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P. D. Whanger
Department of Environmental and Molecular Toxicology
Oregon State University
Corvallis, OR 97331

The statements “Selenium may reduce the risk of certain cancers” and “Selenium may produce anticarcinogenic effects in the body” are supported by scientific evidence. There is significant scientific agreement that daily supplementation with selenium may reduce the risk of some cancers and that selenium is anticarcinogenic. This report will examine epidemiological studies, human clinical trials, animal studies, and in vitro studies on selenium’s relationship to cancer. It will examine the efficacy of different forms of selenium and of different levels of selenium supplementation.

I. Selenium

Selenium is classified in a group VIA of the periodic table of elements which includes the nonmetals, sulfur and oxygen, in the periods above selenium, and the metals, tellurium and polonium, in the period below this element (Combs and Combs, 1986a). By period, selenium lies between the metal arsenic and the nonmetal, bromine. Thus, selenium is considered a metalloid, having both metallic and nonmetallic properties. It has an atomic number of 34 and an atomic weight of 79. Elemental selenium, like its sister elements, sulfur and tellurium, can exist in either an amorphous state or one of three crystalline states.

Elemental selenium can be reduced to the -2 oxidation state (selenide), or oxidized to the +4 (selenite) or +6 (selenate) oxidation states. Hydrogen selenide (H2Se) is a fairly strong acid in aqueous systems. The gas is colorless, has an unpleasant odor, and is highly toxic. At low pH, selenite is readily reduced to the elemental state by mild reducing agents such as ascorbic acid or sulfur dioxide. In its oxidized state (+6), selenium can exist as selenic acid or as selenate salts. Selenic acid is a strong acid. Most selenate salts are soluble in water, in contrast to the corresponding selenite salts and metal selenides. Selenates tend to be rather inert and are very resistant to reduction.

The chemical and physical properties of selenium are very similar to those of sulfur. The two elements have similar outer-valence shell electronic configurations and atomic sizes and their bond energies, ionization potentials and electron affinites are virtually the same. Despite these similarities, the chemistry of selenium and sulfur differ in two respects that distinguish them in biological systems. First, in the biological systems, selenium compounds are metabolized to more reduced states whereas sulfur compounds are metabolized to more oxidized states. The second important difference in the chemical behaviors of these elements is in the acid strengths of their hydrides. The hydride, H2 Se, is much more acidic than is H2S. This difference in acidic strengths is reflected in the dissociation behaviors of the selenohydryl groups of selenocysteine and the sulfhydryl groups on cysteine. Hence, while thiols such as cysteine are predominantly protonated at physiological pHs, the selenohydryl groups of selenols such as selenocysteine are predominantly dissociated under the same conditions.

II. Selenocompounds in plants.

The metabolism of selenocompounds in plants has been summarized (Whanger, 1989). Selenium enters the food chain through incorporation into plant proteins, mostly as selenocysteine and selenomethionine (Semet) at normal selenium levels. However, with elevated selenium levels, Se-methylselenocysteine (SeMCYS) can be the predominant selenocompound. As many as eight other selenocompounds have been identified in plants but their concentrations are usually very low except at high selenium levels. Indicator plants (called selenium accumulators) can accumulate extremely large amounts of selenium, ranging from 1000 to 10,000 Fg selenium per gm because they synthesize mostly nonprotein selenoamino acids (Brown and Shrift, 1981). As much as 80% of the total selenium in some accumulator plants is present as SeMCYS and until recently it was thought to be absent in nonaccumulator plants.

The selenium content of plants is dependent upon the region of growth (summarized by Whanger, 1989). Vegetables such as rutabagas, cabbage, peas, beans, carrots, tomatoes, beets, potatoes, and cucumbers contained a maximum of 6 Fg selenium per gm even when grown on seleniferous soils. Vegetables such as onions and asparagus may accumulate up to 17 Fg selenium per gm when grown on these types of soils. Plants which contain deficient levels of selenium are found in the Pacific Northwest, upper Mid-West, the New England states and along the Atlantic coast of the United States. In other parts of the country such as North and South Dakota, Colorado and Western Nebraska plants may contain high levels of this element. Plants can synthesize organic selenium compounds including Semet from inorganic selenium (Burnell and Shrift, 1977). Because of the uneven global distribution of selenium, disorders of both selenium deficiency and selenium excess are known. For example, China has regions with both the lowest and the highest selenium-containing soil in the world (Yang et al, 1989 a,b). Plants of economic importance do not have a selenium requirement for growth and thus plant selenium is for the health of animals including humans.

Although the data are lacking, synthesis of the nonprotein selenoamino acids by plants probably occurs along pathways normally associated with sulfur metabolism. Conversion of selenocysteine to SeMCYS in accumulators has been shown to involve the transfer of a methyl group from S-adenosylmethionine, analogous to the synthesis of S-methylcysteine (Neuhierl et al, 1999). Even though the primary source of selenium in soil is inorganic, mostly selenate, Astragalus accumulators have been shown to synthesize SeMCYS when supplied with Semet (Chen et al, 1970). The ability of the accumulators to exclude selenoamino acids from proteins has been suggested as a reason for their selenium tolerance. Similar mechanisms apparently operate in selenium enriched plants such as garlic, broccoli, onions and wild leeks where the nonprotein selenoamino, SeMCYS, is the predominant one present.

Most of the selenium in enriched wheat grain (Olson et al, 1970), corn and rice (Beilstein et al, 1991) and soybeans (Yasumoto et al, 1984) is Semet. Semet is the predominant form of selenium in selenium enriched yeast (Ip et al, 2000a). Selenium enriched yeast is the most common source of selenium available commercially (Schrauzer, 2000). The selenoamino acid, Semet, is also available for the public. The major form of selenium is SeMCYS in selenium enriched garlic (Ip et al, 2000a), onions (Cai et al, 1995), broccoli florets (Cai et al, 1995) and sprouts (Finley et al, 2001), and wild leeks (Whanger et al, 2000).

III. Selenocompounds in animals

A brief metabolic pathway for selenium metabolism in animals has been presented (Ip, 1998). Organic selenium such as Semet or inorganic selenium can be converted to a common intermediate, hydrogen selenide. There are two possible pathways for the catabolism of Semet. One is the transsulfuration pathway via selenocystathionine to produce selenocysteine, which in turn is degraded to hydrogen selenide by the enzyme, $-lyase (Mitchell and Benevenga, 1978). The other pathway is the transamination-decarboxylation pathway. It was estimated that 90% of the methionine is metabolized through this pathway and thus could be the major route also for Semet catabolism. SeMCYS is the predominant selenocompound formed in selenium enriched garlic at relatively low concentrations, but γ-glutamyl-Se methyl selenocystine is the predominant one at high selenium concentrations (Dong et al, 2001). Even though this glutamyl derivative may be the predominant one, it is hydrolyzed in the intestinal tract and the absorbed SeMCYS cleaved by a lyase to form methylselenol (Dong et al, 2001). Thus, this glutamyl derivative is metabolized like SeMCYS at the tissue level. SeMCYS is converted to methylselenol directly when cleaved by beta-lyase and unlike Semet it cannot be incorporated nonspecifically into proteins. Since SeMCYS can be converted directly to methylselenol, this is presumably the reason it is more efficacious than other forms of selenium.

When rats are injected with selenite, the majority of the selenium is present in tissues as selenocysteine (Olson and Palmer, 1976; Beilstein and Whanger, 1988). As expected, no Semet was found under the conditions of these studies. In contrast to plants, there is no known pathway in animals for synthesis of Semet from inorganic selenium, and thus they must depend upon plant or microbial sources for this selenoamino acid. However, animals can convert Semet to selenocysteine. One day after injection of Semet there is about three times as much Semet as selenocysteine in tissues, but five or more days afterwards the majority (46-57%) of the selenium is present as selenocysteine (Beilstein and Whanger, 1986).

A total of 24 selenoproteins have been identified in eukaryotes (Gladyshev, 2001). These selenoproteins have been subdivided into groups based on the location of selenocysteine in selenoprotein polypeptides. The first group (called glutathione peroxidase, GPX) is the most abundant and includes proteins in which selenocysteine is located in the N-terminal portion of a relatively short functional domain. These include the four GPXs, selenoproteins P, Pb, W, W2, T T2 and BthD (fromDrosophila). The second group of eukaryotic selenoproteins is characterized by the presence of selenocysteine in C-terminal sequences. These include the three thioredoxin reductases and the G-rich protein from Drosophila. Other eukaryotic selenoproteins are currently placed in the third group that consists of the three deiodinase isozymes, selenoproteins R and N, the 15 kDa selenoprotein and selenophosphate synthetase. The four GPXs are located in different parts of tissues and all detoxify to various degrees hydrogen peroxide and fatty acid derived hydroperoxides and thus are considered antioxidant selenoenzymes. The three deiodinases convert thyroxine to triiodothyronine, thus regulating thyroid hormone metabolism. The thioredoxin reductases reduce intramolecular disulfide bonds and, among other reactions, regenerate vitamin C from its oxidized state. These reductases can also affect the redox regulation of a variety of factors, including ribonucleotide reductase, the glucocorticoid receptor and the transcription factors (Holmgren, 2001). Selenophosphate synthetase synthesizes selenophosphate, which is a precursor for the synthesis of selenocysteine.(Mansell and Berry, 2001). The functions of the other selenoproteins have not been definitely identified.

Selenium is present in all eukaryotic selenoproteins as selenocysteine (Gladyshev, 2001). Semet is incorporated randomly in animal proteins in place of methionine. By contrast, the incorporation of selenocysteine into proteins known as selenoproteins is not random. Thus, by contrast to Semet, selenocysteine does not randomly substitute for cysteine. In fact, selenocysteine has it own triplet code (UGA) and is considered to be the 21st genetically coded amino acid. Interestingly, UGA has a dual role in the genetic code, serving as a signal for termination and also a codon for selenocysteine. Whether it serves as a stop codon or encodes selenocysteine depends upon the location of what is called the selenocysteine insertion sequence (Mansell and Berry, 2001).

A number of reviews have been written on the chemopreventive effects of selenium including most recently those by Combs and Gray (1998), Ganther (1999), Ip (1998), Schrauzer (2000), El-Bayoumy (2001) and Fleming et al (2001). The mechanism for selenium as an anticarcinogenic element is not known but several speculations have been advanced. It is well established that the most effective dose of selenium for cancer protection is at elevated levels, often called supernutritional or pharmacological levels. The suggested mechanisms for cancer prevention by selenium include its effects upon cell cycle (called apoptosis, probably the most accepted possibility), its role in selenoenzymes, its effects upon carcinogen metabolism, its effects upon the immune system, and its specific inhibition of tumor cell growth by certain selenium metabolites.


IV. Epidemiological studies.

There have been a number of epidemiological studies in the United States and throughout the world on the relationship between selenium and cancer. Shamberger and Frost (1969) reported that the selenium status of humans may be inversely related to the risk of some kinds of cancer. Two years later, Shamberger and Willis (1971) in more extensive studies indicated that the mortality due to lymphomas and cancers of the gastrointestinal tract, peritoneum, lung, and breast were lower for men and women residing in areas of the United States that have high concentrations of selenium in forage crops than those residing in areas with low selenium content in the forages. Those studies were supported by a later analysis of colorectal cancer mortality using the same forage data (Clark et al, 1981). A 27-country comparison revealed that total cancer mortality rate and age-corrected mortality due to leukemia and cancers of the colon, rectum, breast, ovary and lung varied inversely with estimated per capita selenium intake (Schrauzer et al, 1977). Similar results were also reported in China, a country where selenium intakes range from deficient to toxic levels (Yu et al, 1985).

Lower selenium levels were found in serum collected from American subjects one to five years prior to diagnosis of cancer as compared to those who remained cancer free during this time (Willett et al, 1983). That association was strongest for gastrointestinal and prostatic cancers. Evidence that low serum selenium is a prediagnostic indicator of higher cancer risk was subsequently shown in studies conducted in Finland (Salonen et al, 1984) and Japan (Ujiie et al, 1998). In additional case-control studies, low serum or plasma selenium were found to be associated with increased risk of thyroid cancer (Glattre et al, 1989), malignant oral cavity lesions (Toma et al, 1991), prostate cancer (Brooks et al, 2001), esophageal and gastric cancers (Mark et al, 2000), cervical cancer mortality rates (Guo et al, 1994) and colorectal adenomas (Russo et al, 1997). A decade long prospective study of selenium status and cancer incidences indicated that initial plasma selenium concentration was inversely related to subsequent risks of both non-melanoma skin cancer and colonic adenomatous polyps (Clark et al, 1993). Patients with plasma selenium levels less than 128 ng/ml (the average normal value) were four times more likely to have one or more adenomatous polyps. An 8-year retrospective case control study in Maryland revealed no significant association of serum selenium level and cancer risk at sites other than the bladder (Helzlsouer et al, 1989), but those with low plasma selenium levels had a 2-fold greater risk of bladder cancer than those with high plasma selenium. In a study with Dutch patients the mean selenium levels were significantly less than that of controls in men, but no differences were found in plasma selenium levels between control women and those with cancer (Kok et al, 1987). No significant associations in three other studies were found between serum selenium concentration and risk to total cancers (Coates et al, 1988) or cancers of the lungs, stomach, or rectum (Nomura et al, 1987 and Kabuto et al, 1994). In other work, significant increases of urinary selenium excretion were found in Mexican women with cervical uterine cancer as compared to controls (Navarrete et al, 2001).

In four studies low toenail selenium values were associated with higher risks of developing cancers of the lung (van den Brandt et al, 1993a), stomach (van den Brandt et al, 1993b), breast (Garland et al, 1995) and prostate (Yoshizawa et al, 1998). In contrast, in four other studies no significant differences were found between cancer cases and controls (Noord et al, 1987, Hunter et al, 1990, Rogers et al, 1991 and Veer et al, 1990). It has been suggested that the reason for those not showing a relationship is because the selenium intakes of most of the subjects tested were below that necessary for protection (Schrauzer, 2000). Obviously these results indicate that many factors must be taken into consideration when evaluating plasma and toenail selenium concentrations in relation to cancer incidence.

V. Human Trials.

In spite of advances in diagnosis and treatment, cancer continues to be a major health burden. With the fear associated with diagnosis of cancer, it is not surprising that the public may have considerable interest in easily implemented measures, such as dietary modification or use of vitamin and trace element supplementation for cancer prevention. Promising results have been obtained, however, to indicate that selenium supplementation is effective in reduction of cancer in humans.

There have been six trials conducted on the effects of selenium supplementation on the incidence of cancer or biomarkers in humans and all of them have shown positive effects of selenium. Three of these were conducted in China and one each in India, Italy and in the United States. The first human intervention trial to prevent cancer with selenium in humans was conducted in Qidong, a region north of Shanghai, China, with a high incidence of primary liver cancer (PLC). Subjects were given table salt fortified with 15 ppm selenium as sodium selenite which provided about 30 to 50 micrograms selenium daily for eight years (Yu et al, 1991, 1997). This resulted in a drop of the PLC incidence to almost one-half (27.2 per 100,000 populations versus 50.4 per 100,000 populations consuming ordinary salt). Upon withdrawal of selenium from the treated group, the PLC incidence began to rise. In a separate study, risk populations receiving selenite salt as a source of selenium also showed a significant reduction in the incidence rate of viral infectious hepatitis, a major predisposing PLC risk factor in this region (Yu et al, 1989). The selenium fortified salt was distributed to the general population of 20,800 persons. Six neighboring townships served as controls and were given normal table salt.

In a second trial, members of families at risk of PLC were either given 200 micrograms selenium daily in the form of high-selenium yeast or a placebo (Yu et al, 1997). During the 2-year study period, 1.26% of the controls developed PLC versus 0.69% in those given selenium enriched yeast. Furthermore, of 226 Hepatitis B surface antigen carriers, seven of 113 subjects in the placebo group developed PLC during four years as opposed to no cases in those taking selenium enriched yeast.

A third human trial on the effects of selenium on cancer was also conducted in China with 3,698 subjects. This intervention trial was conducted from 1984 to 1991 in Linxian, China, a rural county in Henan Province, where the mortalities from esophageal cancer are among the highest in the world (Blot et al, 1993). The results indicated that a treatment containing selenium (50 micrograms Se/day as Se enriched yeast plus vitamin E and $-carotene) produced a modest protective effect against esophageal and stomach cancer mortality among subjects in the general population (Li et al, 1993; Taylor et al, 1994; Blot et al, 1995). Probably the reason for only a modest reduction of cancer by selenium is because only 50 micrograms were given daily in contrast to other studies where up to 200 micrograms were given per day.

In the study conducted in India, 298 subjects were used. One-half of the subjects with precancerous lesions in the oral cavity were supplemented with a mixture of four nutrients [vitamin A, riboflavin, zinc and selenium (100 micrograms daily for six months and 50 micrograms the final six months as selenium enriched yeast)] and compared to controls (also 149 patients) receiving placebos (Prasad et al, 1995). The frequency of micronuclei and DNA adducts were significantly reduced in the supplemented groups at the end of the one year study. The adducts decreased by 95% in subjects taking selenium with all categories of lesions and by 72% in subjects without lesions. No such effects were noted in the placebo group.

In the Italian study subjects were given a mixture called ‘Bio-selenium’ which provided 200 micrograms selenium as L-selenomethionine daily plus zinc and vitamins A, C and E for five years, and compared to those taking a placebo (Bonelli et al, 1998). A total of 304 patients participated in this study and the incidence of metachronous adenomas of the large bowel evaluated. Patients with prior resected adenomatous polyps were used in a randomized trial and new adenomatous polyps were noted. The observed incidence of metachronous adenomas was 5.6% in the group given the ‘Bio-selenium’ mixture versus 11% in the placebo group.

One of the most exciting clinical trials on selenium and cancer in humans was conducted in the United States. A simple experimental design in a double-blind, placebo-controlled trial with 1312 older Americans with histories of basal and/or squamous cell carcinomas of the skin were used (Clark et al, 1996, 1998). The use of a daily oral supplement of selenium enriched yeast (200 µg Se/day) did not affect the risk of recurrent skin cancers. However, supplementation with selenium as selenium enriched yeast reduced the incidence of lung, colon and prostate cancers respectively by 46, 58 and 64%. Restricting the analysis to the 843 patients with initially normal levels of prostate specific antigen, only four cases were diagnosed with cancer in the selenium treated group but 16 cases were diagnosed in the placebo group after a 2-year treatment lag (Clark et al, 1998). Even though Clark et al (1996) did not observe any effect of selenium on skin cancer in their study, the results strongly indicated that other types of skin disorders may be reduced by selenium.

The author is aware of at least three human trials [two in the United States (University of Arizona; and the SELECT trial at NCI; Klein et al, 2001), and one in Europe (PRECISE, Rayman, 2000)] presently under way to confirm the results of this American investigation.

Finally, in another trial, topical application of Semet was effective in protecting against acute ultraviolet irradiation damage to skin of humans (Burke et al, 1992a). Maximal protection appeared to be attained at concentrations between 0.02% and 0.05%.[1][1]

VI. Selenium and tumors in small animals.

There have been more than 100 trials conducted with small animals on the relationship of tumor incidences to selenium status (Combs and Combs, 1986b; Combs and Gray, 1998). Interestingly, the first evidence that selenium may counteract tumors was presented in 1949 where the addition of selenium to a diet for rats significantly reduced tumors caused by ingestion of an azo dye (Clayton and Bauman, 1949). These results were ignored even by these researchers because of the negative image selenium held at that time. The first evidence of the essentiality of selenium was presented in 1957 (Schwarz and Foltz, 1957), at which time selenium was considered a carcinogenic element. A number of reviews on selenium and carcinogensis in animals have been presented which include those by Milner (1985), Ip and Medina (1987) Medina and Morrison (1988) and Whanger (1992). The chemical carcinogens used to produce tumors in liver, mammary gland, colon, skin, lungs, trachea, pancreas and stomach have been summarized (Whanger, 1992). Two thirds of the animal studies showed significant reductions by selenium in the tumor incidence with one-half showing reductions of 50% or more (Combs and Gray, 1998). In the majority of those studies selenium as selenite was used but that may not have been the most effective form (as noted later) to use. Those results with animals and the epidemiological surveys showing a positive relationship between selenium and cancer incidence were the main motivating factors for conducting human trials.

VII. Tissue cultures.

The present research efforts are primarily focused on the mechanism of cancer reduction by selenium and tissue cultures have been used advantageously to study how tumors are reduced by this element. Research with these cultures also indicates that the beta-lyase mediated production of a monomethylated selenium metabolite, namely methylselenol, from SeMCYS is a key step in cancer chemoprevention by this agent (Ip et al, 2000b). In order for SeMCYS to be effective, cells must possess this beta-lyase. One way to get around this is to use methylselenic acid, which is even effective in cells without this lyase. Although several possibilities have been suggested (Combs and Gray, 1998), the evidence indicates that the likely mechanism in which selenium reduces tumors is through its effects upon apoptosis (Unni et al, 2001; Sinha et al, 1999). Methylselenic acid produced a more robust response at one-tenth the concentration of SeMCYS in the inhibition of cell proliferation and the induction of apoptosis in mouse mammary epithelial cells (Ip et al, 2000b). 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 because the beta-lyase enzyme is abundant in many tissues and thus the animal has ample capacity to convert SeMCYS to methylselenol.

Work with the mouse mammary epithelial tumor cells indicate that SeMCYS mediates apoptosis by activating one or more caspases (Unni et al, 2001). Of the caspases, caspase-3 activity appeared to be activated to the greatest extent. Apparently these cells have ample lyases to convert SeMCYS to methylselenol. Further work with these same cells using methylselenic acid produced similar results, providing additional support that monomethylated forms of selenium are the critical effector molecules in selenium mediated growth inhibition in vitro (Sinha et al, 1999). Further research is needed to identify why a monomethylated form of selenium that is required for this effect cannot be fulfilled by other forms of selenium.

VIII. Forms of selenium in foods and supplements.

The efficacy of various selenocompounds using the mammary tumor model has been summarized in Table 1.[2][2] SeMCYS and selenobetaine are the most effective selenocompounds identified thus far against mammary tumorigenesis in animals (table 1). Although selenobetaine is just as effective, SeMCYS is considered to be the most interesting selenocompound because it is the predominant one present in selenium enriched plants such as garlic (Ip et al, 2000a), broccoli florets (Cai et al, 1995) and sprouts (Finley et al, 2001), and onions (Cai et al, 1995). Selenobetaine has never been detected in selenium enriched plants. Therefore, SeMCYS has received the most recent attention as possibly the most useful one for cancer reduction. Except for Semet and selenocystine, the other selenocompounds listed in this table are not present in plants and thus are mostly of academic interest. However, some of them are of therapeutic interest.

Selenobetaine and SeMCYS are good precursors for generating monomethylated selenium (Ip, 1998). Selenobetaine tends to lose a methyl group before scission of the Se-methylene carbon bond to form methylselenol. SeMCYS is converted to methylselenol directly when cleaved by beta-lyase and unlike Semet it cannot be incorporated nonspecifically into proteins. Since these

Table 1. Anticarcinogenic Efficacy of Different Selenium Compounds for reduction of mammary tumors in rats.

Compound Dose of Selenium for 50% Inhibition (ppm)
Se-methylselenocysteine 2
Selenobetaine 2
Selenobetaine methyl ester 2-3
Selenite 3
Selenomethionine 4-5
Selenocystine 4-5
PXSC* 8-10
Triphenylselenonium 10-12
Dimethylselenoxide >10
Trimethylselenonium (No effect at 80 ppm)

*1,4-phenylene bis (methylene) selenocyanate

Data taken from Ip and Ganther, 1993 and Ip et al, 1994a, 1994b.


selenocompounds can be converted directly to methylselenol, this is presumably the reason they are more efficacious than other forms of selenium. Dimethylselenoxide

and selenobetaine methyl ester are converted to dimethylselenide but are less effective for reduction of tumors (Ip, 1998). Trimethylselenonium is essentially not effective in tumor reduction. Thus, there is a negative correlation between the effectiveness of these selenocompounds and the degree of methylation.

Even though Semet is effective against mammary tumors, one disadvantage is that it can be incorporated directly into general proteins instead of converted to compounds which most effectively reduce tumors (Ip, 1998). When this occurs its efficacy for tumor reduction is reduced. For example, when a low methionine diet is fed there is significant reduction in the protective effect of Semet even though the tissue selenium was actually higher in animals as compared to those given an adequate amount of methionine (Ip, 1988). When methionine is limiting, a greater percentage of Semet is incorporated nonspecifically into body proteins in place of methionine because the methionine-tRNA cannot distinguish between methionine and Semet. Feeding diets with Semet to animals as the main selenium source will result in greater tissue accumulation of selenium than other forms of selenium (Ip and Lisk, 1994; Whanger and Butler, 1989). It is not known whether this stored selenium can serve as a reserved pool of this element but the evidence indicates that it is metabolically active (Waschulewski and Sunde, 1988).

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 strategy in this direction was the investigation of enriching garlic with selenium (Ip et al, 1992). The addition of selenium enriched garlic to yield three micrograms selenium per gram diet significantly reduced the mammary tumor incidence in rats from 83% to 33%. Similar to garlic, selenium enriched broccoli also reduced mammary tumors from 90% to 37% (Finley et al, 2001).

Selenium enriched garlic was shown to be twice as effective as selenium enriched yeast in the reduction of mammary tumors (table 2). 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 Semet was the predominant form of selenium in enriched yeast whereas SeMCYS (as the glutamyl derivative) was the predominant form of selenium in enriched garlic (Ip et al, 2000a). The glutamyl derivative is considered a carrier of SeMCYS and both of these compounds were shown to be equally effective in the reduction of mammary tumors (Dong et al, 2001). These results are consistent with those in table 1 where SeMCYS was more effective than Semet for reduction of mammary tumors. The chemical composition of selenocompounds in these two sources of selenium is apparently responsible for this difference in efficacy.

Using another model, selenium enriched broccoli florets (Finley et al, 2000; 2001; Finley and Davis, 2001) as well as enriched broccoli sprouts (Finley et al, 2001) significantly reduced colon tumors in rats. This is intriguing because colon cancer is the third most common newly diagnosed cancer in the United States, resulting in about 55,000 deaths per year due to this type of cancer (American Cancer Society, 2000).

Table 2. Mammary Cancer Prevention by Selenium enriched Garlic or Selenium enriched Yeast in the DMBA and MNU Models

Model Treatment Dietary Selenium (µg/g) Tumor Incidence Total number of Tumors Percentage inhibitiona
DMBA none Se-garlic Se-yeast 0.1 3.0 3.0 26/30 11/30b 19/30c 74 25b 49c 66 34
MNU none Se-garlic Se-yeast 0.1 3.0 3.0 28/30 10/30b 20/30c 80 24b 55c 70 31

a Calculated based on total tumor yield data.

bP < 0.05, compared to the corresponding Se-yeast group.

cP < 0.05, compared to the corresponding control group.

DMBA = dimethylbenz [a] anthracene; MNU = Methylnitrosourea

Taken from Ip et al, 2000a


Selenium enriched broccoli was more effective than selenite, selenate or Semet in the reduction of induced colon carcinogenesis (Feng et al, 1999 and Davis et al, 1999). In contrast, selenite, selenate and Semet were more effective for induction of GPX activity than selenium enriched broccoli (Finley and Davis, 2001). This indicates that the plant converts the selenium to more effective forms for reduction of these tumors and these results emphasize the need to study the effects of selenium in food forms.

Similar to chemically induced colon tumors there were significantly fewer intestinal tumors when mice which have a genetic defect for development of intestinal tumors were fed selenium enriched broccoli (Davis et al, 2002). These results along with data above indicate that selenium enriched broccoli is effective against both chemically and genetically induced intestinal tumors. Data from work with another strain of mice which develop spontaneous intestinal tumors is consistent with these results where selenium deficiency resulted in activation of genes involved in DNA damage (Rao et al, 2001).

IX. Level of selenium necessary for nutritive benefit

The Chinese data have been used almost exclusively to establish the required levels of selenium for nutritive benefit as well as to establish the safe levels for humans (Yang et al, 1989b; Yang and Zhou, 1994). It is fortunate to have a country like China where areas vary from deficient to toxic levels of selenium, and this has made it convenient to collect critical information on the metabolism and effects of various levels of selenium in humans. Significant correlations have been found between daily selenium intake and selenium content of whole blood, plasma, breast milk, and 24 hour urine (Yang et al, 1989a). Highly significant correlations were also found between levels of whole blood selenium and hair selenium, fingernail selenium and toenail selenium, hair selenium and fingernail or toenail selenium, and whole blood selenium and toenail or fingernail selenium. Morphological changes in fingernails were used as the main criterion for clinical diagnosis of selenosis (Yang et al, 1989b). The fingernail changes and loss of hair are the main signs of excess selenium intakes. With excess selenium intakes, the fingernails become brittle and are easily cracked. The data collected on Chinese subjects are summarized in table 3.

An intake of nearly 5 mg of selenium resulted in definite occurrence of selenosis, characterized by hair and nail losses. One suggested reason the subjects were able to tolerate this high level of selenium is because they consumed a high fiber diet. The low adverse effect level of dietary selenium was calculated to range between 1540 and 1600 micrograms daily. However, some effects were noted in individuals with a daily intake of 900 micrograms. The maximum safe dietary selenium intake was calculated to be about 800 micrograms per day, but there were some individuals where an amount of 600 micrograms per day was the maximum safe intake. In order to provide a safety factor, the maximum safe dietary selenium intake was suggested as 400 micrograms per day. A level of about 40 micrograms daily was suggested as the minimum requirement, and an intake of less than 11 micrograms daily will definitely result in deficiency problems. Deficiency of selenium in humans results in a cardiac and muscular disorder called Keshan disease, and deficiency of selenium is thought to be one of the contributing factors to another disorder called Kaschin-Beck disease.

Table 3. Health Effects of Various Levels of Dietary Selenium Intakes

Average AduIt Dietary

Selenium Intakes

(µg/d) (µg/KgBW) Forms Effects on Human Health


*4990 ± 1349 90 Cereal-based plant diet Occurrence of selenosis with hair & nail loss

in seleniferous area

*1660 30 Cereal-based plant diet Adverse effect level (AEL) of dietary Se intake

in seleniferous area

*1540 ± 653 28 Cereal-based plant diet Low adverse effect level of dietary Se intake

in seleniferous area (mean LOAEL)

*×900 17 Cereal-based plant diet Individual low level causes toxicity

in seleniferous area (individual LOAEL)

*819 ± 129 15 Cereal-based plant diet Maximum safe dietary Se intake

in seleniferous atea (NOAEL, mean)

*600 11 Cereal-based plant diet Individual maximum safe dietary Se intake

in seleniferous atea (NOAEL, individual)

400 – Natural Diet Suggested maximum safe dietary Se intake

40 0.7 75% of dietary Se from Suggested adequate dietary Se requirement


< 11 < 0.2 Cereal-based plant diet Prevalence of Keshan disease and

in Keshan disease area Kaschin-Beck disease

*Calculated by regression equation.

Data modified from: Yang and Zhou (1994). .


X. Conclusion.

The RDA for selenium is 55 micrograms for healthy adults, with 40 micrograms selenium as the minimum requirement. Less than 11 micrograms selenium will definitely put people at risk of deficiency that would be expected to cause damage. Daily doses of 100 to 200 micrograms

selenium inhibit genetic damage and cancer development in humans. About 400 micrograms

selenium per day is considered an upper safe limit. Clearly doses above the RDA are needed to

inhibit genetic damage and cancer. Despite concerns about the toxicity of higher dietary levels of selenium, humans consuming up to 600 micrograms of selenium daily appear to have no adverse clinical symptoms.[3][3]

Both animal and human data indicate that more than 100 and up to 200 micrograms of selenium are necessary for greatest reduction of cancer. This is because a methylated form of selenium is necessary for maximum reduction of cancer, and the methylated forms are present at highest levels with elevated intakes of this element. In most human trials, the subjects were supplemented with 200 micrograms selenium per day and in trials where only 50 micrograms were supplemented there was not as much reduction of cancer. Therefore, the selenium requirement for maximum reduction of cancer appears to be at least four times the RDA. However, since only 50 to 200 micrograms additional selenium have been used, it is not possible to indicate which level will give maximum protection. For example, it is not known whether supplemental levels of selenium above 200 micrograms daily will provide any additional protection against cancer.

Selenium enriched yeast is the most common source of selenium available commercially and it also has been the most used selenium source in human trials. Semet is the major form in enriched yeast but SeMCYS is the predominant form in enriched plants such as garlic and broccoli. Selenium enriched garlic was shown to be twice as effective as enriched yeast in reduction of mammary tumors in rats. Apparently, the reason SeMCYS is more effective is because it is converted directly to methylselenol, the suspected biologically active form of selenium for reduction of tumors. However, it is not known whether providing twice as much selenium as enriched yeast will give the same benefits as enriched garlic. Therefore, in addition to enriched yeast, selenium enriched food plants such garlic, broccoli and onions appear also to be an effective and safe method for delivery of selenium to the general population. Nevertheless, regardless of the source of selenium it is apparent that additional intakes of this element by humans will reduce the incidence of cancer.

It has been estimated that one-third of the cancers in humans are environmentally related. The results in this report indicate that on an average there could be 50% reduction of cancer through increased selenium ingestion in humans. If the 50,000 deaths due to colorectal cancer, the 41,800 deaths due to prostate cancer in men, or the 43,300 breast cancer deaths in women could be reduced by one-half with selenium, this would be a very significant contribution to human health.


Phil D. Whanger

Department of Environmental and Molecular Toxicology

Oregon State University

A copy of my curriculum vitae is attached


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[1][1] These results are consistent with some animal data. Hairless mice treated by topical application of selenomethionine (0.02%) or given drinking water with 1.5 micrograms selenium per ml as selenomethionine had significantly less skin damage due to ultraviolet irradiation (Burke et al, 1992b). This is consistent with an earlier study which indicated that dietary selenium (one microgram/g) fed to mice significantly reduced the number of skin tumors induced by two carcinogenic chemicals plus croton oil (Shamberger, 1970).

[2][2] The incidence of breast cancer is greatest of all cancers in women but it is the third highest cause of all cancer deaths (American Cancer Society, 2000), probably reflecting the improved methods for detecting and treatment of breast cancer compared to other cancers . Although usually not mentioned, a small number of men develop breast cancer with even some deaths. About 400 men die of breast cancer each year compared to 43,300 breast cancer deaths in women.

[3][3] The author is aware of a person who consumed one mg of selenium for two years before toxic signs of selenium occurred. Thus this element appears not as toxic as often believed.

Selenium also helps stop damaged DNA molecules from reproducing. In other words, selenium acts to prevent tumors from developing. “It contributes towards the death of cancerous and pre-cancer cells. Their death appears to occur before they replicate, thus helping stop cancer before it gets started,” says Dr. James Howenstine in A Physician’s Guide to Natural Health Products That Work.

In addition to preventing the onset of the disease, selenium has also been shown to aid in slowing cancer’s progression in patients that already have it. According to the Life Extension Foundation, the use of selenium during chemotherapy in combination with vitamin A and vitamin E can reduce the toxicity of chemotherapy drugs. The mineral also helps “enhance the effectiveness of chemo, radiation, and hyperthermia while minimizing damage to the patient’s normal cells; thus making therapy more of a ‘selective toxin,'” says Patrick Quillin in Beating Cancer with Nutrition.

A 1996 study by Dr. Larry Clark of the University of Arizona showed just how effective selenium can be in protecting against cancer. In the study of 1,300 older people, the occurrence of cancer among those who took 200 micrograms of selenium daily for about seven years was reduced by 42 percent compared to those given a placebo. Cancer deaths for those taking the selenium were cut almost in half, according to the study that was published in the Journal of the American Medical Association.

While the study concluded the mineral helped protect against all types of cancer, it had particularly powerful impacts on prostate, colorectal and lung cancers. Jean Carper, in Miracle Cures, called Dr. Clark’s findings an “unprecedented cancer intervention study” that “bumped up the respectability of using supplements against cancer several notches.”

Accordingly, geography can have a significant impact on diet. In Antioxidants Against Cancer, author Ralph Moss PhD, says one theory for why cancer rates are so high in Linxian, China, dubbed “the ‘world capital’ of cancer,” is that the soil is deficient in the essential minerals selenium and zinc. In Earl Mindell’s Supplement Bible, Earl Mindell RPh PhD, suggests part of the reason American men are five times more likely than Japanese men to die from prostate cancer could be because, in general, “the Asian diet contains four times the amount of selenium as the average American diet.”



Introduction:The Promise of the ArtI remember how it was before penicillin. I was a medical student at theend of World War II, before the drug became widely available for civil-ian use, and I watched the wards at New York’s Bellevue Hospital fill tooverflowing each winter. A veritable Byzantine city unto itself, Bellevuesprawled over four city blocks, its smelly, antiquated buildings jammedtogether at odd angles and interconnected by a rabbit warren of under-ground tunnels. In wartime New York, swollen with workers, sailors,soldiers, drunks, refugees, and their diseases from all over the world, itwas perhaps the place to get an all-inclusive medical education. Belle-vue’s charter decreed that, no matter how full it was, every patient whoneeded hospitalization had to be admitted. As a result, beds were packedtogether side by side, first in the aisles, then out into the corridor. Award was closed only when it was physically impossible to get anotherbed out of the elevator.Most of these patients had lobar (pneumococcal) pneumonia. It didn’ttake long to develop; the bacteria multiplied unchecked, spilling overfrom the lungs into the bloodstream, and within three to five days of thefirst symptom the crisis came. The fever rose to 104 or 105 degreesFahrenheit and delirium set in. At that point we had two signs to go by:If the skin remained hot and dry, the victim would die; sweating meantthe patient would pull through. Although sulfa drugs often were effec-tive against the milder pneumonias, the outcome in severe lobar pneu-monia still depended solely on the struggle between the infection and the patient’s own resistance. Confident in my new medical knowledge, Iwas horrified to find that we were powerless to change the course of thisinfection in any way.It’s hard for anyone who hasn’t lived through the transition to realizethe change that penicillin wrought. A disease with a mortality rate near50 percent, that killed almost a hundred thousand Americans each year,that struck rich as well as poor and young as well as old, and againstwhich we’d had no defense, could suddenly be cured without fail in afew hours by a pinch of white powder. Most doctors who have graduatedsince 1950 have never even seen pneumococcal pneumonia in crisis.Although penicillin’s impact on medical practice was profound, itsimpact on the philosophy of medicine was even greater. When Alex-ander Fleming noticed in 1928 that an accidental infestation of the moldPenicillium notatum had killed his bacterial cultures, he made the crown-ing discovery of scientific medicine. Bacteriology and sanitation had al-ready vanquished the great plagues. Now penicillin and subsequentantibiotics defeated the last of the invisibly tiny predators.The drugs also completed a change in medicine that had been gather-ing strength since the nineteenth century. Before that time, medicinehad been an art. The masterpiece—a cure—resulted from the patient’swill combined with the physician’s intuition and skill in using remediesculled from millennia of observant trial and error. In the last two cen-turies medicine more and more has come to be a science, or more accu-rately the application of one science, namely biochemistry. Medicaltechniques have come to be tested as much against current concepts inbiochemistry as against their empirical results. Techniques that don’t fitsuch chemical concepts—even if they seem to work—have been aban-doned as pseudoscientific or downright fraudulent.At the same time and as part of the same process, life itself came to bedefined as a purely chemical phenomenon. Attempts to find a soul, avital spark, a subtle something that set living matter apart from thenonliving, had failed. As our knowledge of the kaleidoscopic activitywithin cells grew, life came to be seen as an array of chemical reactions,fantastically complex but no different in kind from the simpler reactionsperformed in every high school lab. It seemed logical to assume that theills of our chemical flesh could be cured best by the right chemicalantidote, just as penicillin wiped out bacterial invaders without harminghuman cells. A few years later the decipherment of the DNA codeseemed to give such stout evidence of life’s chemical basis that the dou-ble helix became one of the most hypnotic symbols of our age. It seemedthe final proof that we’d evolved through 4 billlion years of chance mo-lecular encounters, aided by no guiding principle but the changelessproperties of the atoms themselves.The philosophical result of chemical medicine’s success has been belief in the Technological Fix. Drugs became the best or only valid treat-ments for all ailments. Prevention, nutrition, exercise, lifestyle, the pa-tient’s physical and mental uniqueness, environmental pollutants—allwere glossed over. Even today, after so many years and millions of dol-lars spent for negligible results, it’s still assumed that the cure for cancerwill be a chemical that kills malignant cells without harming healthyones. As surgeons became more adept at repairing bodily structures orreplacing them with artificial parts, the technological faith came to in-clude the idea that a transplanted kidney, a plastic heart valve, or astainless-steel-and-Teflon hip joint was just as good as the original—oreven better, because it wouldn’t wear out as fast. The idea of a bionichuman was the natural outgrowth of the rapture over penicillin. If ahuman is merely a chemical machine, then the ultimate human is arobot.No one who’s seen the decline of pneumonia and a thousand otherinfectious diseases, or has seen the eyes of a dying patient who’s justbeen given another decade by a new heart valve, will deny the benefits of technology. But, as most advances do, this one has cost us somethingirreplaceable: medicine’s humanity. There’s no room in technologicalmedicine for any presumed sanctity or uniqueness of life. There’s noneed for the patient’s own self-healing force nor any strategy for enhanc-ing it. Treating a life as a chemical automaton means that it makes nodifference whether the doctor cares about—or even knows—the patient,or whether the patient likes or trusts the doctor.Because of what medicine left behind, we now find ourselves in a realtechnological fix. The promise to humanity of a future of golden healthand extended life has turned out to be empty. Degenerative diseases—heart attacks, arteriosclerosis, cancer, stroke, arthritis, hypertension, ul-cers, and all the rest—have replaced infectious diseases as the majorenemies of life and destroyers of its quality. Modern medicine’s incredi-ble cost has put it farther than ever out of reach of the poor and nowthreatens to sink the Western economies themselves. Our cures too oftenhave turned out to be double-edged swords, later producing a secondarydisease; then we search desperately for another cure. And the de-humanized treatment of symptoms rather than patients has alienatedmany of those who can afford to pay. The result has been a sort of medical schizophrenia in which many have forsaken establishment medi-cine in favor of a holistic, prescient type that too often neglects technology’s real advantages but at least stresses the doctor-patient rela-tionship, preventive care, and nature’s innate recuperative power.The failure of technological medicine is due, paradoxically, to its suc-cess, which at first seemed so overwhelming that it swept away all as-pects of medicine as an art. No longer a compassionate healer working atthe bedside and using heart and hands as well as mind, the physician hasbecome an impersonal white-gowned ministrant who works in an officeor laboratory. Too many physicians no longer learn from their patients,only from their professors. The breakthroughs against infections con-vinced the profession of its own infallibility and quickly ossified its be-liefs into dogma. Life processes that were inexplicable according tocurrent biochemistry have been either ignored or misinterpreted. Ineffect, scientific medicine abandoned the central rule of science—revi-sion in light of new data. As a result, the constant widening of horizonsthat has kept physics so vital hasn’t occurred in medicine. The mecha-nistic assumptions behind today’s medicine are left over from the turn of the century, when science was forcing dogmatic religion to see the evi-dence of evolution. (The reeruption of this same conflict today showsthat the battle against frozen thinking is never finally won.) Advances incybernetics, ecological and nutritional chemistry, and solid-state physicshaven’t been integrated into biology. Some fields, such as parapsychol-ogy, have been closed out of mainstream scientific inquiry altogether.Even the genetic technology that now commands such breathless admi-ration is based on principles unchallenged for decades and unconnectedto a broader concept of life. Medical research, which has limited itself almost exclusively to drug therapy, might as well have been wearingblinders for the last thirty years.It’s no wonder, then, that medical biology is afflicted with a kind of tunnel vision. We know a great deal about certain processes, such as thegenetic code, the function of the nervous system in vision, muscle move-ment, blood clotting, and respiration on both the somatic and the cel-lular levels. These complex but superficial processes, however, are onlythe tools life uses for its survival. Most biochemists and doctors aren’tmuch closer to the “truth” about life than we were three decades ago. AsAlbert Szent-Gyorgyi, the discoverer of vitamin C, has written, “Weknow life only by its symptoms.” We understand virtually nothingabout such basic life functions as pain, sleep, and the control of celldifferentiation, growth, and healing. We know little about the wayevery organism regulates its metabolic activity in cycles attuned to thefluctuations of earth, moon, and sun. We are ignorant about nearlyevery aspect of consciousness, which may be broadly defined as the self-interested integrity that lets each living thing marshal its responses toeat, thrive, reproduce, and avoid danger by patterns that range from thetropisms of single cells to instinct, choice, memory, learning, individ-uality, and creativity in more complex life-forms. The problem of whento “pull the plug” shows that we don’t even know for sure how todiagnose death. Mechanistic chemistry isn’t adequate to understandthese enigmas of life, and it now acts as a barrier to studying them.Erwin Chargaff, the biochemist who discovered base pairing in DNAand thus opened the way for understanding gene structure, phrased ourdilemma precisely when he wrote of biology, “No other science deals inits very name with a subject that it cannot define.”Given the present climate, I’ve been a lucky man. I haven’t been agood, efficient doctor in the modern sense. I’ve spent far too much timeon a few incurable patients whom no one else wanted, trying to find outhow our ignorance failed them. I’ve been able to tack against the pre-vailing winds of orthodoxy and indulge my passion for experiment. In sodoing I’ve been part of a little-known research effort that has made anew start toward a definition of life.My research began with experiments on regeneration, the ability of some animals, notably the salamander, to grow perfect replacements forparts of the body that have been destroyed. These studies, described inPart 1, led to the discovery of a hitherto unknown aspect of animallife—the existence of electrical currents in parts of the nervous system.This breakthrough in turn led to a better understanding of bone fracturehealing, new possibilities for cancer research, and the hope of humanregeneration—even of the heart and spinal cord—in the not too distantfuture, advances that are discussed in Parts 2 and 3. Finally, a knowl-edge of life’s electrical dimension has yielded fundamental insights (con-sidered in Part 4) into pain, healing, growth, consciousness, the natureof life itself, and the dangers of our electromagnetic technology.I believe these discoveries presage a revolution in biology and medi-cine. One day they may enable the physician to control and stimulatehealing at will. I believe this new knowledge will also turn medicine inthe direction of greater humility, for we should see that whatever weachieve pales before the self-healing power latent in all organisms. Theresults set forth in the following pages have convinced me that our un-derstanding of life will always be imperfect. I hope this realization willmake medicine no less a science, yet more of an art again. Only then canit deliver its promised freedom from disease.


DR. SEBI was born Alfredo Bowman on November 26, 1933, in the village of Ilanga in Spanish Honduras. He was a self taught pathologist, herbalist, biochemist, and naturalist. He  studied and personally observed herbs in America, Latin America, Africa, and the Caribbean, and developed a unique approach to healing with herbs firmly rooted in over 30 years of practical experience called the African Bio Mineral Balance.

Sebi came to the United States as a self-educated man who was diagnosed with asthma, diabetes, impotency, and obesity. After unsuccessful treatments with conventional doctors, Sebi was lead to an herbalist in Mexico. Finding great healing success from all his ailments, he began creating natural vegetation cell food compounds geared for inter-cellular cleansing and the revitalization of all the cells that make up the human body.

Inspired by the personal healing experience and knowledge he gained, he began sharing the compounds with others, which gave birth to the USHA Research Institute, Dr. Sebi LLC, and the Usha Healing Village located in La Ceiba, Honduras.

Electric foods include live and raw foods. Dr. Sebi divides foods into the following six categories:

  • Live
  • Raw
  • Dead
  • Hybrid
  • Genetically modified
  • Drugs

This plan requires consumers to avoid all seedless fruits, weather resistant crops such as corn, and anything with added vitamins or minerals.

Foods that are recommended include ripe fruit, no starchy vegetables, raw nuts and butter and grains. Leafy greens, quinoa, rye, or Kamut will play a large role in this diet.

Acidic foods such as meat, poultry, seafood, or products containing yeast, alcohol, sugar, iodized salt, or anything that is fried bring negative effects to the human body.

Replacing acidic foods with electric foods will help to heal your body from the negative effects that acidic foods bring.

DR. SEBI’S METHODOLOGY | According to Western medical research, diseases are a result of the host being infected with a “germ”, “virus”, or “bacteria”. In their approach in treating these “infestations”, inorganic, carcinogenic chemicals are employed. Our research immediately uncovers flaws in their premise through basic deductive reasoning. By consistently utilizing the same premise and methods, they have consistently yielded ineffective results. In essence, in the 400-year tradition of the European philosophy of medicine, their approach in treating disease has yet to produce any cures.
In contrast, as we examine the African approach to disease, it diametrically opposes the present Western approach. Specifically, the African Bio-mineral Balance refutes the germ/virus/bacteria premise. Our research reveals that all manifestation of disease finds it genesis when and where the mucous membrane has been compromised. For example, if there is excess mucous in the bronchial tubes, the disease is Bronchitis; if it is in the lungs, the disease is Pneumonia; in the pancreatic duct, it is Diabetes; in the joints Arthritis. All of the African Bio-mineral Balance compounds are comprised of natural plants; which means its constitution is of an alkaline nature.
This is important and instrumental in our success in reversing pathologies- because disease can only exist in an environment that is acid; thus it is inconsistent to utilize inorganic substances when treating disease because they are of an acid base. Only consistent use of natural botanical remedies will effectively cleanse and detoxify a diseased body, reversing it to its intended alkaline state.

– Dr. Sebi | Holistic Healer

Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

Selenium is an essential trace element for mammals due to vital roles played by one or several selenoproteins, which is illustrated by the lethal phenotype of mice lacking the tRNA needed for selenocysteine (Sec; U in one-letter code) incorporation (1). Sec functions as an extraordinarily reactive cysteine homologue; the increased reactivity of oxidoreductases having Sec in place of cysteine is usually regarded as the raison d’etre for selenoproteins, despite their costly and inefficient synthesis machinery (2).

Mammalian thioredoxin (Trx) reductase (TrxR) enzymes are important selenoproteins that, together with Trx and additional Trx-dependent enzymes, carry out several antioxidant and redox regulatory roles in cells. These roles include synthesis of deoxyribonucleotides with ribonucleotide reductase, reduction of peroxides or oxidized methionine residues with peroxiredoxins or methionine sulfoxide reductases, respectively, regulation of several transcription factor or protein kinase activities, as well as regeneration of many low molecular weight antioxidant compounds (see ref. 3 and references therein). The catalytic activities of mammalian TrxR isoenzymes depend on a redox-active Cys-Sec couple within the Cterminal tetrapeptide motif, -Gly-Cys-Sec-Gly-COOH (46). Compared with sulfur, selenium is not only generally more reactive but also exhibits an ≈15% longer bond length, which facilitates formation of a selenenyl-sulfide bridge between the adjacent Cys-Sec residues, which is a necessary intermediate in the catalytic cycle (711). Mutational studies of the mammalian enzyme in which Sec was replaced by Cys showed a marked decrease in the catalytic rate (kcat) for Trx reduction (1012). This result was expected, because disulfide bridges between two sequentially adjacent cysteines are generally not favored (13). Indeed, the disulfide bond between the two Cys residues of the eight-member cyclo-Cys-Cys ring is not compatible with a standard peptide-bond geometry (14).

In Drosophila melanogaster, TrxR is particularly important because its product, Trx(SH)2, in place of glutathione reductase, serves as the principal reductant of glutathione (1516). TrxR of the fruit fly is closely related to mammalian TrxR but carries a redox-active Cterminal -Ser-Cys-Cys-Ser-COOH motif that presumably involves a strained disulfide as a catalytic intermediate within the C-terminal Cys-Cys-sequence. Thus, it is surprising that the kcat for oxidized Trx of the Drosophila enzyme is ≈50% that of the human enzyme for the same substrate (15), which is in strong contrast to the Sec498 → Cys mutants of the mammalian enzyme, which have very low catalytic activity (1012).

In this study, we have demonstrated that very slight changes in the active site of the insect TrxR are sufficient to yield a high catalytic activity without involvement of selenium. The results lead to general questions regarding the role and necessity of Sec in mammalian TrxRs and, possibly, in other selenoproteins as well.

Materials and Methods

Cloning, Expression, and Purification of D. melanogaster TrxR-2 (DmTrxR-2) C-Terminal Mutants. Mutants were designed to yield enzymes with the following C-terminal tetrapeptide sequences: GCCG, SCCG, GCCS, GCCD, DCCG, DCCD, SCSS, GCUG, SCUG, GCUS, and SCUS. These C-terminal mutants of (N-terminally His-tagged) wild-type DmTrxR-1 (National Center for Biotechnology Information accession no. AF301144) were cloned, expressed, and purified by using standard techniques and the recently developed methodology for heterologous expression of mammalian selenoproteins in Escherichia coli (17). Technical details are given in Supporting Methods, which is published as supporting information on the PNAS web site, For an efficient selenoprotein synthesis, the pSUABC-plasmid was used as described in ref. 17, and metabolic labeling with 75Se showed that only Sec-containing mutants incorporated selenium (see Table 1 and Fig. 5, which are published as supporting information on the PNAS web site). Enzyme purity was confirmed by silver-stained SDS/PAGE.

Determination of Enzyme Concentration. Subunit concentrations of the Cys-Cys mutants were determined by measuring absorbance at 462 nm by using an assumed ε462 nm of 11.3 mM–1·cm–1 for the flavoprotein subunits (7). By using the Bio-Rad protein assay procedure with BSA as standard, it was confirmed that all variants of DmTrxR contained stoichiometric amounts of FAD (1 ± 0.2) per 53.9-kDa subunit. Concentrations of Cys-Sec mutants were determined from the selenium concentration as measured by atomic absorption spectroscopy (see Table 1). The concentration of oxidized DmTrx-2, purified as described earlier (18), was measured enzymatically by end-point determination by using excess NADPH and 1 unit/ml of wild-type DmTrxR.

Enzyme Kinetics. All standard assays were carried out at 25°C. Recombinant N-terminally His-tagged DmTrx-2 was prepared as described (18). NADPH was from Biomol (Hamburg, Germany). Buffer T, which was used in most assays consisted of 100 mM potassium phosphate, 2 mM EDTA, 100 μM NADPH, pH 7.4. In all assays, the oxidation of NADPH was monitored as the decrease in absorbance at 340 nm (εNADPH, 340 nm = 6.22 mM–1·cm–1). One unit is defined as the consumption of 1 μmol of NADPH per min. Unless otherwise stated ≈10–20 milliunits/ml of enzyme were used in each assay.

Trx assay. The enzyme samples were added to a cuvette containing buffer T. The reaction was started by adding DmTrx-2 (50 μM final concentration).

GHOST (glutathione as substrate of Trx) assay. Enzyme samples were added to a cuvette containing 1 mM glutathione disulfide in buffer T. The reaction was started by adding oxidized DmTrx-2 (30 μM final concentration).

To minimize the effects of ionic strength due to pH differences, a modified buffer was used for the pH profiles: 400 mM NaCl, 50 mM sodium phosphate, 1 mM EDTA and 100 μM NADPH. The pH was adjusted from 9.0 to 6.0 in ΔpH steps of 0.2 or 0.5 by using HCl.

Methylseleninate reduction assay. Methylseleninic acid (CH3SeO2H) was obtained from PharmaSe (Lubbock, TX). One nanomole (127 mg) was weighed out and dissolved in 500 μl 2 M KOH. Buffer T (500 μl) was added to yield a stock solution with a final concentration of 1 M CH3SeO2K. Enzyme samples were added to a cuvette containing buffer T. The reaction was started by adding methylseleninate (100 μM final concentration).

Hg2+reduction assay. EDTA-free enzyme samples (50–100 milliunits/ml as determined in the GHOST assay) were assayed as described (19).

Titration and Stopped-Flow Experiments. All titrations and stopped-flow experiments were conducted in 100 mM potassium phosphate buffer, pH 7.0, anaerobically, as described (7). The methods of data analysis are outlined in ref. 20.

Reduction of enzymes with NaBH4. DmTrxR mutants were reduced by using a sodium borohydride solution (100 mM in 0.02 M NaOH) in ≈100-fold excess to the enzyme solution under anaerobic conditions. Excess NaBH4 is completely hydrolyzed at neutral pH within a few minutes. The reduced enzyme could be used directly for titrations and stopped-flow experiments.

Stopped-flow-state kinetics. Rapid reaction kinetics were all conducted in a Hi-Tech SF-61DX2 stopped-flow photometer (Hi-Tech, Salisbury, Wiltshire, U.K.) under anaerobic conditions at 10°C. The DmTrxR concentration used was 15–20 μM after mixing. Data from kinetic traces were analyzed by fitting to multiple exponential functions with the program A, which was written by R. Chang, C.Y. Chiu, J. Dinverno, and D. P. B. (University of Michigan).

The reaction of NADPH with oxidized enzyme was carried out in the stopped-flow spectrophotometer. Time-dependent spectra were followed by diode array detection. Single-wavelength kinetic traces were recorded by using a photomultiplier tube. For the oxidative half-reaction, NaBH4-reduced enzyme was prepared and subsequently reacted with oxidized DmTrx-2.

Auranofin Inhibition. Enzyme samples were assayed for their Trx-reduction activity in the absence or presence of 1 μM auranofin (ICN Biochemicals, Aurora, OH) prepared from a 1-mM stock solution in dimethyl sulfoxide.


The two C-terminal redox active cysteines in DmTrxR are flanked by polar serines, rather than by the Gly residues in the corresponding active site of mammalian TrxR. We reasoned that this slight difference in the microenvironment of the C-terminal cysteines might compensate for the tension in the presumably unfavorable disulfide bond of adjacent cysteines (14). Therefore, we expressed mutants of DmTrxR in which the flanking Ser residues were replaced by Gly residues in all possible permutations. We also replaced these residues by Asp residues to introduce groups with negative charges capable of forming hydrogen bonds. Finally, to probe the possible advantage of a Sec residue in the redox-active center, we expressed Cys-to-Sec mutant forms of DmTrxR, by using a rather intriguing methodology, because Sec is incorporated by specific UGA codons in a species-specific manner (217). We achieved good yield of pure enzyme with all mutants (see Table 1 and Fig. 5), and this approach circumvents the potential pitfalls that may arise when comparing enzymes from different species such as humans and Drosophila.

Determination of Kinetic Constants with Different Substrates. All of the variants of DmTrxR were catalytically active (see Fig. 6, which is published as supporting information on the PNAS web site). To the extent that Km is related to Kd, the affinity for Trx appeared to be similar for all mutants, because all Km values were within the range of 2–6 μM (Fig. 1). This finding shows that the changes in the Cterminal tetrapeptide do not significantly affect substrate binding and that all mutants are likely to have an intact quaternary structure capable of binding Trx with similar affinity as the wild-type enzyme. The kcat was, however, highly affected by the sequence of the Cterminal tetrapeptide. By analyzing the GCCG, SCCG, and GCCS mutants, it became apparent that each of the two serine residues present in the wild-type sequence improved the catalytic activity. The second serine (Ser491), however, was far more important in this respect than the first (Ser488). In marked contrast, the kcat values for Trxs of Sec-containing mutants were consistently in the same range as the kcat for the wild-type SCCS enzyme, and were thus largely unaffected by the nature of the flanking residues (Fig. 1). The aspartic acid-containing mutants (DCCG, GCCD, and DCCD) all had a significantly lower kcat values than the wild-type enzyme, some even below the already poorly active GCCG mutant (Fig. 1).

Fig. 1.

Comparison of kinetic parameters of wild-type and mutant DmTrxR. The kcat (Upper) and Km (Lower) values shown here were determined by using DmTrx-2 as a substrate (GHOST assay). The Cterminal tetrapeptide sequences of the respective enzyme are indicated on the x axis. The results for the wild-type enzyme represented by SCCS are highlighted in black. The kcat values shown for the selenoenzymes were normalized for the presence of truncated, inactive enzyme. Note that the wild-type enzyme’s (SCCS) activity does not differ significantly from the kcat values of the Sec mutants.

By using methylseleninate as substrate, the absolute differences between the Cys-Cys and the Cys-Sec variants were more pronounced, because the latter showed a 2- to 5-fold higher turnover per subunit than did the Cys-Cys-homologues. For example, the Sec mutant, SCUS, showed a 5.2-fold higher turnover rate for methylseleninate than did the wild-type enzyme (5.2 s–1 vs. 1 s–1).

Because the Cterminal sequence of mercuric ion reductase is -SCCAG, and thereby exhibits some resemblance with the C terminus of DmTrxR-SCCG, we also analyzed Hg2+-reductase activity of all mutants. Under the conditions used, none of the enzymes however showed any mercuric ion reductase activity above background (data not shown).

pH Profile. The Cys-Sec mutants were found to show little variation in activity within a broad physiological pH range (pH 6.0–9.0), whereas the selenium-free mutants lost significant activity at pH values <7.0. (see Fig. 7, which is published as supporting information on the PNAS web site) The pH profile obtained with the different mutants should in part reflect the pKa values of the C-terminal redox-active residues.

Reductive Half-Reaction. The Cterminal redox-active motif in one subunit of the dimeric high Mr TrxR enzymes receives electrons from two N-terminal redox-active cysteines proximal to an FAD in the other subunit, subsequently transferring these electrons to Trx or other substrates of the enzyme (781121). By using stopped-flow kinetic methods, the rates of these separate events can be determined.

Stopped-flow analysis of both wild-type enzyme (SCCS) and the mutants GCCG, SCCG, and GCCS showed nearly identical first phases during reduction of the flavin with four equivalents of NADPH per subunit (Fig. 2 Inset), with an apparent rate constant, k1 = 150 ± 9 s–1, describing the first step for all four enzymes. The second phase, in which the N-terminal disulfide is reduced by the flavin, was characterized by a rate constant, k2 = 47 ± 14 s–1. The wild-type enzyme exhibited a distinct third phase, with an apparent rate constant, k3, of ≈21 s–1. This phase is most likely attributable to interchange between the N-terminal thiols and the C-terminal disulfide of the enzyme. As the interchange reaction progresses, the charge–transfer interaction between Cys-57 and the FAD decreases, and the absorbance at 462 nm increases slightly. The relative forward and reverse rates of the interchange reaction determine the amount of absorbance increase observed in this step. Thus, the GCCS and SCCG mutant forms showed third phases, but they were smaller than that of the wild-type and were difficult to quantify. Consistent with the interpretation that the third phase is due to interchange between C- and N-terminal cysteine pairs, mutants such as DmTrxR-SSCS and DmTrxR-SCSS, which lack a catalytically functional C-terminal redox-active site, clearly show no third phase, as expected when disulfide–dithiol interchange with the C-terminal group is not possible (22). The kinetic traces at 540 nm, reflecting the state of the charge–transfer complex, (data not shown) were similar in all mutants with rate constants, k1 = 135 s–1 and k2 = 42 s–1.

Fig. 2.

Half-reactions of wild-type enzyme and mutant forms. Each protein was reduced with sodium borohydride. Reduced protein was mixed in the stopped-flow spectrophotometer with various concentrations of oxidized DmTrx. Solid lines represent the reduced enzyme species with no Trx added. Dotted spectra depict the enzyme species after addition of two equivalents of oxidized Trx. In the SCCG case, the dashed line represents three equivalents of Trx, and the dash-dotted line represents five equivalents. For clarity, the spectra of GCCG are shifted by 0.05 units. The absorbance of the GCCS mutant was essentially identical to that of GCCG, whereas the SCCS mutant had essentially the same absorbance as SCCG (data not shown). (Inset) The reductive half-reaction of wild-type enzyme (SCCS) compared with three mutant forms (GCCG, SCCG, and GCCS). The protein was mixed in a stopped-flow spectrophotometer with four equivalents of NADPH per subunit, and the change in absorbance at 462 nm, representing the redox state of the flavin, was recorded over time. All enzymes were used at similar concentrations (≈18 μM subunits). The absorption starting point (0.2) was shifted for the GCCS, SCCG, and GCCG mutants for clarity. Note that the time axis is in logarithmic scale. See text for details.

Oxidative Half-Reaction. Reduction with borohydride yields enzyme having both disulfides reduced to dithiols (EH4). Thus, it is equivalent to enzyme reduced with two equivalents of NADPH, but lacking the NADP+. The spectrum is perturbed due to the charge transfer interaction of the thiolate of Cys-62 with the flavin. Furthermore, a small fraction of the borohydride-reduced enzyme may be present in the fully reduced EH6 form. However, EH6 is unlikely to be formed in vivo due to the low redox potential of the enzyme-bound FAD (22).

Reoxidation on reaction with Trx is indicated by disappearance of the charge-transfer band at 540 nm. The borohydride-reduced enzyme species could not be fully reoxidized, even by the addition of up to five equivalents of oxidized Trx per subunit (Fig. 2). Oxidation is indicated by the loss of charge-transfer absorbance of ≈550 nm and an increase in absorbance of ≈460 nm. Indeed, the mutants with a Gly at position 488 showed only a marginal shift from the fully reduced spectrum toward the spectrum of oxidized enzyme, whereas the forms with Ser at this position led to more pronounced, yet incomplete, reoxidation (Fig. 2). The only spectrum shown for GCCG is that for two equivalents of Trx, but the spectra for three and five equivalents, while of poor quality, were similar. The wild-type enzyme, SCCS, resulted in spectra essentially as shown for SCCG (22). GCCS behaved almost identically to GCCG.

Auranofin Inhibition. Auranofin (1 μM) efficiently inhibited all Cys-Sec mutants, which was similar to the inhibition of mammalian TrxR by this compound (9). The selenium-free mutants were inhibited much less by auranofin (Fig. 3). A considerable difference was observed between the individual mutants, with less inhibition with increasing polarity of the flanking residues in the Cterminal motif.

Fig. 3.

Inhibition by auranofin. Auranofin, an effective inhibitor of human TrxRs, was analyzed for its inhibitory effect on wild-type DmTrxR (highlighted in black) and the DmTrxR mutants. Shown is the percentage of inhibition compared with an auranofin-free control. In all cases, 1 μM auranofin was used and the assays were carried out as in Fig. 1.


This study shows that the polar serine residues present within the Cterminal tetrapeptide sequence of DmTrxR play a key role in making this selenium-free protein almost as active as its mammalian selenoprotein counterpart. It also shows that with the Sec-containing variant of the C-terminal redox-active motif, the impact of the two flanking residues is diminished. How can these pronounced effects of such subtle changes at the active site be explained? The active species in dithiol-disulfide-exchange reactions is the thiolate (or selenolate for Sec). The pKa value of a protein cysteine thiol is typically ≈8.5, unless affected by neighboring residues (23), whereas the pKa of a selenol group is normally ≈5.3 (24). Because of this occurrence, Sec is normally ionized at physiological pH, and in contrast to cysteine, does not require assistance from the protein environment. The pKa of the interchange thiol of the closely related enzyme glutathione reductase is low, due to interactions with a nearby His residue (25). The analogous residue in DmTrxR is His464′, which seems to be positioned exactly as it is in glutathione reductase (Fig. 4A). Furthermore His106, which is also conserved among large TrxRs, is almost certainly involved (Fig. 4A); its modeled position is relative to the Cterminal thiols, just as His464′ is relative to the N-terminal thiols. The pH profiles of the mutants analyzed herein are consistent with the requirement for ionizing the cysteine(s), because increased pH leads to an increase in turnover rate of the Cys-Cys-containing enzyme species up to a plateau above pH 8.0. By contrast, the activity of the Cys-Sec enzymes have very little dependence on pH in the physiological range. The flanking Ser residues in DmTrxR are crucial for dithiol–disulfide-interchange reactions, possibly by kinetically facilitating transient thiolate formation at the C-terminal redox-active cysteines.

Fig. 4.

(A) Computer model of DmTrxR. The protein backbones of the two subunits in the dimeric enzyme are shown as thin green strands (subunit A) and blue ribbons (subunit B). The flexible C-terminal tail of subunit B is shown as a thick blue strand. The FAD (orange) and nicotinamide (gray) of NADP+, bound to subunit A, are represented as stick models. The side chains of the N-terminal and C-terminal redox-active site (Cys57 and Cys62 and Cys489′ and Cys490′, respectively), as well as the putative base catalysts, His106 and His464′, are also shown as stick models, with the sulfurs in yellow and the nitrogens of His in blue. The van der Waals radii of these amino acids are dotted. This model is based on the crystal structure of rat TrxR U498C (21) (Protein Data Bank ID code 1H6V) by using RASWIN V2.7.2 for visualization ( (B) A proposed mechanism for DmTrxR. The dotted arrows between the thiolate of Cys-62 and the flavin indicate charge–transfer interactions. The change in conformation is indicated by the juxtaposition of the two polypeptide chains. See text for other details. (C) Corey–Pauling–Koltun space-filling model of the C terminus of wild-type-DmTrxR in the dithiol state. (D) Corey-Pauling-Koltun space-filling model of the C terminus in the disulfide state. All peptide bonds shown in C and D are trans-configured.

In principle, either cysteine Cys489 or Cys490 could be the thiolate that attacks and reduces the substrate. It has been shown by site-directed mutagenesis and chemical modification studies that Cys490, the penultimate residue of the enzyme, is the thiolate that reacts with both the N-terminal interchange thiol, Cys57, and with Trx (22). The significant differences in kcat values between the SCCG mutant, with ≈30% of wild-type DmTrxR activity and the GCCS-mutant with ≈90%, makes it highly unlikely that the two cysteines in the selenium-free mutants are functionally interchangeable. Consistent with these findings, all mutant forms containing Cys-Sec have essentially the full activity of the wild-type Cys-Cys-enzyme (Fig. 1).

It was surprising to find in the stopped-flow experiments (Fig. 2), that in the enzymes with a serine at position 488, SCCG, and SCCS (wild-type), the flavin was more oxidized than in GCCS, the mutant showing the higher catalytic activity (Fig. 1). The equilibria in Fig. 4Bdescribe the dithiol–disulfide-interchange reactions in the oxidative half-reaction of catalysis. All species, except for species 8, have thiolate-flavin charge-transfer absorbance. Therefore, the equilibria for GCCS result in less species 8 being formed than in SCCG. It is species 8 that is reduced by NADPH. However, this unfavorable equilibrium does not appreciably affect kcat, because the reductive half reaction is not rate limiting in the overall catalysis.

As stated above, both the reduction of Trx by Cys490′ to form a mixed disulfide and the cleavage of the mixed disulfide by Cys489′ require that the thiols be deprotonated; the data indicate that this reaction is promoted by the adjacent Ser residues. The actual acid base catalyst is most likely His464′ in the case of the N-terminal thiols, and His106 for the C-terminal thiols (see above and Fig. 4A). The serine probably assists the deprotonation of either Cys489′ or Cys490′ in various steps of catalysis, as shown in Fig. 4B.

The catalytic mechanism that we propose involves two uncommon configurations as intermediates; that of hydrogen bonding between the Cys residue thiol groups and hydroxyl groups of flanking Ser residues, and a disulfide between two contiguous Cys residues. Although the conformations required for the essential intermediates may seem unfavorable, Corey–Pauling–Koltun space-filling models (Fig. 4 C and D) of the C terminus of wild-type DmTrxR show that interactions, such as those proposed in Fig. 4B, are possible. All peptide bonds are shown in trans-configuration. We have only shown two of the many conformations the peptide permits; many others could be consistent with the proposed mechanism.

The scheme in Fig. 4B shows how the serine residues might potentiate deprotonation (species 1–3). Each Ser-Cys couple serves a specific purpose. We propose that the hydroxyl of the Cterminal Ser forms a hydrogen bond with the neighboring cysteine thiol (Cys490′) to facilitate formation of a reactive thiolate (species 2). This thiolate attacks the disulfide bridge of the substrate, Trx, forming a mixed disulfide and a free thiolate on the substrate, with the thiolate of Trx taking up a proton, probably from His106 (species 3). The thiol of Cys489′ is similarly activated by its adjacent Ser488′ hydroxyl (species 4), and the resulting thiolate of Cys489′ cleaves the intermolecular disulfide between TrxR and Trx, leading to the reduced product, Trx(SH)2, and the C-terminal cysteine residues as a disulfide (species 5). As shown, (22) Cys490′ is also responsible for the dithiol–disulfide interchange with the N-terminal redox-active site, and is promoted by His464′ (species 6 and 7). The x-ray structure of the rat enzyme indicates that the C-terminal tail is quite flexible; the distance between the redox-active Cys497′ and Cys59 could, in that case, be modeled to vary between 3 and 12 Å (20). We suggest that DmTrxR uses this flexibility to optimize interactions of the C-terminal Cys pair alternately with Trx, and with the N-terminal Cys pair as indicated in Fig. 4B (20). The Sec-containing redox-active motif appears to act independent of the nature of the flanking residues, presumably due to the larger atomic radius, a lower pKa, and higher reactivity of selenium (24) compared with sulfur (compare Fig. 4 with Fig. 8, which is published as supporting information on the PNAS web site).

It is tempting to speculate why Diptera, like D. melanogaster and Anopheles gambiae, as well as some other organisms (e.g., Plasmodium falciparum), use selenium-free TrxRs. Alternatively, why do the mammalian TrxR enzymes contain selenium, if a -Ser-Cys-Cys-Ser-COOH motif (as in DmTrxR) is essentially as efficient in Trx reduction as a -Gly-Cys-Sec-Gly-COOH motif? A slightly lowered catalytic activity could easily be compensated for by slightly increased expression of enzyme. In view of their energy-demanding synthesis machineries and dependence on selenium supply, the occurrence of Sec must be explained by some other requirements of the cell. One possibility is that the activity of the Cys-Sec-containing enzymes is less affected by the pH of the surrounding milieu, which would imply that the mammalian Trx system is more resistant to acidic conditions than the corresponding insect system. The importance of pH resistance may have been previously underestimated. Intracellular pH values significantly below neutral as reported for mammalian cells (see e.g., ref. 26) may have imposed a selective genetic pressure that favors Sec over Cys.

Alternatively, the answer might be found in reduction of substrates other than Trx, as impressively indicated by the efficient turnover of non-disulfide compounds, such as the important metabolites, dehydroascorbate, cytosolic ubiquinone, and methylseleninate. These tasks are apparently almost exclusively performed by mammalian (Sec-) TrxRs under physiological conditions (see ref. 3 for a review). The recycling of these intermediates cannot be performed as well with the Cys-Cys-TrxR variants. Thus, it is clear that facilitating the formation of a thiolate cannot fully compensate for the chemical properties of selenium. This finding is revealed, for example, in the reduction of methylseleninate, where the Cys-Sec variants were 3- to 5-fold more efficient than the corresponding Cys-Cys variants. These findings are consistent with the notion that the mammalian Sec-TrxRs have important roles in reduction of low molecular weight substrates and that this might not be as important in insects.

To conclude, we have shown here how a sulfur homolog can approach the catalytic efficiency of a selenium-dependent enzyme. Note that direct sulfur-to-selenium substitution in a selenium-dependent enzyme typically reduces the catalytic efficiency to 5% or less; such dramatic loss of activity has been demonstrated for mammalian TrxR (1012), as well as for all other natural selenoproteins, where this aspect has been studied (27). The lower activity of cysteine, compared with Sec, has therefore been considered a main reason for the evolution and existence of selenoproteins, despite intricate and resource-demanding translation machineries. Our findings, surprisingly, show that Sec is not necessarily required for efficient catalysis of a high Mr TrxR, and that small changes in the active site microenvironment are sufficient to yield high, selenium-independent activity. We therefore propose that Sec may not be essential for a particular enzyme reaction per se, but that this amino acid expands the metabolic capacities in terms of activity toward a wider variety of substrates and over a broader range of pH. This fact alone could explain why organisms that can rely on a continuous and adequate supply of nutritional selenium have either retained or developed selenium-dependent enzymes.


We thank Prof. F. Jakob for helpful comments. This work was supported by Deutsche Forschungsgemeinschaft Grant GR 2028/1-1 (to S.G.), the Karolinska Institute, the Swedish Society of Medicine, and Swedish Cancer Society Project Grants 3775 and 4056 (to E.S.J.A.) and by National Institute of General Medical Sciences Grants GM11106 (to D.P.B.) and GM21444 (to C.H.W.).


  •  To whom correspondence should be addressed at: Biochemie-Zentrum Heidelberg, Heidelberg University, Im Neuenheimer Feld 504, D-69120 Heidelberg, Germany. E-mail:

  • This paper was submitted directly (Track II) to the PNAS office.

  • Abbreviations: Trx, thioredoxin; TrxR, Trx reductase; DmTrxR, Drosophila melanogaster TrxR; Sec, selenocysteine; U, one-letter code for Sec.

  •  This assay is essentially a Trx assay, yet, due to the recycling of oxidized Trx by means of the nonenzymatic reduction of glutathione disulfide, a constant level of Trx is maintained.

  •  Note that mammalian TrxR and DmTrxR share only ≈55% identity. Thus, >200 residues (45%) are different. Attributing the difference in enzymatic activity, as done earlier, solely to the exchange of one amino acid (Sec ⇌ Cys) is therefore an obvious oversimplification. Our results clearly support this point.

  • Received July 18, 2003.