We have shown that autoxidized polyphenolic nutraceuticals oxidize H₂S 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 H₂S- 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 H₂S 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, H₂S 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 H₂S-mediated reduction. Neither 1,2-DB, 1,3-DB, 1,4-DB nor 1,4-BQ reduced polysulfides to H₂S in either 21% or 0% oxygen. Epinephrine and norepinephrine also oxidized H₂S to polysulfides and thiosulfate; dopamine and tyrosine were ineffective. Polyphenones were also examined, but only 2,5-dihydroxy- and 2,3,4-trihydroxybenzophenones oxidized H₂S. These results show that H₂S 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 H₂S and quinones. We propose that polysulfide production by these reactions contributes to the health-promoting benefits of polyphenolic nutraceuticals.

Manganese-centered porphyrins (MnPs), MnTE-2-PyP⁵⁺ (MnTE), MnTnHex-2-PyP⁵⁺ (MnTnHex), and MnTnBuOE-2-PyP⁵⁺ (MnTnBuOE) have received considerable attention because of their ability to serve as superoxide dismutase (SOD) mimetics thereby producing hydrogen peroxide (H₂O₂), and oxidants of ascorbate and simple aminothiols or protein thiols. MnTE-2-PyP⁵⁺ and MnTnBuOE-2-PyP⁵⁺ 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 (H₂S) to hydrogen persulfide (H₂S₂) and longer-chain polysulfides (H₂S_n, n = 3–7). We hypothesized that MnPs may have similar actions on sulfide metabolism. H₂S 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 H₂S and produced H₂S₂ and subsequently longer-chain polysulfides. This reaction appeared to be O₂-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.

Manganese porphyrins (MnPs), MnTE-2-PyP⁵⁺, MnTnHex-2-PyP⁵⁺ and MnTnBuOE-2-PyP⁵⁺, are superoxide dismutase (SOD) mimetics and form a redox cycle between O₂ and reductants, including ascorbic acid, ultimately producing hydrogen peroxide (H₂O₂). We previously found that MnPs oxidize hydrogen sulfide (H₂S) to polysulfides (PS; H₂S_n, n = 2–6) in buffer. Here, we examine the effects of MnPs for 24 h on H₂S metabolism and PS production in HEK293, A549, HT29 and bone marrow derived stem cells (BMDSC) using H₂S (AzMC, MeRho-AZ) and PS (SSP4) fluorophores. All MnPs decreased intracellular H₂S production and increased intracellular PS. H₂S metabolism and PS production were unaffected by cellular O₂ (5% versus 21% O₂), H₂O₂ or ascorbic acid. We observed with confocal microscopy that mitochondria are a major site of H₂S production in HEK293 cells and that MnPs decrease mitochondrial H₂S production and increase PS in what appeared to be nucleoli and cytosolic fibrillary elements. This supports a role for MnPs in the metabolism of H₂S 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.

LCS-1, a putative selective inhibitor of SOD1, is a substituted pyridazinone with rudimentary similarity to quinones and naphthoquinones. As quinones catalytically oxidize H₂S to biologically active reactive sulfur species (RSS), we hypothesized LCS-1 might have similar attributes. Here, we examine LCS-1 reactions with H₂S 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 H₂S in buffer solutions to form RSS, namely per- and polyhydrosulfides (H₂S_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 H₂S to H₂S_3-6. LCS-1 forms monothiol adducts with H₂S, 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 Cys⁵⁷-Cys¹⁴⁶ 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.

Inflammation is a key etiologic component in atherogenesis. Previously we demonstrated that adeno-associated virus (AAV) 2/8 gene delivery of Netrin1 inhibited atherosclerosis in the low density lipoprotein receptor knockout mice on high-cholesterol diet (LDLR-KO/HCD). One important finding from this study was that FOXP3 was strongly up-regulated in these Netrin1-treated animals, as FOXP3 is an anti-inflammatory gene, being the master transcription factor of regulatory T cells. These results suggested that the FOXP3 gene might potentially be used, itself, as an agent to limit atherosclerosis. To test this hypothesis AAV2/8 (AAV)/hFOXP3 or AAV/Neo (control) gene therapy virus were tail vein injected into the LDLR-KO/HCD animal model. It was found that hFOXP3 gene delivery was associated with significantly lower HCD-induced atherogenesis, as measured by larger aortic lumen cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-HCD-treated controls. Moreover these measurements taken from the hFOXP3/HCD-treated animals very closely matched those measurements taken from the normal diet (ND) control animals. These data strongly suggest that AAV/hFOXP3 delivery gave a robust anti-atherosclerosis therapeutic effect and further suggest that FOXP3 be examined more stringently as a therapeutic gene for clinical use.