Abstract
Selenium, recognized as an essential nutrient for human health, is a component of proteins and enzymes required for various biological functions and is currently being used as a feed supplement for livestock in geographical areas that are naturally low in selenium. Selenium is structurally similar to sulfur, replacing the sulfur atom in stoichiometric amounts and thus functions through an association with proteins, termed selenoproteins. In geographic areas low in selenium, there is the potential for animals (including humans) to become selenium deficient and this potential deficiency can be remedied by consumption of exogenous selenium, including selenium-enriched yeast (Saccharomyces cerevisiae) that contains high levels of organic selenium (e.g., selenized yeast). A unique, standardized, registered high selenium food-grade baker’s yeast (S. cerevisiae; Sel-Plex®), was tested in the following battery of Genotoxicity assays; (1) a bacterial reverse mutation test (Ames test); (2) an in vitro mammalian chromosome aberration test; and (3) a mouse micronucleus test. Under the conditions of this assay, Sel-Plex® showed no evidence of mutagenic activity in Salmonella typhimurium, in the bacterial reverse mutation test. Sel-Plex® did not induce significant chromosomal aberrations in cultured human lymphocytes in the in vitro mammalian chromosome aberration test. Sel-Plex® did not statistically increase the frequency or proportion of micronucleated immature erythrocytes in the mouse micronucleus test. Thus, from the studies presented here, the authors conclude that Sel-Plex® is nongenotoxic.
Selenium has been recognized as an essential nutrient for human health for over 40 years, and is a component of proteins and enzymes required for various biological functions, such as antioxidant defense, reduction of inflammation, thyroid hormone production, fertility, and DNA synthesis (Brown and Arthur 2001; Whanger 2002). Selenium has been utilized as a feed supplement for livestock in geographical areas that are naturally low in selenium. It has been proven that selenium deficiencies can also occur in human diets, and may be manifested clinically as muscular weakness and myalgia, fatigue, loss of appetite, cardiomegaly, cardiac arrhythmia, or palpitations (Raisbeck 2000; Kanekura et al. 2005).
The amount of selenium in the human body is not wholly predicated by the amount of selenium in the diet, as its bioavailability is primarily dependent upon the type of selenium, i.e., elemental versus organic, with the latter having much greater bioavailability (Brown and Arthur 2001; Schrauzer 2001). Selenium functions through an association with proteins, termed selenoproteins. As selenium is structurally similar to sulfur, selenium will replace the sulfur atom in stoichiometric amounts in the normal biosynthetic pathways of plants, and is mainly found incorporated into methionine, to form selenomethionine (Sors, Ellis, and Salt 2005). Selenomethionine, synthesized in plants, is randomly incorporated in place of methionine in a variety of proteins in both plants and mammals. Most selenium in animal tissues is present as selenomethionine or selenocysteine (Allan, Lacourciere, and Stadtman 1999; Schrauzer 2003).
Although selenium is considered an essential nutrient, excess consumption of selenium can have toxic effects. Selenium toxicity, termed selenosis, is caused by excessive selenium intake and has been documented in humans in seleniferous regions of China where selenium-contaminated foods are consumed. It is also reported in individuals who consumed excessive amounts of selenium supplements. Symptoms of selenosis include hair loss, loss of nails, skin lesions, diarrhea, and a garlic-like odor from the breath and in body secretions. The level at which selenosis occurs is still under debate. Findings in China indicate that about 3 to 5 mg/day will cause selenosis; however, residents in seleniferous regions of South Dakota have been reported to consume approximately 0.7 mg/day with no toxic symptoms (Yang et al. 1983; Fan and Kizer 1990; Longnecker et al. 1991).
Intake of selenium typically occurs via consumption of selenium-rich plants and animal tissues. Plants grown in selenium-rich soils transform inorganic selenium into selenomethionine. In geographic areas low in selenium, there is the potential for animals (including humans) to become selenium deficient if their diet is limited to the consumption of region-specific plants (or selenium-deficient meat). This potential deficiency can be remedied by consumption of exogenous selenium, including selenium-enriched yeast (McCartney 2005; Power 2005). It has been well documented that fermentation of specific strains of yeast with high selenium levels during their growth phase will incorporate the selenium into the yeast cell; a proteolytic digestion with a nonspecific protease may lead to 90% to 95% recovery of the total amount of selenium from the yeast (Encinar et al. 2003). A method to manufacture a unique, standardized and registered yeast (Saccharoymes cerevisiae) extract that contains high levels of organic selenium (e.g., selenized yeast) has been developed. The incorporation of standardized selenized yeast into food products will decrease the prevalence of potential selenium deficiencies.
Absorption of organic selenium from food is generally believed to be in the range of 80% of the total amount found in food. A number of absorption studies comparing various types of selenium-containing supplements have indicated that selenium in the form of selenized yeast has greater bioavailability than from inorganic selenium sources. The bioavailability may not be as great as from isolated selenomethionine consumption, as selenized yeast also forms a number of other organic selenium compounds. When compared to inorganic selenium, the increased selenium status after selenized yeast consumption is also retained for a longer period (Jacques 2001; McCartney 2005; Power 2005).
Selenized yeast varies significantly from producer to producer and lot to lot on both total selenium content, as well as the preponderance of organoselenium (Encinar et al. 2003; Larsen et al. 2004). It has been reported that for the conductance of some clinical trials, selenium content varies and has varied over the lifetime of the trial; for example, selenomethionine has varied from a low of 27% to values routinely around 54% to 60% (Larsen et al. 2004). Sel-Plex® is standardized to contain a total selenium content of 2000 mg/kg (ppm) and selenomethionine is equal to or greater than 98% of the selenium content.
METHODS AND MATERIALS
Test Articles
Sodium selenite was provided by Alltech (Meath, Ireland). A unique, standardized, registered, high selenium food-grade baker’s yeast (Saccharomyces cerevisiae, S. cerevisiae CNCM 1-3060 extract; Sel-Plex® ), also provided by Alltech,1 was selected after screening in excess of 150 distinct strains of S. cerevisiae for a unique and proprietary strain with the ability to accumulate appreciable levels of selenium to form the final commercial product, Sel-Plex®, which contains S. cerevisiae CNCM 1-3060 with reproducible levels of selenium-containing proteins. The unique S. cerevisiae CNCM 1-3060 strain’s genomic DNA karyotype has been identified via pulsed field electrophoresis and polymerase chain reaction. It is known that yeast strains incorporate selenium into cellular proteins in a strain-specific manner (Encinar et al. 2003), such that the resulting yeast preparation is expected to demonstrate unique bioavailability and thus, unique toxicity or safety. S. cerevisiae CNCM 1-3060 has incorporated selenium from selenium-containing media into cellular proteins. For this reason, the yeast preparation tested in the described studies will be termed “Sel-Plex® .” Sel-Plex® is a spray-dried selenium yeast preparation with a total selenium content of 2000 mg/kg (ppm). The organic selenium content, predominately selenomethionine, is equal to or greater than 98% of the selenium content.
Bacterial Reverse Mutation Test (Ames Test)
Sel-Plex® (batch ES-716) and reference compound, sodium selenite (batch 030301), were evaluated for mutagenic activity in the Ames test using a standard Salmonella typhimurium plate incorporation and preincubation methods (Ames, McCann, and Yamasaki 1975; Maron and Ames 1983), and in accordance with Organization for Economic Co-operation and Development (OECD) guideline number 471 entitled “Bacterial Reverse Mutation Test.” Genotoxicity was assessed in S. typhimurium tester strains TA98, TA100, TA102, TA1535, and TA1537 (courtesy of Dr. B. N. Ames, University of California, Berkeley, CA, USA) in the presence and absence of the postmitochondrial fraction of liver homogenates (S9) from rats pretreated with Aroclor 1254. Toxicity was evaluated based on a decrease in the number of revertant colonies and/or thinning of the bacterial lawn.
Stock bacterial cultures were grown overnight at 37°C in nutrient broth (NB) liquid medium. Sel-Plex® or sodium selenite (0.1 ml) dissolved in water, or reference chemicals, were added to 2 ml of 0.7% agar medium containing a histidine/biotin mixture, 0.1 ml of the overnight bacterial culture, and either 0.5 ml of the S9 mixture or 0.5 ml of phosphate-buffered saline. The contents of the tubes were mixed and poured immediately onto minimal agar plates. Three replicate plates for the test compound and controls were incubated at 37°C for 2 days prior to counting. In the preincubation procedure, 0.1 ml of Sel-Plex®, sodium selenite, or reference compounds was incubated with 0.1 ml of the bacterial culture and 0.5 ml of either phosphate-buffered saline or the S9 mixture, for 60 min at 37°C prior to mixing with the agar medium containing histidine/biotin. The contents were mixed, poured onto agar plates, and incubated as for the standard assay. Three replicate plates per concentration were tested in the preincubation assay. Colonies were counted electronically using an automatic Cardinal counter (Perceptive Instruments, Suffolk, UK).
In Vitro Mammalian Chromosome Aberration Test
Sel-Plex® (batch ES-716) and reference compound, sodium selenite (batch 030301), and control vehicle (0.9% sodium chloride) were evaluated for mutagenic potential in cultured human lymphocytes, in accordance with OECD guideline number 473 entitled “Standard Chromosome Aberration Test.” Genotoxicity was assessed in the presence and absence of postmitochondrial fractions of liver homogenates (S9) prepared from rats pretreated with Aroclor 1254. Exposures were 3, 20, and 44 h. The highest dose level used in the initial test was selected based on pH, osmolality, and solubility. In the main test, reduction of mitotic index in the initial test was also used as a factor to modify the dose selection. For each culture, heparinized whole blood was added to the culture medium containing nitrogen (phytohemagglutinin) and incubated at 37°C for 48 h. The test article was suspended at 55 mg/ml in 0.9% NaCl. The maximum treatment level (with and without S9) in the first test was 5000 μg/ml; and in the main test, the maximum treatment levels were 312.5 μg/ml (without S9) and 1250 μg/ml (with S9).
Mouse Micronucleus Test
Sel-Plex® (batch ES-716) and reference compound, sodium selenite (batch 030301), were evaluated for mutagenic potential in mouse bone marrow cells and in accordance with the OECD guideline 474 entitled “Mammalian Erythrocyte Micronucleus Test.”
Animals
Adult CHS Swiss ICo:OFI (IOPS Caw) mice were obtained from Depre Breeding Centre (Saint Doulchard, France). Animals were acclimatized for at least 7 days before initiation of dosing for the mouse micronucleus test. At dosing, animals were 8 to 12 weeks old. Animals were housed in standard cages with sawdust bedding (SAFE, Villemoisson, Epinay-sur-Orge, France), and a controlled 12-h light/dark cycle. Temperature was maintained at 19°C to 23°C with a relative humidity of 45% to 65%. Potable water and standardized diet, SAFE A04C-10 (SAFE), were provided ad libitum. All animal studies complied with EC Council Directive No. 86/609/EEC, 24NOV1986 (EC 1986) and the appropriate parts of the Animal Welfare Act Regulations, 9 CFR Parts 1, 2, and 3 (Federal Register 1989). These studies also met or exceeded the requirements of OECD Guidelines (1997) and FDA 1993 Redbook II Guidelines (FDA 1993), including OECD (1998) and FDA Principles of Good Laboratory Practices (FDA 2006).
Experimental Procedure
Preliminary toxicity tests were conducted to determine appropriate dose levels for the experiment. For all compounds, groups of five male and female Swiss ICo:OFI mice were administered the test article by gavage. For the Sel-Plex® treatment, groups of male and female mice were dosed with 500, 1000, or 2000 mg/kg/day for 2 days. Three additional groups of male and female mice were administered sodium selenite as a reference compound, at dose levels of 7.5, 15, and 30 mg/kg/day (males) and 3.75, 7.5, and 15 mg/kg/day (females). A negative-control group was administered water (vehicle), and a positive-control group was acutely administered cyclophosphamide (via gavage) at 50/mg/kg. Femoral bone marrow was collected 24 h after the final dosing and smears were fixed and stained according to the method described by Schmid (1975), and modified by Salamone et al. (1980). For each animal, the number of micronu-cleated polychromatic erythrocytes (MPEs) was counted in 1000 polychromatic erythrocytes (PEs). The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was determined after scoring 1000 erythrocytes (Es). To increase statistical power, an additional 2000 PEs were examined for frequency of MPEs and an additional 1000 Es were scored for PE/NE ratio.
The chi-squared test was used to determine within-group heterogeneity, and in the absence of significance, the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 × 2 contingency table to determine the χ2 value. When within-group heterogeneity significance occurred, then that group was compared with the control group using a nonparametric analysis, the Mann-Whitney test (Wilcoxon 1945; Kruskal and Wallis 1952, 1953). The student “t” test was used for the PE/NE ratio comparison, and significance was considered at probability values of p ≤ .05 (Dunn 1964).
RESULTS
Bacterial Reverse Mutation Test (Ames Test)
Preliminary studies indicated there was no toxicity of Sel-Plex® towards any of the strains, with or without S9, at concentrations up to 1000 μg/plate (Table 1). Sodium selenite caused moderate toxicity at dose levels ≥ 1000 μg/plate in strain TA1537 and at 2000 μg/plate in the TA98, TA102, and TA1535 strains (data not shown). The number of revertants in the vehicle and positive controls confirmed the sensitivity of the assay and the metabolizing activity of the S9. Under the conditions of this assay, Sel-Plex® showed no evidence of mutagenic activity in S. typhimurium.
In Vitro Mammalian Chromosome Aberration Test
Sel-Plex® did not induce significant chromosomal aberrations in cultured human lymphocytes after 3-, 20- and 44-h treatments, with or without S9 metabolic activation (Tables 2 and 3). Sodium selenite did not significantly alter the mitotic index (Table 4), but caused a significant increase in the frequency of cells with structural chromosomal aberration after 3 h of sodium selenite treatment without S9 and after 20 h of exposure with S9 (Table 5).
Mouse Micronucleus Test
Sel-Plex® did not statistically increase the frequency or proportion of micronucleated immature erythrocytes at doses of 500, 1000, or 2000 mg/kg/day, compared to vehicle controls (Table 6). Sodium selenite did not exhibit statistically significant changes to either frequency or proportion of immature erythrocytes at lower doses, but treatment with 30 mg/kg sodium selenite increased micronucleated polychromatic erythrocytes in male mice (p < .01).
DISCUSSION
Selenium consumption has been shown to have numerous anticarcinogenic effects, whereas antithetically, other studies have indicated mutagenic, nonmutagenic, and antimutagenic effects of various forms of selenium (Lofrath and Ames 1978; Shamberger 1985; ATSDR 2003; Cemeli et al. 2003; Yu et al. 2004). The differences in the effects of selenium may be based on the form that is presented to the body. The biologically active selenoproteins include several glutathione peroxidase isoforms, iodothyronine 5′-deiodinases, thioredoxin reductases, selenophosphate synthetase and more. All have selenocysteine (SeCys) at their active sites. In order for dietary inorganic or organic selenium forms to be used in selenoprotein synthesis, they must first be converted to hydrogen selenide (H2Se) before being incorporated into SeCys by the cotranslational modification of tRNA-bound serinyl residues. Selenocysteine is inserted into growing polypeptide chains at UGA codons, which normally encode a stop signal (Stadtman 1996; Combs 2001). Selenate is reduced to selenite and this in turn is reduced, via formation of selenodiglutathione, to H2Se (Foster and Sumar 1997). Selenomethionine is activated initially by adenosylation, demethylated and converted to selenocysteine via selenohomocysteine and selenocystathionine, in analogy to methionine (Schrauzer 2000; Jacques 2001). Selenocysteine is then converted to H2Se. Hydrogen selenide not used for selenoprotein synthesis is methylated to yield methylselenol, dimethylselenide, and trimethylselenonium ion. The latter two metabolites are excreted in breath and urine, respectively. Importantly, excess H2Se may be oxidized, leading to the production of superoxide and other reactive oxygen species, which can cause DNA damage (Combs 2001).
The major difference in the metabolism of dietary inorganic and organic forms of selenium lies in the ability of selenomethionine (SeMet) to be incorporated nonspecifically into cellular proteins. Sel-Plex® contains elevated levels of SeMet, which may explain its reduced genotoxicity relative to sodium selenite. Incorporation of SeMet into cellular proteins removes a significant portion of the ingested organic selenium source from circulation, thereby acting as a sink for selenium and reducing its toxic impact (Schrauzer 2001). In contrast, inorganic selenium cannot be efficiently incorporated into cellular proteins and any not used directly for selenoprotein synthesis must remain in circulation until it can be methylated and excreted.
The in vitro clastogenic effects of Sel-Plex® to cultured human lymphocytes indicated that this compound does not induce significant chromosomal aberrations. Moore et al. (1996) found that selenium monosulfide significantly increased bone marrow micronucleated polychromatic erythrocytes, indicative of a weak mutagen. This is contrasted with the organic forms of selenium utilized in the current study, which lacked a mutagenic effect.
Sel-Plex® did not statistically increase the frequency or proportion of micronucleated immature erythrocytes and thus did not cause chromosomal or mitotic aberrations. Previous in vitro work has indicated that preincubation of Chinese hamster lung fibroblasts and rat hepatocellular DNA with selenium actually inhibits the cadmium-induced frequency of micronucleus DNA damage (Hurna, Siklenka, and Hurna 1997; Yu and Chen 2004).
Thus, the safety of Sel-Plex® is further supported by a battery of appropriate genotoxicity tests. Given the history of selenium and S. cerevisiae (Baker’s yeast) consumption, data published in the literature regarding its potential antigenotoxic effects, and from the studies presented here, we conclude that Sel-Plex® is nongenotoxic, even when administered to animals at doses that are significantly higher than those intended for humans.
Footnotes
Figure and Tables
The bacterial reverse mutation test, in vitro mammalian chromosome aberration test, and mouse micronucleus test were conducted at the CIT Safety & Research Laboratories, Evereux, France; Study Director for the bacterial reverse mutation test and in vitro chromosome aberration test was G. Shire, DESS Cellular Engineering, CIT, and the Study Director for the mouse micronucleus test was H. Haddouk, CIT; and the Principle Investigator for the microscopic analysis of the erythrocyte slides was N. Danforth, CIT. All studies were sponsored by Alltech Biotechnology Centre, Meath, Ireland.
1
Yeast strain S. cerevisiae CNCM 1-3060 is a proprietary strain (Alltech Inc. 273-29-2) deposited through an official institute signatory to the Budapest Treaty to the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, F-75724 Paris Cedex 15 on June 24, 2003.