基于肝癌病人CYP2E1代谢活性增高研究亚硝胺类致肝癌作用

Approximately 10% cirrhotic patients can develop into hepatocellular carcinoma (HCC), and more than 80% HCC patients are accompanied by cirrhosis. Although it has been reported that nitrosamines can act as procarcinoges inducing hepatocarcinogenesis, which was supported by animal experiment and human epidemiology study, the correlation of their carcinogenicity and metabolic activity of CYP2E1 has not been determined. Based on the significantly increased CYP2E1 activity in cirrhotic livers of HCC patients in our early studies, we hypothesize that increased CYP2E1 activity may produce enhanced metabolism of nitrosamines to carcinogens, thus leading to HCC.. We have already built up of a bank including 150 normal human livers and 120 cirrhotic livers from HCC patients. The alteration in enzyme kinetics of chlorzoxazone, dimethylnitrosamine and diethylnitrosamine metabolized by CYP2E1, the correlation of their metabolic activity CLint, the relationship between the alteration in CYP2E1 activity and protein content as well as gene polymorphisms would been determined in 150 normal livers and 120 cirrohotic livers from HCC patients. The correlation between metabolic activity of nitrosamines by CYP2E1 and their carcinogenicity will be investigated in wild-type, cyp2e1 gene knock-out and CYP2E1 inhibitor-treated rats to ascertain that increased or decreased CYP2E1 activity can induce or inhibit nitrosamines-promoted hepatocarcinogenesis. The pharmacokinetics of chlorzoxazone will be examined in healthy volunteers, simple liver cirrhotic patients, and HCC patients with cirrhosis to determine changes of CYP2E1 activity in HCC patients. If this hypothesis could be verified, it would provide a new target for early prevention and treatment of HCC.

肝癌病人80%以上伴有肝硬化，肝硬化病人有约10%发展成肝癌。尽管亚硝胺类作为前致癌物引发肝癌已有动物实验和人体流行病学依据，但尚未确定其致癌性与CYP2E1代谢亚硝胺类活性的相关性。根据我们前期发现的肝癌病人肝硬化肝CYP2E1代谢活性明显升高，提出“CYP2E1活性增高，代谢亚硝胺类前致癌物增多，而引发肝癌”的假说。.以前期收集的150例正常人肝和120例肝癌病人肝硬化肝为研究对象，研究CYP2E1代谢氯唑沙宗、二甲基亚硝胺和二乙基亚硝胺的酶动力学改变及机制，并确定其相关性；以正常大鼠、基因敲除大鼠及酶抑制大鼠为研究对象，研究CYP2E1代谢亚硝胺类活性与致癌性之间的相关性，以确定CYP2E1的活性变化与肝癌发生的因果关系；以正常人、单纯性肝硬化病人、肝硬化肝癌病人为研究对象，研究氯唑沙宗的药动学，以确定肝癌病人体内CYP2E1的变化。这可为肝癌病人的早防早治提供新的作用靶点。

肝癌(Hepatocellular carcinoma，HCC)是常见的恶性肿瘤之一，具有恶性度高、预后差、死亡率高等特点。至今尚缺乏有效的治疗药物，因此，其有效防治尤为重要。日常饮食中的亚硝胺类为前致癌物，需在体内经过肝脏 CYP2E1 代谢活化生成致癌物质。本研究以150例正常人肝和120例HCC病人癌旁肝硬化肝组织为研究对象，测定CYP2E1代谢氯唑沙宗、二甲基亚硝胺和二乙基亚硝胺的酶动力学，结果显示HCC病人肝硬化肝CYP2E1代谢氯唑沙宗、二甲基亚硝胺和二乙基亚硝胺的活性均显著升高（分别增高约2倍），且CYP2E1代谢三种物质的活性存在正相关性，相关系数在0.651～0.712之间。其中CYP2E1含量、基因多态性和POR基因多态性及活性对CYP2E1活性无显著性影响。.正常大鼠体内CYP2E1代谢氯唑沙宗与代谢二乙基亚硝胺活性具有较好相关性，相关系数达0.76。采用间断性腹腔注射二乙基亚硝胺的方法制备大鼠肝癌模型，发生率64.9%。于制备模型前、肝硬化及HCC形成期在大鼠体内进行二乙基亚硝胺的毒代动力学研究，结果发现正常大鼠CYP2E1先天活性与HCC发生率和严重程度具有较好相关性，其中低t1/2、低AUC0-t和高CL/F组较高t1/2、高AUC0-t和低CL/F组HCC发生率显著升高；肿瘤结节数、最大肿瘤直径、累积肿瘤直径、最大肿瘤体积及累积肿瘤体积均显著增高/增大。cyp2e1基因敲除和CYP2E1抑制剂氯美噻唑可显著抑制大鼠HCC发生，且抑制后大鼠CYP2E1活性与肝癌严重程度呈正相关；其中抑制后毒代动力学参数 AUC0-t、AUC0-∞、t1/2与最大肿瘤直径及累积肿瘤直径成负相关，CL/F与上述指标均呈正相关。以氯唑沙宗为探针药物，采用体外推体内方法推导出HCC患者CYP2E1活性显著增高，约是正常人的2.15倍。.综上研究，可基本证明“CYP2E1 活性增高，代谢亚硝胺类前致癌物增多，而引发HCC”的假说。这将为HCC的早防早治提供新的靶点，具有重大基础与临床意义。