Selenium and selenoproteins in prostanoid metabolism and immunity.

Selenium (Se) is an essential trace element that functions in the form of the 21st amino acid, selenocysteine (Sec) in a defined set of proteins. Se deficiency is associated with pathological conditions in humans and animals, where incorporation of Sec into selenoproteins is reduced along with their expression and catalytic activity. Supplementation of Se-deficient population with Se has shown health benefits suggesting the importance of Se in physiology. An interesting paradigm to explain, in part, the health benefits of Se stems from the observations that selenoprotein-dependent modulation of inflammation and efficient resolution of inflammation relies on mechanisms involving a group of bioactive lipid mediators, prostanoids, which orchestrate a concerted action toward maintenance and restoration of homeostatic immune responses. Such an effect involves the interaction of various immune cells with these lipid mediators where cellular redox gatekeeper functions of selenoproteins further aid in not only dampening inflammation, but also initiating an effective and active resolution process. Here we have summarized the current literature on the multifaceted roles of Se/selenoproteins in the regulation of these bioactive lipid mediators and their immunomodulatory effects.

Characterization of the human RFX transcription factor family by regulatory and target gene analysis.

BACKGROUND: Evolutionarily conserved RFX transcription factors (TFs) regulate their target genes through a DNA sequence motif called the X-box. Thereby they regulate cellular specialization and terminal differentiation. Here, we provide a comprehensive analysis of all the eight human RFX genes (RFX1-8), their spatial and temporal expression profiles, potential upstream regulators and target genes. RESULTS: We extracted all known human RFX1-8 gene expression profiles from the FANTOM5 database derived from transcription start site (TSS) activity as captured by Cap Analysis of Gene Expression (CAGE) technology. RFX genes are broadly (RFX1-3, RFX5, RFX7) and specifically (RFX4, RFX6) expressed in different cell types, with high expression in four organ systems: immune system, gastrointestinal tract, reproductive system and nervous system. Tissue type specific expression profiles link defined RFX family members with the target gene batteries they regulate. We experimentally confirmed novel TSS locations and characterized the previously undescribed RFX8 to be lowly expressed. RFX tissue and cell type specificity arises mainly from differences in TSS architecture. RFX transcript isoforms lacking a DNA binding domain (DBD) open up new possibilities for combinatorial target gene regulation. Our results favor a new grouping of the RFX family based on protein domain composition. We uncovered and experimentally confirmed the TFs SP2 and ESR1 as upstream regulators of specific RFX genes. Using TF binding profiles from the JASPAR database, we determined relevant patterns of X-box motif positioning with respect to gene TSS locations of human RFX target genes. CONCLUSIONS: The wealth of data we provide will serve as the basis for precisely determining the roles RFX TFs play in human development and disease.

Quantification of low molecular weight selenium metabolites in human plasma after treatment with selenite in pharmacological doses by LC-ICP-MS.

The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were ultrafiltrated using a cut-off value of 3000 Da. The method was validated in aqueous solution as well as plasma using standards of selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenite, and the selenosugar Se-methylseleno-N-acetylgalactosamine (SeGal) for linearity, precision, recoveries, and limits of detection and quantitation with satisfactory results. The method was applied for analysis of a set of plasma samples from cancer patients receiving selenite treatment in a clinical trial. Three LMW selenium compounds were observed. The main compounds, SeGal and selenite were tentatively identified by retention time matching with standards in different chromatographic systems, while the third minor compound was not identified. The identity of the selenosugar was verified by ESI-MS-MS product ion scanning, while selenite was identified indirectly as the glutathione (GSH) reaction product, GS-Se-SG.

Selenium induces a multi-targeted cell death process in addition to ROS formation.

Selenium compounds inhibit neoplastic growth. Redox active selenium compounds are evolving as promising chemotherapeutic agents through tumour selectivity and multi-target response, which are of great benefit in preventing development of drug resistance. Generation of reactive oxygen species is implicated in selenium-mediated cytotoxic effects on cancer cells. Recent findings indicate that activation of diverse intracellular signalling leading to cell death depends on the chemical form of selenium applied and/or cell line investigated. In the present study, we aimed at deciphering different modes of cell death in a single cell line (HeLa) upon treatment with three redox active selenium compounds (selenite, selenodiglutathione and seleno-DL-cystine). Both selenite and selenodiglutathione exhibited equipotent toxicity (IC50 5 µM) in these cells with striking differences in toxicity mechanisms. Morphological and molecular alterations provided evidence of necroptosis-like cell death in selenite treatment, whereas selenodiglutathione induced apoptosis-like cell death. We demonstrate that selenodiglutathione efficiently glutathionylated free protein thiols, which might explain the early differences in cytotoxic effects induced by selenite and selenodiglutathione. In contrast, seleno-DL-cystine treatment at an IC50 concentration of 100 µM induced morphologically two distinct different types of cell death, one with apoptosis-like phenotype, while the other was reminiscent of paraptosis-like cell death, characterized by induction of unfolded protein response, ER-stress and occurrence of large cytoplasmic vacuoles. Collectively, the current results underline the diverse cytotoxic effects and variable potential of redox active selenium compounds on the survival of HeLa cells and thereby substantiate the potential of chemical species-specific usage of selenium in the treatment of cancers.

The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses.

Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology.

Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation.

Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow-/- BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow-/- BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow-/- BMDMs. Furthermore, Selenow-/- BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.

Transport of selenium across the plasma membrane of primary hepatocytes and enterocytes of rainbow trout.

Transport of essential solutes across biological membranes is one of the fundamental characteristics of living cells. Although selenium is an essential micronutrient, little is known about the cellular mechanisms of chemical species-specific selenium transport in fish. We report here the kinetic and pharmacological transport characteristics of selenite and its thiol (glutathione and l-cysteine) derivatives in primary cultures of hepatocytes and isolated enterocytes of rainbow trout. Findings from the current study suggest an apparent low-affinity linear transport system for selenite in both cell types. However, we recorded high-affinity Hill kinetics (K(d)=3.61±0.28 µmol l(-1)) in enterocytes exposed to selenite in the presence of glutathione. The uptake of selenite in the presence of thiols was severalfold higher than uptake of selenite alone (at equimolar concentration) in both hepatocytes and enterocytes. Cellular accumulation of selenium was found to be energy independent. Interestingly, we observed a decrease in selenite transport with increasing pH, whereas selenite uptake increased with increasing pH in the presence glutathione in both cell types. The cellular uptake of selenite demonstrated a pronounced competitive interaction with a structurally similar compound, sulfite. The uptake of selenite as well as its thiol derivatives was found to be sensitive to the anion transport blocker DIDS, irrespective of the cell type. Inorganic mercury (Hg(2+)) elicited an inhibition of selenite transport in both cell types, but augmented the transport of reduced forms of selenite in hepatocytes. Based on the substrate choice and comparable pharmacological properties, we advocate that multiple anion transport systems are probably involved in the cellular transport of selenite in fish.

Water soaking and exogenous enzyme treatment of plant-based diets: effect on growth performance, whole-body composition, and digestive enzyme activities of rohu, Labeo rohita (Hamilton), fingerlings.

A 2 × 2 × 2 factorial experiment was conducted to delineate the main effect of water soaking of plant ingredients, phytase, cellulase, and their interactions on the growth and digestive enzyme activities of Labeo rohita fingerlings. Two basal diets were prepared using water-soaked (S) or unsoaked (US) plant-based ingredients. Feed of US ingredients was supplemented with phytase (U kg(-1)) and cellulase (%) at the level of 0, 0 (C(us)); 500, 0 (T(1)); 0, 0.2 (T(2)); 500, 0.2 (T(3)), and feed of S ingredients at 0, 0 (C(s)); 500, 0 (T(4)); 0, 0.2 (T(5)), and 500, 0.2 (T(6)), respectively. Three hundred and sixty fingerlings were randomly distributed into eight treatments, each with three replicates. Soaking of the ingredients for 24 h significantly reduced the tannin content. However, feeding of S diets did not improve the fish growth. Highest performance was recorded in the T(3) group. A significant interaction between dietary phytase and cellulase was observed for apparent net protein utilization. Tissue crude protein, ether extract, and ash content of the fingerlings were observed highest in the T(3) group. Activities of amylase, protease, and lipase were recorded highest in the T(3) group. Results suggested that soaking of plant-based ingredients reduces tannin content; however, growth and digestive enzyme activities of group fed soaked diet were not improved, possibly due to leaching of soluble nutrients. Probably, a shorter duration soaking may be effective in reducing tannin content and avoiding nutrients leaching.

Selenoprotein P as Biomarker of Selenium Status in Clinical Trials with Therapeutic Dosages of Selenite.

Selenoprotein P (SELENOP) is an established biomarker of selenium (Se) status. Serum SELENOP becomes saturated with increasing Se intake, reaching maximal concentrations of 5-7 mg SELENOP/L at intakes of ca. 100-150 µg Se/d. A biomarker for higher Se intake is missing. We hypothesized that SELENOP may also reflect Se status in clinical applications of therapeutic dosages of selenite. To this end, blood samples from two supplementation studies employing intravenous application of selenite at dosages >1 mg/d were analyzed. Total Se was quantified by spectroscopy, and SELENOP by a validated ELISA. The high dosage selenite infusions increased SELENOP in parallel to elevated Se concentrations relatively fast to final values partly exceeding 10 mg SELENOP/L. Age or sex were not related to the SELENOP increase. Western blot analyses of SELENOP verified the results obtained by ELISA, and indicated an unchanged pattern of immunoreactive protein isoforms. We conclude that the saturation of SELENOP concentrations observed in prior studies with moderate Se dosages (<400 µg/d) may reflect an intermediate plateau of expression, rather than an absolute upper limit. Circulating SELENOP seems to be a suitable biomarker for therapeutic applications of selenite exceeding the recommended upper intake levels. Whether SELENOP is also capable of reflecting other supplemental selenocompounds in high dosage therapeutic applications remains to be investigated.

Ex vivo organotypic culture system of precision-cut slices of human pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, which is mainly due to late diagnosis and profound resistance to treatment. The latter is to a large extent attributed to the tumor stroma that is exceedingly prominent in PDAC and engages in complex interactions with the cancer cells. Hence, relevant preclinical models of PDAC should also include the tumor stroma. We herein describe the establishment and functional validation of an ex vivo organotypic culture of human PDAC that is based on precision-cut tissue slices from surgical specimens and reproducibly recapitulates the complex cellular and acellular composition of PDAC, including its microenvironment. The cancer cells, tumor microenvironment and interspersed remnants of nonneoplastic pancreas contained in these 350 µm thick slices maintained their structural integrity, phenotypic characteristics and functional activity when in culture for at least 4 days. In particular, tumor cell proliferation persisted and the grade of differentiation and morphological phenotype remained unaltered. Cultured tissue slices were metabolically active and responsive to rapamycin, an mTOR inhibitor. This culture system is to date the closest surrogate to the parent carcinoma and harbors great potential as a drug sensitivity testing system for the personalized treatment of PDAC.

Selenite promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells.

Selective targeting of the PML/RARα oncoprotein demonstrates a successful molecular targeted therapy in acute promyelocytic leukemia (APL) with a typical t(15:17) chromosomal translocation. The zinc-thiolate coordination is critical for structural stability of zinc finger proteins, including the PML moiety of PML/RARα. Based on the known interaction of redox-active selenium compounds with thiolate ligands of zinc, we herein have investigated the abrogatory effects of selenite alone or in combination with all-trans retinoic acid on PML/RARα and the possible effects on differentiation in these cells. At pharmacological concentrations, selenite inhibited the proliferation and survival of APL originated NB4 cells. In combination with ATRA, it potentiated the differentiation of NB4 cells without any differentiating effects of its own as a single agent. Concordant with our hypothesis, PML/RARα oncoprotein expression was completely abrogated by selenite. Increased expression of RARα, PU.1 and FOXO3A transcription factors in the combined treatment suggested the plausible basis for increased differentiation in these cells. We show that selenite at clinically achievable dose targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemic cells in combination with ATRA. The present investigation reveals the hitherto unknown potential of selenite in targeted abrogation of PML/RARα in APL cells with prospective therapeutic value.

Redox-active selenium compounds--from toxicity and cell death to cancer treatment.

Selenium is generally known as an antioxidant due to its presence in selenoproteins as selenocysteine, but it is also toxic. The toxic effects of selenium are, however, strictly concentration and chemical species dependent. One class of selenium compounds is a potent inhibitor of cell growth with remarkable tumor specificity. These redox active compounds are pro-oxidative and highly cytotoxic to tumor cells and are promising candidates to be used in chemotherapy against cancer. Herein we elaborate upon the major forms of dietary selenium compounds, their metabolic pathways, and their antioxidant and pro-oxidant potentials with emphasis on cytotoxic mechanisms. Relative cytotoxicity of inorganic selenite and organic selenocystine compounds to different cancer cells are presented as evidence to our perspective. Furthermore, new novel classes of selenium compounds specifically designed to target tumor cells are presented and the potential of selenium in modern oncology is extensively discussed.

Pharmacokinetics and Toxicity of Sodium Selenite in the Treatment of Patients with Carcinoma in a Phase I Clinical Trial: The SECAR Study.

BACKGROUND: Sodium selenite at high dose exerts antitumor effects and increases efficacy of cytostatic drugs in multiple preclinical malignancy models. We assessed the safety and efficacy of intravenous administered sodium selenite in cancer patients' refractory to cytostatic drugs in a phase I trial. Patients received first line of chemotherapy following selenite treatment to investigate altered sensitivity to these drugs and preliminary assessment of any clinical benefits. MATERIALS AND METHODS: Thirty-four patients with different therapy resistant tumors received iv sodium selenite daily for consecutive five days either for two weeks or four weeks. Each cohort consisted of at least three patients who received the same daily dose of selenite throughout the whole treatment. If 0/3 patients had dose-limiting toxicities (DLTs), the study proceeded to the next dose-level. If 2/3 had DLT, the dose was considered too high and if 1/3 had DLT, three more patients were included. Dose-escalation continued until the maximum tolerated dose (MTD) was reached. MTD was defined as the highest dose-level on which 0/3 or 1/6 patients experienced DLT. The primary endpoint was safety, dose-limiting toxic effects and the MTD of sodium selenite. The secondary endpoint was primary response evaluation. RESULTS AND CONCLUSION: MTD was defined as 10.2 mg/m(2), with a calculated median plasma half-life of 18.25 h. The maximum plasma concentration of selenium from a single dose of selenite increased in a nonlinear pattern. The most common adverse events were fatigue, nausea, and cramps in fingers and legs. DLTs were acute, of short duration and reversible. Biomarkers for organ functions indicated no major systemic toxicity. In conclusion, sodium selenite is safe and tolerable when administered up to 10.2 mg/m(2) under current protocol. Further development of the study is underway to determine if prolonged infusions might be a more effective treatment strategy.

Selenium cytotoxicity in cancer.

Selenium is an essential trace element with growth-modulating properties. Decades of research clearly demonstrate that selenium compounds inhibit the growth of malignant cells in diverse experimental model systems. However, the growth-modulating and cytotoxic mechanisms are diverse and far from clear. Lately, a remarkable tumour selective cytotoxicity of selenium compounds has been shown, indicating the potential of selenium in the treatment of cancer. Of particular interest are the redox-active selenium compounds exhibiting cytotoxic potential to tumour cells. These selenium compounds elicit complex patterns of pharmacodynamics and pharmacokinetics, leading to cell death pathways that differ among compounds. Modern oncology often focuses on targeted ligand-based therapeutic strategies that are specific to their molecular targets. These drugs are initially efficient, but the tumour cells often rapidly develop resistance against these drugs. In contrast, certain redox-active selenium compounds induce complex cascades of pro-death signalling at pharmacological concentrations with superior tumour specificity. The target molecules are often the ones that are important for the survival of cancer cells and often implicated in drug resistance. Therefore, the chemotherapeutic applications of selenium offer great possibilities of multi-target attacks on tumour cells. This MiniReview focuses on the tumour-specific cytotoxic effects of selenium, with special emphasis on cascades of cellular events induced by the major groups of pharmacologically active selenium compounds. Furthermore, the great pharmacological potential of selenium in the treatment of resistant cancers is discussed.

Induction of oxidative stress by selenomethionine in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss).

Fish are exposed to environmental selenium predominantly in the form of dietary selenomethionine (SeMet). The present study was designed to investigate the role of oxidative stress in the toxicity of SeMet using isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) as the model experimental system. Cells were exposed to an increasing range of SeMet (0-1000 µM) over 24h, and the time-dependent effects on cell viability, response of enzymatic antioxidants, thiol redox, intracellular calcium balance and caspase-mediated apoptosis were evaluated. SeMet was found to be toxic only at the highest exposure dose (1000 µM), with ∼15% decrease in cell viability. Although modest increases in the activities of antioxidant enzymes were recorded following SeMet exposure, the ratio of reduced to oxidized glutathione decreased in a dose-dependent manner, suggesting a gradual progression towards an oxidative intracellular environment. The peroxidation of membrane lipids also increased with increasing SeMet exposure dose. In addition, a rapid increase in intracellular calcium level and the activation of caspase 3/7 enzymes were recorded at the highest exposure dose, indicating that SeMet at a high exposure dose causes cell death probably via apoptosis. Overall, our study demonstrated that oxidative stress plays a key role in the cytotoxicity of SeMet in fish.

Tissue-specific accumulation and speciation of selenium in rainbow trout (Oncorhynchus mykiss) exposed to elevated dietary selenomethionine.

The toxicity of selenium in fish is influenced by its chemical speciation and the exposure route. In the natural environment, selenium exposure to fish occurs primarily in the form of selenomethionine in diet. Thus, the main objective of this study was to examine the tissue-specific selenium burden and speciation in fish exposed to elevated dietary selenomethionine. Rainbow trout (Oncorhynchus mykiss) were treated with dietary selenomethionine (40 µg g(-1) dry mass) for 2 weeks, and at the end of the exposure different tissue samples were collected to assess the tissue-specific distribution and speciation of selenium. We used synchrotron-based X-ray absorption near edge spectroscopy (XANES) to determine the selenium speciation profile. Selenomethionine, selenocysteine and selenocystine were found to be the predominant form of selenium in all of the tissues; however their relative proportion varied across different tissues. In general, the organs primarily involved in selenium handling in fish (e.g., liver, kidney) accumulated a higher percentage of selenocystine. We also found that dietary selenomethionine exposure resulted into a marked increase in selenium burden of all major tissues in fish including the brain. Collectively, our findings provide new insights into the tissue-specific distribution and speciation of selenium in fish exposed to selenomethionine via diet.

Methylselenol formed by spontaneous methylation of selenide is a superior selenium substrate to the thioredoxin and glutaredoxin systems.

Naturally occurring selenium compounds like selenite and selenodiglutathione are metabolized to selenide in plants and animals. This highly reactive form of selenium can undergo methylation and form monomethylated and multimethylated species. These redox active selenium metabolites are of particular biological and pharmacological interest since they are potent inducers of apoptosis in cancer cells. The mammalian thioredoxin and glutaredoxin systems efficiently reduce selenite and selenodiglutathione to selenide. The reactions are non-stoichiometric aerobically due to redox cycling of selenide with oxygen and thiols. Using LDI-MS, we identified that the addition of S-adenosylmethionine (SAM) to the reactions formed methylselenol. This metabolite was a superior substrate to both the thioredoxin and glutaredoxin systems increasing the velocities of the nonstoichiometric redox cycles three-fold. In vitro cell experiments demonstrated that the presence of SAM increased the cytotoxicity of selenite and selenodiglutathione, which could neither be explained by altered selenium uptake nor impaired extra-cellular redox environment, previously shown to be highly important to selenite uptake and cytotoxicity. Our data suggest that selenide and SAM react spontaneously forming methylselenol, a highly nucleophilic and cytotoxic agent, with important physiological and pharmacological implications for the highly interesting anticancer effects of selenium.

Application of XANES spectroscopy in understanding the metabolism of selenium in isolated rainbow trout hepatocytes: insights into selenium toxicity.

Selenium (Se) is an essential element, but causes toxic effects in fish at a slightly elevated level beyond the threshold. However, the degree of Se toxicity differs depending on the chemical forms of Se (e.g., organic vs. inorganic) to which fish are exposed to. The mechanisms of Se metabolism and toxicity in fish, particularly at cellular level, are poorly understood. The present study was designed to examine the metabolic fate of different seleno-compounds, both inorganic and organic, in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) in primary culture using XANES spectroscopy. In cells exposed to 100 µM of selenate and selenite for 6-24 h, elemental Se was found to be the primary metabolite. Whereas, selenocystine appeared to be the major metabolite in cells exposed to 100 µM seleno-L-methionine for 6-24 h. Interestingly, we recorded L-methionine-γ-lyase activity in S9 fraction of cell lysate-an enzyme that directly catalyzes selenomethionine into methylselenol. We also found concurrent reduction of glutathione (GSH) concentration following reaction of seleno-L-methionine with cellular S9 fraction. Moreover, we observed a rapid increase in cellular reactive oxygen species (ROS) generation with increasing seleno-L-methionine exposure dose (100-1000 µM). These findings indicated the rapid cellular metabolism of seleno-L-methionine into methylselenol at higher exposure dose (≥100 µM), and the occurrence of GSH mediated redox cycling of methylselenol--a process that is known to produce reactive oxygen species (ROS). Overall, our results suggest that inorganic and organic selenium are metabolized through different metabolic pathways in rainbow trout hepatocytes. The findings of our study have important implications for understanding the chemical species-specific differences in Se toxicity to fish.

Selenite causes cytotoxicity in rainbow trout (Oncorhynchus mykiss) hepatocytes by inducing oxidative stress.

Selenium is an essential micronutrient to freshwater fish, but can be very toxic at slightly above the threshold level. The liver is known to be the major site of selenium accumulation and metabolism in fish. Recent evidence from mammalian systems suggests that oxidative damage is an important mechanism of selenium toxicity; however this phenomenon has not been investigated in-depth in fish, either in vivo or in vitro. Therefore, the present study was designed to investigate whether selenium (as selenite) exposure causes cytotoxicity in fish by inducing oxidative stress. We used isolated hepatocytes in primary culture from freshwater rainbow trout (Oncorhynchus mykiss) as the model in vitro experimental system. The 24h LD(50) of selenite to trout hepatocytes was found to be 587 microM. In order to evaluate the dose-dependent response patterns of various oxidative stress parameters, the trout hepatocytes were exposed to three different doses of selenite [50, 100 and 200 microM (corresponding to approximately 10%, 20% and 35% of 24h LD(50))] in addition to control (0 microM of selenite) for 24h. We observed an induction of catalase (CAT) and superoxide dismutase (SOD) activities at 50 and 100 microM of selenite exposure, but not at 200 microM, relative to the control. In contrast, the induction of glutathione peroxidase (GPx) activity was recorded at 100 and 200 microM exposure doses, but not at 50 microM. We also demonstrated that selenite exposure (100-200 microM) increased intracellular ROS formation at an early stage (2h). The reduced to oxidized glutathione ratio (GSH:GSSG) decreased sharply with increasing selenite dose, indicating the loss of cellular reducing capacity. The cellular lipid peroxidation tended to increase with increasing selenite exposure dose, indicating the occurrence of membrane damage. A 20-40% decrease in cell viability was observed at 100 and 200 microM of selenite exposure. The increase in cell death was associated with a significant increase of caspase-3/7 activity, suggesting the induction of apoptosis. Overall, the present study suggests that selenite exposure at high level causes oxidative damage to trout hepatocytes, probably by inducing the imbalance of intracellular glutathione (GSH) redox.

Pre- and post-challenge immuno-haematological changes in Labeo rohita juveniles fed gelatinised or non-gelatinised carbohydrate with n-3 PUFA.

The combined effect of dietary carbohydrate type and n-3 PUFA (EPA+DHA) on pre- and post-challenge haemato-immunological responses in Labeo rohita juveniles was studied. Fish were fed for 67days with six different test diets containing either gelatinised (G) or non-gelatinised (NG) corn (43%) with three levels of n-3 PUFA (0.5%, 1.0% and 2.0%). During the pre-challenge period, significantly higher (P<0.05) NBT, serum lysozyme activity, total protein and globulin content was recorded in the NG carbohydrate fed groups. Highest NBT value was recorded in the groups fed with 1.0% n-3 PUFA, whereas the highest serum lysozyme activity (P<0.05) was recorded at either 0.5% or 2.0% n-3 PUFA fed groups in both the pre- and post-challenge period. Feeding of NG corn significantly increased the total leucocyte count, lysozyme activity, A/G ratio and decreased the total erythrocyte count, haemoglobin, serum total protein and globulin content of L. rohita juveniles during the post-challenge period. Similarly, feeding of n-3 PUFA at any level significantly increased the immunological parameters like lysozyme activity or A/G ratio, whereas total leukocyte count increased due to feeding of either 0.5% or 1.0% n-3 PUFA. The NBT and albumin values remained similar in both the pre- and post-challenge period. After challenge with Aeromonas hydrophila, the highest survival was recorded in the NG carbohydrate fed groups, whereas the lowest survival was recorded in the highest level of n-3 PUFA fed group irrespective of dietary carbohydrate type. Thus, a high level of G carbohydrate as well as n-3 PUFA is found to be immunosuppressive in L. rohita juveniles. NG carbohydrate supplemented with 1.0% n-3 PUFA is found to be optimum to enhance the immunity in L. rohita juveniles.