Characterization of Endogenous and Extruded H₂S and Small Oxoacids of Sulfur (SOS) in Cell Cultures

Abstract

This report characterizes and quantifies endogenous hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS = HOSH, HOSOH) in a panel of cell lines including human cancer (A375 melanoma cells, HeLa cervical cells) and noncancer (HEK293 embryonic kidney cells), as well as E. coli DH5α and S. cerevisiae S288C. The methodology used is a translation of well-studied nucleophilic and electrophilic traps for cysteine and oxidized cysteines residues to target small molecular weight sulfur species; mass spectrometric analysis allows for species quantification. The observed intracellular concentrations of H2S and SOS vary in different cell types, from nanomolar to femtomolar, typically with H2S > HOSOH > HOSH. We propose the term sulfome, a subset of the metabolome, describing the nonproteinaceous metabolites of H2S; the sulfomic index is as a measure of the S-oxide redox status, which gives a profile of endogenous sulfur at different oxidation states. An important observation is that H2S and SOS were found to be continuously extruded into surrounding media against a concentration gradient, implying an active efflux process. Small molecule inhibition of several H2S generating enzymes suggest that SOS are not derived solely from H2S oxidation. Even after successful inhibition of H2S production, cells maintain constant efflux and repopulate H2S and SOS over time. This work proves that these small sulfur oxoacids are generated in cells of all types, and their efflux implies that they play a role in cell signaling and possibly other vascular physiology attributed to H2S.

Expanding the Reactive Sulfur Metabolome: Intracellular and Efflux Measurements of Small Oxoacids of Sulfur (SOS) and H2S in Human Primary Vascular Cell Culture

“…All four cell types displayed similar ranking of species, with HOSOH > H 2 S > HSOH, Figure 1; this ordering is distinctly different than previous measurements in noncancer and cancer lines in which H 2 S > HOSOH > HSOH, with concentrations of the oxidized species dramatically smaller than H 2 S. Table 2 shows normalized ratios of the analytes, which more clearly illustrates differences in S oxidation changes between different cell types. Far higher concentrations of SOS are seen in all four human primary vascular cell lines when comparing to previous data on HEK293, HeLa, and A375 cell lines [54]. The sulfomic index is also presented, a ratio of nucleophilic and electrophilic traps within the analytes, represented as the ratio of S-D concentration divided by the total S concentration, i.e.…”

“…The production of H 2 S is orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar). The distributions of the three analytes, HOSOH > H 2 S > HSOH, demonstrated much higher S-oxidation levels than seen in non-vascular cell lines [ 54 ]. Overall, the smooth muscle cells were found to have almost 25-fold higher concentrations of the endogenous S-analytes as compared to endothelial cells.…”

“…H 2 S, sulfenic (HSOH), and sulfoxylic (HOSOH) acids were trapped by derivatization reactions with nucleophilic and electrophilic reagents to form the products seen in Scheme 1 , as previously described [ 54 ]. The respective concentrations were determined from calculating the selective ion chromatogram peak areas analyzed on a Q Exactive Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Madison, WI, USA) connected to a Thermo Scientific UltiMate 3000 Rapid Separation Quaternary high performance liquid chromatography (HPLC) system (Thermo Scientific, Madison, WI, USA).…”

“…Likewise, cell counting and estimated average cell volumes were used to calculate the concentrations of H 2 S and SOS. With this intent, we translated the LC HD MS method to observe and quantify levels of trappable SOS and H2S in a panel of human cancer and non-cancer cell lines, bacteria, and yeast [54]. The new method involved precipitating and filtering the protein from cell lysates, to select for only small molecule analytes.…”

The antioxidant and oxidant properties of hydropersulfides (RSSH) and polysulfide species

“…Alternatively, inorganic oxidized sulfur species (oxidized H 2 S or oxoacids of sulfur) can be formed which react with RSH to give RSSH species [ 18 ]. Regardless, formation of RSSH from RSH and H 2 S requires an oxidation at some point in the process.…”