Molecular Basis for Antioxidant Enzymes in Mediating Copper Detoxification in the Nematode <i>Caenorhabditis elegans</i>

Antioxidant enzymes play a major role in defending against oxidative damage by copper. However, few studies have been performed to determine which antioxidant enzymes respond to and are necessary for copper detoxification. In this study, we examined both the activities and mRNA levels of SOD, CAT, and GPX under excessive copper stress in Caenorhabditis elegans, which is a powerful model for toxicity studies. Then, taking advantage of the genetics of this model, we assessed the lethal concentration (LC50) values of copper for related mutant strains. The results showed that the SOD, CAT, and GPX activities were significantly greater in treated groups than in controls. The mRNA levels of sod-3, sod-5, ctl-1, ctl-2, and almost all gpx genes were also significantly greater in treated groups than in controls. Among tested mutants, the sod-5, ctl-1, gpx-3, gpx-4, and gpx-6 variants exhibited hypersensitivity to copper. The strains with SOD or CAT over expression were reduced sensitive to copper. Mutations in daf-2 and age-1, which are involved in the insulin/insulin-like growth factor-1 signaling pathway, result in reduced sensitivity to stress. Here, we showed that LC50 values for copper in daf-2 and age-1 mutants were significantly greater than in N2 worms. However, the LC50 values in daf-16;daf-2 and daf-16;age-1 mutants were significantly reduced than in daf-2 and age-1 mutants, implying that reduced copper sensitivity is influenced by DAF-16-related functioning. SOD, CAT, and GPX activities and the mRNA levels of the associated copper responsive genes were significantly increased in daf-2 and age-1 mutants compared to N2. Additionally, the activities of SOD, CAT, and GPX were greater in these mutants than in N2 when treated with copper. Our results not only support the theory that antioxidant enzymes play an important role in copper detoxification but also identify the response and the genes involved in these processes.抗氧化酶（Antioxidant enzymes）在抵御铜诱导的氧化损伤中发挥核心作用。然而，目前针对铜解毒过程中响应并发挥必需功能的抗氧化酶的相关研究仍较为匮乏。本研究以毒性研究的经典模式生物——秀丽隐杆线虫（Caenorhabditis elegans）为对象，检测了过量铜胁迫下其超氧化物歧化酶（SOD）、过氧化氢酶（CAT）与谷胱甘肽过氧化物酶（GPX）的活性及mRNA水平。随后借助该模式生物的遗传操作体系，测定了相关突变株的铜半数致死浓度（LC50）值。 结果显示，处理组的SOD、CAT及GPX活性均显著高于对照组；同时，sod-3、sod-5、ctl-1、ctl-2以及几乎所有gpx基因的mRNA水平在处理组中也显著上调。在受试突变株中，sod-5、ctl-1、gpx-3、gpx-4与gpx-6突变体对铜表现出超敏性；而过表达SOD或CAT的菌株则对铜的敏感性降低。 参与胰岛素/胰岛素样生长因子-1信号通路的daf-2与age-1基因突变，可降低生物体对胁迫的敏感性。本研究发现，daf-2与age-1突变株的铜LC50值显著高于野生型N2线虫；而daf-16;daf-2与daf-16;age-1双突变株的LC50值则显著低于daf-2和age-1单突变株，这表明铜耐受性的降低受DAF-16相关功能调控。与野生型N2相比，daf-2与age-1突变株的SOD、CAT及GPX活性，以及铜响应相关基因的mRNA水平均显著升高；且经铜处理后，这些突变株的上述酶活性同样高于N2对照。 本研究结果不仅证实了抗氧化酶在铜解毒过程中的重要作用，同时明确了参与该过程的响应酶类与相关基因。