Study On The Mechanism Of Tributyltin-induced Apoptosis In Human Amnion Cells At Biochemical Level

As an important organotin chemical, TBT (tributyltin), is mainly used in wood preservation, PVC plastic heat-stabilizer and disinfection of industrial circulating waters. Moreover, as antifouling agent in paints used on the vessels and marine to prevent the adhering of the mollusks is the most important using of TBT. The application of TBT expanded rapidly during the last 50 years, which led to the prolonging of the lifetime of vassels and marine buildings and the decreasing of the expense on dock repairing. Although it brought great economic benefit, it cause serious environmental problem. TBT can not only pollute the marine environment, but also can be accumulated in some edible aquatic organisms. Human exposure mostly arises from consumption of those TBT-contaminated products.Extensive studies have shown that TBT is toxic to the respiratory system, digestive system, neuronal system, immune system and reproductive system of mammals. Immuntoxicity, reproductive toxicity, neurotoxity are mainly involved in the toxicity of TBT. More detailed studies indicated that TBT had an effect on many cellular events and it could affect cell by a rule that high dose induce cell death, low dose induced apoptosis, which represented the cytotoxicity of TBT.Apoptosis is an active, highly regular and gene-directed form of cell death by a finely-established pattern of morphological features and biochemical changes. Apoptosis plays an important role in the control of biological processes such as embryonic development, tissue homeostasis and renewal, and modulation of cell populations. Moreover, turbulence of apoptosis is interrelated with the disease. So that apoptosis is very popular in the life science research.In the past 2 decades, many investigators took up with the mechanism of TBT-induced apoptosis and there have been lots of reports showing that TBT can initiate apoptosis in a variety of cell types. The mechanisms responsible for TBT-induced apoptosis mainly involve the damage to the mitochondrial, the increase of the cytosolic free calcium concentration, the production of ROS, the release of cytochrome c from the mitochondrial membrane into the cytosol and the activation of caspase. However, there have been some conflicting point of views and the exact mechanism of TBT-induced apoptosis has not been clarified until now.Therefore , the present study was undertaken by using the human amnion cells to investigate the effects of TBT on cell viability, apoptosis, cell shape, cytoskeleton, DNA damage, ROS level, cytochrome c, caspase activation and the level of apoptosis related proteins including Bcl-2, Bax and p53, so that to deepen the mechanism of TBT-induced apoptosis.According to the experimental design, the cultured human amnion cells were exposed to different concentration of TBT for different duration. The cell viability was detected by MTT assay. The apoptosis alteration was evaluated by PI/Annexin V-FITC double staining using flow cytometry. The cell shape change was observed directly by light microscope. The cytoskeleton modification was observed by fluorescent microscope after FITC -phalloidin staining. The DNA damage was measured by the single cell gel electrophoresis method. The ROS level was mensurated by DCFH-DA method. The caspase-3 activation was detected by the fluorochrome-labeled inhibitor of caspase-3.The level of cytochrome c and Bcl-2, Bax, p53 protein were determined by the western blot.The results were shown as below:1. TBT exposure induced the decrease of FL cell viability in a dose and time-dependent manner, except the 2 μmol/ group.2. TBT exposure induced apoptosis of FL cells in a dose-dependent manner.3. TBT exposure induced the increase of caspase-3 activation of FL cells in a dose-dependent manner.4. TBT exposure induced the cell shape change and cytoskeleton modification of FL cells in a time and dose-dependent manner.5. TBT exposure induced cytochrome c protein level elevation in FL cells cytosol in a dose-dependent manner.6. TBT exposure induced the rise of ROS level of FL cells in a dose-dependent manner, except the 2 μmol/ group.7. After TBT exposure, the level of Bcl-2 protein of FL cells increased at 1, 2 μ.mol/L group, without significant difference comparing with control. While at 3, 4 μmol/L group, the Bcl-2 protein level was significant decreased. Bax protein level was increased in a dose-dependent manner. As to the Bax/Bcl-2 ratio, there was no obvious change at 1, 2 μmol/L group. But the ratio increased significantly at 3,4 μmol/L group.8. The p53 protein level of FL cells did not changed after exposed to TBT.9. Comet assay showed that TBT exposure could induce the DNA damage of FL cells. Both tail length and tail moment were increased in a dose-dependent manner.Conclusions:1. TBT induced apoptosis in FL cells at low doses in short duration. While at high doses in long duration, TBT exposure led to cell death, which represented its cytotxicity.2. TBT exposure led to the interrelated changes of DNA damage and the ROS levelincrease, which associated with TBT-induced apoptosis. It could be deduced that TBT-induced DNA damage probably attributed to the oxidative stress, meanwhile, TBT-induced DNA damage might be one reason of the apoptosis.3. TBT exposure could down-regulate the protein level of Bcl-2 and up-regulate the protein level of Bax. Moreover, the correlative changes of the ratio of Bax/Bcl-2 and the apoptotic rate revealed the key role of Bcl-2 and Bax in the TBT-induced apoptosis. However, TBT exposure had no effect on the p53 level of FL cell, implying that p53 did not participate in the TBT-induced apoptosis.4. The increase of protein level of cytochrome c in the cytosol and the caspase-3 activation of FL cells after TBT exposure demonstrated the important roles that both cytochrome c and caspase-3 played in TBT-induced apoptosis. The result suggested that TBT exposure could induce the cytochrome c release via the mitochondrial pathway, subsequently the apoptosome was formed by the interaction of cytochrome c and other pro-apoptotic factors, which led the caspase-3 activation and the apoptosis.5. TBT exposure resulted in the cell shape change and cytoskeleton modification, which associated with the alteration of the apoptotic rate. It hinted that the cell shape change was attributed to the cytoskeleton disruption and cytoskeleton played an important role in the TBT-induced apoptosis. Moreover, the cytoskeleton modification might due to the effect of caspase-3.