Roger A. Sunde

Glutathione peroxidase activity in rat lens and other tissues in relation to dietary selenium intake

Abstract Glutathione peroxidase (E.C. 1.11.1.9: glutathione: H 2 O 2 oxidoreductase) activity and selenium concentration were measured in lenses of female rats and their offspring after long-term feeding of either a selenium-supplemented ( 0·1 parts 10 6 ) or selenium-deficient ( 0·02 parts 10 6 ) diet. Long-term selenium deficiency decreased lens glutathione peroxidase activity in parent rats and their offspring to 15 and 14% respectively of supplemented controls. For comparison to lens, glutathione peroxidase was also measured in liver, heart, lung, erythrocytes, kidney, adrenal, testis, and brain of the offspring. Selenium deficiency caused the enzyme to decrease most dramatically in liver (to 0) and least in brain (to 62% of selenium supplemented controls). Although glutathione peroxidase in lens was lower than that in the other organs assayed, it was among the organs more sensitive to depletion caused by selenium deficiency. A short-term selenium deficiency of 8 weeks in newborn lambs had no effect on lens glutathione peroxidase, but the enzyme in organs such as liver was dramatically decreased. Therefore, an extensive period of selenium deficiency appears necessary to affect lens glutathione peroxidase activity, which probably relates to the relatively slow turnover and slow growth of the lens. The possible role of the seleno-enzyme, glutathione peroxidase, in the prevention of cataracts and the relationship of selenium to vitamin E and sulfur-containing amino acids in this regard are discussed.

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.

Selenium regulation of transcript abundance and translational efficiency of glutathione peroxidase-1 and -4 in rat liver.

Glutathione peroxidase (GPX)1 mRNA in rat liver falls dramatically during Se deficiency to levels that are approx. 10% of Se-adequate levels. This regulation is mediated by mRNA stability, and is hypothesized to involve nonsense-mediated mRNA decay. mRNA levels for GPX4 and other selenoproteins are much less regulated by Se status. To evaluate the relative contribution of mRNA abundance versus translational efficiency to overall regulation of GPX1 expression, we quantified GPX1, GPX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts per cell in rat liver. Surprisingly, we found that GPX1 transcripts in Se deficiency are moderately abundant and similar in abundance to GAPDH and other selenoprotein mRNAs; Se supplementation increases GPX1 mRNA so that it is 30-fold higher than GAPDH mRNA. Translational efficiency of GPX1 mRNA is half of that of GPX4. Translational efficiency of GPX1 mRNA increases approx. 20-fold with Se supplementation and appears to switch GPX1 mRNA from nonsense-mediated degradation to translation. This regulatory switch can explain why GPX1 expression is an excellent parameter for assessment of Se status.

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 <or=0.1 microg Se/g diet for most biomarkers. Liver Gpx1 mRNA also decreased to <10% of Se-adequate levels, with a minimum Se requirement at 0.07 microg/g diet. Five selenoprotein mRNA in liver, 4 in kidney, and 2 in muscle decreased to <41% of Se-adequate levels, all with minimum Se requirements at <or=0.07 microg/g diet; the majority of selenoprotein mRNA in each tissue were not significantly regulated by Se status, and 1 selenoprotein, selenophosphate synthetase-2, was upregulated in Se-deficient kidney. Plateau breakpoints for all regulated selenoprotein mRNA were very similar, suggesting that 1 underlying mechanism is in play in Se regulation of selenoprotein mRNA. Lastly, we did not find any selenoprotein mRNA that could be used as biomarkers for super-nutritional/anticarcinogenic levels (up to 0.8 microg Se/g diet) of Se.

Selenium Regulation of the Selenoprotein and Nonselenoprotein Transcriptomes in Rodents

This review discusses progress in understanding the hierarchy of selenoprotein expression at the transcriptome level from selenium (Se) deficiency to Se toxicity. Microarray studies of the full selenoproteome have found that 5 of 24 rodent selenoprotein mRNA decrease to <40% of Se adequate levels in Se deficient liver but that the majority of selenoprotein mRNA are not regulated by Se deficiency. These differences match with the hierarchy of selenoprotein expression, helping to explain these differences and also showing that selenoprotein transcripts can be used as molecular biomarkers for assessing Se status. The similarity of the response curves for regulated selenoproteins suggests one underlying mechanism is responsible for the downregulation of selenoprotein mRNA in Se deficiency, but the heterogeneity of the UGA position in regulated and nonregulated selenoprotein transcripts now indicates that current nonsense mediated decay models cannot explain which transcripts are susceptible to mRNA decay. Microarray studies on the full liver transcriptome in rats found only <10 transcripts/treatment were significantly down- or upregulated by Se deficiency or by supernutritional Se up to 2.0 μg Se/g diet (20× requirement), suggesting that cancer prevention associated with supernutritional Se may not be mediated by transcriptional changes. Toxic dietary Se at 50× requirement (5 μg Se/g diet), however, significantly altered ∼4% of the transcriptome, suggesting number of transcriptional changes itself as a biomarker of Se toxicity. Finally, panels of Se regulated selenoprotein plus nonselenoprotein transcripts predict Se status from deficient to toxic better than conventional biomarkers, illustrating potential roles for molecular biomarkers in nutrition.

Selenium regulation of thioredoxin reductase activity and mRNA levels in rat liver

Mammalian thioredoxin reductase (TRR; NADPH(2):oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) is a new member of the family of selenocysteine-containing proteins. TRR activity in Se-deficient rat liver is reported to decrease to 4.5 to 15% of the activity in Se-adequate rat liver, similar to the fall in Se-dependent glutathione peroxidase-1 activity. Both glutathione peroxidase-1 enzyme activity and mRNA levels decrease dramatically in Se deficiency, whereas glutathione peroxidase-4 activity only decreases to 40% of Se-adequate levels and mRNA level is little affected by Se deficiency. The purpose of these experiments is to study the effect of Se status on TRR mRNA levels and enzyme activity in our well-characterized rat model, and to compare this regulation directly to the regulation of other Se-dependent proteins in male weanling rats fed Se-deficient diets or supplemented with dietary Se for 28 days. In two experiments, TRR activity in Se-deficient liver decreased to 15% of Se-adequate activity as compared to 2% and 40% of Se-adequate levels for GPX1 and GPX4, respectively. Using ribonuclease protection analysis, we found that TRR mRNA levels in Se-deficient rat liver decreased to 70% of Se-adequate levels. This decrease in TRR mRNA was similar to the GPX4 mRNA decrease in Se-deficient liver in these experiments, whereas GPX1 mRNA levels decreased to 23% of Se-adequate levels. This study clearly shows that TRR represents a third pattern of Se regulation with dramatic down-regulation of enzyme activity in Se deficiency but with only a modest decrease in mRNA level. The conservation of TRR mRNA in Se deficiency suggests that this is a valued enzyme; the loss of TRR activity in Se deficiency may be the cause of some signs of Se deficiency.

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

Abstract 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 75 Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced 75 Se incorporation from L[ 75 Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting 75 Se incorporation from [ 75 Se]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.

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 β-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.

SELENOPROTEIN GENE EXPRESSION DURING SELENIUM-REPLETION OF SELENIUM-DEFICIENT RATS

Selenium repletion of selenium-deficient rats with 20 μg selenium/kg body weight as Na2SeO3 was used as a model to investigate the mechanisms that control the distribution of the trace element to specific selenoproteins in liver and thyroid. Cytosolic glutathione peroxidase (cGSHPx), phospholipid hydroperoxide glutathione peroxidase (PHGSHPx), and iodothyronine 5′-deiodinase (IDI) activities were all transiently increased in liver 16 to 32 h after ip injection with selenium. However, only cGSHPx and PHGSHPx activities increased in the thyroid where IDI activity was already increased by selenium deficiency. These responses were owing to synthesis of the seleoproteins on newly synthesised and/or existing mRNAs. The selenoprotein mRNAs in the thyroid gland were increased two- and threefold after the transitory increases in selenoprotein activity. In contrast, there were parallel changes in selenoprotein mRNAs and enzyme activities in the liver, with no prolonged rises in mRNA levels. The organ differences suggest that increased thryotrophin (TSH) concentrations, which are known to induce thyrodial IDI and mRNA, may control the mRNAs for all the thyroidal selenoproteins investigated and be a major mechanism for the preservation of thyroidal selenoproteins when selenium supplies are limited.

Selenium toxicity but not deficient or super-nutritional selenium status vastly alters the transcriptome in rodents

BackgroundProtein 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.ResultsThere 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.ConclusionThis 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.