To determine critically the selenium (Se) requirement for weanling female rats, we used glutathione peroxidase (GSH: H2O2 oxidoreductase, EC 1.11.1.9) (GPX) mRNA and a number of other parameters to assess Se status. Rats were fed a basal torulayeast diet (0.007 micrograms Se/g) supplemented with Se as Na2SeO3 in graded levels from 0 to 0.3 micrograms 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 micrograms 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 +/- 0%, plasma GPX activity was 8 +/- 1%, erythrocyte GPX activity was 40 +/- 3%, liver GPX activity was 2 +/- 1%, and liver GPX mRNA levels were 11-17% of the levels in rats fed 0.1 micrograms Se/g diet. Liver Se concentration and GPX activity in plasma, erythrocytes and liver all reached a plateau breakpoint at or near 0.1 micrograms Se/g diet, indicating that the dietary Se requirement for maximal GPX activity in growing female rats is 0.1 micrograms Se/g diet. Liver GPX mRNA levels reached the plateau breakpoint at 0.05 micrograms 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.

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 6 2% and 15 6 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 39-UTR can completely replace the GPX1 39-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 39-UTR to b-globin mRNA can convey significant Se regulation to b-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 39-UTR and a Sec codon followed by an intron.

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 Na 2 SeO 3 ) (three rats/group) to evaluate classical glutathione peroxidase (GPX1, GSH:H 2 O 2 , 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 Sedeficient 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.

A UGA codon and a selenocysteine insertion sequence in the 3-untranslated region are the only established mRNA elements necessary for selenocysteine (Sec or U) incorporation during translation. These two elements, however, do not universally confer efficient Sec incorporation. The objective of this study was to systematically examine the effect of UGA codon position on efficiency of Sec insertion. In a glutathione peroxidase-1 (F-GPX1) expression vector, the UGA at the native position (U47) was mutated to a cysteine codon, and codons for Ser-7, Ser-12, Ser-18, Ser-29, Ser-45, Ser-93, Cys-154, Val-172, Ser-178, and Ser-195 were individually mutated to UGA and transiently expressed in COS-7 cells.75 Se incorporation at the 11 positions was 31, 72, 54, 105, 90, 100, 146, 135, 13, 11, and 43%, respectively, of 75 Se incorporation at U47, suggesting that Sec is more efficiently incorporated at UGA codons positioned in the middle of the coding region rather than close to the 5 or 3 ends. Ribonuclease protection showed that these differences were not due to differences in mRNA level. When the green fluorescence protein (GFP) coding region was placed in-frame at the 5 or 3 ends of the coding region in F-GPX1 to produce chimeric 50 -51-kDa GFP/GPX1 proteins, Sec incorporation at UGA codons, formerly close to the 5 or 3 ends, was increased to levels comparable to the UGA at U47. Insertion of GFP after the UAA-stop was just as effective in increasing Sec insertion efficiency as GFP inserted before the stop. These studies used a recombinant expression model that incorporated Sec at non-native UGA codons at rates equal to those of endogenous glutathione peroxidase-1 and showed that the efficiency of Sec incorporation can be modulated by UGA position; Sec incorporation at high efficiency appears to require that the UGA be >21 nucleotides from the AUG-start and >204 nucleotides from the selenocysteine insertion sequence element.Mammalian glutathione peroxidase-1 (GPX1 1

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' untranslated region (3'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'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'UTR (pRc/Delta3'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/Delta3'UTR transfectants were compared with pRc/CMV control transfectants. Among the wild type and transfected CHO cells, Se-deficient GPX activity levels averaged 35 +/- 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/Delta3'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'UTR is necessary for Se regulation of GPX mRNA levels in addition to its role in Se incorporation.