Metabolism of Tracer 75Se Selenium From Inorganic and Organic Selenocompounds Into Selenoproteins in Rats, and the Missing 75Se Metabolites.

We now know much about selenium (Se) incorporation into selenoproteins, and there is considerable interest in the optimum form of Se for supplementation and prevention of cancer. To study the flux of 75Se into selenoprotein, rats were fed 0 to 5 µg Se/g diet as selenite for 50-80 d and injected iv with 50 µCi of 75Se-labeled selenite, selenate, selenodiglutathione, selenomethionine, or selenobetaine at tracer levels (~0.5 µg Se). The rats were killed at various times and 75Se incorporation into selenoproteins was assessed by SDS/PAGE. These studies found that there is very rapid Se metabolism from this diverse set of selenocompounds to the common intermediate used for synthesis and incorporation of 75Se into the major selenoproteins in a variety of tissues. No selenocompound was uniquely or preferentially metabolized to provide Se for selenoprotein incorporation. Examination of the SDS/PAGE selenoprotein profiles, however, reveals that synthesis of selenoproteins is only part of the full Se metabolism story. The 75Se missing from the selenoprotein profiles, especially at early timepoints, is likely to be both low-MW and high-MW selenosugars and related precursors, as we recently found in livers of turkeys fed Se-adequate and high-Se diets. Differential metabolism of different selenocompounds into different selenosugar species may occur; these species may be involved in prevention of cancer or other diseases linked to Se status and may be associated with Se toxicity. Additional studies using HPLC-mass spectroscopy will likely be needed to fully flesh out the complete metabolism of selenium.

Minimum Selenium Requirements Increase When Repleting Second-Generation Selenium-Deficient Rats but Are Not Further Altered by Vitamin E Deficiency.

Second-generation selenium-deficient weanling rats fed graded levels of dietary Se were used (a) to study the impact of initial Se deficiency on dietary Se requirements; (b) to determine if further decreases in selenoperoxidase expression, especially glutathione peroxidase 4 (Gpx4), affect growth or gross disease; and (c) to examine the impact of vitamin E deficiency on biochemical and molecular biomarkers of Se status. Rats were fed a vitamin E-deficient and Se-deficient crystalline amino acid diet (3 ng Se/g diet) or that diet supplemented with 100 µg/g all-rac-α-tocopheryl acetate and/or 0, 0.02, 0.05, 0.075, 0.1, or 0.2 µg Se/g diet as Na2SeO3 for 28 days. Se-supplemented rats grew 6.91 g/day as compared to 2.17 and 3.87 g/day for vitamin E-deficient/Se-deficient and vitamin E-supplemented/Se-deficient groups, respectively. In Se-deficient rats, liver Se, plasma Gpx3, red blood cell Gpx1, liver Gpx1 and Gpx4 activities, and liver Gpx1 mRNA levels decreased to <1, <1, 21, 1.6, 49, and 11 %, respectively, of levels in rats fed 0.2 µg Se/g diet. For all biomarkers, ANOVA indicated significant effects of dietary Se, but no significant effects of vitamin E or vitamin E × Se interaction, showing that vitamin E deficiency, even in severely Se-deficient rat pups, does not result in compensatory changes in these biochemical and molecular biomarkers of selenoprotein expression. Se requirements determined in this study, however, were >50 % higher than in previous studies that started with Se-adequate rats, demonstrating that dietary Se requirements determined using initially Se-deficient animals can result in overestimation of Se requirements.

Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo).

The minimum Se requirement for male turkey poults is 0.3 µg Se/g--three times higher than requirements found in rodents--based on liver and gizzard glutathione peroxidase-4 (GPX4) and GPX1 activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only "predicted." Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA) codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.

Blood glutathione peroxidase-1 mRNA levels can be used as molecular biomarkers to determine dietary selenium requirements in rats.

Transcript (mRNA) levels are increasingly being used in medicine as molecular biomarkers for disease and disease risk, including use of whole blood as a target tissue for analysis. Development of blood molecular biomarkers for nutritional status, too, has potential application that parallels opportunities in medicine, including providing solid data for individualized nutrition. We previously reported that blood glutathione peroxidase-1 (Gpx1) mRNA was expressed at levels comparable to major tissues in rats and humans. To determine the efficacy of using blood Gpx1 mRNA to assess selenium (Se) status and requirements, we fed graded levels of Se (0-0.3 microg Se/g as selenite) to weanling male rats. Se status was determined by liver Se concentration and selenoenzyme activity, and selenoprotein mRNA abundance in liver and blood was determined by ribonuclease protection analysis. Liver Se and plasma glutathione peroxidase-3 and liver Gpx1 activities indicated that minimal Se requirements were at 0.08 microg Se/g diet. When total RNA was isolated from whole blood, Gpx1 mRNA in Se-deficient rats decreased to 10% of levels in Se-adequate (0.2 microg Se/g diet) rats. With Se supplementation, blood Gpx1 mRNA levels increased sigmoidally to a plateau with a minimum Se requirement of 0.08 microg Se/g diet, whereas glutathione peroxidase-4 mRNA levels were unaffected. Similarly, Gpx1 mRNA in RNA isolated from fractionated red blood cells decreased in Se-deficient rats to 23% of Se-adequate levels, with a minimum Se requirement of 0.09 microg Se/g diet. Additional studies showed that the preponderance of whole blood Gpx1 mRNA arises from erythroid cells, most likely reticulocytes and young erythrocytes. In summary, whole blood selenoprotein mRNA levels can be used as molecular biomarkers for assessing Se requirements, illustrating that whole blood has potential as a target tissue in development of molecular biomarkers for use in nutrition as well as in medicine.

Selenium requirements are higher for glutathione peroxidase-1 mRNA than gpx1 activity in rat testis.

Selenium (Se) plays a critical role in testis, sperm, and reproduction, and testis Se levels are remarkably maintained in Se deficiency. In most other tissues, Se levels decrease dramatically as do levels of most selenoproteins and levels of a subset of Se-regulated selenoprotein mRNAs. Because of the recent identification of key molecules in the targeted trafficking of Se to the testis, we examined the hierarchy of Se regulation in testis by determining the dietary Se regulation of the full testis selenoproteome in rats fed graded levels of Se (0 to 0.8 microg Se/g) as Na2SeO3 for 28 d. Se status did not significantly affect testis weight or glutathione peroxidase 4 (Gpx4) activity (P>0.05). qRT-PCR analysis of selenoprotein mRNA expression revealed that 21 of the 24 selenoprotein mRNAs and ApoER2 mRNA (the selenoprotein P [Sepp1] receptor) were also not regulated significantly by dietary Se status. In contrast, Gpx1 activity decreased to 28% of Se-adequate levels, and mRNA levels for Gpx1, Sepp1, and Sepw1 (selenoprotein W) decreased significantly in Se-deficient rats to 45, 46, and 55%, respectively, of Se-adequate plateau levels. Overlap of hyperbolic Gpx4 activity and Sepw1 mRNA response curves with testis Se concentration, all with minimum dietary Se requirements<0.016 microg Se/g, showed the priority for synthesis of Gpx4. Higher minimum dietary Se requirements of 0.04 microg Se/g for Gpx1 activity and Sepp1 mRNA, and the even higher minimum dietary Se requirement of 0.08 microg Se/g for Gpx1 mRNA, suggest that the hierarchy of these biomarkers reflects distinct, lower priority pools, cell types, and roles for Se within the testis.

Transcript analysis of the selenoproteome indicates that dietary selenium requirements of rats based on selenium-regulated selenoprotein mRNA levels are uniformly less than those based on glutathione peroxidase activity.

Dietary selenium (Se) requirements in rats have been based largely upon glutathione peroxidase-1 (Gpx1) enzyme activity and Gpx1 mRNA levels can also be used to determine Se requirements. The identification of the complete selenoprotein proteome suggests that we might identify additional useful molecular biomarkers for assessment of Se status. To characterize Se regulation of the entire rat selenoproteome, weanling male rats were fed a Se-deficient diet (<0.01 microg Se/g) supplemented with graded levels of Se (0-0.8 microg/g diet) for 28 d, Se status was determined by tissue Se concentration and selenoenzyme activity, and selenoprotein mRNA abundance in liver, kidney, and muscle was determined by quantitative real-time-PCR. Tissue Se and selenoenzyme biomarkers indicated that minimal Se requirements were

Selenium status highly regulates selenoprotein mRNA levels for only a subset of the selenoproteins in the selenoproteome.

Gpx (glutathione peroxidase)-1 enzyme activity and mRNA levels decrease dramatically in Se (selenium) deficiency, whereas other selenoproteins are less affected by Se deficiency. This hierarchy of Se regulation is not understood, but the position of the UGA selenocysteine codon is thought to play a major role in making selenoprotein mRNAs susceptible to nonsense-mediated decay. Thus in the present paper we studied the complete selenoproteome in the mouse to uncover additional selenoprotein mRNAs that are highly regulated by Se status. Mice were fed on Se-deficient, Se-marginal and Se-adequate diets (0, 0.05 and 0.2 microg of Se/g respectively) for 35 days, and selenoprotein mRNA levels in liver and kidney were determined using microarray analysis and quantitative real-time PCR analysis. Se-deficient mice had liver Se concentrations and liver Gpx1 and thioredoxin reductase activities that were 4, 3 and 3% respectively of the levels in Se-adequate mice, indicating that the mice were Se deficient. mRNAs for Selh (selenoprotein H) and Sepw1 (selenoprotein W) as well as Gpx1 were decreased by Se deficiency to <40% of Se-adequate levels. Five and two additional mRNAs were moderately down-regulated in Sedeficient liver and kidney respectively. Importantly, nine selenoprotein mRNAs in liver and fifteen selenoprotein mRNAs in the kidney were not significantly regulated by Se deficiency, clearly demonstrating that Se regulation of selenoprotein mRNAs is not a general phenomenon. The similarity of the response to Se deficiency suggests that there is one underlying mechanism responsible. Importantly, the position of the UGA codon did not predict susceptibility to Se regulation, clearly indicating that additional features are involved in causing selenoprotein mRNAs to be sensitive to Se status.

Longitudinal selenium status in healthy British adults: assessment using biochemical and molecular biomarkers.

Human selenium (Se) requirements are currently based on biochemical markers of Se status. In rats, tissue glutathione peroxidase-1 (Gpx1) mRNA levels can be used effectively to determine Se requirements; blood Gpx1 mRNA levels decrease in Se-deficient rats, so molecular biology-based markers have potential for human nutrition assessment. To study the efficacy of molecular biology markers for assessing Se status in humans, we conducted a longitudinal study on 39 subjects (age 45 +/- 11) in Reading, UK. Diet diaries (5 day) and blood were obtained from each subject at 2, 8, 17 and 23 weeks, and plasma Se, glutathione peroxidase (Gpx3) enzyme activity, and selenoprotein mRNA levels were determined. There were no significant longitudinal effects on Se biomarkers. Se intake averaged 48 +/- 14 microg/d. Plasma Se concentrations averaged 1.13 +/- 0.16 micromol/l. Plasma Se v. energy-corrected Se intake (ng Se/kJ/d) was significantly correlated, but neither Gpx3 activity v. Se intake (ng Se/kJ/d) nor Gpx3 activity v. plasma Se was significantly correlated. Collectively, this indicates that subjects were on the plateaus of the response curves. Selenoprotein mRNAs were quantitated in total RNA isolated from whole blood, but mRNA levels for Gpx1, selenoprotein H, and selenoprotein W (all highly regulated by Se in rodents), as well selenoprotein P, Gpx3, and phospholipid hydroperoxide glutathione peroxidase were also not significantly correlated with plasma Se. Thus selenoprotein molecular biomarkers, as well as traditional biochemical markers, are unable to further distinguish differences in Se status in these Se replete subjects. The efficacy of molecular biomarkers to detect Se deficiency needs to be tested in Se-deficient populations.

Dietary selenium requirements based on glutathione peroxidase-1 activity and mRNA levels and other Se-dependent parameters are not increased by pregnancy and lactation in rats.

The hierarchy of selenium (Se) requirements for growing rats ranges from <0.01 to 0.1 microg Se/g diet, depending on the choice of Se status parameter. To further evaluate the efficacy of molecular biology markers to determine Se requirements in later periods of the life cycle, which are less amenable to traditional approaches, we studied pregnant and lactating rats. Female weanling rats were fed a Se-deficient diet (<0.01 microg Se/g) or supplemented with graded levels of dietary Se (0-0.3 microg Se/g) for >10 wk, bred, and killed on d 1, 12, and 18 of pregnancy and d 7 and 18 of lactation; Se response curves were determined for 10 parameters including liver glutathione peroxidase (GPX). Growth, and mRNA levels for selenoprotein P, 5'-deiodinase, and GPX4 were not decreased by Se deficiency. GPX4 activity required 0.05 microg Se/g diet for maximum activity, similar to growing rats. Dietary Se requirements for plasma GPX3 activity decreased 33% in pregnancy, but returned during lactation to the requirement of growing rats. The Se requirement for GPX1 activity decreased 25% in pregnancy but not in lactation. GPX1 mRNA required 0.05 microg Se/g diet for maximum levels in both pregnancy and lactation, similar to growing rats. Clearly, Se requirements do not increase during pregnancy and lactation relative to Se requirements in growing rats. Unexpectedly, Se-adequate levels of GPX1 mRNA and activity declined to <40 and 50%, respectively, of nonpregnant Se-adequate levels during pregnancy and lactation, illustrating the need to fully understand biomarkers at all stages of the life cycle.

Selenoprotein mRNA is expressed in blood at levels comparable to major tissues in rats.

Liver glutathione peroxidase-1 (GPX1) mRNA is highly regulated by Se status relative to other parameters, but is of limited use for determining Se requirements in humans. To examine the efficacy of using blood for Se status assessment using molecular biology markers, we used a ribonuclease protection assay (RPA) to study mRNA levels in whole blood relative to 16 other rat tissues. Significant amounts of total RNA (>50 microg) were obtained from 1 mL of whole blood. Total RNA from 28-d postweaning Se-adequate (0.2 microg Se/g diet) male rats was analyzed for GPX1, GPX4, GPX3, thioredoxin reductase-1 (TRR1), and selenoprotein-P (SelP). RPA detected significant mRNA expression for at least 1 selenoprotein in all tissues except pancreas. GPX1 mRNA expression using this mix of RPA probes yielded the highest signal for GPX1 relative to the other selenoprotein signals in all tissues except testis; GPX1 expression was 4th highest in blood and similar to the major organs (liver, 1st; heart, 5th; kidney, 6th). Kidney was highest for GPX3, and testes was highest for GPX4, TRR1, and SelP. This study is the first to report the gene expression pattern for a number of selenoproteins and across a comprehensive set of tissues. The mRNA levels for all selenoproteins in blood were comparable to levels in the major organs, and decreases in blood and liver GPX1 mRNA levels in Se deficiency were similar, supporting potential use of whole blood for assessing Se status using molecular biology markers.

Dietary selenium regulation of glutathione peroxidase mRNA and other selenium-dependent parameters in male rats.

Weanling male rats were fed a basal torula yeast diet (0.007 µg Se/g diet) supplemented with graded levels of Se (0 to 0.2 µg Se/g diet as Na2SeO3) (three rats/group) to evaluate classical glutathione peroxidase (GPX1, GSH:H2O2, oxidoreductase, EC 1.11.1.9) mRNA level as an indicator of intracellular Se status. Growth was followed throughout the dietary treatment and a number of Se-dependent parameters including liver GPX1 mRNA levels were determined after 33 days. Growth was not impaired at any level of dietary Se supplementation. In rats fed the Se-deficient basal diet, liver Se concentration was 5 ± 1%, liver GPXI mRNA levels were 10 ± 2%. plasma GPX activity was 2 ± 1%, erythrocyte GPX activity was 37 ± 1%, and liver GPX activity was 0 ± 2% of the levels in rats fed 0.1 µg Se/g diet; these parameters increased sigmoidally with increasing dietary Se, showing a breakpoint near 0.1 µg Se/g diet. Graphical analysis indicated that the increase in liver GPX1 mRNA level with increasing dietary Se, preceded the increase in liver GPX activity. Se supplementation had no effect on polyadenylated mRNA levels or on ß-actin mRNA levels, demonstrating that Se regulation of GPX1 mRNA is specific. Se-deficient liver selenoprotein P mRNA levels were 69 ± 2% of the levels in rats fed 0.1 µg Se/g diet. We hypothesize that GPX1 mRNA is a primary target of the Se regulatory mechanism, making GPX1 mRNA level a potentially useful indicator of the status of an important intracellular regulatory pool of Se.