Rumen Microorganisms Decrease Bioavailability of Inorganic Selenium Supplements.

Despite the availability of selenium (Se)-enriched trace mineral supplements, we have observed low Se status in cattle and sheep offered traditional inorganic Se supplements. Reasons for this may include inadequate intake or low bioavailability of inorganic Se sources. The objective of this study was to determine whether rumen microorganisms (RMO) alter the bioavailability of Se sources commonly used in Se supplements. Rumen microorganisms were isolated from ewes (n = 4) and incubated ex vivo with no Se (control), with inorganic Na selenite or Na selenate, or with organic selenomethionine (SeMet). Total Se incorporated into RMO and the amount of elemental Se formed were determined under equivalent conditions. Incorporation of Se from Na selenite, Na selenate, or SeMet into RMO was measured as fold change compared with control (no added Se). Incorporation of Se into microbial mass was greater for SeMet (13.2-fold greater than no-Se control) compared with inorganic Se supplements (P = 0.02); no differences were observed between inorganic Na selenate (3.3-fold greater than no-Se control) and Na selenite (3.5-fold greater than no-Se control; P = 0.97). Formation of non-bioavailable, elemental Se was less for RMO incubated with SeMet compared with inorganic Se sources (P = 0.01); no differences were observed between Na selenate and Na selenite (P = 0.09). The clinical importance of these results is that the oral bioavailability of organic SeMet should be greater compared with inorganic Se sources because of greater RMO incorporation of Se and decreased formation of elemental Se by RMO.

Selenoprotein expression and function-selenoprotein W.

Selenoprotein W (SeW) is a small selenoprotein (85 to 88 amino acids) first identified in sheep suffering from selenium deficiency. The levels are highest in muscle, heart (except rodents) spleen and brain. The deduced amino acid sequence has been obtained for mice, rats, monkeys, humans, sheep, pigs, fish and chickens. The sequences of SeW are identical in rats and mice as well as monkeys and humans. In all eight species of animals cysteine is present at residue number 9 and selenocysteine at residue number 13. Residue number 37 is cysteine in six species of animal with fish and chickens as the exceptions. Of those examined, the rodent SeW is the only one containing four cysteines whereas the others contain only two cysteines. Glutathionylaltion has been shown for SeW from rats and monkeys but has not been confirmed for this selenoprotein from the other six animals. The biological function of SeW has not been definitely identified. Evidence has been obtained that it can serve as an antioxidant, responds to stress, involved in cell immunity, specific target for methylmercury, and has thioredoxin-like function.

Microarray analysis of selenium-depleted and selenium-supplemented mice.

Nutritional selenium deficiency is associated with Keshan disease in humans and white muscle disease in ruminant livestock. In this study, mice were fed a selenium-deficient diet for three generations. Female mice from the third depleted generation of these mice were given water containing either no added selenium or 0.1 or 1.0 ppm selenium as sodium selenate; DNA microarrays were used to compare gene expression in the muscle from mice fed the selenium diets to that from mice remaining on the depleted diet. The most prominent expression increases were observed with Ptger2 (a prostaglandin E receptor), Tcrb-V13 (a T-cell receptor beta), Tcf-7 (a T-cell transcription factor), and Lck (lymphocyte protein tyrosine kinase), and the major consistent decrease was Vav2, an oncogene in mice consuming the selenium containing diets.

Deletion analysis of the rodent selenoprotein W promoter.

To identify regulatory elements in the rat selenoprotein W (SeW) promoter, 2090, 1265, 741, and 404 base pair truncations of genomic DNA lying immediately upstream of the SeW coding sequence were cloned into a luciferase reporter vector (pGL3-Basic from Promega, Madison, WI, USA). 3656 and 406 base pair mouse SeW promoter constructs were also compared. SeW promoter activity was assayed in two rat cell lines: L8 muscle cells and C6 brain cells. The SeW promoter was 2-7 times more active (p<0.01) than SV40 promoter. Promoter activity of constructs of the SeW promoter ranging from 200 base pairs to 51 base pairs gradually decreased to zero in brain cells, but fell precipitously to zero in muscle cells. Some truncations stimulated promoter activity, suggesting the full-length promoter may contain binding sites for factors that suppress SeW expression.

Identification of putative transcription factor binding sites in rodent selenoprotein W promoter.

To understand transcriptional regulation of the selenoprotein W (SeW) gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter and antibodies, we observed that specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as to the metal response element (MRE). Although competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore, the SeW gene may be a target for Sp1 whose binding to various regulatory sequences of the SeW promoter may activate or repress the transcription of SeW. The MRE, GRE, AP-1 and LF-A1 sites were also tested but no evidence was obtained for specific binding as indicated by lack of competition with unlabeled probes.

Selenium and its relationship to cancer: an update.

Selenomethionine (Semet) is the major seleno-compound in cereal grains and enriched yeast whereas Se-methylselenocysteine (SeMCYS) is the major seleno-compound in Se-accumulator plants and some plants of economic importance such as garlic and broccoli exposed to excess Se. Animals can metabolize both Semet and SeMCYS. Epidemiological studies have indicated an inverse relationship between Se intake and the incidence of certain cancers. Blood or plasma levels of Se are usually lower in patients with cancer than those without this disorder, but inconsistent results have been found with toenail-Se values and the incidence of cancer. There have been eight trials with human subjects conducted on the influence of Se on cancer incidence or biomarkers, and except for one, all have shown a positive benefit of Se on cancer reduction or biomarkers of this disorder. This is consistent with about 100 small-animal studies where Se has been shown to reduce the incidence of tumours in most of these trials. Se-enriched yeast is the major form of Se used in trials with human subjects. In the mammary-tumour model, SeMCYS has been shown to be the most effective seleno-compound identified so far in reduction of tumours. Several mechanisms have been proposed on the mechanism whereby Se reduces tumours. Even though SeMCYS was shown to be the most effective seleno-compound in the reduction of mammary tumours, it may not be the most effective seleno-compound for reduction of colon tumours.

Selective production of rat mutant selenoprotein W with and without bound glutathione.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x His-tagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10,305 Da) whereas anaerobic conditions produce a glutathione-bound (305 Da) form (10,610 Da). Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography. Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein.

Selenoprotein W gene regulation by selenium in L8 cells.

The effects of selenium on selenoprotein W gene expression were examined in cultured L8 rat skeletal muscle cells. Selenoprotein W contains selenium as selenocysteine in the primary protein structure and levels of this selenoprotein are affected by selenium. Northern blots indicated that there were no significant changes (P < 0.05) in selenoprotein W mRNA levels during cell proliferation and differentiation. Reduction of selenium concentration in the medium decreased the selenoprotein W mRNA levels. Nuclear run-on experiments with isolated L8 nuclei showed the same rate of selenoprotein W mRNA synthesis in cells cultured in either low selenium or selenium supplemented medium, suggesting that the transcription rate of the selenoprotein W gene is independent of selenium. Measurement of the selenoprotein W mRNA half-life in myoblasts treated with the transcription inhibitor, alpha-amanitin, showed that selenoprotein W mRNA levels decreased over time with an estimated half-life of 57 h for cells grown in low selenium medium. Selenium treatment increased the selenoprotein W mRNA half-life 2-fold. These data suggest that selenium stabilizes selenoprotein W mRNA but has no effect on transcription.

Effect of copper, zinc and cadmium on the promoter of selenoprotein W in glial and myoblast cells.

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404 and 1265 bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured C6 (rat glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265 bp (not 404 bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter.

Selenocompounds in plants and animals and their biological significance.

There are several selenocompounds in tissues of plants and animals. Selenate is the major inorganic selenocompound found in both animal and plant tissues. Selenocysteine is the predominant selenoamino acid in tissues when inorganic selenium is given to animals. Selenomethionine is the major selenocompound found initially in animals given this selenoamino acid, but is converted with time afterwards to selenocysteine. Selenomethionine is the major selenocompound in cereal grains, grassland legumes and soybeans. Selenomethionine can also be the major selenocompound in selenium enriched yeast, but the amount can vary markedly depending upon the growth conditions. Se-methylselenocysteine is the major selenocompound in selenium enriched plants such as garlic, onions, broccoli florets and sprouts, and wild leeks.

Urinary selenium and iodine during pregnancy and lactation.

The New Zealand environment is low in selenium and iodine, and is therefore ideally suited for the study of these anionic trace elements. The aim of this study was to determine urinary excretion of selenium and iodine during pregnancy and postpartum as part of an investigation of the influence of pregnancy and lactation on selenium metabolism in women of low selenium status. In a double-blind placebo-controlled study, 35 women in the earliest stages of pregnancy and 17 non-pregnant women were recruited in Dunedin, New Zealand. Eighteen pregnant women received 50 microg selenium as L-selenomethionine, while the others received a placebo daily during pregnancy and 12 months postpartum. The non-pregnant women received the supplement, serving as a positive control. Blood samples and twenty-four hour urine samples were collected monthly during pregnancy and at 3, 6, and 12 months postpartum for analysis of selenium and iodine. Selenium content in plasma and urinary excretion of selenium fell during pregnancy; however, total excretion of selenium was greater during pregnancy than postpartum. Urinary iodine excretion was much lower than reported previously in New Zealand. Due to large intra- and inter-subject variability, no trends in iodide excretion were observed. Factors which influence urinary excretion of selenium include dietary intake, but more closely, plasma concentrations of selenium (which is probably related to total selenium pool), creatinine excretion and therefore lean body mass, and glomerular filtration rate. The exact mechanism and sequence of events remains unclear and future studies incorporating new speciation techniques are necessary.

Selenoprotein W in overexpressed and underexpressed rat glial cells in culture.

Selenium deficiency results in undetectable levels of selenoprotein W (SeW) in muscle but has very little effect upon its content in the brain and thus rat glial cells were studied. Previous work showed that glutathione (GSH) is bound to SeW and this study was undertaken to elucidate its possible antioxidant functions. Full length cDNA of SeW was cloned to inducible LacSwitch expression vector and stably transfected in C6 rat glial cells. After induction, SeW and its mRNA were expressed 22- and 11-fold higher respectively than control. The cDNA coding region of SeW was cloned to the vector in the antisense direction and stably transfected in C6 cells for underexpression of the protein. After induction, SeW expression was reduced to 20% of the control cells. Glutathione peroxidase activity and GSH levels were not significantly different between induced and control cells. There was a greater survival rate of overexpressed than control cells when incubated with 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH), suggesting SeW possibly has an antioxidant function.

Cancer-protective properties of high-selenium broccoli.

Selenium (Se) from high-Se garlic reduces the incidence of chemically induced mammary tumors, and Se from high-Se broccoli reduces colon cancer. However, the ability of Se from high-Se broccoli to protect against mammary cancer has not been tested. Also, the sprout form of broccoli contains many secondary plant compounds that are known to reduce cancer risk, but the anticarcinogenic activity of broccoli sprouts has not been investigated. The present studies examined the ability of high-Se broccoli or high-Se broccoli sprouts to protect against chemically induced mammary or colon cancer. In one experiment, Sprague--Dawley rats that consumed diets containing 3.0 microg of Se/g supplied as high-Se broccoli had significantly fewer mammary tumors than rats fed 0.1 microg of Se as selenite with or without the addition of regular broccoli. In the second experiment, Fisher F-344 rats fed 2.0 microg of Se/g of diet supplied as either high-Se broccoli florets or high-Se broccoli sprouts had significantly fewer aberrant colon crypts than rats fed 0.1 or 2 microg of Se/g of diet supplied as selenite with or without the addition of low-Se broccoli. These data demonstrate that the cancer-protective effect of Se in high-Se broccoli extends to mammary cancer and the protective forms of broccoli against colon cancer include high-Se broccoli sprouts.

Glutathione peroxidase activity and selenoprotein W levels in different brain regions of selenium-depleted rats(1).

Previous studies in selenium (Se)-depleted sheep and rats showed that selenoprotein W (SeW) levels decreased in all tissues except brain. To further investigate this depletion in different parts of the brain, second generation Se-depleted rats were used. Dams consumed a Se-deficient basal diet during gestation and lactation, and deficient rats were obtained by continuation on the same diet. Control rats were fed a diet with 0.1-mg Se/kg diet after weaning. Glutathione peroxidase (GPX) activities were measured for comparative purposes to SeW levels. GPX activity in muscle, skin, spleen, and testis increased about 4-fold with Se repletion and reached a plateau after 6 or 10 weeks, but GPX activity decreased to almost one tenth of the original activity with continuous Se depletion. In contrast, GPX activities increased, rather than declined, in various brain regions (cortex, cerebellum, and thalamus) with time of feeding the deficient diet. An experiment with first generation rats, however, indicated that GPX activity was significantly lower in these three brain regions from rats fed the deficient diet as compared to rats fed the supplemented diet. SeW levels in skin, spleen, muscle, and testis were undetectable in weanling rats, but became detectable after 6 weeks of Se repletion. In contrast, the expression of SeW in cortex, cerebellum, and thalamus was not significantly affected by Se depletion, but increased SeW levels occurred only in thalamus with Se supplementation. The results with GPX using first and second generation rats suggest that there are "mobile" and "immobile" GPX fractions in the brain.

Selenium and the brain: a review.

Similar to other tissues selenium from selenomethionine is deposited in the brain at higher concentrations than selenium in other forms. Vitamin E has a greater effect than selenium in reducing lipid peroxidation in various brain regions. Selenium does not have as great effect on glutathione peroxidase (GPX) activity in the brain as in most other organs. Prolonged selenium and iodine deficiencies will compromise thyroid hormone homeostatus in the brain and this is due to changes in deiodinases activities and lipid peroxidation. Even though selenium deficiency results in reduced GPX activity and selenium content in the brain, there is no reduction in thioredoxin reductase activity or selenoprotein W levels. Selenoprotein P is taken up in greater amounts by the brain but not by other organs in selenium deficient animals, suggesting a critical function of this selenoprotein in this organ. Selenium will influence compounds with hormonal activity (and neurotransmitters) in the brain, and this is postulated to be the reason selenium affects moods in humans and behavior in animals. Even though selenium counteracts the neurotoxicity of mercury, cadmium, lead and vanadium, it causes them to accumulate in the brain, presumably in a nontoxic complex.

Tumorigenesis, metabolism, speciation, bioavailability, and tissue deposition of selenium in selenium-enriched ramps (Allium tricoccum).

Ramps (Allium tricoccum) were grown either in a mixture of vermiculite and peat moss or hydroponically with various concentrations of selenium as sodium selenate. The concentrations used were from 30 to 300 mg of selenium/kg of vermiculite-peat moss or from 10 to 120 mg/L in the hydroponic solutions. Levels as high as 784 mg of selenium/kg were obtained in the ramp bulbs when grown with high levels of selenium in the vermiculite-peat moss, and up to 600 mg of selenium/kg was obtained hydroponically. The predominant form of selenium in the ramp bulbs at all concentrations of selenium was Se-methylselenocysteine, with lower amounts of selenate, Se-cystathionine, and glutamyl-Se-methylselenocysteine. There was a approximately 43% reduction in chemically induced mammary tumors when rats were fed a diet with Se-enriched ramps. Dietary Se-enriched ramps for rats did not result in excessive tissue selenium accumulation or undesirable side effects. Bioavailability studies with rats indicated that selenium in ramps was 15-28% more available for regeneration of glutathione peroxidase activity than inorganic selenium as selenite. Therefore, Se-enriched ramps appear to have potential for the reduction of cancer in humans.

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.

Selenium supplementation of Chinese women with habitually low selenium intake increases plasma selenium, plasma glutathione peroxidase activity, and milk selenium, but not milk glutathione peroxidase activity.

Twenty-one pregnant women living in Xichang County, China, a selenium-deficient area, were divided into two groups and given either a placebo (n = 10) as yeast or selenium-enriched yeast tablets (n = 11) to provide 100 microg selenium per day. This supplementation was begun the last trimester of pregnancy and continued for 3 months after parturition. Plasma selenium levels and glutathione peroxidase (GPX) activity steadily declined in supplemented women, but a curvilinear response occurred in milk selenium and GPX activity in both supplemented and deficient women and in plasma selenium and GPX activity in deficient women. The milk selenium levels were higher in supplemented women but there were no differences in the milk GPX activity between the two groups of women. The plasma alpha-tocopherol concentrations declined after parturition in both groups but no differences were found between the two groups of women. Plasma thiobarbituric acid reactive substances declined in supplemented women but showed a curvilinear response in unsupplemented women, suggesting peroxidative stress in these women. GPX, selenium, and peroxidative responses in plasma and milk following parturition is advocated as a new method to assess selenium status of lactating women.

Selenoprotein W accumulates primarily in primate skeletal muscle, heart, brain and tongue.

The human selenoprotein W coding region with the selenocysteine codon (TGA) changed to a cysteine codon (TGT) was fused to six histidine codons (at its 3' end), cloned into a prokaryotic expression vector (pTrc99a), and the corresponding mutated selenoprotein W was expressed in bacteria. The protein was purified by Ni-NTA agarose column and reverse phase HPLC. Polyclonal antibodies raised against this protein were used in Western blots to determine tissue distribution of selenoprotein W from rhesus monkeys fed a commercial chow. Selenoprotein W was found in several tissues with highest amounts in skeletal muscle and heart (muscle 6 fold greater than liver) and lowest levels in liver, but selenium concentrations were highest in kidneys (10 fold greater than muscle) and lowest in skeletal muscle. Northern blots using a human selenoprotein W cDNA probe indicated that mRNA levels were highest in monkey skeletal muscle and heart (2-2.5 fold greater than in liver), which is similar to the pattern found with a human multiple tissue Northern blot. However, as in the monkey, selenium concentrations were highest in human kidney and lowest in skeletal muscle and heart. Thus, selenoprotein W protein levels correlated with selenoprotein W mRNA levels but not with tissue selenium concentrations.

Selenoprotein W: a review.

Purification of selenoprotein W (Se-W) from rat and monkey muscles was shown to exist in multiple forms: with or without reduced glutathione and/or a 41-Da moiety (identity still unknown). TGA is located at coding position 13 in Se-W complementary DNA (cDNA) from all five species studied (rats, mice, sheep, human and monkey). TGA is also the stop codon in the rodents and sheep cDNA, but TAA is the stop codon in primates. There is an 80% homology of the nucleotide sequence in the coding region among the five species of animals, and the predicted amino acid sequences are 83% identical (rodents identical and primates identical). Se-W levels are highest in muscle, heart and brain from sheep and primates, but very low in rodent hearts. Studies with tissue cultures of muscle and brain cells indicated that selenium influenced Se-W levels. Although the metabolic function of Se-W is unknown, preliminary data suggest that it has an antioxidant function.