重金属镍纳米颗粒的致癌作用及分子机制研究

Nickel is a widely distributed heavy metal. Nickel nanoparticles are a new product with many characteristics, which include a high level of surface energy, high magnetism, low melting point and high surface area. However, concern has been expressed that these same properties of nickel nanoparticles to human health. The most important adverse health effect due to nickel exposure are cancers. While numerous studies have described the carcinogenic effects of nickel compounds, the carcinogenesis of metallic nickel is still unclear. Furthermore, although carcinogenic nickel compounds have been known to disrupt a wide range of cellular processes the precise mechanism by which these exert their carcinogenic effects is not known. Studies showed that nickel nanoparticles induced Reactive oxygen species (ROS) in dose and time dependent manner in cell cultures. Our preliminary studies confirm that JB6 cell apoptosis can be significantly inhibited by the addition of antioxidants of catalase. Cellular signaling transcription factors AP-1 and NF-κB, have been shown to play pivotal roles in tumor initiation, promotion, and progression. Activated AP-1 and NF-κB may result in the expression of target genes involving in neoplastic transformation. In addition, the signal transduction pathways of AP-1 and/or NF-κB are known to be important molecular targets of chemo-preventive strategies. Our previous results show that both metallic nickel fine and nanoparticles induced a dose-related increase of AP-1 and NF-κB transcription activity in JB6 cells after 24 h exposure. Furthermore, metallic nickel nanoparticles elicited stronger stimulation on these two transcription factors than fine particles. Metallic nickel nanoparticles also elicited higher activation of tumor promotion factors Akt and Bcl-2 than fine particles. R-Ras and C-myc are tumor promoter genes. In our previous studies, Western blots show that metallic nickel nanoparticles induced a significant increase of protein expressions of R-Ras and C-myc in a time-dependent manner. Based on these results, metallic nickel nanoparticles may be higher carcinogenic than fine particles. The overall hypothesis of this proposal is that antioxidants, which inhibit oxidative stress, activation of certain transcription factors or/and neoplastic transformation, may lead to protection against nickel particle-induced carcinogenesis. This study will elucidate both induction of oxidative stress and carcinogenesis by nickel particles and role of antioxidants in this pathologic process. Specific Aim 1. Assess the carcinogenesis and difference between nickel fine and nanoparticles. Specific Aim 2.Determine chemopreventive effects of antioxidants on nickel fine and nanoparticle-induced activation of oxidative stress sensitive transcription factors， oncogenes，cell neoplastic transformation and tumor formation in animals.

重金属污染已经成为影响我国人民身体健康的重大危害因素。重金属纳米镍颗粒作为一种新型镍产品，现已迅速在现代工业中得到了应用，由此可能带来的职业和环境危害已不可避免。尽管国际癌症研究组织已将镍的化合物列为确认的人类致癌物，但金属镍颗粒的致癌作用尚未定论；与普通金属镍颗粒相比，纳米金属镍颗粒的致癌作用有无增强作用，则更是一个急待研究阐明的课题；关于镍及其化合物潜在致癌作用的分子机制，尽管有研究表明氧化应激损伤可能起重要作用，但到目前为止，还没有采用抗氧化剂直接拮抗或预防重金属镍诱导细胞恶性转化或动物肿瘤形成的报告。本研究计划采用体外细胞培养及体内动物实验相结合的方法，对纳米及普通金属镍颗粒的潜在致癌作用及分子机制进行对比研究，并探讨抗氧化剂在拮抗和预防金属镍颗粒氧化应激损伤及致癌毒性中的作用。为重金属类物质，尤其为纳米金属镍颗粒的潜在致癌作用的评价、防治和拮抗药物的开发提供科学依据。

作为一种新型产品, 重金属纳米镍颗粒目前已经在工业中得到了广泛应用，其环境和职业危害引起了广泛关注。尽管镍的化合物已经被国际癌症研究组织列为确认人类致癌物，但金属镍颗粒的致癌作用尚未定论。另外，与普通金属镍颗粒相比，纳米金属镍颗粒的致癌作用有无变化，更是一个急待研究阐明的课题。在本科研项目（项目号：81273111）的资助下，项目组历经四年的系统研究，采用体外细胞培养及体内动物实验相结合的方法，对纳米及普通金属镍颗粒的潜在致癌作用及分子机制进行对比研究，并探讨了抗氧化剂在拮抗和预防金属镍颗粒氧化应激损伤及致癌毒性中的作用。动物实验发现纳米金属镍对大鼠肝脏、脾脏、肺脏有更明显的毒性作用，并可引起心脏的病理改变，对肺脏组织可以引起明显的促癌R-Ras和c-myc等基因的表达上调。同时，我们采用细胞培养的方法，对拮抗重金属纳米镍颗粒的抗氧化剂进行了筛选，在加与不加抗氧化剂的情况下研究了重金属纳米镍颗粒的致癌分子机制。结果发现茶多酚比前期发现的过氧化氢酶具有更好的拮抗作用，可以在体外实验中明显拮抗重金属纳米镍颗粒的致癌作用，我们发现氧化应激损伤就是重金属纳米镍颗粒致癌作用的关键因素。用JB6 P+ 细胞，进行了细胞恶性转化实验。在加与不加抗氧化剂茶多酚的条件下，细胞经纳米或普通金属镍颗粒处理后，结果发现，经纳米或普通金属镍颗粒处理后后，细胞可以在软琼脂中生长并形成集落，尤其是纳米镍颗粒处理的细胞更为明显。为鉴定细胞转化的恶性程度，将集落形成细胞进行了裸鼠接种。结果发现，裸鼠皮下细胞接种观察两个月未见明显瘤块形成，说明细胞转化的恶性程度尚未达到在裸鼠体内形成肿瘤的程度。提示，下一步需进一步延长诱导细胞恶性转化的时间。另外，在本项目的资助下，发表SCI论文13篇，国内期刊论文4篇。申请并已经获得发明专利1项、实用新型专利4项。2013年，邀请了德国的Hans-Joachim Galla教授来校进行了学术访问。2014年7月在宁波召开了中德科学家纳米毒理论坛。邀请了美国2013年诺贝尔生理和医学奖获得者托马斯苏德霍夫教授来宁波大学进行了学术访问。2014年资助项目负责人赵进顺教授赴美国CDC进行了学术访问。2016年10月资助了美国CDC三位专家来宁波大学进行了学术访问。