CYP2E1介导多氯联苯代谢活化、致突变性及致癌作用研究

Polychlorinated biphenyls (PCBs) are a group of persistent pollutants and human carcinogens, whose biologic activities require metabolic activation except for a few dioxin-like PCBs. However, the exact metabolic enzyme(s) involved in the metabolic activation of PCBs and the relevant structure-toxicity relationships are up to now unclear; this forms a barrier toward appropriate estimation of the environmental risk assessment of PCBs. Mutagenesis is a major mechanism of carcinogenesis. Our pioneering study have observed varied mutagenicity of more than 20 PCBs following metabolic activation by human CYP2E1, while human CYP 1A1, 1B1, 1A2, and 3A4 did not show significant involvement in the activation. In this project, PCBs will be co-cultured with a human CYP2E1-abundant microsomal activating system, wherein the active metabolites will be identified; our experimental data together with those obtained in our pioneering study will be used to create a 3D-QSAR model for activation of PCBs by human CYP2E1 (as demonstrated by genetic toxicity) by using comparative molecular field analysis (CoMFA) method, for prediction of the mutagenicity of many untested PCBs, and the model will be tested by using experimental data with new congeners. Furthermore, PCBs will be investigated for their abilities to induce gene mutations in the Hprt locus and the sequence changes of the mutated gene in mammalian cells, and the abnormalities in the spindle body and centrioles in cultured human hepatocytes. Finally, the in vivo effects of PCBs will be verified by micronucleus test in mice and hepatic transformation foci test in rats. The achievements from this projest will form a scientific basis for the environmental risk assessment of PCBs and the relevant environmental remediation.

多氯联苯（PCBs）是一类持久性污染物和人类致癌物，通常须代谢活化才具有生物活性；但所涉代谢酶及结构-毒性关系尚不清楚，导致其环境危险性评估困难。致突变作用是化学致癌的主要机制。课题组前期研究发现20余个PCBs被人CYP2E1活化而获得对哺乳动物细胞的致突变性，而CYP1A1、1B1、1A2、3A4则无明显作用。本项目将PCBs与含人CYP2E1的代谢活化系统共培养，鉴定可能的活性代谢物；利用实验所获数据及前期研究已得数据以比较分子场分析（CoMFA）法建立PCBs由CYP2E1活化产生遗传毒性的3D-QSAR模型，以预测更多PCBs的遗传毒性，并用新的实验数据验证。进一步观察PCBs致细胞Hprt位点突变、突变基因序列变化，对人肝细胞纺锤体和中心粒改变和恶性转化的作用；并研究PCBs诱发小鼠骨髓微核及大鼠肝脏转化灶的作用。研究成果将为PCBs的环境危险性评价及环境治理提供科学依据。

多氯联苯（PCBs）是一类持久性有机污染物与人类致癌物，其遗传毒作用依赖于代谢活化，然而其代谢酶特异性、毒作用分子靶点及结构-毒性关系尚不清楚，致其生物危险性评价的局限（主要采用激活芳烃受体的二噁英当量）。本项目提出假说:低氯化及非二噁英样PCBs具有依赖人CYP2E1酶的致突变及致癌癌性。. 本项目采用重组表达人类各种CYP酶的V79衍生细胞系、人肝细胞（L-02系）及人肝细胞瘤细胞（C3A系），以细胞遗传学实验、选择性酶抑制剂及模拟分子对接分析，确证了人类CYP2E1是低氯化及非二噁英样PCBs的主要代谢活化酶，赋予后者致基因突变、染色体断裂及非整倍性作用；而邻位无取代基的二噁英样PCBs主要由（肝外组织表达的）人类CYP1B1酶活化，从而可导致染色体断裂。分子对接研究印证了人CYP2E1对不同PCBs的选择性活化：尽管所有含1~5个氯取代基的PCBs均与活性中心均有亲和力，但二噁英样PCBs作为配体大多与亚铁血红素中铁离子的距离>7.0 Å，不利于电子转移，不符合底物要求。本项目还揭示了PCBs由人CYP2E1酶活化的某些结构-毒性关系，例如邻位氯为单个时PCBs致突变性最强，进一步增多则致突变性愈弱，且这一影响随化合物氯化程度增高而愈加剧烈。此外，发现小鼠和大鼠CYP2E1因活性中心空间太小对绝大多数PCBs不具亲和性（与人类同源酶有种属差异），这与PCBs对重组表达小鼠CYP2E1细胞致突变试验及对幼年小鼠肝脏微核试验的阴性结果相符。此外，我们采用人肝细胞L-02系，探讨了PCBs对不同核受体及CYPs酶表达的影响，发现低氯化及非二噁英样PCBs可通过上调CAR受体表达，而二噁英样PCBs诱导芳烃受体作用极强，二者均增强CYP1A2和CYP3A4表达，从而明显增强黄曲霉毒素B1的遗传毒作用. 此外，鉴于细胞内代谢的特点本项目还探讨了相关遗传毒性实验方案的优化等问题，提出了涉及CYP2E1酶活化的实验中溶剂（DMSO）合理浓度，分析了暴露时程对实验结果的影响。将tranwell与微核试验结合，发现不同前致突变物的酶促代谢活性产物跨膜/距离转运力有明显差异，例如黄曲霉毒素B1与苯并(a)芘；由此可解释它们致癌的不同靶组织分布。