Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply.

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective.

Marginal selenium deficiency down-regulates inflammation-related genes in splenic leukocytes of the mouse.

Moderate selenium deficiency may lead to an impaired capacity to cope with health challenges. Functional effects of suboptimal selenium intake are not fully known, and biomarkers for an insufficient selenium supply are inadequate. We therefore fed mice diets of moderately deficient or adequate selenium intake for 6 weeks. Changes in global gene expression were monitored by microarray analysis in splenic leukocytes. Genes for four selenoproteins, Sepw1, Gpx1, Selh and Sep15, were the most significantly down-regulated in moderate selenium deficiency, and this was confirmed by quantitative polymerase chain reaction (qPCR). Classification of significantly affected genes revealed that processes related to inflammation, heme biosynthesis, DNA replication and transcription, cell cycle and transport were affected by selenium restriction. Down-regulation by moderate selenium deficiency of specific genes involved in inflammation and heme biosynthesis was confirmed by qPCR. Myeloperoxidase and lysozyme activities were decreased in selenium-restricted leukocytes, providing evidence for functional consequences. Genes for 31 nuclear factor (NF)-κB targets were down-regulated in moderate selenium deficiency, indicating an impaired NF-κB signaling. Together, the observed changes point to a disturbance in inflammatory response. The selenoproteins found here to be sensitive to selenium intake in murine leukocytes might also be useful as biomarkers for a moderate selenium deficiency in humans.

Loss of GPx2 increases apoptosis, mitosis, and GPx1 expression in the intestine of mice.

Localization of glutathione peroxidase 2 (GPx2), the gastrointestinal form of GPx's, in the intestinal crypt epithelium points to a specific but so-far unknown function of this particular GPx. Therefore, the consequences of a GPx2 knockout were tested in mice fed a selenium-restricted, Se-adequate, or Se-supplemented diet. An unexpected increase in total GPx activity was found throughout the intestine in selenium-fed GPx2 knockout (KO) animals. Immunohistochemistry revealed a strong increase in GPx1 in the colon and ileum, especially in crypt bases where typically GPx2 is localized. GPx1 mRNA was not enhanced in GPx2 KO, indicating that up-regulation most probably occurs at the translational level. Loss of GPx2 was accompanied by an increase in apoptotic cells at colonic crypt bases, an area essential for the self-renewal of the intestinal epithelium, particularly under selenium restriction. Additionally, mitotic cells increased in the middle parts of the crypts, indicating an extension of the proliferative area. These findings corroborate a role for GPx2 in regulating mucosal homeostasis. In GPx2 KO mice, an increase in GPx1 can only partially compensate for GPx2, even under selenium supplementation, indicating that GPx2 is the major antiapoptotic GPx in the colon. These data explain why spontaneous ileocolitis becomes manifested only if both Gpx2 and Gpx1 are deleted.

Four selenoproteins, protein biosynthesis, and Wnt signalling are particularly sensitive to limited selenium intake in mouse colon.

Selenium is an essential micronutrient. Its recommended daily allowance is not attained by a significant proportion of the population in many countries and its intake has been suggested to affect colorectal carcinogenesis. Therefore, microarrays were used to determine how both selenoprotein and global gene expression patterns in the mouse colon were affected by marginal selenium deficiency comparable to variations in human dietary intakes. Two groups of 12 mice each were fed a selenium-deficient (0.086 mg Se/kg) or a selenium-adequate (0.15 mg Se/kg) diet. After 6 wk, plasma selenium level, liver, and colon glutathione peroxidase (GPx) activity in the deficient group was 12, 34, and 50%, respectively, of that of the adequate group. Differential gene expression was analysed with mouse 44K whole genome microarrays. Pathway analysis by GenMAPP identified the protein biosynthesis pathway as most significantly affected, followed by inflammation, Delta-Notch and Wnt pathways. Selected gene expression changes were confirmed by quantitative real-time PCR. GPx1 and the selenoproteins W, H, and M, responded significantly to selenium intake making them candidates as biomarkers for selenium status. Thus, feeding a marginal selenium-deficient diet resulted in distinct changes in global gene expression in the mouse colon. Modulation of cancer-related pathways may contribute to the higher susceptibility to colon carcinogenesis in low selenium status.

The gastrointestinal microbiota affects the selenium status and selenoprotein expression in mice.

Colonization of germ-free (GF) mice has been shown to induce the gastrointestinal form of the selenium-dependent glutathione peroxidases, GPx2. Since bacterial colonization of the gastrointestinal tract is associated with stress, we aimed to clarify how bacteria affect selenoprotein expression in unstressed conditions. GF and conventional (CV) FVB/NHan(TMHsd) mice were fed a selenium-poor (0.086 ppm) or a selenium-adequate (0.15 ppm) diet for 5 weeks starting from weaning. Each group consisted of five animals. Specific glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) expression was measured in plasma, liver and intestinal sections by activity, protein and mRNA level as appropriate. Under selenium-adequate conditions, selenoprotein expression did not differ in GF and CV mice. Under selenium-limiting conditions, however, GF mice generally contained higher GPx and TrxR activities in the intestine and liver, higher GPx1 protein and RNA levels in the liver, higher GPx2 protein levels in the proximal and distal jejunum and colon and higher GPx1 and GPx2 RNA levels in the colon. In addition, higher selenium concentrations were estimated in plasma, liver and cecum. All differences were significant. It is concluded that bacteria may compete with the host for selenium when availability becomes limiting. A variable association with different microorganisms might influence the daily requirement of mice for selenium. Whether the microbiota also affects the human selenoprotein status appears worthy of investigation.

Glutathione Peroxidase 2 Inhibits Cyclooxygenase-2-Mediated Migration and Invasion of HT-29 Adenocarcinoma Cells but Supports Their Growth as Tumors in Nude Mice.

The selenoprotein gastrointestinal glutathione peroxidase 2 (GPx2) is up-regulated in a variety of cancer cells with thus far unknown consequences. Therefore, two clones of a human colon cancer cell line (HT-29) in which GPx2 was stably knocked down by small interfering RNA (siRNA; siGPx2) were used to test whether cancer-relevant processes are affected by GPx2. The capacity to grow anchorage independently in soft agar was significantly reduced in siGPx2 cells when compared with controls (i.e., HT-29 cells stably transfected with a scramble siRNA). The weight of tumors derived from siGPx2 cells injected into nude mice was lower in 9 of 10 animals. In contrast, in a wound-healing assay, wound closure was around 50% in controls and 80% in siGPx2 cells, indicating an enhanced capacity of the knockdown cells to migrate. Similarly, invasion of siGPx2 cells in a Transwell assay was significantly increased. Migration and invasion of siGPx2 cells were inhibited by celecoxib, a cyclooxygenase-2 (COX-2)-specific inhibitor, but not by alpha-tocopherol. Selenium supplementation of cell culture medium did not influence the results obtained with siGPx2 cells, showing that none of the other selenoproteins could replace GPx2 regarding the described effects. The data show that GPx2 inhibits malignant characteristics of tumor cells, such as migration and invasion, obviously by counteracting COX-2 expression but is required for the growth of transformed intestinal cells and may, therefore, facilitate tumor cell growth. The data also shed new light on the use of selenium as a chemopreventive trace element: a beneficial effect may depend on the stage of tumor development.

Modulation of pregnane X receptor- and electrophile responsive element-mediated gene expression by dietary polyphenolic compounds.

Based on animal models, dietary polyphenols are predicted to be promising chemopreventive agents in humans. Allspice, clove, and thyme extracts as well as defined dietary polyphenolic compounds were, therefore, tested for their ability to activate mechanisms related to phase 1 enzymes, i.e., the PXR-regulated CYP3A4 promoter, and phase 2 enzymes, i.e. the EpRE-regulated promoters of gastrointestinal glutathione peroxidase (GI-GPx) and heme oxygenase-1 (HO-1), examples of Nrf2-regulated genes. From the compounds tested, clove and thyme extracts as well as curcumin and resveratrol activated the PXR. PXR activation correlated with the activation of the CYP3A4 promoter in the case of thyme extract, curcumin, and resveratrol, but not in the case of clove extract. Allspice extract, EGCG, and quercetin did not activate PXR but enhanced CYP3A4 promoter activity. Thyme extract and quercetin activated the EpRE of HO-1. Both significantly activated the GI-GPx promoter, effects that depended on a functional EpRE. Resveratrol did not activate the isolated EpRE but enhanced the GI-GPx promoter activity, whereas clove extract even inhibited it. It is concluded that individual polyphenols as well as polyphenol-rich plant extracts may affect phase 1 and 2 enzyme expression by distinct mechanisms that must be elucidated, before potential health effects can reliably be predicted.

Part of the series: from dietary antioxidants to regulators in cellular signaling and gene regulation. Sulforaphane and selenium, partners in adaptive response and prevention of cancer.

The association of decreased cancer risk with intake of cruciferous vegetables and selenium is stronger than that reported for fruits and vegetables in general. An active constituent in cruciferae is sulforaphane. Chemopreventive effects of both, sulforaphane and selenium have been attributed to an antioxidant action which certainly is too simplicistic. Sulforaphane induces via activation of the Nrf2/Keap1 system phase 2 enzymes that protect against carcinogens and oxidants. Induced enzymes comprise the selenoproteins thioredoxin reductase-1 (TrxR1) and gastrointestinal glutathione peroxidase (GI-GPx, GPx2), which contain antioxidant response elements (ARE) in their promoter regions. Translational realisation of the enhanced transcripts depends on adequate selenium supply, which explains the synergism of Nrf2 activators and selenium. Regarding tumorigenesis the role of TrxR1 is ambiguous: it is essential for fast tumor cell growth but also diminishes vascularisation of tumors. The anticarcinogenic role of GI-GPx is evident from enhanced gastrointestinal tumor formation in gpx2/gpx1 double KO mice.

Is there a future for antioxidants in atherogenesis?

Antioxidants, preferentially those of dietary origin, have for a long time been considered to help against diseases that are presumably aggravated by oxidative stress, such as cardiovascular diseases, cancer, and neurodegenerative disorders. The outcome of clinical trials undertaken to corroborate this hypothesis, however, remained largely inconclusive. Evidence is now emerging that some dietary "antioxidants" influence signaling pathways and the expression of genes relevant in atherosclerosis by mechanisms other than antioxidative ones. By concrete examples we show that (1) vitamin E has gene regulatory functions which might be more important than acting as an antioxidant in vivo, (2) selenium itself is not an antioxidant at all, and even not in general when incorporated into glutathione peroxidases, and (3) a moderate oxidative stress is beneficial rather than detrimental since it can induce defense mechanisms counteracting xenobiotic and oxidative stress. Thus, there is only a future for antioxidants in the prevention of any disease if their real mechanism of action is considered and suitable read-outs and biomarkers are established.

Selenium-dependent enzymes in endothelial cell function.

Glutathione peroxidases and thioredoxin reductases are the main selenoproteins expressed by endothelial cells. These enzymes reduce hydroperoxides, their role in endothelial cell physiology, however, by far exceeds prevention of oxidative damage. Reactive oxygen and nitrogen species, especially superoxide, hydroperoxides, and nitric oxide, are crucial signaling molecules in endothelial cells. Their production is regulated by vascular NAD(P)H oxidases and the endothelial nitric oxide synthase. Their metabolism and physiological functions are coordinated by glutathione peroxidases and the thioredoxin/thioredoxin reductase system. Endothelial selenoproteins are involved in the regulation of the vascular tone by maintaining the superoxide anion/nitric oxide balance, of cell adhesion by controlling cell adhesion molecule expression, of apoptosis via inhibition/activation of apoptosis signal-regulating kinase-1, and of eicosanoid production by controlling the activity of cyclooxygenases and lipoxygenases. Accordingly, they regulate inflammatory processes and atherogenesis. The underlying mechanisms are various and differ between individual selenoproteins. Scavenging of hydroperoxides not only prevents oxidative damage, but also interferes with signaling cascades and enzymes involved. Modulation of proteins by hydroperoxide-driven thiol/disulfide exchange is a novel mechanism that needs to be further investigated. A better understanding of the complex interplay of selenoproteins in regulating endothelial cell functions will help to develop a rationale for an improvement of health by an optimum selenium supply.