Chemopreventive activity of selenocysteine prodrugs against tobacco-derived nitrosamine (NNK) induced lung tumors in the A/J mouse

Prodrugs of L-selenocysteine have potential utility in cancer chemoprevention. This study reports the efficacy of three selenazolidine-4(R)-carboxylic acids, (2-unsubstituted, 2-oxo, and 2-methyl derivatives; SCA, OSCA, and MSCA, respectively) against tobacco-related lung tumorigenesis in a mouse model. Seven days after initiation of an AIN-76A diet supplemented with sodium selenite (5 ppm Se), L-selenomethionine (3.75 ppm Se), Se-methyl-L-selenocysteine (3 ppm Se), L-selenocystine (15 ppm Se), SCA (15 ppm Se), OSCA (15 ppm Se), or MSCA (15 ppm Se), mice received 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; 10 micromol, i.p.). After an additional 16 weeks on the diets, two compounds, OSCA and selenocystine, significantly reduced lung adenoma multiplicity from 7.2 tumors per mouse in the NNK group to 4.5 and 4.6 tumors per mouse, respectively. Neither selenium concentration nor glutathione peroxidase activity in either RBCs or liver served as surrogate indicators of tumor reduction. Hepatic selenium levels were significantly elevated by all selenium-containing compounds except Se-methyl-L-selenocysteine and SCA; RBC selenium levels by all except sodium selenite and MSCA. With the exception of L-selenomethionine, RBC glutathione peroxidase activity was increased along with the elevated selenium levels. Hepatic glutathione peroxidase activity was elevated by all Se-compounds except SCA. The two compounds showing significant tumor reduction (OSCA and selenocystine) were the only two compounds that showed ubiquity of changes, elevating both selenium levels and GPx activity in both liver and RBC.     

Characteristics of selenazolidine prodrugs of selenocysteine: toxicity, selenium levels, and glutathione peroxidase induction in A/J mice

We have previously reported the synthesis and characterization of two new classes of selenazolidine-4(R)-carboxylic acids (2-oxo and 2-methyl-SCAs) (OSCA and MSCA, respectively), as well as the "parent" compound, selenazolidine-4(R)-carboxylic acid (SCA, selenaproline). These compounds were designed as prodrugs of L-selenocysteine with potential application in cancer chemoprevention or other clinical uses. We will be exploring the chemopreventive activity of the new compounds in the well-established A/J mouse model of tobacco-induced lung carcinogenesis. The objectives of the present study were to investigate several fundamental biochemical endpoints after selenazolidine administration compared with other selenium-containing agents. Groups of mice were fed either AIN-76A diet alone or the diet supplemented with the following selenium compounds (ppm Se): sodium selenite (5), L-selenomethionine (3.75), L-selenocystine (15), Se-methyl-L-selenocysteine (3), MSCA (5, 10, or 15), OSCA (5, 10, or 15), or SCA (5, 10, or 15). After 28 days of supplementation, toxicity of the selenazolidines was not evident, as measured by outward appearance and behavior, body and organ weight changes, and histological evaluation of liver and lung tissue. Select treatment groups showed significant increases in selenium levels in blood and tissues. Increased activity of selenium-dependent glutathione peroxidase (GPx) in blood and liver illustrated that the selenazolidines provided a source of biologically-available selenium.     

Incorporation of selenium from selenite and selenocystine into glutathione peroxidase in the isolated perfused rat liver

The erythrocyte-free, isolated perfused rat liver was used to study the incorporation of selenium into glutathione peroxidase. Gel filtration and ion exchange chromatography of liver supernatant demonstrated ⁷⁵Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced ⁷⁵Se incorporation from L[⁷⁵Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting ⁷⁵Se incorporation from [⁷⁵Se]selenite. These results indicate that selenide and selenite are more readily metabolized than is selenocysteine to the immediate selenium precursor used for glutathione peroxidase synthesis, and suggest a posttranslational modification at another amino acid residue, rather than direct incorporation of selenocysteine, as the mechanism for formation of the presumed selenocysteine moiety of the enzyme.     

The bioavailability of various selenium compounds to a marine wading bird

The uptake of dietary selenium (about 3.5 mg/kg AF dry wt) as selenomethionine, selenocystine, selenite, selenate, and fish selenium in the plasma and red blood cells (RBC) of the oystercatcher has been investigated. The birds received the various selenium compounds subsequently, for at least 9 wk. After dietary supplementation of selenocystine, selenite, and selenate, plasma selenium was about 350 μg/L and RBC selenium 2.1 mg/kg dry wt. After supplementation of selenomethionine, the plasma concentration increased to 630 μg/L, and the RBC concentration to 4.1 mg/kg dry wt. When the fodder contained 3.1 mg/kg fish Se, an average plasma and RBC concentration of 415 μg/L and 14.4 mg/kg dry wt, respectively, was measured. The maximal increase of the selenium concentration in the plasma was attained at first sampling, 14 d after a change in dietary selenium (selenomethione or fish Se); the uptake seemed to be a concentration-regulated process. RBC concentrations (γ in mg/kg dry wt) increased with time (X in d) according toY=a?be^?cX . Fifty percent of the total increase was attained within 17d, suggesting that diffusion into the RBC played a role. The selenium concentration in the plasma was positively correlated with the (fish) Se concentration in the fodder; the RBC concentration (60 d after the change in diet) was positively correlated with the plasma concentration. When the diet contained fish Se, the blood selenium concentrations of the captive birds were similar to the concentrations measured in field birds. Fish Se is a yet undetermined selenium compound. The present experiment showed that fish Se differed from selenomethionine, selenocystine, selenite, or selenate in uptake from the food and uptake in the RBC.     

In vitro synthesis of glutathione peroxidase from selenite. Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase.     

Effects of metal ions and selenoamino acids on induction of glutathione peroxidase in mouse neuroblastoma

The induction of glutathione peroxidase in mouse neuroblastoma cells by selenite is enhanced by equimolar amounts of arsenate, arsenite, molybdate, chromic or dichromate ions. At equimolar selenium concentration, selenite, selenocystine and selenomethionine induced glutathione peroxidase activities having the ratios 4:4:1. Protein synthesis inhibitors prevented the induction of glutathione peroxidase by selenite indicating that de novo protein synthesis is required.     

Effect of selenium compounds and thiols on human mammary tumor cells

The effect on cell viability and growth rate of sodium selenite, selenocystine, sodium selenate, and selenomethionine at selenium concentrations of 6.25 and 12.5 uM was studied in vitro on cells of the human mammary tumor cell line HTB123/DU4475. Selenite and selenocystine affected both cell viability and growth rate of the tumor cells at these selenium concentrations. Selenite and selenocystine decreased intracellular glutathione concentrations, but did not affect tumor cell glutathione peroxidase activity. After six days of exposure to either selenate or selenomethionine, the viability of tumor cells remained stable, but cell growth, as measured by numbers of cells, was retarded. Neither selenate nor selenomethionine produced changes in concentrations of intracellular glutathione. The toxic effect of selenite on tumor cells was enhanced by addition of 0.25 mM glutathione to the growth medium. Preincubation of the tumor cells with 62.5 uM buthionine sulfoximine decreased cellular glutathione to 15% of controls at 24 h and enhanced the toxicity of selenite toward the tumor cells. Glutathione, 2-mercaptoethanol, and L-cysteine were all toxic to the tumor cells in a dose-dependent manner.     

In vitro synthesis of glutathione peroxidase from selenite Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [⁷⁵Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [⁷⁵Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [³H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and ⁷⁵Se was incorporated from [⁷⁵Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the ⁷⁵Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [⁷⁵Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase     

Selenium and Glutathione Peroxidase Levels in Lambs Receiving Feed Supplemented with Sodium Selenite or Selenomethionine

Twenty-one 6 months old female lambs were divided into 7 groups and fed a basal diet containing 0.13 mg Se/kg. The basal diet was further supplemented with 0, 0.1, 0.5 or 1.0 mg Se/kg either as sodium selenite or as selenomethionine, and was fed for 10 weeks. Both feed additives produced an increase in the selenium concentration in the tissues analysed. Significant correlations were found between the concentrations of selenomethionine or sodium selenite added to the feed and the subsequent tissue levels. However, the selenium levels seemed to plateau at approximately 0.5 mg Se/kg of supplemented sodium selenite. The total glutathione peroxidase (GSH-Px) activity of the tissues increased when the selenium supplementation increased from 0 to 0.1 mg/kg for both selenium compounds. With further increase in selenium supplementation the GSH-Px activity in the tissues plateaued except in the blood where the activity continued to rise with increasing selenomethionine supplementation. The selenium dependent GSH-Px activity in the liver rose with increasing selenomethionine supplementation, but approached a plateau when 0.1 mg Se/kg as sodium selenite was added to the feed. The selenium concentration in whole blood responded more rapidly to the selenium supplementation than did GSH-Px activity. The experiment indicates that the optimal selenium concentration in the feed is considerably higher than 0.1 mg Se/kg, and that selenium levels of 1.0 mg/kg in the feed do not result in any risk for the animals or the consumers of the products.     

Microbial Transformations of Selenium

Resting cell suspensions of a strain of Corynebacterium isolated from soil formed dimethyl selenide from selenate, selenite, elemental selenium, selenomethionine, selenocystine, and methaneseleninate. Extracts of the bacterium catalyzed the production of dimethyl selenide from selenite, elemental selenium, and methaneseleninate, and methylation of the inorganic Se compounds was enhanced by S-adenosylmethionine. Neither trimethylselenonium nor methaneselenonate was metabolized by the Corynebacterium. Resting cell suspensions of a methionine-utilizing pseudomonad converted selenomethionine to dimethyl diselenide. Six of 10 microorganisms able to grow on cystine used selenocystine as a sole source of carbon and formed elemental selenium, and one of the isolates, a pseudomonad, was found also to produce selenide. Soil enrichments converted trimethylselenonium to dimethyl selenide. Bacteria capable of utilizing trimethylselenonium, dimethyl selenide, and dimethyl diselenide as carbon sources were isolated from soil.     

A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

Interactive effects of organic and inorganic selenium with cadmium and mercury on spermatozoal oxygen consumption and motility in vitro

The effects of cadmium (Cd), mercury (Hg), and three different chemical forms of selenium (Se) (selenite, selenocystine, and selenomethionine) on ram spermatozoal motility and oxygen consumption in vitro were studied over a 4-mo period. Concentrations of 10(-6) to 10(-2) M Cd and Hg were injurious to spermatozoa as indicated by depressed motility and reduced oxygen uptake. Equimolar concentrations of Se as selenite, selenocystine, or selenomethionine counteracted the toxicity of Cd and Hg at low concentrations (10(-5) and 10(-6) M) but not at higher concentrations (10(-4) to 10(-2) M). Gel filtration (Sephadex G-75) of seminal plasma and solubilized sperm prepared from semen incubated with Cd or Hg with or without the Se compounds revealed that Cd or Hg eluted with the void volume proteins in all treatments. Incubation of ram spermatozoa with any of the three chemical forms of Se ranging from 10(-6) to 2.5 X 10(-5) M significantly improved sperm motility and oxygen consumption.     

The metabolism of selenite and selenomethionine in mouse fibroblasts grown in tissue culture

Recent reports have provided evidence that selenium is an essential growth factor for cells grown in tissue culture. The aim of the work reported in this paper was to evaluate mouse fibroblasts as a model for the study of selenium metabolism in mammalian cells. The results showed that transformed mouse lung fibroblasts grown in media containing 9.1% bovine serum did not show a growth response to added selenium as selenite over the range of 10–1000 ng/mL. Uptake of selenium by cells was a direct function of the selenium concentration in the medium. The rate of uptake varied with the time of exposure of the cells to the selenium, and to the form of selenium in the medium. Experiments using radioactive selenium showed that⁷⁵Se from selenite was rapidly absorbed into the cell wall, but slowly incorporated into the soluble protein fraction.⁷⁵Se from selenomethionine was more slowly absorbed into the cells, but once inside, it became rapidly incorporated into soluble cytoplasmic proteins. Cell fractionation and gel filtration procedures established that⁷⁵Se from selenite was rapidly incorporated into glutathione peroxidase (GSHpx), whereas⁷⁵Se from selenomethionine was initially incorporated into a wide spectrum of proteins and only after a longer period did the⁷⁵Se peak become associated with GSHpx. These findings suggest fundamental differences exist in the manner in which mammalian cells initially absorb and metabolize different selenium compounds.     

Effects of selenium supplementation on diet-induced obesity in mice with a disruption of the selenocysteine lyase gene

BACKGROUNDThe amino acid selenocysteine (Sec) is an integral part of selenoproteins, a class of proteins mostly involved in strong redox reactions. The enzyme Sec lyase (SCLY) decomposes Sec into selenide allowing for the recycling of the selenium (Se) atom via the selenoprotein synthesis machinery. We previously demonstrated that disruption of the Scly gene (Scly KO) in mice leads to the development of obesity and metabolic syndrome, with effects on glucose homeostasis, worsened by Se deficiency or a high-fat diet, and exacerbated in male mice. Our objective was to determine whether Se supplementation could ameliorate obesity and restore glucose homeostasis in the Scly KO mice.METHODSThree-weeks old male and female Scly KO mice were fed in separate experiments a diet containing 45 % kcal fat and either sodium selenite or a mixture of sodium selenite and selenomethionine (selenite/SeMet) at moderate (0.25 ppm) or high (0.5–1 ppm) levels for 9 weeks, and assessed for metabolic parameters, oxidative stress and expression of selenoproteins.RESULTSSe supplementation was unable to prevent obesity and elevated epididymal white adipose tissue weights in male Scly KO mice. Serum glutathione peroxidase activity in Scly KO mice was unchanged regardless of sex or dietary Se intake; however, supplementation with a mixture of selenite/SeMet improved oxidative stress biomarkers in the male Scly KO mice.CONCLUSIONThese results unveil sex- and selenocompound-specific regulation of energy metabolism after the loss of Scly, pointing to a role of this enzyme in the control of whole-body energy metabolism regardless of Se levels.     

Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis

The cancer chemopreventive effect of selenium cannot be fully accounted for by the role of selenium as a component of the antioxidant enzyme glutathione peroxidase, which suggests that chemoprevention occurs by another mechanism. Several studies have shown that thiol oxidation and free radical generation occur as a consequence of selenium catalysis and toxicity. In the present study, we evaluated three different selenium compounds; selenite, selenocystamine, and selenomethionine to determine the relative importance of the prooxidative effects of these compounds with regard to their ability to induce apoptosis. The experimental results suggest that, in addition to supporting an increased activity of glutathione peroxidase, an antioxidant function that the three selenium compounds did with equal efficacy, catalytic selenite, and selenocystamine generated 8-hydroxydeoxyguanosine DNA adducts, induced apoptosis and were found to be cytotoxic in mouse keratinocytes. The noncatalytic selenomethionine was not cytotoxic, did not generate 8-hydroxydeoxyguanosine adducts and did not induce cellular apoptosis at any of the selenium concentrations studied. In keratinocytes, apoptosis may be initiated by superoxide (O₂^•−) and oxidative free radicals that are generated by selenite and selenocystamine, but not by selenomethionine.     

The Raman spectra of selenomethionine and selenocystine

The trace element selenium is known to be a part of the enzyme glutathione peroxidase (glutathione-hydrogen peroxide oxidoreductase, E.C.1.11.1.9). Studies have shown that selenium in the enzyme exists in at least two forms or oxidation states. It is probable that selenium has been incorporated into the enzyme as the selenocysteine amino acid. In the present study, the Raman spectra of selenocystine and selenomethionine have been obtained, structural assignments have been verified, and the behavior of the two selenoamino acids have been monitored under varying conditions of oxidative stress. The assignments will assist in the interpretation of the spectrum of the actual enzyme.     

Selenium metabolism in Escherichia coli

Escherichia coli will reduce selenite (SeO 3 2- ) andselenate (SeO 4 2- ) to elemental selenium Se 0 . Seleniumwill also become incorporated intoproteins as part of the amino acids selenocysteine or selenomethionine.The reaction of selenitewith glutathione produces selenodiglutathione (GS-Se-GS). Selenodiglutathioneand itssubsequent reduction to glutathioselenol (GS-SeH) are likely the key intermediatesin the possiblemetabolic fates of selenium. This review presents the possible pathwaysinvolving selenium in E. coli. Identification of intermediates and potentialprocesses from uptake of the toxic oxyanions through to theirdetoxification will assist us inunderstanding the complexities of metalloid oxyanion metabolism in thesebacteria.     

Chemical forms of selenium present in rat and ram spermatozoa

In vivo and in vitro studies were conducted to investigate the chemical forms by ion-exchange chromatography of selenium (Se) present in rat and ovine spermatozoa. After injection with ⁷⁵Se-selenite, the form of ⁷⁵Se in rat sperm was selenocysteine, but selenocysteine and selenomethionine (SeMet) were present in ovine sperm. Presumably, synthesis of SeMet by rumen microbes are responsible for its presence in ovine sperm. In vitro incubation of ram sperm with selenocysteine or SeMet produced no changes, but incubation with selenite produced a compound that eluted one fraction before SeMet from the ion-exchange column. After treatment of this fraction with mercaptoethanol, it eluted in a later fraction upon rechromatography, suggesting it to be selenodicysteine. This compound is apparently formed because of high levels of cysteine in semen. Cysteine, reduced glutathione, and oxidized glutathione were also found in semen. The significance of the results is discussed.     

Antioxidant associated chemoprevention by selenomethionine in murine tumor model

Effectiveness of selenium in different forms like sodium selenite, selenocysteine and selenomethionine has been compared in four different doses, namely 4, 6, 8 and 10 ppm of each, in terms of their bioavailability and prolongation of survival of Dalton's lymphorna (DL) bearing mice. Selenomethionine, at a dose of 8 ppm, was found to be the most bioavailable and least cytotoxic form that was capable of increasing the life span of the tumour bearing hosts maximally (almost two-fold). Benef iciality of selenomethionine has also been studied by observing continuous changes brought about by this compound on the glutathione (GSH) level, glutathione peroxidase (GPx) activity and extent of lipid peroxidation in the hepatic tissue of the tumour bearing hosts, which are indispensable for a cell to function normally and are found to exhibit significantly altered behaviour in neoplastic cells. Selenomethionine caused the maintenance of high steady state GSH level and a normal GPx activity during the fist phase of tumour growth. It also controlled lipid peroxidation during the first 15-20 days following tumour transplantation. These conditions helped in the maintenance of intracellular redox balance, cellular integrity and metabolic rhythms of cells in DL bearing mice receiving selenomethionine.Affiliation