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Development of bioassays for gaseous chemicals using air-liquid interface culture of human lung epithelial cells

利用人肺上皮细胞气液界面培养物开发气态化学物质生物测定法

基本信息

批准号：
13480163
负责人：
SAKAI Yasuyuki
金额：
$ 7.55万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480163/
关键词：
Alveolus Air way ir-liquid interface culture damage to the lung intake Suspended particular matter Cytochrome P450 Numerical simulation 浮遊粒子状物体肺胞上皮肺気道上皮バイオアッセイ有機溶媒

项目摘要

To establish a series of toxicitiy evaluation systems for chemicals or environmental samples in gas phase, we employed air-liquid interface culture (ALIC) of human bronchial (air way) and alveolus epithelial cells as a basic culture method, and investigated their applications to volatile organic compounds and suspended paniculate matter (SPM).First, using a human bronchial epithelial cell line, Calu-3, we developed a simple closed system for direct gas exposure, and tested the toxicity expression of Calu-3 to several organic compounds during 48 hours of loading. The toxicity in ALIC exposure was higher than that in conventional exposure in the liquid phase. The reason was largely explained by numerical estimation that chemical concentration on the cell surface in the liquid culture is lower than that in ALIC culture, in the cause of the diffusion process of molecules in the surface liquid layer. These results indicate that basic concept of the combination of ALIC of lung cells and a si … More mple closed system is promising as a testing cytotoxicrly to gaseous compounds.Second, in vivo mimicking exposure of actual SPM samples to human lung alveolar epithelia was achieved using an air-liquid interface culture of human lung alveolar A549 cells, resulting in successful observation of the toxicity in terms of the cell survivability after 48 hours exposure. In addition, an integrated use of the ALIC-based A549 cell assay for general toxicity and the EROD-based Hep G2 cell assay for PAHs was recommended to evaluate the primary toxicity of SPM. We thereby understood the permeation kinetics of SPM toxicity across the alveolar epithelia in terms of the EROD capacity of Hep G2 cells, and were able to obtain basic information on SPM toxicity in humans.Third, we developed a numerical simulation model that describes elution of chemicals initially adsorbed onto SPM samples, intake and accumulation of these chemicals into alveolus cell layer, and their final permeation to the systemic blood circulation. By focusing on the EROD activity of SPM samples, we quantified the comprehensive toxicity of the relevant samples and used these toxicitis in determining parameters involved in these numerical simulations. In addition, we extended the numerical models in actual situations that reflect low-dose and long-term exposure of SPM to humans across the very thin layer of the alveolar tissue. Less

为了建立一套气相化学物质或环境样品的毒性评价体系，以人支气管（气道）和肺泡上皮细胞的气液界面培养（ALIC）为基础培养方法，研究了其在挥发性有机物和悬浮微粒（SPM）中的应用。首先，使用人支气管上皮细胞系Calu-3，我们开发了一个简单的封闭系统，用于直接气体暴露，并测试了Calu-3在48小时内对几种有机化合物的毒性表达。在液相中，ALIC暴露的毒性高于常规暴露。这在很大程度上可以通过数值估计来解释，液体培养中细胞表面的化学物质浓度低于ALIC培养，这是由于分子在表面液体层的扩散过程。这些结果表明，肺细胞ALIC与单孔封闭系统相结合的基本概念有望作为一种检测气体化合物细胞毒性的方法。其次，利用人肺泡A549细胞的气液界面培养，实现了实际SPM样品对人肺泡上皮的体内模拟暴露，成功观察了暴露48小时后细胞存活率的毒性。此外，建议综合使用基于alic的A549细胞测定一般毒性和基于erod的Hep G2细胞测定多环芳烃来评估SPM的原发性毒性。因此，我们了解了SPM毒性通过Hep G2细胞的EROD能力的肺泡上皮的渗透动力学，并能够获得SPM对人类毒性的基本信息。第三，我们开发了一个数值模拟模型，描述了化学物质最初吸附在SPM样品上的洗脱，这些化学物质的摄入和积累到肺泡细胞层，以及它们最终渗透到全身血液循环。通过关注SPM样品的EROD活性，我们量化了相关样品的综合毒性，并使用这些毒性来确定这些数值模拟中涉及的参数。此外，我们扩展了实际情况下的数值模型，以反映人类在极薄的肺泡组织层上低剂量和长期暴露于SPM。少