Selenium-Based Novel Epigenetic Regulators Offer Effective Chemotherapeutic Alternative with Wider Safety Margins in Experimental Colorectal Cancer.

Colorectal cancer (CRC) is a major cause of morbidity and mortality worldwide. Despite the critical involvement of epigenetic modifications in CRC, the studies on the chemotherapeutic efficacy of various epigenetic regulators remain limited. Considering the key roles of histone deacetylases (HDACs) in the regulation of diverse cellular processes, several HDAC inhibitors are implied as effective therapeutic strategies. In this context, suberoylanilide hydroxamic acid (SAHA), a 2nd-generation HDAC inhibitor, showed limited efficacy in solid tumors. Also, side effects associated with SAHA limit its clinical application. Based on the redox-modulatory and HDAC inhbitiory activities of essential trace element selenium (Se), the anti-carcinogenic potential of Se substituted SAHA, namely, SelSA-1 (25 mg kg-1), was screened for it enhanced anti-tumorigenic role and wider safety profiles in DMH-induced CRC in Balb/c mice. A multipronged approach such as in silico, biochemical, and pharmacokinetics (PK) has been used to screen, characterize, and evaluate these novel compounds in comparison to existing HDAC inhibitor SAHA. This is the first in vivo study indicating the chemotherapeutic potential of Se-based novel epigenetic regulators such as SelSA-1 in any in vivo experimental model of carcinogenesis. Pharmcological and toxicity data indicated better safety margins, bioavailability, tolerance, and elimination rate of SelSA-1 compared to classical HDAC inhibitor SAHA. Further, histological and morphological evidence demonstrated enhanced chemotherapeutic potential of SelSA-1 even at lower pharmacological doses than SAHA. This is the first in vivo study suggesting Se-based novel epigenetic regulators as potential chemotherapeutic alternatives with wider safety margins and enhanced anticancer activities.

Hempseed (Cannabis sativa) offers effective alternative over statins in ameliorating hypercholesterolemia associated nephropathy.

A direct link between hypercholesterolemia (HC) and renal pathologies has been established. Statins, the drugs of choice for HC management, have been associated with various side effects and toxicities, including nephropathy and other renal insults. Thus, natural dietary products based-alternative strategies for HC and associated pathologies are being considered. OBJECTIVES: Based on the unique nutritional composition and numerous health benefits of Hempseeds (Cannabis sativa), currently the potential anti-inflammatory and redox modulatory effects of hempseeds lipid extract (HEMP) against HC associated renal damage were evaluated and compared with statins (Simvastatin) in HFD induced experimental model of HC in rats. DESIGN & METHODS: The hempseed lipid fractions (HEMP) were prepared and their ameliorating effects on HFD induced lipid profiles, renal function markers (RFT), histopathological/morphological changes, renal oxidative stress, and inflammation markers were studied and compared with statins (HFD + STATINS). Further, HEMP-mediated modulation of lipid metabolism mediators (APO-B/E) was studied. RESULTS: Not only, HEMP administration improved the lipid profiles and morphological signs of HC, but it also was safe compared to Simvastatin in terms of hepatic and renal function markers. Further, changes in renal histoarchitecture, biochemical markers of oxidative stress, and expression profiles of lipid metabolism and inflammatory pathways (Cox-1/2, PGDS, PGES) revealed that HEMP positively modulating the redox homeostasis activated the resolution pathways against HC associated renal insults. CONCLUSION: The outcomes of the current study indicated HEMP's ameliorative and therapeutic potential against hypercholesterolemia-associated nephropathies and other systemic effects.

Effect of Zinc on Hepatic and Renal Tissues of Chronically Arsenic Exposed Rats: A Biochemical and Histopathological Study.

Consumption of arsenic-contaminated drinking water has become major global health concern. One of the major mechanism responsible for the toxicity of arsenicals is the generation of oxidative stress. Zinc, a nutritional antioxidant, plays key role in maintaining various cellular pathways. The present study was aimed at elucidating the effects of zinc supplementation on hepatic and renal tissue damage caused by arsenic exposure to rats. Rats were randomly divided into four experimental groups: control; As administered; Zn supplemented; combined zinc; and arsenic supplemented. Arsenic exposure resulted in significantly elevated accumulation of arsenic in the liver and kidney tissue. In the liver, exposure to arsenic reduced the levels of reduced glutathione (GSH), total glutathione (TG), redox ratio, and the activity of superoxide dismutase (SOD), whereas lipid peroxidation (LPO), inflammation markers, and nitric oxide (NO) levels were elevated with no significant change in catalase (CAT) activity. Arsenic exposure also enhanced the serum levels of liver functional indices and histological abnormalities in liver sections. In the kidney, a significant increase in NO levels and decrease in SOD activity was observed, with no significant changes in the rest of the parameters. The administration of zinc- to arsenic-intoxicated animals significantly improved their hepatic function parameters, arsenic burden, and histological changes which were associated with the restoration of enzymatic and non-enzymatic antioxidant defense system as compared to their intoxicated counterparts. In the kidney also, the NO levels and SOD activity were restored. This data reveals that zinc is effective in ameliorating the toxic effects inflicted by chronic arsenic toxicity.

Selenium Ameliorates Ibuprofen Induced Testicular Toxicity by Redox Regulation: Running Head: Se protects against NSAID induced testicular toxicity.

Despite the Cox inhibitory anti-inflammatory and antipyretic effects of most widely used non-steroidal anti-inflammatory drugs (NSAIDs), such as Ibuprofen, their chronic use is associated with a plethora of patho-physiological insults. One such toxic effect on testicular tissues is not well studied and the underlying molecular mechanisms remain unexplored. Thus, the current study is designed to evaluate the antioxidant properties of essential trace element selenium (Se) to ameliorative Ibuprofen associated testicular toxic effects. Adult male Wistar rats were divided into 3 groups and fed on diets containing different concentrations of sodium selenite, viz. 0.01 mg/kg (Se- deficient), 0.2 mg/kg (Se-adequate), or 0.5 mg/kg (Se- supplemented) for 8 weeks. After diet feeding schedule, each group was divided into two subgroups i.e., with or without the treatment of Ibuprofen (120 mg/kg Bw). The protective effect of Se was evaluated by measuring testicular Se and selenoproteins status, spermatogenic markers, histopathology and testicular redox status. Ibuprofen diminished seminal volume, sperm count, sperm motility, which correlated well increased testicular reactive oxygen species. Se deficiency exacerbated these detrimental effects of ibuprofen by increasing oxidative stress. Alternatively, Se supplementation through antioxidant enzymes mediated protective effects. Se as essential antioxidant selenoproteins ameliorates Ibuprofen induced male reproductive toxicity.

Ameliorative effects of hempseed (Cannabis sativa) against hypercholesterolemia associated cardiovascular changes.

BACKGROUND AND AIM: Hypercholesterolemia (HC) is a major risk factor for cardiovascular (CV) diseases, that are the major cause of mortality worldwide. Free radicals mediated oxidative stress is a critical player in HC-associated pathophysiological insults including atherosclerosis. Unwanted side effects associated with statins, COX-2 inhibitors, and other synthetic drugs limit their use. Thus, modulation of oxidative stress during HC using green pharmaceuticals seems an appropriate approach against deleterious CV consequences without noticeable side-effect. In this regard, owing to an abundance of proteins, fiber and optimal ratios of omega 6 PUFA: omega-3 PUFA in Hempseed (HS), we aim to exploit its anti-inflammatory and antioxidant properties to ameliorate HC- associated CV effects. METHODS AND RESULTS: Comparing the antioxidant capacity of protein and lipid fractions of HS using ABTS and DPPH assays, HS was supplemented to high-fat diets (HFD) induced hypercholesterolemic wistar rats. After treatment schedules, lipid profiles, histological and ultrastructural investigations, gene and protein expressions of inflammatory markers, markers of oxidative stress were studied and correlated with biophysical parameters such as ECG and impedance/conductance across the aorta. HS demonstrating in vitro free radical scavenging activity, ameliorated the signs of HC as seen with improved lipid profiles, aortic tissue damage and ECG patterns compared to HFD groups. HS administration also relieved the COX-2 mediated inflammation, which correlated well with the improved redox status in the tissue. CONCLUSIONS: Current study evidently demonstrates that the anti-hypercholesterolemic effects of HS are mediated through redox-sensitive modulation of inflammatory pathways.

Heat induced differential pattern of DNA fragmentation in male germ cells of rats.

Spermatogenesis being a highly dynamic processes is highly vulnerable to various stresses including heat stress. Though, the relationship between physiological temperature and male germ cells is certainly immense, the magnitude of spermatozoal damage after exposure to heat is evidently degree and dose dependent. Further, there are contradictory reports related to germ cells apoptosis in relation to temperatures. Thus, currently the dynamics of temperature and time dependence on germ cell apoptosis were studied by modulating the heat treatment strategies. It was observed that the rate of apoptosis increased initially then decreased with time. The DNA fragmentation in the 10,000×g supernatant of testis homogenate of rats that received heat treatment for 15-min, 30-min as well as 45-min treatment with 15-min intermittent period was found to be almost equal. In various heat treated animals, the apoptosis was found to be maximum after day-1 of treatments, which then followed a decreased pattern. These results indicate that there may be an initial induction of apoptosis in the germ cells, which later primed or programmed the other germ cells to activate protective mechanisms against heat induced DNA damage and thus protecting germ cell population to undergo apoptosis at later durations.

Protective Role of Selenium Against Hemolytic Anemia Is Mediated Through Redox Modulation.

Selenium (Se), an essential trace element and potent nutritional antioxidant, exerts its biological effects through incorporation into selenoproteins like glutathione peroxidase (GPx). Modest decrement in the levels of GPx could be partly responsible for peroxidation of RBCs, which results into hemolytic anemia. Therefore, it is hypothesized that dietary Se, as selenoproteins (GPx), can maintain the homeostasis in RBCs and regulate the erythropoiesis by preventing oxidative stress-mediated hemolysis. Se-deficient (0.01 ppm), Se-adequate (0.1 ppm sodium selenite), and Se-supplemented (0.5 ppm sodium selenite) status were created in Balb/c mice by feeding yeast-based diets for 8 weeks and established by measuring Se levels in plasma and activities, expressions of Se-dependent selenoproteins. Fifty percent of mice from each differential Se group were treated with phenylhydrazine (PHZ, 20 mg/kg, i.p.) to induce hemolytic anemia. Results indicated that PHZ-treated Se-deficient animals demonstrated increased hemolysis, abnormal RBC morphology, increase in Heinz bodies and reticulocytes, and denaturation of hemoglobin to globin precipitates and methemoglobin. Se supplementation protected against these hemolytic changes and makes RBCs less fragile. These findings were consistent with dietary Se concentration-dependent changes in activity and expression of GPx indicating that ROS-mediated oxidative stress is integral to hemolysis. Protective effects of Se supplementation against increased levels of ROS, protein carbonyls, and peroxide damage to membrane lipids and enzymatic antioxidants validated these observations. In conclusion, dietary Se supplementation protected the RBCs against hemolysis by mitigating ROS-mediated oxidative stress.

Redox modulatory protective effects of ω-3 fatty acids rich fish oil against experimental colitis.

Ulcerative colitis (UC), a form of inflammatory bowel disease (IBD), is an immune-modulated disorder characterized by chronic and recurring inflammatory episodes. Oxidative stress and COX pathway of prostaglandin (PG) biosynthesis are indispensable to pathogenesis of UC. Any imbalance between PGs can compromise the mucosal homeostasis, leading to mucosal damage and chronic inflammation. However, blocking these PGs using classical Cox inhibitors such as non-steroidal anti-inflammatory drugs (NSAIDs) can instead aggravate signs of IBD. Therefore, realizing the need for safer and well tolerable alterative treatment approaches, currently, we evaluated the efficacy of n-3 fatty acids rich fish oil (FO) in the resolution of UC. Using a dextran sodium sulfate (DSS) model of experimental colitis, we have demonstrated that supplementation of FO containing 180 mg EPA and 120 mg DHA for 1 month relieved the signs (diarrhea, bloody stools, weight loss) of colitis-associated inflammation. To understand the biophysical changes associated with FO mediated inflammatory regulation, impedance measurement and Fourier transform infrared spectroscopy (FTIR) were done. These changes were also correlated with oxidative stress through markers such as GST, glutathione peroxidase (GPx), LPO, catalase, protein carbonyl content, GR, etc. in colonic mucosa. The modulation of COX mediated pathways in UC-associated inflammation was observed by protein expressions of various pro-inflammatory cytokines such as TNF-α and enzymes of PG synthesis such as COX-2, PGES, TXAS, and anti-inflammatory PGDS. Refuting the earlier reports that suggested the contradictory effects of FO, in the current study, we evidently demonstrated that the protective effects of FO are mediated through molecular mechanisms involving the redox-regulation of metabolism of key lipid metabolites.

Resolution of Cox mediated inflammation by Se supplementation in mouse experimental model of colitis.

UC a form of IBD is a chronic inflammatory disorder of large intestine, with unknown etiology. Reports suggest a critical role of COX-2 dependent prostaglandins (PGs) mediated inflammatory pathway in pathophysiology of UC. However, COX inhibition using NSAIDs exacerbate IBD and thus is not a viable solution. Currently, in DSS induced experimental colitis in mice, we have demonstrated that dietary Se supplementation (0.5ppm as sodium selenite) symptomatically resolves the signs of inflammation in a redox sensitive manner as compared to Se deficient (0.01ppm) conditions, as seen by modulation in oxidative stress markers, morphological changes, histopathological examinations, biochemical studies such as MPO activity, activity of intestinal markers enzymes as well as mRNA and expressions of various pro and anti-inflammatory factors such as, mPGES, hPGDS, TXAS, 15-PGDH, GPX-1 and GPX-2. These findings were validated and correlated with changes in the biophysical parameters such as membrane fluidity, electrical parameters (impedance), transport across the colonic tissue and FTIR. Current study not only concluded that Se at supranutritional concentrations by modulating the redox status relieves the signs of colitis by regulating COX dependent PG biosynthetic pathway, but also sheds light on the biophysical characterization of these inflammatory/resolution pathways involved in UC.

Epigenetic regulation of inflammatory gene expression in macrophages by selenium.

Acetylation of histone and non-histone proteins by histone acetyltransferases plays a pivotal role in the expression of proinflammatory genes. Given the importance of dietary selenium in mitigating inflammation, we hypothesized that selenium supplementation may regulate inflammatory gene expression at the epigenetic level. The effect of selenium towards histone acetylation was examined in both in vitro and in vivo models of inflammation by chromatin immunoprecipitation assays and immunoblotting. Our results indicated that selenium supplementation, as selenite, decreased acetylation of histone H4 at K12 and K16 in COX-2 and TNFα promoters, and of the p65 subunit of the redox sensitive transcription factor NFκB in primary and immortalized macrophages. On the other hand, selenomethionine had a much weaker effect. Selenite treatment of HIV-1-infected human monocytes also significantly decreased the acetylation of H4 at K12 and K16 on the HIV-1 promoter, supporting the down-regulation of proviral expression by selenium. A similar decrease in histone acetylation was also seen in the colonic extracts of mice treated with dextran sodium sulfate that correlated well with the levels of selenium in the diet. Bone-marrow-derived macrophages from Trsp(fl/fl)Cre(LysM) mice that lack expression of selenoproteins in macrophages confirmed the important role of selenoproteins in the inhibition of histone H4 acetylation. Our studies suggest that the ability of selenoproteins to skew the metabolism of arachidonic acid contributes, in part, to their ability to inhibit histone acetylation. In summary, our studies suggest a new role for selenoproteins in the epigenetic modulation of proinflammatory genes.

Crucial role of macrophage selenoproteins in experimental colitis.

Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress proinflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% dextran sodium sulfate in wild-type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.08 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Selenocysteinyl transfer RNA knockout mice (Trsp(fl/fl)LysM(Cre)) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of proinflammatory and anti-inflammatory genes, and modulation of PG metabolites in urine and plasma. Whereas Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an upregulation of expression of proinflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and proresolving effects. Selenoproteins in macrophages protect mice from dextran sodium sulfate-colitis by enhancing 15-PGDH-dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.

Selenium suppresses leukemia through the action of endogenous eicosanoids.

Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here, we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiologic but nontoxic doses. In leukemia CSC, selenium treatment activated ATM-p53-dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR-ABL-expressing CSC revealed that the selenium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production of the cyclooxygenase-derived prostaglandins Δ(12)-PGJ2 and 15d-PGJ2. Accordingly, nonsteroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia.

Evaluation of the stability, bioavailability, and hypersensitivity of the omega-3 derived anti-leukemic prostaglandin: Δ(12)-prostaglandin J3.

Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ(12)-PGJ3, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in in vitro and in vivo models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ(12)-PGJ3. The stability of Δ(12)-PGJ3 was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ(12)-PGJ3 in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ(12)-PGJ3 being a downstream metabolite of PGD3 was tested in vitro using primary mouse bone marrow-derived mast cells (BMMCs) and in vivo mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD2 receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ(12)-PGJ3 was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ(12)-PGJ3 was bioavailable and well absorbed into systemic circulation with a Cmax of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ(12)-PGJ3 did not induce degranulation in BMMCs nor increase histamine. In addition, in vivo testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ(12)-PGJ3 and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ(12)-PGJ3 was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ(12)-PGJ3 failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ(12)-PGJ3 to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.

Δ12-prostaglandin J3, an omega-3 fatty acid-derived metabolite, selectively ablates leukemia stem cells in mice.

Targeting cancer stem cells is of paramount importance in successfully preventing cancer relapse. Recently, in silico screening of public gene-expression datasets identified cyclooxygenase-derived cyclopentenone prostaglandins (CyPGs) as likely agents to target malignant stem cells. We show here that Δ(12)-PGJ(3), a novel and naturally produced CyPG from the dietary fish-oil ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA; 20:5) alleviates the development of leukemia in 2 well-studied murine models of leukemia. IP administration of Δ(12)-PGJ(3) to mice infected with Friend erythroleukemia virus or those expressing the chronic myelogenous leukemia oncoprotein BCR-ABL in the hematopoietic stem cell pool completely restored normal hematologic parameters, splenic histology, and enhanced survival. More importantly, Δ(12)-PGJ(3) selectively targeted leukemia stem cells (LSCs) for apoptosis in the spleen and BM. This treatment completely eradicated LSCs in vivo, as demonstrated by the inability of donor cells from treated mice to cause leukemia in secondary transplantations. Given the potency of ω-3 polyunsaturated fatty acid-derived CyPGs and the well-known refractoriness of LSCs to currently used clinical agents, Δ(12)-PGJ(3) may represent a new chemotherapeutic for leukemia that targets LSCs.

Selenoprotein-dependent up-regulation of hematopoietic prostaglandin D2 synthase in macrophages is mediated through the activation of peroxisome proliferator-activated receptor (PPAR) gamma.

The plasticity of macrophages is evident from their dual role in inflammation and resolution of inflammation that are accompanied by changes in the transcriptome and metabolome. Along these lines, we have previously demonstrated that the micronutrient selenium increases macrophage production of arachidonic acid (AA)-derived anti-inflammatory 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) and decreases the proinflammatory PGE(2). Here, we hypothesized that selenium modulated the metabolism of AA by a differential regulation of various prostaglandin (PG) synthases favoring the production of PGD(2) metabolites, Δ(12)-PGJ(2) and 15d-PGJ(2). A dose-dependent increase in the expression of hematopoietic-PGD(2) synthase (H-PGDS) by selenium and a corresponding increase in Δ(12)-PGJ(2) and 15d-PGJ(2) in RAW264.7 macrophages and primary bone marrow-derived macrophages was observed. Studies with organic non-bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H-PGDS expression and 15d-PGJ(2) production. Treatment of selenium-deficient macrophages with rosiglitazone, a peroxisome proliferator-activated receptor γ ligand, up-regulated H-PGDS. Furthermore, electrophoretic mobility shift assays indicated the presence of an active peroxisome proliferator-activated receptor-response element in murine Hpgds promoter suggesting a positive feedback mechanism of H-PGDS expression. Alternatively, the expression of nuclear factor-κB-dependent thromboxane synthase and microsomal PGE(2) synthase was down-regulated by selenium. Using a Friend virus infection model of murine leukemia, the onset of leukemia was observed only in selenium-deficient and indomethacin-treated selenium-supplemented mice but not in the selenium-supplemented group or those treated with 15d-PGJ(2). These results suggest the importance of selenium in the shunting of AA metabolism toward the production of PGD(2) metabolites, which may have clinical implications.

The regulation of erythropoiesis by selenium in mice.

Redox modulation by antioxidants, such as selenium (Se), has emerged as an important regulator of erythropoiesis. Using Se-deficient (0.04 ppm), Se-adequate (0.1 ppm), and Se-supplemented (0.4 ppm) C57/BL6 mice, we show that Se deficiency caused anemia, when compared to the Se-supplemented and Se-adequate groups. Increased denaturation of hemoglobin, methemoglobin, protein carbonyls, lipid peroxidation, Heinz bodies, and osmotic fragility of erythrocytes were observed in Se-deficient mice. Increased oxidative stress upregulated forkhead transcription factor (FoxO3a) and hypoxia-inducible factor-(HIF)1α in the spleen and kidney of Se-deficient murine as well as in the proerythroblast G1E cells cultured in Se-deficient media. A significant increase in the expression of erythropoietin, a downstream target of HIF1α, and expansion of stress erythroid progenitors (burst forming units-erythroid) were seen in the Se-deficient mice. Despite the increase in erythroid progenitors, lowered reticulocytes suggest a defective erythroid differentiation pathway. While Se deficiency led to increased nuclear levels of active FoxO3a, Se-adequate conditions reversed this effect and increased nuclear export by its binding partner, 14-3-3ßζ, that is under the redox control of selenoproteins. In summary, these results provide insight into the importance of adequate Se nutrition in regulating red cell homeostasis by mitigating oxidative stress-dependent modulation of FoxO3a and HIF1α to effect differentiation of erythroid progenitors.

Synthesis and evaluation of the anti-inflammatory properties of selenium-derivatives of celecoxib.

Celecoxib is a selective cyclooxygenase (COX)-2 inhibitor used to treat inflammation, while selenium is known to down-regulate the transcription of COX-2 and other pro-inflammatory genes. To expand the anti-inflammatory property, wherein celecoxib could inhibit pro-inflammatory gene expression at extremely low doses, we incorporated selenium (Se) into two Se-derivatives of celecoxib, namely; selenocoxib-2 and selenocoxib-3. In vitro kinetic assays of the inhibition of purified human COX-2 activity by these compounds indicated that celecoxib and selenocoxib-3 had identical K(I) values of 2.3 and 2.4µM; while selenocoxib-2 had a lower K(I) of 0.72µM. Furthermore, selenocoxib-2 inhibited lipopolysaccharide-induced activation of NF-κB leading to the down-regulation of expression of COX-2, iNOS, and TNFα more effectively than selenocoxib-3 and celecoxib in RAW264.7 macrophages and murine bone marrow-derived macrophages. Studies with rat liver microsomes followed by UPLC-MS-MS analysis indicated the formation of selenenylsulfide conjugates of selenocoxib-2 with N-acetylcysteine. Selenocoxib-2 was found to release minor amounts of Se that was effectively inhibited by the CYP inhibitor, sulphaphenazole. While these studies suggest that selenocoxib-2, but not celecoxib and selenocoxib-3, targets upstream events in the NF-κB signaling axis, the ability to effectively suppress NF-κB activation independent of cellular selenoprotein synthesis opens possibilities for a new generation of COX-2 inhibitors with significant and broader anti-inflammatory potential.

Diminished reproductive potential of male mice in response to selenium-induced oxidative stress: involvement of HSP70, HSP70-2, and MSJ-1.

The oxidative stress imposed by nutritional variations in selenium (Se) has plausible role in reproductive toxicology and affects the reproductive potential. Also, the expression of heat shock proteins (HSPs) is a highly regulated event throughout the process of spermatogenesis and is modulated by stressful stimuli. This prompted us to investigate the possibility that Se-induced oxidative stress may affect the fertility status by altering the expressions of the constitutive and inducible HSP70 proteins, having crucial role in spermatogenesis. Different Se status-deficient, adequate, and excess, male Balb/c mice were created by feeding yeast-based Se-deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet-feeding schedule, a significant decrease in the Se and glutathione peroxidase (GSH-Px) levels was observed in the Se-deficient group (I), whereas Se-excess group (III) demonstrated an increase. Increased levels of reactive oxygen species, malondialdehyde, and alterations in the redox status in both groups I and III indicated oxidative-stressed conditions. There was an overall reduced fertility status in mice supplemented with Se-deficient and Se-excess diet. The mRNA and protein expression of HSP70 was found to be elevated in these two groups, whereas the expression patterns of HSP70-2 and MSJ-1 demonstrated a reverse trend. In vitro CDC2 kinase assay showed reduced kinase activity in group I and group III. These findings suggest that Se-induced oxidative stress by differentially regulating various HSP70s can affect its downstream factors having crucially important role in differentiation of germ cells and completion of spermatogenesis. Therefore, it can provide an insight into the mechanism(s) by which the oxidative stress-induced reproductive toxicity can lead to increased apoptosis/growth arrest and infertility. This will thus add new dimensions to the molecular mechanism underlying the human male infertility and open new vistas in the development of various chemo-preventive methods.

Selenium variation induced oxidative stress regulates p53 dependent germ cell apoptosis: plausible involvement of HSP70-2.

BACKGROUND: Selenium at altered concentration causes abnormal spermatogenesis and male infertility. However, the exact mechanism behind this is still unexplored. AIMS: It was aimed to investigate if Se induced oxidative stress alters the expressions of testis specific HSP70-2 protein, that is crucial in normal spermatogenesis. The study was extended to delineate the apoptotic process after this change if any. METHODS: To create different Se status-deficient, adequate and excess, male Balb/c mice were fed yeast based Se deficient diet (group I) and this diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III, respectively) for 8 weeks. RESULTS: After the feeding schedule, a dose dependent change in the Se levels and GSH-Px activity was observed in samples of different Se diet fed group animals as reported in earlier studies. Changes in the redox status in both groups I and III indicated oxidative stress conditions. The mRNA and protein expression of HSP70-2 was found to be reduced in group I and III, whereas, the expressions of p53 demonstrated a reverse trend. Increased apoptosis was observed in the group I and III animals as indicated by increased apoptotic index (AI) on the TUNEL stained sections and by DNA fragmentation indicating DNA damage in these groups. CONCLUSION: These findings suggest that Se variations induced oxidative stress leads to germ cell apoptosis and downregulation of HSP70-2. This study suggests that there can be a possible link between these two events and the fate of HSP70-2 in case of oxidative damage can provide an insight into the mechanism(s) by which the nutritional variation induced oxidative stress can affect reproductive potential and thus demonstrates the importance of nutrition at molecular level as well.

Dietary selenium variation-induced oxidative stress modulates CDC2/cyclin B1 expression and apoptosis of germ cells in mice testis.

Oxidative stress has been linked with apoptosis in germ cells and with male infertility. However, the molecular mechanism of oxidative-stress-mediated apoptosis in germ cells has not been clearly defined so far. Because of the involvement of CDC2 and cyclin B1 in cell cycle regulation and their plausible role in apoptosis, the present study aimed to investigate the possibility that selenium (Se)-induced oxidative-stress-mediated modulations of these cell cycle regulators cause DNA damage and apoptosis in germ cells. To create different Se status (deficient, adequate and excess), male Balb/c mice were fed yeast-based Se-deficient diet (Group I) and a deficient diet supplemented with Se as sodium selenite (0.2 and 1 ppm Se in Groups II and III, respectively) for a period of 8 weeks. After the completion of the diet feeding schedule, a significant decrease in Se levels and glutathione peroxidase activity was observed in the Se-deficient group (Group I), whereas the Se-excess group (Group III) demonstrated an increase in Se levels. Increased levels of lipid peroxidation were seen in both Groups I and III when compared to Group II, indicating oxidative stress. The mRNA and protein expressions of both CDC2 and cyclin B1 were found to be significantly decreased in Groups I and III. A decrease in the immunohistochemical localization of these proteins was also observed in spermatogenic cells. The mRNA expressions of apoptotic factors such as Bcl-2, Bax, caspase-3 and caspase-9 were found to be increased in Groups I and III. A decrease in CDC2 kinase activity was also seen in these groups. Increased apoptosis was observed in Group I and Group III animals by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay indicating oxidative-stress-mediated DNA damage. These findings suggest the effect of Se-induced oxidative stress on the cell cycle regulators and apoptotic activity of germ cells, thus providing new dimensions to molecular mechanisms underlying male infertility.