Effect of selenium status on mRNA levels for glutathione peroxidase in rat liver

To determine the effect of Se status on the level of mRNA for Se-dependent glutathione peroxidase (EC 1.11.1.9), rats were fed either a Se-deficient torula yeast diet (less than 0.02 mg Se/kg diet) or a Se-adequate diet (+0.2 mg Se/kg as Na2SeO3) for greater than 135 d. Liver glutathione peroxidase activity was 0.025 for Se-deficient versus 0.615 EU/mg protein for Se-adequate rats. Total liver RNA and polyadenylated RNA were isolated and subjected to Northern blot analysis using a 700 bp DNA probe from cloned murine glutathione peroxidase. Autoradiography showed that Se-deficient liver had 7-17% of the mRNA for glutathione peroxidase present in Se-adequate liver, suggesting that Se status may regulate the level of mRNA for this selenoenzyme.     

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

Mammalian thioredoxin reductase (TRR; NADPH₂: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.     

Selenium deficiency-induced alterations in the vascular system of the rat

To enunciate the mechanisms whereby Se protects against cardiovascular diseases, weanling male Wistar rats were fed deficient (0.022 mg/kg diet) and adequate (0.159 mg/kg diet) Se diets for 14 and/or 39 wk. As the Se content and glutathione peroxidase activity were decreased and the lipid peroxide level was increased, the plasma 6-keto-PGF_1α concentration of the Se-deficient group was markedly decreased in blood and tissues of the Se-deficient rats, as compared with the Se-adequate animals. Furthermore, the Se-deficient group had significantly lower plasma nitric oxide content and vascular nitric oxide synthase activity, higher erythrocyte sedimentation equation K value and aggregation index, and lower erythrocyte deformability than the Se-adequate group. Experimental Se deficiency also resulted in significant increases in serum total cholesterol and low-density lipoprotein cholesterol levels and a significant decrease in serum high-density lipoprotein cholesterol level. These results give some experimental supports to the hypothesis that low Se status and lipid peroxidation are involved in the etiology of cardiovascular diseases.     

Dynamic pathways of selenium metabolism and excretion in mice under different selenium nutritional statuses

The selenoprotein, cellular glutathione peroxidase (cGPx), has an important role in protecting organisms from oxidative damage through reducing levels of harmful peroxides. The liver and kidney in particular, have important roles in selenium (Se) metabolism and Se is excreted predominantly in urine and feces. In order to characterize the dynamics of these pathways we have measured the time-dependent changes in the quantities of hepatic, renal, urinary, and fecal Se species in mice fed Se-adequate and Se-deficient diets after injection of (82)Se-enriched selenite. Exogenous (82)Se was transformed to cGPx in both the liver and kidney within 1 h after injection and the synthesis of cGPx decreased 1 to 6 h and continued at a constant level from 6 to 72 h after injection. The total amount of Se associated with cGPx in mice fed Se-deficient diets was found to be less than in mice fed Se-adequate diets. This finding indicated that cGPx synthesis was suppressed under Se-deficient conditions and did not recover with selenite injection. Excess Se was associated with selenosugar in liver and transported to the kidney within 1 h after injection, and then excreted in urine and feces within 6 h after injection. Any excess amount of Se was excreted mainly as a selenosugar in urine.     

Effects of long-term selenium deficiency on glutathione peroxidase and thioredoxin reductase activities and expressions in rat aorta

The objective of this work was to determine whether long-term selenium (Se) deficiency might affect the antioxidant capacity of rat aorta, and the activities and expressions of glutathione peroxidase (GPx) and thioredoxin reductase (TR) in rat arterial walls. Weanling male Wister rats were fed Se-deficient or Se-adequate diets for 12 months. For the Se supplementation, sodium selenite was supplemented in drinking water (1 microg Se/ml) for 1 month. The aorta isolated from these groups were used to determine activities and mRNA levels. In comparison with the control, the activity and expression of GPx, superoxide dismutase activity and the total antioxidant capacity were significantly decreased in Se-deficient rats arterial walls. Following Se supplementation, they were restored to different extents. The content of malondialdehyde was increased markedly in Se-deficient rats. There seems an inverse relationship between the dietary Se and the activity and expression of TR. A positive relationship exists between dietary Se and the antioxidant capacity of rat arterial walls. The activities and expressions of GPx and TR in arterial walls were regulated by selenium by different mechanisms. Regulation of the expression of TR was mediated by reactive oxygen species, but of GPx by selenium status. The thioredoxin system may be the major cellular redox signaling system in rat arteries, rather than the glutathione system.     

Effect of long-term Se deficiency on the antioxidant capacities of rat vascular tissue

Selenium (Se) is an essential micronutrient in human health and Se deficiency has been incriminated in the etiology of cardiovascular diseases. However, the effect of long-term Se deficiency on the antioxidant capacities of vascular tissue has not been elucidated. This study was to determine whether long-term Se deficiency might affect the antioxidant capacity of rat vascular tissue and whether the diet Se might affect the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TR) in rat vascular tissue. Weanling male Wister rats were fed Se-deficient and Se-adequate diets for 12 mo. Se was supplemented in drinking water (1 μg Se/mL) for 1 mo. The arterial walls isolated from various groups were used in the assay. In comparison with the control, Se-deficient rats exhibited significant decreases of GPx activity and total antioxidant capacity in the arterial wall. Similar decreases appeared in the heart, liver, and kidney. The superoxide dismutase activity was also decreased in the Se-deficient rat’s arterial wall. Followed by Se supplementation, they were restored to different extent. TR activity was decreased in the heart, liver, and kidney, but increased in the arterial wall. The content of malondialdehyde was increased markedly in Se-deficient rats. In conclusion, a positive correlation exists between dietary Se and antioxidant capacity of rat vascular tissue except TR. It seems that the activities of GPx and TR in the rat arterial wall were mediated in different pathways by the Se status.     

Activation of superoxide dismutase in selenium-deficient mice infected with influenza virus.

Selenium (Se) deficiency is associated with decreased activities of Se-dependent antioxidant enzymes, glutathione peroxidase (GPx) and thioredoxin reductase (TR), and with changes in the cellular redox status. We have previously shown that host Se deficiency is responsible for increased virulence of influenza virus in mice due to changes in the viral genome. The present study examines the antioxidant defense systems in the lung and liver of Se-deficient and Se-adequate mice infected with influenza A/Bangkok/1/79. Results show that neither Se status nor infection changed glutathione (GSH) concentration in the lung. Hepatic GSH concentration was lower in Se-deficient mice, but increased significantly day 5 post infection. No significant differences due to Se status or influenza infection were found in catalase activities. As expected, Se deficiency was associated with significant decreases in GPx and TR activities in both lung and liver. GPx activity increased in the lungs and decreased in the liver of Se-adequate mice in response to infection. Both Se deficiency and influenza infection had profound effects on the activity of superoxide dismutase (SOD). The hepatic SOD activity was higher in Se-deficient than Se-adequate mice before infection. However, following influenza infection, hepatic SOD activity in Se-adequate mice gradually increased. Influenza infection was associated with a significant increase of SOD activity in the lungs of Se-deficient, but not Se-adequate mice. The maximum of SOD activity coincided with the peak of pathogenesis in infected lungs. These data suggest that SOD activation in the lung and liver may be a part of a compensatory response to Se deficiency and/or influenza infection. However, SOD activation that leads to increased production of H(2)O(2) may also contribute to pathogenesis and to influenza virus mutation in lungs of Se-deficient mice.     

Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.     

Selenium Regulates Gene Expression for Estrogen Sulfotransferase and Alpha 2U-globulin in Rat Liver

Dietary intake of the essential trace element selenium (Se) regulates expression of genes for seleno-proteins and certain non-Se-containing proteins. However, these proteins do not account for all of Se's biological effects. The objective of this work was to identify additional genes whose expression is regulated by Se. Identification of these genes may reveal new functions for Se or define mechanisms for its biological effects. Weanling male Sprague-Dawley rats were fed a Torula yeast-based Se-deficient basal diet or the same diet supplemented with 0.5 mg Se/kg diet as sodium selenite for 13 weeks. Total RNA was used as template for RNA fingerprinting. Two differentially expressed cDNA fragments were identified and cloned. The first had 99% nucleotide identity with rat liver estrogen sulfotransferase (EST) isoform-6. The second had 99% nucleotide sequence identity with rat liver 2u-globulin. The mRNA levels for both were markedly reduced in Se deficiency. Laser densitometry showed that EST mRNA in Se deficiency was 7.3% of that in Se-adequate rat liver. The level of 2u-globulin mRNA in Se-deficient rat liver was only 12.6% of that in Se-adequate rat liver. These results indicate that dietary Se may play a role in steroid hormone metabolism in rat liver.     

Effect of selenium-induced oxidative stress on the oxidation reduction system and reproductive ability of male mice

The present study was carried out to evaluate the effect of selenium (Se)-induced oxidative stress on the oxidation reduction system and the fertility status of male mice. Different levels of Se, a potent antioxidant, were fed in three separate groups for 8 wk to create the different oxidative stress in mice. A significant decrese in the glutathione peroxidase (GSH-Px) in both liver and testis was observed in the Se-deficient (0.02 ppm) group I, whereas enzyme levels in the Se-excess (1 ppm) group were comparable to the Se-adequate (0.2 ppm) group. Glutathione-S-transferase activity was enhanced in group I in comparison to group II; however, no change was seen in group III. The glutathione reductase and superoxide dismutase activities were decreased in the Se-deficient group, whereas the enzyme levels were significantly increased in the Se-excess group. The fertility status of the animals studied in terms of percentage fertility and litter size showed a significant decrease in the reproductive ability of male mice in group I when compared to group II. No changes in the fertility status of animals were observed in group III. Thus, the data clearly indicate the effect of oxidative stress generated by feeding various Se levels on the oxidation reduction system and, consequently, its effect on the reproductive ability of male mice.     

Effects of dietary selenium and vitamin E concentrations on phospholipid hydroperoxide glutathione peroxidase expression in reproductive tissues of pubertal maturing male rats

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is the second intracellular selenium (Se)-dependent glutathione peroxidase (GSH-Px) identified in mammals. Our objectives were to determine the effect of dietary vitamin E and Se levels on PHGPX activity expression in testis, epididymis, and seminal vesicles of pubertal maturing rats, and the relationship of PHGPX expression with testicular development and sperm quality. Forty Sprague-Dawley male weanling rats (21-d old), were initially fed for 3 wk a torula yeast basal diet (containing 0.05 mg Se/kg) supplemented with marginal levels of Se (0.1 mg/kg as Na₂SeO₃) and vitamin E (25 IU/kg as all-rac-α-tocopheryl acetate). Then, rats were fed the basal diets supplemented with 0 or 0.2 mg Se/kg and 0 or 100 IU vitamin E/kg diet during the 3-wk period of pubertal maturing. Compared with the Se-supplemented rats, those fed the Se-deficient diets retained 31, 88, 67, and 50% of Se-dependent GSH-Px activities in liver, testis, epididymis, and seminal vesicles, respectively. Testes and seminal vesicles had substantially higher (5-to 20-fold) PHGPX activity than liver. Dietary Se deficiency did not affect PHGPX activities in the reproductive tissues, but reduced PHGPX activity in liver by 28% (P < 0.0001). Dietary vitamin E supplementation did not affect PHGPX activity in liver, whereas it raised PHGPX activity in seminal vesicles by 43% (P < 0.005). Neither dietary vitamin E nor Se levels affected body weight gains, reproductive organ weights, or sperm counts and morphology. In conclusion, expression of PHGPX activity in testis and seminal vesicles was high and regulated by dietary Se and vitamin E differently from that in liver.     

Nonsense-mediated decay of mRNA for the selenoprotein phospholipid hydroperoxide glutathione peroxidase is detectable in cultured cells but masked or inhibited in rat tissues

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.     

Effects of Dietary Selenium Deficiency on mRNA Levels of Twenty-One Selenoprotein Genes in the Liver of Layer Chicken

Selenium (Se) is an essential trace element in many life forms due to its occurrence as selenocysteine (Sec) residue in selenoproteins. However, little is known about the expression pattern of selenoproteins in the liver of layer chicken. To investigate the effects of Se deficiency on the mRNA expressions of selenoproteins in the liver tissue of layer chickens, 1-day-old layer chickens were randomly allocated into two groups (n?=?120/group). The Se-deficient group (?Se) was fed a Se-deficient corn–soy basal diet; the Se-adequate group as control (+Se) was fed the same basal diet supplemented with Se at 0.15 mg/kg (sodium selenite). The liver tissue was collected and examined for mRNA levels of 21 selenoprotein genes at 15, 25, 35, 45, 55, and 65 days old. The data indicated that the mRNA expressions of Gpx1, Gpx2, Gpx3, Gpx4, Sepn1, Sepp1, Selo, Sepx1, Selu, Txnrd1, Txnrd2, Txnrd3, Dio1, Dio2, SPS2, Selm, SelPb, Sep15, and Sels were decreased (p?<?0.05), but not the levels of Dio3 and Seli (p?>?0.05). The results showed that the mRNA levels of 19 selenoprotein (except Seli and Dio3) genes in the layer chicken liver were regulated by diet Se level. The present study provided some compensated data about the roles of Se in the regulation of selenoproteins.     

Bioavailability of selenium from veal, chicken, beef, pork, lamb, flounder, tuna, selenomethionine, and sodium selenite assessed in selenium-deficient rats

The bioavailability of selenium (Se) from veal, chicken, beef, pork, lamb, flounder, tuna, selenomethionine (SeMet), and sodium selenite was assessed in Se-deficient Fischer-344 rats. Se as veal, chicken, beef, pork, lamb, flounder, tuna, SeMet, and sodium selenite was added to torula yeast (TY) basal diets to comprise Se-inadequate (0.05 mg Se/kg) diets. Se as sodium selenite was added to a TY basal diet to comprise a Se-adequate (0.10 mg Se/kg), Se-control diet. The experimental diets were fed to weanling Fischer-344 rats that had been subjected to dietary Se depletion for 6 wk. After 9 wk of the dietary Se repletion, relative activity of liver glutathione peroxidase (GSHPx) from the different dietary groups compared with control rats (100%) was: flounder 106%, tuna 101%, pork 86%, sodium selenite 81%, SeMet 80%, beef 80%, chicken 77%, veal 77%, and lamb 58%. Se from flounder was the most efficient at restoring Se concentrations in the liver and skeletal muscle. Se from sodium selenite, SeMet, beef, veal, chicken, pork, lamb, and tuna was not dietarily sufficient to restore liver and muscle Se after 9 wk of recovery following a 6-wk period of Se depletion.     

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.