Mechanisms of mutagenicity and toxicity of sodium selenite (Na2SeO3) in Salmonella typhimurium

The mechanisms of selenite toxicity and mutagenicity in S. typhimurium have been characterized. In contrast to previous reports, selenite toxicity was shown not to involve nonspecific incorporation into protein via the sulfur metabolic pathways. Selenite toxicity was, however, shown to involve its ability to act as an oxidizing agent, primarily through reactions with sulfhydryls. Strains which lack glutathione (GSH) are more sensitive to killing by sulfhydryl reagents. The selenite sensitivity of such a mutant was a biphasic phenomenon. The mutant was much more sensitive than a strain which contained GSH at lower selenite concentrations whereas, at higher concentrations, the mutant was much more resistant to selenite. The mechanism of selenite toxicity at lower concentrations in this mutant thus appeared to involve damage to intracellular sulfhydryls. The sensitization to higher doses of selenite by GSH could be explained by the generation of toxic oxygen species. The in vitro reactions of selenite with both cysteine and GSH readily produced H2O2 and O2-. A S. typhimurium strain which overproduces superoxide dismutase (SOD) and catalase was more resistant to high concentrations of selenite, but not killing by the lower doses. Pretreatment of cells with a nonlethal dose of selenite induced the synthesis of proteins which protected the cells from killing by H2O2 or high doses of selenite. Selenite was also a mutagen in the tester strain TA104, in which a number of other oxidizing agents have also been found to be mutagens. These results were consistent with a model in which the reactions of selenite and intracellular thiols with concomitant production of active oxygen species are the primary causal agents of selenite mutagenicity and toxicity in S. typhimurium.     

Glutathione contributes to the efflux of selenium from hepatoma cells

Selenite is a selenium source for selenoprotein biosynthesis in mammalian cells. Although previous studies have suggested the involvement of glutathione (GSH) and/or thioredoxin reductase in selenite metabolism, intracellular selenite metabolism remains largely unknown. Here, we report that GSH depletion did not affect the amount of selenoprotein in Hepa 1–6 cells, suggesting that GSH does not play a central role in the reduction of selenite in selenoprotein biosynthesis. On the other hand, we found that GSH is involved in the efflux of low-molecular-weight selenium compounds from cells, presumably via the formation of selenodiglutathione. Moreover, selenite inhibited the efflux of a fluorescent bimane-GS conjugate that is mediated by ATP-dependent multidrug-resistant proteins, implying the existence of an active transporter for selenodiglutathione. This is the first report demonstrating that GSH plays a role in selenium excretion from cells by forming a GSH-conjugate, which may contribute to the distribution, detoxification, and homeostasis of selenium in the body.     

Effects of selenite on O2 consumption, glutathione oxidation and NADPH levels in isolated hepatocytes and the role of redox changes in selenite toxicity

Isolated hepatocytes incubated with selenite (30–100 μM) exhibited changes in the glutathione redox system as shown by an increase in O₂ consumption, oxidation of glutathione and loss of NADPH. Selenite (50 μM) raised O₂ consumption within the 1 h and induced an partial depletion of thiols with a concomitant increase in oxidized glutathione, as well as a decrease in NADPH levels within 2 h. With 100 μM selenite more pronounced effects were obtained such as a total depletion of thiols. This concentration of selenite also lysed cells within 3 h. Arsenite, HgCl₂ and KCN prevented the increase in O₂ uptake, counteracted loss of thiols and delayed selenite induced lysis. p-Tert-butylbenzoic acid, an inhibitor of gluconeogenesis, decreased selenite dependent O₂ consumption and potentiated the effect on NADPH levels as well as the toxic effect. Finally, methionine further enhanced O₂ consumption by selenite and also delayed loss of thiols and potentiated selenite toxicity. These results indicated that selenite catalyzed a reduction of O₂ in glutathione dependent redox cycles with NADPH as an electron donor. With subtoxic concentrations of selenite (50 μM) there were indications that O₂ reduction was terminated by selenite biotransformation to methylated metabolites. With toxic concentrations of selenite (100 μM) it appeared that O₂ reduction was eventually limited by the capacity of the cell to regenerate NADPH. It is suggested that a depletion of NADPH mediated the observed cytotoxicity of selenite.     

Genotoxicity of selenite in diploid yeast

Selenite, a chemical of industrial importance and also an antimutagenic/anticarcinogenic agent, was tested for mutagenic and recombinogenic effects in 2 diploid yeast strains, Saccharomyces cerevisiae BZ 34 and D7. Selenite induced gene conversion and toxicity in BZ 34 and a variety of genetic events, viz. back-mutation, gene conversion, mitotic crossing-over, aberrant colony formation and also toxicity in the D7 strain. In both strains, the genetic effects of selenite showed a peak and a decline during 5 h of treatment while its toxicity increased marginally during 1-5 h. In the BZ 34 strain, the presence of glutathione (GSH) during selenite treatment greatly enhanced the convertogenic and toxic effects of selenite.     

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems

A pathway for the synthesis of dimethyl seledine from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30 000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO₂ (93% inhibition by 10^?6 M arsenite in the presence of a 20 000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.     

Influence of alloxan-induced diabetes and selenite treatment on blood glucose and glutathione levels in mice

Many clinical studies reported that diabetic patients had lower glutathione contents in erythrocytes or plasma. Recently, selenium, an essential trace element with well-known antioxidant characteristics, has been found to have insulin-mimetic properties. But seldom information is available about the influence of selenium on glutathione changes induced by diabetes mellitus in animals. Therefore, this study was designed to compare the impacts of selenite treatment on glutathione (GSH) levels of blood and tissues such as brain, kidney, liver, spleen and testis in mice. Four groups were used in this study: a control group, a diabetic group, a selenite-treated normal group and a selenite-treated diabetic group. Selenite was administered to the mice for 4 weeks with an oral dose of 2 mg kg(-1) day(-1) by gavage. The blood glucose level, and GSH level in blood and tissues were determined. The results show that the selenite-treated diabetic group had significantly lower blood glucose levels than the diabetic group. Moreover, alloxan-induced diabetes significantly decreased GSH levels in blood, kidney, liver and testis compared to the controls. Selenite treatment of the diabetic mice only improved the GSH levels in liver and brain. On the other hand, selenite administered to the normal mice reduced GSH levels in the liver compared to the controls. In conclusion, this study suggests that selenite treatment of diabetic mice with an effective dose would be beneficial for the antioxidant system of liver and brain although it exerts a toxic effect on the liver of normal mice.     

Characterization and Distribution of Selenite Reduction Products in Cultures of the Marine Yeast Rhodotorula mucilaginosa-13B

Selenite reduction by fungi is a widespread and ecologically significant phenomenon, but previous studies of fungal isolates have not fully characterized the reduction products. We investigated selenite reduction and the distribution of Se in cultures of the marine yeast Rhodotorula mucilaginosa-13B. Strain 13B reduced a substantial amount of selenite to form amorphous elemental selenium particles. Minor volatilization was also observed. Under the aerobic experimental conditions, intact 13B cultures were required for substantial distribution to the solid and volatile phases. This is the first study to report comprehensive microscopic image data and spectroscopic analyses confirming the accumulation of amorphous Se⁰ particles external and internal to cells of a Rhodotorula strain.     

Interaction of glutathione and sodium selenite in vitro investigated by electrospray ionization tandem mass spectrometry

Selenite has been found to be an active catalyst for the oxidation of sulphhydryl compounds, such as glutathione (GSH). Considering the biological importance of GSH oxidation and the implication of sulphhydryl compounds in selenium poisoning and other biological activities, more information on selenite oxidation of GSH in enzyme-free conditions is desirable. Herein, we describe glutathione and sodium selenite simply mixed in aqueous solutions. The interaction products and transient intermediate are identified and characterized using electrospray ionization (ESI) tandem mass spectrometry. In the first step, GSH directly reacts to form diglutathione (GSSG) and unstable selenodiglutathione (GS-Se-SG). Then selenodiglutathione further reacted with remaining GSH to form diglutathione and elemental selenium, Se(0). As the amount of GSSG significantly increased or acidity of the solution increased, the redox potential of glutathione [E(0')(GSSG/2GSH) approximately -250 mV (NHE)] significantly shifted to the positive direction. This makes the GSSG react with elemental selenium formed in the solution, which can be demonstrated by another unstable intermediate ion identified at m/z 418 by mass spectrometry with the elemental composition of [GSS-Se](-). The reaction mechanism between GSH and sodium selenite has been proposed according to the ESI-MS, NMR and UV-vis spectrometric measurements.     

Degradation of methylmercury by selenium

When methylmercury was incubated in the presence of selenite and reduced glutathione (GSH), the mercury which was extracted into benzene under acidic condition decreased gradually with the elapse of time. This decrease was due to the cleavage of mercury-carbon bond of methylmercury. The reaction did not proceed when selenite or GSH was singly added to the reaction mixture. L-Cysteine, 2-mercaptoethanol and sodium sulfide in place of GSH also were effective for decomposition of methylmercury in combination with selenite, but oxidized glutathione (GSSG) and L-cystine were not. This suggests that reduction of selenite is needed for the degradation of methylmercury. Thus, the effect of reduced metabolites of selenite produced by GSH was investigated. Glutathione selenotrisulfide (GSSeSG) requierd GSH for the degradation of methylmercury, whereas H₂Se possessed a strong activity even in the absence of GSH. This may indicate that H₂Se is involved directly in the conversion of methylmercury to inorganic mercury. This phenomenon found in $\text{in vitro}$ experiments is discussed in relation to the biotransformation of methylmercury.     

Influence of dietary selenite on the binding characteristics of rat serum proteins to mercurial compounds

The effect of dietary selenite on the binding characteristics of serum proteins was investigated with rats. In the control serum, the maximal binding of phenylmercuric acetate (PMA) and methylmercuric chloride (MMC) to rat serum protein was approx. 18 and 9 nmoles per mg protein, respectively. The binding of Hg²⁺ was biphasic and it did not reach a maximum at the concentrations used.Selenite treatment caused a reduction in binding capacity of serum proteins to Hg²⁺ and PMA, and an increase in the binding affinities. However, there were no such changes for the binding of MMC. Selenite protection from mercury toxicity, therefore, acts not only via a change in tissue distribution and a change in the formation of seleno-proteins but, also, via a change in the binding characteristics to some mercury compounds. In the case of methylmercury, a different mechanism of protection must exist as the modification of tissue distribution, its binding to subcellular and soluble proteins and the binding characteristics remained equivocal.     

Temporal changes in tissue glutathione in response to chemical form,dose, and duration of selenium treatment

Selenium has been reported to affect glutathione (GSH) concentrations in short-term animal-feeding experiments. Given the central role that this tripeptide plays in maintaining cellular homeostasis, it was hypothesized that perturbations in glutathione metabolism induced by selenium might account for its cancer chemopreventive activity. In the present study, four experiments were conducted in which the effect of acute, short-, or long-term exposure to selenium was assessed. Selenium was provided as either sodium selenite or D,L-selenomethionine. Selenite was observed to induce a biphasic response in total liver GSH. Injected selenium caused an acute reduction in GSH, whereas short-term feeding (up to 8 wk) increased both total GSH and oxidized glutathione (GSSH), an effect that gradually diminished in magnitude with prolonged feeding. Our data suggest that such changes are unlikely to account for the chemopreventive activity of selenium for the following reasons: Perturbations in glutathione metabolism occurred only at doses of selenite that approached toxicity. These doses are higher than what would be required for producing cancer chemoprevention. The transient nature of these changes also contrasts with the need for a continuous supplementation of selenite in suppression of tumorigenesis. Furthermore, selenomethionine was found to have little activity in altering glutathione metabolism, even though it compares favorably with selenite as a cancer chemopreventive agent. Nonetheless, these findings do not discount the possibility that sulfhydryl compounds, such as glutathione, might be used to modify the toxicity and/or enhance the cancer prophylactic activity of selenium compounds.     

Chemical Forms of Selenium in the Metal-Resistant Bacterium Ralstonia metallidurans CH34 Exposed to Selenite and Selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se⁰). We have studied the kinetics of selenite (Se^IV) and selenate (Se^VI) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se⁰ and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se⁰. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se⁰ was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se^IV was detected as a transient species in the first 12 h after selenate introduction, Se⁰ also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

Selenite Reduction by the Thioredoxin System: Kinetics and Identification of Protein-Bound Selenide

Selenite (SeO₃ ^2?) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/autoradiography using ⁷⁵Se-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).     

Selenium as a sulfhydryl redox catalyst and survey of potential selenium-dependent enzymes

The ability of selenium (Se) to act as a redox catalyst is an important factor in understanding the biological function of selenoproteins in addition to that of GSH peroxidase. Selenocystine at micromolar levels exhibited pseudothiotransferase activity by enhancing the reduction of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) by thiols. In contrast, selenite inhibited the reduction of DTNB by thiols. Selenite was more catalytic than selenocystine in the reduction of cytochrome c by GSH, whereas GSH peroxidase was a weak catalyst. Tissues from Se-deficient and Se-supplemented rats were assayed for activities of GSH-thiotransferase, NADPH cytochrome c reductase, formaldehyde dehydrogenase, and a hypothesized GSH cytochrome c reductase. GSH-thiotransferase activity was significantly increased in the liver of Se-deficient rats. No appreciable activity of this enzyme was found in the kidney of rats from either dietary group. No enzymatic activity for cytochrome c reduction by GSH was detected in cytosols, mitochondria, or microsomes from liver and kidney of Se-deficient or Se-supplemented rats. Formaldehyde dehydrogenase was significantly higher in liver cytosols from Se-supplemented rats than from Se-deficient rats. The higher activity was not attributed to Se-containing proteins, but to an unknown small molecular-weight factor. This study did not support the hypothesis that physiological levels of Se may be involved in sulfhydryl-disulfide exchange reactions in vivo, or that selenium may enhance cytochrome c reduction by GSH in vivo.     

Mode of in vitro interaction of mercuric mercury with selenite to form high-molecular weight substance in rabbit blood

Mode of interaction of mercuric mercury and selenite in rabbit blood was investigated in vitro. After the incubation of rabbit blood with 10^?5 M each of ²⁰³HgCl₂ and Na₂⁷⁵SeO₃, the amounts of both ²⁰³Hg and ⁷⁵Se incorporated into erythrocytes were markedly larger than the case where the blood was treated separately with one of these compounds. Most of ²⁰³Hg and ⁷⁵Se distributed into plasma and erythrocytes were found in high-molecular weight substance(s) (HMWS) fractionated by gel filtration at a molar ratio of 1:1. The ²⁰³Hg and ⁷⁵Se in HMWS found in plasma and erythrocytes were hardly diffusable through the erythrocytes membrane. The formation of the HMWS containing mercury and selenium was observed in stroma-free hemolysate incubated with mercuric chloride and selenite, but not in plasma. Addition of reduced glutathione (GSH) to the plasma, however, gave the HMWS as reaction products containing equimolar amounts of mercury and selenium. Further the binding properties of selenium to proteins were studied in the plasma incubated with selenodiglutathione (GSSeSG) or with selenite in the presence of GSH. The results indicated that GSH, a cellular component, is essential for the formation of an active selenium compound from selenite and that the interaction of mercuric mercury and selenite in plasma in the presence of GSH may occur through the other mechanism than the formation of GSSeSG.     

Effects of glutathione and of cysteine on intestinal absorption of selenium from selenite

The influence of glutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of⁷⁵Se-labeled selenite (10 μmol/L) was investigated in rat jejunum. For comparison, the effect ofl-cysteine (1 mmol/L) on in vivo absorption of⁷⁵Se-labeled selenite was also studied. In the in vitro, uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH.l-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlikel-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occuring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since, GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite.     

ROS mediate selenite-induced apoptosis in colon cancer cells

Selenite-induced oxidative stress and its relationship to mitochondrial apoptosis was studied in human adenocarcinoma HT-29 cells. It is shown that selenite induces caspase-dependent apoptosis, which is mediated by mitochondria via released cytochrome c, apoptosis-inducing factor (AIF) and Smac/Diablo. Selenite activates stress kinases p38 and JNK while suppressing reduced glutathione (GSH) and thioredoxin reductase (TrxR) levels, transiently inducing heme oxygenase (HO-1) system as well as reducing Akt expression. Pre-treatment of cells with selected antioxidants and stress kinase inhibitors significantly prevented selenite-induced cell death, thereby implicating oxidative stress as a direct (Bax) as well as indirect (via kinases) cause of HT-29 cells demise. These results thus demonstrate for the first time active proapoptotic and anti-survival effects of selenite in colon cancer cells.     

Inhibition of Anaplerotic Glutaminolysis Underlies Selenite Toxicity in Human Lung Cancer

Large clinical trials and model systems studies suggest that the chemical form of selenium dictates chemopreventive and chemotherapeutic efficacy. Selenite induces excess ROS production, which mediates autophagy and eventual cell death in non‐small cell lung cancer adenocarcinoma A549 cells. As the mechanisms underlying these phenotypic effects are unclear, the clinical relevance of selenite for cancer therapy remains to be determined. The authors' previous stable isotope‐resolved metabolomics and gene expression analysis showed that selenite disrupts glycolysis, the Krebs cycle, and polyamine metabolism in A549 cells, potentially through perturbed glutaminolysis, a vital anaplerotic process for proliferation of many cancer cells. Herein, the role of the glutaminolytic enzyme glutaminase 1 (GLS1) in selenite's toxicity in A549 cells and in patient‐derived lung cancer tissues is investigated. Using [¹³C₆]‐glucose and [¹³C₅,¹⁵N₂]‐glutamine tracers, selenite's action on metabolic networks is determined. Selenite inhibits glutaminolysis and glutathione synthesis by suppressing GLS1 expression, and blocks the Krebs cycle, but transiently activates pyruvate carboxylase activity. Glutamate supplementation partially rescues these anti‐proliferative and oxidative stress activities. Similar metabolic perturbations and necrosis are observed in selenite‐treated human patients' cancerous lung tissues ex vivo. The results support the hypothesis that GLS1 suppression mediates part of the anti‐cancer activity of selenite both in vitro and ex vivo.     

Genetic and Biochemical Evidence for the Involvement of a Molybdenum-Dependent Enzyme in One of the Selenite Reduction Pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.     

Hydrogenase I of Clostridium pasteurianum functions as a novel selenite reductase

Clostridium pasteurianum's hydrogenase I, an important constitutive metabolic enzyme, has been shown to function as a 'novel selenite reductase'. Selenite reductase activity was found to co-purify with hydrogenase I activity; the fold purification and specific activities for these two activities paralleled each other throughout the purification steps. The highly purified hydrogenase I apparent K(m) for the selenite substrate was 0.2 mM. The stoichiometry for the enzymatic reduction of SeO3(2-) to Se(0) via H2 oxidation, was determined to be 2.3:1 (H2:Se(0)), very close to the theoretical ratio of 2:1 for this reduction reaction. Known electron carriers required for hydrogenase I activity were also found to couple its selenite reductase activity, the most efficient one being ferredoxin. The purified hydrogenase I not only reduced selenite but also tellurite, and its selenite activity was completely inhibited by O2 and CuSO4, potent inhibitors of hydrogenase I activity.