Selenium and inflammatory diseases

Se was once considered a toxin when livestock and poultry suffered from alkali disease after consuming grass containing 10–20 parts per million (ppm) Se. Subsequent studies confirmed the potential for Se poisoning when laboratory rodents supplemented with 5–15 ppm of dietary Se displayed varying degrees of pathology⁽ Reference Moxon and Rhian ^{9 )}. In contrast, others found that Se deficiency (diets containing less than 0·1 ppm Se) caused diseases such as white muscle disease in cattle and lambs⁽ Reference Muth, Oldfield and Remmert ^{10 )} and Keshan disease in human subjects⁽ Reference Chen, Yang and Chen ^{11 )}. Based on these earlier studies, Se is now understood to be an essential micronutrient in the mammalian diet and our knowledge of its metabolism (Fig. 1) and beneficial functions has grown immensely. Current recommendations indicate that the upper tolerable intake of Se is between 90 and 400 µg/d (recommended daily intake between 30 and 55 µg/d) for humans⁽ Reference Monsen ^{12 )} and 0·4 mg/kg body weight in rodents⁽ Reference Abdo ^{13 )}. In a review and meta-analysis of the literature, Huang et al. ⁽ Reference Huang, Shyu and Chen ^{14 )} found that supplementation with Se (between 500 and 2000 µg/d for various durations) in critically ill patients decreased mortality rates associated with sepsis. Additionally, women with normal pregnancies exhibited significantly higher blood Se concentrations compared with women with pre-eclampsia, the leading cause of perinatal and maternal mortality globally⁽ Reference Ghaemi, Forouhari and Dabbaghmanesh ^{15 )}. In a model of inflammatory bowel disease, rats fed a high-Se diet (2 µg/g body weight) for 21 d exhibited decreased colonic tissue necrosis⁽ Reference Tirosh, Levy and Reifen ^{16 )}. It is important to note, however, that not all clinical trials involving Se supplementation improved health outcomes in a significant way. Recently published results from The Selenium and Vitamin E Cancer Prevention Trial (SELECT) showed that Se supplementation (200 µg/d), alone or with vitamin E for a period between 7 and 12 years, did not prevent diseases such as prostate, lung or colon cancers and there were no significant differences in cardiovascular events or diabetes between treatment groups in men⁽ Reference Lippman, Klein and Goodman ^{17 )}. Based on these equivocal findings, it is now clear that more research is required to better understand the underlying mechanisms of Se's beneficial health properties in order to design nutritional intervention strategies that yield more consistent and positive results across a range of human health disorders.

Fig. 1. Selenium metabolism from different dietary sources. Dietary intake sources of selenium include the inorganic selenate and selenite (depicted in the green stars), whereas organic sources (depicted in the red stars) are obtained from animal and plant sources that provide selenium in the form of selenocysteine (Sec), selenomethionine and selenium-methylselenocysteine (Se-methyl-Sec). Inorganic forms of selenium are reduced by thioredoxin reductase (TrxR) and thioredoxin (Trx) or converted to selenodiglutathione (GS-Se-SG) by glutathione disulfide (GSSG), reduced by glutathione reductase to glutathioselenol (GS-SeH), then converted to hydrogen selenide (H₂Se) in a reaction with GSSG. Selenoproteins are broken down by lyases to form H₂Se in intestinal enterocytes. H₂Se can then be converted into selenophosphate by selenophosphate synthase and Sec by selenocysteine synthase for incorporation of Sec into selenoproteins. H₂Se can also be converted into methylated metabolites by methyltransferases which are primarily excreted through exhalation, urine and faeces. GSH, glutathione.

Selenium functions as an antioxidant through the activity of selenoproteins

Although the importance of Se to health is not fully understood, one well-characterised function of Se is its ability to mitigate oxidative stress through antioxidant-functioning selenoproteins (Table 1), including the well-studied glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) families⁽ Reference Hamilton and Tappel ^{18 ,} Reference Sordillo and Aitken ^{19 )}. Oxidative stress occurs when the production of free radicals, including reactive oxygen species (ROS), reactive nitrogen species (RNS), oxidised proteins and oxidised lipids, outweighs an organism's antioxidant capabilities resulting in cellular/tissue damage⁽ Reference Sies ^{20 )}. The GPx and TrxR selenoproteins contain a selenocysteine in their active site making them suitable for oxidation/reduction reactions (Fig. 2). Whereas GPx1 can reduce ROS in the cytoplasm, glutathione peroxidase-4 (GPx4) has the ability to reduce fatty acid hydroperoxides (FAHP) and phospholipid hydroperoxides within cellular membranes (Fig. 2(a))⁽ Reference Kernstock and Girotti ^{21 ,} Reference Thomas, Maiorino and Ursini ^{22 )}. A longer, alternative transcript of GPx4 also was localised to mitochondrial membranes⁽ Reference Pushpa-Rekha, Burdsall and Oleksa ^{23 )} and shown to maintain ATP production during oxidative stress which could have implications on cellular activity and function during disease⁽ Reference Liang, Remmen and Frohlich ^{24 )}. Thioredoxin (Trx) reduces a variety of radicals including lipid hydroperoxides, protein thiols and ROS/RNS. Oxidised Trx is then restored to its reduced form by TrxR selenoproteins (Fig. 2(b)). Selenoproteins W, K and P (Sepw1, Selk, Sepp1) also have been suggested to have antioxidant capabilities, but mechanisms are less understood⁽ Reference Fairweather-Tait, Bao and Broadley ^{25 ,} Reference Jeong, Kim and Chung ^{26 )}.

Fig. 2. General reaction mechanisms for antioxidant glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). (a) GPx catalyses the chemical reduction of lipid peroxides or H₂O₂ to respective alcohols and water by glutathione (GSH) which forms glutathione disulfide (GSSG). Glutathione reductase catalyses the reduction of GSSG back to GSH in the presence of NADPH. (b) Oxidised protein disulfides and other free radicals are reduced to their corresponding thiols by thioredoxin (Trx). TrxR then catalyses the reduction of oxidised Trx in the presence of NADPH.

Table 1. Summary of mammalian selenoproteins with characterised functions*

GPx, glutathione peroxidase; TrxR, thioredoxin reductase; Sepw1, selenoprotein W1; Selk, selenoprotein K; Sepp1, selenoprotein P plasma 1; FAHP, fatty acid hydroperoxide; SelR, selenoprotein R; Sephs2, selenoprotein HS2; Sep15, selenoprotein 15; Selm; selenoprotein M; Seln, selenoprotein N; Sels, selenoprotein S; ER, endoplasmic reticulum; SelH, selenoprotein H; SelI, selenoprotein I; Sepn1, selenoprotein N1; DIO, deiodinase; SelO, selenoprotein O; SelV, selenoprotein V.

* Adapted from Lu & Holmgren⁽ Reference Lu and Holmgren ^{31 )}, Heras et al. ⁽ Reference Heras, Palomo and Madrid ^{104 )} and Kryukov et al. ⁽ Reference Kryukov, Castellano and Novoselov ^{105 )}.

†GPx6 contains a selenocysteine (Sec) in man and a cysteine (Cys) in rodents.

Oxidative stress is a contributing factor in inflammatory disease pathologies including atherosclerosis⁽ Reference Praticò, Tangirala and Rader ^{27 )}, diabetes⁽ Reference Monnier, Mas and Ginet ^{28 )} and mastitis⁽ Reference Ranjan, Swarup and Naresh ^{29 )} among others. There is ample evidence to indicate that selenoproteins can interrupt disease pathogenesis through antioxidant-dependent mechanisms. Numerous studies in human subjects, food-animal species and rodent models demonstrated a negative correlation between measures of selenoprotein activity and disease severity due to oxidative stress⁽ Reference Bellinger, Raman and Reeves ^{30 –} Reference Sordillo ^{32 )}. Direct evidence of the importance of selenoproteins in mitigating oxidative stress was demonstrated in transgenic studies where overexpression of GPx4 significantly reduced lipid peroxidation in atherosclerosis and ischaemia–reperfusion mouse models⁽ Reference Guo, Ran and Roberts ^{33 ,} Reference Dabkowski, Williamson and Hollander ^{34 )}. Several in vitro studies also demonstrated that TrxR1 and selenoprotein P could directly reduce the lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to its corresponding hydroxyl (15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid; 15-HETE)⁽ Reference Björnstedt, Hamberg and Kumar ^{35 –} Reference Rock and Moos ^{37 )}, thus having implications in reducing atherosclerotic lesion formation as a consequence of oxidative stress⁽ Reference George, Afek and Shaish ^{38 )}. Collectively, these studies support the contention that optimally functioning antioxidant selenoproteins may be crucial for reducing excess free radical accumulation and preventing oxidative tissue damage during acute or chronic inflammation.

Role of selenoproteins in cellular redox signalling

Another way in which selenoproteins may protect against immunopathology associated with uncontrolled inflammatory responses is through redox regulation of inflammatory signalling. The redox state of cells or tissues can be defined as the ratio of oxidised and reduced forms of specific redox couples⁽ Reference Schafer and Buettner ^{39 )}. Some redox couples relevant to inflammation include NADP⁺:NADPH, glutathione disulfide:2 glutathiones (GSH), and oxidised thioredoxin (Trx(SS)):reduced thioredoxin (Trx(SH)₂). Thioredoxin and glutathione redox couples function with the help of TrxR and GPx selenoproteins, respectively. Into et al. ⁽ Reference Into, Inomata and Nakashima ^{40 )} found that GSH was capable of modifying nitrosylated forms of the myeloid differentiation factor 88 (MyD88) adaptor protein which enhanced signalling through the toll-like receptor (TLR4) pathway during acute inflammation and resulted in altered IL-8 and IL-6 expression⁽ Reference Into, Inomata and Nakashima ^{40 )}. Mitogen-activated protein kinase (MAPK) signalling also can be affected by redox tone. Apoptosis signal-regulating kinase 1 (ASK-1) is a MAPK intermediate that activates downstream pro-inflammatory and pro-apoptotic signalling cascades⁽ Reference Al-Gayyar, Abdelsaid and Matragoon ^{41 ,} Reference Kataoka, Tokutomi and Yamamoto ^{42 )}. Mammalian Trx is a direct inhibitor of ASK-1 kinase activity and a negative regulator of ASK-1-dependent gene expression⁽ Reference Al-Gayyar, Abdelsaid and Matragoon ^{41 )}. The interaction between ASK-1 and Trx was found to be highly dependent on redox status since oxidation of Trx by ROS results in ASK-1 activation. In contrast, the reduced Trx blocked ASK-1 dependent signalling, indicating a protective role of selenoproteins in regulation of apoptosis during oxidative stress⁽ Reference Saitoh, Nishitoh and Fujii ^{43 )}.

Known as the central regulator of inflammatory gene expression, NF-κB similarly can be redox regulated at several levels. Vunta et al. ⁽ Reference Vunta, Davis and Palempalli ^{44 ,} Reference Vunta, Belda and Arner ^{45 )} reported an association between increased pro-inflammatory NF-κB activation, increased TNFα production and decreased GPx1 activity when macrophages were cultured in Se-deficient media that contained only 6 pmol/ml of Se when compared with cells cultures with 2 nmol/ml of Se⁽ Reference Vunta, Davis and Palempalli ^{44 ,} Reference Vunta, Belda and Arner ^{45 )}. Decreased plasma Se (0·37 (se 0·05) compared with 0·85 (se 0·09) µmol/l)⁽ Reference Allard, Aghdassi and Chau ^{46 )} and decreased selenoprotein synthesis⁽ Reference Gladyshev, Stadtman and Hatfield ^{47 )} in HIV patients were associated with enhanced oxidative stress-induced activation of NF-κB which promoted HIV viral transcription. In the cytoplasm, ROS-mediated activation of NF-κB can be facilitated through activation of protein kinase A (PKAc) which results in release of NF-κB from inhibitor of κB (IKβ)⁽ Reference Jamaluddin, Wang and Boldogh ^{48 )}, and overexpression of Trx caused a decrease in ROS-mediated NF-κB activity⁽ Reference Meyer, Schreck and Baeuerle ^{49 )}. In the nucleus, however, Trx can enhance NF-κB DNA binding by reducing oxidised cysteine resides on NF-κB⁽ Reference Matthews, Wakasugi and Virelizier ^{50 )}. Hirota et al. ⁽ Reference Hirota, Murata and Sachi ^{51 )} showed that reduced Trx is primarily found within the cytoplasm of cells; however, upon oxidant stimulation, Trx migrates to the nucleus to enhance NF-κB-DNA binding. These few examples demonstrate how selenoproteins can both positively and negatively control cell signalling depending on the inflammatory pathway and/or cellular location. Overall, Se nutrition and selenoprotein activity have the potential to improve inflammatory response outcomes in several ways including combating oxidative stress in cells/tissues and through the redox regulation of inflammatory signalling pathways that lead to cytokine/chemokine production. However, another potentially important but less studied mechanism underlying the health benefits of Se may involve the biosynthesis of bioactive lipid mediators that include the eicosanoids (Fig. 3).

Fig. 3. Selenium's potential impact on the regulation of inflammation. Some of the several ways in which inflammation is mediated through selenoproteins include modifying cellular redox tone which has implications on signalling through the NF-κB, mitogen-activated protein kinase (MAPK) and PPARγ pathways, controlling the expression of inflammatory mediators such as cytokines, chemokines, and cyclo-oxygenase (COX) and lipoxygenase (LOX) enzymes. Selenoproteins also combat oxidative stress which could potentially make an impact on COX/LOX enzyme activity and the production of lipid peroxides oxidised non-enzymically by free radicals. Non-enzymic lipid oxidation, COX/LOX expression and COX/LOX activity have been shown to regulate eicosanoid biosynthesis. Selenium has been studied in the context of each of these regulators and the present review focuses specifically on selenium's impact on eicosanoid biosynthesis. ROS, reactive oxygen species.

Regulation of inflammation by eicosanoids

Eicosanoids are a class of lipid mediators that contribute to the orchestration of inflammatory responses. Eicosanoids are synthesised from PUFA substrates primarily found in the cellular membrane including the n-6 arachidonic acid (AA) and linoleic acid (LA) or the n-3 EPA and DHA⁽ Reference Schmitz and Ecker ^{52 )}. These fatty acid substrates are oxidised non-enzymically by free radicals or through different enzymic pathways including the cyclo-oxygenases (COX), lipoxygenases (LOX) and cytochrome P450 pathways to produce both pro-inflammatory and resolving eicosanoids (Fig. 4). Non-enzymic oxidation of AA produces the isoprostane series of PG-like eicosanoids. These lipid mediators have been characterised as biomarkers for oxidative stress⁽ Reference Roberts and Morrow ^{53 )}. As such, they have been quantified in models of inflammatory disease, like atherosclerosis, to identify relationships between disease progression and oxidative damage⁽ Reference Lakshmi, Padmaja and Kuppusamy ^{54 )}. In addition to the isoprostanes, non-enzymic oxidation of AA or LA can also produce hydroperoxide metabolites HPETE or hydroperoxyoctadecadienoic acid (HPODE), respectively, that are enhanced during oxidative stress⁽ Reference Spiteller and Spiteller ^{55 )}. Two isoforms of COX enzymes are involved in the enzymic oxidation pathways. Whereas COX-1 is constitutively expressed in cells, COX-2 expression is inducible during inflammation⁽ Reference Xie, Chipman and Robertson ^{56 ,} Reference Kurumbail, Kiefer and Marnett ^{57 )}. COX catalyse the oxidation of n-6 AA to PGG₂ and PGH₂ ⁽ Reference Dubois, Abramson and Crofford ^{58 )}. From PGH₂, downstream PG synthases produce PGE₂, PGD₂, PGI₂, PGF_2α, among others. Alternatively, thromboxane (TX) synthases convert PGH₂ to thromboxane A₂ (TXA₂) and thromboxane B₂ (TXB₂). Similar to the COX family, there are several isoforms of LOX involved in the enzymic oxidation of fatty acids. For example, 5-LOX catalyses the oxidation of n-6 AA to 5-HPETE which can be further metabolised to produce leukotrienes (LT). Both 15-LOX-1 (12-LOX in mice) and 15-LOX-2 (8-LOX in mice) oxidise AA to 12/15-HPETE⁽ Reference Kuhn and Thiele ^{59 )}. More recent studies have led to the discovery of anti-inflammatory lipoxins (LX) that are produced from the metabolism of 12/15-HPETE intermediates by the 5-LOX pathway⁽ Reference Chiang, Arita and Serhan ^{60 )}. Likewise, 12/15-LOX-1 can oxidise the n-6 LA into 9-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPODE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE)⁽ Reference Kuhn and O'Donnell ^{61 )}. Hydroperoxides can then be reduced to form hydroxyl intermediates (HETE and hydroxy-octadecadienoic acid (HODE)) and further dehydrogenated to form ketone intermediates (oxo-eicosatetraenoic acid (oxoETE) and oxo-octadecadienoic acid (oxoODE))⁽ Reference Yuan, Rapoport and Soldin ^{62 )}. n-3 Fatty acids also can be oxidised by COX and LOX to produce eicosanoids with more anti-inflammatory or resolving properties⁽ Reference Schmitz and Ecker ^{52 )}. EPA is metabolised by 5-LOX and modified forms of COX-2 to produce E-series resolvins (Rv)⁽ Reference Oh, Dona and Fredman ^{63 )}, whereas 12/15-LOX converts DHA to the D-series Rv, protectins (PD1) and the macrophage-specific maresin (MaR₁). During uncontrolled inflammation, a combination of exacerbated production of pro-inflammatory eicosanoids and diminished production of anti-inflammatory eicosanoids prevents full resolution and restoration of homeostasis⁽ Reference Merched, Ko and Gotlinger ^{64 )}. Therefore, the balance between production of pro- and anti-inflammatory eicosanoids is one factor that determines the inflammatory phenotype of a cell/surrounding microenvironment⁽ Reference Serhan and Savill ^{65 )}.

Fig. 4. Eicosanoid biosynthesis pathways. n-3 and n-6 Fatty acids are released from the cellular membrane by phospholipase enzymes. Long-chain PUFA are oxidised either non-enzymically by free radicals or by cyclo-oxygenase-1/2 (COX-1/2), 15-lipoxygenase (15-LOX) and 5-LOX enzymes to produce eicosanoid signalling metabolites. AA, arachidonic acid; AcCOX, aspirin-acetylated cyclo-oxygenase; 15-epi LXA₄, 15-epi lipoxin A₄; Rv, resolvin; F₂-IsoP, PG-like F₂ isoprostanes; TX, thromboxane; 15d-PGJ₂, 15-deoxy-Δ^12,14PGJ₂; LA, linoleic acid; HPETE, hydroperoxyeicosatetraenoic acid; 15-oxoETE, 15-oxo-eicosatetraenoic acid; HETE, hydroxy-eicosatetraenoic acid; MaR₁, maresin; PD, protectin; HODE, hydroxy-octadecadienoic acid; 5-oxoETE, 5-oxo-eicosatetraenoic acid; LT, leukotriene.

Eicosanoid abundance and timing of their production are crucial to successfully initiate and resolve inflammation. Eicosanoid biosynthesis is regulated at several levels and both Se and selenoproteins have been studied in the context of: (1) altering eicosanoid profiles as a function of manipulating dietary Se; (2) feedback loops from other eicosanoids; (3) chemically reducing lipid hydroperoxides; and (4) modifying expression and activity of COX/LOX enzymes (Table 2 and Fig. 5). However, research has just begun to uncover the underlying mechanisms of how Se can influence eicosanoid biosynthesis at each level of regulation.

Fig. 5. Proposed interactions of selenium with eicosanoid biosynthesis pathways. (a) Selenium and selenoproteins interfere with eicosanoid feedback loops. While glutathione peroxidase (GPx)-1 and -4 can reduce fatty acid hydroperoxides (FAHP) to decrease cyclo-oxygenase-2 (COX-2) activity, a buildup of FAHP, when GPx activity is lacking, can also inhibit COX-2. GPx-2 and -4 diminish PGE₂-dependent expression of COX-2. Selenium enhances 15-deoxy-Δ^12,14PGJ₂ (15d-PGJ₂) production which is a ligand for PPARγ. PPARγ signalling enhances haematopoietic PGD₂ synthase (H-PGDS), which synthesises PGD₂, an upstream metabolite of 15d-PGJ₂. AA, arachidonic acid; mPGES-1, microsomal PGE₂ synthase-1. (b) Antioxidant selenoproteins can affect different signalling pathways leading to activation of NF-κB and activator protein-1 (AP-1) and expression of COX, lipoxygenase (LOX) and other inflammatory mediators such as TNFα and macrophage chemoattractant protein-1 (MCP-1). GPx can alter the redox state of the myeloid differentiation factor 88 (MyD88) adaptor protein, when MyD88 is denitrosylated by GPx with glutathione (GSH), signalling is enhanced. Reactive oxygen species (ROS)-mediated phosphorylation of inhibitor of κB (IKβ) can be dampened when antioxidant selenoproteins are present to scavenge ROS. The mitogen-activated protein kinases (MAPK) can also be affected; ROS-mediated oxidation of thioredoxin (Trx) causes its dissociation from apoptosis signal-regulating kinase 1 (ASK-1), enhancing signalling activity. In the nucleus, Trx can reduce oxidised cysteine residues on NF-κB, enhancing DNA binding and transcription. TLR4, toll-like receptor; TrxR, thioredoxin reductase; IKK, IκB kinase; Trx(SS), oxidised Trx; JNK, c-Jun N-terminal protein kinase.

Table 2. The impact of selenium and selenoproteins on eicosanoid biosynthesis

↓, Decrease; ↑, increase; H-PGDS, haematopoietic PGD₂ synthase; mPGES-1, microsomal PGE₂ synthase-1; PGIS, prostacyclin synthase; TXAS, thromboxane A₂ synthase; LTA₄H, leukotriene A₄ hydrolase; COX-2, cyclo-oxygenase-2; 15-LOX, 15-lipoxygenase; GPx4, glutathione peroxidase-4; TXB₂, thromboxane B₂; LTB₄, leukotriene B₄; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; HPODE, hydroperoxyoctadecadienoic acid; HODE, hydroxy-octadecadienoic acid.

Selenium and eicosanoid profiles

Previous studies have documented how dietary Se has an impact on the biosynthesis of eicosanoids in several different species. Following 2 years of supplementation, increased Se in the diet of human subjects (100 µg/d) was correlated with a decreased ratio of urinary 11-dehydro TXB₂:2,3 dinor 6-keto PGF_1α. Increased ratios of TXB₂:6-keto PGF_1α are an indicative biomarker for thrombosis and atherosclerosis⁽ Reference Arnaud, Bost and Vitoux ^{66 )}. Previous research by Meydani⁽ Reference Meydani ^{67 )} and then Haberland et al. ⁽ Reference Haberland, Neubert and Kruse ^{68 )} confirmed that adequate Se intake (300 µg Se/kg and 0·2 ppm, respectively) in rats can decrease the ratio of TXB₂:PGF_1α following short-term (2 months) and long-term (eight generations) of dietary modulation, respectively. In dairy cattle with mastitis, Se-sufficient diets (0·05 mg Se/kg) were associated with decreased pro-inflammatory TXB₂, PGE₂ and LTB₄ eicosanoid production and secretion in milk compared with cows with deficient Se intake after 1 year of dietary interventions⁽ Reference Maddox, Reddy and Eberhart ^{69 )}. Taken together, these results indicate that dietary Se could potentially diminish pro-inflammatory eicosanoid biosynthesis during inflammatory diseases.

Se can also alter feedback loops involved with eicosanoid biosynthesis. One example was reported on the positive feedback loop involving the ability of 15-deoxy-Δ^{12 , 14}PGJ₂ (15d-PGJ₂) to perpetuate anti-inflammatory eicosanoid production by enhancing the expression of its upstream synthesis enzyme in macrophages. Compared with Se deficiency (6 pmol/ml of Se from media FBS (fetal bovine serum) compared with cells supplemented with 250 nm), culturing murine macrophages with Se to maximise GPx activity enhanced 15d-PGJ₂ production; 15d-PGJ₂ is a ligand for PPARγ that, once activated, enhanced H-PGDS (haematopoietic PGD₂ synthase) expression. H-PGDS converts PGH₂ to PGD₂, which is an upstream metabolite of 15d-PGJ₂ ⁽ Reference Gandhi, Kaushal and Ravindra ^{70 )}. Thus, depending on the level of regulation, Se could potentially dampen pro-inflammatory eicosanoid biosynthesis and enhance more anti-inflammatory eicosanoid production; however, more research is needed to determine the specific mechanisms involved at different levels of regulation of eicosanoid biosynthesis and which selenoproteins could have an effect.

Antioxidant-dependent regulation of eicosanoid biosynthesis

There is evidence that certain selenoproteins are at least partially responsible for the ability of Se to modify eicosanoid biosynthesis. A direct cause-and-effect relationship between GPx4 and LT production in cancer cells was previously explored by Imai et al. ⁽ Reference Imai, Narashima and Arai ^{71 )}. At the metabolite level, GPx4 overexpression was shown to reduce FAHP from the 5-LOX pathway (5-HPETE to 5-HETE), thus preventing the production of LTB₄ and C₄ in the leukaemia cell line⁽ Reference Imai, Narashima and Arai ^{71 )}. The proposed mechanism was the antioxidant capabilities of GPx4 and the ability to reduce FAHP to hydroxyl derivatives. Others found that GPx4 reduced 15-HPETE to 15-HETE and pre-incubation of endothelial cells with GPx4 could prevent peroxide formation⁽ Reference Schnurr, Belkner and Ursini ^{72 )}. Both TrxR and Sepp1 also were shown to have lipid hydryperoxidase activity for 15-HPETE, thus supporting the contention that these selenoproteins can function as antioxidant enzymes against highly reactive hydroperoxy intermediates formed during eicosanoid metabolism⁽ Reference Björnstedt, Hamberg and Kumar ^{35 ,} Reference Rock and Moos ^{37 )}. Collectively, these studies suggest that selenoproteins have an important role in protecting cells against oxidative damage caused by lipid hydroperoxides found in the eicosanoid network.

Individual selenoproteins also can modify eicosanoid biosynthesis through controlling the activity of COX/LOX enzymes. Walther et al. described how the Se-containing compound ebselen inhibited 15-LOX activity by altering the oxidation status of the active-site Fe molecule⁽ Reference Walther, Holzhutter and Kuban ^{73 )}. The activation of COX enzymes also requires oxidation of their active site haeme Fe to form a tyrosyl radical that is then capable of oxidising AA and other fatty acid substrates⁽ Reference Marnett, Rowlinson and Goodwin ^{74 )}. GPx1 can inhibit COX enzyme activity by chemically reducing hydroperoxides that could otherwise activate enzymic oxidation⁽ Reference Cook and Lands ^{75 )}. An abundance of eicosanoid metabolites and other radicals, however, can also inhibit the activity of eicosanoid enzymes through what is known as ‘suicide inactivation’, as described for COX⁽ Reference Smith and Lands ^{76 )}, PGI synthase⁽ Reference Wade, Voelkel and Fitzpatrick ^{77 )} and thromboxane A₂ synthase (TXAS)⁽ Reference Jones and Fitzpatrick ^{78 )}. A decrease in COX activity was described in human endothelial cells due to a buildup of peroxides during diminished GPx1 activity⁽ Reference Hampel, Watanabe and Weksler ^{79 )}. These findings suggest that cellular levels of FAHP are critical in COX enzyme activity; both an excess of FAHP or absence of these radicals can result in COX inhibition. This is interesting because GPx-mediated reduction of FAHP could have different effects on COX or LOX activity depending on the accumulation of FAHP. FAHP generated by the 15-LOX pathway were shown to be affected by another selenoprotein in vitro. Sepp1, a selenoprotein present in plasma, was shown to chemically reduce 15-HPETE into 15-HETE⁽ Reference Rock and Moos ^{37 )}. Additionally, Sepp1 decreased the production of free radicals following stimulation with 15-HPETE in vitro ⁽ Reference Rock and Moos ^{37 )}. This study highlighted the antioxidant properties of the plasma selenoprotein, Sepp1, which could have significant implications in preventing oxidative stress associated with vascular inflammatory diseases, such as atherosclerosis.

Redox regulation of eicosanoid biosynthesis

Another way that Se can affect eicosanoid profiles is through the redox regulation of eicosanoid enzyme expression. Pre-treating chondrocytes with physiological levels of selenomethionine (Se-Met) (0·5 µm) for 24 h, for example, decreased IL-1β-induced gene expression of COX-2 and consequent synthesis of PGE₂ ⁽ Reference Cheng, Stabler and Bolognesi ^{8 )}. Hwang et al. showed in mice that supplementation with 30 µg selenate per g body weight for 2 weeks decreased tumour size and COX-2 expression in a model of colon cancer⁽ Reference Hwang, Kim and Surh ^{80 )}. Addition of various supraphysiological doses of Se (250–500 µm) to cultured HT-29 cells dampened extracellular signal-regulated kinase (ERK) signalling following stimulation with a tumour-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), and increased MAPK signalling; both of which decreased COX-2 expression⁽ Reference Hwang, Kim and Surh ^{80 )}. In another model, prostate cancer cells (PC3) pre-treated with sodium selenite (0·5–5 µm) for 24 or 48 h had significantly decreased NF-κB activity, which is another pathway known to control COX-2 expression⁽ Reference Pei, Li and Guo ^{81 )}. As described earlier, the redox control of these signalling pathways can occur at several signalling intermediates. Collectively, these studies support the concept that Se can decrease COX-2 expression, at least in part, through the regulation of various redox-dependent signalling pathways. More research is needed, however, to characterise cause-and-effect relationships identifying where specific selenoproteins could be regulating COX-2 expression through other redox-regulated signalling pathways.

Selenium can affect eicosanoid biosynthesis in cancer models

Inflammatory pathways can play an important role in cancer development through regulation of cell proliferation and migration⁽ Reference Coussens, Zitvogel and Palucka ^{82 )}. For example, eicosanoids can play an important role in tumorigenesis by regulating apoptosis and proliferation of cancer cells⁽ Reference Sheng, Shao and Morrow ^{83 ,} Reference Avis, Hong and Martínez ^{84 )} and Se may exert anti-cancerous properties through the manipulation of eicosanoid signalling. For example, Ghosh et al. ⁽ Reference Ghosh ^{85 )} reported that supplementation with various Se doses (0–3 µm) for 72 h induced apoptosis of LNCaP human prostate cancer cells but not of normal PrEC prostate cells⁽ Reference Ghosh ^{85 )}. Additionally, they noted that stimulation of LNCaP with 5-LOX-derived eicosanoids, 5-HETE and 5-oxoETE (5-oxo-eicosatetraenoic acid), reversed Se's apoptotic effect and enhanced growth of cancerous cells, thus indicating that 5-LOX-derived eicosanoids may play a role in promoting cancerous cell growth in prostate cancer⁽ Reference Ghosh ^{85 )}. Other researchers explored the relationship between specific selenoproteins and eicosanoid regulation in models of colon cancer. In GPx2-silenced HT-29 colon cancer cells, an increase in COX-2 and microsomal PGE₂ synthase-1 enzyme expression with a concomitant increase in PGE₂ production was reported⁽ Reference Banning, Florian and Deubel ^{5 )}. The authors proposed that GPx2 disrupted the positive feedback loop of PGE₂-dependent expression of COX-2, representing a unique role specific for GPx2 in the colon cancer model⁽ Reference Banning, Florian and Deubel ^{5 )}. This same feedback loop also was studied in the context of GPx4 and a fibrosarcoma cancer model. In L29 fibrosarcoma tumour cells, overexpression of GPx4 prevented tumour growth, decreased COX-2 expression and PGE₂ production, and abrogated PGE₂-dependent COX-2 expression⁽ Reference Heirman, Ginneberge and Brigelius-Flohé ^{86 )}. These studies provide examples in cancer models that the redox-regulating properties of certain selenoproteins could decrease pro-inflammatory eicosanoid production and reduce inflammatory-dependent tumour progression.

Selenium's effect on eicosanoid biosynthesis in CVD models

Atherosclerosis is another inflammatory-based disease that remains the leading cause of death in the developed world⁽ Reference Lloyd-Jones, Adams and Carnethon ^{1 )}. As such, interest is growing in understanding how Se may be beneficial in CVD models. Oxidative stress plays a significant role in the aetiology of cardiovascular lesion development by promoting the production of oxidised lipoproteins (oxLDL) and lipids such as the non-enzymically oxidised eicosanoids, PG-like F₂ isoprostanes (F₂-IsoP)⁽ Reference Lakshmi, Padmaja and Kuppusamy ^{54 )}. These radicals, oxLDL in particular, are recognised and internalised by circulating monocytes which initiate foam cell development and macrophage infiltration into blood vessels⁽ Reference Quinn, Parthasarathy and Fong ^{87 )}. The lipid hydroperoxide scavenging GPx4 was overexpressed in a mouse model of atherosclerosis (ApoE^−/– mice) which resulted in decreased overall atherosclerotic lesion development⁽ Reference Guo, Ran and Roberts ^{33 )}. The mechanisms behind the protective effect of GPx4 in this study were thought to be enhanced through GPx4's antioxidant capabilities to decrease the accumulation of hydroperoxide radicals and diminish oxidative stress. In support of this theory, both F₂-IsoP production and accumulation of intercellular and secreted hydroperoxides were significantly decreased in GPx4-overexpressing mouse aortic endothelial cells compared with atherosclerotic cells⁽ Reference Guo, Ran and Roberts ^{33 )}. When mitochondrial GPx4 was overexpressed in a mouse ischaemia–reperfusion model, researchers documented significantly increased cardiac function and decreased lipid peroxidation⁽ Reference Dabkowski, Williamson and Hollander ^{34 )}. In another atherosclerosis model, ApoE^−/– and GPx1 double knockout mice exhibited significantly increased atherosclerotic lesion development, suggesting that GPx1 may also play a role in disease progression⁽ Reference Lewis, Stefanovic and Pete ^{88 )}. Taken together, these data suggest that GPx could be a potential therapeutic target during heart disease due to their antioxidant properties and their capability to reduce lipid hydroperoxides and other radicals to less reactive lipid alcohols.

In addition to the antioxidant properties of selenoproteins, other possible mechanisms to explain Se's protective effects in an atherosclerosis disease model were examined. For example, Paniker et al. explored the impact of fatty acid substrate availability and downstream eicosanoid enzymic expression⁽ Reference Panicker, Swathy and John ^{89 )}. In their study, sodium selenite (8 µg/100 g body weight) supplementation for 30 d in isoproterenol-induced myocardial infarction in rats decreased LOX activity, leukotriene A₄ hydrolase (LTA₄H) expression, and LTB₄ production in monocytes⁽ Reference Panicker, Swathy and John ^{89 )}. Se supplementation also decreased the amount of NEFA in the heart which can serve as substrates for LOX enzymic pathways. The expression of LTA₄H was diminished and resulted in decreased LTB₄ concentrations. By diminishing the expression of LTA₄H, the intermediate lipid metabolite LTA₄ is prevented from being metabolised to the more pro-inflammatory eicosanoid LTB₄, and preserved for the biosynthesis of resolving eicosanoids, such as LXA₄. Although the mechanism behind the decrease in LTA₄H in Se-treated animals was not explored, evidence suggests that specific enzymic pathways are potential target for Se-mediated treatment of uncontrolled inflammation. The current findings support the concept that antioxidant selenoproteins could play a role in controlling both non-enzymic and COX/LOX-mediated oxidation of lipid mediators during CVD. Further research is needed, however, to determine which antioxidant selenoproteins are most critical for regulating eicosanoid biosynthesis and lipid peroxide-mediated disease progression.

Selenium's impact on eicosanoids in specific cell-types: endothelial cells

Since many different cell types function in concert during inflammation, studies have focused on characterising the effects of Se on single-cell cultures to determine their role in inflammatory disease. Endothelial cells are an important component of the immune system. They are the barrier between the blood and tissue, regulate immune cell trafficking, and have been the focus of a number of studies on Se nutrition and eicosanoid biosynthesis. Confirmation that selenoprotein expression within endothelial cells is essential to survival was demonstrated when targeted knock out of selenoproteins in murine endothelial cells resulted in embryonic death due to haemorrhaging and erythrocyte immaturity⁽ Reference Shrimali, Weaver and Miller ^{90 )}. The ability of Se to reduce lipid radical accumulation in endothelial cells was explored in early studies by Cao et al. ⁽ Reference Cao, Reddy and Sordillo ^{91 )}. Se-deficient bovine aortic endothelial cells cultured in the presence of only 0·01 ppm Se were characterised by a significant decrease in GPx1 activity with concomitant increases in 15-HPETE and TXB₂ compared with cells supplemented with 10 ng/ml sodium selenite⁽ Reference Cao, Reddy and Sordillo ^{91 )}. The same group then explored the association between diminished Se status of endothelial cells and the ability of 15-HPETE to elicit signs of oxidative stress⁽ Reference Trigona, Mullarky and Cao ^{36 )}, enhanced adhesion molecule expression⁽ Reference Sordillo, Streicher and Mullarky ^{92 )}, higher rates of apoptosis⁽ Reference Sordillo, Weaver and Cao ^{93 )} and dampened expression of PGI₂ ⁽ Reference Weaver, Maddox and Cao ^{94 )}. Collectively, these studies support the concept that the antioxidant ability of selenoproteins is necessary to mitigate the pro-inflammatory effects of 15-HPETE and reduce endothelial cell death as a consequence of oxidative stress. Evidence also supports a direct effect of TrxR in controlling oxidative stress and inflammation in vascular endothelial cells. Trigona et al. examined the role that TrxR activity may have on the differential regulation of the antioxidant enzyme haeme oxygenase-1 (HO-1) in 15-HPETE-challenged endothelial cells⁽ Reference Trigona, Mullarky and Cao ^{36 )}. Silencing TrxR expression and activity prevented the compensatory increase in HO-1 when endothelial cells were stimulated with 15-HPETE. Additional experiments demonstrated that HO-1 induction was dependent on the TrxR redox activity since restoring intracellular levels of reduced Trx was sufficient to increase HO-1 expression when endothelial cells were cultured in Se-deficient media (less than 0·1 ppm Se)⁽ Reference Trigona, Mullarky and Cao ^{36 )}. This area requires more attention in future research, especially in the context of 15-LOX activity and redox-regulation of signalling that controls 15-LOX-derived metabolite formation as there are some conflicting reports of the role of this pathway in disease progression. Whereas some researchers have found that enhancing 15-LOX enzyme activity leads to resolving eicosanoid production⁽ Reference Serhan and Savill ^{65 )}, others have found enhanced pro-inflammatory effects⁽ Reference Rydberg, Krettek and Ullström ^{95 )}. It will be necessary to identify how selenoproteins, such as TrxR1, affect the balance of pro- and anti-inflammatory eicosanoids as a function of 15-LOX activity in endothelial cells to better understand their role in inflammatory responses.

Impact of selenium on eicosanoids in specific cell-types: leucocyte function

Lymphocytes are critical responders to inflammatory stimuli. They play a major role in inflammatory-based diseases including CVD by producing chemoattractants such as macrophage chemoattractant protein-1 (MCP-1) to enhance macrophage infiltration⁽ Reference Takahashi, Takeya and Sakashita ^{96 )}. Lymphocytes are also important sources of eicosanoids and were studied in the context of Se nutrition. One group found significant decreases in eicosanoid production from lymphocytes obtained from rats fed a Se-deficient diet containing only <0·05 mg Se/kg⁽ Reference Cao, Weaver and Chana Reddy ^{97 )}. The underlying mechanism behind the decrease in eicosanoid biosynthesis was proposed to be that Se-deficient lymphocytes had significantly diminished phospholipase D activation which is responsible for liberating fatty acid substrates from cellular membranes. Future studies should focus on determining how antioxidant selenoproteins can specifically affect the expression and activity of phospholipases, potentially through redox regulation, and how this may affect the eicosanoids produced during inflammation.

Macrophages are especially crucial in pathogen recognition and orchestration of inflammation. Since macrophages synthesise copious amount of ROS to aid in pathogen destruction, they rely on selenoprotein antioxidants to reduce excess radicals that have the potential to cause self-damage⁽ Reference Carlson, Yoo and Sano ^{98 )}. Macrophages were acknowledged as a key cell type in the early development of atherosclerosis because they are responsible for recognising and ingesting oxidised lipoproteins (oxLDL)⁽ Reference Quinn, Parthasarathy and Fong ^{87 )}. Macrophages were the focus of several reports characterising eicosanoid regulation as a function of Se status. Prabhu et al. were interested in exploring the relationship between Se nutrition and the pro-inflammatory signalling pathway, NF-κB⁽ Reference Prabhu, Zamamiri-Davis and Stewart ^{99 )}. These investigators described an association between enhanced NF-κB activity in macrophages cultured in media containing only 6 pmol/ml of Se when compared with cells supplemented with 2 nmol/ml of sodium selenite⁽ Reference Prabhu, Zamamiri-Davis and Stewart ^{99 )}. Additional studies proved that a significant increase in COX-2 enzyme expression during Se deficiency was mediated through increased NF-κB activity⁽ Reference Zamamiri-Davis, Lu and Thompson ^{100 )}. In contrast, Se supplementation (20–50 µm) was able to decreased NF-κB activation and COX-2 expression through the toll-like receptor (TLR4) pathway⁽ Reference Youn, Lim and Choi ^{101 )}. In microglial cells (macrophages specific to the central nervous system and brain), pre-treating cells with Se-containing compounds (0–10 µm) decreased lipopolysaccharide (LPS)-induced NF-κB activation, COX-2 expression and PGE₂ production⁽ Reference Nam, Koketsu and Lee ^{102 )}. Collectively, these studies suggest that Se, through the activity of antioxidant selenoproteins, could mediate eicosanoid biosynthesis by controlling NF-κB-dependent COX-2 expression. Other signalling pathways also may be involved in regulating COX-2 expression and the subsequent metabolism of lipids through this pathway. For example, LPS-stimulated macrophages cultured in Se-supplemented media (0·1 µm-sodium selenite) led to a significant decrease in LPS-induced expression of COX-2 and TNF-α by inhibition of the MAPK signalling pathway⁽ Reference Vunta, Belda and Arner ^{45 )}. Additional experiments demonstrated that mice maintained on a Se-deficient diet had significant increases in LPS-mediated infiltration of lung macrophages when compared with animals maintained on a Se-adequate diet⁽ Reference Vunta, Belda and Arner ^{45 )}. One way that Se status was suggested to alter macrophage inflammatory properties was through changes in the profile of COX-derived eicosanoids. Macrophages cultured in Se-supplemented media (0·1 µm-sodium selenite) demonstrated a time-dependent increase in the production of 15d-PGJ₂ which is an endogenous inhibitor of NF-κB activation⁽ Reference Vunta, Davis and Palempalli ^{44 )}. Recently, reports showed that downstream eicosanoid synthase enzymes also are affected by selenoproteins⁽ Reference Gandhi, Kaushal and Ravindra ^{70 )}. Se supplementation (0·1 µm) enhanced macrophage expression of H-PGDS and the subsequent increase in Δ¹²-PGJ₂ and 15d-PGJ₂ production. These effects where mediated by selenoproteins as confirmed by silencing selenoprotein expression through selenophosphate synthatase 2 in macrophages. On the other hand, microsomal PGE₂ synthase and thromboxane A₂ synthase (TXAS) were decreased during Se supplementation⁽ Reference Gandhi, Kaushal and Ravindra ^{70 )}. Together, these studies have begun to demonstrate the association between antioxidant selenoproteins and different levels of eicosanoid regulation in macrophages through several different mechanisms including modification of signalling (i.e. NF-κB, MAPK) to affect COX/LOX expression, manipulating downstream eicosanoid synthase expression, altering the production of specific eicosanoids, and disrupting eicosanoid feedback loops. However, more research is warranted to determine which specific selenoproteins are responsible for these effects in order to gain a better understanding of where in the eicosanoid cascade that Se nutritional intervention may be possible.