INVESTIGATION INTO PARAQUAT CYTOTOXICITY

百草枯细胞毒性研究

• 批准号: 3253191
• 负责人: MICHAEL J KELNER
• 金额: $ 15.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3253191
• 关键词: RNA biosynthesis; antioxidants; catalase; complementary DNA; copper; cytotoxicity; drug adverse effect; drug metabolism; drug resistance; enzyme inhibitors; enzyme linked immunosorbent assay; enzyme mechanism; glutathione transferase; manganese; messenger RNA; metalloenzyme; metallothionein; molecular cloning; monoclonal antibody; nucleic acid sequence; oligonucleotides; paraquat; protein biosynthesis; superoxide dismutase; transfection; transposon /insertion element; western blottings; zinc

This project will investigate the mechanism of paraquat cytotoxicity. Despite a consensus that oxygen and the redox properties of paraquat are involved, the ultimate cytotoxic event and the protective role of individual anti-oxidant enzymes have not been identified. This project will investigate these events by a novel approach. The ability of anti- oxidant enzymes to protect against paraquat cytotoxicity will be studied on an individual basis. This will be accomplished by coupling the cDNA of these enzymes to suitable expression vectors. Anti-oxidant enzymes to be studied include copper-zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, catalase, metallothionein, and glutathione transferases. Two different, selectable, expression vectors will be constructed for each enzyme. One vector will use the noninducible SV promoter, and the other will use the inducible mouse metallothionein (mMT). Preliminary results with a copper-zinc superoxide dismutase expression vector [SV-CuZnSOD-SVneo] indicate the enzyme is produced in large quantities and paraquat resistance develops. The increase in resistance does not correlate to superoxide dismutase activity, but to increase activity of each enzyme against paraquat will be compared to others to determine which enzyme is most effective. The induction of the anti-oxidant enzymes copper-zinc superoxide dismutase, manganese superoxide dismutase, and catalase by paraquat will be studied to determine if the increase occurs by gene amplification, alterations in mRNA, or protein synthesis. In contrast to these anti-oxidant enzymes, glutathione peroxidase appears to be selectively inhibited by paraquat. The mechanism of cell line. This cell line appears to be resistant to paraquat on the basis of increased glutathione peroxidase activity, and not other anti-oxidant enzymes (such as superoxide dismutase), again suggesting that glutathione peroxidase plays an important role in detoxifying paraquat. The mechanism and cellular level (DNA, RNA, protein synthesis) by which this cell continues to express glutathione peroxidase, even when exposed to paraquat, will be determined.

本课题旨在研究百草枯的细胞毒性机制。 尽管人们一致认为，氧气和百草枯的氧化还原特性 参与，最终的细胞毒性事件和保护作用， 尚未鉴定出单独的抗氧化酶。 这个项目 将用一种新的方法来研究这些事件。 抗- 将研究氧化酶对百草枯细胞毒性的保护作用， 个人基础。 这将通过偶联以下的cDNA来实现： 将这些酶转化到合适的表达载体中。 抗氧化酶是 研究包括铜锌超氧化物歧化酶，锰超氧化物 歧化酶，谷胱甘肽过氧化物酶，过氧化氢酶，金属硫蛋白，和 谷胱甘肽转移酶。 两种不同的可选择的表达载体 将为每种酶构建。 一种载体将使用不可诱导的 SV启动子，另一个将使用可诱导的小鼠金属硫蛋白 （mMT）。 铜锌超氧化物歧化酶的初步结果 表达载体[SV-CuZnSOD-SVneo]表明该酶是在 对百草枯产生抗药性。 的增加 抗性与超氧化物歧化酶活性无关， 将比较每种酶对百草枯增加的活性， 其他人则确定哪种酶最有效。 抗氧化酶铜锌超氧化物歧化酶的诱导， 锰超氧化物歧化酶和过氧化氢酶的百草枯将研究， 确定增加是否通过基因扩增、 mRNA或蛋白质合成。 与这些抗氧化酶相反， 谷胱甘肽过氧化物酶似乎受到百草枯的选择性抑制。 细胞系的机制。 这种细胞系似乎对 百草枯的基础上增加谷胱甘肽过氧化物酶的活性，而不是 其他抗氧化酶（如超氧化物歧化酶），再次表明 谷胱甘肽过氧化物酶在解毒过程中起着重要作用 百草枯。 机制和细胞水平（DNA，RNA，蛋白质合成） 这种细胞继续表达谷胱甘肽过氧化物酶，即使 百草枯的影响将被确定。