Study On The Induction Effects Of Pentachlorophenol On Vitellogenin And Heat Shock Protein In Chinese Rare Minnow(Gobiocypris Rarus)

Environmental endocrine disruptors (EEDs) can interfere with the normal body endocrine system and may affect the organism growth, development, reproduction and other life activities, which has become one of the hot issues relating to human survival and reproduction. Pentachlorophenol (PCP) belongs one of EEDs for priority testing chemicals, which was used as a cheap and efficient pesticide, wood preservatives and herbicide widely used in the world, and it had been used to kill oncomelania and control schistosomiasis for a long time in China’s middle and lower reaches of the Yangtze River11provinces, cities and autonomous regions wide, PCP had finally resulted in persistent water pollution by entering the water ecological system. PCP has a strong carcinogenic, teratogenic, mutagenic effect, and a much more influnce on the organism’s reproductive system. At present, the effect of PCP on the organism of endocrine disruption is increasingly obvious, but the estrogen effect always exists a discrepancy, and there are less studies on the molecular mechanism of specific endocrine disruption of PCP, and the available sensitive molecular biomarkers for dectecting the PCP are also less. As unigue aquatic organisms of China, the chinese rare minnow (Gobiocypris rarus) has better sensitivity and reproducibility of experiments compared with zebrafish (Danio rerio), so it is an ideal material on testing the chemical toxicity and environmental water toxicity experiment. Therefore, this study selected Gobiocypris rarus as research object, in order to reveal the estrogenic effects and mechanism of PCP, adopting the water exposure, the induction effects of PCP on vitellogenin in Gobiocypris rarus were studied from organization, protein and genetic level respectively. At the same time, through the research of PCP on Rare Minnow heat shock protein induction effect, the sensitive and accurate molecular biomarkers were screened for detecting PCP. The results mainly include the following components:1. In order to study the expression and distribution of VTG in different organs and tissues of Gobiocypris rarus and to study the induction effect of PCP on VTG distribution in different tissues of Gobiocypris rarus, immunohistochemistry method was used to investigate the VTG localization in major organs and tissues of Gobiocypris rarus. The adult male, female Gobiocypris rarus and juvenile were exposed to PCP at16,80,160μg·L-1respectively, while setting blank, solvent control (DMSO<0.01%) and17α-ethynylestradiol (EE2) for14,21,28d. Using immunohistochemical method in blank control group of Gobiocypris rarus, the VTG protein expression and distribution were studied in vivo liver, kidney, spleen, intestine, gonads, brain, muscle tissue. The results showed that VTG was synthesized in liver cells of Gobiocypris rarus, and got to other tissues through blood circulation. It was found that VTG protein only distributed in the liver, kidney, spleen, intestine, gonad of female Gobiocypris rarus, and any tissue of young or male Gobiocypris rarus showed no distribution. Using immunohistochemistry detection, to monitor the situation of induced expression of VTG by the PCP and EE2exposure in liver, kidney, spleen, intestine, gonads, brain, muscle tissues of young and male Gobiocypris rarus. It was found that both PCP and EE2exposures could induce their liver, kidney, spleen, intestine tissues to produce VTG protein, but the effect of PCP was weaker than EE2, and the production of VTG protein was related with the concentration effect and time effect of PCP exposure. It could be concluded that the abnormal expression and distribution of VTG in juvenile and male Gobiocypris rarus can be applied to environmental assessment of estrogen effects. And it is proved that PCP has estrogenic effects from organizational level.2. In order to accurately detect VTG protein induction content when different concentrations of PCP exposure on male and female Gobiocypris rarus accurately, the two ways of liver homogenate and collection of serum combined with the enzyme-linked immunosorbent assay (ELISA) technique were used. The results showed that40,80,120,160μg·L-1PCP exposure for21d could significantly induce the liver and serum of male and female Gobiocypris rarus VTG protein, and had a significant concentration effect (P<0.01). The studies suggested that liver organization homogenate of Gobiocypris rarus combined with ELISA also could accurately detect the estrogen effects of PCP. The VTG protein of Gobiocypris rarus could be used as candidate sensitive biomarkers for decting PCP. And it is proved that PCP has estrogenic effects from protein level.3. In order to reveal the mechanism of estrogen effect of PCP, Real-time PCR was used to detect the expression of estrogen related key genes (ERα、ERβ1、ERβ2、 VTG Ⅰ、VTG Ⅱ) in the liver of male and juvenile Gobiocypris rarus exposed to PCP at different concentrations (8,16,80,160μg·L-) for21d. DMSO (<0.01%) and17α-ethynylestradiol (EE2,50ng·L-1) were set as solvent control and positive control, respectively. The research found that different concentrations of PCP could inhibit the mRNA expression of ERa gene in the liver of male and juvenile Gobiocypris rarus (P <0.05), induce the mRNA expression of ERβ1、ERβ2(P<0.05). But EE2had the opposite effect, it could induce the mRNA expression of ERα gene(P<0.01), inhibite the mRNA expression of ERβ1、ERβ2gene(P<0.05). While, PCP and EE2could significantly induce the mRNA expression of VTG Ⅰ、VTGⅡ gene(P<0.01), but the effect of PCP was much weaker than EE2. The studies suggested that PCP and EE2might play the estrogen effects by regulation the expression of different ER gene, and then induce the expression of VTG gene. Also, it is proved that PCP has estrogenic effects from gene level.4. Using the degenerate primers and RT-PCR technology from the liver of Gobiocypris rarus, HSP70and HSP90gene sequence fragments were colned. HSP70gene fragment had1783bp sequence, ecoding594amino acids, submitted to Genbank and received the seguence number:HQ897964; HSP90gene fragment had1995bp sequence, ecoding665amino acids, submitted to Genbank and received the seguence number:HQ897963. The research found that HSP70and HSP90gene of Gobiocypris rarus were highly conservative by the amino acid sequence aligment. They had the closer relationship with Tanichthys albonubes by creating phylogenetic trees. Moreover, the mRNA expressions of HSP70and HSP90in various tissues of Gobiocypris rarus were examined. The date showed that HSP70gene was all detected but different in tested tissues. The highest were found in the gill and heart tissues. While HSP90gene was all expressed in tested tissues but the fins, the highest was found in the liver tissue. The studies suggested that the cloned HSP70and HSP90gene of Gobiocypris rarus can lay a basis of study the molecular biomarker for detecting the environmental endocrine disruptors.5. Using Real-time PCR technique, mRAN expression of HSP70and HSP90gene were detected in the liver of Gobiocypris rarus which were exposed at different concentrations and times of PCP. The studies found that PCP could significantly induce the expression of HSP70and HSP90gene, had concentration and time effects.8、16、80、160μg·L-1PCP exposure for7d, the mRNA expression of HSP70gene were induced with the increasing PCP concentrations, the regression equation was: y=0.0408x+1.6692; HSP90gene expression increased after the first drop with increasing concentrations of PCP, the regression equation was: y=0.0002x2+0.0311x+1.1563.80μg·L-1PCP exposure for0.5、1、3、5、7d, the mRNA expression of HSP70gene also were induced with the increasing PCP concentrations, the regression equation was:y=0.7216x+1.1606; HSP90gene expression also increased after the first drop with the increasing concentrations of PCP, the regression equation was:v=-0.0917x2+0.8292x+1.0386. The studies suggested that HSP70and HSP90gene of Gobiocypris rarus can be used as the biological toxicity assessment and new molecular biomarkers for early warning of PCP pollution.