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Review |
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon
Address reprint requests to: P. D. Whanger, PhD, Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331. E-mail: phil.whanger{at}orst.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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Key words: selenomethionine, selenocysteine, Se-methylselenocysteine, nonprotein selenoamino acids, selenite, selenate, plants, yeast, animals
Key teaching points:
• Selenocompounds are found in differing types and amounts depending on the food source and selenium content of the substrate on which the food is grown.
• Conjugation of selenium to different amino acids alters both its bioavailability and metabolism.
• Selenite should not be used as a representative selenocompound because its behavior is dissimilar from that of selenate or organically-bound forms.
• The anticancer effects of the various selenium compounds vary manyfold.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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The initial work was done with toxic wheat [7–9] and corn [3,9]. As far as could be determined, selenite was first used as a control selenium compound in rat studies with toxic wheat in 1935 [10], and it was subsequently used in studies with swine [11,12], horses [12,13], mules and cattle [12]. It was not indicated in any of these studies why selenite was selected as the selenocompound, and the assumption is that it was the most readily available and cheapest source. This apparently set the stage for the extensive use of selenite as the "standard" selenocompound in many of the studies during the next several decades, and one purpose of this review is to examine the optimum chemical form of selenium to use in biological studies. Selenite has been used as the standard selenocompound in selenium bioavailability studies [14] and in most animal studies dealing with the relationships of selenium to carcinogenesis [15,16].
Reactions of Selenite
There are a number of reactions of selenite which are not shared by other forms of selenium as summarized in Table 1. Selenite can easily be reduced to elemental selenium by such mild reducing agents as ascorbate [15]. Tea [17] and rumen microbes [18] reduce selenite to insoluble forms. Presumably the reason selenite causes cataracts [19] in young rats is because it promotes peroxidation. The lower absorption of selenite [20] than other forms is consistent with results showing significantly less absorption with ligated loops [21]. Similar patterns were noted for suckling rats. The uptake and retention by suckling rat pups were most rapid for selenomethionine (70%), followed by selenate (51%) and least for selenite (29%) when added to infant formulas [22]. This difference was explained by a higher retention of selenium as selenite in the stomach and small intestinal wall of pups given the selenite supplement. This is postulated to be due to low diffusibility during digestion [23] and greater binding to brush border membrane vesicles [24]. The greater variation in absorption of selenite than selenate from the digestive tract [25] is speculated to be due to greater instability of selenite. There is lucigenin-dependent chemiluminescence with reduced glutathione (GSH) only with selenite [26], and it reacts with cysteine and GSH to form selenotrisulfides [27,28]. There are many metabolites from selenite during absorption which are not present with selenate or selenomethionine [29].
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Simultaneous vascular plus luminal perfusion of rat small intestine previously used to measure zinc absorption [31] was employed to study the absorption of various selenocompounds [29]. The transfer of selenium as selenomethionine and selenate to vascular effluents was respectively 2.4 fold and 1.5 fold greater than the transfer of selenite selenium. The selenium uptake as selenomethionine was significantly higher than selenium absorption as selenate or selenite. Selenium as selenate was transferred into the vascular effluent at a significantly greater rate than selenium given as selenite. Marked differences in metabolites during absorption were found with the three selenocompounds. When selenite was given, it represented only 14% of the overall quantity of selenium, and the selenotrisulfide metabolites, as a percentage of total selenium absorbed, were selenodicysteine, 10; mixed selenotrisulfide of cysteine and reduced glutathione, 14; selenodiglutathione, 8. Unidentified components composed 14% of total absorbed selenium. In contrast, selenate was transferred to the vascular effluent largely intact. The total absorbed selenium comprised 89% selenate, 1% selenotrisulfides and 9% unknown. Similar to selenate, there was little metabolism of selenomethionine during absorption. Protein-bound species were 7% of total absorbed selenium, selenite was less than 1%, and the remainder was selenomethionine. Therefore, there is extensive metabolism of selenite during absorption, but not of either selenate or selenomethionine.
Selenocompounds in Plants
The selenocompounds identified in plants have been summarized by Whanger [33]. These include selenate, selenite, selenocystine, selenomethionine, selenohomocysteine, Se-methylselenocysteine, 
-glutamyl-selenocystathionine, selenomethionine selenoxide, -glutamyl-Se-methylselenocysteine, selenocysteineselenic acid, Se-proponylselenocysteine selenoxide, Se-methylselenomethionine, selenocystathionine, dimethyl diselenide, selenosinigrin, selenopeptide and selenowax. Until a few years ago, the lability of selenocompounds and the problem of artifacts which arose during isolation procedures have hampered the identification of selenocompounds. Unfortunately some compounds like selenite can easily change forms by mere contact with metal containers. Precautions concerning checks on purity of radioactive selenium compounds, exposure of selenium to metallic utensils, storage of selenocompounds, some chromatographic problems with selenocompounds, the affinity of some volatile selenium for glass, and some precautions for identification of selenocompounds have been reviewed [34].
Indicator plants such as the Astragalus species can accumulate extremely large amounts of selenium, ranging from 1000 to 10,000 µg selenium per gram, because they synthesize mostly nonprotein selenoamino acids [35]. As much as 80% of the total selenium in some accumulator plants is present as Se-methylselenocysteine, and until recently it was thought to be absent in nonaccumulator plants. The nonprotein selenoamino acids which have been identified in selenium accumulator plants are Se-methylselenocysteine, selenocystathionine, Se-methylselenomethionine, -glutamyl-Se-methylselenocysteine, -glutamyl-selenocystathionine, selenopeptides, and selenohomocysteine. Very little, if any, selenite has been detected in any of these accumulator plants.
The selenium content of plants is dependent upon the area of growth (summarized in [33]), and vegetables such as rutabagas, cabbage, peas, beans, carrots, tomatoes, beets, potatoes and cucumbers contained a maximum of 6 µg selenium per gm even when grown on seleniferous soils. Other vegetables such as onions and asparagus were found to contain up to 17 µg selenium per gram when grown on these types of soils. Seleniferous soils are found mostly in the Rocky Mountain states [33], and plants growing in these areas will contain more selenium than those growing in other areas of the United States. For example, the selenium content in wheat, barley, corn, oats and rye can range up to 30 µg selenium per gram when grown in North and South Dakota, Nebraska, Kansas and Colorado as compared to less than 0.1 µg selenium per gram when grown in other areas of the country. The known selenium content in soil and plants in various countries of the world has been recently published [36].
Except for onions, garlic tubers, beets, cabbage and tomato leaves, information on the chemical forms of selenium in these plants is not available (discussed in [33]). Information on the chemical forms of selenium in forage plants is meager, but one of the predominant forms appears to be selenate. Selenium as selenate in 24 native plants of the United States was reported to range from 5% to 92% of the total selenium. Selenite was absent in all of these plants except for one species (Atriplex confertifolia). Even in this species, selenite was present at only 3% of the total selenium. One week after application of selenite to leaf surfaces of brome grass, less than 11% of the applied selenium remained as selenite [37], suggesting that this form of selenium does not remain in the plant. Therefore, selenate selenium appears to be the major form when humans consume vegetables not intentionally enriched with high levels of this element.
Selenium in Enriched Plants and Yeast
Current information on the chemical forms of selenium in enriched vegetables or supplements is summarized in Table 2. The chemical forms of selenium in plants or plant products can vary markedly. From 1% to 50% of the selenium was selenate in 20 different vegetables grown on sludge-amended soil [38]. Up to 44%, 38%, 40%, 50%, 40%, 48% and 39% of the selenium was selenate in broccoli florets, broccoli leaves, cucumbers, beet leaves, cabbage leaves, garlic tubers and tomato leaves, respectively. Most of the selenium in enriched wheat grain [39,40], corn and rice [41] and soybeans [42] is selenomethionine. When grassland legumes were grown in selenium-laden soils (at various concentrations), the percentage of selenium as selenomethionine was always highest at all levels of soil selenium [43]. Over 50% of the selenium in wheat straw was selenate [39], but no selenite was detected. Selenomethionine is the predominant form of selenium in most selenium enriched yeast products, but this can vary markedly [44–47]. The major form of selenium is Se-methylselenocysteine in Astragalus and selenium enriched garlic [45,47–50], onions [47,48], broccoli florets [48] and sprouts [51], and wild leeks [52]. At low selenium concentrations, Se-methylselenocysteine is the major form present but at elevated concentrations -glutamyl-Se-methylselenocysteine is the predominant one, and the hypothesis has been advanced that it serves primarily as a carrier of Se-methylselenocysteine [53]. Phytoplankton is the only plant found thus far that contains significant levels of extractable selenium as selenite [54]. However only 15% of the total selenium was extracted by an aqueous solution so that, when corrected for poor extraction, selenite was still a minor component. Since selenite is the form of selenium added to commercial preparations, this accounts for its being essentially the only form detected [54].
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Metabolic Aspects of Various Forms of Selenium
After 4.5 years of supplementation of American subjects with selenium enriched yeast, the incidence of lung, colon and prostate cancers was surprisingly reduced by 46%, 58% and 64%, respectively [62]. Even though the selenium contents of each batch of yeast were reported to be determined, if the selenium composition varied greatly between batches as suggested in Tables 2 and 3, this could raise some questions about the significance of the results. These results, however, are consistent with more than 150 animal studies showing an inverse relationship between selenium intake and the incidence of chemically and virally induced tumors [15,16]. Obviously much more work is needed to establish the relationship of selenium to cancer in humans.
Based on animal data, selenium enriched yeast may not be the most effective source of selenium for reduction of cancer in humans. The efficacy of various selenocompounds using the mammary tumor model has been summarized in Table 4. Se-methylselenocysteine and selenobetaine are the most effective selenocompounds identified thus far against mammary tumorigenesis. Although selenobetaine is just as effective, Se-methylselenocysteine is considered to be the most interesting selenocompound because it is the predominant one present in selenium enriched plants such as garlic, broccoli florets and sprouts, onions and wild leeks (Table 2). Therefore, this selenocompound has received the most recent attention as possibly the useful one for cancer reduction. Except for selenomethionine and selenocystine, the other selenocompounds listed in this table are not present in plants and thus are mostly of academic interest. However, a number of them are of therapeutic interest.
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Even though selenomethionine is effective against mammary tumors, one disadvantage of it is that it can be directly incorporated into general proteins instead of compounds which most effectively reduce tumors. When this occurs, its efficacy for tumor reduction may be reduced. For example, when a low methionine diet is fed there is significant reduction in the protective effect of selenomethionine even though the tissue selenium was actually higher in animals as compared to those given an adequate amount of methionine [67]. When methionine is limiting, a greater percentage of selenomethionine is incorporated nonspecifically into body proteins in place of methionine because the methionine-tRNA cannot distinguish between methionine and selenomethionine. Feeding diets with selenomethionine to animals as the main selenium source will result in greater tissue accumulation of selenium than other forms of selenium [68,69]. It is not known whether this stored selenium can serve as a reserved pool of selenium, but the evidence indicates that it is metabolically active [70].
With the knowledge of the effects of these selenocompounds as anticarcinogenic agents, it was of interest to investigate the most appropriate methods for delivery to the general population. One obvious approach was to investigate additional methods for expeditious ways to deliver these protective agents through the food system. One approach in this direction was the investigation of enriching garlic with selenium [71]. The addition of selenium enriched garlic to yield three µg selenium per gram of diet significantly reduced the mammary tumor incidence in rats from 83% to 33%. Interestingly, plain garlic reduced the tumor incidence to 60%, suggesting that there are other components in this plant besides selenium which will reduce the tumor incidence. Similar to garlic, regular broccoli also reduced mammary tumors, from 90% to 57% [51], suggesting that there are also components in broccoli in addition to selenium which will counteract tumors.
In the rat mammary carcinogenesis model, premalignant tissues known as intraductal proliferations are detectable within a few weeks after a carcinogenic insult [72]. Se-methylselenocysteine stimulated apoptosis by threefold to fourfold, and this increase was evident in both small and large intraductal proliferations. These data suggest that exposure to Se-methylselenocysteine blocks clonal expansion of premalignant lesions at an early stage.
Using another model, selenium enriched broccoli florets [73–75], as well as enriched broccoli sprouts [51], have been shown to reduce colon tumors in rats. This is intriguing because colon cancer is the third most common newly diagnosed cancer in the United States [74]. However, different results were found with various forms of selenium. Selenium enriched broccoli was most effective, selenite and selenate were intermediate, but selenomethionine was ineffective in the reduction of aromatic amine-induced colon carcinogenesis [76,77]. Selenite, selenate and selenomethionine were more effective for induction of glutathione peroxidase activity than selenium enriched broccoli, but the enriched broccoli was more effective in the reduction of colon tumors, indicating that selenium bioavailability for enzyme induction is not correlated with its efficacy for cancer reduction [78]. Furthermore, the addition of selenite to regular broccoli florets or sprout powder was ineffective in the reduction of colon tumors [51], indicating that the plant converts the selenium to more effective forms for reduction of these tumors. These results emphasize the need to study the effects of selenium in food forms.
Selenium enriched yeast is the most popular selenium supplement presently available to the general public, but selenium enriched garlic was shown to be twice as effective as enriched yeast in the reduction of mammary tumors [45]. The total number of tumors as well as the incidence of tumors was reduced to a greater extent by enriched garlic than enriched yeast. Chemical speciation of selenium in these two products indicated that selenomethionine was the predominant form of selenium in enriched yeast, whereas Se-methylselenocysteine (as the glutamyl derivative) was the predominant form of selenium in enriched garlic [45]. These results are consistent with those in Table 4, where pure compounds were used for reduction of mammary tumors. The fact that pure selenocompounds reduced tumors in the mammary tumor model (Table 4), but not in the colon tumor model, suggests that different results may be obtained depending upon the model used.
Tissue cultures have been advantageously used to study tumor reduction. Research indicates that the beta-lyase mediated production of a monomethylated selenium metabolite, namely methylselenol, from Se-methylselenocysteine is a key step in cancer chemoprevention by this agent [79]. In order for Se-methylselenocysteine to be effective, cells must possess this beta-lyase. One way to get around this is to use methylseleninic acid, which is even effective in cells without this lyase. The evidence indicates that the mechanism in which selenium reduces tumors is through its effects on apoptosis [80–84]. Methylseleninic acid produced a more robust response at one-tenth the concentration of Se-methylselenocysteine in the inhibition of cell proliferation and the induction of apoptosis in mouse mammary epithelial cells [79]. Apparently these cells have low levels of the beta-lyase. Interestingly the distinction between these two compounds disappears in vivo, where their cancer chemopreventive efficacies were found to be very similar. The reason for this is that the beta-lyase enzyme is abundant in many tissues and thus the animal has ample capacity to convert Se-methylselenocysteine to methylselenol.
Work with mouse mammary epithelial tumor cells indicates that Se-methylselenocysteine mediates the cleavage of poly (ADP-ribose) polymerase which results in apoptosis by activating one or more caspases [80]. Of the caspases, caspase-3 activity appeared to be activated to the greatest extent. Apparently these cells have ample lyases to convert Se-methylselenocysteine to methylselenol. Further work with these same cells using methylseleninic acid produced similar results, providing additional support that monomethylated forms of selenium are the critical effector molecules in selenium-mediated growth inhibition in vitro [83]. Consistent with in vivo results (Table 4), dimethyl selenide, a putative metabolite of methylselenol, was inactive. Furthermore, exposure of human umbilical vein endothelial cells to methylseleninic acid led to DNA fragmentation and caspase-mediated cleavage of poly (ADP-ribose) polymerase, which is a biochemical hallmark of apoptosis [81]. Apoptosis induced by methylseleninic acid is associated with an increased phosphorylation of P38 MAPK in addition to the dephosphorylation of several kinases. Further research is needed to identify the reasons for a monomethylated form of selenium that is required for this effect which cannot be fulfilled by other forms of selenium. In work by other researchers, Se-methylselenocysteine reduced the total protein kinase C in mouse mammary epithelial tumor cells [82]. Evidence was obtained with HL-60 cells that a reactive oxygen species was an important mediator in apoptosis induced by Se-methylselenocysteine [84], indicating that other factors influence the effects of these selenocompounds.
Conclusions
An examination of the literature did not yield a valid scientific reason to use selenite as a standard compound in selenium investigations, and its extensive use has been apparently due to tradition because of the initial studies on selenium toxicity. This chemical form of selenium is not found in any biological material at very high levels and in the majority of cases it was not even detectable. Admittedly, selenite has been used extensively to prevent selenium deficiency disorders in livestock production, and there is thus no question, although variable, it is biologically effective. The purpose of this review is not to discredit its biological effectiveness, but to question its use as a representative selenocompound. Selenate is the most predominant inorganic form found in both animal and plant tissues. Selenomethionine is the predominant form of selenium found in cereal grains, soybeans and grassland legumes. Se-methylselenocysteine is the predominant form of selenium present in selenium accumulator plants and in selenium-enriched vegetables such as garlic, broccoli, onions and wild leeks. Of those studied, Se-methylselenocysteine has been shown to be the most effective selenocompound against chemically induced mammary tumors [44,63]. It is interesting that such plants as garlic, onions, broccoli and leeks apparently produce Se-methylselenocysteine as a defense against selenium toxicity, but this in turn is beneficial to animals and presumably humans for reduction of tumors. Therefore, it would appear that if researchers want to simulate the results likely to be obtained with selenium-enriched plants, similar selenocompounds to those found in these various products must be used. However, it should be remembered that the use of pure selenocompounds may not be relevant because of interactions not only with other selenocompounds, but also with other compounds in the plant, but the use of the major selenocompound(s) likely to be found in such biological materials would appear paramount. Hence, if an inorganic selenium compound is desired, then selenate should be used. Selenomethionine should be the compound of choice if results similar to the selenium in cereal grains, grassland legumes and soybeans are desired. Se-methylselenocysteine should be the compound of choice if reactions analogous to selenium accumulator plants or selenium enriched vegetables are desired. It was indicated in a recent review that ideally selenium should be supplemented in the form it occurs naturally in foods [30].
The instability of selenite is apparently responsible for many of its reactions which are not shared by other forms of selenium. Charles Boone (personal communication) at the National Cancer Institute, Bethesda, MD, indicated that in 1986 it was decided not to use selenite in any of their investigations because of its instability. Greater drip loss of breast meat was found with broilers fed selenite in the diet as compared to organic selenium [85], and the comment was "five years onward: no more selenite!" The author predicted sodium selenite use will decline in the animal feed industry. The present reviewer suggests that it also should disappear from use in selenium research as a representative selenocompound. The common comments of reviewers of papers on selenium and carcinogenesis where selenite was not used are that, because this form of selenium has been used in the past, it should be continued for the sake of continuity. This reviewer could hardly disagree more! It is time to change the attitude toward selenite; traditional use is not justification for continued use.
There is a need for greater investigation of the chemical forms of selenium in biological materials with the latest techniques available. Until the last decade, paper chromatography, chromatography on ion exchange resins, high voltage paper electrophoresis and thin layer chromatography have been the predominant methods for the speciation of selenium (summarized in [33]). While these methods have been useful, with the development of more sensitive methods, additional detailed studies would appear to be paramount. These more sensitive methods include ion-pair liquid chromatography with inductively coupled plasma-mass spectrometry or with electrospray ionization-mass spectrometry, or chromatography with interfaced atomic mass spectrometry and atomic emission spectral detection. Additional information about the chemical forms of selenium in animals tissue is needed. From the present information, when inorganic selenium is given to animals, selenocysteine is the main selenocompound formed, but little or no information is available on other selenocompounds in animal tissues. When selenomethionine is consumed by animals, this amino acid is the predominant one deposited in tissues initially, but there is conversion to selenocysteine with time afterwards. However, information on other forms of selenium during this conversion process is lacking. More detailed information is warranted on the selenocompounds in plants of economic importance, and the use of more sensitive techniques should fill this gap. It is now apparent that the total selenium content is no longer sufficient to fully evaluate the potential biological effects of an enriched source of selenium. The greater effectiveness of Se-methylselenocysteine than other selenocompounds against carcinogenesis is an example of this type of much needed information.
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FOOTNOTES |
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Received October 3, 2001. Accepted January 18, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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