Products of Sulfide/Selenite Interaction Possess Antioxidant Properties, Scavenge Superoxide-Derived Radicals, React with DNA, and Modulate Blood Pressure and Tension of Isolated Thoracic Aorta

Selenium (Se), an essential trace element, and hydrogen sulfide (H2S), an endogenously produced signalling molecule, affect many physiological and pathological processes. However, the biological effects of their mutual interaction have not yet been investigated. Herein, we have studied the biological and antioxidant effects of the products of the H2S (Na2S)/selenite (Na2SeO3) interaction. As detected by the UV-VIS and EPR spectroscopy, the product(s) of the H2S-Na2SeO3 and H2S-SeCl4 interaction scavenged superoxide-derived radicals and reduced ·cPTIO radical depending on the molar ratio and the preincubation time of the applied interaction mixture. The results confirmed that the transient species are formed rapidly during the interaction and exhibit a noteworthy biological activity. In contrast to H2S or selenite acting on their own, the H2S/selenite mixture cleaved DNA in a bell-shaped manner. Interestingly, selenite protected DNA from the cleavage induced by the products of H2S/H2O2 interaction. The relaxation effect of H2S on isolated thoracic aorta was eliminated when the H2S/selenite mixture was applied. The mixture inhibited the H2S biphasic effect on rat systolic and pulse blood pressure. The results point to the antioxidant properties of products of the H2S/selenite interaction and their effect to react with DNA and influence cardiovascular homeostasis. The effects of the products may contribute to explain some of the biological effects of H2S and/or selenite, and they may imply that a suitable H2S/selenite supplement might have a beneficial effect in pathological conditions arisen, e.g., from oxidative stress.

Selenium (Se) is a relatively rare but an essential trace element for humans, plants, and microorganisms. Se, which exerts multiple and complex effects on human health, is known as an antioxidant due to its presence in 25 selenoproteins in the form of selenocysteine amino acid. Both beneficial and detrimental effects of Se deficiency and/or supplementation are well known. The biological effects of Se compounds (selenite, selenate, selenocysteine, and selenomethionine) on cardiac oxidative damage, heart disease, cancer prevention, immunity, diabetes, neuroregeneration, or dementia have been reported [12][13][14][15][16][17]. However, the beneficial effect of Se supplementation for men's health is still a controversial issue [18][19][20][21][22][23]. Selenite, which is a common Se supplement, is considered as a promising anticarcinogen [24][25][26]. It can induce apoptosis in cancer cells through the production of reactive oxygen species (ROS) leading to oxidative stress [27,28]. However, Se compounds were also found to damage DNA in healthy cells [29] and therefore may not be considered as a suitable protective agent against cancer and/or other chronic diseases. Actually, they can cause or advance some kinds of cancers [30,31]. The exact mechanisms of the beneficial and toxic effects of Se are not yet fully understood, giving rise to further uncertainty about its potential use in nutrition supplements and/or clinical treatment.
Se and H 2 S are present in living organisms, and each one has beneficial and/or toxic effects through its interaction mostly with ROS [1,2,[30][31][32][33]. However, the biological effects of products of the H 2 S/selenite interaction are not yet known, namely, their involvement in the production and/or inhibition of ROS, reaction with DNA, or influence on cardiovascular system. Therefore, we have studied the effects of products of the H 2 S/selenite interaction on O 2 ⋅− and ⋅ cPTIO radicals, DNA cleavage, tension of isolated aortic rings, and rat blood pressure (BP). We found that the products have significant biological effects that differ from those caused by H 2 S or by selenite on their own. These results may contribute to the understanding of possible coupled biological effects of H 2 S and Se.
2.1.4. Sulfide. Na 2 S (100 mmol L −1 stock solution, Dojindo SB01, Japan) was dissolved in argon degassed deionized H 2 O, stored at −80°C, and used immediately after thawing. Na 2 S dissociates in aqueous solution and reacts with H + to yield H 2 S, HS − , and a trace of S 2− . We use the term H 2 S to describe the total mixture of H 2 S, HS − , and S 2− forms. The stock concentration was checked by UV-VIS spectroscopy: by the absorbance of 1000x diluted stock solution at 230 nm (ε 230 nm = 7700 mol −1 L cm −1 , diluted by deionized water) and also by the reduction of 100 μmol L −1 DTNB by 2000x diluted stock solution (1 H 2 S molecule generates 2 TNB − equivalents, ε 412 nm = 14,100 mol −1 L cm −1 , measured in 1 mmol L −1 phosphate buffer), according to Nagy et al. [34].

UV-VIS of ⋅ cPTIO.
To obtain 1 mL of the working solution, 10 or 100 μL of stock solution of the compounds studied was added to the appropriate volume (990 or 900 μL, respectively) of 100 mmol L −1 sodium phosphate buffer (at given pH, 37°C) containing the final concentrations of 100 μmol L −1 ⋅ cPTIO and 100 μmol L −1 DTPA. UV-VIS absorption spectra (900-190 nm) were recorded every 30 s for 20 to 40 min with a Shimadzu 1800 (Kyoto, Japan) spectrometer at 37°C. The ⋅ cPTIO extinction coefficient of 920 mol −1 L cm −1 at 560 nm was used. The reduction of the ⋅ cPTIO radical was determined as the decrease of the absorbance at 560 nm (absorption maximum of ⋅ cPTIO in VIS range) or at 358 nm after subtracting the absorbances at 730 or at 420 nm, respectively [5,35].
To study the involvement of O 2 in the H 2 S/SeO 3 2− -induced reduction of the ⋅ cPTIO radical, 10 mmol L −1 Na 2 S in H 2 O, 10 mmol L −1 Na 2 SeO 3 in H 2 O, and 102 μmol L −1 ⋅ cPTIO in the 100 mmol L -1 sodium phosphate buffer, supplemented with 100 μmol L −1 DTPA (pH 7.4; 37°C), were deaerated with argon for 10 min at 37°C. The compounds were mixed in a closed UV-cuvette, and the UV-VIS spectra were recorded. The O 2 concentration in the deaerated samples was 3-5%, confirmed with an oxygen electrode (OXELP, SYS-ISO2, WPI, USA). In all UV-VIS experiments, H 2 O was used as a blank.
2.3. EPR of the ⋅ BMPO Adducts. To study the ability of H 2 S/SeO 3 2− to scavenge the O 2 ⋅− radical or its derivatives produced in DMSO/KO 2 solution, sample preparation and EPR measurements were conducted in accordance with previously reported protocols [5]. The solution (final concentrations) of BMPO (20 mmol L −1 ), DTPA (100 μmol L −1 ) in sodium phosphate buffer (50 mmol L −1 , pH 7.4) was incubated for 1 min at 37°C; an aliquot of the compound was added, followed by saturated KO 2 /DMSO solution (10% v/v DMSO/final buffer) 3 s later. The sample was mixed for 5 s and transferred to a standard cavity aqueous EPR flat cell. The first EPR spectrum was recorded 2 min after the addition of KO 2 /DMSO solution at 37°C. The sets of individual EPR spectra of the ⋅ BMPO spin adducts were recorded as 15 sequential scans, each 42 s, with a total time of 11 min. Each experiment was repeated at least twice. EPR spectra of the ⋅ BMPO spin adducts were measured on a Bruker EMX spectrometer, X-band~9. 2.4. Plasmid DNA Cleavage. pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs Inc., N3033L) was performed as reported previously [5,36]. In this assay, all samples contained 0.2 μg pDNA in sodium phosphate buffer (25 mmol L −1 sodium phosphate, 50 μmol L −1 DTPA, pH 7.4, 37°C). After addition of compounds, the resulting mixtures were incubated for 30 min at 37°C. All concentrations listed in the section were final in the samples. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Samples were electrophoresed in TBE buffer (89 mmol L −1 Tris, 89 mmol L −1 boric acid, and 2 mmol L −1 EDTA) at 5.5 V cm −1 for 2 h; gels were stained with GelRed™ Nucleic Acid Gel Stain and photographed using a UV transilluminator. Integrated densities of all pBR322 forms in each lane were quantified using the TotalLab TL100 image analysis software to estimate pDNA cleavage efficiency (Nonlinear Dynamic Ltd., USA). 2.6. Functional Study of Isolated Thoracic Aorta. Normotensive Wistar Kyoto (WKY) rats (307 ± 4:3 g) were killed by decapitation after a brief anesthetization with CO 2 , and the thoracic aorta was isolated as described in our previous study [38]. The changes in isometric tension were measured by the electromechanical transducers (FSG-01, MDE, Budapest, Hungary). The resting tension of 1 g was applied to each ring and maintained throughout a 45 to 60 min of equilibration period until stress relaxation no longer occurred. Changes in thoracic aorta tension were followed by noradrenaline (NA; 1 μmol L −1 ) precontracted arterial rings after a stable plateau was achieved.

Guide for the
2.7. Functional Study of Rat Blood Pressure. Male Wistar rats (n = 10; 350 ± 40 g) were from the Department of Toxicology and Laboratory Animal Breeding at Dobra Voda, Slovak Academy of Sciences, Slovakia. The rats were housed under a 12 h light-12 h dark cycle, at a constant humidity (45-65%) and temperature (20-22°C), with free access to standard laboratory rat chow and drinking water. The Institute of Experimental Pharmacology and Toxicology, Centre of Experimental Medicine, Slovak Academy of Sciences, provided veterinary care. The tranqualizer xylazine (Rometar) was purchased from Zentiva (Czech Republic), and the anesthetic combination of tiletamine+zolazepam (Zoletil 100) was acquired from Virbac (France). All other chemicals were purchased from Sigma-Aldrich. Experiments were carried out as previously described [39]. Rats were anesthetized with Zoletil 100 (tiletamine+zolazepam, 80 mg kg -1 , i.p.) and Rometar (xylazine, 5 mg kg -1 , i.p.). During the anesthesia, BP, heart rate, and reflex responses to mechanical stimuli were monitored. The animals were under anesthesia during the whole experiment and were euthanized with an overdose of Zoletil via jugular vein at the end of the surgical procedure. All experiments were supervised and performed under the same experimental conditions. 2.8. Blood Pressure Measurement. The right jugular vein was cannulated to administer compounds under anesthesia as described above. The left arteria carotis communis was cannulated for inserting the fiber optic microcatheter pressure transducers (FISO LS 2F Harvard Apparatus, USA). The analog signal was digitalized at 10 kHz, filtered at 1 kHz, and recorded by DEWESoft 6.6.7 (GmbH, Austria). The signal was evaluated 5 s before and 10 min after compound administration. After stabilization of BP (10-20 min), the compounds were administered into the right jugular vein as a bolus of 500 μL kg −1 over 15 s period. The solution of the H 2 S/SeO 3 2− mixture (10/5 in mmol L −1 ) was prepared as follows: to 123.5 μL of 100 mmol L −1 phosphate buffer, 100 μmol L −1 DTPA, 14 μL of 1 mol L −1 HCl was added, followed by 62.5 μL of 40 mmol L −1 Na 2 SeO 3 in 0.9% NaCl, and finally 50 μL of 100 mmol L −1 Na 2 S in H 2 O was added. The pH of the buffered mixture was 7.4. The mixture was incubated for 40 ± 10 s at 23°C before i.v. administration. Unbuffered H 2 S/SeO 3 2− mixture (10/5 in mmol L −1 ) was prepared, when 0.9% NaCl was used instead of phosphate buffer and HCl. The pH of the unbuffered mixture was~11 measured by a pH paper indicator.
2.9. Statistical Analysis. Unless otherwise stated, data are represented as the means ± S:E:M. Statistical significance was determined by Student's t-test or one-way ANOVA followed by the multiple comparison test. Differences between means were considered significant at * P ≤ 0:05. Data analysis and plot construction were carried out using SigmaPlot 12 (Systat Software GmbH).

H 2 S
Interacts with Na 2 SeO 3 and SeCl 4 , but Not with Na 2 SeO 4 , to Form Initial Reactive Intermediate(s), which Reduce the ⋅ cPTIO Radical. Since the antioxidant properties of H 2 S/SeO 3 2− products are unknown, we have used the ⋅ cPTIO radical to study the reducing properties of the products of H 2 S/SeO 3 2− interaction. The ⋅ cPTIO radical is stable in aqueous solution, and its formation and reduction can be monitored by the UV-VIS spectrophotometry at 358 or 560 nm. Even in the presence of up to 100 μmol L −1 H 2 S or 100-400 μmol L −1 SeO 3 2− , the absorbance (ABS) of the radical at 358 and 560 nm decreases only by <7% after 40 min, indicating that neither H 2 S nor SeO 3 2− on its own reduces this radical ( Figure 1). In contrast, once H 2 S (25-100 μmol L −1 ) was added to the ⋅ cPTIO/SeO 3 2− (100/2.5-400 μmol L −1 ) mixture, or SeO 3 2− was added to ⋅ cPTIO/H 2 S, the absorbances at 358 and 560 nm decreased rapidly over the time (≤30 s), indicating a possible formation of strong reducing agent(s) which fastly and efficiently reduced the ⋅ cPTIO radical (Figures 1 and 2). Similar results were obtained when SeCl 4 , but not Na 2 SeO 4 , was used instead of SeO 3 2− (Figures S1 and S2a). The reduction of ⋅ cPTIO followed a bell-shaped dependence on the concentration of SeO 3 2− at a constant ⋅ cPTIO/H 2 S concentration (Figure 2(a)), with a maximum radical scavenging activity at an H 2 S : SeO 3 2− ratio of roughly 4 : 1. The ability of H 2 S to reduce ⋅ cPTIO in the presence of SeO 3 2− increased with the increasing H 2 S concentration (Figure 2(b)) and followed also a bell-shaped dependence on pH (Figures 3 and S3).
If H 2 S and SeO 3 2− were preincubated for different periods of time before the addition to ⋅ cPTIO, it clearly resulted in the highest radical scavenging activity, which was subsequently lost over the time (Figure 4). An H 2 S/SeO 3 2− (100/100 in μmol L −1 ) mixture preincubated for ≥1 min prior to ⋅ cPTIO addition did not reduce ⋅ cPTIO, demonstrating that later products of the reaction of sulfide with SeO 3 2− could not be responsible for the reduction of the radical and that the relevant active species were formed swiftly, in less than 1 min and have a short lifetime, as recently suggested [40]. Notably, formation of these active early intermediates to reduce ⋅ cPTIO was prolonged with the increase of the H 2 S/SeO 3 2− ratio ( Figure 4(e)). At H 2 S/SeO 3 2− concentration of 100/25 in μmol L −1 and 5 min preincubation time, the mixture still possessed around 50% potency to reduce ⋅ cPTIO (Figure 4(e)). This timing, once more, accounts for a rapidly formed selenosulfide intermediate as beeing the ultimate responsible for this radical scavenging activity, a species also possibly being sensitive to oxidation over prolonged time periods (Figure 4).
While the available evidence is in accordance with the formation of HSSeSH as the main reactive species, there are other chalcogen-based candidates which are good reducing agents, namely, hydrogen selenide (H 2 Se), hydroselenide anion (HSe − ), selenide (Se 2− ), persulfides, and polysulfides (S x 2-) [41][42][43]. Interestingly, O 2 does not seem to play a major role in the H 2 S/SeO 3 2− -induced reduction of the ⋅ cPTIO radical probably due to slower kinetics of interaction of reactants and/or intermediates with O 2 in comparison to the rate of ⋅ cPTIO reduction [44][45][46][47][48]. Under argon flushed conditions, reduction of the ⋅ cPTIO radical was neither enhanced nor suppressed significantly, hence ruling out any major involvement of H 2 Se, as this selenium compound is highly sensitive to O 2 ( Figure S2b). ) Radical. We aimed to ascertain whether the initial products of the H 2 S/SeO 3 2− interaction are able to scavenge other radicals, i.e., O 2 ⋅− or its derivatives. The interactions with O 2 ⋅− were studied with the EPR spin trap method based on the reaction of this dioxygen radical with BMPO to form the ⋅ BMPO-OOH adduct [49]. This assay was chosen due to biological and mechanistical reason; O 2 ⋅− is a simple radical reduced by one-electron transfer.   Figure S5). This indicates that the production of highly active species, products of the H 2 S/SeO 3 2− interaction, was time-dependent: they appeared within a few seconds after addition of SeO 3 2− to the H 2 S solution and their effects diminished after few minutes of interaction.

EPR of
From our previous studies of O 2 ⋅− reaction with BMPO [5], we assumed that the EPR spectra of the ⋅ BMPO adducts were superposed on the ⋅ BMPO-OOH/OH radicals with minor contribution from the ⋅ BMPO-C radical. Therefore, we simulated the spectra using hyperfine coupling constants for ⋅ BMPO-OOH, ⋅ BMPO-OH, and ⋅ BMPO-C (derived from DMSO) radicals. The means of hyperfine coupling constants used were as follows: ⋅ BMPO-OOH1 (black) a N = 13:30 ± 0:03 G, a H = 11:7 ± 0:1 G; ⋅ BMPO-OOH2 (red) a N = 13:24 ± 0:03 G, a H = 9:4 ± 0:1 G; ⋅ BMPO-OH1 (green) a N = 13:7 ± 0:3 G, a H = 12:3 ± 0:4 G; ⋅ BMPO-OH2 (yellow) a N = 13:6 ± 0:2 G, a H = 15:3 ± 0:1 G; and ⋅ BMPO-C (blue) a N = 15:2 ± 0:1 G, a H = 21:5 ± 0:1 G. The constants are similar to those reported by Zhao et al. [49]. The simulation revealed that O 2 ⋅− was trapped in the control and in the presence of  , since H 2 O 2 now takes on the role of SeO 3 2− as an oxidant, with the simultaneous formation of the ⋅ OH radicals (Figures 6(b1) and 6(b2)). Further evidence for the involvement of radicals may come from the fact that dimethylsulfoxide (DMSO), a known ⋅ OH scavenger [50,51] frequently employed as solvent in biology, is able to interfere with the damage to DNA (Figures 6(c1) and 6(c2)). 3 2− Modulates Tension of Isolated Thoracic Aorta. As some of our in vitro assays indicated an antioxidant activity of the H 2 S/SeO 3 2− mixture, and H 2 S is known to promote relaxation of blood vessels [52], the impact of the H 2 S/SeO 3 2− mixture on the isolated thoracic aorta was examined. The thoracic aorta was precontracted by noradrenaline (NA) (1 μmol L −1 ). After a stabile plateau of the contraction was reached (Figure 7(a)), SeO 3 2− (100 μmol L -1 ) showed only negligible activity, while Na 2 S (200 μmol L −1 ) significantly relaxed the aortic rings, in agreement with our previous studies [38]. A simultaneous addition of SeO 3 2− (100 μmol L −1 ) and H 2 S (200 μmol L −1 ) resulted once more in a biphasic activity profile, where a minor relaxation was noticed first, followed by a pronounced contraction (Figure 7). It is supposed that the contraction effect may result also from the antioxidant properties of the mixture, similarly as it has been reported for ascorbate [53]. 3 2− Modulates Rat Systolic and Pulse Blood Pressure. Since the products of the H 2 S/SeO 3 2− interaction modulated the tension of the thoracic aorta, we subsequently studied whether the products influence blood pressure (BP). Intravenous (i.v.) administration of 5 μmol kg −1 SeO 3 2− had only minor effects on BP (Figure 8(g)). The administration of 10 μmol kg −1 of Na 2 S transiently decreased and increased BP (Figures 8(a) and 8(g)), as observed in our previous study [54]. The stock solution of the H 2 S/SeO 3 2− mixture (20/10 in mmol L −1 ) prepared in 0.9% NaCl was colorless and had pH~11. However, when the mixture was prepared in solution with pH 7.4, it had orange color with an absorption maximum at 570 nm ( Figure S6), indicating formation of the sulfur-selenium complexes [40]. The i.v. administration of the mixture H 2 S/SeO 3 2− (10/5 μmol kg −1 , pH~7.4), in comparison to H 2 S alone, inhibited both BP decrease and increase (Figures 8(b) and 8(g)). The effects of the mixture were less pronounced at pH~11, being the effects at this pH similar to those observed for H 2 S alone (Figures 8(c) and 8(g)).

H 2 S/SeO
The studied compounds influenced pulse BP, as an important parameter of cardiovascular system reflecting arterial stiffness [55,56]. The administration of 5 μmol kg −1       (a2-h2) show details of the accumulated first ten spectra of the (a1-h1) sets. The intensities of the time-dependent EPR spectra (a1-h1) and detailed spectra (a2-h2) are comparable; they were measured under identical EPR settings. EPR modulation amplitude 0.15 mT. (a3-h3) Comparison of the integral intensity of individual components of simulated BMPO+O 2 ⋅− without (control) and with chalcogen species shown in (a1-h1). The first five EPR spectra were accumulated and used for simulation. The data represent the means of n = 2; standard error was ≤10% of the mean value. Simulated relative intensities of the two conformers of the radicals: ⋅ BMPO-OOH1 (black), ⋅ BMPO-OOH2 (red), ⋅ BMPO-OH1 (green), ⋅ BMPO-OH2 (yellow), and ⋅ BMPO-C (blue). SeO 3 2− intravenously had minor effects on pulse BP (Figure 8(h)). The administration of 10 μmol kg −1 of Na 2 S transiently increased and later decreased pulse BP (Figures 8(d) and 8(h)) [54]. The i.v. administration of the mixture H 2 S/SeO 3 2− (10/5 in μmol kg −1 , pH~7.4), with comparison to H 2 S alone, eliminated pulse BP increase, but did not affect pulse BP decrease (Figures 8(e) and 8(h)). The effects of the mixture were less pronounced at pH~11 and were similar to those observed for H 2 S alone (Figures 8(f) and 8(h)). The inefficiency of the H 2 S/SeO 3 2− products at pH~11 may be connected with the minor effect of the mixture on the ⋅ cPTIO radical reduction at high pH (Figures 3(e) and 3(f)). This may imply that the reduction properties of the mixture could be responsible for their effects on systolic and pulse BP. The results confirm that the products of the H 2 S/SeO 3 2− interaction depend on pH and influence differently the cardiovascular system. In conclusion, the reactivity and biological activity of the H 2 S/SeO 3 2− interaction products prepared at pH 7.4 differ from those of Na 2 S alone.

Discussion and Conclusions
Overall, our studies demonstrate that the two suspected and commonly used "antioxidants," H 2 S and SeO 3 2− , are not necessarily typical reducing agents, such as ascorbic acid or tocopherol, when employed on their own. Interestingly, these two chalcogen agents, when added together, rapidly activate each other and form a cascade of considerably more reactive, often reducing species, supposing the involvement of inorganic HSSeSH and polysulfides S x 2− , which may account for some of the observed biological actions. The nature of some of these intermediate reactive sulfur and/or selenium species was suggested in a recently published review [40].
The fast and efficient reduction of the ⋅ cPTIO radical by H 2 S/SeO 3 2− products (Figures 1 and 4) supports the notion that the initial intermediate(s) formed by the reaction of H 2 S with SeO 3 2− are responsible for this kind of action. The bell shape and the maximum radical scavenging activity of H 2 S : SeO 3 2− at a ratio of~4 : 1 ( Figure 2) may indicate the suggested formation of (HSS) 2 Se [40]. The kinetics and efficiency of the H 2 S/SeO 3 2− products to reduce ⋅ cPTIO ( Figure 3) point out to complex pH-dependent chemical and radical reactions of the species.
Reactions of H 2 S or polysulfides with SeO 3 2− and/or SeCl 4 were reported [40,[57][58][59]. They point towards a rapid conversion of SeO 3 2− and SeCl 4 to an intermediate, probably HSSeSH, and a subsequent, slower reductive elimination of this intermediate to elemental (mixed) chalcogen particles and disulfides [40]. However, to our knowledge, there is no information about the formation and detection of HSSeSH in cells or its cytoprotective or other biological effect. The first synthesis of H-S-Se-S-H (1.3-dithiatriselane) was reported by Hahn and Klünsch in 1994, but the stability and reactivity in aqueous solution were not investigated. Solid HSSeSH has a melting point at −40°C. It was one component of a mixture of H 2 S 2 Se n , prepared by the interaction of 2 H 2 S with Se 2 Cl 2 [60].
The EPR data ( Figure 5) once more confirm that SeO 3 2− on its own is not an antioxidant; it becomes activated by reduction, for instance, by H 2 S, which concurrently is activated by oxidation. The mutual redox activation is fast, and, as in the case of the ⋅ cPTIO radical scavenging, the pristine mixture of H 2 S and SeO 3 2− is most active, with a decrease of activity over the time, pointing once more at simple H 2 S x or H 2 S x Se, and notably HSSeSH, but not an S x Se y , as being responsible for this kind of activity.
We found that the products of this described H 2 S/SeO 3 2− interaction have several noteworthy biological effects, involving ROS scavenging, modulation of the redox state, reaction with DNA, tensing isolated aorta, and influencing BP and pulse BP (Figures 6-8). These effects obviously need to be investigated further and in considerably more detail and were not present or were less pronounced when H 2 S or selenite acted alone. The properties of the products of the H 2 S/SeO 3 2− interactions significantly depended on the H 2 S/SeO 3 2− molar ratio, pH, and preincubation time. The combination of these variables makes the work with H 2 S/SeO 3 2− very complicated, and these facts should be taken into account at the time of designing in vitro and in vivo experiments. These properties of the products may explain the previously published beneficial and contrasting toxic Se  , H 2 S, and H 2 S/SeO 3 2− on NA (1 μmol L −1 )-precontracted rings of rat thoracic aorta. The rings were exposed to bolus dose of H 2 S (200 μmol L −1 , relaxation), SeO 3 2− (100 μmol L −1 , nonsignificant contraction), and that of the mixture H 2 S/SeO 3 2− (100 μmol L −1 of SeO 3 2− immediately followed by 200 μmol L −1 H 2 S). The SeO 3 2− /H 2 S mixture had a biphasic activity; firstly, it significantly relaxed the aorta, which was followed by significant contraction (b). Asterisks mark the statistical significance of H 2 S/SeO 3 2− mixture vs. H 2 S ( * * P < 0:01, * * * P < 0:001).
effects, for example, in conditions of oxidative stress and cancer [12-14, 18, 20, 22, 23, 30]. H 2 S is endogenously produced in vivo in most, if not in all, cells, and H 2 S donors are commonly used in biological experiments, and they are considered to be applied in medicine. Our results suggest that in biological experiments with selenite, in its nutrition supplement and clinical use, effects of the H 2 S/SeO 3 2− interaction should be considered. While SeO 3 2− is used widely as a nutritional supplement already, one may, in the future, wish to spice it up with some reduced sulfur. Natural spices such as garlic and onions contain suitable sulfide releasing agents, such as diallyltrisulfide (DATS) and diallyltetrasulfide (DATTS), which both occur naturally in garlic, or dipropyltrisulfide and dipropyltetrasulfide, both present in onions [61]. Our results imply that application research of suitable H 2 S/SeO 3 2− supplements may lead to the beneficial effects in pathological conditions arising, e.g., from ROS overproduction. (a, d)) and its mixture with 5 μmol kg −1 SeO 3 2− prepared at pH~7.4 (b, e) and pH~11 (c, f) solution on BP (a, b, c) and pulse BP (d, e, f). Transient changes of rat BP (g) and pulse BP (h) after i.v. bolus administration of SeO 3 2− (5 μmol kg −1 , empty column), H 2 S (10 μmol kg −1 , empty coarse column) and their mixture (SeO 3 2− /H 2 S, 5/10 in μmol kg −1 ) prepared at pH~7.4 (grey column) and pH~11 (grey coarse column). Data are presented as means ± SD; n = 5-10. To test a statistical significance between group differences, we used one-way ANOVA followed by Dunnett's test for multiple comparisons. Hence, we also observed the biphasic effect of Na 2 S in our previous study [54]; we compared a set of "first part" and "second part" effects of SeO 3 2− , H 2 S/SeO 3 2− at pH~7.4, and H 2 S/SeO 3 2− at pH~11.0 to the corresponding effect of Na 2 S on systolic or pulse blood pressure. Only the mixture of H 2 S/SeO 3 2− prepared at pH~11.0 was able to generate similar decrease and subsequent increase or vice versa in systolic blood pressure or pulse blood pressure as the H 2 S, respectively. Asterisks mark the statistical significance as follows: * * P < 0:01, * * * P < 0:001, and * * * * P < 0:0001.