| Butyl benzyl phthalate (BBP) is one of the most important representative chemical of phthalate esters (PAEs) and it's known as an environmental endocrine disruptor (EEDs). BBP is widely used as a plasticizer, flexibilizer, trager and additive, automobile, lubricant, cosmetics, clothing, pesticides, and so on. Because BBP was not covalently linked to the plastic polymer during processing, it can leach from the plastic matrix and migrate into the environment while the plastic products being used or discarded, providing opportunity for widespread exposure, and it is harm to the health of population. The most important route of human exposure to BBP is via food which main source is PVC food packaging materials.In recent years, the toxicity of BBP has been a great concern. It is estimated that exposure dose of BBP for adults is 8μg/kg/d of mean and 20μg/kg/d of maximum, while the exposure of infants and children is approximately three times of adults. And a study found, Lvelisse Colon of Puerto Rico University examined blood samples of 41 premature development of girls and 35 normal girls in the Puerto Rican island by GC/MS, and found that the concentration of BBP, DMP, DEP, DBP, DEHP was higher than the control group excluding the interference of others factors. He also inferred that the premature of girls in the locality may be related with PAEs. Therefore, the toxic effect of BBP can not be ignored.Many scholars believe that BBP as an estrogen-like substances combines with corresponding hormone receptor after it gets into the body, which affect the growth, development and behavior of human. For example, human who has been long-term chronic exposure to BBP can lead to endocrine disorders, reproduction disfunction, and so on. BBP also can produce inter-generational impact through the placenta and lactation. In previous studies of our department, BBP exhibited some effects on the neurobehavioral development of rats in the F1 generation. The male offspring were more sensitive to BBP than the female.In summary, the study for the pollution and hazard to BBP still has some limitations. Therefore, it is necessary for it to deeply investigate the mechanism of toxic action. In present study, it investigates the effect of BBP on estrogenic action in human neuroblastoma cells (SH-SY5Y cells) in vitro using SH-SY5Y cells as a model and will provide a scientific basis for further consummating the mechanism of neurotoxicity induced by BBP extensively.PartⅠEffect of BBP on toxicity of SH-SY5Y cells1,Effect of BBP on the activity of SH-SY5Y cells Objective: To investigate the effect of BBP on the activity of SH-SY5Y cells. Methods: The cell activity was measured by the method of CCK-8. Results: (1) The exposure time of cell was determined for 24 h, the dose of ICI182,780 and E2 was 1μmol/L, 0.1μmol/L, respectively; (2) The cell survival rate of BBP group was 113.10 %, 114.76 % at the dose of 0.01, 0.1μmol/L respectively, which were significantly higher than the control group. The cell survival rate of 0.1μmol/L E2 group was 115.45 %, also significantly higher than control. The cell survival rate of BBP group at the dose of 30, 300μmol/L respectively was significantly lower than the control group; (3) BBP and E2 can promote the proliferation of SH-SY5Y cells, and ICI182,780 could reverse the role of BBP and E2 in cell proliferation.Conclusion: There were deferent effects between low-dose BBP and high-dose BBP, the effect of BBP may be mediated by ER.2,Effect of BBP on the apoptosis of SH-SY5Y cells Objective: To investigate the effect of BBP on the apoptosis of SH-SY5Y cells and initially investigate the mechanism of BBP induced apoptosis in SH-SY5Y cells. Methods: Apoptosis was examined by flow cytometric analysis.Results: (1) BBP caused cell apoptosis rate significantly higher than control group at the dose of 100, 300μmol/L (P <0.05). (2) The early and total apoptosis rate of BBP and E2 which dose were 0.01, 0.1μmol/L and 0.1μmol/L respectively was significantly lower than the control group (P <0.05). The groups which cooperated ICI182,780 with BBP or E2, could induce the apoptosis rate significantly higher (P <0.05).Conclusion: Low-dose BBP can promote the proliferation of SH-SY5Y cells and decrease the cell apoptosis rate, while high-dose BBP inhibited the growth of SH-SY5Y cells and promoted it apoptosis, ICI182,780 can induce BBP or E2 causing apoptosis.PartⅡEffect of BBP on the estrogen secretion of SH-SY5Y cellsObjective: To investigate the effect of BBP on secreting estrogen content of SH-SY5Y cellsMethods: Estrogen content in cell culture supernatant which BBP induced the SH-SY5Y cells was examined by ELSIA assay. Results: BBP caused the content of estrogen secretion in the SH-SY5Y cells significantly higher than control group at the dose of 0.01, 0.1, 10μmol/L (P <0.05).The groups which combined ICI182,780 with BBP could restrain the action significantly.Conclusion: Low-dose BBP can increase the content of estrogen secretion in the SH-SY5Y cells, which may be related with the ER.PartⅢEffect of BBP on aromatase of SH-SY5Y cells Objective: To investigate the effect of BBP on aromatase of SH-SY5Y cells and its possible mechanism.Methods: The aromatase activity of SH-SY5Y cells was measured by the method of ELISA assay; the expression of aromatase was examined form protein and gene level by the method of immunofluorescence, Flow Cytometry indirect labeling, Western blotting and real-time PCR.Results: (1) BBP caused aromatase activity in the SH-SY5Y cells significantly higher than control group at the dose of 0.01, 0.1μmol/L (P <0.05); the groups which cooperated ICI182,780 with BBP could restrain the action significantly. (2) The expression of aromatase is mainly in the cytoplasm of the SH-SY5Y cells by immunofluorescence; Results of indirect labeling technique by Flow Cytometry showed that the expression of aromatase in 0.1, 1μmol/L BBP group was significantly higher than that in the control group (P < 0.05). Effects on protein expression: the content of aromatase in 0.01, 0.1, 30μmol/L BBP group increased significantly (P<0.05); Effects on gene expression: aromatase expression in the 0.01, 0.1, 30μmol/L BBP group also increased, and the differences were significant as compared to control (P<0.05); (3) ICI182,780 can reverse the effect of BBP on aromatase in the SH-SY5Y cells.Conclusion: In SH-SY5Y cells exposure to low-dose BBP increased aromatase activity and the expression of aromatase, ICI182,780 also played a role in reversing the changes of aromatase activity and the expression of aromatase, indicating that the toxic effect of BBP on SH-SY5Y cells may be mediated by ER. Part IV Effect of BBP on estrogen receptors of SH-SY5Y cells Objective: To investigate the effect of BBP on estrogen receptors of SH-SY5Y cells.Methods: Real time-PCR was used to detect the expression of ERαand ERβmRNA levels of SH-SY5Y cells.Results: BBP at the dose of 0.01μmol/L can significantly decease the expression of ERβand consistent with the effect of E2. There were no significant differences in the ERβgene expression of SH-SY5Y cells between doses (0.1, 1, 10μmol/L) of BBP groups and control group, the gene expression of ERβin 30, 100, 300μmol/L BBP group was significantly higher than that in the control group (P < 0.05); In this experiment, we did not detect the expression of ERαgene. Conclusion: There may be different impacts of ERβmRNA in different doses of BBP on SH-SY5Y cells, suggesting that the mechanism may be different.Part V Effect of BBP on the expression of CREB in the SH-SY5Y cells Objective: To investigate the impact of BBP on the expression of CREB protein in SH-SY5Y cells.Methods: Western blotting was used to detect expression of CREB protein in SH-SY5Y cells.Results: The exposure to BBP at the dose of 0.01, 0.1, 30, 100μmol/L increased the expression of CREB protein in SH-SY5Y cells, the difference was statistically significant (P <0.05). The group which combined ICI182,780 with 0.1μmol/L BBP significantly inhibited this effect compared with 0.1μmol/L BBP group (P <0.05).Conclusion: Effect of BBP on the expression of CREB was consistent with the effect on aromatase; there may be some connection between the two, this need to be further investigated. |
PDF Full Text Request