Uremic Toxin Lanthionine Interferes with the Transsulfuration Pathway, Angiogenetic Signaling and Increases Intracellular Calcium.

(1) The beneficial effects of hydrogen sulfide (H2S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H2S biosynthesis, previously used as a marker for H2S production, is dramatically increased in circulation in uremia, while H2S release is impaired. Thus, lanthionine could be classified as a novel uremic toxin. Our research was aimed at defining the mechanism(s) for lanthionine toxicity. (2) The effect of lanthionine on H2S release was tested by a novel lead acetate strip test (LAST) in EA.hy926 cell cultures. Effects of glutathione, as a redox agent, were assayed. Levels of sulfane sulfur were evaluated using the SSP4 probe and flow cytometry. Protein content and glutathionylation were analyzed by Western Blotting and immunoprecipitation, respectively. Gene expression and miRNA levels were assessed by qPCR. (3) We demonstrated that, in endothelial cells, lanthionine hampers H2S release; reduces protein content and glutathionylation of transsulfuration enzyme cystathionine-ß-synthase; modifies the expression of miR-200c and miR-423; lowers expression of vascular endothelial growth factor VEGF; increases Ca2+ levels. (4) Lanthionine-induced alterations in cell cultures, which involve both sulfur amino acid metabolism and calcium homeostasis, are consistent with uremic dysfunctional characteristics and further support the uremic toxin role of this amino acid.

Alterations in sulfur amino acid metabolism in mice treated with silymarin: a novel mechanism of its action involved in enhancement of the antioxidant defense in liver.

It has been known that silymarin exhibits protective activity against oxidative liver injury induced by various hepatotoxicants, but the underlying mechanism of its beneficial action remains unclear. We determined the alterations in sulfur-containing amino acid metabolism induced by silymarin in association with its effects on the antioxidant capacity of liver. Male mice were treated with silymarin (100 or 200 mg/kg, p. o.) every 12 h for a total of 3 doses, and sacrificed 6 h after the final dosing. The hepatic methionine level was increased, but the activity and protein expression of methionine adenosyltransferase were decreased by silymarin in a dose-dependent manner. S-Adenosylmethionine or homocysteine concentration was not changed, whereas the sulfur-containing metabolites generated from homocysteine in the transsulfuration pathway including cystathionine, cysteine, and glutathione were increased significantly. Cystathionine ß-synthase was induced, but cysteine dioxygenase was downregulated, both of which would contribute to the elevation of cysteine and its product, glutathione, in liver. Oxygen radical scavenging capacity of liver cytosol against peroxyl radical and peroxynitrite was increased, and also hepatic lipid peroxidation was diminished in the silymarin-treated mice. Taken together, the results demonstrate that silymarin enhances hepatic glutathione generation by elevating cysteine availability via an increment in cysteine synthesis and an inhibition of its catabolism to taurine, which may subsequently contribute to the antioxidant defense of liver.

The oxidized thiol proteome in fission yeast--optimization of an ICAT-based method to identify H2O2-oxidized proteins.

Major intracellular disulfide formation is prevented in the cytosol by potent reducing systems. However, protein thiols can be oxidized as a consequence of redox-mediated physiological reactions or due to the unwanted toxicity of reactive oxygen species. In addition, the reactivity of cysteine residues towards peroxides is used by H(2)O(2) sensors in signal transduction pathways in a gain-of-function process to induce transcriptional antioxidant responses. Thus, the Schizosaccharomyces pombe peroxiredoxin Tpx1 and the transcription factor Pap1 are sensors of H(2)O(2) meant to promote cell survival. In an attempt to compare signaling events versus global thiol oxidation, we have optimized thiol-labeling approaches to characterize the disulfide proteome of fission yeast in response to added H(2)O(2). We propose a method based on (i) freezing the redox state of thiols with strong acids prior to cell lysis; (ii) blocking thiol groups with iodoacetamide, and reversibly oxidized thiols with heavy and light isotope-coded affinity tags (ICAT) reagents; and (iii) quantifying individual relative protein concentrations with stable-isotope dimethyl labeling. We have applied this highly sensitive strategy to provide a map of H(2)O(2)-dependent oxidized thiols in fission yeast, and found Tpx1 and Pap1 as some of the major targets.

Possible roles of sulfur-containing amino acids in a chemoautotrophic bacterium-mollusc symbiosis.

Invertebrate hosts of chemoautotrophic symbionts face the unique challenge of supplying their symbionts with hydrogen sulfide while avoiding its toxic effects. The sulfur-containing free amino acids taurine and thiotaurine may function in sulfide detoxification by serving as sulfur storage compounds or as transport compounds between symbiont and host. After sulfide exposure, both taurine and thiotaurine levels increased in the gill tissues of the symbiotic coastal bivalve Solemya velum. Inhibition of prokaryotic metabolism with chloramphenicol, inhibition of eukaryotic metabolism with cycloheximide, and inhibition of ammonia assimilation with methionine sulfoximine reduced levels of sulfur-containing amino acids. Chloramphenicol treatment inhibited the removal of sulfide from the medium. In the absence of metabolic inhibitors, estimated rates of sulfide incorporation into taurine and thiotaurine accounted for nearly half of the sulfide removed from the medium. In contrast, amino acid levels in the nonsymbiotic, sulfide-tolerant molluscs Geukensia demissa and Yoldia limatula did not change after sulfide exposure. These findings suggest that sulfur-containing amino acids function in sulfide detoxification in symbiotic invertebrates, and that this process depends upon ammonia assimilation and symbiont metabolic capabilities.

Effects of high-dose folic acid and pyridoxine on plasma and erythrocyte sulfur amino acids in hemodialysis patients.

In this investigation, sulfur amino acids (sAA) and sulfhydryls were determined in the plasma and erythrocytes (RBC) of 10 uremic patients on regular hemodialysis (HD) treatment and 10 healthy subjects, before and after supplementation with 15 mg/d of folic acid and 200 mg/d of pyridoxine for 4 wk. The basal total plasma concentrations of homocysteine (Hcy), cysteine (Cys), cysteinylglycine (Cys-Gly), gamma-glutamylcysteine (gamma-Glu-Cys), glutathione (GSH), and free cysteinesulfinic acid (CSA) were significantly higher in HD patients when compared to healthy subjects, whereas methionine (Met) and taurine (Tau) concentrations were the same in the two groups. HD patients showed significantly higher RBC levels of Hcy and Cys-Gly, whereas the RBC concentrations of Met, Cys, Tau, and GSH were not different from those in the healthy subjects. The plasma concentrations of sAA and sulfhydryls differed compared with RBC levels in the healthy subjects and HD patients. In both groups, supplementation with high doses of folic acid and pyridoxine reduced the plasma Hcy concentration. In addition, increased plasma concentrations of Cys-Gly and GSH were found in the HD patients and of CSA in the healthy subjects. After vitamin supplementation, the RBC concentrations of Hcy, Cys, and GSH increased and that of Tau decreased in healthy subjects. The only significant finding in RBC of HD patients was an increase in GSH levels after supplementation. This study shows several RBC and plasma sAA and sulfhydryl abnormalities in HD patients, which confirms earlier findings that RBC and plasma pools play independent roles in interorgan amino acid transport and metabolism. Moreover, high-dose supplementation with folic acid and pyridoxine significantly reduced Hcy levels, but did not restore the sAA and sulfhydryl abnormalities to normal levels. The increase that was observed in GSH after vitamin supplementation may have a beneficial effect in improving blood antioxidant status in uremic patients. Finally, the findings of elevated plasma Cys levels correlating to the elevated plasma Hcy levels in the presence of elevated plasma CSA levels, both before and after vitamin supplementation, led to the hypothesis that a block in decarboxylation of CSA is linked to hyperhomocysteinemia in end-stage renal failure.