The Study On The Hepatotoxicity Of Typical Chlorophenol Chemicals On Rare Minnow And Its Mechanisms

The chlorophenol chemicals (CPs) are toxic environmental pollutants that are considered to be ubiquitous, of all the CPs, 2,4,6-trichlorophenol (TCP) and Pentachlorophenol (PCP) had been heavily used to control termites and protect wood from fungal-rot and wood-boring insects throughout the world. The CPs 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 rodents carcinogen. In current reports it suggested that residue of PCP in liver and kidney, may enhance toxicity and carcinogenicity of PCP, since it is capable of inducing oxidative damage,and other results indicated that the exposure to rats at high levels of PCP will lead to increase the body temperature, liver defects, damage to the immune system and reproductive defects, while severe exposure of PCP could cause an acute and occasionally fatal illness mainly due to PCPs cytotoxicity, but the correlated molecular damage mechanisms has not been clear clarification.Recently, with the development of molecular biology and it's application in the environmental toxicology research,to research the principles and techniques for protein expression under polluted environment have long been of concern. Previous studies have mostly focused on the single toxicity respect of these chemicals and there is rarely report so far about the effect on global protein profiles exposed to CPs.Taking Gobiocypris rarus as the test animals, this study was designed to investigate the hepatotoxicity of chlorophenols on fish in genetic transcription, protein and enzyme activity levels from both in vitro and in vivo respectively. And this dissertation is to discuss the molecular mechanism of the toxic effect following the CPs exposure, the toxicoproteomic approach and the real–time PCR analysis method were applied to identify proteins differentially expressed in the livers of rare minnow. The results will provide useful data for not only the assessment of the potential risk of Chlorophenols, but also the understanding of their mechanism of action. The main results are as follows:1. The system of primary cultural hepatocytes from rare minnow was successfully established, and the hepatocyte of rare minnow were also extracted for primary culture, whereby the inhibition rate and DNA damage of hepatocyte exposed to these materials were examined by MTT assay and single-cell gel electrophoresis (SCGE), respectively. Results show that: Each group observed a inhibition effect on hepatocyte, furthermore, in each group, the low-dose exposure (0.5, 2.5μg·L-1) showed a obvious dose-effective and a significant inhibition effect compared with control group when reaching medium and high dose (25, 50μg·L-1). The SCGE results show that is a significant increase in DNA damage at PCP exposed dose of 12.5, 50μg·L-1 (p<0.01), TCP and joint exposure dose of 12.5μg·L-1 (p<0.05). There is significant (p<0.01, p<0.05) difference between the damage rate of these treated groups and that of control group, suggesting that the PCP and TCP have cytotoxicity and can induce the hepatocyte DNA strand to break in vitro.2. For further understanding of mechanisms of action and identify the potential protein biomarkers for CPs exposure, two-dimensional electrophoresis coupled with mass spectrometry (MALDI-TOF-MS) was used to identify the differentially expressed proteins in the livers of rare minnow following TCP exposure of 1, 10, 100μg·L-1, and PCP exposure of 0.5, 5, 50μg·L-1 under flow-through conditions for period time of 28d, respectively. The pathological of liver was associated with a hypertrophy of hepatocytes and damages to cellar structure by pathological observation, and the tissue somatic indices were also analysis.After comparison of the protein profiles between treated and control groups, 39 and 35 protein spots were found altered in abundance (>2-fold) from PCP and TCP treated groups, respectively. Matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (MALDI-TOF-MS) analysis allowed the unambiguous identification, and the 30 protein spots were identified successfully. The identified proteins were then matched to specific biological processes or functions by searching Gene Ontology (GO Terms) using Uniprot/Swissprot database and submitted to ingenuity, we classified those proteins manually to a variety of cellular biological processes, according to that took part in a variety of cellular biological processes such as: transport, metabolism of lipid, response to oxidative stress, protein repair, oxidative phosphorylation and other related processes. And the results show that the gonadosomatic index (GSI) was significantly greater than that of control, while, the hepatosomatic index (HSI) for female and male contained statistically differences compared to control groups. PCP increased LDH and GST activity in liver of Gobiocypris rarus, and decreased SOD activity, these results indicate that exposure to Chlorophenols may lead to generation of free radicals, depletion of antioxidants and peroxidation of lipids, thus resulting in oxidative stress in liver of the Gobiocypris rarus. 3. The transcriptional analysis of six mRNA encoding proteins altered by PCP exposure in the proteome analysis was determined by real-time PCR, such as Mitochondrial ATP synthase, Steroid Sulfotransferase-like protein, Liver-basic fatty acid binding protein b and Vitellogenin were observed to be potential biomarker in toxicological studies of rare minnow following PCP exposure, which should be the main focus of studies on mechanism of PCP toxicity in the future.In conclusion, the system of primary cultural hepatocytes from Gobiocypris rarus was successfully established. The in vitro analysis using the primary cultural hepatocyte was carried out to confirm that the PCP and TCP have cytotoxicity can induce the hepatocyte DNA strand to break. Based on the observed histopathological changes, and the differentially expressed proteins and the mRNA level in the livers of Gobiocypris rarus following TCP and PCP exposure in vivo, we can concluded that the molecular damage mechanisms of toxicity effect of CPs mainly by the interfering the fatty acid transporting, the mitochondrial ATP synthase and the related fundamental regulation processes. In addition, the discrepant results between mRNA and protein levels suggested that complicated regulatory mechanisms of gene expression were implicated in the response to chlorophenols exposure.