Synergy between sulforaphane and selenium in the up-regulation of thioredoxin reductase and protection against hydrogen peroxide-induced cell death in human hepatocytes.

Dietary isothiocyanates and selenium are chemopreventive agents and potent inducers of antioxidant enzymes. It has been previously shown that sulforaphane and selenium have a synergistic effect on the upregulation of thioredoxin reductase-1 (TrxR-1) in human hepatoma HepG2 cells. In this paper, further evidence is presented to show that sulforaphane and selenium synergistically induce TrxR-1 expression in immortalised human hepatocytes. Sulforaphane was found to be more toxic toward hepatocytes than HepG2 cells with IC50=25.1 and 56.4 µM, respectively. Sulforaphane can protect against hydrogen peroxide-induced cell death and this protection was enhanced by co-treatment with selenium. Using siRNA to knock down TrxR-1 or Nrf2, sulforaphane (5 µM)-protected cell viability was reduced from 73% to 46% and 34%, respectively, suggesting that TrxR-1 is an important enzyme in protection against hydrogen peroxide-induced cell death. Sulforaphane-induced TrxR-1 expression was positively associated with significant levels of Nrf2 translocation into the nucleus, but co-treatment with selenium showed no significant increase in Nrf2 translocation. Moreover, MAPK (ERK, JNK and p38) and PI3K/Akt signalling pathways were found to play no significant role in sulforaphane-induced Nrf2 translocation into the nucleus. However, blocking ERK and JNK signalling pathways decreased sulforaphane-induced TrxR-1 mRNA by about 20%; whereas blocking p38 and PI3K/AKT increased TrxR-1 transcription. In summary, a combination of sulforaphane and selenium resulted in a synergistic upregulation of TrxR-1 that contributed to the enhanced protection against free radical-mediated oxidative damage in human hepatocytes.

Selenium supplementation attenuates procollagen-1 and interleukin-8 production in fat-loaded human C3A hepatoblastoma cells treated with TGFbeta1.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and hepatic steatosis. Non-alcoholic steatohepatitis (NASH) is a serious consequence of NAFLD where chronic tissue damage and inflammation result in fibrosis which may progress to cirrhosis. Transforming growth factor beta1 (TGFbeta1), proinflammatory cytokines and oxidative stress are thought to play crucial roles in the pathogenesis of these conditions. The contributions of individual liver cell types to fibrogenesis remain controversial and the influence of selenium status has not been investigated. METHODS: In this study we have used a cell culture model of fat-loading using oleate-treated human hepatoblastoma (C3A) cells to investigate how fat-loading and selenium status might influence the production of collagen in response to TGFbeta1. The secretion of inflammatory cytokines was also investigated, together with the epithelial character of the treated cells. RESULTS: We found that in response to treatment with TGFbeta1, C3A cells produced mRNA encoding the pro-alphaI chain of procollagen I, secreted procollagen I peptide, up-regulated production of the proinflammatory cytokine interleukin-8 (IL-8) and the mesenchymal marker vimentin, and down-regulated albumin production. Most of these responses were considerably enhanced when cells were fat-loaded with oleate and were attenuated by selenium addition at a dose that optimised the expression of thioredoxin reductase and glutathione peroxidase. CONCLUSIONS: Our data establish that both fat-loading and suboptimal selenium status enhance collagen and IL-8 production by C3A hepatocytes in response to TGFbeta1, possibly as part of an epithelial to mesenchymal transition. GENERAL SIGNIFICANCE: These findings suggest that the hepatocyte may be an important contributor to the pathogenesis of fibrosis associated with NAFLD.

Randomized controlled trial of the effect of selenium supplementation on thyroid function in the elderly in the United Kingdom.

BACKGROUND: Thyroid function depends on the essential trace mineral selenium, which is at the active center of the iodothyronine deiodinase enzymes that catalyze the conversion of the prohormone thyroxine (T(4)) to the active form of thyroid hormone, triiodothyronine (T(3)). OBJECTIVE: Because selenium intake in the United Kingdom has fallen during the past 25 y, we wanted to determine whether current selenium status might be limiting conversion of T(4) to T(3) in the elderly, in whom marginal hypothyroidism is relatively common. DESIGN: We investigated the effect of selenium supplementation in a double-blind, placebo-controlled trial in 501 elderly UK volunteers. Similar numbers of men and women from each of 3 age groups, 60-64 y, 65-69 y, and 70-74 y, were randomly allocated to receive 100, 200, or 300 microg Se/d as high-selenium yeast or placebo yeast for 6 mo. As part of the study, plasma selenium, thyroid-stimulating hormone, and total and free T(3) and T(4) were measured. Data from 368 euthyroid volunteers who provided blood samples at baseline and 6 mo were analyzed. RESULTS: Although selenium status at baseline correlated weakly with free T(4) (r = -0.19, P < 0.001) and with the ratio of free T(3) to free T(4) (r = 0.12, P = 0.02), we found no evidence of any effect of selenium supplementation on thyroid function, despite significant increases in plasma selenium. However, baseline plasma selenium in our study (x: 91 microg/L) was somewhat higher than in previous supplementation studies in which apparently beneficial effects were seen. CONCLUSION: We found no indication for increasing selenium intake to benefit T(4) to T(3) conversion in the elderly UK population.

Lack of effect of oral selenite on p53 associated gene expression during TL01 therapy of psoriasis patients.

Selenium (Se) has protective properties against ultraviolet (UV)-induced changes in skin cells in vitro but little is known about such activity in human subjects. In the present study, seven patients with psoriasis ingested 400 microg of sodium selenite daily during a 4 week course of whole-body narrow-band UVB (TL01) therapy while six more psoriasis patients, similarly irradiated, ingested a placebo. Skin biopsies, collected at the start and end of the phototherapy were analysed for phosphorylated p53, Fas, Bcl-2, Bax and oxidized guaninosine, and for numbers of Langerhans and sunburn cells. Following the TL01 therapy, no significant difference was observed for any of these markers when the Se group was compared with the placebo group of patients, although p53 and Bcl-2 expression decreased in the Se supplemented group.

Genetic polymorphisms in the human selenoprotein P gene determine the response of selenoprotein markers to selenium supplementation in a gender-specific manner (the SELGEN study).

Selenium (Se), a micronutrient essential for human health, is incorporated into at least 25 selenoproteins including selenoprotein P (SePP), which transports Se within the body. This research identified two single nucleotide polymorphisms (SNPs) in the SePP gene, one in the coding region (position 24731, causing an Ala to Thr change) and one in the 3'untranslated region (position 25191). Their frequency was similar in Caucasian, Chinese, and South Asian populations. Prospectively genotyped volunteers were supplemented for 6 wk with 100 microg sodium selenite/day. Blood samples were analyzed for plasma Se and selenoprotein biomarkers at baseline, after supplementation, and during a washout period. Plasma Se, SePP, and glutathione peroxidase 3 (GPx3) levels increased with supplementation. Baseline plasma Se content depended on both SePP genotypes and body mass index. Presupplementation SePP concentration was associated with gender and genotype at SNP 24731 and postsupplementation concentration with SNP 25191. Both SNPs and gender were associated with differences in GPx3 activity, plasma, and erythrocyte thioredoxin reductase 1 concentrations and lymphocyte glutathione peroxidase 1 and 4 activities and concentrations. In conclusion, the data reveal two common functional SNPs within the human SePP gene that may predict behavior of biomarkers of Se status and response to supplementation and thus susceptibility to disease.

Sulforaphane, erucin, and iberin up-regulate thioredoxin reductase 1 expression in human MCF-7 cells.

Isothiocyanates (ITCs) found in cruciferous vegetables are potentially important anticarcinogenic phytochemicals for many types of cancers including breast cancer. In this study, we have shown that three isothiocyanates, sulforaphane, erucin, and iberin, are potent inducers of thioredoxin reductase 1 (TrxR1) in human breast cancer MCF-7 cells. Sulforaphane, erucin, and iberin at 1 microM induce TrxR1 mRNA 2-3-fold within 8 h of treatment, and induce mRNA 5-7-fold with 12 microM ITC treatments. Selenium did not affect sulforaphane-induced TrxR1 mRNA levels, but significantly enhanced both TrxR1 protein expression (up to 9-fold in erucin treatment) and corresponding activities. These results suggest that dietary ITCs are important factors in the regulation of redox status through the induction of the selenoprotein thioredoxin reductase.

Selenium and endocrine systems.

The trace element selenium (Se) is capable of exerting multiple actions on endocrine systems by modifying the expression of at least 30 selenoproteins, many of which have clearly defined functions. Well-characterized selenoenzymes are the families of glutathione peroxidases (GPXs), thioredoxin reductases (TRs) and iodothyronine deiodinases (Ds). These selenoenzymes are capable of modifying cell function by acting as antioxidants and modifying redox status and thyroid hormone metabolism. Se is also involved in cell growth, apoptosis and modifying the action of cell signalling systems and transcription factors. During thyroid hormone synthesis GPX1, GPX3 and TR1 are up-regulated, providing the thyrocytes with considerable protection from peroxidative damage. Thyroidal D1 in rats and both D1 and D2 in humans are also up-regulated to increase the production of bioactive 3,5,3'-tri-iodothyronine (T3). In the basal state, GPX3 is secreted into the follicular lumen where it may down-regulate thyroid hormone synthesis by decreasing hydrogen peroxide concentrations. The deiodinases are present in most tissues and provide a mechanism whereby individual tissues may control their exposure to T3. Se is also able to modify the immune response in patients with autoimmune thyroiditis. Low sperm production and poor sperm quality are consistent features of Se-deficient animals. The pivotal link between Se, sperm quality and male fertility is GPX4 since the enzyme is essential to allow the production of the correct architecture of the midpiece of spermatozoa. Se also has insulin-mimetic properties, an effect that is probably brought about by stimulating the tyrosine kinases involved in the insulin signalling cascade. Furthermore, in the diabetic rat, Se not only restores glycaemic control but it also prevents or alleviates the adverse effects that diabetes has on cardiac, renal and platelet function.

Selenium in the immune system.

Selenium as an essential component of selenocysteine-containing protein is involved in most aspects of cell biochemistry and function. As such, there is much potential for selenium to influence the immune system. For example, the antioxidant glutathione peroxidases are likely to protect neutrophils from oxygen-derived radicals that are produced to kill ingested foreign organisms. When the functions of all selenoproteins are described, only then will it be possible to fully understand their role in maintaining optimal immune function.

Synergy between sulforaphane and selenium in the induction of thioredoxin reductase 1 requires both transcriptional and translational modulation.

Thioredoxin reductases (TrxRs) catalyse the NADPH-dependent reduction of thioredoxin and play an important role in multiple cellular events related to carcinogenesis including cell proliferation, apoptosis and cell signaling. We have used human hepatoma HepG2 cells to examine the regulation of TrxRs by isothiocyanate (sulforaphane) and selenium (Se). We show that TrxR1 mRNA, but not TrxR2 mRNA, is induced up to 4-fold by sulforaphane, and this increase was abolished by actinomycin D, a transcription inhibitor. Se, in the form of sodium selenite, induced TrxR1 at the translational level, as shown by an increase in protein (2.1-fold) and activity (4.8-fold), but not mRNA. In combination, sulforaphane and Se synergistically induced TrxR1 protein (5.5-fold), activity (13-fold) and mRNA (6.5-fold). Although Se does not induce TrxR1 mRNA, Se can delay the degradation of sulforaphane-induced TrxR1 mRNA. Modulation of TrxR1 mRNA by sulforaphane was glutathione and protein kinase C-dependent, as L-buthionine-S,R-sulfoximine (a specific inhibitor of glutathione synthesis), and the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine, significantly reduced the induction. The combination of sulforaphane and Se also efficiently protected HepG2 cells from paraquat-induced cell death, whereas sulforaphane-only and Se-only treatments showed very little if any protective effect. These results demonstrate that synergy can result from a combination of induction at the levels of transcription and translation.

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 microM 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 microM 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 microM) than selenium-deficient cells (LC(50) 175 microM). 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 and the regulation of cell signaling, growth, and survival: molecular and mechanistic aspects.

In the past 30 years, it has been recognized that dietary selenium (Se) is essential for the normal function of many of the systems of the body. Furthermore, low Se intake can have deleterious effects on several aspects of human and animal health. The importance of Se is characterized in its role as a constituent of several key antioxidant and redox enzyme families. Most of the effects of Se are probably mediated by selenoproteins, which have the micronutrient covalently incorporated into the protein. The purpose of this review is to examine basic mechanisms by which Se regulates cell growth, gene transcription, cell signaling, and cell death. We start with the historical background to Se. The synthesis and function of selenoproteins are described, followed by details of the dietary sources of Se and Se status in different parts of the world, together with the clinical effects of Se deficiency and toxicity. We consider some aspects of the molecular mechanisms by which Se modulates cell growth, intracellular signaling, and gene transcription.