| 其他摘要 | 本研究根据目前我国渤海重金属和多环芳烃的污染现状，选择具有环境指示功能的贝类菲律宾蛤仔为研究对象，克隆了多种与I相、II相解毒代谢相关的基因，包括细胞色素P450（CYP450）和7种谷胱甘肽硫转移酶（GST）的同工酶基因。研究了Cd、Cu和BaP作用下菲律宾蛤仔组织内重金属累积规律、抗氧化酶活力变化，解毒代谢酶基因表达调控规律以及代谢产物响应规律，探讨重金属和BaP对菲律宾蛤仔的分子致毒机理，为多指标体系生物标志物环境检测技术的建立提供理论依据。论文获得如下一系列的结果和认识：1. 菲律宾蛤仔解毒代谢相关基因克隆与表达分析本研究克隆了菲律宾蛤仔多个I相与II相解毒代谢相关基因的全长，包括细胞色素P450（命名为CYP414A1）和GST的7种同工酶基因（分别命名为VpGSTS1、VpGSTS2、VpGSTS3、VpGSTO、VpGSTM、VpGSTR和VpGSTMi）。CYP414A1属于CYP450超家族中一个新的家族，与脊椎动物CYP2家族的同源性最高。7种GST同工酶基因中有三种属于Sigma（VpGSTS1、VpGSTS2和VpGSTS3）家族，其余四种分别属于Omega（VpGSTO）、Mu（VpGSTM）、Rho（VpGSTR）和Microsomal（VpGSTMi）家族。这7种GST同工酶基因分别与软体动物、鱼类和其他无脊椎动物的GST具有较高的序列相似性（38%-63%）。组织表达分析发现，克隆得到的解毒代谢酶基因大部分主要表达于菲律宾蛤仔的肝胰腺组织中，与软体动物肝胰腺是污染物解毒和代谢主要场所的观点相一致。2. 菲律宾蛤仔多指标体系生物标志物对重金属暴露的响应本研究测定了Cd和Cu暴露24、48和96 h后蛤仔肝胰腺和鳃中重金属的积累量。结果发现，蛤仔肝胰腺与鳃组织对重金属Cd和Cu的积累随暴露浓度与暴露时间的增加而增加，具有明显的剂量效应与时间效应。菲律宾蛤仔对Cd的富集速率大于Cu，且肝胰腺中重金属的蓄积量高于鳃。重金属暴露下蛤仔肝胰腺和鳃组织中SOD、GPx与GST活力在大部分情况下均显著升高，但在暴露的某些时段 SOD与GST活力被高浓度Cd与Cu显著抑制。这三种抗氧化酶中SOD对Cd暴露比较敏感，而SOD与GST活力变化对Cu暴露都比较敏感。利用荧光定量 PCR 方法，分析了菲律宾蛤仔CYP414A1与VpGSTs同工酶基因在不同浓度重金属暴露下的时序表达情况。CYP414A1基因mRNA在10与40 μg/L Cd暴露48与96 h后其表达量均显著升高（最高表达量为对照组的2.8倍），但在10与40 μg/L Cu暴露下，其基因表达被显著抑制（最低表达量为对照组的0.4倍）。大部分VpGSTs 同工酶基因mRNA表达量对Cd暴露响应不太明显，其中VpGSTS2基因的转录水平被显著抑制（最低为对照组的0.07倍）。Cu暴露后，VpGSTS2在转录水平的响应最为突出，其mRNA表达量最高达到对照组的11倍。另外VpGSTS1、VpGSTS3与VpGSTM基因的表达量也被显著诱导（为对照组表达水平的2.5-4.8倍）。VpGSTO与VpGSTMi基因表达在Cu暴露下被显著抑制（最低可达对照组的0.07倍）。代谢组学研究表明，重金属暴露引起菲律宾蛤仔鳃组织内代谢图谱发生显著变化，并持续至48或96 h。根据渗透调节物质、柠檬酸循环中间产物以及氨基酸等代谢物含量的上调或下调，推测重金属暴露引起机体能量代谢异常和渗透调节紊乱。其中，镉暴露组中谷氨酸盐含量的显著升高表明Cd具有一定的神经毒性。另外，通过对比发现，代谢产物变化对Cu的响应更加敏感和持久。3. 菲律宾蛤仔多指标体系生物标志物对BaP暴露的响应5与50 μg/L BaP暴露菲律宾蛤仔24、48及96 h后对其组织内抗氧化酶活力产生了一定的影响。蛤仔肝胰腺内抗氧化酶指标变化并不显著，而鳃组织内SOD、GST活力被BaP显著诱导，并表现出一定的时间效应和剂量效应关系。关于解毒代谢酶基因表达调控方面，菲律宾蛤仔在50 μg/L BaP暴露96 h后，CYP414A1基因表达量显著升高，为对照组水平的5.6倍。7种GST同工酶基因中， VpGSTS2和VpGSTR基因表达对BaP暴露最敏感，二者mRNA表达量的最高值均为对照组的6.5倍。同样，VpGSTS3和VpGSTM基因表达量也显著升高，为对照组表达水平的1.7-3.8倍。代谢组学研究表明，50 μg/L BaP暴露24 h能引起蛤仔鳃组织内渗透调节紊乱和厌氧代谢，并在48 h恢复至正常组水平。另外，BaP两个剂量组代谢产物在整个暴露过程中均表现出完全相反的趋势，这可能是由于BaP暴露对菲律宾蛤仔具有一定的毒物兴奋效应。上述结果表明，重金属和BaP在菲律宾蛤仔体内的富集产生了大量的活性氧产物，对机体造成严重的氧化压力，从而刺激SOD和GST迅速合成来清除和转运活性氧产物。重金属和BaP暴露对解毒代谢酶基因表达调控主要表现为两种形式，即表达的诱导和表达的阻遏。本文所研究的I相与II相解毒代谢酶基因在蛤仔应对环境污染物的氧化压力和代谢解毒过程中发挥着巨大的作用，其中CYP414A1、VpGSTS2、VpGSTS3、VpGSTM、VpGSTR基因对Cu或BaP暴露响应最为敏感，具有作为监测海洋环境污染生物标志物的潜能。代谢组学研究表明，重金属和BaP暴露均引起菲律宾蛤仔鳃组织内代谢图谱发生显著变化，主要表现为能量代谢异常和渗透调节紊乱。通过比较三种污染物作用后蛤仔代谢产物的响应情况，发现Cu暴露引起蛤仔鳃组织代谢谱的变化最敏感和持久。; Based on the contaminated situation of heavy metals and polycyclic aromatic hydrocarbons (PAHs) at the Bohai coast, we studied the toxic mechanisms of heavy metals and PAHs on Manila clam, Venerupis philippinarum. Several phase I and phase II enzymes genes were identified from V. philippinarum, such as cytochrome P450 (CYP450) and seven glutathione S-transferases isoforms (GSTs). Heavy metals accumulation, antioxidant enzymes activity, xenobiotic metabolizing enzymes gene expression, and the response of metabolites were investigated in order to illustrate the toxic effect of heavy metals and BaP, and provide a theoretic support for the multi biomarkers selection in heavy metals and PAHs biomonitoring. Details of the thesis were listed as followed.1. Molecular cloning and expression of xenobiotic metabolizing enzyme genes from V. philippinarumIn this study, we reported the cloning and characterization of a new cytochrome P450 gene (designated as CYP414A1) and seven GST isoforms gene (designated as VpGSTS1, VpGSTS2, VpGSTS3, VpGSTO, VpGSTMi, VpGSTM and VpGSTR) from V. philippinarum. CYP414A1 was named by the CYP Nomenclature Committee as a member of a new subfamily. Blast and phynogenetic analysis indicated a close relationship between the CYP414A1 and CYP2 family from some vertebrates. In addition, the GST isoforms gene were classified into five different classes, including three in Sigma (VpGSTS1, VpGSTS2 and VpGSTS3) and one each in Omega (VpGSTO), Mu (VpGSTM), Rho (VpGSTR) and the microsomal GST isoenzyme (VpGSTMi). The seven GST isoforms showed high similarities with those from fishes, mollusks and other invertebrates (38%-63%). At basal levels of tissue expression, most genes indentified in this study were highly expressed in hepatopancreas compared with those in other tissues, which was consistent with the viewpoint that hepatopancreas was the major organ for detoxification of xenobiotics. 2. Responses of multi biomarkers in V. philippinarum to the exposure of heavy metalsIn this study, we investigated the bioaccumulation of heavy metals in the hepatopancreas and gills of clams, which were treated with Cd and Cu for 24, 48 and 96 h. The results suggested that there were obvious time- and does-dependent responses. The clam showed higher bioaccumulation ability to Cd than Cu, and the heavy metal concentrations in hepatopancreas were greater than those in gills. Additionally, the activities of superoxide dismutase (SOD), glutathione peroxidases (GPx) and GST were significantly up-regulated in hepatopancreas and gills of V. philippinarum at different concentrations and time intervals, while the activities of SOD and GST dropped significantly at high concentrations of Cd and Cu in some time intervals. Among these, SOD showed higher susceptibility to Cd exposure; for Cu exposure, both SOD and GST were highly induced, demonstrating their great potential as biomarkers for environmental biomonitoring studies. Moreover, the temporal expression profiles of hepatopancreas CYP414A1 and GSTs transcript to different concentrations of Cd and Cu exposure were measured by quantitative real-time RT-PCR. The expression level of CYP414A1 was significantly up-regulated by 10 and 40 μg/L Cd (with a maximal response of 2.8-fold), but sharply depressed to 0.4-fold of control group by Cu. No significant change was observed in the mRNA expression of most GST isoforms at the tested Cd concentrations throughout the time course, with the exception that the expression level of VpGSTS2 dropped obviously to 0.04-fold of control level. Cu exposure affected the mRNA expression of GST isoforms differentially. More notably, the expression of VpGSTS2 was significantly higher than those of the other up-regulated GSTs isoforms, with a maximal response of 11 fold. In addition, VpGSTS1, VpGSTS3 and VpGSTM also showed significant increases (2.5-4.8 folds) after exposure to 10 and 40 μg/L of Cu. With respect to VpGSTO and VpGSTMi, the expression level decreased significantly to 0.07 fold of the control group.Metabolomics analysis showed significant separations between the control and the heavy metals treated groups during 48 or 96 h of exposure. Both Cd and Cu could cause the disturbances in energy metabolism and osmotic regulation based on the increase or decrease of metabolic biomarkers such as osmolytes, intermediates of the Krebs cycle and amino acids. In the Cd-exposed samples, the elevation of glutamate was differentially observed and it was suggested the biomarker of neurotoxicity. By contrast, the metabolic responses in Cu-treated groups were more sensitive and constant than those in Cd-treated groups.3. Responses of multi biomarkers in V. philippinarum to the exposure of BaP.According to our study, the antioxidant system in the hepatopancreas of BaP-treated groups exhibited no modulation at all time points. However, the activities of SOD and GST in gills were significantly induced with a dose- and time-dependent manner. Moreover, CYP414A1 mRNA expression was significantly induced by 50 μg/L BaP (with a maximal response of 5.6-fold). Among the seven GST isoforms, VpGST2 and VpGSTR mRNA expression displayed the most notable increases, both with the highest expression level of 6.5 fold. Likewise, VpGSTS3 and VpGSTM mRNA expression were significantly induced with 1.7-3.8 fold increases. Metabolomics analysis suggested that 50 μg/L BaP induced the disturbances of energy metabolism and osmotic regulation at 24 h, and the recovery of metabolic response was found after 48 and 96 h of exposures. In addition, the contrary metabolic differences between the low and high doses of BaP-exposed groups were observed throughout the time course, which was likely caused by the hormesis of BaP in clam.In conclusion, the accumulation of Cd, Cu and BaP in the tissue of clams induced excessive production of reactive oxygen species, resulting in a serious oxidative stress, and then the activities of SOD or GST were rapidly up-regulated to mitigate such oxidative damage. The expression profiles of CYP414A1 and GST isoforms appeared to be selectively up or down regulated, which enhanced our understanding of the detoxification and antioxidant roles of phase I/phase II enzymes. Of them, CYP414A1, VpGSTS2, VpGSTS3, VpGSTM and VpGSTR exhibited significant up-regulation, indicating their good biomarker potential for Cu or BaP pollution in marine environment. Metabolomics analysis suggested that heavy metals and BaP exposure caused significant metabolic changes, such as the disturbances of energy metabolism and osmotic regulation. By contrast, the metabolic responses in Cu-treated groups were more sensitive and constant than those in Cd and BaP treated groups. |
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