75Se Uptake Research Articles

All Regions of Mouse Brain Are Dependent on Selenoprotein P for Maintenance of Selenium

The brain and testis retain selenium better than other tissues during selenium deficiency. Studies of mice with selenoprotein P (Sepp1) deleted (Sepp1−/− mice) showed that brain and testis selenium levels are largely dependent on Sepp1. Therefore, we examined tissue selenium in mice fed varying amounts of selenium and in Sepp1−/− mice to characterize better the role(s) of Sepp1. Mice were fed a selenium-deficient diet for 8 wk supplemented with selenium as selenite from none to 0.25 mg/kg diet and tissue selenium was measured. Brain and testis maintained their selenium better than did liver, kidney, and muscle when dietary selenium was limiting but testis selenium fell sharply in the group fed the deficient diet. Brain retained its selenium well, even in the group fed the deficient diet. After intravenous injection of 75Se-Sepp1 into Sepp1−/− and Sepp1+/+ mice, qualitative differences between brain and testis 75Se uptake were noted, further suggesting differences in their uptake of selenium from Sepp1. Finally, selenium was measured in brain regions of Sepp1−/− and Sepp1+/+ mice fed the diet supplemented with 1 mg selenium/kg and Sepp1+/+ mice fed the deficient diet. Deletion of Sepp1 and selenium deficiency each lowered selenium a similar amount in cortex, midbrain, brainstem, and cerebellum. Selenium in the hippocampus was lowered by deletion of Sepp1 but not by selenium deficiency. These results suggest that Sepp1 is more important for maintaining selenium in the hippocampus than in other brain regions. They also confirm the position of the brain at the apex of the organ selenium hierarchy.

Bioconcentration Mechanism of Selenium by a Coccolithophorid, Emiliania huxleyi

We investigated the uptake and bioconcentration of the essential element selenium by a coccolithophorid, Emiliania huxleyi, using [75Se]selenite. The time course of 75Se uptake showed a biphasic pattern, namely a primary phase and a subsequent secondary phase. The primary and secondary phases are due to a rapid selenite uptake process that attained a stationary level within 2 min and a slow Se-accumulation process that continued at a constant rate for 4 h or longer, respectively. Kinetic analysis revealed that the selenite uptake process consists of two components, one saturable and one linearly related to substrate concentration. The Km of the saturable component was 29.8 nM selenite; the uptake activity of this component was suppressed by inhibitors of ATP biogenesis, suggesting that selenite uptake is driven by a high-affinity, active transport system. During a 6-h incubation of cells with [75Se]selenite, 70% of the intracellular 75Se was incorporated into low-molecular-mass compounds (LMCs), and 17% was incorporated into proteins, but [75Se]selenite was barely detectable. A pulse-chase experiment demonstrated that the 75Se that had accumulated in LMCs was transferred into proteins. When the syntheses of amino acids and proteins were each separately inhibited, 75Se incorporation into LMCs and proteins was decreased. These results suggest that E. huxleyi rapidly absorbs selenite, filling a small intracellular pool. Then, Se-containing LMCs are immediately synthesized from the selenite, creating a pool of LMCs that are then metabolized to selenoproteins.

Kinetic analyses of mitochondrial 75selenium uptake in Trigonella foenum-graecum seedlings exposed to selenium and mimosine.

Uptake of (75Se) added in vitro was followed in mitochondria isolated from Trigonella foenum-graecum seedlings grown under different Se status (0.5-1.0 ppm) and with added mimosine (0.1 mM). Uptake of 75Se followed with added Na2 75SeO3 upto 20 microM in the medium was nonlinear in all the groups. Kinetic analyses of the uptake of 75Se for 1 min were carried out for all the groups. The results indicated a cooperative effect during Se transport. Graphical analyses using the Hill plot and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Scatchard plots were biphasic, suggesting the probable presence of two classes of binding sites. The presence of succinate or ATP in the incubation medium inhibited 75Se uptake by 40%. Studies with mitochondrial respiratory inhibitors indicated the uptake to be energy independent. A decrease in the uptake of 75Se by 40% effected by HgCl2, N-ethyl maleimide, and iodoacetate confirmed the interaction of active thiols in the process. The present study confirms the controlled nature of 75Se uptake by plant mitochondria.

Mitochondrial selenium-75 uptake and regulation revealed by kinetic analysis.

Uptake of Na2(75)SeO3 by mitochondria of the larvae of the insect C. cephalonica reared at different dietary selenium (Se) levels revealed: 1. A proportional increase in the uptake with externally added Na2(75)SeO3 in concentrations upto 25.32 microM; and 2. At each added selenite concentration, an increase up to 60 min, with linearity up to 15-30 min. A differential affinity for Na2(75)SeO3 was elicited in the mitochondrial protein fractions of different dietary Se groups and correlated well with the pattern and the ratio of distribution of incorporated 75Se in protein to nonprotein fractions. Kinetic studies on 75Se uptake by whole mitochondria negated passive diffusion of selenite and revealed a trend of negative cooperativity, confirmed by Hill and Scatchard plots. Half saturation value was estimated to be approx 13 nmole Se/mg mitochondrial protein. Scatchard plot for 75Se uptake was biphasic and the high affinity binding sites were estimated to be around 5 nmole/mg mitochondrial protein. Calculated dissociation constants revealed maximal affinity for 75Se in the 1.5 ppm group (KSe 0.0034 nM) and minimal in the basal group (KSe 0.007 nM). In the mitochondria of all the three dietary Se groups, the estimated low affinity sites amounted to be 15-19 nmole/mg mitochondrial protein. Inherent Se in the mitochondria of the high Se group positively enhanced the incorporation of 75Se in the mitochondrial protein fraction. About 20-30% of the total uptake was indicated to be energy linked as revealed by studies with respiratory inhibitors. Addition of sulfite and sulfate (5-25 microM) in the medium, inhibited 75Se uptake by 35-55%, suggestive of the involvement of the dicarboxylate port. Thiol interactive 75Se uptake was confirmed by the inhibition mediated by mersalyl and NEM up to 50-70%. The study revealed thiol-selenite interactions of metabolic significance during selenite uptake.

Kinetic analysis of 75selenium uptake by mitochondria of germinating Vigna radiata of different selenium status.

Earlier studies in our laboratory demonstrated the beneficial role of Se in Vigna radiata, a Se-deficient legume, during germination, as reflected in growth-related parameters and specific uptake of 75Se. Uptake of Na2(75)SeO3, added in vitro by mitochondria isolated from seedlings germinated in control (without Se), and Se-supplemented groups (0.5, 1.0, and 2.0 ppm Se) indicated a proportional increase in the uptake with added Na2(75)SeO3, in concentrations up to 25 microM. The uptake of 75Se, increased linearly with time up to 15 min and a definite efflux followed at 30 min. The results were indicative of cooperative effects during Se transport. Kinetic analyses of the uptake of 75Se during time intervals of 15 and 30 min were carried out both in the whole mitochondria and the mitochondrial protein fractions. Graphical analyses using Lineweaver-Burk plot, Hill plot, log [v] vs log [A] and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Hill coefficient (nH) values were estimated to be around 0.7-0.8. Scatchard plots for 75Se uptake were biphasic, suggesting the probable presence of two classes of binding sites. The number of high and low affinity binding sites were estimated to be around 4-7 and 26-30 nmol/mg protein, respectively. Studies with mitochondrial respiratory inhibitors indicated about 10-20% of the total 75Se uptake to be energy dependent. Inhibition of 75Se uptake by about 60-70% by sulfate and sulfite (5-25 microM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of 75Se by 40-60% effected by CdCl2, HgCl2, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of 75Se uptake by mitochondria of V. radiata emerges from the present study.

Effect of selenium compounds on murine B16 melanoma cells and pigmented cloned pB16 cells.

The effects of selenium compounds such as sodium selenite, sodium selenate, seleno-DL-cystine and seleno-DL-methionine (100 microM and 10 microM) on B16 and pigmented cloned pB16 murine melanoma cells were investigated in vitro. At the tested concentrations, B16 cells showed a greater sensitivity to the toxic effects of sodium selenite and seleno-DL-cystine than pB16 cells, whereas no decrease of B16 and pB16 cell number was observed after incubation with sodium selenate or seleno-DL-methionine. Glutathione (GSH) percentages were strongly decreased only by selenite and seleno-DL-cystine; it was marked more in B16 than in pB16 cells. The pretreatment of B16 cells with a GSH depleting agent (10 microM buthionine-[S,R]-sulfoximine) did not significantly influence the cytotoxic effects of selenite and seleno-DL-cystine. On both cell populations, GSH preincubation (50 microM) enhanced the cytotoxicity of selenite whereas the survival of seleno-DL-cystine treated cells was increased. Glutathione peroxidase (GSH-Px) activity in B16 cells was more sensitive than in pB16 cells to the activating effect of selenite, and particularly of seleno-DL-cystine: however, cell-free controls indicated that activation was mainly due to glutathione reductase. The rate of 75Se (as sodium selenite) uptake in both cell populations was maximal within the first hour of incubation, with a preferential accumulation in the cytosol; after 24 h of incubation, the amount of 75Se in cytosol and pellet was approximately the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of mucosal uptake of selenium from selenite by some thiols at various sites of rat intestine.

The influence of several thiols (conc. 1 mmol/L) on mucosal uptake of 75Se from 75Se-labeled selenite (conc. 10 mumol/L) across the brush border of rat jejunum and cecum was investigated in vitro using a short-term uptake technique. L-Cysteine (L-Cys) stimulated 75Se uptake in the mid- and distal jejunum and cecum, but not in the proximal jejunum. The effect was maximal in the distal jejunum. D-Cys was less effective in the jejunum and similarly effective in the cecum. L-Leucine (L-Leu) and L-glutamic acid significantly reduced the stimulatory effect of L-Cys on Se uptake in the distal jejunum, whereas the respective effect of D-Cys was not diminished by L-Leu. Cysteamine stimulated mucosal 75Se uptake at all intestinal sites tested, whereas the effect of mercaptopyruvate was restricted to the distal jejunum. Thioglycolate also enhanced 75Se uptake in the distal jejunum. The stimulatory effects of L-Cys, mercaptopyruvate, and thioglycolate were Na(+)-dependent, whereas the effect of cysteamine also occurred in the absence of Na+. Mercaptosuccinate, D-penicillamine, ergothioneine, and thiosulfate did not enhance mucosal 75Se uptake. It is concluded from these findings that the reaction of some thiols with selenite results in Se compounds that are rapidly absorbed by the intestinal epithelium through various Na(+)-dependent and Na(+)-independent mechanisms. The high bioavailability of Se from selenite found by others might thus be the result of the presence of thiols in the gastrointestinal tract.

Effect of selenium on the growth of three human colon cancer cell lines

The effects of selenium were investigated on three human colon cancer cell lines: Caco 2, HRT 18, and HT 29. At low concentrations (10-100 nM), selenium stimulated cell growth in serum-free medium. Thus, selenium is an essential trace element for cell proliferation. At higher concentrations, selenium inhibited cell growth. The rate of 75Se uptake was the same in all of the cell lines studied, but the quantity incorporated differed. GSH-Px activity was dependent on the selenium content of the medium. DNA and protein synthesis paralleled the growth curve. Comparison with the curve of viability revealed that selenium inhibited cell growth in two ways: by inhibiting DNA synthesis, without affecting cell viability, and, at higher doses, by cytotoxicity.

Uptake of 75Se-Selenite by Brush Border Membrane Vesicles from Chick Duodenum Stimulated by Vitamin D

Brush border membrane vesicles were isolated from mucosal homogenates of duodena from normal, rachitic and vitamin D-treated rachitic chicks using a discontinuous sucrose gradient, and further purified by glycerol gradient centrifugation. In vitro uptake of 75Se-selenite by purified brush border membrane vesicles was studied using a rapid filtration technique. The time course of 75Se uptake was non-linear; rapid initial binding was followed by a gradual decrease in the rate of uptake until an equilibrium value was reached at 60-120 min. The initial binding at 36 s was not affected by selenite concentration in the incubation buffer, while the fractional rate of uptake between the 36 s and 2 min time periods was clearly lower with 1 mM Se than with 4-100 microM Se. 75Se uptake did not show any dependency on the external Na-gradient, nor could it be inhibited by other anions (arsenate, phosphate). Treatment of rachitic chicks either with cholecalciferol (500 Iu, 72 h) or with 1,25(OH)2-cholecalciferol (0.5 microgram given 16 h prior to isolation of the vesicles) significantly enhanced 75Se uptake. A threefold excess of mannitol in the outside buffer reduced 75Se uptake by vesicles from vitamin D-deficient and D-treated chicks 60% and 35% respectively, but had no effect on vesicles from vitamin D-treated chicks preloaded with 75Se. Neither saponin treatment nor excess cold selenite could release the label from the vesicles preloaded with 75Se. These data are compatible with the hypothesis that selenite easily crosses the brush border membrane into the intravesicular space and, once inside, is tightly bound by the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

A developmental study of rat sperm and testis selenoproteins.

Essentially all of the selenium in the rat spermatozoon is bound to a polypeptide of Mr 15,000-17,000 confined to the capsule that surrounds the sperm mitochondria. Isoelectric focussing of isolated 75Se-labelled, carboxymethylated mitochondrial capsule protein (MCP) reveals the presence of at least four radioactive components, with a predominant charge isomer at pI4.6. The sperm selenoprotein appears to be identical with MCP, as judged by the exact coincidence of radioactivity and protein stain during two-dimensional electrophoresis. The temporal pattern of 75Se-labelling of rat caput epididymal spermatozoa after intratesticular 75Se injection suggests that maximum incorporation of 75Se into MCP occurs in step 7-step 12 spermatids and that 75Se uptake ceases during step 15 of spermiogenesis. The developmental appearance of sperm selenoprotein in rat testis therefore appears to lag several days behind that reported for MCP in mouse testis, suggesting the presence of selenium-free MCP in immature germ cells. SDS gel electrophoretic analysis of testis subcellular fractions 24 h after 75Se injection into rat testis at 21, 28 and 90 days of age indicates that sperm selenoprotein first appears in very low concentration during late meiosis and that its concentration increases sharply during early spermiogenesis. Additional 75Se-labelled polypeptides were detected on the gels, most of them of higher molecular weight than MCP. At least two of these (Mr 47,000 and 54,000) displayed a marked decrease in labelling between 5 and 24 h after injection into adult testis, coincident with a comparable increase in 75Se-labelled MCP, indicating that they may be precursors of MCP.

Selenium compounds in the fathead minnow ( Pimephales promelas)—I. Uptake, distribution, and elimination of orally administered selenate, selenite and l-selenomethionine

1. 1. Treatment of fathead minnows ( Pimephales promelas) with either [ 75Se]selenate, -selenite or- l-selenomethionine by gavage at 20 ng Se/g resulted in organ uptake and early distribution patterns which differed significantly between compounds. The greatest differences in uptake between compounds was observed in liver tissue which accumulated much less [ 75Se]selenate than either selenite or l-selenomethionine. 2. 2. The 75Se burdens and relative distribution among the various organs were nearly identical during the elimination phase for [ 75Se]selenate and -selenite. This suggests that selenium derived from these compounds converge to a common metabolic pool. 3. 3. The whole body T 1 2 , rate of 75Se uptake and magnitude of 75Se accumulation were generally greater for [ 75Se]selenomethionine than the inorganic forms. 4. 4. Selenium-75 was present in the bile following the oral administration of each compound. 5. 5. The partitioning of selenate and selenite into the plasma and cellular fraction of blood differs with both the compound and time following exposure.

Erythrocyte selenium-75 uptake as a measure of selenium status in weaner sheep, and its relationship to erythrocyte glutathione peroxidase activity.

The relationship between in vitro erythrocyte 75Se uptake (75Se uptake) and erythrocyte glutathione peroxidase (EGSHPx) activity was examined in weaner sheep during periods of selenium depletion and repletion, to determine whether 75Se uptake was better correlated than EGSHPx activity to the development of weaner nutritional myopathy. In the 2 trials conducted, only 3 of 45 Merino wether weaners developed clinical myopathy and histological lesions in skeletal muscles. The 75Se uptake values and EGSHPx activities in these 3 sheep were no different from those in the unaffected sheep. There was a significant negative correlation between 75Se uptake values and EGSHPx activities over the entire period of the trials. It could not be demonstrated that 75Se uptake was any better correlated than EGSHPx activity to the development of nutritional myopathy, and it was concluded that EGSHPx activity indicated selenium status better than 75Se uptake in weaner sheep.

Age dependence of selenite uptake in rat eye lenses

75Se-selenite was administered to young and adult rats in subcutaneous injection or added into the medium in which eye lenses were incubated. The 75Se uptake in lenses of the young was significantly higher in comparison with adults both in vivo and in vitro. The in vivo 75Se uptake, referred to unit of lens weight, was almost 500 times higher in young animals while in vitro it was only five times higher, which apparently reflects the age-dependent differences in the structure and function of membranes and/or system barriers.

The value of 75Se-selenomethionine scanning in solitary nodules of the thyroid gland.

This paper describes the use of 75Se-selenomethionine in the study of thyroid nodules. Making use of the different main gamma energies of 75Se and 125I and the short physical half-life of 99Tcm, sequential comparison scanning was carried out in a series of 48 patients with solitary thyroid nodules in which histology was established. Preliminary phantom studies and measurements on a biopsy specimen established that the threshold of detectability of a tumour was 1·5 cm diameter for 75Se and 0·5 cm for 125I. The absorbed dose for the activities used was estimated to be 4·5 rads to the liver and 1·05 rad to the gonads from 150 μCi of 75Se and 30–105 rads to the thyroid gland from 75 μCi of 125I. Eight out of 11 malignant conditions gave positive 75Se-selenomethonine scans. There were only four positive scans in 32 benign conditions and three of these were attributable to physiological or pathological change. The value of the complementary scans was shown in solitary nodules. Diffuse uptake of 75Se-selenomethionine indicated a generalized change in the thyroid. There was no established relationship between the pathology and the intensity of the 75Se uptake. In one case of follicular carcinoma the 45-minute uptake of 75Se was estimated to be 4·8 per cent of the dose above background. The operation specimen contained 18 μCi 125I and 4·6 μCi 75Se per 100 g corrected to scan dates.

Selenium metabolism: III. Serum proteins, lipoproteins and liver injury

The amount of carrier SeO 4 2− had a significant effect on the distribution of 75Se, after 75SeO 4 2− inhection, in the serum lipoproteins and serum proteins. This effect could not be due to simple dilution of 75Se by the carrier Se. After the administration of 75SeO 4 2−, the lipid fraction of serum lipoproteins was firmly labeled with 75Se both in the rat and in man. There was a higher association of 75Se with α 2 globulins than with other serum protein fractions both in the rat and in man. In patients with liver disease, which was associated with alcoholism, there was an increased binding of 75Se by serum lipoproteins after the injection of 75SeO 4 2− of high specific activity. In a patient with cirrhosis hepatoma the 75Se concentration was the highest in the kidney and the largest amount was in the liver 8 days after injection. No significant 75Se uptake was found in the hepatoma.

Selenium metabolism. II. Effect of injected selenium compounds and of liver injury.

SummaryA 17.5-fold increase of carrier SeO4 did not alter the urinary excretion of 75Se, increased its respiratory excretion and hepatic uptake, but decreased its uptake in other viscera. 75Se accumulation in the liver and pancreas was similar after 75Se-methionine and after 75SeO4 injections. After CCl4 therapy urinary excretion, and renal and pulmonary uptake of 75Se increased. In choline deficient cirrhotic rats urinary excretion of 75Se decreased and visceral, though not the hepatic, uptake of 75Se increased. However, SeO4 supplements reversed these changes in the cirrhotic rats with the exception of the increased renal uptake of 75Se. Over 80% of the non-dialyzable 75Se in the liver accumulated in the mitochondrial and supernatant fractions. The increase of carrier SeO4 was accompanied by an increase of 75Se in the nuclear fraction but did not change the distribution of 75Se in the mitochondrial, microsomal and supernatant fractions. The 75Se uptake increased in the nuclear fraction after CCl4 therap...