| IntroductionOver the past few years, the application of pesticides is more and more in our country. While the pesticides with high toxicity are completely banned, the use of pesticides with middle toxicity increased substantially, such as fipronil applied in the central region of our country. Fipronil, named"Regent", belonging to phenyl pyrazole insecticides, is a broad-spectrum insecticide compared with the pyridine insecticides, especially for the pests that cannot been killed by organic phosphorus, carbamate and synthetic pyrethroid pesticides. It is a pesticide of middle toxicity and high biological activity. The toxicity of fipronil is considerable with pyrethroid pesticides. It acts on gamma amino butyric acid (GABA) receptors to prevent the inhibition of GABA. As a new insecticide in the world, a distinc disadvantage is that fipronil is highly toxic to aquatic organisms, such as shrimp and crab, so it is can not be used in these areas.Reactive oxygen species (ROS) are important free radicals including oxygen ions and inorganic peroxide. It is reported that appropriate level of ROS can inhance the immune system, while excess ROS are harmful for organisms. It is imposed oxidative stress in organisms when exc ess free radicals are directly or indirectly induced by environmental factors or exogenous chemical substances. Apoptosis is influenced by the level of ROS, expression of Bcl-2 protein and concentration of Ca2+.Bcl-2 protein is a protein related to apoptosis, expressed by Bcl-2 gene, can reduce the rate of cell apoptosis. The specific mechanism of Bcl-2 protein is still not clear now, but most resrarchers believe that Bcl-2 protein is related with anti-oxidation and inhibion of Ca2+ movement.This study aims to investigate the level of ROS and the expression of Bcl-2 protein in PC12 cells that are treated with fipronil. At the same time, insight of the toxicity mechanism may be gained.MethodsThere were five groups in this experiment, control group and four dose groups. The dose groups were 7.81×10-7, 3.13×10-6, 1.25×10-5 and 5.00×10-5mol/L.1. The effect of fipronil on the survival rate of PC12 cells of these four dose groups after 12h, 24h and 48h treatment was detected by Methyl Thiazolyl Tetrazolium (MTT).2. The effect of fipronil on the apoptosis and necrosis of PC12 cells of 3.13×10-6, 1.25×10-5 and 5.00×10-5mol/L three dose groups after 24h treatment was detected by Morphology.3. The effect of fipronil on the apoptosis rate of PC12 cells of 3.13×10-6, 1.25×10-5 and 5.00×10-5mol/L three dose groups after 24h treatment was detected by flow cytometer (FCM).4. The expression of Bcl-2 protein in PC12 cells of 3.13×10-6, 1.25×10-5 and 5.00×10-5mol/L three dose groups after 24h treatment was measured by immunofluorescence.5. Chemical colorimetry was applied to measure the impact of fipronil on the content of the activity of ROS in PC12 cells.Results1. The experiment of the cell survival rates indicated that there was no significant toxic effect of four dose groups after 12h treatment. But the survival rates of four dose groups were lower than the control group after 24h and 48h treatment, the toxic effect was statistically significant. 2. We found that the number of apoptotic cells of 3.13×10-6 mol/L group was more than the control group examined by fluorescence microscope, and the number of dead cells of 5.00×10-5mol/L group was more than the control group.3. The apoptotic rates of PC12 cells was higher in the 3.13×10-6 mol/L group than the control group mersured by FCM, and the dead rates of PC12 cells was higher in the 5.00×10-5mol/L group than the control group.4. Immunofluorescence cytochemistry experiment demonstrated that the level of Bcl-2 expression was reduced in the 3.13×10-6 mol/L group than the control group.5. Chemical colorimetry showed that after exposure to fipronil for 4h, the level of ROS of 3.13×10-6 mol/L group in PC12 cells increased much more than the control group, the difference was statistically significant. But after exposure to fipronil for 24h, the activities of ROS in the 5.00×10-5mol/L group reduced much more than the control group, also the difference was statistically significant.ConclusionsFipronil is a middle toxicity pesticide, with neuro toxicity. It can reduce the survival rate of PC12 cells. At low dosage, fipronil can increase the apoptotic rates of PC12 cells by reducing the expression of Bcl-2 protein and increasing the level of ROS. At high dosage, fipronil only increases the amount of necrotic cells. The specific mechanism requires further investigation. |
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