Effect of high-dose sodium selenite therapy on polymorphonuclear leukocyte apoptosis in non-Hodgkin's lymphoma patients

The present study was undertaken to explore the effect of the administration of high doses of sodium selenite on apoptosis in polymorphonuclear leukocytes in patients with non-Hodgkin's lymphoma. Thirty patients with newly diagnosed non-Hodgkin's lymphoma were randomly divided into two groups. Group I was treated with chemotherapy and group II received 0.2 mg/kg/d sodium selenite in addition to chemotherapy. Flow cytometry was used for the monitoring of apoptosis on peripheral blood neutrophils at the time of diagnosis and after treatment in both groups of patients. Sodium selenite administration resulted in a significant reduction in neutrophils apoptosis (82+/-10% vs 32+/-18%, p<0.05) and this was associated with significant reduction in infection rate following chemotherapy (67% vs 20%, p<0.05). Also, significant improvement in cardiac ejection fraction was observed (62+/-4% vs 69+/-5% p<0.05). It is concluded that sodium selenite administration at the dosage chosen acts as a cytoprotective agent, alleviating side effects and immunosuppressive effects of cytotoxic chemotherapeutic agents.     

Induction of apoptosis by sodium selenite in human acute promyelocytic leukemia NB4 cells: involvement of oxidative stress and mitochondria.

The mechanisms involved in the anti-carcinogenic activity of selenium remained to be elucidated. In the present study, we examined sodium selenite induced apoptosis and oxidative stress in human acute promyelocytic leukemia cell lines (NB4). Cell growth and viability were assessed by trypan blue exclusion and cell counting; apoptosis by DNA electrophoresis and analysis of intracellular DNA contents; reactive oxygen species and reduced glutathione in the cell were measured by lucigenin dependent chemoluminescent (CL) test and spectrophotometer; mitochondrial transmembrane potential was measured by flow cytometry. Sodium selenite could inhibit the growth and induce apoptosis of NB4 cells. Sodium selenite could increase the production of reactive oxygen species (ROS) in NB4 cells and decrease the level of intracellular reduced glutathione, but caused no change in the activity of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx). Sodium selenite enhanced the collapse of mitochondrial transmembrane potential (MTP), in parallel with the production of ROS. Finally antioxidant N-acetylcysteine (NAC) could inhibit the ROS production, MTP collapse and apoptosis in NB4 cells. Our results suggested that sodium selenite could induce apoptosis of NB4 cells through mitochondrial change mediated by production of reactive oxygen species within the cells.     

Sodium Selenite Improves Folliculogenesis in Radiation-Induced Ovarian Failure: A Mechanistic Approach

Radiotherapy is a major factor contributing to female infertility by inducing premature ovarian failure (POF). Therefore, the need for an effective radioprotective agent is evident. The present study investigated the mechanism of potential radioprotective effect of sodium selenite on radiation-induced ovarian failure and whether sodium selenite can stimulate in-vivo follicular development in experimental rats. Immature female Sprague-Dawely rats were either exposed to gamma-radiation (3.2 Gy, LD₂₀), once and/or treated with sodium selenite (0.5 mg/kg), once daily for one week before irradiation. Follicular and oocyte development, apoptotic markers, proliferation marker as well as oxidative stress markers were assessed 24-h after irradiation. In addition, fertility assessment was performed after female rats became completely mature at two months of age. Sodium selenite significantly enhanced follicular development as compared to the irradiated group. Sodium selenite significantly reversed the oxidative stress effects of radiation that was evidenced by increasing in lipid peroxide level and decreasing in glutathione level, and glutathione peroxidase (GPx) activity. Assessment of apoptosis and cell proliferation markers revealed that caspase 3 and cytochrome c expressions markedly-increased, whereas, PCNA expression markedly-decreased in the irradiated group; in contrast, sodium selenite treatment prevented these alterations. Histopathological examination further confirmed the radioprotective efficacy of sodium selenite and its in-vivo effect on ovarian follicles’ maturation. In conclusion, sodium selenite showed a radioprotective effect and improved folliculogenesis through increasing ovarian granulosa cells proliferation, estradiol and FSH secretion, and GPx activity, whilst decreasing lipid peroxidation and oxidative stress, leading to inhibition of the apoptosis pathway through decreasing the expressions of caspase 3 and cytochrome c.     

Biological effects of a nano red elemental selenium.

A novel selenium form, nano red elemental selenium (Nano-Se) was prepared by adding bovine serum albumin to the redox system of selenite and glutathione. Nano-Se has a 7-fold lower acute toxicity than sodium selenite in mice (LD(50) 113 and 15 mg Se/kg body weight respectively). In Se-deficient rat, both Nano-Se and selenite can increase tissue selenium and GPx activity. The biological activities of Nano-Se and selenite were compared in terms of cell proliferation, enzyme induction and protection against free racial-mediated damage in human hepatoma HepG2 cells. Nano-Se and selenite are similarly cell growth inhibited and stimulated synthesis of glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and thioredoxin reductase (TR). When HepG2 cells were co-treated with selenium and glutathione, Nano-Se showed less pro-oxidative effects than selenite, as measured by cell growth. These results demonstrate that Nano-Se has a similar bioavailability in the rat and antioxidant effects on cells.     

Comparative study on sodium transport and Na+,K+-ATPase activity in Caco-2 and rat jejunal epithelial cells: effects of dopamine

The present study reports on the effects of dopamine on sodium transepithelial transport and Na+,K+-ATPase activity in Caco-2 cells, a human epithelial intestinal cell line which undergoes enterocyte differentiation in culture, and jejunal epithelial cells from 20 day old Wistar rats. Addition of amphotericin B to the mucosal side stimulated Isc in a concentration dependent manner (Caco-2 cells, EC50=0.9 [0.5, 1.7] microM; rat jejunum, EC50=7.4 [0.8; 70.1] microM). The presence of 1 microM dopamine did not change the effect of amphotericin B in Caco-2 cells, but produced a significant (P<0.05) decrease in the maximal effect of amphotericin B in the rat jejunum. Dopamine (1 microM), added to the serosal side, did not change the Isc profile in Caco-2 cells, but produced a significant increase in the rat jejunum. This effect was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). Basal Na+,K+-ATPase activity (in nmol Pi mg protein(-1) min(-1)) in Caco-2 cells (49.5+/-0.2) was similar to that observed in isolated rat jejunal epithelial cells (52.3+/-3.4). Dopamine (1 microM) significantly (P<0.05) decreased Na+,K+-ATPase activity in rat jejunal epithelial cells, but failed to inhibit Na+,K+-ATPase in Caco-2 cells. This effect of dopamine was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). The specific binding of [3H]-Sch 23390 to the rat intestinal mucosa was saturable with an apparent dissociation constant (KD) of 2.4 (0.4; 4.5) nM and maximum receptor density of 259.8+/-32.6 fmol/mg protein. No significant specific binding of [3H]-Sch 23390 was observed in membranes from Caco-2 cells. In conclusion, the results obtained show that D1-like receptor mediated effects of dopamine in the rat jejunum on sodium absorption are absent in Caco-2 cells, most probably because this cell line does not express D1-like dopamine receptors, which ultimately are responsible for the inhibitory effect of the amine upon intestinal Na+,K+-ATPase.     

Comparison of glutathione peroxidase 1 and iodothyronine deiodinase 1 mRNA expression in murine liver after feeding selenite or selenized yeast

The experiment was conducted to compare the effect of different selenium sources on the expression of glutathione peroxidase 1 (GPx1) and iodothyronine deiodinase 1 (Dio1) mRNA in mice by quantitative real-time PCR. A total of 60 male Kunming mice at average body weight of 20 g were allotted to three groups in a randomized complete block design, namely two treatments and one control. Mice in Group 1 were fed a basal diet as control, while mice in Groups 2 and 3 were fed the basal diet supplemented with 0.1 mg/kg selenium as sodium selenite or selenized yeast, respectively. Whole feeding experiment lasted for 30 d. At the end of the feeding trial, liver mRNA levels of GPx1 and Dio1 were determined by quantitative real-time PCR, as well as growth performance, body composition, blood and GPx activity were determined. The results showed that no significant differences in overall growth performance and body composition, including body weight, body length, heart weight, kidney weight and liver weight, were found between the experimental groups (P>0.05). Blood GPx activity increased in all of the selenium supplemented groups compared with control group (P<0.01). However, blood GPx activity in selenized yeast group was higher than that in sodium selenite group (P<0.05). Liver mRNA levels of GPx1 and Dio1 also increased in the two selenium supplemented groups compared with the control group (P<0.05), while there was no significant difference between the sodium selenite and selenized yeast groups (P>0.05). In conclusion, selenium increased the mRNA expression of GPx1 and Dio1 genes in murine liver, and there was no significant difference between the organic or inorganic form of selenium used.     

Influence of SelS gene silence on β-Mercaptoethanol-mediated endoplasmic reticulum stress and cell apoptosis in HepG2 cells

Selenoprotein S (SelS), a transmembrane selenoprotein, may be related to the response of endoplasmic reticulum (ER) stress. In this report, the influence of selenite supplementation and SelS gene silence on β-mercaptoethanol (β-ME)-mediated ER stress and cell apoptosis in HepG2 cells were examined. The results showed that SelS protein expression was markedly increased by 10 mM β-ME and 100 nM sodium selenite in HepG2 cells. GRP78 protein level was significantly increased after treatment with 10 mM β-ME in HepG2 cells, which suggested that β-ME was also an ER stress inducer. Meanwhile, β-ME (10 mM) was found to induce cell apoptosis, which was alleviated obviously when cells were pretreated with 100 nM selenite before exposure to β-ME. Moreover, the suppression of SelS gene by siRNA could aggravate HepG2 cell apoptosis induced by β-ME significantly. In conclusion, these results suggested that β-ME, also an ER stress agent, could induce cell apoptosis, and SelS may play an important role in protecting cells from apoptosis induced by ER stress in HepG2 cells.     

Mechanistic studies on protopanaxadiol, Rh2, and ginseng (Panax quinquefolius) extract induced cytotoxicity in intestinal Caco-2 cells

Certain ginsenosides, also known as triterpene glycosides, have been recently reported to have a characteristic effect on cultured intestinal and leukemia cell growth. Ginsenoside aglycones 20(S)-protopanaxadiol (PD), 20(S)-protopanaxatriol (PT), and ginsenoside Rh2 have been identified as having a strong effect on reducing cell viability. Furthermore, ginsenoside Rh2 is thought to be a rare ginsenoside not found in all ginseng products. Rather, Rh2 has been recently reported to be a breakdown product of thermal processing of North American ginseng. In this study, pure ginsenosides PD, PT, Rh2 standards and an enriched Rh2 fraction derived from ginseng leaf were tested in cultured Caco-2 cells for relative cytotoxic potency. PD and Rh2 LC50 were similar after 24 to 72 h, whereas a drop in PT LC50 occurred later at 48 and 72 h. Furthermore, PD and Rh2 affected membrane integrity as indicated by LDH secretion earlier than PT and the enriched Rh2 fraction (P < or = 0.05). Ginsenoside Rh2 showed the greatest (P < or = 0.05) build up of necrotic cells (18.3 +/- 0.1%) at the respective LC50 after 24 h and PD (21.3 +/- 0.3%) showed the largest effect after 44 h of exposure. The effect on apoptotic cells at 44 h of treatment were significantly different (P < or = 0.05) for Rh2 (21 +/- 0.4%), PD (14.6 +/- 0.1%), enriched Rh2 leaf fraction (9.9 +/- 0.6%), and PT (2.3 +/- 0.1%) treatments. Caco-2 caspase-3 activity was different between ginsenoside exposure; Rh2 (10.6 +/- 0.3 nM pNA) had the greatest (P < or = 0.05) activity followed by the enriched Rh2 leaf fraction (8.3 +/- 0.2 nM pNA), PT (7.3 +/- 0.3 nM pNA). The PD (4.8 +/- 0.04 nM pNA) treatment was similar to untreated cells (4.3 +/- 0.05 nM pNA) in caspase-3 activity. These results show variable bioactive response in cultured intestinal cell to specific ginsenosides and an enriched Rh2 North American ginseng extract which may be explained on basis of hydrophobic/hydrophilic balance.     

Arsenic suppresses necrosis induced by selenite in human leukemia HL-60 cells

Selenium, an essential trace element for humans, has been shown to have anticancer effects. Arsenic, a possibly essential ultratrace element for humans, has been used in the treatment of leukemia. Anticancer effects of selenium and arsenic have been related to their ability to induce apoptosis. Because humans are exposed to diverse trace elements simultaneously, it is important to learn their interrelationship. In this study, we demonstrate that sodium selenite (Na₂SeO₃) causes apoptosis at 3 μM and necrosis at high concentrations (>3 μM) in HL-60 cells. Similarly, both sodium arsenite (NaAsO₂) at 50 μM and sodium arsenate (Na₂HAsO₄) induce apoptosis at 500 μM and necrosis at higher concentrations (>50 μM and >500 μM, respectively) in HL-60 cells. Arsenite/arsenate, but not selenite, enhances AP-1 DNA-binding activity. This finding indicates different mechanisms through which apoptosis is induced by these two elements. Interestingly, we observed that HL-60 cell necrosis induced by a high concentration (>3 μM) of selenite was essentially inhibited by arsenic (50 μM of NaAsO₂ or 500 μM of Na₂HAsO₄), which resulted in a net effect of apoptosis. Because AP-1 DNA-binding activity was not induced in the presence of a combination of necrotic amount of selenite and apoptotic amount of arsenite/arsenate, the observed apoptosis apparently was through the mechanism used by selenite. Our results suggest, for the first time, that the toxic necrotic effect of selenite can be neutralized by arsenite/arsenate at the cellular level. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

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.     

Goniothalamin induces apoptosis in vascular smooth muscle cells

Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.     

Epidermal growth factor, insulin, and estrogen stimulate development of prolactin-secreting cells in cultures of GH3 cells

Pituitary tumor GH3 cells synthesize and secrete both growth hormone (GH) and prolactin (PRL). Morphological and functional changes of GH3 cells induced by epidermal growth factor (EGF, 10 nM), insulin (300 nM), and estradiol-17beta (E2, 1 nM) were studied. Treatment of cultures of GH3 cells for 6 days with EGF, insulin, or E2 alone, and with EGF plus E2 did not affect the total number of GH3 cells, but a combination of EGF, insulin, and E2 decreased the total number of GH3 cells compared with control treatment. DNA-synthesizing cells were detected by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake. EGF, E2, or a combination of EGF, insulin, and E2 significantly decreased the proportion of BrdU-labeled cells (21.1+/-1.7%, 21.0+/-1.4%, 18.2+/-1.3%; P<0.05, P<0.05, P<0.01, respectively) compared with control treatment (28.6+/-1.5%), but insulin did not (31.4+/-2.4%). Immunocytochemical analysis of GH3 cells cultured in 5% fetal calf serum-supplemented medium (control) showed that about 70% of all GH3 cells were GH-immunoreactive cells (GH-ir cells), apparently containing only GH, and 14% were mammosomatotrophs (MS cells), containing both GH and PRL, while PRL-immunoreactive cells (PRL-ir cells), containing only PRL, were not detected. No GH or PRL immunoreactivity could be detected in the remaining cells (15%). EGF decreased the proportion of GH-ir cells. The effects of EGF were enhanced by simultaneous exposure to insulin and E2; this decreased the proportion of GH-ir cells to about 20% of the total GH3 cells and significantly increased the proportion of MS cells to 300% of controls. Treatment with EGF plus insulin, EGF plus E2, or a combination of EGF, insulin, and E2 all stimulated the appearance of PRL-ir cells. Exposure to EGF caused a significant decrease in GH mRNA (P<0.01) and a significant increase in PRL mRNA (P<0.05). These observations suggest that EGF is closely involved in differentiation of PRL-ir cells from GH-ir cells via MS cells in GH3 cell cultures. Cytosine arabinoside (10(-7) M), an inhibitor of cell division, did not affect the changes in proportion of the three cell types induced by treatment with a combination of EGF, insulin, and E2. It is therefore probable that the transdifferentiation does not require mitosis of the GH3 cells.     

Osteoblastic MG-63 cell differentiation, contraction, and mRNA expression in stress-relaxed 3D collagen I gels

The present study was designed to evaluate the apoptotic efficacy of selenium (Se) under glutathione-deprived conditions. Testicular cells were used as a model to assess the above. For the study, cells were maintained for 4 h under various treatments; control (media only), selenium (0.5 microM and 1.5 microM), BSO (20 nM), selenium + BSO (0.5 microM Se + 20 nM BSO and 1.5 microM Se + 20 nM BSO). The treated cells were harvested for various estimations viz. viability, GSH, GSSG, redox ratio, ROS generation and integrity of DNA. mRNA was extracted for RT-PCR analysis of JNK, p38, caspase 3 and Bcl-2. It was observed that the cell viability decreased concomitant with the decrease in GSH levels, increase in GSSG levels and increase in the generation of ROS in the combined treatment group in comparison to control and individual treatments. Also, there was an increase in the mRNA expression of JNK and p38 MAPK along with an increase in caspase 3 expression and decrease in Bcl-2 expression. The integrity of DNA was also found to be altered in the combined treatment. Thus, the results presented in this work agree with those earlier reports in a notion that sodium selenite causes apoptosis and the toxicity of selenite is mediated by increase of intracellular ROS. Also, reduction in endogenous GSH along with selenite treatment is associated with increased apoptosis, increased expression of p38 and JNK MAPK, decreased Bcl-2 expression, and increase in caspase-3 expression. Our data indicates that GSH participates in apoptosis in testicular cells and that depletion of this molecule may be critical in predisposing these cells to apoptotic cell death.     

Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand

Neutrophils release soluble Fas ligand (sFasL), which can induce apoptosis in certain Fas-bearing cell types (Liles WC, Kiener PA, Ledbetter JA, Aruffo A, and Klebanoff SJ. J Exp Med 184: 429-440, 1996). We hypothesized that neutrophils could induce alveolar epithelial apoptosis via release of sFasL. A549 pulmonary adenocarcinoma cells expressed surface Fas and underwent cell death (10 +/- 7% viability) and DNA fragmentation (354 +/- 98% of control cells) when incubated with agonistic CD95/Fas monoclonal antibody (P < 0.05). Coincubation with human neutrophils induced significant A549 cell death at 48 (51 +/- 9% viability; P < 0.05) and 72 h (25 +/- 10%; P < 0.05) and increased DNA fragmentation (178 +/- 42% of control cells; P < 0.05), with morphological characteristics of apoptosis. The addition of antioxidants did not inhibit apoptosis. sFasL concentrations were maximally increased in coculture medium at 24 h (4.9 +/- 0.7 ng/ml; P < 0.05). Neutrophil-induced A549 cell apoptosis was blocked by inhibitory anti-Fas (42 +/- 6% of control cells; P < 0.05) and anti-FasL monoclonal antibodies (29 +/- 3%; P < 0.05). Human neutrophils and Fas similarly affected murine primary alveolar epithelial cell bilayers, and caspase activation occurred in response to Fas exposure. We conclude that neutrophils undergoing spontaneous apoptosis induce A549 cell death and DNA fragmentation, independent of the oxidative burst, that is mediated by sFasL.     

Metabolism of subtoxic level of selenite by double-perfused small intestine in rats

Intestinal metabolism of the subtoxic level of selenite in rats was investigated using a double-perfusion system, which is an in situ, in vitro preparation in which the intestinal lumen and its vasculature are perfused simultaneously. The toxicity of sodium selenite was determined by inhibition of 3-O-methyl glucose (3MG) absorption and by histological examination. Levels of 1.2 mM selenite were required to significantly (p<0.05) reduce 3MG intestinal absorption (58±11%, mean±SD). Cation-exchange chromatography was used to determine the chemical forms of Se from selenite after using luminal concentrations of 1–200 μM in vascular perfusates. The chemical forms were selenite, selenodiglutathione (GS-Se-SG), mixed selenoglutathione plus cysteine (GS-Se-CYS), selenodicysteine (CYS-Se-CYS), protein-bound Se, and unidentified selenocompounds. Selenite was the predominant selenocompound found in vascular perfusate, but protein-bound Se was the predominant metabolite from selenite present in the vascular effuents. There was a corresponding increase of all metabolites with increased levels of selenite with time of absorption, but not with increased concentration of luminal selenite.     

Intracellular reduction of selenite into glutathione peroxidase. Evidence for involvement of NADPH and not glutathione as the reductant

Selenium (Se) in selenite is present in an oxidized state, and must be reduced for it to be incorporated as selenocysteine into selenoenzymes such as glutathione peroxidase (GPx). In vitro, Se, as in selenite, can be reduced utilizing glutathione (GSH) and glutathione reductase (GRed). We determined the effects of decreasing GSH levels, inhibiting GRed activity, and decreasing cellular NADPH on the selenite-dependent rate of GPx synthesis in cultured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD)-deficient cell line. A novel statistical analysis method was developed (using Box Cox transformed regression and a bootstrap method) in order to assess the effects of these manipulations singly and in combinations. Buthionine sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GRed activity and methylene blue (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinations on GPx activity. Decreasing the GSH level (< 5% of control) did not have an effect on the selenite-dependent rate of GPx synthesis in PC3 or CHO cells, but did have a small inhibitory effect on the rate of GPx synthesis in E89 cells. Inhibiting GRed activity was also associated with either no effect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decreasing NADPH levels in cells treated with MB was associated with a large decrease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of control in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BCNU were not synergistic in any of the cell lines. The effects of BSO plus MB were synergistic in G-6-PD-deficient E89 cells, but not in PC3 or CHO cells. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that are eventually incorporated into GPx. For cells with abnormal ability to generate NADPH, lowering the GSH levels had a small effect on selenite-dependent GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx.     

Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death

To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H₂O₂-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H₂O₂, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak-1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H₂O₂-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 μM H₂O₂ was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 μM H₂O₂. Meanwhile, H₂O₂ induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulation of Bcl-2 (P < 0.05). When lymphocytes were pretreated with Se before treated with H₂O₂, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P < 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulated Bcl-2 (P < 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress.     

Somatostatin inhibition of phosphoinositides turnover in isolated rat acinar pancreatic cells: interaction with bombesin.

The effects of somatostatin-14 and bombesin on [3H]inositol phosphate accumulation were studied in 24 h myo-[3H]inositol-prelabeled cultured rat acinar cells. Bombesin, 10 nM, stimulated basal formation of phosphatidyl monophosphate (InsP1), phosphatidyl 4,5-biphosphate (InsP2) and inositol 1,4,5-triphosphate (InsP3) by 128 +/- 5.2%, 147 +/- 10% and 155 +/- 5%, respectively. At 5 s, the ED50 value for InsP3 stimulation was 0.70 +/- 0.2 nM. This stimulation was partly blocked (64 +/- 0.04% inhibition) by 10 ng/ml Bordetella pertussis toxin. In contrast to bombesin, somatostatin, 10 nM, inhibited basal InsP1, InsP2 and InsP3 formation. At 5 s, the inhibition degree for InsP3 was 18 +/- 2.5% and the IC50s values 1 +/- 0.09 nM, 1 +/- 0.12 nM and 0.07 +/- 0.005 nM for InsP1, InsP2 and InsP3, respectively. Bombesin-stimulated InsP3 formation was also inhibited by somatostatin. At 5 s, the inhibition degree was 85 +/- 3.5% at 10 nM and the IC50 value, 0.10 +/- 0.05 nM. Furthermore, somatostatin inhibition of bombesin stimulation was partly blocked (66 +/- 4% inhibition) by Bordetella pertussis toxin. These data therefore suggest that the acinar pancreatic cells contain a somatostatin receptor exerting a negative control on basal and bombesin receptor-stimulated phosphatidyl inositol turnover.