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、高活性和剧毒的分子。硫化物毒性的最广为人知和最具代表性的机制是在极低的(微摩尔)硫化物浓度下对线粒体细胞色素c氧化酶(COX)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COX以外的近20种酶，对血红蛋白的损伤，抑制肌肉收缩，以及与神经元信号通路的相互作用-甚至可能作为神经调节剂本身。考虑到硫化物的潜在毒性，也许令人惊讶的是，各种海洋无脊椎动物是世界各地栖息地的特有物种，在这些栖息地，硫化物的浓度可能比推定的有毒水平高出一千倍。这项提议中的实验将检验这样一种假设，即这些无脊椎动物暴露在硫化物中会导致不可逆转的线粒体损伤，导致通过自噬摄取受损的线粒体，这是细胞在专门的消化室中隔离和降解自己的细胞器和细胞质的一般机制。这一过程是为了最大限度地减少线粒体损伤造成的广泛细胞死亡。拟议的实验将利用海洋多毛类二枝甘油的体腔细胞(“红细胞”)。整个动物(随后将从其中提纯体腔细胞)和分离的体腔细胞将暴露在硫化物和其他线粒体毒素中，无论有没有自噬抑制剂。这项拟议的工作包含四个具体目标：1)确定硫化物暴露后假定的自噬间隔的诱导和消失的时间进程；2)证明诱导的间隔确实包含自噬和线粒体的特征；3)证明硫化物暴露引起线粒体损伤，在体外和体内表现为不可逆的线粒体去极化；和4)证明硫化物暴露会导致细胞死亡，较低的硫化物浓度会导致细胞死亡，较高的硫化物浓度会在体外和体内引起坏死。这项建议中的实验将形成两个长期研究项目的基础：1)硫化物在动物细胞中的毒性机制(S)和适应硫化物的动物减少这种毒性的策略；2)环境应激源对线粒体自噬和生物发生(即线粒体周转率)的影响。如果这一假设得到验证，那么适应硫化物的环节动物暴露的组织很可能正在经历极大的线粒体损伤、自噬和生物发生，这些到目前为止还没有被注意到，值得进一步研究。此外，受极端环境条件影响的其他动物可能也是如此，这些极端环境条件可能会导致线粒体损伤，如高温、紫外线辐射增加、极端pH、高氧和许多有毒污染物。最后，该项目将为实验室环境中的几名本科生和一名研究生提供教育、培训、研究经验和财政支持，这些学生具有加强妇女和少数群体参与研究的强大传统。

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.