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Harsh acid oxidation of activated charcoal transforms an insoluble carbon-rich source into water-soluble, disc structures of graphene decorated with multiple oxygen-containing functionalities. We term these pleiotropic nano-enzymes as "pleozymes". A broad redox potential spans many crucial redox reactions including the oxidation of hydrogen sulfide (H 2 S) to polysulfides and thiosulfate, dismutation of the superoxide radical (O 2 - *), and oxidation of NADH to NAD + . The oxidation of H 2 S is predicted to enhance protein persulfidation-the attachment of sulfur to cysteine residues. Persulfidated proteins act as redox intermediates, and persulfidation protects proteins from irreversible oxidation and ubiquitination, providing an important means of signaling. Protein persulfidation is believed to decline in several neurological disorders and aging. Importantly, and consistent with the role of persulfidation in signaling, the master antioxidant transcription factor Nrf2 is regulated by Keap1's persulfidation. Here, we demonstrate that pleozymes increased overall protein persulfidation in cells from apparently healthy individuals and from individuals with the mitochondrial protein mutation responsible for Friedreich's ataxia. We further find that pleozymes specifically enhanced Keap1 persulfidation, with subsequent increased accumulation of Nrf2 and Nrf2's antioxidant targets.

Down syndrome (DS) is a multisystemic disorder that includes accelerated aging caused by trisomy 21. In particular, overexpression of cystathionine-β-synthase (CBS) is linked to excess intracellular hydrogen sulfide (H 2 S), a mitochondrial toxin at higher concentrations, which impairs cellular viability. Concurrent overexpression of superoxide dismutase 1 (SOD1) may increase oxidative stress by generating excess hydrogen peroxide (H 2 O 2 ) while also mitigating the toxic H 2 S burden via a non-canonical sulfide-oxidizing mechanism. We investigated the phenotypic variability in basal H 2 S levels in relation to DS B lymphocyte cell health and SOD1 in H 2 S detoxification. The H 2 S levels were negatively correlated with the DS B lymphocyte growth rates but not with CBS protein. Pharmacological inhibition of SOD1 using LCS-1 significantly increased the H 2 S levels to a greater extent in DS cells while also decreasing the polysulfide products of H 2 S oxidation. However, DS cells exhibited elevated H 2 O 2 and lipid peroxidation, representing potential toxic consequences of SOD1 overexpression. Treatment of DS cells with a pleiotropic carbon nanozyme (pleozymes) decreased the total oxidative stress and reduced the levels of the H 2 S-generating enzymes CBS and 3-mercaptopyruvate sulfurtransferase (MPST). Our results indicate that pleozymes may bridge the protective and deleterious effects of DS SOD1 overexpression on H 2 S metabolism and oxidative stress, respectively, with cytoprotective benefits.

LCS-1, a putative selective inhibitor of SOD1, is a substituted pyridazinone with rudimentary similarity to quinones and naphthoquinones. As quinones catalytically oxidize H 2 S to biologically active reactive sulfur species (RSS), we hypothesized LCS-1 might have similar attributes. Here, we examine LCS-1 reactions with H 2 S and SOD1 using thiol-specific fluorophores, liquid chromatography-mass spectrometry, electron paramagnetic resonance (EPR), UV-vis spectrometry, and oxygen consumption. We show that LCS-1 catalytically oxidizes H 2 S in buffer solutions to form RSS, namely per- and polyhydrosulfides (H 2 S n , n = 2-6). These reactions consume oxygen and produce hydrogen peroxide, but they do not have an EPR signature, nor do they affect the UV-vis spectrum. Surprisingly, LCS-1 synergizes with SOD1, but not SOD2, to oxidize H 2 S to H 2 S 3-6 . LCS-1 forms monothiol adducts with H 2 S, glutathione (GSH), and cysteine (Cys), but not with oxidized glutathione or cystine; both thiol adducts inhibit LCS-1-SOD1 synergism. We propose that LCS-1 forms an adduct with SOD1 that disrupts the intramolecular Cys 57 -Cys 146 disulfide bond and transforms SOD1 from a dismutase to an oxidase. This would increase cellular ROS and polysulfides, the latter potentially affecting cellular signaling and/or cytoprotection.

1,4-naphthoquinones (NQs) catalytically oxidize H 2 S to per- and polysufides and sulfoxides, reduce oxygen to superoxide and hydrogen peroxide, and can form NQ-SH adducts through Michael addition. Here, we measured oxygen consumption and used sulfur-specific fluorophores, liquid chromatography tandem mass spectrometry (LC-MS/MS), and UV-Vis spectrometry to examine H 2 S oxidation by NQs with various substituent groups. In general, the order of H 2 S oxidization was DCNQ ~ juglone > 1,4-NQ > plumbagin >DMNQ ~ 2-MNQ > menadione, although this order varied somewhat depending on the experimental conditions. DMNQ does not form adducts with GSH or cysteine (Cys), yet it readily oxidizes H 2 S to polysulfides and sulfoxides. This suggests that H 2 S oxidation occurs at the carbonyl moiety and not at the quinoid 2 or 3 carbons, although the latter cannot be ruled out. We found little evidence from oxygen consumption studies or LC-MS/MS that NQs directly oxidize H 2 S 2-4 , and we propose that apparent reactions of NQs with inorganic polysulfides are due to H 2 S impurities in the polysulfides or an equilibrium between H 2 S and H 2 S n . Collectively, NQ oxidation of H 2 S forms a variety of products that include hydropersulfides, hydropolysulfides, sulfenylpolysulfides, sulfite, and thiosulfate, and some of these reactions may proceed until an insoluble S 8 colloid is formed.

Hydrogen sulfide (H 2 S) is a noxious, potentially poisonous, but necessary gas produced from sulfur metabolism in humans. In Down Syndrome (DS), the production of H 2 S is elevated and associated with degraded mitochondrial function. Therefore, removing H 2 S from the body as a stable oxide could be an approach to reducing the deleterious effects of H 2 S in DS. In this report we describe the catalytic oxidation of hydrogen sulfide (H 2 S) to polysulfides (HS 2+n - ) and thiosulfate (S 2 O 3 2- ) by poly(ethylene glycol) hydrophilic carbon clusters (PEG-HCCs) and poly(ethylene glycol) oxidized activated charcoal (PEG-OACs), examples of oxidized carbon nanozymes (OCNs). We show that OCNs oxidize H 2 S to polysulfides and S 2 O 3 2- in a dose-dependent manner. The reaction is dependent on O 2 and the presence of quinone groups on the OCNs. In DS donor lymphocytes we found that OCNs increased polysulfide production, proliferation, and afforded protection against additional toxic levels of H 2 S compared to untreated DS lymphocytes. Finally, in Dp16 and Ts65DN murine models of DS, we found that OCNs restored osteoclast differentiation. This new action suggests potential facile translation into the clinic for conditions involving excess H 2 S exemplified by DS., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H 2 S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H 2 S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H 2 S to both inorganic and organic hydroper-/hydropolysulfides (R 2 S n , R=H, Cys, GSH; n = 2-4) and organic sulfoxides (GS n OH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H 2 S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H 2 S.

Significance: Nutraceuticals are ingested for health benefits, in addition to their general nutritional value. These dietary supplements have become increasingly popular since the late 20th century and they are a rapidly expanding global industry approaching a half-trillion U.S. dollars annually. Many nutraceuticals are promulgated as potent antioxidants. Recent Advances: Experimental support for the efficacy of nutraceuticals has lagged behind anecdotal exuberance. However, accumulating epidemiological evidence and recent, well-controlled clinical trials are beginning to support earlier animal and in vitro studies. Although still somewhat limited, encouraging results have been suggested in essentially all organ systems and against a wide range of pathophysiological conditions. Critical Issues: Health benefits of "antioxidant" nutraceuticals are largely attributed to their ability to scavenge oxidants. This has been criticized based on several factors, including limited bioavailability, short tissue retention time, and the preponderance of endogenous antioxidants. Recent attention has turned to nutraceutical activation of downstream antioxidant systems, especially the Keap1/Nrf2 (Kelch like ECH associated protein 1/nuclear factor erythroid 2-related factor 2) axis. The question now becomes, how do nutraceuticals activate this axis? Future Directions: Reactive sulfur species (RSS), including hydrogen sulfide (H 2 S) and its metabolites, are potent activators of the Keap1/Nrf2 axis and avid scavengers of reactive oxygen species. Evidence is beginning to accumulate that a variety of nutraceuticals increase cellular RSS by directly providing RSS in the diet, or through a number of catalytic mechanisms that increase endogenous RSS production. We propose that nutraceutical-specific targeting of RSS metabolism will lead to the design and development of even more efficacious antioxidant therapeutic strategies. Antioxid. Redox Signal. 38, 68-94.

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H2S) using RSS-specific fluorophores, liquid chromatography-mass spectrometry, UV-Vis absorption spectrometry, oxygen-sensitive optodes, thiosulfate-specific nanoparticles, HPLC-monobromobimane derivatization, and ion chromatographic assays. We show that NQs, catalytically oxidize H2S to per- and polysulfides (H2Sn, n = 2−6), thiosulfate, sulfite and sulfate in reactions that consume oxygen and are accelerated by superoxide dismutase (SOD) and inhibited by catalase. The approximate efficacy of NQs (in decreasing order) is, 1,4-NQ ≈ juglone ≈ plumbagin > 2-methoxy-1,4-NQ ≈ menadione >> phylloquinone ≈ anthraquinone ≈ menaquinone ≈ lawsone. We propose that the most probable reactions are an initial two-electron oxidation of H2S to S0 and reduction of NQ to NQH2. S0 may react with H2S or elongate H2Sn in variety of reactions. Reoxidation of NQH2 likely involves a semiquinone radical (NQ·−) intermediate via several mechanisms involving oxygen and comproportionation to produce NQ and superoxide. Dismutation of the latter forms hydrogen peroxide which then further oxidizes RSS to sulfoxides. These findings provide the chemical background for novel sulfur-based approaches to naphthoquinone-directed therapies.

Hydrogen sulfide has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious and neuropathological diseases have been undertaken. Here we review the possible direct targets of H 2 S in mammals. H 2 S directly interacts with reactive oxygen/nitrogen species and is involved in redox signaling. H 2 S also reacts with hemeproteins and modulates metal-containing complexes. Once being oxidized, H 2 S can persulfidate proteins by adding -SSH to the amino acid cysteine. These direct modifications by H 2 S have significant impact on cell structure and many cellular functions, such as tight junctions, autophagy, apoptosis, vesicle trafficking, cell signaling, epigenetics and inflammasomes. Therefore, we conclude that H 2 S is involved in many important cellular and physiological processes. Compounds that donate H 2 S to biological systems can be developed as therapeutics for different diseases.

In the canonical pathway for mitochondrial H 2 S oxidation electrons are transferred from sulfide:quinone oxidoreductase (SQR) to complex III via ubiquinone (CoQ 10 ). We previously observed that a number of quinones directly oxidize H 2 S and we hypothesize that CoQ 10 may have similar properties. Here we examine H 2 S oxidation by CoQ 10 and more hydrophilic, truncated forms, CoQ 1 and CoQ 0 , in buffer using H 2 S and polysulfide fluorophores (AzMC and SSP4), silver nanoparticles to measure thiosulfate (H 2 S 2 O 3 ), mass spectrometry to identify polysulfides and O 2 -sensitive optodes to measure O 2 consumption. We show that all three quinones concentration-dependently catalyze the oxidization of H 2 S to polysulfides and thiosulfate in buffer with the potency CoQ 0 >CoQ 1 >CoQ 10 and that CoQ 0 specifically oxidizes H 2 S to per-polysulfides, H 2 S 2,3,4 . These reactions consume and require oxygen and are augmented by addition of SOD suggesting that the quinones, not superoxide, oxidize H 2 S. Related quinones, MitoQ, menadione and idebenone, oxidize H 2 S in similar reactions. Exogenous CoQ 0 decreases cellular H 2 S and increases polysulfides and thiosulfate production and this is also O 2 -dependent, suggesting that the quinone has similar effects on sulfur metabolism in cells. Collectively, these results suggest an additional endogenous mechanism for H 2 S metabolism and a potential therapeutic approach in H 2 S-related metabolic disorders., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The ability to detect oxygen availability is a ubiquitous attribute of aerobic organisms. However, the mechanism(s) that transduce oxygen concentration or availability into appropriate physiological responses is less clear and often controversial. This review will make the case for oxygen-dependent metabolism of hydrogen sulfide (H 2 S) and polysulfides, collectively referred to as reactive sulfur species (RSS) as a physiologically relevant O 2 sensing mechanism. This hypothesis is based on observations that H 2 S and RSS metabolism is inversely correlated with O 2 tension, exogenous H 2 S elicits physiological responses identical to those produced by hypoxia, factors that affect H 2 S production or catabolism also affect tissue responses to hypoxia, and that RSS efficiently regulate downstream effectors of the hypoxic response in a manner consistent with a decrease in O 2 . H 2 S-mediated O 2 sensing is then compared to the more generally accepted reactive oxygen species (ROS) mediated O 2 sensing mechanism and a number of reasons are offered to resolve some of the confusion between the two.

Nutraceutical polyphenol catechins in green tea oxidize H 2 S to polysulfides (PS) in buffer and in cells thereby conveying their cytoprotective effects. Here we measure H 2 S oxidation in buffer and HEK293 cells by over-the-counter nutraceuticals, blueberry, bilberry and cranberry, and by polyphenols, cyanadin (Cya), quercetin (Que), rosmarinic acid (RA) and resveratrol (Res). H 2 S and PS were measured with specific fluorophores, AzMc and SSP4 respectively, and thiosulfate (TS) production was measured in buffer using silver nanoparticles (AgNPs). All compounds increased polysulfide production from H 2 S in buffer and increased polysufides in cells. Decreasing oxygen from 100% to 21% and 0% progressively decreased PS production by Que and RA in buffer and Que decreased PS production in cells incubated in 5% O 2 compared to 21% O 2 . Que, RA and Res, but not Cya, increased TS production from H 2 S in 21% O 2 but not in 0% O 2 . Superoxide dismutase did not affect PS production from H 2 S by Que or TS production from H 2 S by Que, RA or Res, whereas catalase inhibited TS production by all three polyphenols. Conversely, these polyphenols only slightly reduce a mixed polysulfide (K 2 S n ) or thiosulfate to H 2 S in 0% O 2 . Collectively, our results suggest that polyphenols are autoxidized to a semiquinone radical and that this, in turn, oxidizes H 2 S to a thiyl radical from which polysulfides and thiosulfate derived. They also suggest that this is catalyzed by a semiquinone radical and it is independent of either superoxide or hydrogen peroxide concomitantly produced during polyphenol autoxidation. The polysulfides produced in these reactions are potent antioxidants and also initiate a variety of downstream cytoprotective effector mechanisms. It is also possible that H 2 S can be regenerated from the thiosulfate produced in these reactions by other cellular reductants and reused in subsequent reactions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

"Nothing in biology makes sense except in the light of evolution" (Theodosius Dobzhansky) and "For such a large number of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied" (August Krogh); dictums that can be used to illustrate the past and provide a guide to the future. Although sulfur was integral in the origin of life, and nearly seven-eights of subsequent evolution, its physiological importance is largely overlooked because much of contemporary life it is based on oxygen and the adherent problems associated with oxygen deficit (hypoxia) or excess (oxidative stress). This graphical review will summarize sulfur's role in evolution and make a case that many of the regulatory activities attributed to oxygen and reactive oxygen species (ROS) can also be ascribed to reactive sulfur species (RSS). ROS and RSS are chemically similar and signal via identical cysteine residues on regulatory proteins and have identical downstream effector responses. Antioxidant mechanisms, generally attributed to the advent of an oxic existence, actually appeared over 2 billion years prior, in sulfur metabolizing organisms. Recent evidence suggests they are active in sulfur metabolism to this day. Understanding these aspects of ROS and RSS suggests that alternative mechanisms for oxidant/antioxidant pathways and therapies must be considered. As oxygen and reduced sulfur do not coexist, either in cells or the environment, it is also important to design and conduct experiments in oxygen levels that are physiologically relevant. For every experiment there are optimal conditions under which it must be studied., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We have shown that autoxidized polyphenolic nutraceuticals oxidize H 2 S to polysulfides and thiosulfate and this may convey their cytoprotective effects. Polyphenol reactivity is largely attributed to the B ring, which is usually a form of hydroxyquinone (HQ). Here, we examine the effects of HQs on sulfur metabolism using H 2 S- and polysulfide-specific fluorophores (AzMC and SSP4, respectively) and thiosulfate sensitive silver nanoparticles (AgNP). In buffer, 1,4-dihydroxybenzene (1,4-DB), 1,4-benzoquinone (1,4-BQ), pyrogallol (PG) and gallic acid (GA) oxidized H 2 S to polysulfides and thiosulfate, whereas 1,2-DB, 1,3-DB, 1,2-dihydroxy,3,4-benzoquinone and shikimic acid did not. In addition, 1,4-DB, 1,4-BQ, PG and GA also increased polysulfide production in HEK293 cells. In buffer, H 2 S oxidation by 1,4-DB was oxygen-dependent, partially inhibited by tempol and trolox, and absorbance spectra were consistent with redox cycling between HQ autoxidation and H 2 S-mediated reduction. Neither 1,2-DB, 1,3-DB, 1,4-DB nor 1,4-BQ reduced polysulfides to H 2 S in either 21% or 0% oxygen. Epinephrine and norepinephrine also oxidized H 2 S to polysulfides and thiosulfate; dopamine and tyrosine were ineffective. Polyphenones were also examined, but only 2,5-dihydroxy- and 2,3,4-trihydroxybenzophenones oxidized H 2 S. These results show that H 2 S is readily oxidized by specific hydroxyquinones and quinones, most likely through the formation of a semiquinone radical intermediate derived from either reaction of oxygen with the reduced quinones, or from direct reaction between H 2 S and quinones. We propose that polysulfide production by these reactions contributes to the health-promoting benefits of polyphenolic nutraceuticals.

Significance: Oxidative stress in moderation positively affects homeostasis through signaling, while in excess it is associated with adverse health outcomes. Both activities are generally attributed to reactive oxygen species (ROS); hydrogen peroxide as the signal, and cysteines on regulatory proteins as the target. However, using antioxidants to affect signaling or benefit health has not consistently translated into expected outcomes, or when it does, the mechanism is often unclear. Recent Advances: Reactive sulfur species (RSS) were integral in the origin of life and throughout much of evolution. Sophisticated metabolic pathways that evolved to regulate RSS were easily "tweaked" to deal with ROS due to the remarkable similarities between the two. However, unlike ROS, RSS are stored, recycled, and chemically more versatile. Despite these observations, the relevance and regulatory functions of RSS in extant organisms are generally underappreciated. Critical Issues: A number of factors bias observations in favor of ROS over RSS. Research conducted in room air is hyperoxic to cells, and promotes ROS production and RSS oxidation. Metabolic rates of rodent models greatly exceed those of humans; does this favor ROS? Analytical methods designed to detect ROS also respond to RSS. Do these disguise the contributions of RSS? Future Directions: Resolving the ROS/RSS issue is vital to understand biology in general and human health in particular. Improvements in experimental design and analytical methods are crucial. Perhaps the most important is an appreciation of all the attributes of RSS and keeping an open mind.

Matcha and green tea catechins such as (-)-epicatechin (EC), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) have long been studied for their antioxidant and health-promoting effects. Using specific fluorophores for H 2 S (AzMC) and polysulfides (SSP4) as well as IC-MS and UPLC-MS/MS-based techniques we here show that popular Japanese and Chinese green teas and select catechins all catalytically oxidize hydrogen sulfide (H 2 S) to polysulfides with the potency of EGC > EGCG >> EG. This reaction is accompanied by the formation of sulfite, thiosulfate and sulfate, consumes oxygen and is partially inhibited by the superoxide scavenger, tempol, and superoxide dismutase but not mannitol, trolox, DMPO, or the iron chelator, desferrioxamine. We propose that the reaction proceeds via a one-electron autoxidation process during which one of the OH-groups of the catechin B-ring is autooxidized to a semiquinone radical and oxygen is reduced to superoxide, either of which can then oxidize HS - to thiyl radicals (HS • ) which react to form hydrogen persulfide (H 2 S 2 ). H 2 S oxidation reduces the B-ring back to the hydroquinone for recycling while the superoxide is reduced to hydrogen peroxide (H 2 O 2 ). Matcha and catechins also concentration-dependently and rapidly produce polysulfides in HEK293 cells with the potency order EGCG > EGC > EG, an EGCG threshold of ~300 nM, and an EC 50 of ~3 μM, suggesting green tea also acts as powerful pro-oxidant in vivo. The resultant polysulfides formed are not only potent antioxidants, but elicit a cascade of secondary cytoprotective effects, and we propose that many of the health benefits of green tea are mediated through these reactions. Remarkably, all green tea leaves constitutively contain small amounts of H 2 S 2 ., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The use of mesenchymal stem cells (MSCs) for treatment during ischemia is novel. Hydrogen sulfide (H 2 S) is an important paracrine mediator that is released from MSCs to facilitate angiogenesis and vasodilation. Three enzymes, cystathionine-beta-synthase (CBS), cystathionine-gamma-lyase (CSE), and 3-mercaptopyruvate-sulfurtransferase (MPST), are mainly responsible for H 2 S production. However, it is unclear how these enzymes impact the production of other critical growth factors and chemokines. We hypothesized that the enzymes responsible for H 2 S production in human MSCs would also critically regulate other growth factors and chemokines., Materials and Methods: Human MSCs were transfected with CBS, MPST, CSE, or negative control small interfering RNA. Knockdown of enzymes was confirmed by polymerase chain reaction. Cells were plated in 12-well plates at 100,000 cells per well and stimulated with tumor necrosis factor-α (TNF-α; 50 ng/mL), lipopolysaccharide (LPS; 200 ng/mL), or 5% hypoxia for 24 h. Supernatants were collected, and cytokines measured by multiplex beaded assay. Data were compared with the Mann-Whitney U-test, and P < 0.05 was significant., Results: TNF-α, LPS, and hypoxia effectively stimulated MSCs. Granulocyte colony-stimulating factor (GCSF), epidermal growth factor, fibroblast growth factor, granulocyte/monocyte colony-stimulating factor (GMCSF), vascular endothelial growth factor, and interferon gamma-inducible protein 10 were all significantly elevated when CSE was knocked down during TNF-α stimulation (P < 0.05). Knockdown of MPST during LPS stimulation more readily increased GCSF and epidermal growth factor but decreased GMCSF (P < 0.05). CBS knockdown decreased production of GCSF, fibroblast growth factor, GMCSF, and vascular endothelial growth factor (P < 0.05) after hypoxia., Conclusions: The enzymes that produce H 2 S in MSCs are also responsible for the production of other stem cell paracrine mediators under stressful stimuli. Therefore, reprogramming MSCs to endogenously produce more H 2 S as a therapeutic intervention could also critically impact other paracrine mediators, which may alter the desired beneficial effects., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aim: Numerous studies have shown that H 2 S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H 2 S also serves in extended oxygen sensing., Methods: Here, we compare the effects of extended exposure (24-48 hours) to varying O 2 tensions on H 2 S and polysulphide metabolism in human embryonic kidney (HEK 293), human adenocarcinomic alveolar basal epithelial (A549), human colon cancer (HTC116), bovine pulmonary artery smooth muscle, human umbilical-derived mesenchymal stromal (stem) cells and porcine tracheal epithelium (PTE) using sulphur-specific fluorophores and fluorometry or confocal microscopy., Results: All cells continuously produced H 2 S in 21% O 2 and H 2 S production was increased at lower O 2 tensions. Decreasing O 2 from 21% to 10%, 5% and 1% O 2 progressively increased H 2 S production in HEK293 cells and this was partially inhibited by a combination of inhibitors of H 2 S biosynthesis, aminooxyacetate, propargyl glycine and compound 3. Mitochondria appeared to be the source of much of this increase in HEK 293 cells. H 2 S production in all other cells and PTE increased when O 2 was lowered from 21% to 5% except for HTC116 cells where 1% O 2 was necessary to increase H 2 S, presumably reflecting the hypoxic environment in vivo. Polysulphides (H 2 S n , where n = 2-7), the key signalling metabolite of H 2 S also appeared to increase in many cells although this was often masked by high endogenous polysulphide concentrations., Conclusion: These results show that cellular H 2 S is increased during extended hypoxia and they suggest this is a continuously active O 2 -sensing mechanism in a variety of cells., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The biological effects of oxidants, especially reactive oxygen species (ROS), include signaling functions (oxidative eustress), initiation of measures to reduce elevated ROS (oxidative stress), and a cascade of pathophysiological events that accompany excessive ROS (oxidative distress). Although these effects have long been studied in animal models with perturbed ROS, their actions under physiological conditions are less clear. I propose that some of the apparent uncertainty may be due to confusion of ROS with endogenously generated reactive sulfur species (RSS). ROS and RSS are chemically similar, but RSS are more reactive and versatile, and can be stored and reused. Both ROS and RSS signal via oxidation reactions with protein cysteine sulfur and they produce identical effector responses, but RSS appear to be more effective. RSS in the form of persulfidated cysteines (Cys-S-S) are produced endogenously and co-translationally introduced into proteins, and there is increasing evidence that many cellular proteins are persulfidated. A number of practical factors have contributed to confusion between ROS and RSS, and these are discussed herein. Furthermore, essentially all endogenous antioxidant enzymes appeared shortly after life began, some 3.8 billion years ago, when RSS metabolism dominated evolution. This was long before the rise in ROS, 600 million years ago, and I propose that these same enzymes, with only minor modifications, still effectively metabolize RSS in extant organisms. I am not suggesting that all ROS are RSS; however, I believe that the relative importance of ROS and RSS in biological systems needs further consideration., Competing Interests: Competing interestsThe author declares no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Manganese porphyrins (MnPs), MnTE-2-PyP 5+ , MnTnHex-2-PyP 5+ and MnTnBuOE-2-PyP 5+ , are superoxide dismutase (SOD) mimetics and form a redox cycle between O 2 and reductants, including ascorbic acid, ultimately producing hydrogen peroxide (H 2 O 2 ). We previously found that MnPs oxidize hydrogen sulfide (H 2 S) to polysulfides (PS; H 2 S n , n = 2-6) in buffer. Here, we examine the effects of MnPs for 24 h on H 2 S metabolism and PS production in HEK293, A549, HT29 and bone marrow derived stem cells (BMDSC) using H 2 S (AzMC, MeRho-AZ) and PS (SSP4) fluorophores. All MnPs decreased intracellular H 2 S production and increased intracellular PS. H 2 S metabolism and PS production were unaffected by cellular O 2 (5% versus 21% O 2 ), H 2 O 2 or ascorbic acid. We observed with confocal microscopy that mitochondria are a major site of H 2 S production in HEK293 cells and that MnPs decrease mitochondrial H 2 S production and increase PS in what appeared to be nucleoli and cytosolic fibrillary elements. This supports a role for MnPs in the metabolism of H 2 S to PS, the latter serving as both short- and long-term antioxidants, and suggests that some of the biological effects of MnPs may be attributable to sulfur metabolism.

The health benefits of lipoic acid (LA) are generally attributed to mitigating the harmful effects of reactive oxygen species (ROS). ROS are chemically similar to reactive sulfur species (RSS) and signal through identical mechanisms. Here we examined the effects of LA on RSS in HEK293 cells using H 2 S and polysulfide (PS) specific fluorophores, AzMC and SSP4. We show that LA concentration-dependently increased both H 2 S and PS. Physioxia (5% O 2 ) augmented the effects of LA on H 2 S production but decreased PS production. Thiosulfate, a known substrate for reduced LA, and an intermediate in the catabolism of H 2 S enhanced the effects of LA on H 2 S and PS production. Inhibiting peroxiredoxins with conoidin A and gluraredoxins with tiopronin augmented the effects of LA on PS and H 2 S, respectively while decreasing glutathione with buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased the stimulatory effect of LA on H 2 S production but augmented LA's effect on PS. Aminooxyacetate (AOA) and propargylglycine (PPG), inhibitors of H 2 S production from cysteine partially inhibited LA augmentation of H 2 S production and further decreased the LA effect when applied concurrently with BSO and DEM. The selective and cell-permeable H 2 S scavenger, SS20, inhibited the effects of LA on cellular H 2 S. Estimates of single-cell H 2 S production suggest that 0.1-0.2% of O 2 consumption is used to metabolize H 2 S and these requirements may increase to 1-2% with 1 mM LA. Collectively, these results suggest that LA rescues H 2 S from irreversible oxidation and that the effects of LA on RSS directly confer antioxidant, anti-inflammatory and cytoprotective responses. They also suggest that TS may be an effective supplement to increase the efficacy of LA in clinical settings., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Manganese-centered porphyrins (MnPs), MnTE-2-PyP 5+ (MnTE), MnTnHex-2-PyP 5+ (MnTnHex), and MnTnBuOE-2-PyP 5+ (MnTnBuOE) have received considerable attention because of their ability to serve as superoxide dismutase (SOD) mimetics thereby producing hydrogen peroxide (H 2 O 2 ), and oxidants of ascorbate and simple aminothiols or protein thiols. MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ are now in five Phase II clinical trials warranting further exploration of their rich redox-based biology. Previously, we reported that SOD is also a sulfide oxidase catalyzing the oxidation of hydrogen sulfide (H 2 S) to hydrogen persulfide (H 2 S 2 ) and longer-chain polysulfides (H 2 S n , n = 3-7). We hypothesized that MnPs may have similar actions on sulfide metabolism. H 2 S and polysulfides were monitored in fluorimetric assays with 7-azido-4-methylcoumarin (AzMC) and 3',6'-di(O-thiosalicyl)fluorescein (SSP4), respectively, and specific polysulfides were further identified by mass spectrometry. MnPs concentration-dependently consumed H 2 S and produced H 2 S 2 and subsequently longer-chain polysulfides. This reaction appeared to be O 2 -dependent. MnP absorbance spectra exhibited wavelength shifts in the Soret and Q bands characteristic of sulfide-mediated reduction of Mn. Taken together, our results suggest that MnPs can become efficacious activators of a variety of cytoprotective processes by acting as sulfide oxidation catalysts generating per/polysulfides.

Life began in a ferruginous (anoxic and Fe 2+ dominated) world around 3.8 billion years ago (bya). Hydrogen sulfide (H 2 S) and other sulfur molecules from hydrothermal vents and other fissures provided many key necessities for life's origin including catalytic platforms (primordial enzymes) that also served as primitive boundaries (cell walls), substrates for organic synthesis and a continuous source of energy in the form of reducing equivalents. Anoxigenic photosynthesis oxidizing H 2 S followed within a few hundred million years and laid the metabolic groundwork for oxidative photosynthesis some half-billion years later that slightly and episodically increased atmospheric oxygen around 2.3 bya. This oxidized terrestrial sulfur to sulfate which was washed to the sea where it was reduced creating vast euxinic (anoxic and sulfidic) areas. It was in this environment that eukaryotic cells appeared around 1.5 bya and where they evolved for nearly 1 billion additional years. Oxidative photosynthesis finally oxidized the oceans and around 0.6 bya oxygen levels in the atmosphere and oceans began to rise toward present day levels. This is purported to have been a life-threatening event due to the prevalence of reactive oxygen species (ROS) and thus necessitated the elaboration of chemical and enzymatic antioxidant mechanisms. However, these antioxidants initially appeared around the time of anoxigenic photosynthesis suggesting a commitment to metabolism of reactive sulfur species (RSS). This review examines these events and suggests that many of the biological attributes assigned to ROS may, in fact, be due to RSS. This is underscored by observations that ROS and RSS are chemically similar, often indistinguishable by analytical methods and the fact that the bulk of biochemical and physiological experiments are performed in unphysiologically oxic environments where ROS are artifactually favored over RSS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Umbilical mesenchymal stem cells (USC) have been shown to reduce illness in animal models of necrotizing enterocolitis (NEC), possibly through the paracrine release of hydrogen sulfide (H 2 S). We hypothesized that animals treated with USCs with inhibited H 2 S synthesis would exhibit more severe disease., Methods: NEC was induced in five-day-old mouse pups by formula feeding and hypoxic and hypothermic stress. Experimental groups received intraperitoneal injection of either saline vehicle or 80,000cells/gram of one of the following cell types: USC, USCs with negative-control siRNA, or USCs with targeted siRNA inhibition of the H 2 S-producing enzymes. Pups were monitored by clinical assessment and after euthanasia, intestine and lung histologic injury were scored. Tissue was homogenized, and concentrations of IL-6, IL-10, and VEGF were determined by ELISA. For statistical analysis, p<0.05 was considered significant., Results: Animals treated with negative-control siRNA USCs were significantly improved compared to vehicle. Clinical sickness scores as well as intestinal and lung histologic injury scores in the targeted siRNA groups were significantly worse when compared to the negative-control siRNA group. IL-6, IL-10, and VEGF had varying patterns of expression in the different groups., Conclusion: Inhibition of H 2 S production in USCs reduces the beneficial effects of these cells during therapy in experimental NEC., Level of Evidence: Animal studies are typically described as "foundational evidence" without a true level assigned., Type of Study: Animal Study., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Elaborate antioxidant pathways have evolved to minimize the threat of excessive reactive oxygen species (ROS) and to regulate ROS as signaling entities. ROS are chemically and functionally similar to reactive sulfur species (RSS) and both ROS and RSS have been shown to be metabolized by the antioxidant enzymes, superoxide dismutase and catalase. Here we use fluorophores to examine the effects of a variety of inhibitors of antioxidant pathways on metabolism of two important RSS, hydrogen sulfide (H 2 S with AzMC) and polysulfides (H 2 S n , where n = 2-7, with SSP4) in HEK293 cells. Cells were exposed to inhibitors for up to 5 days in normoxia (21% O 2 ) and hypoxia (5% O 2 ), conditions also known to affect ROS production. Decreasing intracellular glutathione (GSH) with l-buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased H 2 S production for 5 days but did not affect H 2 S n . The glutathione reductase inhibitor, auranofin, initially decreased H 2 S and H 2 S n but after two days H 2 S n increased over controls. Inhibition of peroxiredoxins with conoidin A decreased H 2 S and increased H 2 S n , whereas the glutathione peroxidase inhibitor, tiopronin, increased H 2 S. Aminoadipic acid, an inhibitor of cystine uptake did not affect either H 2 S or H 2 S n . In buffer, the glutathione reductase and thioredoxin reductase inhibitor, 2-AAPA, the glutathione peroxidase mimetic, ebselen, and tiopronin variously reacted directly with AzMC and SSP4, reacted with H 2 S and H 2 S 2 , or optically interfered with AzMC or SSP4 fluorescence. Collectively these results show that antioxidant inhibitors, generally known for their ability to increase cellular ROS, have various effects on cellular RSS. These findings suggest that the inhibitors may affect cellular sulfur metabolism pathways that are not related to ROS production and in some instances they may directly affect RSS or the methods used to measure them. They also illustrate the importance of carefully evaluating RSS metabolism when biologically or pharmacologically attempting to manipulate ROS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Reactive sulfur species (RSS) such as H 2 S, HS • , H 2 S n , (n = 2-7) and HS 2 •- are chemically similar to H 2 O and the reactive oxygen species (ROS) HO • , H 2 O 2 , O 2 •- and act on common biological effectors. RSS were present in evolution long before ROS, and because both are metabolized by catalase it has been suggested that "antioxidant" enzymes originally evolved to regulate RSS and may continue to do so today. Here we examined RSS metabolism by Cu/Zn superoxide dismutase (SOD) using amperometric electrodes for dissolved H 2 S, a polysulfide-specific fluorescent probe (SSP4), and mass spectrometry to identify specific polysulfides (H 2 S 2 -H 2 S 5 ). H 2 S was concentration- and oxygen-dependently oxidized by 1μM SOD to polysulfides (mainly H 2 S 2 , and to a lesser extent H 2 S 3 and H 2 S 5 ) with an EC 50 of approximately 380μM H 2 S. H 2 S concentrations > 750μM inhibited SOD oxidation (IC 50 = 1.25mM) with complete inhibition when H 2 S > 1.75mM. Polysulfides were not metabolized by SOD. SOD oxidation preferred dissolved H 2 S over hydrosulfide anion (HS - ), whereas HS - inhibited polysulfide production. In hypoxia, other possible electron donors such as nitrate, nitrite, sulfite, sulfate, thiosulfate and metabisulfite were ineffective. Manganese SOD also catalyzed H 2 S oxidation to form polysulfides, but did not metabolize polysulfides indicating common attributes of these SODs. These experiments suggest that, unlike the well-known SOD-mediated dismutation of two O 2 •- to form H 2 O 2 and O 2 , SOD catalyzes a reaction using H 2 S and O 2 to form persulfide. These can then combine in various ways to form polysulfides and sulfur oxides. It is also possible that H 2 S (or polysulfides) interact/react with SOD cysteines to affect catalytic activity or to directly contribute to sulfide metabolism. Our studies suggest that H 2 S metabolism by SOD may have been an ancient mechanism to detoxify sulfide or to regulate RSS and along with catalase may continue to do so in contemporary organisms., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

It is now well established that hydrogen sulfide (H 2 S) is an effector of a wide variety of physiological processes. It is also clear that many of the effects of H 2 S are mediated through reactions with cysteine sulfur on regulatory proteins and most of these are not mediated directly by H 2 S but require prior oxidation of H 2 S and the formation of per- and polysulfides (H 2 S n , n = 2-8). Attendant with understanding the regulatory functions of H 2 S and H 2 S n is an appreciation of the mechanisms that control, i.e., both increase and decrease, their production and catabolism. Although a number of standard "conventional" pathways have been described and well characterized, novel "unconventional" pathways are continuously being identified. This review summarizes our current knowledge of both the conventional and unconventional., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental importance in cellular function as a regulator of apoptosis, inflammation, and perfusion. Mechanisms of endogenous H2S signaling are poorly understood; however, signal transmission is thought to occur via persulfidation at reactive cysteine residues on proteins. Although much has been discovered about how H2S is synthesized in the body, less is known about how it is metabolized. Recent studies have discovered a multitude of different targets for H2S therapy, including those related to protein modification, intracellular signaling, and ion channel depolarization. The most difficult part of studying hydrogen sulfide has been finding a way to accurately and reproducibly measure it. The purpose of this review is to: elaborate on the biosynthesis and catabolism of H2S in the human body, review current knowledge of the mechanisms of action of this gas in relation to ischemic injury, define strategies for physiological measurement of H2S in biological systems, and review potential novel therapies that use H2S for treatment.

Fluorescence spectroscopy and microscopy have been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and, more recently, reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm, which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), iron protoporphyrin IX (hemin), and copper protoporphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3',6'-di( O -thiosalicyl)fluorescein, SSP4), and H 2 S (7-azido-4-methylcoumarin, AzMC) previously activated by H 2 O 2 , a mixed polysulfide (H 2 S n , n = 1-7) and H 2 S, respectively. All except albumin concentration dependently inhibited fluorophore fluorescence and absorbed light between 450 and 550 nm, suggesting that the inhibitory effect was physical not catalytic. Catalase inhibition of fluorescein fluorescence was unaffected by sodium azide, dithiothreitol, diamide, tris(2-carboxyethyl)phosphine (TCEP), or iodoacetate, supporting a physical inhibitory mechanism. Catalase and TBAP augmented, then inhibited DCF fluorescence, but only inhibited SSP4 and AzMC fluorescence indicative of a substrate-specific catalytic oxidation of DCF and nonspecific fluorescence inhibition of all three fluorophores. These results suggest caution must be exercised when using any fluorescent tracers in the vicinity of metal-centered porphyrins.

Significance: Oxidative stress is thought to account for aberrant redox homeostasis and contribute to aging and disease. However, more often than not, administration of antioxidants is ineffective, suggesting that our current understanding of the underlying regulatory processes is incomplete. Recent Advances: Similar to reactive oxygen species and reactive nitrogen species, reactive sulfur species are now emerging as important signaling molecules, targeting regulatory cysteine redox switches in proteins, affecting gene regulation, ion transport, intermediary metabolism, and mitochondrial function. To rationalize the complexity of chemical interactions of reactive species with themselves and their targets and help define their role in systemic metabolic control, we here introduce a novel integrative concept defined as the reactive species interactome (RSI). The RSI is a primeval multilevel redox regulatory system whose architecture, together with the physicochemical characteristics of its constituents, allows efficient sensing and rapid adaptation to environmental changes and various other stressors to enhance fitness and resilience at the local and whole-organism level., Critical Issues: To better characterize the RSI-related processes that determine fluxes through specific pathways and enable integration, it is necessary to disentangle the chemical biology and activity of reactive species (including precursors and reaction products), their targets, communication systems, and effects on cellular, organ, and whole-organism bioenergetics using system-level/network analyses., Future Directions: Understanding the mechanisms through which the RSI operates will enable a better appreciation of the possibilities to modulate the entire biological system; moreover, unveiling molecular signatures that characterize specific environmental challenges or other forms of stress will provide new prevention/intervention opportunities for personalized medicine. Antioxid. Redox Signal. 00, 000-000.

Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases evolved when metabolism was largely sulfur-based, long before O 2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H 2 S n , the sulfur analog of H 2 O 2 , hydrogen sulfide (H 2 S) and other sulfur-bearing molecules using H 2 S-specific amperometric electrodes and fluorophores to measure polysulfides (H 2 S n ; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H 2 S n , but did not anaerobically generate H 2 S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H 2 S and in so doing acted as a sulfide oxidase with a P 50 of 20mmHg. H 2 O 2 had little effect on catalase-mediated H 2 S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H 2 O 2 rapidly and efficiently expedited H 2 S metabolism in both normoxia and hypoxia suggesting H 2 O 2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H 2 S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H 2 S in the presence of O 2 . H 2 S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H 2 S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase reductase activity independent of peroxide dismutation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

In lung epithelial cells, hypoxia decreases the expression and activity of sodium-transporting molecules, thereby reducing the rate of transepithelial sodium absorption. The mechanisms underlying the sensing of hypoxia and subsequent coupling to sodium-transporting molecules remain unclear. Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule whose intracellular concentrations critically depend on oxygen levels. Therefore, it was questioned whether endogenously produced H2S contributes to hypoxic inhibition of sodium transport. In electrophysiological Ussing chamber experiments, hypoxia was established by decreasing oxygen concentrations in the chambers. Hypoxia concentration dependently and reversibly decreased amiloride-sensitive sodium absorption by cultured H441 monolayers and freshly dissected porcine tracheal epithelia due to inhibition of basolateral Na(+)/K(+)-ATPase. Exogenous application of H2S by the sulfur salt Na2S mimicked the effect of hypoxia and inhibited amiloride-sensitive sodium absorption by both tissues in an oxygen-dependent manner. Hypoxia increased intracellular concentrations of H2S and decreased the concentration of polysulfides. Pretreatment with the cystathionine-γ-lyase inhibitor d/l-propargylglycine (PAG) decreased hypoxic inhibition of sodium transport by H441 monolayers, whereas inhibition of cystathionine-β-synthase (with aminooxy-acetic acid; AOAA) or 3-mercaptopyruvate sulfurtransferase (with aspartate) had no effect. Inhibition of all of these H2S-generating enzymes with a combination of AOAA, PAG, and aspartate decreased the hypoxic inhibition of sodium transport by H441 cells and pig tracheae and decreased H2S production by tracheae. These data suggest that airway epithelial cells endogenously produce H2S during hypoxia, and this contributes to hypoxic inhibition of transepithelial sodium absorption., (Copyright © 2016 the American Physiological Society.)

The health benefits of garlic and other organosulfur-containing foods are well recognized and have been attributed to both prooxidant and antioxidant activities. The effects of garlic are surprisingly similar to those of hydrogen sulfide (H2S), which is also known to be released from garlic under certain conditions. However, recent evidence suggests that polysulfides, not H2S, may be the actual mediator of physiological signaling. In this study, we monitored formation of H2S and polysulfides from garlic oil in buffer and in human embryonic kidney (HEK) 293 cells with fluorescent dyes, 7-azido-4-methylcoumarin and SSP4, respectively and redox activity with two redox indicators redox-sensitive green fluorescent protein (roGFP) and DCF. Our results show that H2S release from garlic oil in buffer requires other low-molecular-weight thiols, such as cysteine (Cys) or glutathione (GSH), whereas polysulfides are readily detected in garlic oil alone. Administration of garlic oil to cells rapidly increases intracellular polysulfide but has minimal effects on H2S unless Cys or GSH are also present in the extracellular medium. We also observed that garlic oil and diallyltrisulfide (DATS) potently oxidized roGFP in buffer but did not affect DCF. This appears to be a direct polysulfide-mediated oxidation that does not require a reactive oxygen species intermediate. Conversely, when applied to cells, garlic oil became a significant intracellular reductant independent of extracellular Cys or GSH. This suggests that intracellular metabolism and further processing of the sulfur moieties are necessary to confer antioxidant properties to garlic oil in vivo., (Copyright © 2016 the American Physiological Society.)

Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that are chemically and biochemically similar to reactive sulfide species (RSS) derived from one-electron oxidations of hydrogen sulfide to elemental sulfur. Both ROS and RSS are endogenously generated and signal via protein thiols. Given the similarities between ROS and RSS, we wondered whether extant methods for measuring the former would also detect the latter. Here, we compared ROS to RSS sensitivity of five common ROS methods: redox-sensitive green fluorescent protein (roGFP), 2', 7'-dihydrodichlorofluorescein, MitoSox Red, Amplex Red, and amperometric electrodes. All methods detected RSS and were as, or more, sensitive to RSS than to ROS. roGFP, arguably the "gold standard" for ROS measurement, was more than 200-fold more sensitive to the mixed polysulfide H2Sn(n = 1-8) than to H2O2 These findings suggest that RSS may be far more prevalent in intracellular signaling than previously appreciated and that the contribution of ROS may be overestimated. This conclusion is further supported by the observation that estimated daily sulfur metabolism and ROS production are approximately equal and the fact that both RSS and antioxidant mechanisms have been present since the origin of life, nearly 4 billion years ago, long before the rise in environmental oxygen 600 million years ago. Although ROS are assumed to be the most biologically relevant oxidants, our results question this paradigm. We also anticipate our findings will direct attention toward development of novel and clinically relevant anti-(RSS)-oxidants., (Copyright © 2016 the American Physiological Society.)

The chemical versatility of sulfur and its abundance in the prebiotic Earth as reduced sulfide (H2S) implicate this molecule in the origin of life 3.8 billion years ago and also as a major source of energy in the first seven-eighths of evolution. The tremendous increase in ambient oxygen ∼ 600 million years ago brought an end to H2S as an energy source, and H2S-dependent animals either became extinct, retreated to isolated sulfide niches, or adapted. The first 3 billion years of molecular tinkering were not lost, however, and much of this biochemical armamentarium easily adapted to an oxic environment where it contributes to metabolism and signaling even in humans. This review examines the role of H2S in evolution and the evolution of H2S metabolism and signaling., (©2016 Int. Union Physiol. Sci./Am. Physiol. Soc.)

In pulmonary epithelia, β-adrenergic agonists regulate the membrane abundance of the epithelial sodium channel (ENaC) and, thereby, control the rate of transepithelial electrolyte absorption. This is a crucial regulatory mechanism for lung liquid clearance at birth and thereafter. This study investigated the influence of the gaseous signaling molecule hydrogen sulfide (H2S) on β-adrenergic agonist-regulated pulmonary sodium and liquid absorption. Application of the H2S-liberating molecule Na2S (50 μM) to the alveolar compartment of rat lungs in situ decreased baseline liquid absorption and abrogated the stimulation of liquid absorption by the β-adrenergic agonist terbutaline. There was no additional effect of Na2S over that of the ENaC inhibitor amiloride. In electrophysiological Ussing chamber experiments with native lung epithelia (Xenopus laevis), Na2S inhibited the stimulation of amiloride-sensitive current by terbutaline. β-adrenergic agonists generally increase ENaC abundance by cAMP formation and activation of PKA. Activation of this pathway by forskolin and 3-isobutyl-1-methylxanthine increased amiloride-sensitive currents in H441 pulmonary epithelial cells. This effect was inhibited by Na2S in a dose-dependent manner (5-50 μM). Na2S had no effect on cellular ATP concentration, cAMP formation, and activation of PKA. By contrast, Na2S prevented the cAMP-induced increase in ENaC activity in the apical membrane of H441 cells. H441 cells expressed the H2S-generating enzymes cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, and they produced H2S amounts within the employed concentration range. These data demonstrate that H2S prevents the stimulation of ENaC by cAMP/PKA and, thereby, inhibits the proabsorptive effect of β-adrenergic agonists on lung liquid clearance., (Copyright © 2015 the American Physiological Society.)

Significance: Although oxygen (O2)-sensing cells and tissues have been known for decades, the identity of the O2-sensing mechanism has remained elusive. Evidence is accumulating that O2-dependent metabolism of hydrogen sulfide (H2S) is this enigmatic O2 sensor., Recent Advances: The elucidation of biochemical pathways involved in H2S synthesis and metabolism have shown that reciprocal H2S/O2 interactions have been inexorably linked throughout eukaryotic evolution; there are multiple foci by which O2 controls H2S inactivation, and the effects of H2S on downstream signaling events are consistent with those activated by hypoxia. H2S-mediated O2 sensing has been demonstrated in a variety of O2-sensing tissues in vertebrate cardiovascular and respiratory systems, including smooth muscle in systemic and respiratory blood vessels and airways, carotid body, adrenal medulla, and other peripheral as well as central chemoreceptors., Critical Issues: Information is now needed on the intracellular location and stoichometry of these signaling processes and how and which downstream effectors are activated by H2S and its metabolites., Future Directions: Development of specific inhibitors of H2S metabolism and effector activation as well as cellular organelle-targeted compounds that release H2S in a time- or environmentally controlled way will not only enhance our understanding of this signaling process but also provide direction for future therapeutic applications.

Hydrogen sulfide (H2S) signaling has been implicated in physiological processes in practically all organ systems studied to date. At times the excitement of this new field has outpaced the technical expertise or practical knowledge with which to accurately assess these advancements. Recently, the myriad of proposed H2S actions has spawned interest in using indicators of H2S metabolism, especially plasma H2S concentrations, as a means of identifying a variety of pathophysiological conditions or to predict clinical outcomes. While this is a noteworthy endeavor, there are a number of contraindications to this practice at this time. First, there is little consensus regarding normal, i.e., "physiological" concentrations of H2S in either plasma or tissue. In fact, it has been shown that the methods most often employed for these measurements are associated with substantial artifact. Second, interactions, or presumed lack thereof, of H2S with other biomolecules (e.g., O2, H2O2, pH, etc.) or analytical reagents (e.g., reducing reagents, N-ethylmaleimide, phenylarsine, etc.) are often assumed but not evaluated. Third, the experimental design and/or statistical analyses may not be sufficient to justify using H2S concentration in tissue or blood as a predictive biomarker of pathophysiology. In this study, we first briefly review the problems associated with plasma and tissue H2S measurements and the associated errors and we provide some simple methods to evaluate whether the data obtained is physiologically relevant. Second we provide a brief analysis of H2S interactions with the above biomolecules. Third, we provide a statistical tool with which to determine the clinical applicability of H2S measurements. It is hoped that these points will provide a rational background for future work., (Copyright © 2014 Elsevier Inc. All rights reserved.)

H2S derived from organic thiol metabolism has been proposed serve as an oxygen sensor in a variety of systems because of its susceptibility to oxidation and its ability to mimic hypoxic responses in numerous oxygen-sensing tissues. Thiosulfate, an intermediate in oxidative H2S metabolism can alternatively be reduced and regenerate H2S. We propose that this contributes to the H2S-mediated oxygen-sensing mechanism. H2S formation from thiosulfate in buffers and in a variety of mammalian tissues and in lamprey dorsal aorta was examined in real time using a polarographic H2S sensor. Inferences of intracellular H2S production were made by examining hypoxic pulmonary vasoconstriction (HPV) in bovine pulmonary arteries under conditions in which increased H2S production would be expected and in mouse and rat aortas, where reducing conditions should mediate vasorelaxation. In Krebs-Henseleit (mammalian) and Cortland (lamprey) buffers, H2S was generated from thiosulfate in the presence of the exogenous reducing agent, DTT, or the endogenous reductant dihydrolipoic acid (DHLA). Both the magnitude and rate of H2S production were greatly increased by these reductants in the presence of tissue, with the most notable effects occurring in the liver. H2S production was only observed when tissues were hypoxic; exposure to room air, or injecting oxygen inhibited H2S production and resulted in net H2S consumption. Both DTT and DHLA augmented HPV, and DHLA dose-dependently relaxed precontracted mouse and rat aortas. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions and that this is not only a ready source of H2S production but also serves as a means of recycling sulfur and thereby conserving biologically relevant thiols.

Hydrogen sulfide (H2S) metabolism has been proposed as the oxygen (O2) sensing mechanism coupling hypoxia to effector responses in a variety of tissues including vascular and chemoreceptor cells. Implicit in this sensing system is a mechanism for regulating intracellular H2S concentration, presumably through oxidation. However, verification of this mechanism, or any other pathway of H2S signaling has been hampered by the lack of suitable methods for measuring intracellular concentration and distribution profiles. Here, intracellular H2S concentration profiles are modeled using simple monoexponential diffusion equations and current knowledge of H2S biosynthetic and metabolic pathways. The models predict that; (1) while both mitochondrial oxidation and simple diffusion out of the cell can reduce H2S concentration, the former is considerably more effective as an effector of intracellular H2S and (2) exogenously applied H2S may have unanticipated effects on endogenous signaling processes. In addition, these models provide additional support for mitochondrial H2S oxidation as the key couple in H2S-mediated O2 sensing., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The ability to monitor oxygen (O2) availability and delivery is crucial to an animal's survival. Vertebrates have a number of O2 'sensing' cells that monitor environmental oxygen and ensure adequate delivery to the tissues. While there is little doubt that these cells perform important homeostatic functions, there is little consensus on how a change in O2 concentration, or partial pressure (pO2), is transduced into a physiological response. We recently proposed that the metabolism of hydrogen sulfide (H2S) functions as the O2 sensor in a variety of tissues. In this mechanism, the concentration of biologically active H2S is regulated by the simple balance between constitutive H2S production and its oxidation by mitochondria. This hypothesis is supported by a number of experimental observations in a wide range of O2 sensing tissues: 1) exogenous H2S produces the same physiological response as hypoxia; 2) cellular H2S production is inversely related to pO2 at physiologically relevant pO2s; 3) agonists and antagonists of H2S biosynthesis augment and inhibit hypoxic responses, respectively; and 4) H2S and hypoxia appear to act via common effector pathways. The reciprocal relationship between H2S and O2 also has a long evolutionary history suggesting these gases have been inexorably intertwined throughout evolution. The intent of this review is to elaborate on the mechanism of H2S-mediated O2 sensing.

Hydrogen sulfide, H2S, is a colorless gas with a strong odor that until recently was only considered to be a toxic environmental pollutant with little or no physiological significance. However, the past few years have demonstrated its role in many biological systems and it is becoming increasingly clear that H2S is likely to join nitric oxide (NO) and carbon monoxide (CO) as a major player in mammalian biology. In this review, we have provided an overview of the chemistry and biology of H2S and have summarized the chemistry and biological activity of some natural and synthetic H2S-donating compounds. The naturally occurring compounds discussed include, garlic, sulforaphane, erucin, and iberin. The synthetic H2S donors reviewed include, GYY4137; cysteine analogs; S-propyl cysteine, S-allyl cysteine, S-propargyl cysteine, and N-acetyl cysteine. Dithiolethione and its NSAID and other chimeras such as, L-DOPA, sildenafil, aspirin, diclofenac, naproxen, ibuprofen, indomethacin, and mesalamine have also been reviewed in detail. The newly reported NOSH-aspirin that releases both NO and H2S has also been discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The role of the gaseous mediator hydrogen sulfide (H2S) in hemorrhagic shock is still a matter of debate. This debate is emphasized by the fact that available literature data on blood and tissue H2S concentrations vary by three orders of magnitude, both under physiological conditions as well as during stress states. Therefore, in a rat model of unresuscitated, lethal hemorrhagic shock, Van de Louw and Haouzi tested the two hypotheses of whether blood and tissue H2S levels would increase due to the shock-related tissue hypoxia, and whether vitamin B12 would attenuate organ injury and improve survival as a result of enhanced H2S oxidation. Hemorrhage did not affect the blood and tissue H2S content, and, despite the increased capacity to oxidize H2S, vitamin B12 did not affect any parameter of shock severity. The authors concluded that H2S concentrations cannot be used as a marker of shock, most probably as a result of tissue's capacity to oxidize H2S even under conditions of severe oxygen debt. This research paper elegantly re-adjusts the currently available data on blood and tissue H2S levels, and thereby adds an important piece to the puzzle of whether H2S release should be enhanced or lowered during stress conditions associated with tissue hypoxia.

The concept that non-respiratory gases, such as nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H(2)S) functioned as signaling moieties is a relatively recent development, due in part to their ephemeral existence in biological tissues. However, from an evolutionary perspective these gases dominated the prebiotic and anoxic Earth and were major contributors to the origin of life and the advent of eukaryotic animals. As Earth's oxygen levels rose, NO, CO and H(2)S disappeared from the environment and cells began to utilize their now well-developed metabolic pathways to compartmentalize and regulate these three gases for signaling purposes. Ironically, many of the signaling pathways have become now intimately involved in regulating oxygen delivery and their evolution has continued well into the vertebrates. This review examines the role NO, CO and H(2)S played in early life and their regulatory roles in oxygen delivery during the course of vertebrate evolution., (Copyright © 2012 Elsevier B.V. All rights reserved.)