 8.20±0.36 and 8.20±0.40 for 12 and 24 h respectively, compared with that for 8 h group, P〉0.05）. The results also showed that MMP diminished with dose of TCE. Under TEM, mitochondria in TCE-treated group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae; but in control group, mitochondrial structure was integrated, with uniform matrix and visible mitochondria cristae. Conclusions TCE could inhibit mitochondrial metabolic enzyme, reduce ATP production, diminish MMP, and destroy ultrastructure of mitochondria in KC, all these contributing to the cytotoxicity of TCE.