Se-deficient Cells Research Articles

Extracellular Vesicles Derived from Selenium-Deficient MAC-T Cells Aggravated Inflammation and Apoptosis by Triggering the Endoplasmic Reticulum (ER) Stress/PI3K-AKT-mTOR Pathway in Bovine Mammary Epithelial Cells.

Selenium (Se) deficiency disrupts intracellular REDOX homeostasis and severely deteriorates immune and anti-inflammatory function in high-yielding periparturient dairy cattle. To investigate the damage of extracellular vesicles derived from Se-deficient MAC-T cells (SeD-EV) on normal mammary epithelial cells, an in vitro model of Se deficiency was established. Se-deficient MAC-T cells produced many ROS, promoting apoptosis and the release of inflammatory factors. Extracellular vesicles were successfully isolated by ultrahigh-speed centrifugation and identified by transmission electron microscopy, particle size analysis, and surface markers (CD63, CD81, HSP70, and TSG101). RNA sequencing was performed on exosomal RNA. A total of 9393 lncRNAs and 63,155 mRNAs transcripts were identified in the SeC and SeD groups, respectively, of which 126 lncRNAs and 955 mRNAs were differentially expressed. Furthermore, SeD-EV promoted apoptosis of normal MAC-T cells by TUNEL analysis. SeD-EV significantly inhibited Bcl-2, while Bax and Cleaved Caspase3 were greatly increased. Antioxidant capacity (CAT, T-AOC, SOD, and GSH-Px) was inhibited in SeD-EV-treated MAC-T cells. Additionally, p-PERK, p-eIF2α, ATF4, CHOP, and XBP1 were all elevated in MAC-T cells supplemented with SeD-EV. In addition, p-PI3K, p-Akt, and p-mTOR were decreased strikingly by SeD-EV. In conclusion, SeD-EV caused oxidative stress, thus triggering apoptosis and inflammation through endoplasmic reticulum stress and the PI3K-Akt-mTOR signaling pathway, which contributed to explaining the mechanism of Se deficiency causing mastitis.

Selenium supplementation through Se-rich dietary matrices can upregulate the anti-inflammatory responses in lipopolysaccharide-stimulated murine macrophages

ABSTRACTThe accessibility of selenium from naturally enriched sources such as cereals crops can potentially be used as selenium supplements to support nutritional requirements. Dietary selenium supplementation, as Se-rich wheat extracts, on RAW264.7 macrophage cells enhanced the antioxidant capacity via augmentation of cellular selenoprotein glutathione peroxidase 1 (GPx-1) expression in the absence or presence of lipopolysaccharide (LPS) treatment. Cells were supplemented with Se in the form of sodium selenite (SS), seleniferous wheat extract (SeW) and seleniferous wheat extract with rMETase treatment (SeW + rMET) at three different concentrations. Cells supplemented with SS and SeW + rMET showed increase in GPx-1 expression as compared to SeW-treated cells. SeW + rMET, further, downregulated the LPS-induced expression of cyclooxygenase-2, microsomal PGE synthase-1 and inducible nitric oxide synthase w.r.t. Se-deficient cells, while the expression of haematopoietic PGD synthase was upregulated. This demonstrates SeSup effectively modulates the expression of inflammatory responses, indicating the potential benefits of dietary selenium supplementation.

Interplay between the chalcone cardamonin and selenium in the biosynthesis of Nrf2-regulated antioxidant enzymes in intestinal Caco-2 cells

Selenoenzymes and nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated phase II enzymes comprise key components of the cellular redox and antioxidant systems, which show multiple interrelations. Deficiency of the micronutrient selenium (Se) and impaired biosynthesis of selenoproteins have been reported to result in induction of Nrf2 target genes. Conversely, transcription of the selenoenzymes glutathione peroxidase 2 (GPx2) and thioredoxin reductase 1 (TrxR1) is up-regulated upon Nrf2 activation. Here, we have studied the interplay between Se and the secondary plant metabolite cardamonin, an Nrf2-activating chalcone, in the regulation of Nrf2-controlled antioxidant enzymes. Se-deficient and Se-repleted (sodium selenite-supplemented) human intestinal Caco-2 cells were exposed to cardamonin. Uptake of cardamonin by the Caco-2 cells was independent of their Se status. Cardamonin strongly induced gene expression of GPx2 and TrxR1. However, cardamonin treatment did not result in elevated GPx or TrxR activity and protein levels, possibly relating to a concomitant down-regulation of O-phosphoseryl-tRNA(Sec) kinase (PSTK), an enzyme involved in translation of selenoprotein mRNAs. On the other hand, induction of the Nrf2-regulated enzyme heme oxygenase 1 (HO-1) by cardamonin was diminished in Se-replete compared to Se-deficient cells. Our findings suggest that cardamonin interferes with the biosynthesis of Nrf2-regulated selenoenzymes, in contrast to the Nrf2-activating isothiocyanate compound sulforaphane, which has been shown earlier to synergize with Se-mediated cytoprotection. Conversely, the cellular Se status apparently affects the cardamonin-mediated induction of non-selenoprotein antioxidant enzymes such as HO-1.

Modulation of thioredoxin reductase-2 expression in EAhy926 cells: Implications for endothelial selenoprotein hierarchy

Background We examined the expression of the mitochondrial selenoenzyme TrxR2 in the endothelial cell line EAhy926 under conditions known to modify its cytoplasmic counterpart TrxR1. Methods Cells were cultured with varying concentrations of selenite, sulforaphane or the Ca 2+ ionophore A23187 for 72-h, prior to assay of TrxR concentration and activity. Further cultures underwent prolonged (7-day) Se-depletion before selenoprotein measurement. Results In Se-deficient cultures, neither Se, A23187 or sulforaphane affected TrxR2 concentration, while these treatments induced TrxR1 concentration ( p < 0.05). When co-incubated, optimal concentrations of Se (40 nM) and sulforaphane (4 μM) only modestly increased TrxR2 protein (∼ 1.3-fold), compared with TrxR1 (∼ 4-fold). In Se-deficient cells, TrxR activity was unaffected by sulforaphane or A23187. Prolonged Se-depletion caused a comparatively small reduction in TrxR2 (66% TrxR2 retained) against TrxR1 and glutathione peroxidase-1 activity (38% and 17% retained, respectively). Conclusions The relative resistance of TrxR2 to Se-deprivation and induction by sulforaphane and A23187 suggests TrxR2 lies near the top of the selenoprotein hierarchy in EAhy926 cells and exhibits near maximum expression under a range of culture conditions. In Se deficiency an inactive (possibly truncated) TrxR1 is produced in response to stimulus by sulforaphane and A23187. General significance These observations underpin a likely critical antioxidant role for TrxR2 and TrxR1 in the endothelium.

Regulation of expression of apolipoprotein A-I by selenium status in human liver hepatoblastoma cells

Cardiomyopathy is common to areas with low selenium (Se) intake and in patients receiving total parenteral nutrition. Although controversial, a few studies have suggested a protective role for Se in coronary heart disease on the basis of modulation of high-density lipoproteins (HDL). In this study, the role of Se as a positive regulator of expression of a key HDL, apolipoprotein A-I (apoA-I), has been evaluated in human hepatoblastoma (HepG2) cell culture model. We further examined if the transcription of apoA-I, driven by the nuclear hormone receptor, peroxisome-proliferator activated receptor, PPARalpha, was trans-repressed by the presence of the oxidative stress-responsive transcription factor, NF-kappaB. Modulation of expression of apoA-I and activation of nuclear NF-kappaB subunit p65 and PPARalpha by Se status were evaluated by Western blot and luciferase-based assays. Interaction of p65 with PPARalpha was evaluated by immunoprecipitation. HepG2 cultured in media with Se (100 nM) demonstrated an increase in the expression of apoA-I when compared to Se-deficient cells. A similar trend was also seen in mice that were supplemented with 0.4 ppm of Se as sodium selenite. Treatment of Se-supplemented cells with bacterial lipopolysaccharide (LPS) showed induction of apoA-I. Supplementation of hepatocytes with Se decreased the nuclear levels of p65, which prevented its interaction with PPARalpha to modulate apoA-I transcription. Our results suggest that supplementation of hepatocytes with Se mitigates oxidative stress-dependent repression of apoA-I expression by suppressing the NF-kappaB pathway, which allows PPARalpha to effectively drive the expression of apoA-I.

In vitro effects of selenium deficiency on West Nile virus replication and cytopathogenicity

BackgroundSelenium (Se) deficiency plays an important role in viral pathogenesis. To understand the effects of Se deficiency on West Nile virus (WNV) infection, we analyzed cytopathogenicity, apoptosis and viral replication kinetics, using a newly developed Se-deficient cell culture system.ResultsBoth Vero and SK-N-SH cells grown in Se-deficient media exhibited a gradual loss of glutathione peroxidase (GPx1) activity without any significant effect on cell growth and viability. In SK-N-SH cells, Se deficiency had no effect on the expression of key antioxidant enzymes, including manganese- and copper-zinc superoxide dismutase (MnSOD and CuZnSOD), catalase and inducible nitric oxide synthase, whereas Vero cells demonstrated a significant increase in the expression of MnSOD and an overall increase in oxidative stress (OS) at day 7 post-induction of Se deficiency. At 2 days after infection with WNV, CPE and cell death were significantly higher in WNV-infected Se-deficient Vero cells, compared to WNV-infected control cells. Furthermore, WNV-induced apoptosis was significantly heightened in Se-deficient cells and was contributed by loss of mitochondrial membrane potential and increased caspase activity. However, no significant difference was found in WNV copy numbers between control, Se-adequate and Se-deficient cell cultures.ConclusionOverall results demonstrate that the in vitro Se-deficient model can be used to study responses of WNV to this essential nutrient. Although Se deficiency has no in vitro effect on WNV replication kinetics, adequate Se is presumably critical to protect WNV-infected cells against virus-induced cell death.

Selenium is critical for the regulation of tumor suppressor and pro‐inflammatory gene expression in human colon Caco‐2 cells

The essential role of Se in growth of most mammalian cells is well recognized but certain cancer cells appear to have acquired a survival advantage under conditions of Se‐deficiency. We generated Se‐deficient colon Caco‐2 cells by gradually reducing serum in the media because serum contains a trace amount of Se, which is sufficient to maintain the expression of cellular selenoproteins such as glutathione peroxidase (GPx). The GPx activity of Se‐deficient Caco‐2 cells was 10.8 mU/mg protein compared to 133.6∼146.3 mU/mg protein in Caco‐2 cells supplemented with 500 nmol/L selenite, Se‐ (methyl)selenocysteine or selenomethionine (three tested Se chemical forms) after 7‐ day culture in serum free media. Interestingly, there were not detectable differences on cell growth, cell cycle progression between Se‐deficient and Se‐supplemented cells. However, cancer signal pathway‐specific array assay and the real time PCR analysis demonstrated that Se increased the expression of humoral defense gene (A2M) and tumor suppressor‐related genes (IGFBP3, HHIP) but decreased pro‐inflammatory gene (CXC L9, HSPB2) expression at cellular nutritional Se doses, even though colon Caco‐2 cells were resistant to Se deprivation.

Size effect of elemental selenium nanoparticles (Nano-Se) at supranutritional levels on selenium accumulation and glutathione S-transferase activity

It has been shown that 36 nm Nano-Se has lower toxicity than selenite or selenomethionine, but these forms of selenium (Se) all possess similar ability to increase selenoenzyme levels. The size of nanoparticles plays an important role in their biological activity: as expected, 5–200 nm Nano-Se can directly scavenge free radicals in vitro in a size-dependent fashion. However, in Se-deficient cells and Se-deficient mice, the size effect of Nano-Se on increasing selenoenzymes and liver Se disappears unexpectedly. We hypothesize that under conditions of Se deficiency, the avidity of Se uptake mechanisms may be increased to maintain the biosynthesis of selenoenzymes, which are fundamental for redox homeostasis. This increased avidity may override the potential advantage of small size Nano-Se seen under Se-replete conditions, thereby eliminating the size effect. Once selenoenzymes have been saturated, Se uptake mechanisms may downregulate; accordingly, the size effect of Nano-Se can then reappear. To test this hypothesis, Se-deficient mice were administered either 36 or 90 nm Nano-Se at supranutritional doses, in both a short-term model and a single-dose model. Under these conditions, Nano-Se showed a size effect on Se accumulation and glutathione S-transferase (GST) activity. A size effect of Nano-Se was found in 15 out of 18 total comparisons between sizes at the same dose and time in the two models. Furthermore, the magnitude of the size effect was more prominent on Se accumulation than on GST activity. GST is strictly regulated by transcriptional and translational mechanisms, so its increase in activity normally does not exceed 3-fold. In contrast, the homeostasis of Se accumulation is not as tightly controlled. In the present experiments, GST activity had reached or was approaching saturation, but liver Se was far below saturation. Therefore, our results strongly suggest that the saturation profile of the tested biomarker has an impact on the size effect of Nano-Se. Since both GST and small molecular weight selenocompounds accumulated in vivo are important intermediates for chemoprevention by Se, our results also suggest that Nano-Se should be most effective as a chemopreventive agent at smaller particle size.

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3′UTR, or a combination of the TrxR1 5′ and 3′UTRs. Cell lines expressing constructs with the TrxR1 3′UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3′UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5′UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5′ or 3′UTR sequences when assessed using luciferase reporter constructs.

Selenium is critical for cancer‐signaling gene expression but not cell proliferation in human colon Caco‐2 cells

Selenium (Se) is a potential anticarcinogenic nutrient, and the essential role of Se in cell growth is well recognized but certain cancer cells appear to have acquired a survival advantage under conditions of Se-deficiency. To understand the molecular basis of Se-anticancer effects at nutritional doses (nmol/L) for cultured cells, we generated Se-deficient colon Caco-2 cells by gradually reducing serum in media because serum contains a trace amount of Se. The glutathione peroxidase (GPx) activity of Se-deficient Caco-2 cells was 10.8 mU/mg protein compared to 133.6 approximately 146.3 mU/mg protein in Caco-2 cells supplemented with 500 nmol/L selenite, SeMSC or SeMet (three tested Se-chemical forms) after 7-d culture in serum free media. Interestingly, there were no detectable differences in cell growth, cell cycle progression between Se-deficient cells and cells supplemented with 500 nmol/L Se. To examine differential cancer signaling-gene expression between Se-deficient and Se-supplemented cells, we employed a cancer signal pathway-specific array assay coupled with the real time PCR analysis. Our data demonstrate that although Caco-2 cells are resistant to Se deprivation, Se may exert its anticancer property through increasing the expression of humoral defense gene (A2M) and tumor suppressor-related genes (IGFBP3, HHIP) while decreasing pro-inflammatory gene (CXC L9, HSPB2) expression.

Selenium supplementation acting through the induction of thioredoxin reductase and glutathione peroxidase protects the human endothelial cell line EAhy926 from damage by lipid hydroperoxides

The human endothelial cell line EAhy926 was used to determine the importance of selenium in preventing oxidative damage induced by tert-butyl hydroperoxide ( tert-BuOOH) or oxidised low density lipoprotein (LDLox). In cells grown in a low selenium medium, tert-BuOOH and LDLox killed cells in a dose-dependent manner. At 555 mg/l LDLox or 300 μM tert-BuOOH, >80% of cells were killed after 20 h. No significant cell kill was achieved by these agents if cells were pre-incubated for 48 h with 40 nM sodium selenite, a concentration that maximally induced the activities of cytoplasmic glutathione peroxidase (cyGPX; 5.1-fold), phospholipid hydroperoxide glutathione peroxidase (PHGPX;1.9-fold) and thioredoxin reductase (TR; 3.1-fold). Selenium-deficient cells pre-treated with 1 μM gold thioglucose (GTG) (a concentration that inhibited 25% of TR activity but had no inhibitory effect on cyGPX or PHGPX activity) were significantly ( P<0.05) more susceptible to tert-BuOOH toxicity (LC 50 110 μM) than selenium-deficient cells (LC 50 175 μM). This was also the case for LDLox. In contrast, cells pre-treated with 40 nM selenite prior to exposure to GTG were significantly more resistant to damage from tert-BuOOH and LDLox than Se-deficient cells. Treatment with GTG or selenite had no significant effect on intracellular total glutathione concentrations. These results suggest that selenium supplementation, acting through induction of TR and GPX, has the potential to protect the human endothelium from oxidative damage.

Selenium deficiency increases the expression of inducible nitric oxide synthase in RAW 264.7 macrophages: role of nuclear factor-kappaB in up-regulation.

The inducible isoform of nitric oxide synthase (iNOS) is implicated in atherosclerosis, malignancy, rheumatoid arthritis, tissue and reperfusion injuries. A key determinant of the pro-oxidant versus protective effects of NO is the underlying redox status of the tissue. Selenoproteins, such as glutathione peroxidases (GPxs) and thioredoxin reductases, are key components of cellular defence and promote optimal antioxidant/oxidant balance. In this study, we have investigated the relationship between Se status, iNOS expression and NO production in Se-deficient and Se-supplemented RAW 264.7 macrophage cell lines. The cellular GPx activity, a measure of Se status, was 17-fold lower in Se-deficient RAW 264.7 cells and the total cellular oxidative tone, as assessed by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate, was higher in the Se-deficient cells than the Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation of these cells in culture, we found significantly higher iNOS transcript and protein expression levels with an increase in NO production in Se-deficient RAW 264.7 cells than the Se-supplemented cells. Electrophoretic mobility-shift assays, nuclear factor-kappaB (NF-kappaB)-luciferase reporter assays and Western blot analyses indicate that the increased expression of iNOS in Se deficiency could be due to an increased activation and consequent nuclear localization of the redox-sensitive transcription factor NF-kappaB. These results suggest an inverse relationship between cellular Se status and iNOS expression in LPS-stimulated RAW 264.7 cells and provide evidence for the beneficial effects of dietary Se supplementation in the prevention and/or treatment of oxidative-stress-mediated inflammatory diseases.

Nuclear factor-κB mediates over-expression of cyclooxygenase-2 during activation of RAW 264.7 macrophages in selenium deficiency

Selenium (Se) is an essential micronutrient for all mammalian species and is associated with a variety of physiological functions, notably immune system, in the form of selenoproteins. Inadequate Se nutrition has been linked to various diseases, including rheumatoid arthritis, cardiomyopathy, and cancer. Important to this discussion is that cyclooxygenase-2 (COX-2) is over-expressed in all the aforesaid pathologies; however, a casual relationship between Se status and COX-2 expression remains to be established. The present study is based on the hypothesis that oxidant stress, a consequence of Se deficiency, lowers the activation potential of the redox-sensitive transcription factor, NF-κB, and that the activated NF-κB is required for the altered expression of COX-2. To test this hypothesis, we have investigated the relationship between Se status and COX-2 expression in response to LPS stimulation in RAW 264.7, a macrophage-like cell line. In Se-deficient cells, the Se-dependent glutathione peroxidase activity (Se-GPx), a measure of Se status, was markedly reduced and the overall oxidative stress was significantly higher than Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation, we found 2-3-folds higher COX-2 protein expression as well as higher PGE 2 levels in Se-deficient cells than Se-supplemented cells. In comparison, COX-1 protein expression was not affected by either LPS stimulation or Se status. Following LPS stimulation, the nuclear localization of NF-κB was significantly increased in Se-deficient macrophages, thereby leading to increased expression of COX-2. This is the first report demonstrating an inverse relationship between Se status and the expression of COX-2.

Increased 15-HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells

Selenium (Se) is an integral component of glutathione peroxidase and is able to detoxify peroxides that can affect arachidonic acid (AA) metabolism, thereby influencing eicosanoid biosynthesis. This study investigated the effects of oxidant stress, a consequence of Se deficiency, on eicosanoid formation and important key enzyme expression in bovine aortic endothelial cells (BAEC). Bovine aortic endothelial cells cultured in Se-deficient media and stimulated with tumor necrosis factor α or H 2O 2 produced significantly less prostacyclin (PGI 2) and more 15-hydroxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and thromboxane than Se-supplemented BAEC. Additionally, reverse transcription polymerase chain reaction and immunoblotting determined that the mRNA and protein levels of the eicosanoid forming enzymes cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), and PGI synthase were not significantly changed. The addition of 15-HPETE to Se-supplemented BAEC inhibited the production of PGI 2 suggesting that the accumulation of lipid hydroperoxides during Se-deficiency may be the underlying factor in the altered eicosanoid production during Se deficiency. Furthermore, inhibition of COX and addition of PGH 2 to Se-deficient or Se-supplemented BAEC still resulted in lower PGI 2 formation by Se-deficient cells. Together, these results suggest that Se deficiency modifies eicosanoid production by affecting the activity of key enzymes, particularly PGI synthase, rather than their transcription or translation.

Altered eicosanoid biosynthesis in selenium-deficient endothelial cells

Selenium (Se) is an integral part of the Se-dependent glutathione peroxidase (Se-GSH-Px) catalytic domain. By modulating the cellular levels of fatty acid hydroperoxides, Se-GSH-Px can influence key enzymes of arachidonic acid cascade, in this case cyclooxygenase (COX) and lipoxygenase (LOX). To investigate this phenomenon, the effects of cellular Se status on the enzymatic oxidation of arachidonic acid were investigated in bovine mammary endothelial cells (BMEC), which were cultured in either Se-deficient (−Se) or Se-adequate (+Se) media. When stimulated with calcium ionophore A23187, BMEC produced eicosanoids of both COX and LOX pathways. Compared with the Se-adequate cells, the production of prostaglandin I 2 (PGI 2), prostaglandin F 2 (PGF 2α), and prostaglandin E 2 (PGE 2) was significantly decreased in Se-deficient cells, whereas the production of thromboxane A 2 (TXA 2) was markedly increased in the −Se BMEC cultures. Although the enzymatic oxidation of arachidonic acid by the LOX pathway was found to be relatively less than by the COX pathway, the BMEC cultured in −Se media produced significantly more 15-hydroperoxyeicosatetraenoic acid (15-HPETE) than the +Se cells produced. Based on these results, we postulate that cellular Se status plays an important regulatory role in the enzymatic oxidation of arachidonic acid by the COX and LOX pathways. The altered eicosanoid biosynthesis, especially the overproduction of 15-HPETE, in −Se BMEC may be one of the underlying biochemical phenomena responsible for vascular dysfunction during Se deficiency.

Thyroid stimulating hormone and selenium supply interact to regulate selenoenzyme gene expression in thyroid cells (FRTL-5) in culture

In the absence of a sodium selenite supplement, FRTL-5 cells showed a reduced activity of cytosolic glutathione peroxidase (cGSH-Px), a marker of selenium status, indicating the cells were Se-deficient. Se-deficient cells showed a 65% reduction in cGSH-Px mRNA abundance but little change in abundance of either phospholipid hydroperoxide glutathione peroxidase or type 1 deiodinase (IDI) mRNA. In Se-replete cells increased thyroid stimulating hormone (TSH) caused a small decrease in IDI abundance but in Se-deficient cells TSH caused a large increase. The results indicate an interaction between TSH and Se status in the regulation of thyroid selenoenzyme synthesis.

The protective role of glutathione peroxidase in apoptosis induced by reactive oxygen species.

Selenium-dependent glutathione peroxidase (GPx) plays a protective role in oxidative stress-induced apoptosis. In this study, we demonstrated that MDBK cells, a bovine renal epithelial cell line, exhibited internucleosomal DNA fragmentation characteristic of apoptotic cell death under selenium-deficient conditions with lower doses of hydrogen peroxide (H2O2) than under selenium-supplemented ones. This was due to a decreased amount of GPx in the cells under selenium-deficient conditions, because other antioxidative enzyme activities were not affected by the selenium supplementation. Cumene hydroperoxide also induced DNA fragmentation in selenium-deficient cells but no ladder formation was observed. Flow cytometric analysis showed that selenium-deficient cells were less capable of scavenging intracellular peroxides after exposure to exogenous H2O2 than selenium-supplemented ones. In contrast, there was no difference in viability between selenium-supplemented and non-supplemented cells in cell survival after exposure to menadione, which activates the electron transport system and increases intracellular superoxide radicals. Clofibrate, a peroxisomal proliferator and an inducer of catalase (CAT), partially protected both Se-deficient and Se-supplemented cells from exogenous H202. We concluded that selenium-deficient cells were more easily brought to apoptotic cell death by peroxides, but not by superoxide radicals, than selenium-supplemented ones and that CAT could compensate for the depletion of GPx to a certain degree by scavenging H2O2.

Extracellular Glutathione Peroxidase mRNA and Protein in Human Cell Lines

Extracellular glutathione peroxidase (E-GPx) and cellular glutathione peroxidase (C-GPx) are selenoenzymes encoded by two distinct genes. Using specific immunoprecipitations of [75Se]selenium metabolically labeled human cell lines in culture, it was found that Caco-2, Hep3B, Hep G2, and Caki-2 synthesize C-GPx and E-GPx and secrete E-GPx. HBL-100, BT-20, and MCF-7 synthesize only C-GPx. The relationship between Se status (as determined by C-GPx activity) and E-GPx and C-GPx mRNA steady-state levels was investigated in Hep G2, Caco-2, and Caki-2. The most Se-deficient Hep G2, Caco-2, and Caki-2 cells had 8.7 ± 2.6, 11.2 ± 4.9, and 9.4 ± 5.0%, respectively, of C-GPx activity of the replete cells. The steady-state levels of mRNA were measured by Northern and slot blot hybridization analysis. By Northern analysis, a single band was present at 1.0 and 1.80 kb for C-GPx and E-GPx mRNA, respectively, in all three cell lines. Scanning densitometry of the blots revealed that the most Se-deficient cells had 30-50% C-GPx mRNA and 60-80% E-GPx mRNA of the replete cells. It is concluded that, in addition to previously examined human cell lines, Hep3B and Caco-2 make and secrete E-GPx while HBL-100 and BT-20 do not. The slightly reduced levels of C-GPx and E-GPx mRNA in Se-deficient human cell lines can only partially account for the decreased C-GPx activity in Se-deficient human cell lines.

Modulation of glutathione peroxidase expression by selenium: effect on Human MCF-7 breast cancer cell transfectants expressing a cellular glutathione peroxidase cDNA and doxorubicin-resistant MCF-7 cells

We have studied the effect of selenium on the expression of a cellular glutathione peroxidase, GSHPx-1, in transfected MCF-7 cells and in doxorubicin-resistant (Adrr) MCF-7 cells. A GSHPx-1 cDNA with a Rous Sarcoma virus promoter was transfected into a human mammary carcinoma cell line, MCF-7, which has very low endogenous cytosolic glutathione (GSH) peroxidase activity and no detectable message. The transfectant with the highest GSH peroxidase activity among the isolates, MCF-7H6, was characterized. Adrr MCF-7 cells, a subline of MCF-7 cells, also has elevated GSH peroxidase activity. GSH peroxidase expressed by MCF-7H6 and Adrr MCF-7 cells is similar to the endogenous GSHPx-1 based on molecular weight, immunoreactivity, and metabolic labeling with 75Se. MCF-7H6 and Adrr MCF-7 cells grown in Se-deficient media had 2.6 +/- 2.4 (mean +/- S.D.) and 4.2 +/- 3.6 units/mg protein of GSH peroxidase specific activity, respectively. Se supplementation increased GSH peroxidase activity in a concentration- and time-dependent fashion. Enzymatic activity reached a level of 164 +/- 62 in MCF-7H6 cells and 114 +/- 27 in Adrr MCF-7 cells within 5 days of growth in media supplemented with 30 nM Se. Northern analysis revealed that Se-deficient MCF-7H6 cells expressed 2.1 +/- 0.4-fold less GSHPx-1 mRNA than their Se-sufficient counterparts. Similarly, Se-deficient Adrr MCF-7 cells expressed 3.3 +/- 1.8-fold less GSHPx-1 mRNA than their Se-supplemented counterparts after the quantity of mRNA was normalized with beta-actin. These studies suggest that modulation of GSH peroxidase activity by Se in both MCF-7H6 transfectants expressing pRSV-GSHPx-1 and Adrr MCF-7 cells expressing endogenous GSHPx-1 occurs largely at the translational level, and to a lesser degree at the level of mRNA, possibly by stabilizing GSHPx-1 mRNA since the transfected cDNA in MCF-7H6 cells has only 5 nucleotides 5' to the AUG initiation codon.

Tert-butyl hydroperoxide-induced hemolysis of alpha-tocopherol-decreased erythrocytes from selenium-deficient and selenium-adequate rats.

The protective function of alpha-tocopherol, glutathione (GSH), and glutathione peroxidase (GSH-Px) from tert-butyl hydroperoxide (t-BuOOH)-induced hemolysis was studied with the erythrocytes from male Wistar rats fed selenium (Se)-adequate or -deficient diet for 3 months. By the preincubation with a water-soluble radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), at 10 mM for 6 h at 37 degrees C, alpha-tocopherol levels of the erythrocytes were decreased to 40% of the original level, that is, to the level insufficient for supporting the normal functions of the erythrocytes. With the Se-deficient cells, the hemolysis proceeded rapidly irrespective of the presence or absence of GSH in the incubation medium, and irrespective of the presence or absence of AAPH in the preincubation medium. Contrarily, GSH suppressed the hemolysis of Se-adequate cells which were preincubated with and without AAPH. These results are consistent with the notion that Se serves as the prime, important defense mechanism in the t-BuOOH-induced hemolysis through the activity of GSH-Px. Either alpha-tocopherol or GSH by itself, or both by themselves, may not play so significant a role as Se does in suppressing the hemolysis.