INVESTIGATION INTO PARAQUAT CYTOTOXICITY

百草枯细胞毒性研究

• 批准号: 3253190
• 负责人: MICHAEL J KELNER
• 金额: $ 15.26万
• 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/3253190
• 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.

本项目将对百草枯的细胞毒性机制进行研究。 尽管人们一致认为氧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 具体的抗氧化酶还没有确定。这个项目 将以一种新的方法调查这些事件。抗病毒的能力 将研究防止百草枯细胞毒性的氧化酶 以个人为基础。这将通过偶联cdna来实现。 将这些酶转化到合适的表达载体上。抗氧化酶有待进一步研究 研究内容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。两个不同的、可选择的表达载体 将为每种酶构建。一个向量将使用不可诱导的 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检测结果 表达载体[SV-CuZnSOD-SVneo]表明该酶是在 产生大量的百草枯抗药性。这一增长 抗药性与超氧化物歧化酶活性无关，而与 每种酶对百草枯的活性增加将与 其他人决定哪种酶是最有效的。 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对百草枯的影响 确定增加是否是通过基因扩增、基因改变 信使核糖核酸或蛋白质合成。与这些抗氧化酶相反， 谷胱甘肽过氧化物酶似乎被百草枯选择性地抑制。 细胞系的作用机制。该细胞系似乎对 基于谷胱甘肽过氧化物酶活性增加的百草枯，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中起重要作用 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，即使当 暴露于百草枯，将被确定。