| ?Background?Paraquat(PQ, also named N, N?-dimethyl-4, 4?-bipyridinium dichloride), as a classical herbicide, has been widely used in the world. But after using for a long time, it was found that PQ had a very high toxicity, and its mortality rate could reach more than 90% after poisoning. According to the survey from the poisoning control center in the United States, people died from PQ poisoning accounted for the first in pesticide poisoning in 2008. Studies had shown that PQ could produce a large amount of reactive oxygen species(ROS) in various ways. Excessive ROS can lead to oxidative stress, which can even lead to multiple organ dysfunction. Liver is an important target organ for PQ poisoning, which may cause severe damage and even failure. Up to now, the specific mechanism of liver injury induced by PQ is still not clear.Mitochondrion, as an important organelle of metabolism in cell, can participate in the ATP production, ROS generation, apoptosis, etc. In the process of ATP production, there is a small amount of electron which inevitably leaks from mitochondrial electron transport chain. All kinds of ROS originated from mitochondria are called mitochondrial reactive oxygen species(mt ROS). Recent studies show that intracellular ROS have a compartmentalized distribution in cells. The biological effects of ROS from different sources are often discordancy. For example, in addition to the same effect of leading to oxidative damage as ROS, mt ROS also have anti-aging protective effects. Studies also find that the increase of ROS induced by PQ is mainly derived from the mitochondria. However, it is not clear that whether mt ROS is directly involved in liver damage induced by PQ. As a kind of pesticides, PQ is an exogenous chemical or xenobiotic to cells. Cells often have a relatively sensitive or tolerant phase for exogenous compounds. The cell cycle is divided into four phases which are G1, S, G2 and M phase. The cells cultured in vitro are a mixture of all phase cells. Obviously, it is difficult to discern which phase is more sensitive to PQ toxicity than others and to determine the exact mechanism of toxic effects of PQ. Similarly, comprehensive effects of mixture cells will also cover the sensitive phase, and decrease the sensitivity of detection results. Therefore, the research of PQ sensitive phase and its poisoning mechanism are very important not only for clarifing the toxic effects of PQ, but also for studying toxicity mechanisms of other xenobiotics.?Objective?1. To observe the changes of ROS and mt ROS in the process of asynchronous hepatocytes injury induced by PQ and to investigate the relationship between liver injury, ROS and mt ROS. 2. To establish an ideal cell synchronization model and determine the phase of cell cycle. Based on synchronization, we conduct further research on the relationship between PQ induced liver injury, ROS and mt ROS. 3. By comparing the effects of oxidative and apoptotic damage induced by PQ in asynchronous and synchronous hepatocytes, we determine whether synchronization has an important role in the mechanism of PQ poisoning.?Methods?1. To investigate the relationship between ROS, mt ROS and damage induced by PQ in asynchronized cells : AML12 cells were treated with PQ for 24 h which ha d concentrations of 0, 25, 50, 100, 200 and 300 ?mol·L-1, respectively; the changes of cell viability were detected by MTT method. The effect of PQ on apoptosis was evaluated by staining Annexin V-FITC/PI. DCFH-DA staining method combined with flow cytometry was used to measure the level of ROS. The changes of mt ROS in cells were observed by laser scanning confocal microscopy with mitochondria targeted Mito SOX staining and the level of mitochondrial membrane potential was measured by JC-1 staining method. In order to determine it is ROS or mt ROS that results in cell viability damage and apoptosis, the changes of ROS, mt ROS, cell viability, apoptosis rate and membrane potential were analyzed by Pearson correlation analysis. 2. To establish an ideal cell synchronized model and determine the phase of cell cycle: RO-3306 was used to screen the best synchronized method by the bidirectional selection of synchronized and apoptosis rates. After the synchronized treatment, the cells were collected and fixed with 70% alcohol and the cell cycle was detected by PI method with flow cytometry. After the synchronization model was determined, the synchronized cells were released at different time points. According to the results of cell cycle detection and morphological observation, the G1, S, G2 and M phase of cell cycle were determined, respectively. 3. To further study the relationship between PQ, mt ROS and ROS in hepatocyte injury induced by PQ in synchronous cells: After Hep G2 cells were synchronized to different cell cycle phases and treated with PQ for 2h, PQ was replaced with the fresh medium to cultivate for 24 h. Then, the changes in ROS and mt ROS levels were detected just as above. Finally, we compared the PQ-induced damage, ROS and mt ROS in different phase cells.?Results?1. PQ obviously damages cell viability dose dependently. PQ with a concentration of 300 ?mol·L-1 can significantly induce apoptosis in asynchronized cells. Low dose of PQ increases mitochondrial membrane potential, while high dose of PQ reduces it. 2. PQ promotes the production of ROS which lead to the imbalance of redox homeostasis in cells. The lower concentration of PQ increases the level of mt ROS, but the higher concentration decreases it. Therefore, PQ has a dose-effect relationship with the generation of mt ROS. That is to say, different doses of PQ have different effects on mt ROS. 3. The correlation analysis reveals that there is a correlation between the level of total ROS and the apoptosis, viability damage induced by PQ. However, mt ROS have no correlation. 4. With more than 96% of synchronization rate and very low apoptosis rate, RO-3306 block method was selected from all the synchronization methods. Cell cycle distribution was detected by releasing different time points. After the release of the 0, 8 and 14 h, the cells are in the G2, S and M phase, respectively. Combined with morphological observation, the cells were found to enter the M phase after the release of 1h. After treated with PQ, the cells in different cell cycle phases show different sensitivities to PQ. The viability damage of cells in G2 phase is the most serious in four cell cycle phases, Compared to G1 and S phases, G2 and M phases are more sensitive to apoptosis induced by PQ. 5. After PQ treatment in synchronized cells and detection of intracellular ROS and mt ROS, the most obvious increase of ROS level is in the G2 phase followed by M, G1 and S phase. Contrary to ROS change, the level of mt ROS in S phase increases the most significantly, followed by G1, M and G2 phase. 6. In synchronous cells, we can not only verify the results obtained in the asynchronized cells which intracellular ROS are involved in the process of hepatocyte injury induced by PQ, but also find that the sensitive phase of PQ toxicity is the G2 phase and that is contrary to mt ROS change, etc.?Conclusions?1. In asynchronized cells, PQ can lead to the increase of ROS which mediate the damage of hepatocytes. The low dose of PQ increases the mt ROS content, while the high dose reduces that. Mt ROS may not directly be involved in the process of liver damage induced by PQ. 2. PQ can lead to different toxic effects for the cells in different cell cycle phases and the cells in G2 phase are the most sensitive to PQ toxicity, which is related to the increased ROS and the decreased mt ROS levels in cells. 3. By comparing the effects of oxidative damage and apoptosis induced by PQ in both asynchronized and synchronized cells, we find that synchronization treatment can improve the sensitivity of the detection index and amplify the toxic effects of PQ, which is of great significance for studies of the toxicity mechanism of PQ and other exogenous chemicals. |
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