Selenium metabolism in cancer cells: The combined application of XAS and XFM techniques to the problem of selenium speciation in biological systems

Determining the speciation of selenium in vivo is crucial to understanding the biological activity of this essential element, which is a popular dietary supplement due to its anti-cancer properties. Hyphenated techniques that combine separation and detection methods are traditionally and effectively used in selenium speciation analysis, but require extensive sample preparation that may affect speciation. Synchrotron-based X-ray absorption and fluorescence techniques offer an alternative approach to selenium speciation analysis that requires minimal sample preparation. We present a brief summary of some key HPLC-ICP-MS and ESI-MS/MS studies of the speciation of selenium in cells and rat tissues. We review the results of a top-down approach to selenium speciation in human lung cancer cells that aims to link the speciation and distribution of selenium to its biological activity using a combination of X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM). The results of this approach highlight the distinct fates of selenomethionine, methylselenocysteine and selenite in terms of their speciation and distribution within cells: organic selenium metabolites were widely distributed throughout the cells, whereas inorganic selenium metabolites were compartmentalized and associated with copper. New data from the XFM mapping of electrophoretically-separated cell lysates show the distribution of selenium in the proteins of selenomethionine-treated cells. Future applications of this top-down approach are discussed.Claire M. Weekley, Jade B. Aitken, Lydia Finney, Stefan Vogt, Paul K. Witting, and Hugh H. Harri

The combined application of XAS and XFM techniques to the problem of selenium speciation in biological systems

Includes Notice of erratumSe is an essential element that is incorporated into proteins as the 21st amino acid, selenocysteine. The observation of inverse relationships between serum Se status and the incidence of various diseases, including cancer and cardiovascular disease, has led to the consideration of dietary Se supplementation as a disease prevention measure. However, the results of clinical trials into the use of Se supplementation in disease prevention have been mixed. An important aspect of Se in biology is that its metabolism and biological activity differ with speciation. Thus the choice of Se compound is an important factor when considering dietary Se supplementation. The metabolism, speciation and biological activity of Se in vivo must be understood in order to choose the most efficacious Se compound(s) for disease prevention. This thesis describes the application of two synchrotron-based X-ray techniques, X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM), to the study of Se speciation and distribution in cells and in vivo. The combined application of XAS and XFM was found to be of great utility in determining Se speciation in biological systems. With complementary biochemical data, XAS and XFM studies provided insight into the differing fates and biological activities of dietary Se compounds. Initially, human cancer cells were treated with the dietary Se compounds selenomethionine (SeMet), methylselenocysteine (MeSeCys) or selenite. Evidence from XAS and XFM indicates that SeMet was largely incorporated into proteins in place of methionine, hence its low toxicity. Conversely, the diselenide species present in MeSeCys-treated cells were indicative of oxidative stress leading to cell death. Finally, a time-course study of selenite speciation in cancer cells indicated the formation of diselenide species concomitant with a decrease in cell viability. Large proportions of elemental Se and selenotrisulfide species were also observed in selenite-treated cells. XFM revealed that the inorganic Se species were distributed in a punctate manner throughout the cytosol in association with Cu. Biochemical assays and further XAS studies indicated that the increase in Cu concentration and its association with Se in selenite-treated cells was found to have at least two origins: the upregulation of SOD1 in response to selenite-mediated superoxide generation, and the formation of Cu-Se bonds. Tissues harvested from rats fed high Se (as selenite) diets were used to study Se supplementation in vivo using the same XAS and XFM methods that were applied in the cell culture studies. Speciation in this more complex system could not be determined as readily as in cancer cells. Nonetheless, it was observed that even though the overall speciation of Se in these tissues differed from its speciation in selenite-treated cells, the presence of Se-S and Se- Se species was common to both cells and rat tissues. In the kidneys of rats fed a high selenite diet, Se and Cu were found to be colocalised to a greater extent than was observed in selenitetreated cells. However, this spatial relationship appeared to be unrelated to SOD1 expression and evidence for Cu-Se bonding was weak. Further investigation into the origin of the association between Se and Cu in these tissues is required.Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 201

Methylselenocysteine Treatment Leads to Diselenide Formation in Human Cancer Cells: Evidence from X-ray Absorption Spectroscopy Studies

The selenoamino acids methylselenocysteine (MeSeCys) and selenomethionine (SeMet) have disparate efficacies as anticancer agents. Herein, we use X-ray absorption spectroscopy to determine the chemical form of selenium in human neuroblastoma cells. Cells treated with MeSeCys contain a significant diselenide component, which is absent from SeMet-treated cells and suggests that metabolites of MeSeCys are capable of altering the redox status of the cells. The differences in the speciation of Se in the selenoamino acid-treated cells may provide insight into the differing anticancer activities of MeSeCys and SeMet

Selenium inhibits renal oxidation and inflammation but not acute kidney injury in an animal model of rhabdomyolysis

Acute kidney injury (AKI) is a manifestation of rhabdomyolysis (RM). Extracellular myoglobin accumulating in the kidney after RM promotes oxidative damage, which is implicated in AKI. AIM: To test whether selenium (Se) supplementation diminishes AKI and improves renal function. RESULTS: Dietary selenite increased Se in the renal cortex, as demonstrated by X-ray fluorescence microscopy. Experimental RM-stimulated AKI as judged by increased urinary protein/creatinine, clusterin, and kidney injury molecule-1 (KIM-1), decreased creatinine clearance (CCr), increased plasma urea, and damage to renal tubules. Concentrations of cholesterylester (hydro)peroxides and F₂-isoprostanes increased in plasma and renal tissues after RM, while aortic and renal cyclic guanidine monophosphate (cGMP; marker of nitric oxide (NO) bioavailability) decreased. Renal superoxide dismutase-1, phospho-P65, TNFα gene, MCP-1 protein, and the 3-chloro-tyrosine/tyrosine ratio (Cl-Tyr/Tyr; marker of neutrophil activation) all increased after RM. Dietary Se significantly decreased renal lipid oxidation, phospho-P65, TNFα gene expression, MCP-1 and Cl-Tyr/Tyr, improved NO bioavailability in aorta but not in the renal microvasculature, and inhibited proteinuria. However, CCr, plasma urea and creatinine, urinary clusterin, and histopathological assessment of AKI remained unchanged. Except for the Se++ group, renal angiotensin-receptor-1/2 gene/protein expression increased after RM with parallel increases in MEK1/2 inhibitor-sensitive MAPkinase (ERK) activity. INNOVATION: We employed synchrotron radiation to identify Se distribution in kidneys, in addition to assessing reno-protection after RM. CONCLUSION: Se treatment has some potential as a therapeutic for AKI as it inhibits oxidative damage and inflammation and decreases proteinuria, albeit histopathological changes to the kidney and some plasma and urinary markers of AKI remain unaffected after RM.Anu Shanu, Ludwig Groebler, Hyun Bo Kim, Sarah Wood, Claire M. Weekley, Jade B. Aitken, Hugh H. Harris, and Paul K. Wittin

Fear of fatness and drive for thinness in predicting smoking status in college women

Recent research has identified fear of fatness (FF) as a related yet distinct construct from drive for thinness (DT). Whereas DT may be associated with need for approval and an “approach” tendency, FF may be more strongly related to avoidance of disapproval and an avoidant problem-solving style. Although no research has directly compared the influence of FF vs. DT with regard to smoking behavior, FF and DT might represent distinct motivations for smoking. We predicted that both FF and DT would be significantly associated with cigarette smoking, but that FF would be a stronger predictor of smoking behavior, even after controlling for variables such as body mass index (BMI) and nicotine dependence. Participants (N = 289) were female college undergraduate students. Daily smokers had the highest scores on measures of DT and FF, followed sequentially by infrequent smokers, “triers,” and never smokers. More frequent smokers also reported greater levels of body dissatisfaction and eating pathology than less frequent and never-smokers. Hierarchical regression analyses showed that greater DT predicted higher likelihood of smoking on a daily basis; however, higher FF predicted fewer cigarettes smoked per day. FF and DT may each play a role in the relationship between eating pathology and smoking, but they might be differentially related to specific smoking patterns. Both FF and DT and their coinciding coping styles should be further researched in the role of smoking initiation and maintenance

Selenite-mediated production of superoxide radical anions in A549 cancer cells is accompanied by a selective increase in SOD1 concentration, enhanced apoptosis and Se-Cu bonding

Selenite may exert its cytotoxic effects against cancer cells via the generation of reactive oxygen species (ROS). We investigated sources of, and the cellular response to, superoxide radical anion (O2 ·−) generated in human A549 lung cancer cells after treatment with selenite. A temporal delay was observed between selenite treatment and increases in O2 ·− production and biomarkers of apoptosis/necrosis, indicating that the reduction of selenite by the glutathione reductase/NADPH system (yielding O2 ·−) is a minor contributor to ROS production under these conditions. By contrast, mitochondrial and NADPH oxidase O2 ·− generation were the major contributors. Treatment with a ROS scavenger [poly(ethylene glycol)-conjugated superoxide dismutase (SOD) or sodium 4,5-dihydroxybenzene-1,3-disulfonate] 20 h after the initial selenite treatment inhibited both ROS generation and apoptosis determined at 24 h. In addition, SOD1 was selectively upregulated and its perinuclear cytoplasmic distribution was colocalised with the cellular distribution of selenium. Interestingly, messenger RNA for manganese superoxide dismutase, catalase, inducible haem oxygenase 1 and glutathione peroxidase either remained unchanged or showed a delayed response to selenite treatment. Colocalisation of Cu and Se in these cells (Weekley et al. in J. Am. Chem. Soc. 133:18272–18279, 2011) potentially results from the formation of a Cu–Se species, as indicated by Cu K-edge extended X-ray absorption fine structure spectra. Overall, SOD1 is upregulated in response to selenite-mediated ROS generation, and this likely leads to an accumulation of toxic hydrogen peroxide that is temporally related to decreased cancer cell viability. Increased expression of SOD1 gene/protein coupled with formation of a Cu–Se species may explain the colocalisation of Cu and Se observed in these cells.Claire M. Weekley, Gloria Jeong, Michael E. Tierney, Farjaneh Hossain, Aung Min Maw, Anu Shanu, Hugh H. Harris, Paul K. Wittin

Which form is that? The importance of selenium speciation and metabolism in the prevention and treatment of disease

The biological activity of selenium is dependent upon its speciation. We aim to integrate selenium speciation and metabolism into a discussion of the mechanisms by which selenium exerts its biological activity. First, we present the current status of selenium in the prevention of cancer, cardiovascular and neurodegenerative diseases with particular attention paid to the results of major chemoprevention trials involving selenium supplementation. A comprehensive review of the current understanding of the metabolism of common dietary selenium compounds - selenite, selenomethionine, methylselenocysteine and selenocystine - is presented, with discussion of the evidence for the various metabolic pathways and their products. The antioxidant, prooxidant and other mechanisms of the dietary selenium compounds have been linked to their disease prevention and treatment properties. The evidence for these various mechanisms -in vitro, in cells and in vivo- is evaluated with emphasis on the selenium metabolites involved. We conclude that dietary selenium compounds should be considered prodrugs, whose biological activity will depend on the activity of the various metabolic pathways in, and the redox status of, cells and tissues. These factors should be considered in future laboratory research and in selecting selenium compounds for trials of disease prevention and treatment by selenium supplementation.Claire M. Weekley and Hugh H. Harri