New assay for the measurement of selenoprotein P as a sepsis biomarker from serum

Selenium (Se) is incorporated into selenoproteins as the 21st proteinogenic amino acid selenocysteine. Serum Se concentrations decline during critical illness and are indicative of poor prognosis. Serum Se is mainly contained in the hepatically derived selenoprotein P (SePP) which controls the expression of antioxidative selenoproteins. Here, we describe the development of an immunoluminometric sandwich assay that uses two polyclonal sheep antihuman SePP antibodies. After assessing the stability of the analyte, we determined SePP concentrations in samples from healthy individuals and patients with sepsis. The analytical detection limit was 0.016 mg SePP/L serum. The assay was linear on dilution. SePP was stable in serum at room temperature for at least 24 h and resistant to six freeze-thaw cycles. Median SePP concentration in healthy individuals was 3.04 mg SePP/L serum (25th–75th percentiles, 2.6–3.4 mg/L) which corresponded to 98.4 μg Se/L serum. The interlaboratory CV was <20% for SePP values >0.06 mg/L. There was no association with gender, but concentrations differed between young and older individuals. Median SePP concentrations were significantly (P<0.0001) decreased in patients with sepsis (n=60) compared to healthy controls (n=318). Since SePP contains the major fraction of serum Se, we conclude that downregulation of SePP biosynthesis or removal of circulating SePP from blood underlies the negative acute phase response of serum Se in critical illness.     

Dietary selenium regulation of transcript abundance of selenoprotein N and selenoprotein W in chicken muscle tissues

Selenium (Se), selenoprotein N (SelN) and selenoprotein W (SelW) play a crucial role in muscle disorders. Se status highly regulates selenoprotein mRNA levels. However, few attempts have been performed on the effect of dietary Se supplementation on muscle SelN and SelW mRNA levels in birds. To investigate the effects of Se on the regulation of SelN and SelW mRNA levels in muscle tissues, one-day-old male chickens were fed either a commercial diet or a Se-supplemented diet containing 1.0, 2.0, 3.0 or 5.0 mg/kg sodium selenite for 90 days. Muscle tissues (breast, flight, thigh, shank and cardiac muscles) were collected and examined for Se content and mRNA levels of SelN and SelW. Moreover, Selenophosphate synthetase-1 (SPS-1) and selenocysteine-synthase (SecS) mRNA levels were analyzed. Significant increases in SelN mRNA levels were obtained in breast, thigh and shank muscles treated with Se, with maximal effects at 3.0 mg Se/kg diet, but 2.0 mg Se/kg diet resulted in peak levels of Sel N mRNA in flight muscles. Changes in SelW mRNA abundance in thigh and shank muscles increased in response to Se supply. After reaching a maximal level, higher Se supplementation led to a reduction in both SelN and SelW mRNAs. However, SelN and SelW mRNA levels displayed a different expression pattern in different skeletal and cardiac muscles. Thus, it suggested that skeletal and cardiac muscles SelN and SelW mRNA levels were highly regulated by Se supplementation and different muscle tissues showed differential sensitivity. Moreover, Se supplementation also regulated the levels of SPS1 and SecS mRNAs. The mRNA levels of SPS1 and SecS were enhanced in the Se supplemented groups. These data indicate that Se regulates the expression of SelN and SelW gene and affect the mRNA levels of SecS and SPS1.     

Concentration of selenium in the whole blood and the thyroid tissue of patients with various thyroid diseases

We investigated the possible differences between the concentrations of selenium in the whole blood and thyroid tissue of patients with thyroid disease. The study comprises 41 women with nodular goiter, 19 women and 2 men with thyroid cancer, 18 women with Graves’ disease, and 7 women with thyroiditis. The concentration of selenium was determined by the TRXRF method. The lowest mean selenium level was achieved in the whole blood of women with Graves’ disease and the highest in the whole blood of healthy people. In the thyroid cancer tissue, we found the lowest concentration of selenium and the highest in the thyroid gland of women with nodular goiter and Graves’ disease. The low selenium levles in the thyroid tissue may increase thyroid cancer risk.     

The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX

Background

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.

Methods

Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.

Results

In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.

Conclusions

Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.

General significance

Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.     

Characterization and expression analysis of seven selenoprotein genes in yellow catfish Pelteobagrus fulvidraco to dietary selenium levels

BackgroundSelenium (Se) appears in the selenoproteins in the form of selenocysteine (Sec) and is important for the growth and development of vertebrates. The present study characterized seven selenoproteins, consisting of the GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3 cDNAs in various tissues of yellow catfish, explored their regulation to dietary Se addition.Methods3′ and 5′ RACE PCR were used to clone full-length cDNA sequences of seven selenoprotein genes (GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3). Their molecular characterizations were analyzed, including conservative motifs and the SECIS elements. The phylogenetic trees were generated through neighbor-joining (NJ) method with MEGA 6.0 with 1000 bootstrap replications. Quantitative real-time PCR was used to explore their mRNA tissue distribution in the heart, anterior intestine, dorsal muscle, head kidney, gill, liver, brain, spleen and mesenteric fat. Yellow catfish (mixed sex) were fed diets with dietary Se contents at 0.03 (low Se), 0.25 (adequate Se) and 6.39 (high Se) mg Se/kg, respectively, for 12 weeks, and their spleen, kidney, testis and brain were used for the determination of the mRNA levels of the seven selenoproteins.ResultsThe seven selenoproteins had similar domains to their corresponding members of other vertebrates. They were widely expressed in nine tissues, including heart, liver, brain, spleen, head kidney, dorsal muscle, mesenteric fat, anterior intestine and gill, but showed tissue-dependent expression patterns. Dietary Se addition affected the expression of the seven genes in spleen, kidney, testis and brain tissues of yellow catfish.ConclusionTaken together, our study demonstrated the characterization, expression and regulation of seven selenoproteins, which increased our understanding of the biological functions of Se and selenoproteins in fish.     

Umbilical cord blood and placental mercury,selenium and selenoprotein expression in relation to maternal fish consumption

Seafood is an important source of nutrients for fetal neurodevelopment. Most individuals are exposed to the toxic element mercury through seafood. Due to the neurotoxic effects of mercury, United States government agencies recommend no more than 340 g (12 oz) per week of seafood consumption during pregnancy. However, recent studies have shown that selenium, also abundant in seafood, can have protective effects against mercury toxicity. In this study, we analyzed mercury and selenium levels and selenoprotein mRNA, protein, and activity in placenta of a cohort of women in Hawaii in relation to maternal seafood consumption assessed with dietary surveys. Fish consumption resulted in differences in mercury levels in placenta and cord blood. When taken as a group, those who consumed no fish exhibited the lowest mercury levels in placenta and cord blood. However, there were numerous individuals who either had higher mercury with no fish consumption or lower mercury with high fish consumption, indicating a lack of correlation. Placental expression of selenoprotein mRNAs, proteins and enzyme activity was not statistically different in any region among the different dietary groups. While the absence of seafood consumption correlates with lower average placental and cord blood mercury levels, no strong correlations were seen between seafood consumption or its absence and the levels of either selenoproteins or selenoenzyme activity.     

Protective effects of finasteride on the pulmonary immune response in a combined model of trauma-hemorrhage and polymicrobial sepsis in mice

Literature supports findings about a gender specific outcome following multiple trauma. Male sex hormones such as dihydrotestosterone (DHT) exert deleterious effects on the posttraumatic immune response whereas increased estradiol concentrations are correlated with improved outcome. Pretreatment with the 5α-reductase inhibitor finasteride resulted in an improved outcome following trauma-hemorrhage (TH) in mice. The present study tested the hypothesis that finasteride exerts beneficial effects on the posttraumatic immune response also in a combined setting of TH and sepsis when administered during the resuscitation process.

Material and Methods

Male C57BL/6N-mice were subjected to TH (blood pressure, 35 mm Hg, 60 min) followed by finasteride application and fluid resuscitation. Thereafter, finasteride was administered every 12 h. 24 h after TH, sepsis was induced by cecal ligation and puncture (CLP) or sham operation was performed. Plasma cytokines (MIP-1α, MIP-1β, TNF-α, MCP-1, IL-6), productive capacity by alveolar macrophages (AM) and systemic estradiol levels were determined 4 h thereafter. The expression of pro-inflammatory mediators in lung tissue was evaluated by PCR. Pulmonary infiltration of PMN was determined by immunohistochemical staining.

Results

Finasteride treatment resulted in a reduced posttraumatic cytokine secretion of AM as well as in a decreased concentration of MCP-1 and MIP-1β in lung tissue. Systemic estradiol levels were increased following finasteride treatment.

Conclusion

Finasteride mediates salutary effects on the pulmonary immune response using a therapeutical approach following TH–CLP in mice. Thus, finasteride might represent a relevant therapeutic substance following major trauma also in the clinical setting.     

Copper and selenium status as biomarkers of neonatal infections

Neonatal infections are a major risk factor for neonatal mortality. A reliable diagnosis of early-onset sepsis (EOS) is hampered by the variable clinical presentations of the children. We hypothesized that changes in the Se or Cu status, or the biomarkers selenoprotein P (SELENOP) or ceruloplasmin (CP) alone or in combination may be informative of EOS.We generated a new human CP-specific non-competitive immunoassay (ELISA) suitable of analysing small sample volumes and validated the method with a commercial CP source. Using this novel CP assay, we analysed a case-control study of EOS (n = 19 control newborns, n = 18 suspected cases). Concentrations of Se, Cu, SELENOP, CP, interleukin-6 (IL-6), and C-reactive protein (CRP) along with the Cu/Se and CP/SELENOP ratios were evaluated by correlation analyses as biomarkers for EOS. Diagnostic value was estimated by receiver operating characteristic (ROC) curve analyses.The new CP-ELISA displayed a wide working range (0.10–6.78 mg CP/L) and low sample requirement (2 μL of serum, EDTA-, heparin- or citrate-plasma). Plasma CP correlated positively with Cu concentrations in the set of all samples (Pearson r = 0.8355, p < 0.0001). Three of the infected neonates displayed particularly high ratios of Cu/Se and CP/SELENOP, i.e., 3.8- to 6.9-fold higher than controls. Both the Cu/Se and the CP/SELENOP ratios correlated poorly with the early infection marker IL-6, but strongly and positively with the acute-phase protein CRP (Cu/Se-CRP: Spearman ϱ = 0.583, p = 0.011; CP/SELENOP-CRP: ϱ = 0.571, p = 0.013). The ROC curve analyses indicate that a combination of biomarkers for the Se and Cu status do not improve the early identification of EOS considerably.This study established a robust, highly precise, partly validated and scalable novel CP sandwich ELISA suitable for basic and clinical research, requiring minute amounts of sample. The ratio of circulating CP/SELENOP constitutes a promising new composite biomarker for detection of EOS, at least in a subset of severely diseased children.     

The effects of di(2-ethylhexyl) phthalate and/or selenium on trace element levels in different organs of rats

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer for synthetic polymers, is known to have endocrine disruptive potential, reproductive toxicity, and induces hepatic carcinogenesis in rodents. Selenium (Se) is a component of several selenoenzymes which are essential for cellular antioxidant defense and for the functions of mammalian reproductive system. The present study was designed to investigate the effects of DEHP exposure on trace element distribution in liver, testis, and kidney tissues and plasma of Se-deficient and Se-supplemented rats. Se deficiency was produced by feeding 3-week old Sprague-Dawley rats with ≤0.05 mg Se/kg diet for 5 weeks, and supplementation group were on 1 mg Se/kg diet. DEHP treated groups received 1000 mg/kg dose by gavage during the last 10 days of feeding period. Se, zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn) levels were measured by inductively coupled plasma mass spectrometry (ICP-MS). Se supplementation caused significant increases in hepatic, renal, and testicular Se levels. With DEHP exposure, plasma Se and Zn, kidney Se, Cu and Mn levels were significantly decreased. Besides, liver Fe decreased markedly in all the DEHP-treated groups. Liver and kidney Mn levels decreased significantly in DEHP/SeD group compared to both DEHP and SeD groups. These results showed the potential of DEHP exposure and/or different Se status to modify the distribution pattern of essential trace elements in various tissues, the importance of which needs to be further evaluated.     

Differences in the expression of LPS-receptors are not responsible for the sex-specific immune response after trauma and hemorrhagic shock

Several studies demonstrated a sex-specific cytokine secretion by macrophages following trauma-hemorrhage (T-H) and incubation with lipopolysaccharide A (LPS). Although LPS is known to act via the receptors CD14 and TLR4 on macrophages, it remains unknown whether differences in LPS receptor expression in males and females may be responsible for the gender-specific LPS induced cytokine response following (T-H). To study this, male and proestrus female mice (C3H/HeN) were subjected to trauma (laparotomy) followed by hemorrhage or sham operation. At 2 h thereafter, SMphi and PMphi were harvested and cultured for 2 h. The expression of CD14 and TLR4 was measured by flow cytometry on unstimulated SMphi and PMphi as well as after LPS stimulation. The results indicate that the expression of CD14 and TLR4 on SMphi and PMphi from female and male mice was similar in sham-operated animals and after (T-H). Incubation of macrophages with LPS did not alter CD14 and TLR4 expression in the study groups. Thus, the sex specific LPS induced cytokine secretion after (T-H) is not caused by differences in LPS receptor expression on Mphi of male and female mice.     

Endocrine and metabolic regulation of renal drug transporters

Renal xenobiotic transporters are important determinants of urinary secretion and reabsorption of chemicals. In addition to glomerular filtration, these processes are key to the overall renal clearance of a diverse array of drugs and toxins. Alterations in kidney transporter levels and function can influence the efficacy and toxicity of chemicals. Studies in experimental animals have revealed distinct patterns of renal transporter expression in response to sex hormones, pregnancy, and growth hormone. Likewise, a number of disease states including diabetes, obesity, and cholestasis alter the expression of kidney transporters. The goal of this review is to provide an overview of the major xenobiotic transporters expressed in the kidneys and an understanding of metabolic conditions and hormonal factors that regulate their expression and function. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:407–421, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21435     

Dietary selenium deficiency and supplementation differentially modulate the expression of two ER-resident selenoproteins (selenoprotein K and selenoprotein M) in the ovaries of aged mice: Preliminary data

In the present report, we determined the impact of dietary selenium (Se) deficiency and supplementation on the expression of two ER-resident selenoproteins i.e., Selenok and Selenom in the ovaries of aging mice. The mRNA expression of Selenok and Selenom (RT-qPCR) was significantly higher in the ovaries of mice fed diets supplemented with inorganic (ISe-S: 0.33 mg Se/kg) and organic (OSe-S: 0.33 mg Se/kg) Se compared to those fed a Se-deficient (Se-D: 0.08 mg Se/kg) diet and both Se-adequate (ISe-A: 0.15 mg Se/kg and OSe-A: 0.15 mg Se/kg) diets. Similarly, the protein signals of SELENOK (immunofluorescence assay) were also significantly higher in the Se-supplemented groups compared to those fed Se-D and Se-adequate (ISe-A and OSe-A) diets. Meanwhile, the rate of in vitro-produced blastocysts developing from MII oocytes was also evaluated and it was revealed that this rate was significantly higher in the Se-supplemented mice compared to those fed a Se-D diet. Altogether, the dietary Se supplementation increased the expression of Selenok (also its protein expression) and Selenom in the ovaries of aging mice, potentially contributing to an improved developmental potential of in vitro-matured M II oocytes.     

Effects of selenium supplementation on selenoprotein gene expression and response to influenza vaccine challenge: a randomised controlled trial

Background

The uncertainty surrounding dietary requirements for selenium (Se) is partly due to limitations in biomarkers of Se status that are related to health outcomes. In this study we determined the effect of different doses and forms of Se on gene expression of selenoprotein S (SEPS1), selenoprotein W (SEPW1) and selenoprotein R (SEPR), and responses to an immune function challenge, influenza vaccine, were measured in order to identify functional markers of Se status.

Methods and Findings

A 12 week human dietary intervention study was undertaken in 119 volunteers who received placebo, 50, 100 or 200 µg/day Se-enriched yeast (Se-yeast) or meals containing unenriched or Se-enriched onions (50 µg/day). Gene expression was quantified in RNA samples extracted from human peripheral blood mononuclear cells (PBMC''s) using quantitative RT-PCR. There was a significant increase in SEPW1 mRNA in the Se-enriched onion group (50 µg/day) compared with the unenriched onion group. SEPR and SEPW1 did not change significantly over the duration of the supplementation period in the control or Se-yeast groups, except at week 10 when SEPW1 mRNA levels were significantly lower in the 200 µg/day Se-yeast group compared to the placebo group. Levels of SEPS1 mRNA increased significantly 7 days after the influenza vaccine challenge, the magnitude of the increase in SEPS1 gene expression was dose-dependent, with a significantly greater response with higher Se supplementation.

Conclusions

This novel finding provides preliminary evidence for a role of SEPS1 in the immune response, and further supports the relationship between Se status and immune function.

Trial Registration

ClinicalTrials.gov [{"type":"clinical-trial","attrs":{"text":"NCT00279812","term_id":"NCT00279812"}}NCT00279812]     

Gender dimorphism in regulation of plasma proteins in streptozotocin‐induced diabetic rats

In the present study, we examined differentially regulated plasma proteins between healthy control and streptozotocin (STZ)‐induced male and female diabetic rats by 2DE‐based proteomic analysis. Animal experiments revealed that significantly lower plasma insulin levels were observed in female diabetic rats, consequently resulting in higher blood glucose levels in female diabetic rats. Importantly, plasma levels of sex hormones were significantly altered in a gender‐dependent manner before and after STZ treatment. Results of the animal experiment indicated the existence of sexual dimorphism in the regulation of plasma proteins between healthy control and diabetic rats. Plasma proteome analysis enabled us to identify a total of 38 proteins showing sexual dimorphic regulation patterns. In addition, for the first time, we identified several differentially regulated plasma proteins between healthy control and diabetic rats, including apolipoprotein E, fetuin B, α‐1‐acid glycoprotein, β‐2‐glycoprotein 1, 3‐hydroxyanthranilate 3,4‐dioxygenase, and serum amyloid P‐component. To the best of our knowledge, this is the first proteomic approach to address sexual dimorphism in diabetic animals. These proteomic data on gender‐dimorphic regulation of plasma proteins provide valuable information that can be used for evidence‐based gender‐specific clinical treatment of diabetes.     

Effects of selenium and serum on selenoprotein W in cultured L8 muscle cells

When rat L8 muscle cells were cultured to examine the effects of serum and selenium concentration on selenoprotein W levels and glutathione peroxidase (GPX) activities, no significant differences (P > 0.05) were found in selenoprotein W levels and GPX activities during differentiation. With three different forms of selenium, selenoprotein W levels and GPX activities were shown to increase in L8 myotubes cultured in media with these selenocompounds. Selenite was utilized more efficiently than selenocysteine for both selenoprotein W and GPX activity, but selenium as selenomethionine was less available. Both the protein content and mRNA levels for selenoprotein W were affected by the selenium content of the media. Northern blot data indicated that the expression of selenoprotein W mRNA increased significantly when L8 myotubes were cultured with selenium (P > 0.05). L8 myotubes cultured in 10% calf serum (CS) versus 2% CS with or without addition of 10 m selenium indicated that the increase of selenoprotein W level in L8 myotubes cultured with higher serum concentration (10% CS) is due to the higher selenium concentration in media rather than serum itself.     

mTOR-dependent regulation of PHLPP expression controls the rapamycin sensitivity in cancer cells

PHLPP belongs to a novel family of protein phosphatases that serve as negative regulators of Akt. There are two isoforms, PHLPP1 and PHLPP2, identified in this family. Our previous studies indicated a tumor suppressor role of both PHLPP isoforms in colon cancer. Here we report that the expression of PHLPP is controlled by mTOR-dependent protein translation in colon and breast cancer cells. Treating cells with rapamycin or knockdown of mTOR using RNAi results in a marked decrease of PHLPP protein expression. In contrast, stable knockdown of TSC2, a negative regulator of mTOR activity, increases PHLPP expression. The rapamycin-mediated down-regulation of PHLPP is blocked by expression of a rapamycin-insensitive mutant of p70S6K. In addition, depletion of 4E-BP1 expression by RNAi results in an increase of PHLPP expression and resistance to rapamycin-induced down-regulation. Moreover, inhibition of mTOR activity by amino acid or glucose starvation reduces PHLPP expression in cells. Functionally, we show that rapamycin-mediated inhibition of PHLPP expression contributes to rapamycin resistance in colon cancer cells. Thus, our studies identify a compensatory feedback regulation in which the activation of Akt is inhibited by up-regulation of PHLPP through mTOR, and this mTOR-dependent expression of PHLPP subsequently determines the rapamycin sensitivity of cancer cells.