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Effects of PBDE-47 on p53 Function and Activation in SH-SY5Y Apoptosis

Author: KuangGang
Tutor: WangAiGuo
School: Huazhong University of Science and Technology
Course: Occupational and Environmental Health
Keywords: PBDE-47 SH-SY5Y Apoptosis Methylation p53
CLC: R114
Type: Master's thesis
Year: 2010
Downloads: 16
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Abstract

Polybrominated diphenyl ethers(PBDEs), a new kind of brominated flame retardants(BFRs), have been widely used in various fields of social production and civil life for its unique advantages, such as low price and applicationamount, high antiflaming rate, nice thermostabilization and so on. It has been reported that PBDEs is a new kind of persistent organic pollution(sPOPs), and has been found in large numbers of environmental mediums and biological materials. During the past decades, there is a sharp increase in its capacity in environment. Several health hazards have been confirmed in vitro and in vivo experiments, including hepatotoxicity, reproductive toxicity, neurotoxicity and developmental toxicity, endocrine disrupting effect and carcinogenesis.Tumor suppression factor p53, which encodes a 53kD protein, is a key member of tumor-suppression genes. Wild type P53 is a monitor in cell cycles, and can block the cell cycle when DNA is damaged in order to make the impaired DNA repaired. There are two distinct outcomes after the cell cycle arrest, DNA repair or apoptos is when the repair is invalid. Studies suggest that there are mutations in p53 gene in large numbers of neoplasms, which translates the wild type p53 into mutant p53. The mutant p53 encodes a mutant protein of P53, whose normal biological fuction is eliminated. The mutant protein not only miss the normal biological function of P53, but also can distrub the function of wild type P53, and translates the normal cells into cancer cells at last.Apoptosis, which is also called programed cell death, is a kind of automatic cell death controlled by genes in order to maintain the internal environment homeostasis. Results show that a doses of PBDE-47 could induce the apoptosis of SH-SY5Y. In our primany experiments we found that there was an increase in the expression of Fas and Cytochrome C while a downregular outcome was found in Pro-Caspase 8 and Pro-Caspase 3. Although the Polychlorinate biphenyls(PCBs), the structural similar POPs with PBDE-47, has been found to accumulate P53 protein in cytoplasm and activate the p53-dependent apoptosis pathway, the same effects in PBDE-47 is unknown. So the further study is still in demand.DNA methylation, an important chemical modification method in Epigenetics in eukaryotic genomes, is the process that the methyl donor-S-adenosylmethionine(SAM)transmits its methyl to the 5th carbon atom of the cytosin in CpG dinucleotide in DNA, which transforms the normal cytosin to 5-methylcytosine(5mC). The whole process is catalyzed by the DNA methytransferse(Dnmts), a key element in the modification process. It is known to all that DNA methylation has a profound effect on gene expression. There is a negative correlation between mathylation level and expression in genomes, which means there is a transcriptional inhibition in hypermethylation status and a transcriptional activation in hypomethylation state.So far, it is not clear whether PBDE-47 could induce the accumulation of p53 in SH-SY5Y and whether there is a relationship between neurotoxicity and p53 methylation. In order to illustrate the relationship between the accumulation of p53 and the apoptosis induced by PBDE-47 in SH-SY5Y cells, we conduct the experiments to observe the levels of accumulation of p53 and the methylation in p53 promoter in vitro with SH-SY5Y cells exposed to PBDE-47.Objective To investigate the effects of PBDE-47 on the mRNA and protein expression levels of p53 in SH-SY5Y cells. Methods SH-SY5Y cells were exposed to different concentrations of PBDE-47( 1, 5, 10μmol/L)for 24 h in vitro, mRNA and protein expression levels were determined. Results Compared with control group, the mRNA expression level in 1, 5, 10μmol/L PBDE-47-treated groups were significantly higher(p<0.05), especially, the 10μmol/L group was higher than that of in 1 and 5μmol/L groups(p<0.05). Compared with control group, the protein expression levels in 5 and 10 μmol/L groups were significantly higer(p<0.05), and 10μmol/L group was significantly higher than 5μmol/L group(p<0.05). There is no significant difference between control and 1μmol/L group (p>0.05). Conclusion PBDE-47 could induce the expression of p53 mRNA and protein in SH-SY5Y cells.PartⅡEffect of PBDE-47 on methylation level in p53 promoter areaObjective To analyze the methylation level of p53 promoter in SH-SY5Y cells by using the bisulfite sequencing. Methods The genomic DNA was extracted from SH-SY5Y cells after exposing to different concentrations of PBDE-47(1, 5, 10μmol/L)for 24 h, the extracted DNA was treated by bisulfite sodium, then the target sequence was amplified by polymerse chain reaction. After gene clone technology, plasmid sequence was sequenced. Results Target DNA segment is 255 bp in legth, there are 9 cytosines(C)change into thymidines(T). The same results are also seen in 1, 5, 10μmol/L PBDE-47 treated groups respectively.. Conclusion The methylation level in p53 promoter has no marked change, so it is hard to illuminate whether the demethylation mechanism is involved in the activation of p53.

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