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.
