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Functionality and genomics of selenium and vitamin E supplementation in ruminants

Abstract. Selenium (Se) and vitamin E are essential micronutrients for animal health and production. The major function of both Se and vitamin E is to prevent the oxidative damage of biological membranes and they can influence growth, reproduction, immune function, health, and product quality in ruminants. Both Se and vitamin E are important for maintaining low cellular and systemic concentrations of reactive oxygen species and lipid hydroperoxides, to ensure optimum cellular function. Discovery of various selenoproteins and vitamin Eresponsive genes has contributed significantly to improving our understanding about multiple functions of Se and vitamin E. There is evidence that these functions extend beyond the classical antioxidant properties to immunomodulation and intracellular cell signalling and gene regulation. Research in recent years has also shown that supranutritional supplementation of Se and vitamin E is required to improve the performance of ruminants under certain stressful conditions such as heat stress and during transition period. Considering the growing awareness among consumers of the benefits of antioxidant-rich food, there is a great opportunity for the livestock industries to focus on producing antioxidant-enriched milk and meat products or functional foods. The present review focuses on the recent developments in understanding multiple functions of Se and vitamin E at the cellular and molecular level and the effects of supranutritional supplementation on ruminant performance. In addition, the paper also articulates the potential opportunities to produce functional foods enriched with antioxidants, and underlines the need for optimum supplementation of these micronutrients for efficient ruminant production.

Introduction Selenium (Se) and vitamin E are essential micronutrients for animal health and production (McDowell et al. 1996; Hefnawy and Tortora-Perez 2010; Suttle 2010; Willshire and Payne 2011; Politis 2012; Chauhan et al. 2014c). The metabolic functions of Se and vitamin E are closely linked and they act synergistically. Vitamin E is the most important chain-breaking, lipid-soluble antioxidant, which very efficiently can scavenge reactive oxygen species (ROS) and lipid hydroperoxides, converting them into non-reactive forms (Hidiroglou et al. 1992a), whereas Se is an important structural component of glutathione peroxidase (GPX; Rotruck et al. 1973) and protects against oxidative damage by catalysing the destruction of H2O2 or decomposition of lipid hydroperoxides. Thus, the major function of both Se and vitamin E is

to prevent the oxidative damage of biological membranes by neutralising ROS (such as e.g. superoxide, hydroxyl radical, nitric oxide and hydro peroxides) normally produced during aerobic metabolism (Machlin and Bendich 1987; Halliwell and Gutteridge 1990). Under normal conditions, the production of free radicals and the concomitant damage at cellular and tissue levels are controlled by cellular antioxidant systems (Halliwell 1999). However, when the production of free radicals is much faster than their neutralisation by the antioxidant system, oxidative balance is perturbed, resulting in oxidative stress (OS), leading to oxidative damage (Machlin and Bendich 1987; Lykkesfeldt and Svendsen 2007). There are already comprehensive reviews on functions and metabolism of Se and vitamin E in ruminants (Hidiroglou et al. 1992a, 1992b; Miller et al. 1993; Finch and Turner 1996; McDowell et al. 1996), with more recent reviews focussing on reproduction, immune responses and product quality (Rooke et al. 2004; Spears and Weiss 2008; Hefnawy and Tortora-Perez 2010; Willshire and Payne 2011; Politis 2012; Chauhan et al. 2014c; Liu et al. 2014). The premise of the present paper is to review the recent developments in understanding the functionality of Se and vitamin E at the mammalian cellular and molecular levels, particularly in ruminants. The effects of supranutritional (higher than the recommended) supplementation of Se and vitamin E on ruminant performance under certain environmental and metabolic challenges, and the genomics of these effects, will also be discussed.

Selenium For many years, before discovery of the essentiality of Se for animal health, Se was best known for its toxic effects. The importance of Se in animal physiology was established with the association of its deficiency with vitamin E and white muscle disease (Muth et al. 1958). However, a specific biochemical role of Se as a structural component was not understood until the discovery of GPX (Rotruck et al. 1973) and it is now well established that Se is an important structural component of specific proteins called selenoproteins (SePs) (glutathione peroxidases and thio-redoxin reductases). Selenium, along with vitamin E, is an integral component of the antioxidant network of the cell (Fig. 1; Machlin and Bendich 1987; McDowell et al. 1996), and is important in maintaining low concentrations of ROS. Since the discovery of GPX, the essentiality of Se has been further elucidated, taking on more complex perspectives with the identification of various classes of other SeP (Behne and Kyriakopoulos 2001; McKenzie et al. 2002; Kryukov et al. 2003; Suttle 2010).

Selenoproteins

Since the discovery of GPX (Rotruck et al. 1973), more than 30 SePs have been identified as being expressed in the mammalian body (Table 1; Behne and Kyriakopoulos 2001; Kryukov et al. 2003). Se is incorporated as seleno-cysteine residues into SePs and is ionised, unlike sulfur, at normal pH of the body. By virtue of this property of Se, seleno-enzymes are able to participate in redox reactions (Kryukov et al. 2003; Rooke et al. 2004). Among these SePs, the functions of three major categories of SePs, namely, GPXs, thioredoxin reductases (TRs) and iodothyronine deiodinases (DI), have been well recognised in ruminants (Behne and Kyriakopoulos 2001; Suttle 2010). To some extent, the breadth and magnitude of the effects of Se status on animal growth, immune function, reproduction and mortality can be explained by the involvement of SePs in many metabolic pathways (NRC 2007).

Glutathione peroxidases (GPXs) Glutathione peroxidases include cytosolic or classical glutathione peroxidase (GPX1), gastrointestinal glutathione peroxidase (GPX2), extracellular or plasma glutathione peroxidase (GPX3) and phospholipid hydroperoxide glutathione peroxidase (GPX4). GPX1 is present in the cytosol of nearly all tissues and catalyses the reduction of H2O2 and various soluble organic peroxides (Behne and Kyriakopoulos 2001). Recent research has suggested that reduced GPX1 activity may be compensated by other components of the antioxidant system under normal conditions, but that the protective effects of GPX1 are critical during stressful conditions (Behne and Kyriakopoulos 2001; Rooke et al. 2004; Suttle 2010). GPX2 is tissue specific, being expressed in the intestinal mucosa and is important in defence against ingested lipid hydroperoxides or those endogenously produced during stressful conditions and high metabolic demands such as during the transition period (Suttle 2010). GPX3 is present in extracellular fluid, with the greatest abundance of GPX3 mRNA found in kidney and lungs (Behne and Kyriakopoulos 2001; Kryukov et al. 2003), plausibly to protect the renal tubules and the alveolar capillaries, respectively (Suttle 2010). GPX4 is mainly associated with membranes and unlike other GPXs, GPX4 can directly reduce hydroperoxides and, hence, has a major protective role against oxidative damage of biomembranes. While GPX4 is believed to be responsible for substitution of vitamin E by Se, there is a unique role of GPX4 in spermatogenesis, indicating the importance of Se in reproduction (Arthur and Beckett 1994; Ursini et al. 1999; Beckett and Arthur 2005).

Thioredoxin reductases (TRs)

Thioredoxin reductases (TRs) are a family of flavoenzymes. TR1 (cytosolic) and TR2 (mitochondrial) are ubiquitous in distribution, while TR3 is mainly localised in the testis. TRs have a wide range of functions, including regulation of transcription by regulating oxidation and reduction in the cellular membranes (Fig. 2; McKenzie et al. 2002; Suttle 2010), recycling of vitamins C and E (Fig. 1), calcium absorption (Moreno-Reyes et al. 2006) and prevention of muscular dystrophy (Surai et al. 2006). Selenium is essential for the enzymatic activity of TRs because selenocysteine is indispensable for enzyme function (Gladyshev et al. 1996; Gromer et al. 1998). The DNA-binding ability of transcription factors NF-kB (Matthews et al. 1992), AP-1, and the glucocorticoid receptors can be regulated by TRs (reviewed by McKenzie et al. 2002). As mentioned above, TRs also play an important role in regeneration of a-tocopherol (vitamin E) from a-tocopheroxyl radical (vitamin E radical) and can also reduce dehydro-ascorbic acid to ascorbic acid (vitamin C) (Fig. 1; McKenzie et al. 2002; Rimbach et al. 2010).

Iodothyronine deiodinases (DI) Another function of Se was recognised with the identification iodothyronine deiodinases (DI) that catalyse the activation of thyroid hormones. Type 1 DI (DI1) is responsible for conversion of thyroxine (T4) to triiodothyronine (T3) and plays an important role in regulation of metabolic rate, growth and immune responses. Selenium deprivation can increase the ratio of T4 to T3 in the plasma of cattle (Awadeh et al. 1998), whereas Se supplementation increases plasma T3 concentrations in calves (Wichtel et al. 1996) and sheep (Rock et al. 2001). Type 2 DI (DI2) can also convert T4 to T3 and is predominant in nonruminants. Type 3 DI (DI3), found in placenta, catalyses the conversion of T4 and T3 to inactive metabolites (Salvatore et al. 1995). An adequate supply of Se is important to ensure the sufficient expression of DI and basal metabolic rate and physiological processes such as survival under cold stress and immune responses (Hefnawy and Tortora-Perez 2010; Suttle 2010).

Distribution of Se and the expression of different selenoproteins Another interesting contribution of recent Se research is the enhanced understanding of the hierarchical distribution of Se and the expression of various SePs in different tissues. In Sedepletion studies, the prioritisation determined that certain organs (brain, endocrine and reproductive organs) still retained Se, whereas Se was lost rapidly from liver and muscle, the latter explaining why white muscle disease is the most common condition of Se deficiency

(Hefnawy and Tortora-Perez 2010; Rooke et al. 2004). In these tissues, GPX4 synthesis takes priority over GPX1 and GPX3 (Behne and Kyriakopoulos 2001). Similarly, during limited Se availability, selenoenzymes in the central organs are synthesised as a priority, while the synthesis of blood GSH-Pxs are the last priority under these conditions. It is therefore reasonable to expect the higher requirements of Se for better health and immune responses, because blood GSH-Pxs are very important for an effective defence against pathogens. Respiratory burst (generating superoxide and H2O2) is the major defence mechanism adopted by neutrophils and macrophages to kill bacteria. However, GSH-Pxs and TRxs are required to protect the selfdamage to immune cells from these ROS (McKenzie et al. 2002; Rooke et al. 2004). Therefore, sufficient Se is important to ensure robust immune responses against the invading pathogens. To conclude this section, considerable progress has been achieved in Se research in recent years and the identification of various SePs has laid the foundation for understanding multiple functions of Se. However, the control switch(s) at the cellular or molecular level that regulates Se distribution and the prioritising of selenoenzyme expression still need to be elucidated. It is clear that sufficient Se is essential to ensure the expression of various selenoenzymes to maximise their beneficial effects.

Vitamin E Vitamin E consists of mixtures of tocopherols and tocotrienols synthesised in plants. Tocopherol homologues (a, b, g, d) have a fully saturated 16-carbon isoprenoid side chain, whereas tocotrienols have a similar isoprenoid chain with three double bonds (Fig. 3). Among the various tocopherol homologues, a- tocopherol is the most biologically active (Rimbach et al. 2002a). There are eight possible stereoisomers of a-tocopherol. Four stereoisomers possess the 2R configuration (RRR, RRS, RSS and RSR) and the other four stereoisomers possess the 2S configuration (SSS, SRR, SSR and SRS; Fig. 2). The naturally occurring form of a-tocopherol (RRR-a-tocopherol) possesses 3R configuration, whereas the synthetic form (commercially available) of a-tocopherol (all-rac a-tocopherol) contains all eight possible stereoisomers (Politis 2012). The major function of vitamin E is to oxidative damage to cell membranes (Putnam and Comben 1987). Polyunsaturated fatty acids in cell membranes are the prime targets of ROS produced during the normal aerobic metabolism (Bekhit et al. 2013). If not scavenged by the antioxidants, ROS accumulation leads to OS and, ultimately, results in oxidative damage of lipids, proteins and other major molecules in the cell (Lykkesfeldt and Svendsen 2007; Celi 2011), disrupting membrane integrity and normal function of the cell (Trevisan et al. 2001). Vitamin E exerts its antioxidant action by

donating the hydrogen atom from the phenolic hydroxyl group to render free radicals less reactive and this process forms a stable lipid species (Rimbach et al. 2010). As mentioned above, excess free radicals react with the lipids to generate highly reactive lipid peroxyl radicals, which, in turn, can trigger further damage by forming more free radicals and this chain reaction goes on if not controlled. Thus, vitamin E prevents the propagation of a chain of radical damage and, hence, is called a chain-breaking antioxidant. However, it is important to appreciate that vitamin E does not work in isolation, rather it is one of the various components of the antioxidant system of the cell (Fig. 2).

Supplementation of vitamin E and Se in ruminants Vitamin E and Se act synergistically to prevent oxidative damage of cells (Hoekstra 1975). Supplementation of Se and vitamin E has been advised for many years in animals with deficiency symptoms of these micronutrients, to prevent white muscle disease, to improve the reproductive efficiency and immune responses, and to control mastitis in dairy cows and lipid peroxidation of beef and meat (Hogan et al. 1993; Miller et al. 1993; McDowell et al. 1996; Smith et al. 1997; Politis et al. 2004; Willshire and Payne 2011; Politis 2012). Given that multiple functions of vitamin E and Se have been recognised, recent research has focussed on the supranutritional (above the recommended) supplementation of these micronutrients.