Development of methods for quantitative determination of the total and reactive polysulfides: Reactive polysulfide profiling in vegetables.

Alliaceous and cruciferous vegetables are rich in bioactive organosulfur compounds, including polysulfides, which exhibit a broad spectrum of potential health benefits. Here, we developed novel, accurate, and reproducible methods to quantify the total polysulfide content (TPsC) and the reactive polysulfide content (RPsC) using liquid chromatography-electrospray ionization-tandem mass spectrometry, and analyzed the reactive polysulfide profiles of 22 types of fresh vegetables, including onions, garlic, and broccoli. Quantitative analyses revealed that onions contained the largest amounts of polysulfides, followed by broccoli, Chinese chive, and garlic. A strong positive correlation was observed between the TPsC and RPsC, whereas only a moderate positive correlation was found between the total sulfur content and TPsC. These results suggest that reactive polysulfide profiling can be a novel criterion for evaluating the beneficial functions of vegetables and their derivatives, which may lead to an understanding of the detailed mechanisms underlying their bioactivities.

SOD1 is an essential H2S detoxifying enzyme.

Although hydrogen sulfide (H2S) is an endogenous signaling molecule with antioxidant properties, it is also cytotoxic by potently inhibiting cytochrome c oxidase and mitochondrial respiration. Paradoxically, the primary route of H2S detoxification is thought to occur inside the mitochondrial matrix via a series of relatively slow enzymatic reactions that are unlikely to compete with its rapid inhibition of cytochrome c oxidase. Therefore, alternative or complementary cellular mechanisms of H2S detoxification are predicted to exist. Here, superoxide dismutase [Cu-Zn] (SOD1) is shown to be an efficient H2S oxidase that has an essential role in limiting cytotoxicity from endogenous and exogenous sulfide. Decreased SOD1 expression resulted in increased sensitivity to H2S toxicity in yeast and human cells, while increased SOD1 expression enhanced tolerance to H2S. SOD1 rapidly converted H2S to sulfate under conditions of limiting sulfide; however, when sulfide was in molar excess, SOD1 catalyzed the formation of per- and polysulfides, which induce cellular thiol oxidation. Furthermore, in SOD1-deficient cells, elevated levels of reactive oxygen species catalyzed sulfide oxidation to per- and polysulfides. These data reveal that a fundamental function of SOD1 is to regulate H2S and related reactive sulfur species.

Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics.

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate L-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

Basic characterization of 64Cu-ATSM as a radiotherapy agent.

64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) is a promising radiotherapy agent for the treatment of hypoxic tumors. In an attempt to elucidate the radiobiological basis of 64Cu-ATSM radiotherapy, we have investigated the cellular response patterns in vitro cell line models. Cells were incubated with 64Cu-ATSM, and the dose-response curves were obtained by performing a clonogenic survival assay. Radiation-induced damage in DNA was evaluated using the alkali comet assay and apoptotic cells were detected using Annexin V-FITC and propidium iodide staining methods. Washout rate and subcellular distribution of 64Cu in cells were investigated to further assess the effectiveness of 64Cu-ATSM therapy on a molecular basis. A direct comparison of subcellular localization of Cu-ATSM was made with the flow tracer analog Cu-pyruvladehyde-bis(N4-methylthiosemicarbazone). In this study, 64Cu-ATSM was shown to reduce the clonogenic survival rate of tumor cells in a dose-dependent manner. Under hypoxic conditions, cells took up 64Cu-ATSM and radioactive 64Cu was highly accumulated in the cells. In the 64Cu-ATSM-treated cells, DNA damage by the radiation emitted from 64Cu was detected, and inhibition of cell proliferation and induction of apoptosis was observed at 24 and 36 h after the treatment. The typical features of postmitotic apoptosis induced by radiation were observed following 64Cu-ATSM treatment. The majority of the 64Cu taken up into the cells remained in the postmitochondrial supernatant (the cellular residue after removal of the nuclei and mitochondria), which indicates that the beta- particle emitted from 64Cu may be as effective as the Auger electrons in 64Cu-ATSM therapy. These data allow us to postulate that 64Cu-ATSM will be able to attack the hypoxic tumor cells directly, as well as potentially affecting the peripheral nonhypoxic regions indirectly by the beta- particle decay of 64Cu.