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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 (

The hierarchy of selenium (Se) requirements for growing rats ranges from 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 [micro]g 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 GPXl activity decreased 25% in pregnancy but not in lactation. GPX1 mRNA required 0.05 [micro]g 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 GPXl mRNA and activity declined to KEY WORDS: * biomarkers * gene expression * molecular biology * RDA * ribonuclease protection

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/[micro]g) were obtained from 1 mL of whole blood. Total RNA from 28-d postweaning Se-adequate (0.2 [micro]g 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. KEY WORDS: * biomarkers * glutathione peroxidase * molecular biology * RDA * requirements

The sequencing and annotation of the human genome and the genomes of other model organisms offer new tools and new opportunities for human nutrition research in the 21st century. Basic research continues to be the key foundation for formulating solid nutrition recommendations for the public, but the basis for establishing human nutrient recommendations today suffers because of lack of good biomarkers and because of weak federal funding for nutrition research. In the context of this post-human genome-sequencing era, tantalizing opportunity exists in seven areas--four basic and three applied: 1) identification of molecular biomarkers for nutrient status; 2) characterization of single polynuclear polymorphisms (SNPs) associated with nutrition; 3) development of a national genome array nutrition database; 4) use of models at all phylogenetic levels for nutrition research; 5) application of post-genome-sequencing tools to study diet and human health, including phytonutrients and genetically modified foods; 6) application of these tools to the role of nutrition in pathogenesis of human disease; and 7) development of a funding base for research on exercise and human health. KEY WORDS: * exercise * genome array database * National Research Initiative * nutrient biomarkers * single polynuclear polymorphisms.

Classical glutathione peroxidase (GPX) mRNA levels fall dramatically in selenium (Se)-deficient animals, but it is not known whether this mechanism is related to the mRNA 3[prime] untranslated region (3[prime]UTR) sequences that have been shown to direct Se incorporation. In this study, we used recombinant GPX constructs to investigate the role of the GPX 3[prime]UTR in Se regulation of GPX mRNA levels in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with GPX (pRc/GPX), GPX lacking the 3[prime]UTR (pRc/[Delta]3[prime]UTR) or the pRc/CMV vector alone, and GPX activity and GPX mRNA levels were determined in stable transfectants grown in low Se basal medium with a range of added Se concentrations. We identified two pRc/GPX transfectants with significantly elevated GPX activity levels compared with pRc/CMV transfectants. The elevated GPX expression did not dramatically shift the amount of Se that was sufficient for GPX activity to reach the Se-adequate plateau level (100 nmol/L added Se). As expected, GPX activity was not significantly different when pRc/[Delta]3[prime]UTR transfectants were compared with pRc/CMV control transfectants. Among the wild type and transfected CHO cells, Se-deficient GPX activity levels averaged 35 [+ or -] 5% of Se-adequate levels. Selenium-deficient levels of endogenous GPX mRNA as well as recombinant pRc/GPX mRNA averaged 54-58% of Se-adequate levels; 3-4 nmol/L added Se was sufficient for maximal GPX mRNA levels. In contrast, pRc/[Delta]3[prime]UTR mRNA levels in the unsupplemented cells remained at Se-adequate levels and showed no distinct Se regulation. These studies demonstrate that the GPX 3[prime]UTR is necessary for Se regulation of GPX mRNA levels in addition to its role in Se incorporation. KEY WORDS: selenium; glutathione peroxidase; gene expression; nutrient requirements; Chinese hamster ovary cells

To determine critically the selenium (Se) requirement for weanling female rats, we used glutathione peroxidase (GSH: [H.sub.2][O.sub.2] oxidoreductase, EC 1.11.1.9) (GPx) mRNA and a number of other parameters to assess Se status. Rats were fed a basal torula-yeast diet (0.007 [[micro]gram] Se/g) supplemented with Se as [Na.sub.2]Se[O.sub.3] in graded levels from 0 to 0.3 [[micro]gram] Se/g diet for 32 d (3 rats/group). Selenium supplementation had no effect on growth, showing that the Se requirement for growth is less than 0.007 [[micro]gram] Se/g diet, whereas other parameters showed significant increases with Se supplementation. In rats fed the Se-deficient basal diet, liver Se concentration was 4 [+ or -] 0%, plasma GPX activity was 8 [+ or -] 1%, erythrocyte GPX activity was 40 [+ or -] 3%, liver GPX activity was 2 [+ or -] 1%, and liver GPX mRNA levels were 11-17% of the levels in rats fed 0.1 [[micro]gram] Se/g diet. Liver Se concentration and GPX activity in plasma, erythrocytes and liver all reached a plateau breakpoint at or near 0.1 [[micro]gram] Se/g diet, indicating that the dietary Se requirement for maximal GPX activity in growing female rats is 0.1 [[micro]gram] Se/g diet. Liver GPX mRNA levels reached the plateau breakpoint at 0.05 [[micro]gram] Se/g diet, showing that the minimum dietary Se requirement for maximal GPX mRNA levels in female rats is half of the Se requirement for maximal GPX activity. This experiment demonstrates that GPX mRNA can be used to determine the dietary Se requirement; the gap between the dietary Se necessary for maximal GPX mRNA and that for maximal GPX activity may represent an evolutionarily derived biological margin of safety. J. Nutr. 126: 2260-2267, 1996. INDEXING KEY WORDS: * gene expression * glutathione peroxidase * nutrient requirements * rats selenium

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) and classical glutathione peroxidase (GPX1) are encoded by separate genes with only about 40% amino acid and nucleic acid sequence identity. To determine the response of tissue PHGPX expression to dietary Se level and to compare these responses with those for GPX1, weanling male rats were fed amino acid diets containing from 2 (-Se) to 130 (+Se) [[micro]gram] Se/kg diet or a torula diet containing 5 and 190 [[micro]gram] Se/kg diet as [Na.sub.2]Se[O.sub.3] for 28 d. Tissues were analyzed for PHGPX and GPX1 activity and mRNA. There was no effect of Se on growth. In -Se rats, GPX1 activity was reduced to 1% in liver and 4-9% in heart, kidney and lung compared with +Se rats; PHGPX activity was reduced only to 25-50% in these four tissues. The Se response curves indicated that the dietary Se requirement to reach plateau liver PHGPX activity was half that required for plateau GPX activity. In -Se rats, liver and heart GPX1 mRNA levels were reduced to 6 and 12%, respectively, whereas PHGPX mRNA was not significantly affected by Se deficiency. Notably, 65 [[micro]gram] Se/kg diet resulted in plateau liver GPX1 mRNA levels but not plateau GPX activity. Testis had the lowest GPX activity and GPX1 mRNA of all tissues examined, but had 15-fold higher PHGPX activity and 45-fold higher PHGPX mRNA levels when compared with liver. There was no significant effect of dietary Se on testis GPX1 and PHGPX mRNA levels. This study demonstrates that these two selenoperoxidases are differentially regulated by dietary Se. Differences in Se regulation of mRNA levels in liver and heart were even more pronounced than for enzyme activity. The lack of any significant effect of reduced dietary Se on PHGPX mRNA levels suggests that there are detailed underlying molecular mechanisms whereby Se status regulates GPX1 mRNA levels but not PHGPX mRNA levels. INDEXING KEY WORDS: gene expression; glutathione peroxidase; selenium deficiency; mRNA; rats

Classical glutathione peroxidase (GPX) is a useful Se-dependent parameter for determining Se status, but loss of GPX activity alone cannot explain the full effects of Se deficiency. The recent identification of type 1 thyroxine 5 [prime]-deiodinase as a Se-dependent enzyme provides a new potentially critical role for Se. To develop a model of impaired growth due to Se deficiency, second generation deficient weanling rats were fed a Se-deficient amino acid diet with adequate vitamin E and methionine. Initial growth rates of deficient males and females were 53 and 63%, respectively, of rats fed 0.1 pg Se/g diet. In short-term experiments with deficient males, liver Se and GPX activity were reduced 99%, liver glutathione-s-transferase activity was increased 114%, plasma thyroxine concentrations were increased 67%, plasma triiodothyronine was decreased 23% and the plasma triiodothyronine:thyroxine ratio was decreased 55%, compared with rats fed 0.2 [[micro]gram] Se/g diet. When deficient rats were injected on d 14 with 0, 1, 5 or 10 [[micro]gram] Se/100 g, rats grew 4.45, 7.62, 7.17 and 9.05 g/d, respectively, over the next 7 d. Injection with 10 [[micro]gram] Se/100 g restored plasma thyroxine and triiodothyronine concentrations 7 d later. Rats injected with 1 [[micro]gram] Se/100 g rat had significantly altered plasma thyroxine and triiodothyronine concentrations 1 but not 7 d after injection. Infusion of Se-deficient rats with 438 ng triiodothyronine/d for 7 d restored plasma triiodothyronine concentrations but did not increase growth rate compared with rats infused with saline. This model produced a significant growth depression that was significantly reversed by as little as 1 [[micro]gram] Se/100 g rat, but not by triiodothyronine infusion, suggesting that other Se-dependent roles in addition to 5 [prime]-deiodinase and GPX are necessary for adequate growth. INDEXING KEY WORDS: growth; rats; selenium; thyroxine 5 [prime]-deiodinase; glutathione peroxidase

Abstract Background Protein and mRNA levels for several selenoproteins, such as glutathione peroxidase-1 (Gpx1), are down-regulated dramatically by selenium (Se) deficiency. These levels in rats increase sigmoidally with increasing dietary Se and reach defined plateaus at the Se requirement, making them sensitive biomarkers for Se deficiency. These levels, however, do not further increase with super-nutritional or toxic Se status, making them ineffective for detection of high Se status. Biomarkers for high Se status are needed as super-nutritional Se intakes are associated with beneficial as well as adverse health outcomes. To characterize Se regulation of the transcriptome, we conducted 3 microarray experiments in weanling mice and rats fed Se-deficient diets supplemented with up to 5 μg Se/g diet. Results There was no effect of Se status on growth of mice fed 0 to 0.2 μg Se/g diet or rats fed 0 to 2 μg Se/g diet, but rats fed 5 μg Se/g diet showed a 23% decrease in growth and elevated plasma alanine aminotransferase activity, indicating Se toxicity. Rats fed 5 μg Se/g diet had significantly altered expression of 1193 liver transcripts, whereas mice or rats fed ≤ 2 μg Se/g diet had < 10 transcripts significantly altered relative to Se-adequate animals within an experiment. Functional analysis of genes altered by Se toxicity showed enrichment in cell movement/morphogenesis, extracellular matrix, and development/angiogenesis processes. Genes up-regulated by Se deficiency were targets of the stress response transcription factor, Nrf2. Multiple regression analysis of transcripts significantly altered by 2 μg Se/g and Se-deficient diets identified an 11-transcript biomarker panel that accounted for 99% of the variation in liver Se concentration over the full range from 0 to 5 μg Se/g diet. Conclusion This study shows that Se toxicity (5 μg Se/g diet) in rats vastly alters the liver transcriptome whereas Se-deficiency or high but non-toxic Se intake elicits relatively few changes. This is the first evidence that a vastly expanded number of transcriptional changes itself can be a biomarker of Se toxicity, and that identified transcripts can be used to develop molecular biomarker panels that accurately predict super-nutritional and toxic Se status.