Cl^-homeostasis failure and abnormal cell migration by endocrine disruptor cause damage to nervous system

[Cl~(-)]i的测定和细胞迁移利用体内电极法，将EGFP基因导入胚胎第14天(E14)小鼠。电极化后，小鼠经羊水暴露三丁基氯化锡(1μL)。染毒后3-4天，观察小鼠的情况，结果如下：1.四氯化碳1M：小鼠萎缩死亡。小鼠双侧完好，也死亡。2.500 mm-100 mm：小鼠萎缩死亡。小鼠双侧完好存活。3.10 mm：小鼠死亡，但外观正常。4.1 mm：小鼠存活，细胞迁移正常。四叔丁基氯暴露对氯离子稳态的影响。采用革兰西丁穿孔膜片钳方法，测定[Cl2+]_i TBTCl暴露细胞(30 nM，3天)和完整细胞(0.1%DMSO，3天)。两组细胞的[Cl~-]_i均正常。观察了TBTCl暴露对细胞内GABA受体表达的影响。采用逆转录聚合酶链式反应的方法分析α1-5、、β1-3、、γ1-3、、δ、ε、和β-肌动蛋白受体亚单位的表达。低浓度的三氯甲烷(30 NM)对膜电位和突触电流无明显影响。低浓度的四丁基氯的毒性作用似乎较慢。

Measurement of [Cl^-]i and cell miglationUsing in vivo electropolation, we transfected eGFP prasmid embryonic day 14 (E14) mice. After electropolation, mice were exposed tributyltin chloride (TBTCl ; 1μl) via amniotic fluid. 3-4 days after exposure TBTCl, mice were observed their conditions.Results were as follows ;1.TBTCl 1M : Mice were dead with atrophy. Both side of mice, which were intact, also dead.2.500 mM-100 mM : Mice were dead with atrophy. Both side of mice, which were intact, survived.3.10 mM : Mice were dead but looks were normal.4.1 mM : Mice sere survived and cell miglation was normal.The effect of TBTCl exposure on Cl^-homeostasis.Using gramicidin perforate patch-clamp method, we measured [Cl^-]_i TBTCl exposed cell (30 nM, 3days) and intact cell (0.1 % DMSO, 3 days). Both cell group had normal [Cl^-]_i.The effect of TBTCl exposure on GABA receptor expressionmRNAs of TBTCl exposed cell and control cell were harvested. Using RT-PCR method, expression of GABAA receptor subunit (α1-5、β1-3、γ1-3、δ、ε、and β-actin)were analysed.The acute effect of TBTCl on culture cell.Using patch-clamp method and Ca2+ imaging, neurons were observed effect of TBTCl application. At low concentration of TBTCl application (30 nM), there were no effect on membrane potantial and synaptic current. It seems that toxicity at low concentration of TBTCl acts slowly.