Figure 1.
Structure of selenium compounds of interest in the present paper.
Figure 2.
GC-Mass spectrum of methylselenol.
Positive ion LDI-TOF mass spectrum in the 270–360 m/z region of the precipitated silver salts. A) In absence of Trx1, no peaks corresponding to [Ag2SeCH3]+ ion were detected. B) However, in the presence of Trx1, the spectrum shows the [Ag2SeCH3]+ ion (peaks in the range 304.8–314.8 m/z) overlapped with more abundant Ag2-containing clusters. In particular, low-intensity peaks at 305.8, 307.8 and 309.8 m/z (designated by arrows) denote the presence of selenium in the [Ag2SeCH3]+ molecule.
Figure 3.
A–D. Oxidation of NADPH by the thioredoxin and glutaredoxin systems in the presence of selenite and GS-Se-SG, catalyzed by SAM.
Reaction catalyzed by the thioredoxin system was performed in 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, and 200 µM NADPH. A) The reaction mixture contained 2 µM human Trx1 and 50 nM mammalian TrxR1. SAM, 4 mM (♦) Selenite (5 µM) (○), SAM and selenite, 4 mM and 5 µM respectively (without Trx1 in the reaction) (•), SAM and selenite (Δ). B) Reaction catalyzed by the glutaredoxin system was performed in TE-buffer, containing 200 µM NADPH, 6 µg/mL GR, 1 µM human Grx1 and 50 µM GSH. SAM, 4 mM (♦) Selenite (5 µM) (○), SAM and selenite, 4 mM and 5 µM respectively (without Grx1 in the reaction) (•), SAM and selenite, 4 mM and 5 µM respectively (Δ). C) The thioredoxin system with: 5 µM GS-Se-SG (without Trx1 in the reaction) (♦), 5 µM GS-Se-SG (•), 5 µM GS-Se-SG and SAM (without Trx1 in the reaction) (○), 5 µM GS-Se-SG and SAM (Δ). D) The glutaredoxin system with: 15 µM GS-Se-SG (♦), GS-Se-SG and SAM (•), GS-Se-SG and 1 µM Grx1 (○), GS-Se-SG, SAM and 1 µM Grx1 (Δ).
Table 1.
Peroxidase activity.
Table 2.
Peroxidase activity.
Figure 4.
Inhibition of thioredoxin mediated protein disulfide reductase activity.
The method was performed as described by Kumar et al. [6]. The reaction mixture contained 80 mM HEPES buffer, pH 7.6, 3 mM EDTA and 0.7 mM NADPH. TrxR1 and Trx1 were added to a final concentration of 8 nM and 1 µM respectively. The measurements were performed with the following selenite concentrations (1, 5 and 10 µM). The amount of SH-group formed was measured at 412 nm. Grey bars: Addition of selenite at varying concentrations. White bars: Addition of both selenite and SAM (4 mM). Student t-test, dependent by samples, was used for statistical analysis (*p<0.05, **p<0.01).
Figure 5.
A–D. Total selenium accumulation, extracellular and intracellular thiols after treatment with various selenium compounds.
Selenium accumulation in ng/mg protein after 5 h treatment with A) 5 µM selenite +/− SAM and MSG B) 5 µM GS-Se-SG +/− SAM and MSG measured by GF-AAS analysis. C) Total extracellular thiol content after 5 h treatment with 5 µM selenite +/− SAM and MSG. (*p<0.05 compared to control) and D) total intracellular thiol content following 5 h treatment with selenite (5 µM) +/− SAM was determined by the DTNB assay. Statistical analysis was performed by one-way ANOVA (95% confidence interval) followed by Tukey-Kramer multiple comparison test. (*p<0.05, **p<0.01 and ***p<0.001 compared to controls, °p<0.01 compared to selenium treated cells).
Figure 6.
A–H. Cytotoxicity of selenium compounds in the presence of SAM.
Cell viability was measured by XTT after 24 h incubation with selenium treatment, combined with SAM, DTNB and MSG. Cells were pretreated with MSG (60 mM) followed by treatment with selenium compounds +/− SAM (500 µM) A) Selenite (5 µM), B) GS-Se-SG (5 µM), C) Seleno-DL-cystine (100 µM). D) SAM toxicity was determined by clonogenic assay. Cells were treated for 8 h with 500 µM SAM, washed and re-seeded, in triplicates. After 9 days, clones were stained and counted. E) Viability over time (0–48 h) after pretreatment with MSG followed by addition of selenite +/− SAM. F) Selenium accumulation in ng/mg protein after 24 h treatment (same concentration of all compounds as in E) measured by GF-AAS analysis. G) Comparison of toxicity between selenite (5 µM) +/− SAM and MSA (5 µM) after 24 h of treatment. H) Representative morphological changes associated with the treatments of selenite (5 µM), selenite +/− SAM and MSA (5 µM) for 20 h. In D, Student t-test was performed to verify the statistical significance between two groups. One-way ANOVA (99.9% confidence interval) followed by Tukey-Kramer multiple comparison test was performed to determine statistical significance in A–C, F (**p<0.01 and ***p<0.001 compared to controls, °p<0.01 and °°p<0.001 compared to selenium treated cells). In E, two-way ANOVA (95% confidence interval) was performed, followed by Bonferroni multiple comparison test. (*p<0.05 and ***p<0.001, compared to control at selected time point). In Fig. G, one-way ANOVA was used, followed by Student-Newman-Keuls multiple comparison test (95% confidence interval, *p<0.05 compared to selenite treatment).
Figure 7.
Effect of selenium compounds on superoxide production.
H157 cells were stained with hydroethidine and treated for 5 h with selenite (5 µM) +/− SAM (500 µM), and MSA (5 µM) before detection of accumulated superoxide produced by FACS analysis, as described under materials and methods.
Figure 8.
Proposed schematic overview of the spontaneous methylation of selenide to methylselenol.
A schematic diagram showing the individual role of selenite, thioredoxin and glutaredoxin system and SAM in redox cycling of selenium intermediate metabolites. Selenite is reduced to hydrogen selenide either by thioredoxin or glutaredoxin system (reaction 1) [8], [9]. The same reaction can be catalyzed either by glutathione or cysteine, resulting into the same final end product. Hydrogen selenide can successively be oxidized by oxygen to form superoxide radical or undergo redox cycling mediated by thioredoxin or glutaredoxin system (reaction 2) [8], [9]. However, hydrogen selenide can spontaneously react with SAM to form methylselenol (reaction 3). Subsequently, the thioredoxin and glutaredoxin system participate in the redox cycling of methylselenol in a similar way (reaction 4) with that of hydrogen selenide and generate reactive oxygen species. Under reducing environment, monomethylselenol may act as a radical scavenger because of its superior nucleophilicity compared to its counterpart hydrogen selenide.