A nontoxic dose of chrysotile induces a malignant phenotype tendency of human pleural mesothelial cells MeT-5A, short- term and long-term exposures induce different features of ROS
Authors: Yuan Xiuyuan , Zhang Min , Li Rui , Gao Yanan , Xiao Yun , Yu Min , Jia Zhenyu , Ju Li , Li Junxian , Zhang Fangfang , Zhu Lijin 
Published: December 2023

Abstract
Chrysotile products are widely used in daily life, and a large amount of inhalable dust can be generated during the production process. At present, there is still controversy in the international community about the safety of chrysotile fibers, and it is not clear whether inhalation of chrysotile dust will cause mesothelioma. In our study, a lower dose(5µg/cm2) of chrysotile was used to explore the toxicity of short-term and long-term exposure to chrysotile asbestos. In this study, three time points of short-term exposure (24h, 48h, 72h) and long-term exposure of 28w were selected to infect human mesothelial cells MeT-5A to detect the malignant phenotypic changes, including cells proliferation, migration, invasion, cycle and apoptosis levels, as well as changes in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), to evaluate the carcinogenicity of chrysotile and its molecular mechanism in the carcinogenic process of mesothelioma. The results showed that MeT-5A cells showed a certain degree of malignant phenotype after short-term exposure to chrysotile. After 28 weeks of long-term exposure, the cells were anchor-independent manner, and transformed cells (Asb-T MeT-5A) were successfully established. In addition, the CCK-8 experiment was used to detect the cell proliferation ability, and the scratch experimentand Transwell were used to evaluate the cell migration and invasion ability. Flow cytometry is used to detect cell cycle and apoptosis, and flow cytometry is used to detect cell ROS and MMP. The results showed that the migration and invasion capabilities of MeT-5A cells exposed to short-term exposure were significantly enhanced (p < 0.05). The number of cells in G1 was significantly lower than that of the control group, but the number of apoptotic cells was significantly higher than that of the control group. Through the transformation of chrysotile, the proliferation, migration and invasion ability of Asb-T MeT-5A cells was significantly enhanced (p < 0.01). The results of flow cytometry showed that the number of cells in G1 in the Asb-T MeT-5A group was significantly lower than that of the control group,and the number of apoptotic cells in the Asb-T MeT-5A group was significantly lower than that of the control group. ROS and MMP level detection results showed that the ROS level of MeT-5A cells exposed to short-term exposure increased, while the ROS of transformed cells Asb-T MeT-5A decreased. The results of the MMP of cells treated at different times were consistent, all showed increasing trend (p < 0.05). Chrysotile can induce the malignant transformation of MeT-5A cells, enhance the proliferation, migration and invasion ability of MeT-5A cells, and reduce the number of G1 phase and apoptotic cells. Chrysotile asbestos can change the ROS and membrane potential levels of MeT-5A cells.