Effect of physicochemical character differences on the genotoxic potency of kaolin.
Effect of physicochemical character differences on the genotoxic potency of kaolin.
复制标题
DOI：
10.1186/s41021-017-0075-y
复制
发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Totsuka Y
中科院分区：
文献类型：
--
作者：
Kato T;Toyooka T;Ibuki Y;Masuda S;Watanabe M;Totsuka Y
高岭土是一种白色粘土矿物，化学成分为Al2Si2O5(OH)4，具有不同晶体结构的多种高岭土被用于工业、化妆品和医学领域。为了评估理化特性差异对高岭土遗传毒性的影响，研究中使用了两种类型的高岭土：具有光滑球形晶体的高岭土-S 和具有薄假六边形板簇的高岭土-P。 ICR 小鼠气管内滴注高岭土（0.05 和 0.2 毫克/小鼠），并在其肺部进行彗星测定。两种高岭土均在小鼠肺部显示出 DNA 损伤，但高岭土-P 的 DNA 损伤效力远高于高岭土-S。为了阐明不同基因毒性效力的机制，我们使用流式细胞术 (FCM) 分析检查了这两种类型高岭土在肺泡上皮 A549 和巨噬细胞样 RAW264 细胞中的掺入率和 ROS 生成。与高岭土-S 相比，高岭土-P 表现出更高的哺乳动物细胞掺入率和 ROS 生成率。特别是，RAW264细胞积极掺入高岭土，并产生ROS，而在A549细胞中几乎没有观察到ROS产生。此外，使用 ELISA 方法对炎症细胞因子进行定量，以进一步了解高岭土的遗传毒性效力差异。暴露于两种高岭土后，培养基中白细胞介素-1β (IL-1β) 和肿瘤坏死因子-α (TNF-α) 的浓度增加，但在高岭土-P 的情况下，这些炎症细胞因子显着升高。基于这些发现，基因毒性效力的差异可能会影响免疫细胞的掺入率。此外，免疫细胞和上皮细胞可能密切相互作用，导致体内基因毒性的出现。为了阐明上皮细胞和免疫细胞之间的相互作用，将A549和RAW264共培养，并且RAW264细胞仅暴露于高岭土，然后将A549应用于FCM分析和彗星测定。与单一培养物相比，在暴露于高岭土的 RAW264 细胞中，A549 细胞中观察到的 DNA 损伤显着增加。从这些观察结果表明，高岭土对上皮细胞的遗传毒性机制是通过巨噬细胞的激活。因此，人们认为上皮细胞和免疫细胞之间的相互作用对于评估细颗粒物的遗传毒性非常重要。我们还在此表明，上皮细胞和免疫细胞的共培养模型可以用作评估细颗粒物（包括纳米材料）的肺部遗传毒性的合适模型，作为体内模拟系统。
Kaolin is white clay mineral with the chemical composition Al2Si2O5(OH)4, and many varieties of kaolins having different crystal structures are utilized in industrial, cosmetic and medical fields. To evaluate the effect of physicochemical character differences on the genotoxicity of kaolin, two types of kaolin, kaolin-S with smooth, sphere-shaped crystals, and kaolin-P with clusters of thin pseudohexagonal plates, were used in the study. ICR mice were intratracheally instilled with the kaolins (0.05 and 0.2 mg/mouse), and comet assay was performed on their lungs. Both kaolins showed DNA damage in the lungs of the mice, however the DNA damaging potency was much higher with kaolin-P than that with kaolin-S. In order to clarify the mechanisms for the different genotoxic potency, we examined the incorporation rate and ROS generation of these two types of kaolin in alveolar epithelial A549 and macrophage-like RAW264 cells, using flow cytometric (FCM) analysis. Kaolin-P showed a higher incorporation rate into the mammalian cells and ROS generation than that of kaolin-S. Especially, RAW264 cells aggressively incorporated kaolins, and generated ROS, whereas almost no ROS generation was observed in A549 cells. In addition, inflammatory cytokines were quantified, using the ELISA method, to understand further genotoxic potency differences of kaolins. Concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the media were increased by exposure to both kaolins, but in the case of kaolin-P, these inflammatory cytokines were significantly elevated. Based on these findings, differences of genotoxic potency may contribute to incorporation rates into immune cells. Furthermore, it is likely that immune cells and epithelial cells might closely interact with each other for the appearance of genotoxocity in vivo. In order to clarify the interaction between epithelial and immune cells, A549 and RAW264 were co-cultured and RAW264 cells only were exposed to kaolins, then subsequently A549 was applied to FCM analysis and comet assay. DNA damage observed in the A549 cells markedly increased in the presence of kaolin-exposed RAW264 cells compared to the single culture. From these observations, it is suggested that mechanisms of kaolin genotoxicity against epithelial cells are through the activation of macrophage cells. Therefore, it is thought that interactions between epithelial and immune cells would be very important for evaluation of the genotoxicity of fine particulate matter. We also showed here that co-culture models of epithelial and immune cells could be used as suitable models for evaluation of lung genotoxicity of fine particulate matter, including nanomaterials, as in vivo mimicking systems.