The Effect Of Steroidogenic Enzyme Key Genes Exposed To PCP By RNAi In MCF-7

The chlorophenol chemicals (CPs) are toxic environmental pollutants that are considered to be ubiquitous due to improper disposal, of all the CPs, pentachlorophenol (PCP) had been heavily used to control termites and protect wood. Acute exposure to high doses of these chlorophenols especially PCP is often lethal. PCP is stable and persistent in the environment, including air, water and soil, and it also can be absorbed into the body by ingestion, inhalation and through the skin, which has been associated with an increased risk of human and rodent carcinogen. The mechanisms of toxicity induced by PCP to mammals and humans have been studied in vivo and in vitro. PCP may be a promoter of carcinogenesis in rodents, the malignant lymphoma and leukemia have been associated with occupational exposures to PCP in humans and endocrine disrupting activities, which may affect the serum testosterone levels in crucian carp. What is more, exposure to high levels of PCP has been reported to result in increase in body temperature, liver defects, damage to the immune system, disruption of normal sexual, cognitive, physical and emotional development. Currently, regarding the toxicity and potential carcinogenicity of PCP, many scholars, at home or abroad, have some reports; while little study has reported about the endocrine distuption mechanism of PCP, this aspect of the study is still not clear. This study selected the MCF-7that expressed positive estrogen receptor alpha and U2-OS cell as our study targets, when MCF-7and U2-OS were exposed to different concentration PCP to study cell proliferation and the genes expression for steroidogenic enzyme key genes. Besides that, this research that combing RNA interference technology and plasmid transfection study the the genes expression for steroidogenic enzyme key genes in MCF-7cell, when MCF-7that the gene ERa was interfered were exposed to different concentration PCP. Based on the above, the pathway to endocrine disruption of PCP were discussed, either receptor pathway or non-receptor pathway. This study will want to reveal the endocrine disruption mechanism of PCP from molecular and cellular levels. The contents of this study include the following components:1. In order to study the toxicology of PCP in MCF-7and U2-OS and be sure the median lethal concentration of PCP in the both cell exposed to PCP for48hours. MCF-7and U2-OS cells were exposed to different PCP concentrations medium for24hours and48hours,200μmol·L-1acted as the highest PCP exposure concentration, the dilution factor is2, eight PCP exposure concentrations were selected on turn. The method of MTT was used to analyze the cell proliferation and be sure the median letha concentration of both cells exposed to PCP48h. The results showed that PCP have inhibited the growth of both cells and have presented concentration effect. The median lethal concentration of MCF-7and U2-OS were97.08μmol·L-1and90.23μmol·L-1, respectively.2. In order to detect the gene expression for steroidogenic enzyme key genes in MCF-7and U2-OS cells after PCP exposure and to reveal the endocrine disruption mechanism of PCP. According to the result of cell toxicology,6.25μmol·L-1,12.5μmol·L-1,25μmol·L-1and50μmol·L-1were selected PCP exposed concentrations, MCF-7and U2-OS were exposed to different PCP concentrations for48hours. Real-time PCR was used to detect the mRNA expression of steroidogenic enzyme key genes, such as CYP17, CYP19, StAR and17β-HSD12. The results showed that PCP have inhibited the gene expressions of CYP17, StAR and17β-HSD12and have demonstrated the endocrine disruption mechanism of PCP for changing the concentration and ratio of absolute and relative of hormone. CYP19have different expressions in both cell, it ranged from reducing to increasing in MCF-7cell; while it has induced in U2-OS cell. That discrepancy may be existed tissues and cells.3.In order to further reveal the endocrine disruption mechanism of PCP either through receptor pathway or non-receptor pathway (steroidogenic enzyme key gene expressions). RNA interference technology was used, interference plasmid for ERa gene was transfected in MCF-7cell. Four ERa gene interference sequences were designed and constructed to plasmid pGPU6/GFP/Neo, and one negative sequence was designed acted as negative control. The validity of recombinants plasmid were analyzed by sequencing and then these recombinants were transfected to MCF-7cell, positive clones were screened by G418. Real-time PCR and western blotting were used to detect the interference efficient at mRNA and protein levels in transfected recombinants MCF-7cell and then the cell of the highest of interference efficient be sure.4. Real-time PCR were used to detect the gene expressions of steroidogenic enzyme key genes CYP17, CYP19, StAR and17β-HSD12in MCF-7cell that ERa gene was interfered. The results showed that the mRNA of CYP17and StAR were inhibited at low PCP exposure concentrations, and were induced at high PCP exposure concentrations. At6.25μmol·L-1and50μmol·L-1PCP, the mRNA of CYP19was induced, but at12.5μmol·L-1PCP, the mRNA of CYP19was inhibited. A comparison of the interference of ERα gene indicated that PCP inhibited the expression of steroidogenic enzyme key genes by other signal pathway to disrupt the endocrine system at low PCP exposure concentration; while the interaction of PCP and ERa disrupted the endocrine system at high PCP exposure concentration. According to above studies, the endocrine disruption mechanism of PCP were revealed from cellular and molecular level.