A Second Look at Sulfide Toxicity: Intracellular Inclusions, Mitochondrial Damage and Cell Death in Sulfide-Adapted Annelids

硫化氢是一种天然存在的高活性和高毒性分子。硫化物毒性的最知名和最佳表征机制是在极低（微摩尔）硫化物浓度下对线粒体细胞色素c氧化酶（考克斯）的可逆抑制。此外，硫化物还有其他潜在的毒性机制，包括抑制考克斯以外的近20种酶、损害血红蛋白、抑制肌肉收缩以及与神经元信号传导途径相互作用--甚至本身可能是一种神经调节剂。考虑到硫化物的潜在毒性，可能令人惊讶的是，各种海洋无脊椎动物是世界各地硫化物浓度可能比假定的毒性水平高出一千倍的栖息地特有的。本提案中的实验将检验这样的假设，即这些无脊椎动物中的硫化物暴露仍然会导致不可逆的线粒体损伤，导致通过自噬摄入受损的线粒体，自噬是细胞在专门的消化室中隔离和降解其自身细胞器和胞质溶胶的一般机制。这一过程的提出是为了尽量减少广泛的细胞死亡，否则将导致线粒体损伤。拟议的实验将利用体腔细胞（“红细胞”）从海洋多毛类动物甘油二鳃。整个动物（随后将从中纯化体腔细胞）和分离的体腔细胞将暴露于硫化物和其他线粒体毒素，有和没有自噬抑制剂。本研究的主要目的是：1）确定硫化物暴露后自噬区室的诱导和消失的时间过程; 2）证明诱导区室确实包含自噬和线粒体的特征; 3）证明硫化物暴露导致线粒体损伤，表现为体外和体内不可逆的线粒体去极化;以及4）证明硫化物暴露会导致细胞死亡，在体外和体内，较低浓度的硫化物会导致细胞凋亡，较高浓度的硫化物会导致细胞坏死。本提案中的实验将为两个长期研究项目奠定基础：1）硫化物在动物细胞中的毒性机制以及适应硫化物的动物用于降低这种毒性的策略，和2）环境应激对线粒体自噬和生物发生的影响（即，“线粒体周转率”）。如果这一假设得到验证，那么硫化物适应环节动物的暴露组织可能会大大增加线粒体损伤，自噬和生物合成，这些都是迄今为止未被注意到的，值得进一步研究。此外，这也可能适用于其他受极端环境条件影响的动物，这些条件可能导致线粒体损伤，例如高温，紫外线辐射增加，pH极端，高氧和许多有毒污染物。最后，该项目将在一个具有加强妇女和少数民族研究机会的悠久传统的实验室环境中，向几名本科生和一名研究生提供教育、培训、研究经验和财政支助。

Hydrogen sulfide is a naturally occurring, highly reactive and highly toxic molecule. The most well-known and best characterized mechanism of sulfide toxicity is its reversible inhibition of mitochondrial cytochrome c oxidase (COX) at very low (micromolar) sulfide concentrations. In addition, sulfide has other potential mechanisms of toxicity, which include inhibition of almost 20 enzymes besides COX, damage to hemoglobin, inhibition of muscle contraction, and interaction with neuronal signaling pathways - potentially even as a neuromodulator itself. Given sulfide's potential toxicity, it is perhaps surprising that a variety of marine invertebrates are endemic to habitats worldwide where the sulfide concentration can be a thousand times higher than the presumed toxic level. The experiments in this proposal will test the hypothesis that such sulfide exposures in these invertebrates nonetheless cause irreversible mitochondrial injury that leads to ingestion of the injured mitochondria by autophagy, which is a general mechanism by which cells sequester and degrade their own organelles and cytosol within specialized digestive compartments. This process is proposed to minimize the widespread cell death that would otherwise result from mitochondrial injury.The proposed experiments will utilize coelomocytes ("red blood cells") from the marine polychaete Glycera dibranchiata. Whole animals (from which the coelomocytes will subsequently be purified) and isolated coelomocytes will be exposed to sulfide and other mitochondrial toxins, both with and without inhibitors of autophagy. The proposed work contains four specific aims: 1) Determine the time course for the induction and disappearance of the presumed autophagy compartments following sulfide exposure; 2) demonstrate that induced compartments do indeed contain signatures of both autophagy and mitochondria; 3) demonstrate that sulfide exposure causes mitochondrial injury, as represented by irreversible mitochondrial depolarization both in vitro and in vivo; and 4) demonstrate that sulfide exposure causes cell death, with lower sulfide concentrations causing apoptosis, and higher concentrations causing necrosis both in vitro and in vivo.The experiments in this proposal will form the foundation for two long-term research projects: 1) the mechanism(s) of sulfide toxicity in animal cells and the strategies used by sulfide-adapted animals to reduce this toxicity, and 2) the effects of environmental stressors on mitochondrial autophagy and biogenesis (i.e., the rate of "mitochondrial turnover"). If the hypothesis is validated, then exposed tissues of sulfide-adapted annelids are likely undergoing greatly increased mitochondrial injury, autophagy and biogenesis that have thus far gone unnoticed and are worthy of further investigation. Furthermore, this may also be true for other animals affected by extreme environmental conditions with the potential to cause mitochondrial injury, such as high temperature, increased UV radiation, pH extremes, hyperoxia and many toxic pollutants. Finally, this project will provide education, training, research experience and financial support to several undergraduate students and one graduate student in a laboratory environment having a strong tradition of enhancing research access to women and minorities.