Oxidative Stress And Apoptosis Induced By Microcystin-LR On Chinese Hamster Ovary Cells

Microcystins (MCs) are cyclic heptapeptides which are composed of seven amino acids. MCs are the most common group of cyanotoxins and more than80variants are known. Microcystin-LR (MC-LR) is one of the most abundant and toxic variant. MCs could enter into the body of human through the food chain and water, form a serious threat to the life and health of people.Gonad was the second target organ of MCs. Some studies proved MCs may have reproductive toxicity. At present, the reports about the toxic effect induced by the MCs in reproductive system were still few. especially in female reproductive system. The mechanisms were still unclear. However, studies have found that MC-LR can present in large amounts in the egg and can transfer to descendent. Moreover, MC-LR was endocrine disruptors, can influence the endocrine system, and play a role like estrogen in fish and mammalian cells. Therefore, exploring the toxic effects of MC-LR in the female reproductive system is very important.In this study, Chinese hamster ovary (CHO) cells were used and exposed to MC-LR. And then, the change of cytoactive, oxidative stress and apoptosis effects induced by microcystin-LR were measured. The aim of this study was to illuminate the reproductive toxicity effects of MC-LR on molecular level.Methods:1. Subcultured CHO cell model was established in this study.2. MTT method was used to determine the change of cytoactive induced by MC-LR on CHO cells:CHO cells were treated with different concentrations of MC-LR (0μg/ml,1μg/ml,5μg/ml,10μg/ml,15μg/ml) for24h. Cell survival rates were measured by MTT method.3. Colony formation test:CHO cells were treated with different concentrations of MC-LR (0μg/ml,2.5μg/ml,5μg/ml,10μg/ml) for24h. And then the colony forming efficiencies were calculated. The effects of MC-LR on single cell proliferation of CHO cell were assessed. 4. The levels of catalase (CAT)、malondialdehyde (MDA) and reactive oxygen species (ROS) in CHO cells were detected after exposed to MC-LR. After CHO cells were treated with different concentrations of MC-LR (0μg/ml,2.5μg/ml,5μg/ml,10μg/ml) for24h. ROS, MDA and CAT were detected by DCFH-DA method,TBA method and colorimetric determination, respectively.5. JC-1was used to detect the change of mitochondrial membrane potential in CHO cells after being exposed to different concentrations of MC-LR(0μg/ml,2.5μg/ml,5μg/ml,10μg/ml) for24h.6. The change of caspase-3activity induced by different concentrations of MC-LR (0μg/ml.2.5μg/ml,5μg/ml,10μg/ml) in CHO cells was measured by cleavage of the caspase-3substrate(Ac-DEVD-pNA for caspase-3).7. The apoptosis of CHO cells induced by different concentrations of MC-LR (0μg/ml,2.5μg/ml,5μg/ml.10μg/ml) was measured by Annexin V-PI staining combined with flow cytometry.Results:1. The results of MTT showed that after CHO cells exposed to different concentrations of MC-LR (0μg/ml,1μg/ml,5μg/ml,10μg/ml,15μg/ml) for24h, the cytoactive decreased and cytomorphology changed gradually with concentration of MC-LR increasing. Differences were statistically significant between control group and groups of5μg/ml,10μg/ml and15μg/ml (P<0.05). The results of Colony formation test were consistent with the MTT results. With the increase of MC-LR concentration, colony count (%of control) decreased. The results indicated that MC-LR can inhibit CHO cell activity.2. The results showed that intracellular CAT activity reduced, ROS generation and lipid peroxidation increased significantly compared with the control group (P<0.05) after exposed to MC-LR (0μg/ml,2.5μg/ml,5μg/ml,10μg/ml) for24h.3. After being exposed to MC-LR (0μg/ml,2.5μg/ml,5μg/ml,10μg/ml) for24h, JC-1detective results showed that MC-LR could lead to the onset of mitochondrial permeability transition (MPT) and mitochondrial depolarization. With the concentration of MC-LR increased, the mitochondrial membrane potential (MMP) decreased. The differences between the control group and experimental groups were statistically significant (P<0.05).4. After CHO cells were treated with MC-LR for24h, caspase-3activity was dose-dependent enhancement. And the differences between the control group and experimental groups were statistically significant (P<0.05).5. The apoptosis effect induced by MC-LR was measured using Annexin V-PI staining combined with flow cytometry. The results demonstrated that the cell apoptosis rate of experimental groups was increased in a dose-dependent manner. The differences between the control group and experimental groups were statistically significant (P<0.05).Conclusion:1. Exposure to MC-LR could induce oxidative stress generation in CHO cell.2. MC-LR could lead the further toxicity effects on CHO cells in vitro, depress cellular viability and cause cells to undergo apoptosis. resulting in the reproductive toxicitv in female rats.