Oxygen is essential for all metazoans to survive, with one known exception 1 . Decreased O 2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions [2][3][4][5] . Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O 2 concentrations without confounding effects since C. elegans obtain O 2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system 6 . Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O 2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxiainducible transcription factor [6][7][8][9] . C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O 2 7,10. Hypoxia is a general term for "less than normal O 2 ". Normoxia (normal O 2 ) can also be difficult to define. We generally consider room air, which is 210,000 ppm O 2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O 2 concentrations from 5-12% (50,000-120,000 ppm O 2 ) 11 . In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O 2 12. However, hif-1 does not play a role in the response to lower concentrations of O 2 (anoxia, operational definition <10 ppm O 2 ) 13 . In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases 10 . The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O 2 .Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O 2 . The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia 10,14 . This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Hydrogen sulfide (H 2 S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H 2 S improves survival of ischemia/reperfusion. We have previously shown that exposure to H 2 S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H 2 S metabolism. SQRD-1 is generally considered important to detoxify H 2 S. Here, we show that SQRD-1 is also required to maintain protein translation in H 2 S. In sqrd-1 mutant animals, exposure to H 2 S leads to phosphorylation of eIF2␣ and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H 2 S or in hif-1(ia04) mutants that die when exposed to low H 2 S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H 2 S-induced phosphorylation of eIF2␣. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H 2 S, but not in wild-type animals. We speculate that SQRD-1 activity in H 2 S may coordinate proteostasis responses in multiple cellular compartments. Hydrogen sulfide (H 2 S)3 is an endogenously produced gas molecule with roles in signaling, neuromodulation, and vasodilation (reviewed in Ref. [1][2][3][4]. Treatment with exogenous H 2 S improves outcome in multiple mammalian models of ischemia/ reperfusion injury (5). However, H 2 S is also toxic at high concentrations, provoking immediate apnea and loss of consciousness that can result in death (6). Industrial exposure to H 2 S is the second-leading cause of death by inhalation, behind only carbon monoxide. The mechanistic differences between beneficial and toxic effects of H 2 S are poorly understood.Sulfide-quinone oxidoreductase (SQRD) is a highly conserved mitochondrial protein that oxidizes cellular H 2 S by transferring electrons to the mitochondrial electron transport chain and adding sulfane sulfur atoms to free sulhydryl moieties (Fig. 1A) (7-9). Isolated mitochondria from chicken liver and human cells can generate ATP when exposed to H 2 S as a result of SQRD activity, which is considered an important aspect of cellular sulfide detoxification (10 -12). However, it is now clear that protein activity can be regulated by post-translational modification by sulfide, and this may be an important aspect of the cellular signaling roles of H 2 S (2, 4). SQRD is therefore positioned to modulate both signaling and toxicity of H 2 S in animals.The nematode Caenorhabditis elegans has a single orthologue of SQRD, sqrd-1. SQRD-1 localizes to mitochondria and is essential for animals to survive exposure to even low concentrations of H 2 S (13). Here, we show SQRD-1 activity is required to prevent activation of the integrated stress response upon exposure to H 2 S. We found that the translation initiation factor eIF2␣ is phosphorylated by both PEK-1 and GCN-2 kinases in sqrd-1 mu...

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that can be cytoprotective, especially in conditions of ischemia/reperfusion injury. However, H2S is also toxic, and unregulated accumulation or exposure to environmental H2S can be lethal. In Caenorhabditis elegans, the hypoxia inducible factor (hif-1) coordinates the initial transcriptional response to H2S, and is essential to survive exposure to low concentrations of H2S. We performed a forward genetic screen to identify mutations that suppress the lethality of hif-1 mutant animals in H2S. The mutations we recovered are specific for H2S, as they do not suppress embryonic lethality or reproductive arrest of hif-1 mutant animals in hypoxia, nor can they prevent the death of hif-1 mutant animals exposed to hydrogen cyanide. The majority of hif-1 suppressor mutations we recovered activate the skn-1/Nrf2 transcription factor. Activation of SKN-1 by hif-1 suppressor mutations increased the expression of a subset of H2S-responsive genes, consistent with previous findings that skn-1 plays a role in the transcriptional response to H2S. Using transgenic rescue, we show that overexpression of a single gene, rhy-1, is sufficient to protect hif-1 mutant animals in H2S. The rhy-1 gene encodes a predicated O-acyltransferase enzyme that has previously been shown to negatively regulate HIF-1 activity. Our data indicate that RHY-1 has novel, hif-1 independent, function that promotes survival in H2S.

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