Study On Selenoproteins Selenoproteins Biology Essay

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The vast majority of mammalian selenoproteins incorporate Se in the form of the 21st amino acid, selenocysteine (Bock, 2001; Hatfield and Gladyshev, 2002). In many cases, selenium is present at a catalytic center of an enzyme responsible for redox reactions (Stadtman, 2000; 2001). In selenoprotein mRNA, the stop codon UGA encodes SeCys insertion, requiring a specialized group of transcription factors to translate it properly (see Berry et al.2001 and Copeland, 2003 for review). The process of SeCys incorporation is highly selenium dependent, requiring both cis and trans acting components that lead to a hierarchy of Se-protein expression (Allan et al., 1996; Sunde, 2001). Se deficiency often results in a lack of selenoprotein expression through altered mRNA stability and protein translation (Chu et al., 1990; Allan et al., 1999).

Selenocysteine incorporation requires a SeCys charged tRNA containing the UCA codon (Diamond et al., 1981; Leinfelder et al., 1988). The SeCys tRNA has methylated and non-methylated isoforms, indicating a possible mechanism for mediating the hierarchy of Se incorporation into protein (Jameson and Diamond, 2004)

Although the eukaryotic synthesis of SeCys tRNA is not yet fully characterized, a partial picture of the process has emerged. Selenocysteine is synthesized on a tRNA initially charged with serine (Carlson et al., 2001), which is phosphorylated to form a phosphoseryl tRNA. Anactivated selenium group (most likely monoselenophosphate, formed by a selenophosphate synthetase) is then exchanged for the phosphate group, completing the synthesis of SeCys tRNA (see Review by Hatfield and Gladyshev, 2002).

In order to incorporate SeCys at the UGA codon, a cis-acting selenocysteine insertion sequence (SECIS) is located in the 3' untranslated region (3'UTR) of the mRNA (Berry et al., 1991). The sequence of SECIS is not highly conserved among eukaryotes but they are structurally similar across species, forming a stem-loop (Martin and Berry, 2001). The SECIS binding protein 2 (SBP2) which has been shown to bind with SECIS, is able to interact with ribosomes (Copeland et al., 2001). It is believed that these characteristics allow SBP2 to alter the ribosomes reading of the genetic code such that UGA leads to SeCys incorporation (Copeland et al., 2001).

A recent paper by Kryukov et al., (2003) reported 25 distinct putative selenoproteins, identified by a computer algorithm based on the presence of the SeCys codon and a SECIS element. Although not all of these may be active genes, it is exciting to consider these potential subjects of selenoprotein research next to the known and characterized selenoproteins (n=14, based on Lei, 2001).

Among the well characterized selenoproteins (Burk and Hill, 1993; Sunde, 1994; Stadtman, 1996; Flohe et al., 1997),


Glutathione is a thiol containing tripeptide (Glu-Cys-Gly) which serves as a cofactor for the glutathione peroxidases. Glutathione and other low molecular weight thiols have the advant age in redox reactions of being easily oxidized and regenerated. Due to these characteristics, glutathione can play an essential role in many biochemical and pharmacological reactions (Mates, 2000; Locigno and Castronovo, 2001; Paolicchi et al., 2002). Some of the important roles of glutathione are: reduction or inactivation of ROS and RNS by formation of glutathione disulfide (GSSG) and conjugation of reduced glutathione (GSH) with a xenobiotic and subsequent elimination as a mercapturic acid (Berlett and Stadtman, 1997; Hayes et al., 1999; Strange et al., 2001). The functions of the glutathione system in redox balance and in xenobiotic detoxification demonstrate the importance of understanding this system in a physiological context and make it an interesting potential target for genetic and therapeutic manipulation.

2.4. 1. Glutathione Peroxidases

(In the past few years, the role of oxygen-free radicals, known as "reactive oxygen species" (ROS) and "reactive nitrogen species" (RNS), has been intensively studied in experimental and clinical medicine [B. Halliwell (1999) J. L. Avanzo (2001)].The effect of reactive oxygen and nitrogen species is balanced by the action of non-enzymatic antioxidants, as well as antioxidant enzymes. The most efficient non-enzymatic antioxidant involves Vitamin C, Vitamin E, carotenoids, thiols, natural flavonoids and other compounds, while enzymatic antioxidants include superoxide dismutase, catalase and glutathione peroxidase.

The endogenous activity of antioxidant enzymes may be modulated by nutritional factors such as some trace elements. Minerals as Cu, Mn, Zn, Se and Fe have demonstrated to be important cofactors for the regulation of the antioxidant enzyme activity. [M. Valko, C. J. Rhodes,J. (2006)].

Glutathione peroxidase (GPx) is an enzymatic antioxidant which presents two forms: one of them is selenium-independent while the other is selenium-dependent. The properties of both GPx allow them to eliminate peroxidases as potential substrates for Fenton reaction [M. Valko, C. J. Rhodes,J. (2006)]. For these reasons, and taking into account the narrow boundary between toxicity and essentiality for Se, this element has received a considerable attention as essential micronutrient.

The mechanism by which selenium exerts its beneficial effects on health may be through selenoproteins. The amino acid selenocysteine is involved in the synthesis of diverse selenoenzyme such as glutathione peroxidase (reducing peroxides) [R.Brigelius-Flohe. 1999) P. J. Crack,(2001)], iodothyronine deiodinases (regulating thyroid hormone activity), and thioredoxin reductases (regenerating antioxidant systems) [6, 7, 8]. Compared to these well characterized enzymes, many functions of a growing number of selenoproteins remain unclear [9]. Also, selenium replaces sulphur in methionine to form selenomethionine, which can be incorporated non-specifically into proteins [10]. Furthermore, antioxidant and antitumor activities have been reported for Se containing low-molecular -weight compounds (both natural and synthesized) [11]. Therefore, low or suboptimal levels of selenium intake were associated with a wide variety of human diseases such as heart disease, cystic fibrosis and several types of cancer [12]. The work of Clark and co-workers carried out the study of selenium compounds as a relevant group of cancer chemo-preventive agents.[13] Anticarcinogenic activity has been attributed to some selenium organic species, [13, 14] and because selenium can only be obtained from food, is very important to known selenium dietary sources. The National Research Council has established a Recommended Dietary Allowance (RDA) of selenium for humans, 55 and 70 µg/day for men and women, respectively [15]. Humans take most of the selenium from cereals, fish, meat, and dairy products [16]. Nutritional supplements have also been recommended to increase daily selenium intake; however, recent studies shown that the amount of selenium in over the counter supplements can be much lower than advertised. [17] Therefore, one focus seems to be on increasing selenium intake through natural sources.

The processes of oxidation and reduction are necessary in the body. This gain or removal of an electron keeps many of the life processes working. As respiration occurs in animals, which is defined as the process from which cells derive energy in the form of ATP from the reactions of hydrogen and oxygen, they often produce various peroxides. These peroxides, including hydrogen peroxide, can be harmful to the body as they can lead to generation of free radicals, which can damage or destroy cells [97](Arthur, 2000).

The collective enzymes, known as the glutathione peroxidases (GSH-Px), are hydroperoxidases, and their function is to protect the body from these harmful peroxides (Arthur, 2000). The primary function of these enzymes is to catalyze a reaction that removes hydrogen peroxide from erythrocytes via reduced glutathione.

The reduced glutathione is made via the enzyme, glutathione reductase, from oxidized glutathione. This reduction process also requires NADPH, which is provided by the pentose phosphate pathway. The general reaction for GSH-Px, as described by Rotruck et al. (1973), is shown below. In this schematic, ROOH is any hydrogen or lipid peroxide, GSH is glutathione in the reduced form, ROH is the reduced peroxide, GSSG is oxidized glutathione (Rotruck et al., 1973; Levander, 1986; Sunde, 1997).


Mills (1957) first described the activity of glutathione peroxidase, and it was hypothesized that its function was to protect red blood cells from oxidative hemolysis. In 1973, Rotruck et al. suggested that Se was an integral part of glutathione peroxidase, which Flohe et al. (1973) later demonstrated. Since that time, six different glutathione peroxidase enzymes have been elucidated. Four of these require selenocysteine at their active site for proper function, while the other two only require cysteine at their active site and thus, are not Se-dependent. The following discussion will focus on the four Se-dependent glutathione peroxidase enzymes.

The GSH-Px that Mills described in 1957 is the classical cytosolic glutathione peroxidase (GPX-1) present in all cells of the body. It can metabolize hydrogen peroxide and several organic peroxides, such as cholesterol and long-chain fatty acid peroxides (Sunde, 1997). The GPX-1 enzyme is a tetrameric protein with four identical subunits, and each subunit contains one selenocysteine (Arthur, 2000). This enzyme is very specific for glutathione as a reducing substrate, and as such it often is discussed in relation with glutathione reductase activity (Sunde, 1997; Arthur, 2000).

Shortly after the discovery of GPX-1, Chow and Tappel (1974) indicated that GPX activity in plasma responded quickly to Se deficiency and resupplementation. This plasma GPX activity was originally thought to be due to GPX-1 leaking from the liver or other organs, but it did not react with antibodies to GPX-1 purified from red blood cells (Takahashi and Cohen, 1986). This lack of reactivity indicated that this GPX was a distinct enzyme, and ultimately it was determined to be a glycoprotein with distinct extracellular functions (Sunde, 1997; Arthur, 2000). Similar to GPX-1, it has a tetrameric protein structure, but it shares only 40 to 50% amino acid homology to GPX-1 (Arthur, 2000).

Glutathione Peroxidase 4

Phospholipid hydroperoxide glutathione peroxidase or GPx4 (EC was discovered by Ursini et al.(1982) as a selenoprotein in pig liver extract with the ability to protect cellular lipids against peroxidation and to reduce phosphatidylcholine hydroperoxides. Being distinct from cellular glutathione peroxidase-1 (GPx1) (Schuckelt et al., 1991), GPx4 is expressed as three isoforms with alternate start codons and exons: a 23 kDa form (with a 27 amino acid mitochondrial targeting sequence that is later cleaved), a 20 kDa non-mitochondrial form and a 34 kDa sperm nucleus form (with an alternate first exon) (Pushpa-Rekha et al., 1995; Arai et al., 1996). Compared with other selenoperoxidases, GPx4 shares approximately 30 to 40 % nucleotide identity (Imai and Nakagawa, 2003). The enzyme functions as a monomer rather than a tetramer (as in the case of other GPx proteins), and it is the only GPx that is able to reduce phospholipid hydroperoxides (Ursini et al., 1985). Nutritionally, GPx4 is much more resistant to dietary Se deficiency than the other GPx enzymes, particularly GPx1. When liver GPx1 activity and protein are reduced to nearly zero in selenium-depleted rodents (Weitzel et al., 1990; Lei et al., 1995; Bermano et al., 1996), liver GPx4 activity maintains approximately 20% of the selenium adequate levels (Weitzel et al.,1990; Thompson et al., 1998).

The fourth Se-dependent GPX enzyme (GPX-4) is significantly different from the GPX-1 enzyme. The GPX-4 has a monameric protein structure compared with the tetrameric structure of G PX-1, and it is not glutathione specific, such that it can use phospholipid hydroperoxides as substrates (Sunde, 1997; Arthur, 2000). Because of its unique structure, GPX-4 is believed to be able to bind to a wider range of substrates than the other GPX enzymes. Although its function has not been clearly defined, Godeas et al. (1994) indicated that GPX-4 may protect cellular membranes from hydroperoxides via the cytosol by rolling along membrane surfaces and mitochondrial intermembrane spaces.

Other Selenoproteins

Besides the glutathione peroxidase family of enzymes, there are several other proteins that also must have Se in the form of selenocysteine to function properly.

These include three forms of the deiodinase enzyme necessary for the formation of the thyroid hormone, thyroxine; the plasma selenoproteins P; and the muscle selenoprotein W.

The thyroxine 5'-deiodinase-1, 2, and 3 (DI1, DI2, and DI3) enzymes are responsible for deiodinating thyroxine or reverse triiodothyronine to make triiodthyronine or diiodothyronine, respectively (Sunde, 1997 ). More than 90% of the circulating triiodothyronine in the plasma is produced by DI1, which was first identified as a selenoenzyme by Arthur et al. (1990) and Behne et al. (1990). The other deiodinase enzymes, DI2 and DI3, are most prevalent in the central nervous system and brown adipose tissue (Sunde, 1997).

Selenium Deficiency In Poultry

Although the requirement for Se often is met by the natural feedstuffs in poultry diets, there are several detrimental conditions that can result in poultry when dietary Se is deficient. Exudative diathesis, pancreatic fibrosis, and impaired reproduction are observed if the Se level in the diet is deficient. Exudative diathesis and pancreatic fibrosis, which are discussed in detail below, have a major difference in the form of Se needed to alleviate their deficiency signs. Reproductive impairment, on the other hand, does not seem to be specific in the form of Se needed to alleviate its deficiency signs (Underwood and Suttle, 1999).

Exudative diathesis is characterized by a general edema due to atypical permeability of the capillary walls (Under wood and Suttle, 1999). It first appears on the breast, wing, and neck as greenish-blue discoloration due to fluid accumulation under the ventral skin. Abnormal growth rate and high mortality are common in flocks with exudative diathesis, and Hartley and Grant (1961) indicated that this condition usually occurs between 3 and 6 weeks of age. Noguchi et al. (1973a) reported that either Se or vitamin E could prevent exudative diathesis. In a subsequent study, Noguchi et al. (1973b) reported that dietary Se is directly related to GPX-3 activity and the prevention of exudative diathesis. Selenium in the form of SS or selenocysteine provides the most protection from exudative diathesis (Cantor et al. 1975a,b).

Pancreatic fibrosis results from a severe Se deficiency in poultry, and it causes atrophy of the pancreas, as well as poor growth and feathering (Thompson and Scott, 1969). Bunk and Combs (1980) reported that appetite depression associated with this condition is negated within hours of Se supplementation.

Furthermore, Noguchi et al. (1973a) indicated that the pancreatic lesions, which become apparent by 6 days of age, return to normal within 2 weeks after the onset of Se supplementation. High dietary vita min E cannot alleviate this condition as pancreatic fibrosis results in a secondary vitamin E deficiency due to impaired formation of lipid bile micelles, which are necessary for the absorption of vitamin E (Thompson and Scott, 1969). Selenium in the form of SM protects poultry from pancreatic fibrosis more efficiently than SS or selenocysteine (Cantor et al.


Impaired reproduction in females also can result from Se deficiency. Cantor and Scott (1974) reported that egg production and hatchability were reduced in laying hens fed diets with reduced levels of Se, and Latshaw et al. (1977) indicated that hatchability was the most sensitive criteria of Se deficiency in hens. Furthermore, Jensen (1968) reported that low dietary Se impaired fertile egg hatchability and chick viability in Japanese quail.

Use Of Selenium For Poultry

Selenium is a dietary essential nutrient for poultry (NRC, 1994). The Se requirement for the laying hen ranges from 0.05 to 0.08 ppm depending on daily feed intake while the broiler's requirement is 0.15 ppm (NRC, 1994). Natural feedstuffs often will meet these requirements, but as mentioned before, there is considerable variation in Se content of natural feedstuffs. Therefore, it is common practice in the poultry industry in the U.S. to supplement the diet with some form of Se. The maximum level of Se supplementation allowed in poultry diets is 0.30 ppm (NRC, 1994; AAFCO, 2003). This supplementation has historically come from inorganic sources of Se, primarily SS, but in 2000, the FDA approved the use of SY. There have been several reports comparing the use of organic Se with inorganic Se in broilers and laying hens, which will be discussed below.

Selenium In The Diets Of Broilers

The response to dietary Se supplementation has been somewhat variable. Several researchers reported that Se supplementation increased growth performance (Thompson and Scott, 1969; Bunks and Combs, 1980; Cantor et al., 1982; Echevarria et al., 1988b) while several others have reported no effect (Miller et al., 1972; Shan and Davis, 1994; Edens et al., 2001; Spears et al., 2003). Only Echevarria et al. (1988a) reported a negative effect of Se on growth performance, and they were feeding very high levels of SS (3, 6, or 9 ppm), which could be toxic to broilers. None of the research has reported a difference in growth performance due to source (organic versus inorganic).

The results of Se supplementation on tissue Se concentrations are fairly consistent when diets are supplemented with Se. There are several reports of Se supplementation increasing breast, liver, or plasma Se levels (Scott and Thompson, 1971; Cantor et al., 1982; Echevarria et al., 1988a,b; and Spears et al., 2003).

Furthermore, Cantor et al. (1982) and Spears et al. (2003) both indicated that organic Se increased tissue Se levels more than inorganic Se or a diet with no supplemental Se. The published results on plasma GPX-3 activity are variable. Cantor et al. (1982) and Spears et al. (2003) both reported that plasma GPX-3 activity was increased when diets were supplemented with Se, regardless of source. However, in a second trial, Spears et al. (2003) indicated that plasma GPX-3 was increased more by SS supplementation than by SM. Only Cantor et al. (1975) indicated no differences in plasma GPX-3 when broilers were fed SS, SM, or no supplemental Se.

Reactive oxygen species

In the process of normal aerobic metabolism, reactive oxygen species (ROS) are formed. Enzymatic systems are a very important means by which cells can maintain these potentially damaging molecules within concentrations needed for normal cellular function. At normal concentrations, ROS such as superoxide, hydrogen peroxide and lipid hydroperoxides are able to regulate activities of kinases, transcription factor s and apoptotic factors in addition to contributing to the normal function of many other metabolic and signaling systems. Chronically increased levels of ROS can contribute to pathological states including cancer and cardiovascula r disease (Finkel, 1998; Rhee, 1999; Thannickal and Fanburg, 2000; Nomura et al., 2001) while acute increases in ROS can lead to massive damage of biomolecules (e.g. protein, DNA and lipids) and consumption of reducing molecules (e .g. NADPH) leading to cellular apoptosis or necrosis and, on a whole animal scale, death (Bus et al., 1974; Smith, 1977; Witschi et al., 1977; Cagen and Gibson, 1977; Keeling and Smith, 1982; Burk, 1991; Sunde, 1994; Ho et al., 1997; Berlett and Stadtman, 1997; Cheng et al., 1998; De Haan et al., 1998; Cheng et al., 1999).

Selenium-dependent glutathione peroxidases 1 and 4 and copper, zinc-dependent superoxide dismutase 1 are three important micronutrient dependent enzymes in coping with oxidative stress (McCord and Fridovich, 1969; Flohe et al., 1973; Rotruck et al., 1973).

Copper, Zinc and the Superoxide Dismutases

Copper and zinc serve as components of proteins important for cytosolic antioxidant defense. A primary example of this is copper, zinc superoxide dismutase (SOD1, EC, which catalyzes the dismutation of hydrogen peroxide to oxygen and hydrogen peroxide which is then reduced to water by the action of the selenoprotein glutathione peroxidases

(see Figure 1.1 and Figure 1.2 ) (McCord and Fridovich, 1969; Flohe et al.,1973; Rotruck et al., 1973).

In addition to its activity in oxidant defense, copper, as a redox active metal can also participate in the production of free radicals by interaction with thiols (e.g. reduced glutathione) and oxygen. In situations of high intracellular hydroperoxide concentrations, copper (and more classically, iron) also has the potential to participate in Fenton reactions (Figure 1.3) which produce hydroxyl radicals (Oshino et al., 1973). Due to its potentially damaging reactivity, there are high levels of copper scavenging proteins in the cell, maintaining free copper ion concentrations at near zero (Rae et al., 1999). Among these scavenging proteins are the metal binding proteins, the metallothioneins, the expression of which can be induced by copper (Murata et al., 1999). Copper plays a vital role as a co-factor for a number of metalloenzymes including:

• Cu/Zn superoxide dismutase (antioxidant defense),

• cytochrome c oxidase (mitochondrial respiration),

• lysyl oxidase (formation of connective tissue),

• tyrosinase (melanin synthesis)

• ceruloplasmin (iron homeostasis) (Pena et al., 1999; Shim and Harris, 2003)

Many of the symptoms associated with copper deficiency are a consequence of decreased activity of copper-dependent enzymes (Prohaska, 1991; Milne and Nielsen, 1996; Turnlund et al., 1997; Kehoe et al., 2000) although overt copper deficiency is rare in humans.

Originally known as erythrocuprein, copper, zinc superoxide dismutase (SOD1) (EC was the first SOD identified (McCord and Fridovich, 1969). Now three distinct superoxide dismutases are known in mammals, with their genomic structure, cDNA, and proteins described. Two of these SOD isoforms have copper and zinc in their catalytic center, cytosolic SOD1 and extracellular SOD3. SOD1 is a homodimer of about 32kDa (Chang et al., 1988; Keller et al., 1991; Crapo et al., 1992; Liou et al.,1993). First detected in human plasma, lymph, ascites, and cerebr ospinal fluids (Marklund et al., 1982, 1986), SOD3 functions as a homotetramer of 135 kDa (Marklund, 1982). The third SOD isoform, MnSOD or SOD2, contains manganese (Mn) as a cofactor and has been localized to the mitochondria of aerobic cells (Weisiger and Fridovich, 1973).

Plasma Alanine Aminotransferase

Plasma alanine aminotransferase (ALT) is a frequently used in clinical settings to assess liver function. Activity of ALT can increase rapidly in the plasma in the presence of xenobiotics that cause liver necrosis (e.g. AP and DQ) (Daniel and Gage, 1966; Prescott, 1980; Flanagan et al., 1995). ALT Stored in hepatocytes is released when hepatocytes are acutely damaged. Increases in plasma concentrations of this enzyme provide important evidence of hepatocyte damage (Rosenthal et al., 1997).

In vertebrate animal species, selenium is the pivotal element in an enzyme, iodothyronine deiodinase (types I, II, and III), which is needed for the conversion of the thyroid hormone, thyroxine (T4) to triiodothyronine (T3) (Edens and Gowdy, 2005). If T4 is not converted, deficiencies can arise causing hypothyroidism, which leads to numerous metabolic disorders that have numerous signs such as extreme fatigue, goiter, miscarriages, mental slowing, and cretinism (Edens and Gowdy, 2005).

Shamberger and Frost (1969) were the first to indicate that selenium helped to reduce cancer mortality rates in their 1969 study. Since that time, there have been numerous experimental and epidemiologic studies to investigate this hypothesis. Several scientific studies suggest that an increased risk of cancer occurs as a result of low concentrations of selenium in the diet (Schrauzer et. al., 1977; Clark et. al., 1991; Combs and Gray, 1998; Rayman and Clark, 2000).

Evidence has been amassed to suggest strongly that selenium should be taken to aid in the prevention of many types of cancer. One study involved 1312 patients who daily took 200 ug of selenium yeast or low-selenium yeast placebo for 4.5 years. Selenium supplementation was shown to reduce the total mortality and mortality from cancers as well as the incidence of lung colorectal and prostate cancer by 46%, 58%, and 64% respectively when the subjects were seen 6.4 years after the ingestion of the supplements (Clark et. al., 1996) Nevertheless, the lack of appropriate apoptosis in selenium deficient animals might exacerbate the growth of neoplastic tumors (Edens et. al., 2007). Other research has shown a link between HIV/AIDS and selenium (Raymond, 2000; Jacques, 2006). In one study, the author concluded that selenium supplements offer a low-cost, simple and safe treatment for people with HIV (Jacques, 2006). Hurwitz and colleagues included 174 subjects using both genders. The subjects consumed 200 μg/day of selenium from high-selenium yeast or a placebo for 9 months. Selenium supplementation decreased HIV-1 viral load (Hurwitz, 2007).

The effect of selenium supplementation on the immune function in healthy adults has been investigated. Twenty-two adults, who had relatively low plasma concentrations of selenium, were randomly assigned to consume either 50 or 100 μg/day of selenium from either sodium selenite or a placebo for 15 weeks. Sodium selenite supplements increased the plasma concentration of selenium, the exchangeable selenium pool, and glutathione peroxidase activity in both lymphocyte phospholipids and cytosol (Broome et. al., 2004).

In a study on the impact of selenium type II diabetes, 56 people with type II diabetes were randomized to receive either 960 μg/day selenium or a placebo for three months. Ten nondiabetics also participated in the study as the controls. Selenium supplements significantly increased plasma selenium concentrations and red-cell selenium glutathione peroxidase activity. Selenium supplements reduced nuclear factor- kappa B (NF-kB) activity in people with type II diabetes to a level near that of the nondiabetic controls proving that selenium helps protect against type II diabetes (Faure et. al., 2004). The decrease in NF-kB probably is significant because it has been associated with increased apoptosis in many tissues including muscle and soft tissues such as pancreas and immunologically active tissues.

Figure1.1. Diagram showing the relationship among various human diseases and the influence of sustained elevation in production of reactive oxygen (ROS) and reactive nitrogen (RNS) species metabolites.

Figure 1.1 summarizes a few of the things that selenium does to aid human and animal health by controlling reactive oxygen and nitrogen species reactions via their neutralization. As shown in Table 1.1, selenium and/or vitamin E deficiencies can cause many different disorders leading to diseases shown in Figure 1.1.

It may be possible that the Se intake needed for saturation of some of the antioxidative selenoenzymes in muscle cells (Gpx 1, Gpx 4, thioredoxin reductase and selenoprotein W) is higher than needed for saturation of selenoenzymes in blood plasma and blood cells. Higher level of antioxidant enzymes will lead to reduction of the rate of lipid peroxidation, which means reduction of the rate of EPA, DPA and DHA degradation by peroxidation.

Another possibility may be that the oxidation of EPA, DPA and DHA in mitochondria may be reduced as a consequence of selenide replacing sulphide in mitochondrial iron-sulphur enzymes. This might change the kinetic properties of these enzymes, since a change of the Se--/S--ratio at the sulphide positions must be expected to affect the standard redox potential for the Fe++/Fe+++ equilibrium. Fe++ is bound more strongly to selenide than to sulphide ions as illustrated by the much lower solubilityproduct of FeSe (10-26) [Buketov EA, 1964] compared with FeS (4 Ã- 10-19) [Sienko MJ,1974.]. A higher selenide/sulphide ratio in the iron- sulphur clusters would therefore be expected to enhance the stability of ferrous iron, i.e. to enhance the standard redox potential for the Fe++ /Fe+++ equilibrium. It might be speculated that this could lead to enhancement of the rate of\mitochondrial NADH oxidation, other factors being equal. This might in turn enhance the rate of electron flow from NADH to cytochrome c oxidase, leading to enhancement of the rate of O2 reduction by the latter enzyme. Enhancement of the rate of O2 reduction might in turn lead to reduction of the intracellular O2 partial pressure in most organs, and to reduction of the rates of reactive oxygen species (ROS) production and lipid peroxidation.

The rate of peroxidation of polyunsaturated fatty acid groups depends on the number of double bonds per fatty acid molecule [Chow CK, (Ed); 2000.]. The number of double bonds is especially high in EPA, DPA and DHA, making these fatty acids particularly vulnerable to peroxidative attack. More oxidizing conditions (e.g. higher rate of ROS production) must therefore be expected to affect the rates of EPA, DPA and DHA peroxidation more strongly than it affects the rates of peroxidation of fatty acids with a smaller number of double bonds, such as LA.

Iron is a very important catalyst of lipid peroxidation reactions [Pryor WA; 1976¼Œ Oubidar M, 1996]. It might be speculated that a higher selenide/sulphur ratio in iron sulphur proteins also might affect the concentration of iron that is bound to small molecules and functions as a catalyst of peroxidation reactions. This is another putative mechanism, by which a higher selenide/sulphide ratio in mitochondrial enzymes might lead to reduction of the rate of lipid peroxidation reactions, with this effect being especially pronounced for those fatty acids that have the largest number of double bonds.

An increase in the very long chain omega-3 fatty acids EPA, DPA and DHA in muscle by a diet rich in Se is highly interesting. Pending further investigations, it is necessary to keep all possibilities open regarding the possible mechanisms. In humans the concentration of these fatty acids are also dependent both on the rates of intake plus synthesis and degradation. The conversion of ALA to EPA, DPA and DHA is reported to be low in humans [Burdge GC, 2002]. If poor selenium status should lead to enhancement of the rate of EPA, DPA and DHA degradation or reduced synthesis, this will interact synergistically with low dietary intake of these fatty acids. An increased intake of Se in the diet might thus have practical implications by increasing the level of these valuable fatty acids. A health benefit of high Se intakes on disorders related to lack of long chain omega-3 fatty acid might be suggested.

Growth Performance

The research of different Se source for their potential use as additives in poultry was still increasing [ 17-19]. It was clear from our studies that the administration of Se via the basal diet had beneficial effect on avian broiler performance. In the present research, FCR was significantly reduced in groups of Se treatment compared with that of the control. Similar results were observed by Mahmoud and Edens [20] who demonstrated that the FCR of broiler chickens (Gallus gallus) is affected by dietary Se level. Similar improvements in growth performance had been reported for poultry receiving Se [ 21]. However, there was no significant difference among the treatment groups (T-1, T-2, and T-3) with different source and concentrations of Se. This indicated that the forms and quantity of Se was only one of the factors improving the DWG and FCR of avian broilers.

Poultry diets deficient in selenium result in poor growth and development, increased mortality, reduced egg production, decreased hatchability, pancreatic fibrosis, and muscle myopathies [22-24]. The present research result proved this point, and the control groups fed with basal diet unsupplemented with any forms of Se showed the symptoms of selenium deficiency such as lower survival rate, DWG, and higher FCR. The minimum level of supplemental selenium to sustain growth and performance in broiler chickens was 0.1 mg kg−1 according to the National Research Council. However, the Se content of basal diet was only 0.055 ± 0.007 mg kg−1 and lower than the standard. In contrast, no significant survival increases were detected, and the survival rates of all the groups supplemented with Se (T-1, T-2, and T-3) were 97.33%, 98.00%, and 96.67%, respectively, after 42 days feeding. It indicated that the nano-Se had the same biological functions as sodium selenite in avian broilers. Moreover, no remarkable significance was observed between T-2 and T-3 in the present study, and it suggested from the opposite side that the addition of 0.5 mg kg−1 nano-Se was acceptable in avian feeding.

It was obvious that the tissues with Se content were markedly increased as the dietary Se level increased. Similar results were observed in Rohman laying hens by Pan et al. [25] who reported that breast muscles and whole body, liver, kidney, spleen selenium concentrations were higher in the groups given selenium compared with that of the control. Animal studies have demonstrated that the liver is the major target organ of selenium accumulation [ 26]. In the present study, higher Se content was observed in liver than in muscle across all treatments.

A substantial research has also defined an important role for Se in antioxidant defense.

Se is important for the control of oxidative stress, and therefore the redox state of the cell, due to its incorporation as selenocysteine into GSH-Px [27] and thioredoxin reductase [ 28].

In this study, broilers fed a diet deficient in Se showed decreased GSH-Px activity in serum and liver. By Supplementation of the diet with Se, both sodium selenite and nano-Se, increased the GSH-Px activity. However, GSH-Px activity was not linearly related to the concentration of the dietary nano-Se. This was not in agreement with the previous studies which showed that the GSH-Px activity increased as a logarithmic function of the dietary selenium (sodium selenite and selenomethionine) level [29]. However, it was difficult to directly assess different studies using Se because the efficacy of a Se application depended on many factors such as species composition and viability, administration level, application method, frequency of application, overall diet, bird age, overall farm hygiene, and environmental stress factors. Essentially, there was no difference in GSH-Px activities both in serum and liver from broilers fed equal gram-atoms of selenium as sodium selenite and as nano-Se in the present research. This indicated that the form of Se was only one of the factors promoting the GSH-Px activity of avian broilers.

Based on the findings of our study, nano-Se could serve as another Se form andsuccessfully improved DWG, FCR, survival rate, tissue Se content, and the GSH-Px activity of avian broilers compared with the control. Furthermore, different tissue Secontents were observed in the groups fed with different concentration nano-Se. However, no significant differences were found in DWG, FCR, survival rate, and GSH-Px activity of serum and liver across all treatments fed with 0.2 mg kg − 1 sodium selenite (T-1), 0.2 mg kg −1 nano-Se (T-2), and 0.5 mg kg − 1 nano-Se (T-3), respectively. The addition, however, of a different form of Se, especially selenomethionine, the predominant chemical form of organic selenium in feedstuffs, and nano-Se to avian broilers in general requires further research to compare the bioavailability and clearly understand the functional mechanism between the Se and animals. Moreover, modern molecular techniques should be applied to study whether there are other metabolic pathways of nano-Se which differed from sodium selenite and/or selenomethionine.

The results of this present study clearly indicated that Se treated with supplemented diet could improve the FCR of broiler chickens (Table 3), thus dietary Se supplementation is necessary. Similar results were observed by Mahmoud and Edens (2005), who demonstrated that the FCR of broiler chickens (Gallus gallus) is affected by dietary Se level. Poultry diets deficient in selenium result in poor growth and development, increased mortality, reduced egg production, decreased hatchability, pancreatic fibrosis, and muscle myopathies (Walter and Jensen, 1963; Scott et al., 1967). The present research result proved this point from the opposite side and it might be associated with the quantity of Se in diet. Control fed with basal diet unsupplemented any forms Se did not show the symptoms of selenium deficiency in present study and might be associated with the experimental days, which was only 21 and very much less than the other researches (Combs, 1994; Surai, 2002a; Zuberbuehler et al., 2006). Furthermore, the National Research Council established the minimum level of supplemental selenium to sustain growth and performance in broiler chickens to be 0.1 mg kg−1 National Research Council (1994). The Se concentration of basal diet was also up to 0.08± 0.004 mg kg−1 and it might be another reason to explain that the control fed with basal diet did not show the symptoms of selenium deficiency in present study.

Selenium has a large number of biological functions in the animals and the most important and known action is its antioxidant effect because it forms selenocysteine, part of the active center of the glutathione peroxidase enzyme (Levander and Burk, 1994). The results of trials 1 and 2 in the present study were also showed higher GSH-Px activity whichever in plasma and liver of broiler chickens compared with control (Table 5).

However, these data did not agree with Payne and Southern (2005) who reported the GSH-Px activity was not affected by Se source or concentration. It might be associated with the breed of broilers and experimental phase and the experiment was started at d 0 and lasted for 49 d in broilers (Ross Ã- Ross) (Payne and Southern, 2005). Further-more, selenium as the active core of GSH-Px in broiler chickens might be presumed from the results of this research (Mahmoud and Edens, 2003). On the other hand, in the cell GSH-Px plays an important function, because the reduced form of this enzyme reduces the hydrogen peroxide and lipidic hydroperoxides at the level of the cytosol and mitochondrial matrix (Roch et al., 2000). This element is also included in other functionally active selenoproteins as the type1 iodothyronine 5-deiodinase which interacts with iodine to prevent abnormal hormone metabolism (Foster and Sumar, 1997). The effect of selenium on FCR of broiler chickens might be associated with these forenamed functions of Se.

The results of this present study to compare the effects of supplementing a poultry basal diet with different selenium sources on broiler chickens showed that the concentration of muscles Se in trial 2 treated with selenium yeast was higher (P<0.05) than that in trial1 treated with sodium selenite (Table 4). Similar results were observed in Rohman laying hens by Pan et al. (2007) , who reported that breast muscles and whole-body, liver, kidney, spleen selenium concentrations were highest in the groups given selenium yeast compared with that of given sodium selenite and the control. It indicated that sodium selenite and selenium yeast had different metabolic methods

respectively, although both inorganic and organic forms cross the intestinal barrier. In general, animal study trials demonstrate that bioavailability of organic forms of Se was higher than that obtained for inorganic forms (Levander, 1983; Smith and Picciano, 1987; Surai, 2002b; Pan et al., 2007), as was also observed in human studies (Favier, 1993; Thomson and Robinson, 1993). The selenium yeast as the organic forms of Se can be stored in a protein pool when the methionine is limited or catabolized with the release of Se which passes to another pool. However, the inorganic forms (sodium selenite) go directly into the pool, from which independent of its origin, all the Se is used in the synthesis of selenoproteins as the GSH-Px and the excess is excreted. Thus, Se bioavailability depended not only on its absorption by the intestine but also on its conversion to a biologically active form (Foster and Sumar, 1995).

In vivo studies, one effective way to estimate the bioavailability is by the determination of the GSH-Px activity (Favier, 1993) in blood, which have demonstrated as previously indicated for blood samples, that organic forms enhance the activity of this enzyme com-pared with selenate or selenite, which can be correlated with the fact that different Se forms follow distinct metabolic pathways in the organism (Thomson et al., 1982; Mahan, 1999).

In the present study, The GSH-Px activity in plasma of broiler chickens was remarkably higher (P<0.05) in trial 2 supplemented with selenium yeast compared with trial 1 supplemented with sodium selenite (Table 5). Thus, this result also suggested the bioavailability of organic forms of Se (selenium yeast) was higher than that obtained for inorganic forms (sodium selenite).

In summary, the current study demonstrated that different Se source (sodium selenite and selenium yeast) treated with supplemented diet could improve the FCR, glutathione peroxidase activities and tissues selenium content of broiler chickens. Moreover, organic forms of Se (selenium yeast) gave better results than that of inorganic forms (sodium selenite) in some results determined in the current study.

Approximately half or 40% of whole-body selenium is in GSH-Px and its presence increases enzyme activity 100-1000 fold (Burk, 2002). Enzyme GSH-Px with catalase and superoxide dismutase and nonenzymatic molecules (glutathione, vitamins A, E, C, uric acid, bilirubin, etc.) are mayor determinants of tissue susceptibility to oxidant injury (Michiels et al., 1994). Oxidant injury can results from the increased generation of reactive oxygen specie s and/or from decrease in antioxidant defend (Ivanova and Ivanov, 2000). Reactive oxygen species interact with a number of cellular components. The damage manifests as the peroxidation of membrane polyunsaturated fatty acid chains and disrupts the cohesive lipid bilayer arrangement and structural organisation (Yu, 1994).

Amino acids, the building blocks of peptides and protein macromolecules, are also targets of free radical attack. It results in modification of DNA and carbonyla tion and loss of sulfhydryls in proteins, among other changes. Carbonyl modifications of proteins occur in certain amino acid residues present near transition metal-binding sites. After oxidative modification, the protein becomes highly sensitive to proteolytic degradation, and in the case of enzymes they are converted to catalytically inactive or less active, more termolabile forms (Stadtman and Oliver, 1991) .

Nutritional factors can influence the sensitivity of tissues to oxidative stress and effects tend to be most marked in the case of nutritional deficiencies which are generalized in nature or involved in the biochemical processes which determine tissue antioxidant status. Starvation was associated with complex pattern of antioxidative enzyme and nonenzymatic molecule alterations, the nature of which varied with the particular tissue studied (Cho et al., 1981; Go din and Wohaieb, 1988; Di Simplicio et


Iodothyronine Deiodinases. Iodothyronine Deiodinases (ID) are the second largest group of selenoproteins. The three deiodinases (Type I, II, and III ID) control the local

availability and concentration of the active thyroid hormone, 3,3',5-triiodothyronine (T3). These enzymes catalyze the conversion of thyroxin (T 4) to T3 (Type I and II ID) or the deiodination of T4 and T3 to non-active metabolites (Type III ID). These three isoenzymes are encoded by different genes and have tissue and development-specific patterns of expression and regulation (Kohrle et al ., 2000). In general, ID is ranked higher in priority for available Se supply than is cytosolic GSH-Px and was similar in ranking to that for GSH-Px-PH and selenoprotein P (Kohrle, 2000). Because thyroid hormone controls growth, development, differentiation and many metabolic reactions, Se is believed to be involved in regulation of those functions as well.

In Se-deficient animals, activity of ID I is low. This results in plasma T4 increase while T3 is decreased. A role for Se in type I deiodinase and thyroid hormone metabolism has many implications. The reduced activity of thyroid hormone explains why Se-deficient animals grow more slowly as effects of this hormone are mainly anabolic (Arthur, 1993). Reduced ID I activity in the pituitary is associated with lower levels of growth hormone in Se-deficient animals (MacPherson, 1994).

Thyroid hormone activity is a key factor in animal tolerance to cold stress. A well-known thyroid hormone function is the heat-producing increase in oxygen consumption of tissues in response to cold temperatures. Involvement of thyroid hormones in feathering has long been reported. The active T3 is known to be intimately involved in feather development.

During periods of feather growth, basal metabolic rate increases to provide energy for feather production and to keep the bird warm. Thyroid hormone levels increase and in response the bird increases heat production. If Se is limiting, T3 levels might be expected to be lower. As feather cover increases, the basal metabolic rate falls and heat production is diminished because oxidative metabolism decreases (Edens, 2000).

Immunology. Se deficiency has been reported to decrease both cellular and humoral immune function in man and laboratory animals (Combs and Combs, 1986).The knowledge of specific mechanisms in lives tock is less detailed than in laboratory animals although the increase in susceptibility to disease in deficient livestock is well documented (Maas, 1998). Sordillo et al., (1997) reported that Se deficiency is an established risk factor in mastitis incidence and has been correlated with decreased bactericidal activity of neutrophils and the severity of mastitis infection.

Selenium in the Immune System

The body fights disease organisms, cancers, and foreign substances by its immune system. The immune system is generally divided into two interactive parts named as innate or non-specific immunity and adaptive or specific/acquired immunity (Parkin and Cohen 2001).

Innate cellular immunity includes cellular elements consisting of macrophages, leukocytes, natural killer cells (NKC) and dendritic cells (Delves and Roitt 2000) (Fig.1).

The innate immunity also includes some components with recognition molecules such as C - reactive protein, serum amyloid protein and mannose-binding protein as acute phase proteins and helps activates the complement system for phagocytosis and cell lysis (Delves and Roitt 2000). These molecules help to distinguish host cells from invaders and facilitate phagocytosis and removal of the intruder. Secretions of pro-inflammatory cytokines (IL-1, IL-6, IL-12; TNFα,) leukotrienes, prostaglandins and reactive oxidative species (ROS) are increased by stimulated phagocytic cells (Ryan-Harshman and Aldoori 2005). The NKC lyse cancerous cells and pathogen-infected cells in response to macrophage-driven cytokines and interferons which help to arrest infections (Delves and Roitt 2000). The dendretic cells (DCs) are also activated by interferon-γ (IFNγ) and serve as antigen-presenting cells and activate naive T cells to initiate immune responses in the absence of formulated immunological memories of the antigen (Ryan-Harshman and Aldoori 2005).

Adaptive immunity is a defense system that strengthens innate immunity (Fig.1.1)(Parkin and Cohen 2001). When infection occurs for the second time , the B and T memory cells quickly activate the immune system (Parkin and Cohen 2001). The T lymphocytes represent the major portion of the cells of specific immunity (Delves and Roitt 2000). T lymphocytes originate in bone marrow and mature in thymus while B lymphocytes originate and mature in bone marrow (Delves and Roitt 2000). Both types of lymphocytes have receptors that differentiate self from non self and identify antigens specific to infective agents (Delves and Roitt 2000). On the other hand, humoral immunity is facilitated by antibodies secreted in B- cells and this immunity is highly protective against extracellular pathogens (Albers, Antoine et al. 2005) . The antibodies bind with antigen on the surface of pathogens and facilitate destruction by macrophages (Albers, Antoine et al. 2005).

Fig.1.1: Overview of the Immune System (Wintergerst, Maggini et al. 2007)

The role of Selenium in immune function

The immune system is dependent upon several processes which include production of reactive oxidative molecules (i.e. protection against microbial pathogens), organized and coordinated functions of adhesion molecules and production of soluble mediators such as eicosanoids and cytokines and receptors (McKenzie, S. Rafferty et al. 1998). Se likely influences these immune processes at all stages as it is important for optimum function of both the innate and adaptive immune systems (McKenzie, S. Rafferty et al. 1998). The production of ROS is important for microbicidal activity of immune cells, such as neutrophils, as released in the respiratory burst reaction (McKenzie, Arthur et al. 2002; Arthur 2003). Excessive production of ROS, however, is lethal. In small amounts, the ROS help to attack microbial agents by generating inflammation, but excessive and prolonged generation of these reactive species may cause damage to the host (McKenzie, Arthur et al. 2002; Arthur 2003; McKenzie, Beckett et al. 2006). The antioxidant system of the host is used as a defense against excessive ROS. The first evidence on the role of Se in immune function was derived, in 1959, from a study in dogs injected with 75Se which incorporated the isotope into a leukocyte protein (Hoffmann 2007). The protein which was observed then was later identified to be cytoplasmic glutathione peroxidase (cGSH-Px) (Rotruck, Pope et al. 1973). In sheep and in humans, Se has been found to be concentrated in tissues such as spleen, liver and lymph nodes which are involved in immune response (Spallholz 1990). The finding in 1973 that Se was required for the activity of the selenoenzyme cGSH-Px provided some insight into a mechanism by which Se exerted its biological functions including its impact on the immune system. cGSH-Px detoxifies harmful ROS such as organic hydroperoxides, as well as hydrogen peroxide, which are produced during cellular respiration (Spallholz 1990; Sunde 1990; Arthur, Bermano et al. 1996; Foster 1997; Rayman 1997). Other types of GSH-PXs, as well as other selenoenzymes and selenoproteins also play preventive roles against oxidative damage to cells in the body (Spallholz 1990). Throughout the 1970's and the 1980's, there was marked progress in research on the immunostimulatory effects of Se, as summarized by Spallholz (Spallholz 1990).

Research in the 1980's demonstrated the immunological protective roles of Se through modulation of antibody and complement production. Research has shown that Se

intensifies immunological responses to several types of immunogens such as tetanus toxoid, typhoid toxin and sheep red blood cells (Spallholz 1990). On the other hand,

when Se was deficient in the host, it has been associated with failure of neutrophil responses, reduction of neutrophil numbers, reduced antibody production to sheep red blood cells, enhanced H2O2 discharge by phagocytes, decreased antibody titers to bacterial and mycotic antigens and decreased natural killer cell activity (McKenzie, Beckett et al. 2006). Se supplementation as sodium selenite in drinking water (2.5µg of Se/ml), on the other hand, boosted the immunity from vaccination against malaria by increasing antibody-producing B-cell numbers and T-cell dependent antibody production with elevated concentration of Se in neutrophils and GSH-Px activity in lymphocytes (Desowitz and Barnwell 1980). In some instances, however, toxic levels of Se

supplements have been shown to decrease immunity (Spallholz 1990), which probably indicates the need for an optimal dose of Se for enhanced stimulation of the immune system.

The current studies also show that adequate dietary Se is essential for both innate and adaptive immune responses (Wang, Wang et al. 2009). Se deficiency affects several immune response pathways including impairment of leukotriene B4 synthesis, which assists in neutrophil migration to inflammatory sites (Arthur 2003). Similarly, a decrease in the humoral immune response (immunoglobulin production) was shown in Se deficiency both in rats and humans (Arthur 2003). For example, in Se deficiency, markers of the humoral immune system such as IgM, IgG and IgA titers were decreased in rats, while IgG and IgM titers were found to be lower in humans (Arthur 2003).

Overall, the role of Se, as an essential nutrient, for immune response is well recognized both in animals and humans (McKenzie, S. Rafferty et al. 1998; Arthur 2003). The GSH-Px facilitates the antioxidant function of Se to minimize harmful effects of lipid hydroperoxides and hydrogen peroxide (Arthur 2000). Different peroxidases function in different parts of cells and tissues (Arthur 2000; Pfeifer, Conrad et al. 2001). For example, the GSH-Px functions in the extracellular space, the cell cytosol and in cell membranes as in the gastrointestinal tract and influences the immune response of the host. In addition, the thioredoxin reductase (TR) (Miller, Walker et al. 2001), and

selenoprotein P and W also provide antioxidant functions (McKenzie, Arthur et al. 2002; McKenzie, Beckett et al. 2006). All selenoproteins with antioxidant functions have roles in the immune system (McKenzie, Arthur et al. 2002). As these selenoproteins are present in all cells, it may be possible that Se affects cellular activities through antioxidant functions and regulation of the redox-active proteins (McKenzie, Beckett et al. 2006). Thus, Se has a role in the control of several metabolic functions and specific processes that enhance the immune system. The specific immune challenges, however, determine which functions of Se will be involved in the immune response.

From the studies discussed above, information on the specific dose of Se recommended to promote optimal immune response is lacking. After the first study on the role of Se in the immune system in 1959, several studies were undertaken to establish the relationship between Se and immune response both in animal and human studies. Most of the studies used different chemical forms and doses of Se which made it difficult to interpret results and draw conclusions. This study, as part of an experimental study on selenium and bone, thus, has assessed the immune response of mice stimulated by low dose and slow release lipopolysaccharide (LPS) and supplemented with dietary Se to investigate the effect of different doses of dietary Se supplementation as sodium selenite on immune response.

We used slow-release Lipopolysaccharide (E. coli Serotype 0127) pellets to provide a consistent dose of LPS for 28 days (Innovative Research of America, Sarosota, FL) and these pellets were implanted using the method of Smith et al (Smith, Lerner et al. 2006). This provided low grade inflammation and we measured selected inflammatory and immune markers in mice supplemented with dietary Se. Based upon the evidence outlined above, Se modulates inflammation in several ways. Accordingly, several studies have been carried out to assess the extent to which Se down-regulates excessive inflammatory responses to prevent further impacts of inflammation. Most of these studies used high grade inflammation models and to our knowledge, no prior study has been carried out to assess the impact of Se on low grade inflammation. Therefore, the purposes of this study were first to determine if increasing levels of Se prevented LPS-induced alterations in numbers of selected immune cells and in biochemical markers and secondly if these effects of Se were associated with alterations in expression of selected pro-inflammatory cytokines.