In Vitro Cytotoxicity of Trastuzumab (Tz) and Se-Trastuzumab (Se-Tz) against the Her/2 Breast Cancer Cell Lines JIMT-1 and BT-474.

Her/2+ breast cancer accounts for ~25% mortality in women and overexpression of Her/2 leads to cell growth and tumor progression. Trastuzumab (Tz) with Taxane is the preferred treatment for Her/2+ patients. However, Tz responsive patients often develop resistance to Tz treatment. Herein, redox selenides (RSe-) were covalently linked to Tz using a selenium (Se)-modified Bolton-Hunter Reagent forming Seleno-Trastuzumab (Se-Tz; ~25 µgSe/mg). Se-Tz was compared to Tz and sodium selenite to assess the viability of JIMT-1 and BT-474 cells. Comparative cell viability was examined by microscopy and assessed by fluorometric/enzymatic assays. Se-Tz and selenite redox cycle producing superoxide (O2•-) are more cytotoxic to Tz resistant JIMT-1 and Tz sensitive BT-474 cells than Tz. The results of conjugating redox selenides to Tz suggest a wider application of this technology to other antibodies and targeting molecules.

Selenomethionine and Methioninase: Selenium Free Radical Anticancer Activity.

Colloidal selenium, was first used to treat cancer as early as 1911 in both humans and mice. Selenium was identified as the toxic component in forage plants of sheep, cattle, and horses in the 1930s. The animal toxicity of selenium compounds was determined to be from the metabolism by animals of the elevated concentrations of Se-methylselenocysteine and selenomethionine in plants. The metabolism of both Se-methylselenocysteine and selenomethionine by animals gives rise to the metabolite, methylselenide (CH3Se-), which if in sufficient concentration oxidizes thiols and generates superoxide and other reactive oxygen species. Cancer cells that may overly express methionine gamma-lyase, or beta-lyase (methioninase), by induced viral genomic expression, are susceptible to free radical-induced apoptosis from selenomethionine or Se-methylselenocysteine supplementation.

Cytotoxicity of Selenium Immunoconjugates against Triple Negative Breast Cancer Cells.

Within the subtypes of breast cancer, those identified as triple negative for expression of estrogen receptor α (ESR1), progesterone receptor (PR) and human epidermal growth factor 2 (HER2), account for 10⁻20% of breast cancers, yet result in 30% of global breast cancer-associated deaths. Thus, it is critical to develop more targeted and efficacious therapies that also demonstrate less side effects. Selenium, an essential dietary supplement, is incorporated as selenocysteine (Sec) in vivo into human selenoproteins, some of which exist as anti-oxidant enzymes and are of importance to human health. Studies have also shown that selenium compounds hinder cancer cell growth and induce apoptosis in cancer cell culture models. The focus of this study was to investigate whether selenium-antibody conjugates could be effective against triple negative breast cancer cell lines using clinically relevant, antibody therapies targeted for high expressing breast cancers and whether selenium cytotoxicity was attenuated in normal breast epithelial cells. To that end, the humanized monoclonal IgG1 antibodies, Bevacizumab and Trastuzumab were conjugated with redox selenium to form Selenobevacizumab and Selenotrastuzumab and tested against the triple negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231 as well as a normal, immortalized, human mammary epithelial cell line, HME50-5E. VEGF and HER2 protein expression were assessed by Western. Although expression levels of HER2 were low or absent in all test cells, our results showed that Selenobevacizumab and Selenotrastuzumab produced superoxide (O2•-) anions in the presence of glutathione (GSH) and this was confirmed by a dihydroethidium (DHE) assay. Interestingly, superoxide was not elevated within HME50-5E cells assessed by DHE. The cytotoxicity of selenite and the selenium immunoconjugates towards triple negative cells compared to HME-50E cells was performed in a time and dose-dependent manner as measured by Trypan Blue exclusion, MTT assay and Annexin V assays. Selenobevacizumab and Selenotrastuzumab were shown to arrest the cancer cell growth but not the HME50-5E cells. These results suggest that selenium-induced toxicity may be effective in treating TNBC cells by exploiting different immunotherapeutic approaches potentially reducing the debilitating side effects associated with current TNBC anticancer drugs. Thus, clinically relevant, targeting antibody therapies may be repurposed for TNBC treatment by attachment of redox selenium.

Selenium-mediated arsenic excretion in mammals: a synchrotron-based study of whole-body distribution and tissue-specific chemistry.

Arsenicosis, a syndrome caused by ingestion of arsenic contaminated drinking water, currently affects millions of people in South-East Asia and elsewhere. Previous animal studies revealed that the toxicity of arsenite essentially can be abolished if selenium is co-administered as selenite. Although subsequent studies have provided some insight into the biomolecular basis of this striking antagonism, many details of the biochemical pathways that ultimately result in the detoxification and excretion of arsenic using selenium supplements have yet to be thoroughly studied. To this end and in conjunction with the recent Phase III clinical trial "Selenium in the Treatment of Arsenic Toxicity and Cancers", we have applied synchrotron X-ray techniques to elucidate the mechanisms of this arsenic-selenium antagonism at the tissue and organ levels using an animal model. X-ray fluorescence imaging (XFI) of cryo-dried whole-body sections of laboratory hamsters that had been injected with arsenite, selenite, or both chemical species, provided insight into the distribution of both metalloids 30 minutes after treatment. Co-treated animals showed strong co-localization of arsenic and selenium in the liver, gall bladder and small intestine. X-ray absorption spectroscopy (XAS) of freshly frozen organs of co-treated animals revealed the presence in liver tissues of the seleno bis-(S-glutathionyl) arsinium ion, which was rapidly excreted via bile into the intestinal tract. These results firmly support the previously postulated hepatobiliary excretion of the seleno bis-(S-glutathionyl) arsinium ion by providing the first data pertaining to organs of whole animals.

Selenium-enriched garlic and cabbage as a dietary selenium source for broilers.

This study determined the selenium (Se) bioavailability from Se-enriched garlic and cabbage using broiler chickens. Se-enriched garlic (18.5 mg of Se/kg) and cabbage (101.5 mg of Se/kg) were produced by soil enrichment using selenate. Conventional and Se-enriched garlic and cabbage were dried, ground, and added to broiler chick diets. Ninety-six broiler chickens at 1 day of age were assigned to four dietary treatments: NC (cabbage + garlic), PC (cabbage + garlic + selenomethionine, 0.5 mg of Se/kg of diet), GS (cabbage + Se-enriched garlic, 0.5 mg of Se/kg of diet), and CS (garlic + Se-enriched cabbage, 0.5 mg of Se/kg of diet), with six replicates per treatment and four birds per cage. Birds were fed the experimental diets for 4 weeks and slaughtered to obtain blood and tissues: white (breast) muscle, dark (thigh) muscle, liver, and feathers. All excreta were collected weekly, dried, and ground for Se analysis. Bird weight gain and feed intake were measured weekly. Total Se content and glutathione peroxidase (GPX) activity in liver and plasma were measured. Total liver Se content of the PC birds (0.876 mg of Se/kg) was the highest (P < .05). The CS (0.693 mg of Se/kg) and GS (0.627 mg of Se/kg) birds had higher (P < .05) total liver Se than the NC birds (0.514 mg of Se/kg). Plasma GPX activity of the PC birds was highest (P < .05), and that of CS and GS birds was higher (P < .05) than the NC birds. Liver GPX activity of the PC birds was higher (P < .05) than all other treatments. Bioavailability of Se to broiler chickens was not different (P > .05) among PC (65.2%), CS (61.2%), and GS (70.7%) birds. This study indicates that the Se from Se-enriched garlic and cabbage is highly bioavailable and can potentially be beneficial in enhancing Se status and GPX activity.

Toxicity and mutagenicity of selenium compounds in Saccharomyces cerevisiae.

Selenium (Se) is an essential trace element for humans, animals and some bacteria which is important for many cellular processes. Se's bio-activity is mainly influenced by its chemical form and dose. The use of Se supplements in the human diet emphasizes the need to establish both the beneficial and detrimental doses of each Se compound. We have evaluated three different Se compounds, sodium selenite (SeL), selenomethionine (SeM) and Se-methylselenocysteine (SeMC), with respect to their potential DNA damaging effects. The budding yeast Saccharomyces cerevisiae was used as a model system to test the toxic and mutagenic effects as well as the DNA double-strand breakage potency of these Se compounds in both exponentially growing and stationary yeast cells. Only SeL manifested any significant toxic effects in the yeast which were more pronounced in the exponentially growing cells than in those cells in the stationary phase of growth. The toxic effects of SeL were however accompanied with the pro-mutagenic effects in the stationary cell phase of growth. The toxic and mutagenic effects of SeL are likely associated with the ability of this compound to generate DNA double-strand breaks (DSB). We also show that SeL significantly increased frame-shift mutations, especially 1-4 bp deletions, in the CAN1 mutational spectrum of the yeast genome when compared to untreated control. We propose that SeL is acting as an oxidizing agent in S. cerevisiae producing superoxide and oxidative damage to DNA accounting for the observed DSB and cell death.

Prevention of bacterial colonization of contact lenses with covalently attached selenium and effects on the rabbit cornea.

PURPOSE: Although silicone hydrogel materials have produced many corneal health benefits to patients wearing contact lenses, bacteria that cause acute red eye or corneal ulcers are still a concern. A coating that inhibits bacterial colonization while not adversely affecting the cornea should improve the safety of contact lens wear. A covalent selenium (Se) coating on contact lenses was evaluated for safety using rabbits and prevention of bacterial colonization of the contact lenses in vitro. METHODS: Contact lenses coated with Se were worn on an extended-wear schedule for up to 2 months by 10 New Zealand White rabbits. Corneal health was evaluated with slit-lamp biomicroscopy, pachymetry, electron microscopy, and histology. Lenses worn by the rabbits were analyzed for protein and lipid deposits. In addition, the ability of Se to block bacterial colonization was tested in vitro by incubating lenses in a Pseudomonas aeruginosa broth followed by scanning electron microscopy of the contact lens surface. RESULTS: The covalent Se coating decreased bacterial colonization in vitro while not adversely affecting the corneal health of rabbits in vivo. The Se coating produced no noticeable negative effects as observed with slit-lamp biomicroscopy, pachymetry, electron microscopy, and histology. The Se coating did not affect protein or lipid deposition on the contact lenses. CONCLUSION: The data from this pilot study suggest that a Se coating on contact lenses might reduce acute red eye and bacterial ulceration because of an inhibition of bacterial colonization. In addition, our safety tests suggest that this positive effect can be produced without an adverse effect on corneal health.

Arsenic and selenium in human hair: a comparison of five countries with and without arsenicosis.

The selenium (Se) content of the diet and/or selenium supplements might have an ameliorating effect on arsenic (As) toxicity as recently shown by Wang et al., Yang et al., and as reviewed by Spallholz et al.. The underlying principles of the ameliorating effect is the complexation of Se with As forming the seleno-bis (S-glutathionyl) arsinium ion excreted in bile and the complexation of Se with As in tissues forming nontoxic insoluble selenides. Additional protection afforded by Se supplementation from arsenicosis could be the elevation of glutathione peroxidase activity reducing the oxidative stress induced by As. The present study assessed the status of Se and As in hair by neutron activation analysis (NAA). Human hair samples were collected from the United States, Canada, The People's Republic of China (PRC), Bangladesh, and Nepal, the latter two countries now engaged in a struggle to find relief from human arsenicosis resulting from extensive domestic groundwater contamination by As. No statistically significant differences were observed in the samples between the Se and As content of hair from, Lubbock, Texas (USA) or Winnipeg, Canada. The concentration of As in all hair samples analyzed correlated (r = 0.960, p < 0.001) with the amount of As in the drinking water. Selenium levels in hair were highest from Nepal. The results demonstrate the viability of hair as a noninvasive biomonitor in assessing aspects of dietary Se and environmental As exposure. The hair data confirmed the known low intake of Se in the Keshan disease area of the PRC, the very high accumulation in hair of As from subjects consuming contaminated groundwaters, and an adequate Se status in subjects from North America consuming municipal water of low As content. The high As content of hair from people in Bangladesh is the result of a high As consumption from contaminated water compounded by a less than desirable intake of Se. From Nepal, the As content of hair corresponded to the known low and high intake of As from contaminated groundwater. The very high Se content found in all hair samples from Nepal might be the result of the use of henna.

Environmental hypothesis: is poor dietary selenium intake an underlying factor for arsenicosis and cancer in Bangladesh and West Bengal, India?

To reduce the incidence of dysentery, cholera and other water-borne diseases and mortality of people drinking from surface contaminated sources of water, the World Bank and United Nations Children's Fund began to sink tube wells into the underlying aquifers of Bangladesh and West Bengal, India, in the 1970s. Many of the tube wells were drilled into underground aquifers that provided microbiologically clean water that was later determined to contain arsenic (As). As contamination of drinking water is a problem of natural occurrence throughout the world and domestic water often exceeds the World Health Organization limit of 50 microg As/l in the countries of Bangladesh, West Bengal, India and Nepal as well as other areas occupying much of the Ganges-Brahmaputra delta. It is estimated that as many as one-half of these tube wells discharge water with sufficient amounts of As to produce arsenicosis, i.e. As toxicity in the human population. Access to clean As free water is the priority of most organized relief efforts. Where As free domestic water cannot be provided, an improved diet and/or dietary supplements may ameliorate As toxicity or prevent its toxicity all together. The dietary status of the essential human trace element, selenium (Se) may be adversely affected by a chronic excessive ingestion of As. As added to animal diets has been known to counteract Se toxicity in animals since the 1930s. It is reasoned therefore, that high levels of chronic As ingestion from well water by people within the delta will accelerate the excretion of Se lowering the body's content of this essential trace element. Excessive Se excretion owing to Se/As complexation may add to the likelihood of As being more toxic and carcinogenic over time, due to the oxidative stress imposed by the excessive As and low Se ingestion. Because of the unique environment of the Ganges-Brahmaputra delta in which millions of people are presently exposed to As, we ask the question: are low dietary Se ingestion and accelerated Se depletion by As possible contributing factors to arsenicosis?

Methioninase and selenomethionine but not Se-methylselenocysteine generate methylselenol and superoxide in an in vitro chemiluminescent assay: implications for the nutritional carcinostatic activity of selenoamino acids.

Methylselenol from selenium metabolism is postulated to be and most experimental evidence now indicates that it is the selenium metabolite responsible for the dietary chemoprevention of cancers. Using the recombinant enzyme methioninase, methylselenol-generating chemiluminesence by superoxide (O2*-) is shown to be catalytically produced from L-selenomethionine and D,L-selenoethionine, but not from methionine or L-Se-methylselenocysteine (SeMC). Methylselenol enzymaticaly generated by methioninase activity from the substrate selenomethionine arises from an initial putative selenium radical as measured by chemiluminesence in the absence of glutathione (GSH). In the presence of GSH, superoxide was generated as measured by chemiluminesence and superoxide dismutase inhibition of chemiluminescence. Ascorbic acid also quenched the chemiluminesence from the activity of methioninase with selenomethionine. Methylselenol and other redox cycling selenium compounds are almost assuredly accountable for inducing cell-cycle arrest and apoptosis in cancer cells in vitro and in vivo. Methylselenol generated from selenomethionine by methioninase is catalytic alone in oxidizing thiols, i.e. GSH, generating superoxide and inducing oxidative stress in direct proportion to its concentration. Se-methylselenocysteine in vivo is very likely carcinostatic in like manner to selenomethionine by generating methylselenol from other enzymatic activity, i.e. beta-lyase or amino acid oxidases.

Selenium toxicity: cause and effects in aquatic birds.

There are several manners in which selenium may express its toxicity: (1) an important mechanism appears to involve the formation of CH(3)Se(minus sign) which either enters a redox cycle and generates superoxide and oxidative stress, or forms free radicals that bind to and inhibit important enzymes and proteins. (2) Excess selenium as selenocysteine results in inhibition of selenium methylation metabolism. As a consequence, concentrations of hydrogen selenide, an intermediate metabolite, accumulate in animals and are hepatotoxic, possibly causing other selenium-related adverse effects. (3) It is also possible that the presence of excess selenium analogs of sulfur-containing enzymes and structural proteins play a role in avian teratogenesis. L-selenomethionine is the most likely major dietary form of selenium encountered by aquatic birds, with lesser amounts of L-selenocysteine ingested from aquatic animal foods. The literature is suggestive that L-selenomethionine is not any more toxic to adult birds than other animals. L-Selenomethionine accumulates in tissue protein of adult birds and in the protein of egg white as would be expected to occur in animals. There is no suggestion from the literature that the levels of L-selenomethionine that would be expected to accumulate in eggs in the absence of environmental concentration of selenium pose harm to the developing embryo. For several species of aquatic birds, levels of Se as selenomethionine in the egg above 3 ppm on a wet weight basis result in reduced hatchability and deformed embryos. The toxicity of L-selenomethionine injected directly into eggs is greater than that found from the entry of L-selenomethionine into the egg from the normal adult diet. This suggests that there is unusual if not abnormal metabolism of L-selenomethionine in the embryo not seen when L-selenomethionine is present in egg white protein where it likely serves as a source of selenium for glutathione peroxidase synthesis in the developing aquatic chick.