Role of AQP9 in transport of monomethyselenic acid and selenite.

AQP9 is an aquaglyceroporin with a very broad substrate spectrum. In addition to its orthodox nutrient substrates, AQP9 also transports multiple neutral and ionic arsenic species including arsenic trioxide, monomethylarsenous acid (MAsIII) and dimethylarsenic acid (DMAV). Here we discovered a new group of AQP9 substrates which includes two clinical relevant selenium species. We showed that AQP9 efficiently transports monomethylselenic acid (MSeA) with a preference for acidic pH, which has been demonstrated in Xenopus laevis oocyte following the overexpression of human AQP9. Specific inhibitors that dissipate transmembrane proton potential or change the transmembrane pH gradient, such as FCCP, valinomycin and nigericin did not significantly inhibit MSeA uptake, suggesting MSeA transport is not proton coupled. AQP9 was also found to transport ionic selenite and lactate, with much less efficiency compared with MSeA uptake. Selenite and lactate uptake via AQP9 is pH dependent and inhibited by FCCP and nigericin, but not valinomycin. The selenite and lactate uptake via AQP9 can be inhibited by different lactate analogs, indicating that their translocation share similar mechanisms. AQP9 transport of MSeA, selenite and lactate is all inhibited by a previously identified AQP9 inhibitor, phloretin, and the AQP9 substrate arsenite (AsIII). These newly identified AQP9 selenium substrates imply that AQP9 play a significant role in MSeA uptake and possibly selenite uptake involved in cancer therapy under specific microenvironments.

Effects of diphenyl diselenide on depressive-like behavior in ovariectomized mice submitted to subchronic stress: involvement of the serotonergic system.

RATIONALE: The transition to menopause is associated with an increased risk of depressed mood. OBJECTIVES: This study was conducted to investigate whether diphenyl diselenide [(PhSe)2] treatment could reduce the effects of postmenopausal depression-like behavior in ovariectomized female mice submitted to subchronic stress exposure. METHODS: Mice were divided into four groups: sham, (PhSe)2, ovariectomy (OVX), and OVX + (PhSe)2. Animals were ovariectomized/sham-operated and subjected to stress session once a day for 7 days from the fifth to the 11th day after OVX. The behavioral tests (open field, tail suspension (TST), and forced swimming (FST)) were performed on the 14th day after OVX. Mice were treated orally once a day with vehicle (canola oil, 10 ml/kg) or (PhSe)2 (10 mg/kg; 10 ml/kg) 30 min before being exposed to subchronic stress, or from the 11th to the 14th day. Paroxetine (8 mg/kg i.p.) and pargyline (30 mg/kg i.p.) were used as positive controls. The involvement of serotonergic receptor subtypes in the antidepressant-like effect of (PhSe)2 was assessed in the FST using WAY 100635 (0.1 mg/kg s.c.), ritanserin (1 mg/kg i.p.), and ondansetron (1 mg/kg i.p.) as serotonergic antagonists. Monoamine oxidase (MAO) A and B activities were also determined. RESULTS: The prolongation of immobility time in TST and FST in OVX mice submitted to subchronic stress was prevented by (PhSe)2 treatment. Ritanserin and ondansetron blocked the antidepressive-like effect of (PhSe)2, suggesting the involvement of 5-HT(2A/2C) and 5-HT3 receptor subtypes. Both paroxetine and pargyline were effective in reducing the immobility time of stressed OVX mice in the FST. No alterations in locomotor activity were observed. Although (PhSe)2 had inhibited in vitro MAO-A and MAO-B activities, none of the groups presented alterations neither in ex vivo MAO-A nor in MAO-B activity. CONCLUSIONS: (PhSe)2 treatment could influence mood and behavior, indicating a promising role of this organoselenium compound in the management of postmenopausal depressive symptoms.

Opposing regulation of histamine-induced calcium signaling by sodium selenite and ebselen via alterations of thiol redox status.

Elevated blood histamine plays a role in the pathogenesis of atherosclerosis. Calcium signaling mediates histamine action in endothelial cells. Selenium (Se) is a dietary essential trace element for humans. Se compounds in different oxidation states were found to exhibit an opposing effect on the histamine-induced calcium signaling in the ECV304 cell line. When Se in the form of sodium selenite was added in the cell culture, the reactivity of the histamine H(1)-receptor was increased as reported in our previous paper. We here show that as a culture supplement, sodium selenite enhanced the activity of selenoprotein thioredoxin reductase (TrxR) and the calcium response to histamine stimulation, which were reversed by treating the cells with gold thioglucose, a nucleophilic drug that selectively modifies thiolate/selenolate groups. Sodium selenite most likely caused a reductive shift in the thiol/disulfide redox balance through increasing TrxR activity. In contrast, when the cells were treated with Se in the form of ebselen, a thiol oxidant with peroxidase-like activity, histamine-induced calcium release and calcium entry were significantly suppressed. This effect appeared related to the thiol-directed modification rather than the peroxidase-like activity of ebselen, because this inhibitory effect was not replicated by increasing cellular peroxidase activity. Thus, the opposing effects of sodium selenite and ebselen on histamine-induced calcium signaling are achieved, at least in part, through their opposite actions in modulating the thiol/disulfide redox state.

Pro-oxidant action of diphenyl diselenide in the yeast Saccharomyces cerevisiae exposed to ROS-generating conditions.

Organoselenium compounds have a potential thiol peroxidase-like activity. Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. Using TRAP assay of chemiluminescense we have shown that diphenyl diselenide clearly possesses a pro-oxidant property. For an investigation on the mechanisms of this property, we used mutant strains of Saccharomyces cerevisiae defective in antioxidant defenses, i.e. in superoxide dismutase, in biosynthesis of glutathione, and the transcription factor yAP-1-lacking yap 1 mutant that cannot activate genes of the oxidative stress response. Exposure of growing cultures to the drug increased cell sensitivity to oxidizing agents. The pro-oxidant effect was independent of the metabolic condition or of the oxidative mutagen tested. N-acetylcysteine, a precursor of glutathione biosynthesis, could neutralize the pro-oxidant effects of diphenyl diselenide by stimulating an increase of endogenous glutathione biosynthesis or by directly binding to the drug. Vitamin E (Trolox), a known antioxidant, was also able to protect S. cerevisiae against the pro-oxidant effect of diphenyl diselenide. In vitro assays showed that diphenyl diselenide interacts non-enzymatically with the thiol group of glutathione.

Organochalcogens effects on delta-aminolevulinate dehydratase activity from human erythrocytic cells in vitro.

Organochalcogens are important intermediates and useful reagents in organic synthesis, which can increase human exposure risk to these chemicals in the workplace. As well, there are a number of reported cases of acute toxicity following organochalcogen ingestion of vitamins and dietary supplements. Since, the erythrocytic delta-ALA-D activity could be an important indicator of toxicity this report investigated the organochalcogens effects on blood delta-ALA-D in vitro. To investigate a possible involvement of cysteinyl groups in the inhibitory actions of diphenyl diselenide, diphenyl ditelluride and Ebselen (4-100 micro M), the effects of thiol reducing agents (0-3 mM) or zinc chloride (0-2 mM) were examined. Diphenyl ditelluride, diphenyl diselenide and Ebselen inhibited in a concentration-dependent manner delta-ALA-D activity from human erythrocytes. Ebselen was lesser delta-ALA-D inhibitor than (PhSe)(2) and (PhTe)(2), whereas the diorganoyldichalcogenides displayed similar inhibitory potency towards delta-ALA-D. Dithiothreitol, a hydrophobic SH-reducing agent, was able to reactivate and to protect inhibited delta-ALA-D. The pre-incubation of blood with the inhibitors changed considerably the reversing potency of thiols. From these findings we suggest that organochalcogens inactivate in vitro human erythrocyte delta-ALA-D by an interaction with the sulfhydryl group essential of the enzyme activity.

Ebselen prevents early alcohol-induced liver injury in rats.

Oxidants have been shown to be involved in alcohol-induced liver injury. Moreover, 2-phenyl-1,2-benzisoselenazole-3(2H)-one (ebselen), an organoselenium compound and glutathione peroxidase mimic, decreases oxidative stress and protects against stroke clinically. This study was designed to test the hypothesis that ebselen protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g/kg/d) continuously for up to 4 weeks using the intragastric enteral feeding protocol developed by Tsukamoto and French. Ebselen (50 mg/kg twice daily, intragastrically) or vehicle (1% tylose) was administered throughout the experiment. Mean urine ethanol concentrations were not significantly different between treatment groups, and ebselen did not affect body weight gains or cyclic patterns of ethanol concentrations in urine. After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (37 +/- 5 IU/l) by enteral ethanol (112 +/- 7 IU/l); ebselen blunted this increase significantly (61 +/- 8 IU/l). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver (pathology score: 4.3 +/- 0.3). In contrast, these pathological changes were blunted significantly by ebselen (pathology score: 2.5 +/- 0.4). While there were no significant effects of either ethanol or ebselen on glutathione peroxidase activity in serum or liver tissue, ebselen blocked the increase in serum nitrate/nitrite caused by ethanol. Furthermore, ethanol increased the activity of NF-kappaB over 5-fold, the number of infiltrating neutrophils 4-fold, and the accumulation of 4-hydroxynonenal over 5-fold. Ebselen blunted all of these effects significantly. These results indicate that ebselen prevents early alcohol-induced liver injury, most likely by preventing oxidative stress, which decreases inflammation.

Induction of apoptosis by selenite and selenodiglutathione in HL-60 cells: correlation with cytotoxicity.

Effects of selenite and selenodiglutathione, an initial metabolite of selenite, on the induction of apoptosis and cytotoxicity were investigated in human promyelocytic leukemia HL-60 cells. Treatment of selenite or selenodiglutathione resulted in concentration-dependent cytotoxicity, measured by lactate dehydrogenase leakage assay, and by tetrazolium salt reduction assay. Selenodiglutathione has been shown to exert more cytotoxic effect than selenite in both assay systems. Time-course study of cellular selenium uptake suggests that the higher cytotoxicity of selenodiglutathione be largely due to faster and greater selenium uptake rate. Treatment with selenite or selenodiglutathione also induced apoptosis in a dose-dependent manner, as detected by enzyme-linked immunosorbent assay and by DNA fragmentation assay. The dose-response data of apoptosis induced by selenite or selenodiglutathione were similar to those of cytotoxicity, implicating a relationship between the induction of apoptosis and cytotoxicity. Zn, which is a well-known inhibitor of apoptosis, dose-dependently blocked not only the induction of apoptosis, but also the membrane damage induced by selenium, corroborating this hypothesis. It was noted that the inhibition of apoptosis by Zn exerted little protective effect on cytotoxicity at higher concentrations of selenium, compared with a perfect protective effect at low concentration of selenium. These results suggest that cytotoxicity induced by selenium may be partially correlated with apoptosis.

Effect of ebselen on bovine and rat nitric oxide synthase activity is modified by thiols.

The inhibition of nitric oxide synthase (NOS) by ebselen, 2-phenyl-1,2-benzisoselenazole-3(2H)-one, was reversed by the addition of 10(-5) M dithiothreitol, suggesting that ebselen reacts with a critical thiol group of NOS in the inhibitory mechanism. In the presence of 10(-4) to 10(-3)M dithiothreitol, ebselen dose-dependently enhanced NOS activity, implicating another interaction of ebselen with NOS under these conditions. Thus, the effect of ebselen on the NOS activity is modified by thiols.

Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione.

Both human recombinant 5-lipoxygenase (EC 1.13.11.34) and 15-lipoxygenase (EC 1.13.11.33, mammalian enzyme) purified from rabbit reticulocytes were inhibited in the absence of glutathione (GSH) by submicromolar concentrations of the seleno-organic compound ebselen. These concentrations were comparable to those of the enzymes. Soybean lipoxygenase-1 (EC 1.13.11.33, plant enzyme) was not inhibited, whereas prostaglandin endoperoxide synthase-1 (EC 1.14.99.1) was inhibited only at much higher concentrations of ebselen (IC50 = 37.7 +/- 4.3 microM). The action of ebselen on reticulocyte 15-lipoxygenase (IC50 = 0.17 +/- 0.01 microM) was studied in detail. Inhibition occurred instantaneously and appeared to be reversible and was largely abolished by a 20-fold molar excess of GSH over ebselen. In the presence of 1 mM GSH 50% inhibition was observed only at ebselen concentrations as high as 234 +/- 27 microM. 13S-hydroperoxy-9Z, 11E-octadecadienoic acid, the lipoxygenase product formed from linoleic acid, augmented the inhibitory effect at low concentrations and caused a partial reversal at high concentrations. A variety of derivatives or structural analogues of ebselen were also tested and proved to be either inactive or weaker inhibitors of 15-lipoxygenase. We have concluded that the potent inhibition of 15-lipoxygenase by ebselen is due neither to GSH peroxidase-like activity nor to lowering of the hydroperoxide tone. The pharmacological implications of these unique characteristics of the action of ebselen on lipoxygenases are then discussed.

Carboxyebselen a potent and selective inhibitor of endothelial nitric oxide synthase.

Ebselen (Ebs) a glutathione peroxidase like agent has been recently described as an inhibitor of nitric oxide synthase (NOS). Presently, we report that carboxyebselen (HOOC-Ebs), a hydrophyllic derivative of Ebs inhibits NOS present in enzymatic preparations from bovine endothelium, porcine cerebella, and murine spleen, however, it is both more potent and more selective for the constitutive endothelial NOS than Ebs. Unlike Ebs, HOOC-Ebs (0.1-30 microM) causes a concentration-dependent endothelium-independent relaxations of rings of rabbit aorta. The mechanism of this relaxation remains unknown and it is attenuated by glutathione (GSH, 30-300 microM) and N-acetyl-L-cysteine (NAC, 30-300 microM). The vasorelaxant activity of acetylcholine (Ach, 0.1-1 microM) in aortic rings exposed to low concentrations of HOOC-Ebs (0.1-1 microM) or rings exposed to 10 microM HOOC-Ebs after their pretreatment with GSH or NAC (30-300 microM) remained unchanged. The lack of activity of HOOC-Ebs as a NOS inhibitor in intact endothelial cells contrasts the effectiveness of Ebs in this respect.

Inhibition of endothelial nitric oxide synthase by ebselen. Prevention by thiols suggests the inactivation by ebselen of a critical thiol essential for the catalytic activity of nitric oxide synthase.

NO synthase (NOS) is a unique P-450-type enzyme containing both a reductase and a heme domain on a single polypeptide. We show that ebselen [Ebs, 2-phenyl-1,2-benzisoselenazol-3-(2H) one], a nontoxic selenoorganic compound known to break a cysteine thiolate/Fe bond of some of P-450 enzymes, is a relatively selective inhibitor of endothelial isoform of NOS. In rings of rabbit aorta, Ebs irreversibly blocked both the basal as well as acetylcholine- or calcium ionophore A23187-stimulated release of nitric oxide with an IC50 of 6 microM. In homogenates of bovine aortic endothelial cells, Ebs inhibited the activity of NOS, assayed by monitoring conversion of L-[2,3-3H]arginine to L-[2,3-3H]citrulline, with an IC50 of 8.5 microM. The inhibitory action of Ebs was prevented by glutathione, N-acetyl-L-cysteine or dithiothreitol (30-500 microM). The prevention by thiols of Ebs-induced inhibition of NOS suggests that these are competing with a thiol group of NOS that is essential for the catalytic activity of the enzyme. The consequence of the presence of thiols is the "trapping" of Ebs in the form of inactive selenyl sulfides. Consistent with the proposed mechanism of action of Ebs is lack of activity of diselenide of Ebs, which also demonstrates that the action of Ebs is independent of its glutathione peroxidase-like activity. In comparison to endothelial preparations, IC50 values of Ebs for inhibition of soluble isoforms of NOS present in homogenates of porcine cerebellum and of spleens obtained from lipopolysaccharide-treated rats were more than 30-fold higher.