| Background:Asbestos is a naturally occurring fibrous substances with thermal, heat and sound insulating purposes. Asbestos have been widely used in residential and commercial applications for several thousand years and the related products have been more than3000. They are widely in automotive, chemical and industrial commerce. Although asbestos has an important role in the industrial development, but asbestos can cause pulmonary fibrosis, bronchogenic carcinoma, and mesothelioma with long-term exposure. Asbestos is divided into two categories:chrysotile fiber (CF) and amphibole fibers. The amphibole fibers have more identified pathogenicity than chrysotile fiber.It is controversial for carcinogenicity on chrysotile asbestos. Nowadays, our policy has restricted the applications of amphibole fibers and advocated the safe use of chrysotile. Recently, considering the carcinogenicity of asbestos, various asbestos substitutes have been industrial applied widely. They called man-made mineral fibers, and also known as man-made vitreous fibers (Man-Made Vitreous Fibers, MMVF). They commonly calssfied as glass wool (GW), rock wool (RW),slag wool, refractory ceramic fibers (RCF) and so on. However, studies have shown that asbestos substitutes have similar characteristics to some forms of asbestos, it is urgent to evaluate the safety of these asbestos substitutes in vitro and in vivo. Though results from studies in vitro do not offer precise assessment of toxicity of the fibers, they may provide valuable information such as biological effects of asbestos and its substitutes. APE/Ref-1, the multifunction DNA repair/redox enzyme, is involved in the repair of DNA damage as well as in the transcriptional regulation of genes. Poly (ADP-ribose) polymerase-1 (PARP-1) was recognized as monitoring elements to DNA damage which also involved in many important life events such as apoptosis, transcription, DNA repair, cell death, et al.It is controversial for carcinogenicity on chrysotile asbestos. Nowadays, Some developed countries have restricted industrial and residential applications of asbestos. Recently, various asbestos substitutes have been industrial applied widely. They called man-made mineral fibers, and also known as man-made vitreous fibers (Man-Made Vitreous Fibers, MMVF). They commonly calssfied as glass wool (GW), rock wool (RW),slag wool, refractory ceramic fibers (RCF) and so on. However, studies have shown that asbestos substitutes have similar characteristics to some forms of asbestos, it is urgent to evaluate the safety of these asbestos substitutes in vitro and in vivo. Though results from studies in vitro do not offer precise assessment of toxicity of the fibers, they may provide valuable information such as biological effects of chrysotile and its substitutes. APE/Ref-1, the multifunction DNA repair/redox enzyme, is involved in the repair of DNA damage as well as in the transcriptional regulation of genes. Poly (ADP-ribose) polymerase-1(PARP-1) was recognized as monitoring elements to DNA damage which also involved in many important life events such as apoptosis, transcription, DNA repair, cell death, et al.The study on DNA damage, apoptosis and ROS generation in Human bronchial epithelial cells (BEAS-2B) and human pleuralmesothelial cell (Met-5A) exposed to chrysotile and its substitutes fibers may be to illustrate the pathogenesis. This subject intends to construct PARP-1and APE/Ref-1deficient cell line of human bronchial epithelial cell and human pleural mesothelial cell using RNA interference technology to illuminate the molecular mechanisms induced by RCF1, GW1, RWland chrysotile fibers.Objective:To evaluate cytotoxicity of BEAS-2B and Met-5A cells induced by RCF1, GW1, RW1and chrysotile fibers. To evaluate the role of APE/Ref-1and PARP-1in BEAS-2B and Met-5A cells after fiber exposure.Methods: 1. The cells of Met-5A and BEAS-2B were cultured in vitro. Cells were treated with RCF1, GW1, RW1and chrysotile fibers for different time, and then the cell viability was determined by MTT, DNA damage and DNA damage repair were studied by comet assay, and the apoptosis of cells was determined by flow cytometry analysis. Intercellular ROS concentration was determined by measure fluorescence of fluorescent probe DHR123and DCFH-DA. Morphological changes of fiber-treated cells visualized under scanning electron microscopy.Pre-treatment of cells with ROS scavengers, N-acetyl-L-cysteine (NAC) for2h prior to exposure to fibers to observe the effect of apoptosis, DNA damage.2. RT-PCR and Western Blot were used to detect the level of caspase-3, caspase-9, APE/Ref-1and PARP-1mRNA and protein in the fiber-treated cells.3. PARPl-deficient and APE/Refl-deficient cell line of Met-5A and BEAS-2B were constructed using RNA interference technology, and then used for tool cells in the following researches.4. In cells silencing APE/Refl and PARP1, apoptosis and DNA damage were observed in the deficient cells exposed to fibers.Results:1. BEAS-2B and Met-5A cells were exposed to various concentrations, ranging from5to200μg/cm2, of RCF1, GW1, RW1and chrysotile fibers for24h,48h and72h. Cell proliferation corresponded to a increase compared to the unexposed cells at low doses. At high dose (20,40,100and200μg/cm2), there were a clearly dose-and time-response decrease in cell viability.2. Under SEM, cells exposed to fibers at dose of20μg/cm2were found microvilli loss, cell smoothing up and phagocytosis.3. Cells exposed to0-20μg/cm2fibers exhibited a clear and time-dependent and dose-dependent increase in DNA damage (P<0.05). Repair of DNA damage could be seen clearly in fibers-treated cells for3hours post removal of fibers. NAC could inhibit the parameter of DNA damage.4. The percentage of apoptosis was higher in cells exposed to RCF1, RW1, GW1and chrysotile than that exposed to GW1. ROS generation in cells exposed to RCF1, RW1, GW1and chrysotile, but unexposed group was not obvious. NAC effectively decreased the rate of apoptosis and production of ROS.5. The level of caspase-3, caspase-9mRNA and protein were increased in BEAS-2B and Met-5A cells treated by RCF1, GW1, RW1and chrysotile, comparing with the unexposed cells. The level of APE/Ref-1and PARP-1mRNA and protein were show a time-dependent increase and then decrease.6.PARPl-deficient Met-5A and BEAS-2B were constructed by RNAi technology and also constructed APE/Refl-deficient in Met-5A and BEAS-2B cell line. RT-PCR and western blotting were used to identify the silencing effect. mRNA expression level decreased by70～80%, and protein was absent more than80%. The silencing effect was good and stable.7. Comparing with normal cells, the gene-deficient cells were more sensitive to the DNA damage and apoptotic protein caused by different fibers.Conclusions:1. The effects of DNA damage, apoptosis and oxidative stress induced by RCF1, GW1, RW1and chrysotile fibers were showed in BEAS-2B and Met-5A cells. ROS play an important role in cytotoxic effect.2. The caspases-3and caspases-9were involved in the pathway of apoptosis of BEAS-2B and Met-5A cells treated with chrysotile and its substitute fibers.3. PARP-1and APE/Ref-1deficiency increases the sensitivity of DNA damage and expression of apoptotic protein of Met-5A and BEAS-2B to RCF1, GW1, RW1and chrysotile fibers. PARP-1and APE/Ref-1plays an important role in the cellular damage caused by RCF1, GW1, RW1and chrysotile fibers.4. PARP1-deficient and APE/Refl-deficient cell line was successfully constructed by RNAi technology mediated by lentivirus, and the silence effect was remarkable and stable, and could be used as an effective tool cell for further functional research. |
