Selenoprotein T protects against cisplatin-induced acute kidney injury through suppression of oxidative stress and apoptosis.

Previously, selenoprotein T (SelT) expression was shown to be induced in nervous, endocrine, and metabolic tissues during ontogenetic and regenerative processes. However, whether SelT plays a critical role in renal diseases remains unclear. Here, we explored the role of SelT in cisplatin-induced acute kidney injury (AKI). Results revealed that SelT was highly expressed in renal tubules, but its expression was significantly reduced in cisplatin-induced AKI. Importantly, knocking down of SelT expression in kidney cells in vitro resulted in cisplatin-induced cell apoptosis, as indicated by the elevation of cleaved-PARP and Bax expression, Caspase-3 activity, and number of TUNEL-positive cells. Moreover, SelT silencing-induced reactive oxygen species (ROS) production, accompanied by a decrease in intracellular superoxide dismutase (SOD) and catalase (CAT) activity and increase in malondialdehyde (MDA) content. Notably, the protein and mRNA levels of Nox4 were increased in response to SelT downregulation. Furthermore, suppression of Nox4 expression by GKT137831 partially alleviated SelT knockdown-induced ROS generation and cell apoptosis in cisplatin-treated kidney cells. Taken together, our findings provide the first evidence that SelT protects against cisplatin-induced AKI by suppression of oxidative stress and apoptosis.

Activation of TGR5 alleviates inflammation in rheumatoid arthritis peripheral blood mononuclear cells and in mice with collagen II­induced arthritis.

Rheumatoid arthritis (RA) is characterized by chronic inflammatory synovitis resulting in progressive joint destruction. Persistent synovial inflammation is induced by activation of various inflammatory cells. G­protein­coupled bile acid receptor 1 (TGR5) is a G­protein­coupled receptor activated by various bile acids, which has been reported to act as a key adaptor in regulating various signaling pathways involved in inflammatory responses and a diverse array of physiological processes, including bile acid synthesis, lipid and carbohydrate metabolism, carcinogenesis, immunity and inflammation. In the present study, TGR5 expression was detected in RA peripheral blood mononuclear cells (PBMCs), and its association with clinical disease activity, histological synovitis severity and radiological joint destruction was analyzed. Subsequently, the role and potential underlying mechanisms of TGR5 in the PBMCs of patients with RA and mice with collagen II­induced arthritis (CIA) were investigated. PBMCs were obtained from 50 patients with RA and 40 healthy controls (HCs). The mRNA and protein expression levels of TGR5 were detected in PBMCs via reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and immunofluorescence staining, respectively. Additionally, the levels of proinflammatory cytokines were analyzed by RT­qPCR and enzyme­linked immunosorbent assay (ELISA). The activation of nuclear factor­κB (NF­κB) and IκB kinase a was determined via western blot analysis. The anti­arthritic and anti­inflammatory effects of LCA on mice with CIA were then investigated. The arthritis score was assessed, and the protein levels of proinflammatory cytokines in the plasma of mice were detected via ELISA. TGR5 mRNA expression was significantly downregulated in the PBMCs of patients with RA compared with in those of the HCs (0.53±0.58 for patients vs. 1.49±0.83 for HCs; P<0.001); similar findings were observed at the protein level. The mRNA expression levels of TGR5 in the PBMCs of patients with RA with a high 28­Joint Disease Activity Score (DAS28) were significantly decreased compared with in patients with a low DAS28 (0.81±0.65 for low score vs. 0.35±0.46 for high score; P=0.002). Furthermore, TGR5 expression was significantly correlated with the levels of C­reactive protein (r=­0.429; P=0.002) and the DAS28 (r=­0.383; P=0.006). RT­qPCR and ELISA analyses indicated that lithocholic acid (LCA, 10 mg/kg/day) attenuated lipopolysaccharide­induced proinflammatory cytokine production via inhibition of NF­κB activity in the PBMCs of patients with RA. In addition, the arthritis score was significantly decreased in LCA­treated CIA mice compared with in non­treated CIA mice. The increased production of tumor necrosis factor­α, interleukin (IL)­1ß, IL­6 and IL­8 was significantly reduced in the plasma of LCA­treated CIA mice compared with the control. In conclusion, TGR5 may contribute to the inflammation of PBMCs in patients with RA and mice with CIA.

The Proteomic Profile of Keratinocytes and Lymphocytes in Psoriatic Patients.

PURPOSE: Psoriatic skin lesions are associated with chronic inflammation related to immune cell activity. Therefore, the aim of this study is to compare changes in the proteome of psoriatic keratinocytes and lymphocytes. EXPERIMENTAL DESIGN: A proteomics approach is used to analyze the expression of proteins in keratinocytes and lymphocytes from psoriatic patients and healthy controls. RESULTS: As a result 2119 proteins for keratinocytes and 1235 proteins for lymphocytes are identified. Psoriatic keratinocytes has 68 downregulated and 7 upregulated proteins and psoriatic lymphocytes has 106 downregulated and 67 upregulated proteins compared to healthy individuals. The list of downregulated proteins includes proteins involved in antioxidant homeostasis and, transcription regulation; upregulated proteins are involved in glycolytic processes and translation. These changes are accompanied by an increased level of 4-Hydroxynonenal-protein adducts; control cells are characterized by 4-Hydroxynonenal-Lysine adducts formed with structural and binding proteins, while in psoriatic cells 4-Hydroxynonenal-Lysine, 4-Hydroxynonenal-Histidine, and 4-Hydroxynonenal-Cysteine adducts with various molecular function proteins occur. CONCLUSIONS AND CLINICAL RELEVANCE: This study highlights the changes in psoriatic keratinocytes and lymphocytes that can be directly involved in the development of psoriasis. In both cell types the most significant changes are associated with upregulation of phosphoglycerate mutase 1 and downregulation of thioredoxin reductase.

Overexpression of Endogenous Anti-Oxidants with Selenium Supplementation Protects Trophoblast Cells from Reactive Oxygen Species-Induced Apoptosis in a Bcl-2-Dependent Manner.

The human placenta provides life support for the developing foetus, and a healthy placenta is a prerequisite to a healthy start to life. Placental tissue is subject to oxidative stress which can lead to pathological conditions of pregnancy such as preeclampsia, preterm labour and intrauterine growth restriction. Up-regulation of endogenous anti-oxidants may alleviate placental oxidative stress and provide a therapy for these complications of pregnancy. In this study, selenium supplementation, as inorganic sodium selenite (NaSel) or organic selenomethionine (SeMet), was used to increase the protein production and cellular activity of the important redox active proteins glutathione peroxidase (GPx) and thioredoxin reductase (Thx-Red). Placental trophoblast cell lines, BeWo, JEG-3 and Swan-71, were cultured in various concentrations of NaSel or SeMet for 24 h and cell extracts prepared for western blots and enzyme assays. Rotenone and antimycin were used to stimulate mitochondrial reactive oxygen species (ROS) production and induce apoptosis. Trophoblast cells supplemented with 100 nM NaSel and 500 nM SeMet exhibited significantly enhanced expression and activity of both GPx and Thx-Red. Antimycin and rotenone were found to generate ROS when measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, and selenium supplementation was shown to reduce ROS production in a dose-dependent manner. Rotenone, 100 µM treatment for 4 h, caused trophoblast cell apoptosis as evidenced by increased Annexin V binding and decreased expression of Bcl-2. In both assays of apoptosis, selenium supplementation was able to prevent apoptosis, preserve Bcl-2 expression and protect trophoblast cells from mitochondrial oxidative stress. This data suggests that selenoproteins such as GPx and Thx-Red have an important role in protecting trophoblast cells from mitochondrial oxidative stress and that selenium supplementation may be important in treating some placental pathologies.

Intrauterine growth restriction in neonatal piglets affects small intestinal mucosal permeability and mRNA expression of redox-sensitive genes.

Neonates with intrauterine growth restriction (IUGR) show lower efficiency of nutrient utilization compared to normal birth weight (NBW) newborns. This study was conducted using neonatal piglets as a model to test the hypothesis that IUGR affects the intestinal barrier function, intestinal structure, and antioxidant system development during the suckling period. The small intestinal mucosae were obtained from IUGR and NBW littermates in the suckling period (d 0, 3, 8, and 19 postnatal). The epithelial barrier function was assessed by FITC-dextran 4 (FD4) and horseradish peroxidase (HRP) fluxes across the epithelium, histomorphologic measurements, and expression of tight-junction proteins. Redox status represented by the glutathione disulfide/glutathione ratio and malondialdehyde concentrations was determined, whereas mRNA expressions of some redox-sensitive proteins were quantified. Results showed that IUGR piglets exhibited a 2-fold higher intestinal permeability in the proximal small intestine on d 0 (P < 0.05), and this difference between IUGR and NBW piglets was widened to 3 and 4 times for FD4 and HRP, respectively (P < 0.05), on d 3. In accordance, expression of occludin was down-regulated at the transcriptional level in IUGR piglets at d 0 and 19 (P < 0.01). Furthermore, the transcription of heme oxygenase 1, catalase, and thioredoxin reductase genes was down-regulated in IUGR piglets, mainly on postnatal d 0 and 19 (P < 0.01). It appears that IUGR subjects have a lower capacity to mount an antioxidant response in the early postnatal period. Collectively, these results add to our understanding of the mechanisms responsible for intestinal dysfunction in IUGR neonates.

Molecular profiling and functional insights of rock bream (Oplegnathus fasciatus) thioredoxin reductase 3-like molecule: investigation of its transcriptional modulation in response to live pathogen stress.

The thioredoxin (Trx) system plays a significant role in cellular antioxidative defense by dismutating the surpluses of reactive oxygen species. Thus, the role of thioredoxin reductase (TrxR) cannot be ignored, owing to its participation in initiating the Trx enzyme cascade. Here, we report the identification and molecular characterization of a teleostean TrxR (RbTrxR-3) ortholog that showed high similarity with the TrxR-3 isoforms of other vertebrates. The complete RbTrxR-3 coding sequence comprised 1800 nucleotides, encoding a 600-amino acid protein with a predicted molecular mass of ~66 kDa. RbTrxR-3 consisted of 16 exons separated by 15 introns and had a total length of 12,658 bp. In silico analysis of the RbTrxR-3 protein sequence revealed that it possesses typical TrxR domain architecture. Moreover, using multiple sequence alignment and pairwise sequence alignment strategies, we showed that RbTrxR-3 has high overall sequence similarity to other teleostean TrxR-3 proteins, including highly conserved active site residues. Phylogenetic reconstruction of RbTrxR-3 affirmed its close evolutionary relationship with fish TrxR-3 orthologs, as indicated by its clustering pattern. RbTrxR-3 transcriptional analysis, performed using quantitative polymerase chain reaction (qPCR), showed that RbTrxR-3 was ubiquitously distributed, with the highest level of mRNA expression in the blood, followed by the gill, and liver. Live bacterial and viral stimuli triggered the modulation of RbTrxR-3 basal transcription in liver tissues that correlated temporally with that of its putative substrate, rock bream thioredoxin1 under the same conditions of pathogenic stress. Finally, resembling the typical function of TrxR protein, purified recombinant RbTrxR-3 showed detectable dose-dependent thiol reductase activity against 5,5'-dithiobis (2-nitrobenzoic) acid. Taken together, these results suggest that RbTrxR-3 plays a role in the host Trx system under conditions of oxidative and pathogenic stress.

Effect of CDNB on filarial thioredoxin reductase : A proteomic and biochemical approach.

Thioredoxin reductase plays a crucial role in the maintenance of cellular redox homeostasis. In this study, we have targeted TrxR in Setaria cervi, a bovine filarial parasite using its inhibitor CDNB. It caused significant decrease in the motility and viability of these parasites leading to their death. Inhibition of TrxR leads to the downregulation of the antioxidant system followed by generation of oxidative stress in these parasites. The increased ROS level induced lipid peroxidation and protein carbonyl formation which might alter the mitochondrial membrane permeability leading to release of cytochrome c. CDNB significantly downregulated the level of ced-9 and activity of tyrosine phosphatases, cytochrome c oxidase. It also upregulated ced-3, homolog of mammalian caspase 3 suggesting initiation of intrinsic pathway of apoptosis. The proteomic profile of CDNB treated parasites showed marked alteration in abundance of different protein spots with 20% downregulated and 13% unregulated spots in comparison to control parasites. We observed a downregulation in the glycolytic enzymes such as enolase, PGK, and GAPDH thereby blocking the ATP formation in the parasite. This study suggests that TrxR inhibition disrupts the cellular homeostasis thereby generating oxidative stress followed by mitochondrial mediated apoptosis in filarial parasites leading to the death of the parasites. BIOLOGICAL SIGNIFICANCE: Lymphatic filariasis is one of the most prevalent tropical diseases caused by tissue dwelling parasitic nematodes viz., Wuchereria bancrofti, Brugia malayi and Brugia timori. Currently available antifilarial drugs effectively eliminate larval stages of the parasite but are ineffective against the adult worms. Therefore, there is an urgent need for finding proteins/enzymes which play a crucial role in the persistence of these parasites. Our study for the first time reports the important role played by S. cervi TrxR in its survival. Thus, suggesting filarial TrxR as a potent chemotherapeutic target against lymphatic filariasis. This would help in screening of new compounds having macrofilaricidal activity.

Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins.

Plant redox-related proteins were overexpressed using a genetic codon substitution downstream of the translation initiation codon. This method significantly improved recombinant protein expression levels of Arabidopsis chloroplastic thioredoxins and cytosolic nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (E.C. 1.8.1.9) in Escherichia coli. Using these proteins, the in vitro chloroplastic thioredoxins-reduction system was reconstituted in an NADPH-dependent manner. This system could convert the five classes of chloroplastic Arabidopsis thioredoxins and two chloroplastic Spinach thioredoxins to their reduced forms, independent of dithiothreitol and the photosynthetic electron transport system.

Inflammation, oxidative stress and L-fucose as indispensable participants in schistosomiasis-associated colonic dysplasia.

BACKGROUND: Schistosomiasis is a parasitic disease causing chronic ill health in humans with a serious consequences for socio-economic development in tropical and subtropical regions. There is also evidence linking Schistosoma mansoni to colonic carcinoma occurrence. The aim of this study was to evaluate some inflammatory and oxidative stress biomarkers, as well as L-fucose as linkers between intestinal schistosomiasis and colonic dysplasia development in mice. MATERIALS AND METHODS: This study was conducted upon 80 mice that were divided the control group (10 non infected mice) and infected group which was subdivided into 7 sub-groups (10 mice each) according to the time of sacrifaction in the post infection (p.i.) period, 10 mice being sacrificed every two weeks from 6 weeks p.i. to 18 weeks p.i. Tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), and pentraxin 3 (PTX3) levels were estimated by immunoassay. The L-fucose level, and thioredoxin reductase (TrxR) and lactate dehydrogenase (LDH) activities were also evaluated in colonic tissue. RESULTS: The current study revealed statistically significant elevation in the studied biochemical markers especially at 16 and 18 weeks p.i. The results were confirmed by histopathological examination that revealed atypical architectural and cytological changes in the form of epithelial surface serration and nuclear hyper-chromatizia at 14, 16 and 18 weeks p.i. CONCLUSIONS: inflammation, oxidative stress and L-fucose together may form an important link between Schistosomal mansoni infection and colonic dysplasia and they can be new tools for prediction of colonic dysplasia development in experimental schistosomiasis.

Biochemical characterization of thioredoxin reductase from Babesia bovis.

This paper addresses the identification, cloning, expression, purification and functional characterization of thioredoxin reductase from Babesia bovis, the etiological agent of babesiosis. The work deals with in vitro steady state kinetic studies and other complementary analyses of the thioredoxin reductase found in the pathogenic protist. Thioredoxin reductase from B. bovis was characterized as a homodimeric flavoprotein that catalyzes the NADPH-dependent reduction of Trx with a high catalytic efficiency. Moreover, the enzyme exhibited a disulfide reductase activity using DTNB as substrate, being this activity highly sensitive to inhibition by Eosin B. The thioredoxin reductase/thioredoxin system can reduce oxidized glutathione and S-nitrosoglutathione. Our in vitro data suggest that antioxidant defense in B. bovis could be supported by this enzyme. We have performed an enzymatic characterization, searching for targets for rational design of inhibitors. This work contributes to the better understanding of the redox biochemistry occurring in the parasite.

Insights into the fish thioredoxin system: expression profile of thioredoxin and thioredoxin reductase in rainbow trout (Oncorhynchus mykiss) during infection and in vitro stimulation.

Production of reactive oxygen species (ROS) is the first biological response during a disease outbreak and after injury. ROS are highly reactive molecules that can either endanger cell homeostasis or mediate cell signaling in several physiological pathways, including the immune response. Thioredoxin (Trx) and thioredoxin reductase (TrxR) are the essential components of the thioredoxin system, one of the main intracellular redox systems and are therefore important regulators of ROS accumulation. Through the regulation of the intracellular redox milieu, the thioredoxin system plays a key role within the immune system, linking immunology and free radical science. In this study we have firstly identified TrxRs in fish and used this new sequence information to reevaluate the evolution of the thioredoxin system within the vertebrate lineage. We next measured the expression of rainbow trout (Oncorhynchus mykiss) Trx and TrxR transcripts during infection in vivo and in vitro after stimulation of a macrophage cell line and primary macrophage cultures with pathogen associated molecular patterns (PAMPs). Our results showed that both Trx and TrxR were induced during infection at the transcriptional level, confirming their likely involvement in the innate immune response of fish. Since TrxRs are selenium-containing proteins (selenoproteins), we also measured the modulation of their expression upon organic and inorganic selenium exposure in vitro. TrxR was found to be responsive to selenium exposure in vitro, suggesting that it may represent a key mediator in the selenium modulation of innate immunity. In conclusion, our study highlights the need to investigate the involvement of the cell antioxidant pathways, especially the thioredoxin system, within the immune system of vertebrate species.

Effects of diphenyl diselenide on methylmercury toxicity in rats.

This study investigates the efficacy of diphenyl diselenide [(PhSe)2] in attenuating methylmercury- (MeHg-)induced toxicity in rats. Adult rats were treated with MeHg [5 mg/kg/day, intragastrically (i.g.)] and/ or (PhSe)2 [1 mg/kg/day, intraperitoneally (i.p.)] for 21 days. Body weight gain and motor deficits were evaluated prior to treatment, on treatment days 11 and 21. In addition, hepatic and cerebral mitochondrial function (reactive oxygen species (ROS) formation, total and nonprotein thiol levels, membrane potential (ΔΨm), metabolic function, and swelling), hepatic, cerebral, and muscular mercury levels, and hepatic, cerebral, and renal thioredoxin reductase (TrxR) activity were evaluated. MeHg caused hepatic and cerebral mitochondrial dysfunction and inhibited TrxR activity in liver (38,9%), brain (64,3%), and kidney (73,8%). Cotreatment with (PhSe)2 protected hepatic and cerebral mitochondrial thiols from depletion by MeHg but failed to completely reverse MeHg's effect on hepatic and cerebral mitochondrial dysfunction or hepatic, cerebral, and renal inhibition of TrxR activity. Additionally, the cotreatment with (PhSe)2 increased Hg accumulation in the liver (50,5%) and brain (49,4%) and increased the MeHg-induced motor deficits and body-weight loss. In conclusion, these results indicate that (PhSe)2 can increase Hg body burden as well as the neurotoxic effects induced by MeHg exposure in rats.

Maintenance of S-nitrosothiol homeostasis plays an important role in growth suppression of estrogen receptor-positive breast tumors.

INTRODUCTION: Protein denitrosylation by thioredoxin reductase (TrxR) is key for maintaining S-nitrosothiol (SNO) homeostasis, although its role in tumor progression is unknown. Therefore, the present study aimed to assess the role of altered SNO homeostasis in breast cancer cells. METHODS: The impairment of SNO homeostasis in breast cancer cells was achieved with the highly specific TrxR inhibitor auranofin and/or exposure to S-nitroso-L-cysteine. S-nitrosylated proteins were detected using the biotin switch assay. Estrogen receptor (ER) alpha knockdown was achieved using RNA silencing technologies and subcellular localization of ERα was analyzed by confocal microscopy. The Oncomine database was explored for TrxR1 (TXNRD1) expression in breast tumors and TrxR1, ER and p53 expression was analyzed by immunohistochemistry in a panel of breast tumors. RESULTS: The impairment of SNO homeostasis enhanced cell proliferation and survival of ER+ MCF-7 cells, but not of MDA-MB-231 (ER-, mut p53) or BT-474 (ER+, mut p53) cells. This enhanced cell growth and survival was associated with Akt, Erk1/2 phosphorylation, and augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-α. The specific silencing of ERα expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation in MCF-7 cells potentiated the pro-proliferative effect of impaired SNO homeostasis. Moreover, the subcellular distribution of ERα was altered, with a predominant nuclear localization associated with phosphorylation at Thr311 in those cells with impaired SNO homeostasis. The impairment of SNO homeostasis also expanded a cancer stem cell-like subpopulation in MCF-7 cells, as indicated by the increase of percentage of CD44+ cells and the augmented capability to form mammospheres in vitro. Notably, ER+ status in breast tumors was significantly associated with lower TXNDR1 mRNA expression and immunohistochemical studies confirmed this association, particularly when p53 abnormalities were absent. CONCLUSION: The ER status in breast cancer may dictate tumor response to different nitrosative environments. Impairment of SNO homeostasis confers survival advantages to ER+ breast tumors, and these molecular mechanisms may also participate in the development of resistance against hormonal therapies that arise in this type of mammary tumors.

Polymorphisms in thioredoxin reductase and selenoprotein K genes and selenium status modulate risk of prostate cancer.

Increased dietary intake of Selenium (Se) has been suggested to lower prostate cancer mortality, but supplementation trials have produced conflicting results. Se is incorporated into 25 selenoproteins. The aim of this work was to assess whether risk of prostate cancer is affected by genetic variants in genes coding for selenoproteins, either alone or in combination with Se status. 248 cases and 492 controls from an EPIC-Heidelberg nested case-control study were subjected to two-stage genotyping with an initial screening phase in which 384 tagging-SNPs covering 72 Se-related genes were determined in 94 cases and 94 controls using the Illumina Goldengate methodology. This analysis was followed by a second phase in which genotyping for candidate SNPs identified in the first phase was carried out in the full study using Sequenom. Risk of high-grade or advanced stage prostate cancer was modified by interactions between serum markers of Se status and genotypes for rs9880056 in SELK, rs9605030 and rs9605031 in TXNRD2, and rs7310505 in TXNRD1. No significant effects of SNPs on prostate cancer risk were observed when grade or Se status was not taken into account. In conclusion, the risk of high-grade or advanced-stage prostate cancer is significantly altered by a combination of genotype for SNPs in selenoprotein genes and Se status. The findings contribute to explaining the biological effects of selenium intake and genetic factors in prostate cancer development and highlight potential roles of thioredoxin reductases and selenoprotein K in tumour progression.

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.

Selenium homeostasis and induction of thioredoxin reductase during long term selenite supplementation in the rat.

Selenium is a candidate treatment for liver tumour prevention in chronic liver disease. In this study, we have studied selenium uptake, distribution and accumulation in rats provided with water containing tumour-preventive doses of sodium selenite for 10 weeks. Male Fischer 344 rats were given drinking water containing 1 µg/mL or 5 µg/mL sodium selenite. Selenium levels were monitored in serum and liver tissue over the 10-week period, and the kinetics of induction of the redox-active cytosolic selenoenzyme thioredoxin reductase were followed. Selenite exposure via drinking water caused a dose-dependent increase in blood and liver selenium levels, with plateaus at 6 and 8 weeks, respectively. These plateaus were reached at the same level of selenium regardless of dose, and no further accumulation was observed. A selenium-dependent increase in the activity of TrxR1 in parallel with the increase in liver selenium levels was also seen, and the induction of TrxR1 mRNA was seen only during the first three days of treatment, when the levels of selenium in the liver were increasing. Sodium selenite at 1 and 5 µg/mL did not affect body weight or relative liver mass. We concluded that long-term treatment with selenite did not cause accumulation of selenium and that the activity of TrxR1 in the liver rose with the selenium levels. We therefore suggest that sodium selenite at doses up to 5 µg/mL could be used for long-term tumour prevention.

Thioredoxin and thioredoxin reductase expression in thyroid cancer depends on tumour aggressiveness.

Thyroid cancer is the second most common malignancy following breast cancer in Arab females. Thioredoxin (TRX) is a small multi-functional redox protein with both intracellular and extracellular functions. The protein exists in either a reduced form (thioredoxin-SH2) or an oxidized form (thioredoxin-S2). TRX acts as an enhancement for growth factors and stimulates the growth of cancer cells. In this study of thyroid neoplasms, involving 121 female and 62 male patients, expression of TRX and TRX-R was studied using purified mouse anti-human TRX monoclonal antibody and anti-human TRX-R antiserum from rabbits, respectively. In order to delineate tumour cell growth, proliferating cell nuclear antigen (PCNA) polyclonal antibody was used. Compared to normal thyroid tissue, expression of TRX and TRX-R was increased in the cytoplasm and nuclei of thyroid cancer cells. Furthermore, TRX expression correlated with that of TRX-R. Of the 183 thyroid neoplasms investigated, overexpression of TRX-R was found in different types of neoplasms. The majority of carcinomas showed a correlation between strongly positive TRX and TRX-R expression and neoplastic cellular proliferation, as measured by PCNA. This indicates that increased TRX and TRX-R expression may be associated with tumourigenesis by acting as an autocrine growth stimulus. This study suggests that TRX immunoreactivity in thyroid tumours is a function of malignancy and cancer progression. In addition, secreted TRX can also act as an extracellular growth factor for both normal and tumour cells and enhance the sensitivity of the cells. Furthermore, this study emphasizes the potential benefits of anti-TRX/TRX-R agents in cancer therapeutics in the treatment of thyroid cancer.

Statin-induced liver injury involves cross-talk between cholesterol and selenoprotein biosynthetic pathways.

Statins have become the mainstay of hypercholesterolemia treatment. Despite a seemingly clear rationale behind their use, the inhibition of HMG-CoA reductase, these compounds have been shown to elicit a variety of unanticipated and elusive effects and side effects in vivo. Among the most frequently noted side effects of statin treatment are elevations in liver enzymes. Here, we report our finding that atorvastatin, cerivastatin, and lovastatin at clinically common concentrations induce a selective, differential loss of selenoprotein expression in cultured human HepG2 hepatocytes. The primarily affected selenoprotein was glutathione peroxidase (GPx), whose biosynthesis, steady-state expression level, and catalytic activity were significantly reduced with 10 to 100 nM concentrations of the different compounds. Messenger RNA levels of GPx1 and GPx4 were unaffected by statin treatment, pointing at a post-transcriptional mechanism of selenoprotein suppression. Although statins at selenoprotein-modulatory doses were not cytotoxic by themselves, they induced a significantly increased sensitivity of the cells to peroxides, an effect that was largely reversible by supraphysiological concentrations of selenite. We conclude that statins inhibit the expression of inducible selenoproteins by preventing the mevalonate-dependent maturation of the single human selenocysteine-tRNA and may thereby evoke an increased vulnerability of the liver to secondary toxins. Selenoprotein modulation might constitute an important mechanism of statins to bring forth their clinical effects.

Expression pattern of a chloroplast NADPH-dependent thioredoxin reductase in Chlorella vulgaris during hardening and its interaction with 2-Cys peroxiredoxin.

A chloroplastic NADPH-dependent thioredoxin reductase gene was identified from Chlorella vulgaris and designated CvNTRC. Mature CvNTRC protein (mCvNTRC) was expressed in Escherichia coli, and it showed both NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx)-like dithiol-disulfide oxidoreductase activities. The transcript of CvNTRC increased throughout 24-h hardening, whereas the encoded protein amount and total NTR activity decreased once and then increased during hardening. By in vitro pull-down assay, a 21.2-kDa protein bound to mCvNTRC was isolated and identified as a 2-Cys peroxiredoxin (2-Cys Prx) based on the N-terminal sequence. These data suggest that CvNTRC is maintained at a constant level during hardening and functions as an antioxidant with 2-Cys Prx in the acquisition of freezing tolerance of Chlorella.

Entamoeba histolytica: response of the parasite to metronidazole challenge on the levels of mRNA and protein expression.

Entamoeba histolytica remains a cause of significant morbidity and mortality in many countries. Although metronidazole has been used for the treatment of amoebiasis for several decades, little is known on how the amoebae react to this challenge on the levels of mRNA and protein expression. In this study, we examined their response using a focused microarray, quantitative RT-PCR, and two-dimensional gel electrophoresis (2DE). The amoebae modestly increased the levels of mRNA coding for superoxide dismutase, peroxiredoxin, ferredoxin, thioredoxin reductase, and the galactose/N-acetylgalactosamine specific lectin light and heavy chains. The mRNAs encoding actin and the 70kDa and 101kDa heat shock proteins were decreased. All the changes occured within 1h of exposition, with very little further changes. In addition, the proteome revealed only very few changes. Taken together, E. histolytica appears to make only modest mRNA and protein expression changes when confronted with an unknown chemical stress.