Study On PCP Toxicity On HeLa Cells And Mechanism Of Endocrine Disruption

Recently, the environmental endocrine disruptors' harm to human has attracted public and academe's attention, it has been research hot of the toxicology in the world.The growth,development,breed of aquatic organism were menaced and aquatic ecosystem were destroyed by pentachlorophenol (PCP), PCP could enter into body by food chain and produce potential danger.Hence, it is very important to research cytotoxicity of PCP at the cellar level by mulitiple evaluate index.So far, the specific mechanism of PCP endocrine disruption is not clear.Estrogen receptor(ER) has two subtypes:ERαand ERβ, but PCP exercises the role of endocrine disruption by combining with one of ERa and ERβor two subtypes,which has not been got. It is studied PCP exercises the role of endocrine disruption whether to combine with ERa or not in order to farther explain the specific mechanism of PCP endocrine disruption. This experiment is divided into two parts.The first part:the toxic effects of PCP on HeLa cells were studied by mulitiple evaluation index by measuring cell morphology, inhibition rate of cell proliferation, DNA damage, lactate dehydrogenase(LDH) leakage rate and SOD activity in cell culture fluid.We confirmed that 10-5 mol/L PCP has started to effect HeLa cell morphology by observing cell morphology; we comfirmed that when the number of cell inoculation is 6000～10000 cells/well it can reflect experimental results primely, so it can establish basis for follow-up experiments; HeLa cell proliferation rate was measured by MTT assay, the IC50 of PCP is 66.59βmol/L. When HeLa cells were exposed to 16.47μmol/L PCP for 24 h, cell proliferation was inhibited evidently, it indicated mitochondria was damaged, with increasing of PCP concentration, inhibition rate of cell proliferation increased, the mitochondria damage's degree increased.HeLa cells were exposed to 6.25μmol/L,12.5μmol/L,25μmol/L PCP for 24 h and 36 h, HeLa cells DNA damage were detected by single cell gel electrophoresis. The results showed that whether exposure to 24 h or 36 h, three doses of PCP did not cause HeLa cells tail DNA% and tail monment to increase significantly, it showed that PCP didn't damage HeLa cells DNA.At the exposure time(12h,24h,36h,48 h) for HeLa cells, LDH leakage rate in 12.5μmol/L PCP was low, with increasing of PCP concentration, LDH leakage rate gradually increased, the difference of the PCP concentration group and 12.5μmol/L PCP was significant(p<0.05), LDH leakage rate of 200μmol/L PCP was 90%.12.5μmol/L PCP damaged HeLa cells membrane significantly, at certain PCP concentration(experimental concentration:25μmol/L PCP and 50μmol/L PCP), exposure time and LDH leakage rate of HeLa cells showed a certain relation of time-effect. LDH leakage rate of HeLa cells was one of the sensitive indicators evaluating the toxicity of PCP.Experimental results showed that 12.25μmol/L,17.5μmol/L and 25μmol/L PCP concentration groups compared with the control group, SOD activity in cell culture fluid were significantly difference(p<0.05), as the PCP concentration increased, SOD activity in cell culture fluid decreased, there is a certain concentration-effect relationship. SOD activity in cell culture fluid in exposure time(12 h,24 h,36 h,48 h) at certain PCP concentration(experimental concentration:25μmol/L PCP and 17.5μmol/L PCP), exposure time and SOD activity in cell culture fluid showed a certain relation of time-effect. SOD activity in cell culture fluid in compared with LDH leakage rate was more sensitive indicator to evaluate the toxicity of PCP on the HeLa cells.The second part:a preliminary study found that PCP is a kind of weak estrogenic activity of endocrine disruptors by the establishment of hERa cell screening system.That PCP combined with ERa formed ligand-receptor complex, the comformation changed and the complexs became homodimer complexes. The homodimer complexes could combine with estrogen response element in DNA molecule, activated luciferase gene of the ERE regulation, luciferase gene transcribed mRNA, mRNA could further translated into luciferase, it can indirectly reflect PCP endocrine disruption. PCP could compete with EE2 combing with ERa, as the concentration of PCP increased, the ability of competing with EE2 combing with ERa increased, the level of inducing Luc expression decreased at the same time.These experimental results showed HeLa cells exposed to PCP could change the cell morphology, 10-4 mol/L PCP resulted in death of most HeLa cells; 16.47μmol/L PCP has caused mitochondrial injury; PCP didn't damage HeLa cells DNA; Cell LDH leakage rate and SOD activity in cell culture fluid were both sensitive indicators to evaluate toxicity of PCP effecting HeLa cells. PCP is a kind of weak estrogenic activity of endocrine disruptors, PCP could exercise the role of endocrine disruption by combing with ERa.