Effect of physicochemical character differences on the genotoxic potency of kaolin

Tatsuya Kato, Tatsushi Toyooka, Yuko Ibuki, Shuichi Masuda, Masatoshi Watanabe, Yukari Totsuka

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.