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REVIEW ARTICLEClaire M. Weekley and Hugh H. HarrisWhich form is that? The importance of selenium speciation and metabolism in the prevention and treatment of disease

www.rsc.org/chemsocrev Volume 42 | Number 23 | 7 December 2013 | Pages 8801–9198

Chemical Society Reviews

8870 Chem. Soc. Rev., 2013, 42, 8870--8894 This journal is c The Royal Society of Chemistry 2013

Cite this: Chem. Soc. Rev.,2013,42, 8870

Which form is that? The importance of seleniumspeciation and metabolism in the prevention andtreatment of disease

Claire M. Weekley* and Hugh H. Harris

The biological activity of selenium is dependent upon its speciation. We aim to integrate selenium speciation

and metabolism into a discussion of the mechanisms by which selenium exerts its biological activity. First, we

present the current status of selenium in the prevention of cancer, cardiovascular and neurodegenerative

diseases with particular attention paid to the results of major chemoprevention trials involving selenium

supplementation. A comprehensive review of the current understanding of the metabolism of common

dietary selenium compounds – selenite, selenomethionine, methylselenocysteine and selenocystine – is

presented, with discussion of the evidence for the various metabolic pathways and their products. The

antioxidant, prooxidant and other mechanisms of the dietary selenium compounds have been linked to

their disease prevention and treatment properties. The evidence for these various mechanisms – in vitro,

in cells and in vivo – is evaluated with emphasis on the selenium metabolites involved. We conclude that

dietary selenium compounds should be considered prodrugs, whose biological activity will depend on

the activity of the various metabolic pathways in, and the redox status of, cells and tissues. These factors

should be considered in future laboratory research and in selecting selenium compounds for trials of

disease prevention and treatment by selenium supplementation.

1 Introduction

Selenium is a micronutrient that is of potential use in the preven-tion and treatment of disease. It has been a rapid turnaround forthe element: after being discovered and named by Berzelius in1817, selenium was identified in the 1930s as the cause of livestockdeath in some parts of the US due to its high levels in cerealgrains.1 In the 1950s its essentiality to mammals was recognised2

and in 1973 the identification of the first selenoprotein, glutathioneperoxidase (GPx), was reported.3,4 At present, 25 selenoproteins,containing selenocysteine (SeCys) residues at their active sites, areknown in humans with roles as antioxidants (GPxs), in seleniumtransport (selenoprotein P), in maintaining intracellular redoxstatus (thioredoxin reductases) and in thyroid hormone production(iodothyronine deiodinases), amongst other functions; some ofwhich are yet to be determined. The essentiality of selenium isfurther highlighted by the high incidence of Keshan disease, acardiomyopathy, and Kashin–Beck disease, an osteoarthropathyin areas of endemic selenium deficiency.5,6

Selenium intake varies worldwide (ranging between 7 and4990 mg per day) and is often dependent on the selenium

content of the soils in which crops are grown. The averagerecommended daily intake of selenium for adults is 53 mg perday for women and 60 mg per day for men.7 The Australian andNew Zealand guidelines recommend 60 mg per day for adultwomen and 70 mg per day for adult men, with an upper limit of400 mg per day.8 Early trials with selenised yeast at doses up to800 mg of selenium per day for several years showed no adverseeffects.9 Trial participants on doses up to 1600 mg per day for anaverage of 12 months have reported symptoms of selenosis: garlicbreath, brittle hair and nails and stomach upset.1,10 There isrecent evidence that long-term administration of as little as200 mg per day selenium (as SeMet or selenised yeast) is associatedwith the increased incidence of alopecia and dermatitis11 andtype 2 diabetes.12

An inverse relationship between selenium status and theincidence of various diseases has been observed in epidemiologicalstudies: these studies in addition to laboratory studies and sele-nium’s reputation as an antioxidant have lead to the hypothesis thatselenium supplementation may be useful in disease prevention.However, studies of selenium supplementation in disease preven-tion have presented mixed results, and concerns about seleniumtoxicity persist. Understanding the efficacy of selenium in diseaseprevention and treatment is further complicated by the effects of thespeciation and metabolism of the ingested compounds on thepreventative, therapeutic and toxic mechanisms of selenium.

School of Chemistry and Physics, The University of Adelaide, SA 5005, Australia.

E-mail: [email protected]; Fax: +61-8-8313-4358;

Tel: +61-8-8313-4248

Received 24th July 2013

DOI: 10.1039/c3cs60272a

www.rsc.org/csr

Chem Soc Rev

REVIEW ARTICLE

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This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42, 8870--8894 8871

A number of authors have recently reviewed different aspectsof selenium speciation and biological activity including thepharmacology of synthetic organoselenium compounds,3,4,13,14

the antioxidant and prooxidant properties of inorganic seleniumand oxo-selenium compounds5,6,15 and the speciation of seleniumin food and supplements and their metabolism in mammals.7,16

Each of the aforementioned reviews offers a comprehensivesummary of one or two of the aspects of selenium speciation,metabolism or the prophylactic and therapeutic mechanismsof selenium.

Herein, we aim to draw all three of these important aspectsof selenium biochemistry together (speciation, metabolism andmechanisms of biological action) in respect to the most commondietary selenium compounds: selenite, selenomethionine (SeMet),methylselenocysteine (MeSeCys) and selenocystine (CysSeSeCys).In doing so, we hope to link the speciation of the ingestedcompounds and their metabolites to the their biological activitiesin mammals, with particular emphasis on the antioxidant andprooxidant activities of low molecular weight selenium compoundsand their roles in cancer, cardiovascular and neurodegenerativediseases. We will highlight gaps in the knowledge of seleniummetabolism and biological activity and comment on the outlook forselenium in the prevention and treatment of disease.

2 Selenium and disease2.1 Evidence for a role for selenium in disease prevention

Evidence from observational studies conducted across fourdecades, and reviewed elsewhere,8,17,18 has linked low serumselenium levels to higher cancer risk. There is increasingevidence that the relationship between selenium status andthe incidence of disease is U-shaped across a narrow range ofserum selenium concentrations, rather than a linear relation-ship.7,9 A prospective study of all-cause mortality and cancermortality in US adults identified a nonlinear relationshipbetween serum selenium levels and these outcomes: at lowselenium levels (o130 ng mL�1) mortality was decreased andat higher levels (>150 ng mL�1) a small increase in mortality wasobserved.19 The Netherlands Cohort Study identified an inverserelationship between toenail selenium status (as a measure oflong-term selenium exposure) and the risk of advanced prostatecancer.20 In a population with low selenium concentrations(mean levels between 63.2 and 77.1 ng mL�1), a recent Polishcase-control study found a linear relationship between serumselenium levels and lung and laryngeal cancers.21 Furtherevidence of a role for selenium compounds in cancer preven-tion and treatment is derived from laboratory studies: recent studieshave found that methylated selenium compounds (MeSeCys andmethylseleninic acid, MeSeA) inhibit the progression and metastasisof prostate cancer and extend survival in a mouse model of thecancer;22 pre- and post-application of selenite reduced the numberand size of hepatic nodules induced by a carcinogen in rats;23 andMeSeA supplementation reduced the spontaneous metastasis ofLewis lung carcinoma in mice.24

Observational studies have provided some evidence of alink between selenium status and cardiovascular diseases.

A case-control study found that serum selenium status wasinversely related to the instance of myocardial infarction anddeath from cardiovascular disease in a Finnish population.25 Ina Spanish population, serum selenium concentrations werelower in patients with acute myocardial infarction or ischaemiccardiomyopathy.26 A prospective study of German patients withsuspected coronary artery disease found that low GPx1 activityin erythrocytes was associated with an increased risk of cardio-vascular events; a weak, but significant, correlation wasobserved between GPx1 activity and selenium levels.27 However,in a US population with much higher serum selenium levels, aprospective study (the same study that found a correlationbetween all-cause and cancer mortality and selenium status)found no relationship between cardiovascular mortality andselenium status.19 Furthermore, some studies have foundadverse relationships between selenium status and risk factorsfor cardiovascular disease: high serum selenium levels wereassociated with hypertension28 and high serum lipid levels29 inUS populations. In contrast, supplementation with 100 mg perday or 200 mg per day selenium as selenised yeast significantlyreduced plasma lipid levels in participants with relatively lowbaseline selenium status.30 There is evidence of a link betweenselenium status and cardiovascular disease in animal studies:in rats fed a high selenium diet, selenium supplementationreduced the size of myocardial infarct by 30%.31

There is even greater uncertainty about the relationshipsbetween selenium status (both in the brain and in serum)and neurodegenerative diseases, partly due to the difficulty inaccurately determining concentrations of selenium in thebrain.32,33 A decline in plasma selenium has been linked tocognitive decline in an elderly cohort,34 although neuronal cellshave been found to be sensitive to selenium toxicity, relativeto prostate and cancer cells.35 Epidemiological studies arelacking, but there is experimental evidence that selenium maymitigate the effects of neurodegenerative diseases. Seleniumdeficiency in a transgenic mouse model of Alzheimer’s diseaseamyloidosis led to an elevated deposition of amyloid plaquescompared to mice on a Se-adequate diet over an 19 monthperiod.36 In a rat model of sporadic dementia of Alzheimer’stype, selenite supplementation prevented oxidative damage,morphological changes and cognitive decline37 and organo-selenium supplementation reverted oxidative stress and memoryimpairment.38 A diet supplemented with selenised yeast (1 ppmselenium in feed) decreased the amyloid burden and oxidativedamage in a mouse model of Alzheimer’s disease.39 In severalAlzheimer’s disease models, selenate reduced tau phosphorylation,which has a pathogenic role in the disease, and reduced memoryand motor deficits and neurodegeneration.40,41

2.2 Mixed results of selenium supplementation trials

The results of observational studies and laboratory experimentsand the antioxidant properties of selenium, both of seleno-proteins and low molecular weight selenium compounds, havemade it an attractive element to investigate with regard to theprevention and treatment of disease. Diseases in which oxida-tive stress is implicated, including cancer, cardiovascular and

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neurodegenerative diseases, are particularly attractive targetsof selenium supplementation. A meta-analysis of studies intothe relationship between selenium status and coronary heartdisease found that observational studies show an inverseassociation between selenium status and the risk of coronaryheart disease, but results from a small number of randomisedcontrolled trials were inconclusive.42 More recently, a Cochranereview found there was no statistically significant effect of seleniumsupplementation on all-cause mortality, cardiovascular diseasemortality or cardiovascular disease events in a meta-analysis of12 randomised controlled trials.43 A second Cochrane review foundevidence for an inverse association between selenium status andthe risk of some cancers (prostate and bladder), but warned thatthe results should be treated with caution, noting also the incon-sistent results of the few randomised controlled trials of seleniumsupplementation.44 Meta-analyses of observational studies havefound that selenium status is inversely associated with lung,45

bladder46 and prostate47 cancers. Meta-analyses that also includerandomised controlled trials of selenium supplementation havefound an inverse association between selenium status and the riskof prostate48,49 and lung50 cancer, the latter amongst populationswith low baseline selenium status.

A recent review concluded that there is currently noevidence, from the small number of studies in humans, for a rolefor selenium in the treatment of Alzheimer’s disease, but that itmay be useful in prevention.33 The prevention of Alzheimer’sdisease by vitamin E and selenium (PREADViSE) trial was con-ducted as an adjunct to the selenium and vitamin E cancerprevention trial (SELECT), which itself was terminated early (aswill be discussed shortly) and converted into an exposure trial. Dueto the termination of SELECT, and other problems with thePREADViSE trial, the effect of selenium supplementation onthe incidence of Alzheimer’s disease is unlikely to be determinedwhen the study data are unblinded.51 The lack of evidence thatselenium supplementation can prevent cardiovascular disease43

and the limited evidence for its role in cancer prevention44 arecontrary to the substantial evidence for the biological activities ofselenium and the benefits of selenium supplementationobserved in laboratory studies, particularly in regard to cancer.

There is potential for selenium compounds in disease treat-ment: selenium compounds are being investigated as potentialpharmaceuticals.14 Ebselen, an organoselenium compound,holds promise as a neuroprotective agent that has been shownto improve the outcome of acute ischaemic stroke in patientstreated shortly after onset.52 Selenium compounds, such asMeSeCys, MeSeA and CysSeSeCys, may sensitise cancer cells tochemotherapeutic drugs.53,54 In cell culture experiments, MeSeA incombination with doxorubicin, caused apoptosis in doxorubicin-resistant MCF-7 breast cancer cells.55 In the same cells, CysSeSeCysand auranofin caused synergistic enhancement of apoptosisby targeting TrxR.54 MeSeCys and tamoxifen have been shownto synergistically inhibit tumour growth in MCF-7 breastcancer xenografts.56

The contrast between laboratory studies and the resultsof human intervention trials suggests that the potential forselenium compounds in disease prevention and treatment is

yet to be realised. Attempts to understand the mechanisms ofselenium in disease prevention and treatment are complicatedby many factors, including the relative contribution of seleno-proteins and low molecular weight selenium compounds, thetoxicity of selenium compounds and the varying metabolismand biological activities of different selenium species.

3 Selenium speciation and diseaseprevention: a tale of two trials3.1 Results of the NPC trial and SELECT

A comparison of the results of two clinical trials into theprevention of cancer by selenium supplementation clearlydemonstrates the difficulty in understanding what role seleniummay play in disease prevention. The results of the trials under-score the importance of considering the speciation of theselenium supplement and the selenium status of individualsand populations. The nutritional prevention of cancer (NPC)trial is the seminal study into selenium and cancer prevention.The initial results of the trial showed that selenium supple-mentation (200 mg per day as selenised yeast) significantlyreduced total cancer mortality, total cancer incidence and theincidence of prostate, lung and colorectal cancers without anyeffect on the primary endpoint, nonmelanoma skin cancer.57

These results have driven research into the use of selenium incancer prevention for more than a decade, with both lowmolecular weight selenium compounds and selenoproteinsscrutinised in laboratory experiments. In transgenic mice withreduced selenoprotein expression, both selenoproteins and lowmolecular weight selenium compounds were found to beimportant in cancer prevention,58 but this review will primarilyfocus on the role of low molecular weight selenium compounds.The involvement of low molecular weight selenium compoundsis suggested by the fact that the baseline plasma seleniumlevels of almost all of the participants of the NPC trial wereabove that required to maximise GPx levels (70–90 ng mL�1),59,60

and lay within the range at which selenoprotein P (SelP)is thought to be maximised (between 110 ng mL�1 and125 ng mL�1 selenium).61 (Selenoproteins have roles in cancerprevention and promotion and the relationship betweenselenoproteins and cancer has been the subject of recentreviews by Hatfield et al.62 and Davis et al.,63 but the topic isbeyond the scope of this review.)

Reanalysis of the NPC trial results, after a longer period offollow-up, showed that the reductions in lung and prostatecancer incidences were restricted to participants with low(o123 ng mL�1), but nutritionally adequate, baseline serumselenium levels.64,65 Selenium supplementation was also asso-ciated with an increased risk of type 2 diabetes.12 Anotherclinical trial began in 2001: SELECT was designed to studythe effect on prostate cancer incidence of 200 mg selenium perday SeMet supplementation alone and with vitamin E.66 Thetrial was discontinued earlier than planned as there was noevidence of benefit, nor any possibility of a benefit, from eitherof the supplements.11 The authors noted a slightly increased

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risk, albeit nonsignificant, in type 2 diabetes in the seleniumsupplementation group,11 and a significantly increased risk ofprostate cancer in the vitamin E supplementation group.67 Thefailure of SELECT to show any benefit of selenium or vitamin Esupplementation is a result consistent with doubts over theability of antioxidant supplements to prevent mortality andreduce cancer incidence in healthy people68,69 and people withvarious diseases.70,71 A meta-analysis of randomised controlledtrials of selenium supplementation found that results of thetrials were inconsistent and concluded that there was not yetany convincing evidence that selenium supplementation canprevent cancer44 and Stranges et al. argue that seleniumsupplementation for disease prevention is not justified in lightof the lack of evidence of efficacy in disease prevention and thepotential risks for cardiometabolic health.72 (The risks ofselenium supplementation to cardiometabolic health alsoremain uncertain: a trial of up to 300 mg per day selenium asselenised yeast found no diabetogenic effect after 6 months’supplementation73).

3.2 Lessons from the NPC trial and SELECT

The role of selenium in biology is complex, extending beyondits antioxidant capability, and is not fully understood. Inreflecting on the different outcomes of SELECT and the NPCtrial a number of factors have been discussed by commentatorsincluding dose and baseline selenium status74,75 and chemicalspeciation.74 The mean baseline serum selenium level in theNPC trial was 114 ng mL�1 versus 135 ng mL�1 in SELECT. TheNPC trial participants with baseline selenium levels below121.6 ng mL�1 experienced significant reductions in totalcancer incidence with selenium supplementation, but thosewith baseline selenium levels greater than 121.6 ng mL�1 had anon-significant elevated incidence of cancer with seleniumsupplementation.59 The serum selenium status of NPC partici-pants supplemented with selenium averaged 190 ng mL�1;57

levels up to a mean of 250 ng mL�1 were recorded duringSELECT.11 Rayman et al. argue that the baseline seleniumstatuses and outcomes for the participants in the NPC trialand SELECT suggest that selenium supplementation has ‘‘aU-shaped dose-response relationship between selenium intakeand cancer risk.’’75 A meta-analysis of a few small trials ofselenium and bladder cancer suggests that there is an inverserelationship between selenium status and cancer incidence,46

while a meta-analysis of selenium supplementation and pros-tate cancer by Hurst et al. suggests that selenium supplementa-tion is beneficial only over a narrow range of selenium status:49

there is a clear need to test the relationship between seleniumdose and cancer risk.

Most relevant to this review is the question of how seleniumspeciation may have contributed to the results of the trials.The selenised yeast used in the NPC trial was only analysedafter the trial’s conclusion and the tablets collected froma number of batches and stored under sub-optimal conditionsvaried widely in the proportion of SeMet: 18–69% as determinedby Larsen et al.76 and 7–25% as determined by Uden et al.77

who also found selenite, g-glutamyl-methylselenocysteine

(g-glutamyl-MeSeCys) and Se-adenosylhomocysteine, which are mostlikely degradation products. In both studies, large selenium fractionsremain unidentified. Compared to the mixture of seleniumcompounds that were presumed present in the selenised yeastof the NPC trial, SELECT used a single form of selenium. SeMetwas chosen for SELECT because it is the major component ofselenised yeast (the composition of which could not be exactlyreplicated from the NPC trial), it was known to suppressmalignant prostate cells in vitro,78 and it was considered asafer and more practical choice than selenite and methylatedselenium compounds.66 However, in comparing the outcomesof the NPC trial and SELECT, we are reminded that seleniumcompounds differ in their metabolism, mechanisms of actionand therefore biological activity. As Hatfield and Gladyshevargue,74 a better understanding of selenium biology is requiredin order to better target selenium supplementation to diseaseprevention and treatment. Knowledge of the metabolic products ofselenium compounds, and their biological activities, is crucial tounderstanding selenium biology, and perhaps resolving some of theinconsistencies between the epidemiological and laboratory dataand the results of selenium supplementation trials (the majority oflab experiments use selenite, and increasingly methylated seleniumcompounds, and yet yeast and SeMet are the preferred forms ofselenium in trials). To this end, we review the current under-standing of the metabolism, mechanisms of action and bio-logical activities of the most common selenium compoundsavailable in supplements and foods – selenite, SeMet, MeSeCysand CysSeSeCys – and consider their prospective use in diseaseprevention and treatment.

4 Selenium speciation in foods andsupplements

Selenite, SeMet, MeSeCys and CysSeSeCys (which will bereferred to as dietary selenium compounds) are the mostintensively studied selenium compounds in laboratory andepidemiological studies into disease prevention and treatment.They are also found in foods. A brief discussion of seleniumspeciation in the diet is required here, but more extensivereviews of selenium speciation in foods are available.16,79,80

The structures of the selenium compounds referred to through-out this review can be found in Fig. 1.

4.1 Selenium in foods

Organic forms of selenium dominate the speciation of grains,legumes and vegetables, although selenate is also observed insome of these foods. Generally, grains and legumes are bettersources of SeMet than MeSeCys and the Allium and Crussiferaefamilies including garlic, onions and broccoli are better sourcesof MeSeCys (and related species) than SeMet.16,79,80,81 SeCys,CysSeSeCys and numerous other organic selenium species havealso been found in these foods. Rice is an important source ofselenium for much of the world’s population and the dominantspecies is SeMet (B80%) with small amounts of MeSeCys,SeCys and selenite.82,83


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4.2 Selenium in yeast and supplements

Selenite and SeMet supplements are available to the public.Selenised yeast is a popular selenium supplement and its use assuch has been reviewed by Rayman.9 SeMet is most often themajor component of selenised yeast with selenite constituting aminor component (usually no more than 1%, although up to5% has been observed).76,77,84,85 The balance of selenium is

made up by small proportions of organic compounds includingMeSeCys, its pre-curser g-glutamyl-MeSeCys, and CysSeSeCys.The form of selenium in selenised yeast tablets will depend onthe manufacturer and has been shown to change over years ofstorage and with storage conditions.86 As such the phrase ‘selenisedyeast’ does not indicate a standard formula, but encompassesa range of selenium species in various proportions with SeMet

Fig. 1 Structures of the selenium compounds referred to within this review.


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usually the major component. The complex, poorly characterisedmixture of selenium compounds in the selenised yeast used inthe NPC trial left open to conjecture which form(s) of seleniumwere responsible for the positive results of the trial. Efforts toimprove the characterisation of selenium in selenised yeast andfoods, both selenium-enriched and unenriched, and to under-stand the bioavailability of the compounds continue.87–89

Knowledge of the potential benefit or harm that might bederived from different forms of selenium used as long-term dietarysupplements becomes more important as selenium-enriched foodsand selenium supplements become more popular.

5 The metabolic routes of seleniumcompounds

The dietary selenium compounds differ significantly in theirmetabolic pathways and in their abilities to produce variousselenium metabolites. The biological activities of seleniumcompounds are exerted via their metabolites. Thus the routes bywhich each of the dietary selenium compounds are metabolisedand the relative abundance of their metabolites is entwined withthe efficacy of selenium compounds in disease prevention andtreatment. Hence the need for the characterisation of seleniumspeciation, not only in selenised yeast and selenium-enrichedfoods, but in cells and tissues.90 Given the ability of all of thedietary selenium compounds to generate selenoproteins andmethylated selenium compounds for excretion, it has beendeduced that the metabolic pathways intersect at a commonmetabolite, widely held to be hydrogen selenide (H2Se: hydrogenselenide ion (HSe�) at physiological pH). The common seleniumintermediate has sometimes been referred to as a ‘selenide pool’,but given the reactivity of selenides with oxygen91 and metals92

the intermediate is unlikely to exist freely in such quantities asthe descriptor ‘pool’ would suggest. It is convenient to separateselenium metabolism into two parts: downstream of the putativeHSe� intermediate where the selenoprotein synthesis, methyla-tion and excretion pathways are common to all dietary seleniumcompounds; and upstream of the selenide intermediate wherethe metabolic pathways of each compound are unique. Oneselenium metabolite straddles the divide between upstream anddownstream metabolites: methylselenol (MeSeH: MeSe� at physio-logical pH) can be produced both upstream and downstream ofHSe� as will be discussed in due course.

5.1 Downstream metabolites

The downstream metabolic pathways consist of selenoproteinsynthesis, methylation and the generation of selenosugars forexcretion. Selenoprotein synthesis is unique amongst proteinsyntheses due to the insertion of a SeCys residue at the UGAcodon, which is read as a stop codon in the synthesis of otherproteins. Several factors are required in order for the UGA codonto be read as a SeCys insertion codon, including a unique tRNA(tRNA[Ser]Sec) upon which SeCys is synthesized.93 The seleniumrequired for the synthesis of selenocysteyl-tRNA[Ser]Sec fromphosphoseryl-tRNA[Ser]Sec is provided by monoselenophosphate

(H2SePO3�), which can be synthesised in vitro from the reaction

of HSe� and ATP catalysed by selenophosphate synthetase;providing further evidence for HSe� as the common seleniumintermediate.94–96

Two distinct pathways are available for excretion of selenium:the methylation pathway and the selenosugar pathway. Thebiological significance of selenosugars is unknown,97 butmethylation is considered a detoxification pathway due to thelow toxicity of the methylated metabolites.98,99 The methylationpathway generates dimethyl selenide (DMSe), which is excreted viathe breath,100 and trimethylselenonium (TMSe+) in the urine.101

Methyltransferases, including thiol-S-methyltransferase andthioether-S-methyltransferase, are responsible for the enzymatictransfer of methyl groups from S-adenosyl-L-methionine (SAM)to HSe� and its subsequent methylated metabolites; the pre-sumed metabolite, MeSe�, and the excretory compounds DMSeand TMSe+.102–105 The methylation reactions are in equilibriumwith demethylation reactions facilitated by unidentifieddemethyltransferases, with demethylation from MeSe� to HSe�

occurring at the greatest rate of the three possible demethylationreactions.106,107 TMSe+ was considered the major urinary seleniummetabolite until the identification of selenosugars includingthe major selenosugar Se-methylseleno-N-acetylgalactosamine(MeSeGalNAc) and minor species Se-methylseleno-N-acetyl-glucosamine (MeSeGluNAc) and Se-methylselenogalactosamine(MeSeGalNH2).97,108,109 The aforementioned selenosugars arethought to be derived from the reductive methylation ofglutathionylseleno-N-acetylgalactosamine (GSSeGalNAc).108,110 Ithas been suggested that TMSe+, having been misidentified in thepast, is a trace or, at best, minor selenium metabolite.111 A smallstudy into urinary metabolites identified TMSe+, MeSeGalNAc andMeSeGalNH2 in urine samples from human volunteers givenselenite and found that TMSe+ was present as a trace metabolitein most subjects, but also appeared as a significant metabolite inothers, suggesting that the excretory metabolism of seleniumcan vary widely amongst individuals.112

5.2 Upstream metabolites

The upstream metabolic pathways of the dietary seleniumcompounds converge on HSe�, but are very distinct. Selenitewas the first selenium compound to undergo extensive investiga-tion of its metabolism. Ganther et al. studied the metabolism ofselenite in a series of experiments in cell-free mouse liver extractsand in vitro systems. Firstly, it was shown that the reduction ofselenite to the excretory product DMSe occurs in mammaliantissues and that it is dependent on the availability of glutathione(GSH) and the presence of enzymes.113 The reaction of seleniteand GSH was shown to produce selenodiglutathione (GSSeSG),114

which could then be reduced by NADPH and glutathione reduc-tase (GR) to glutathione selenenylsulfide (GSSeH); both GSSeSGand GSSeH decomposed to elemental selenium.115 Finally, anacid-volatile selenide, tentatively identified as HSe�, was producedin a system containing selenite, GSH, NADPH and GR, as wellas in nonenzymatic systems: it was suggested that the selenidecould act as a precursor for the biosynthesis of other seleniumcompounds.116 More recently, selenite and GSSeSG were shown


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to be reduced, presumably to HSe�, by thioredoxin reductase(TrxR), and the thioredoxin (Trx) and glutaredoxin (Grx) systems,to which the selenium compounds are more reactive than toGSH, at least in vitro.105,117,118 GSSeSG has been identified in ratliver cytosol119 and pig epithelial cell homogenates along withanother selenotrisulfide, cysteine-glutathione selenotrisulfide(CysSSeSG).120 Elemental selenium has been identified in a lungcancer cell line after exposure to selenite,121 but selenopersulfideand HSe� are too reactive for direct identification. Althoughselenite has been identified intact in cell culture,122 it is generallyunderstood to be rapidly metabolised, as observed in rats123 andother cell culture studies,121 particularly given the high levels ofGSH in cells.

The metabolism of the selenoamino acids differs significantlyfrom the inorganic selenite. SeMet has a high bioavailability andlow toxicity and is therefore a popular form for dietary supple-mentation. As the selenium analogue of methionine (Met),SeMet can be non-specifically incorporated into proteins inplace of Met and thus the proteins act as a SeMet store untilthey are degraded. SeMet is transformed into SeCys via the trans-sulfuration pathway that is also responsible for the synthesis ofcysteine (Cys) from Met (Fig. 2).124,125 The final steps in the

multi-step synthesis of SeCys from SeMet involve the synthesisof selenocystathionine (SeCysta) from selenohomocysteine(SeHCys) and serine by cystathionine-b-synthase, followedby the a,g-elimination of selenocysteine by cystathionine-g-lyase.124

It has been shown that more SeCys was produced in rats supple-mented with SeMet when additional Met was also included inthe diet;126 providing indirect evidence that the storage ofSeMet in proteins prevents its efficient metabolism to SeCys.Methionine-g-lyase is known to cleave SeMet to MeSe�,a-ketobutyrate and ammonia in bacteria by an a,g-elimination,127

but the extent to which this occurs in mammals is questionable.The results from the incubation of SeMet in rat liver super-natant suggests that g-lyase activity is insignificant, at least inthose tissues.128

Selenocysteine has been shown, in tissue homogenates, toundergo lysis by selenocysteine Se-conjugate b-lyases (also knownas S-conjugate b-lyases), with HSe� identified, indirectly, as theselenium product of this reaction.134,138 Thus the metabolismof SeMet is linked to the presumed common intermediate.Analogous to the adventitious incorporation of SeMet intoproteins in place of Met, SeCys can be incorporated in placeof Cys and released upon degradation of these proteins andselenoproteins. SeCys can also be produced by the reduction ofCysSeSeCys: it is a substrate for TrxR and the Trx131 and Grx105

systems. In the intestinal cytosol of mice, CysSeSeCys wasreduced by GSH to selenocysteine-glutathione selenenylsulfide(CysSeSG; not to be confused with the selenotrisulfide CysSSeSGidentified in selenite-treated porcine epithelial cells that was alsodenoted CysSeSG by those authors120), which is presumablyreduced by GSH or GR to SeCys.129,130

MeSeCys (and other selenocysteine Se-conjugates) can be cleavedto MeSe� (and other selenols) by selenocysteine Se-conjugateb-lyases132,133 and thus directly produces MeSe� rather thanindirectly via methylation of HSe� (as is the case for otherdietary selenium compounds). Like HSe�, MeSe� has not beendirectly identified in cell or animal models due to its highvolatility and reactivity, however the cleavage of MeSeCys toMeSe� has been indirectly monitored in rat liver supernatant inthe same experiment in which g-lyase cleavage of SeMet toMeSe� was not detectable.128 Thus there is a stark differencebetween the metabolism of the selenoamino acids MeSeCysand SeMet. MeSeCys is not adventitiously incorporated intoselenoproteins and is likely to be readily available for b-lyasecleavage to MeSe�, whereas SeMet is adventitiously incorpo-rated into selenoproteins and the alternative g-lyase pathway toMeSe�may be only a minor pathway. MeSe� is considered a keymetabolite in the anti-cancer activity of selenium135,139 and sothe relative abilities of MeSeCys and SeMet to produce thismetabolite are important when considering selenium supple-mentation. The activity of b-lyase, however, is a limiting factorin MeSeCys-generated MeSe� and so a simplified form, methyl-seleninic acid (MeSeA), is often used in experiments in place ofMeSeCys. It has been proposed that MeSeA is reduced byintracellular thiols to MeSe� from a selenoxide (MeSe(O)SR),methylselenenic acid (MeSeOH) and methylselenenylsulfides(MeSeSR).135,140,141 (Sinha et al. also suggest that MeSeCys can

Fig. 2 The metabolic pathways of the dietary selenium compounds. The schemeis the result of in vitro and some in vivo experiments conducted by a myriad ofresearchers over the past 50 years, which is described in detail in the text. Thetrans-sulfuration pathway was described by Esaki et al.124 The reduction ofCysSeSeCys has been described by several authors,105,129–131 as has the selenitereduction pathway (see text for more detail). The b-lyase cleavages of MeSeCysand SeCys to MeSe� and HSe�, respectively, were described by Andreadouet al.132 and Rooseboom et al.,133 and Esaki et al.134 The cleavage of SeMet toMeSe� was first observed by Esaki et al.127 The pathway for the reduction ofMeSeA to MeSe� was postulated by Ip et al.135 The transamination pathwayswere described by Commandeur et al.136 and Pinto et al.137 The selenosugarsynthesis pathway was proposed by Kobayashi et al.108 See the review byTuranov et al.93 for more detail on the selenoprotein synthesis pathway, whichrequires monoselenophosphate (H2SePO3

�).94–96 See text for more detail on themethylation and excretion pathways. Abbreviations: DeMeT, demethyltrans-ferases; MeT, methyltransferases; SPS, selenophosphate synthetase.


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be oxidised to a selenoxide that can be reduced by thiols toMeSe�.141) Ip et al. found that while MeSeA was better atinducing apoptosis in mouse mammary hyperplastic endothelialcells, the difference in efficacy of MeSeA and MeSeCys disappearedin rat mammary tumour models where b-lyase was likely to beactive.135 The relevance of rat models in the b-lyase cleavage ofMeSeCys has come into question by Rooseboom et al. who foundthat b-lyase activity was higher in the hepatic cytosol of rats than inthe renal cytosol, whereas the reverse was true in human tissuecytosols.133 Furthermore, b-lyase activity in the human hepaticcytosol was substantially lower than in corresponding rat cytosol.

A final, interesting, transformation of organic selenium com-pounds is transamination that results in the production of selenoa-keto acids from MeSeCys, other SeCys conjugates and SeMet.137

MeSeCys is both a b-lyase and transaminase substrate of glut-amine transaminase K (GTK)136 and SeMet is a substrate ofglutamine transaminase L (GTL)137 and these transaminationreactions generate b-methylselenopyruvate (MSP) and a-keto-g-methylselenobutyrate (KMSB), respectively. As observed for therelative abilities of SeMet and MeSeCys to generate MeSe�, theirseleno a-keto acid generation abilities also differ in cell culture.142

The metabolic routes of the selenium compounds (describedabove and summarised in Fig. 2) have been established largely fromin vitro studies. Similarly, the selenium metabolites that havebeen unequivocally identified (unequivocal identification definedby Gammelgaard et al. as identification by the use of a combinationof ICP-MS and molecular MS or NMR spectroscopy) have all beenidentified in vitro.16 The speciation of metabolites in the methylationand excretion pathways are relatively straightforward to determine inhuman urine, but even the identity of selenosugars was establishedonly in the last decade. Upstream metabolites are more difficult tocharacterise as they are retained and further metabolised in cellsand tissues. In terms of the prophylactic and therapeutic activities ofselenium, these upstream metabolites are the most efficacious andtherefore knowledge of their in vivo distribution and abundance is ofgreat importance. An in vitro understanding of selenium metabolismis inadequate when an appreciation of the form of selenium thatis transported to cells and tissues and its further metabolisminside different cells and tissues is required. An in vivo under-standing of the activities of the various metabolic pathways andthe relative abundance of selenium metabolites is necessary forunderstanding the disease prevention and treatment potential ofdietary selenium compounds.

6 Mechanisms by which selenium exerts itsbiological activities

The many different metabolic pathways described above generate adiverse array of selenium metabolites that are responsible forselenium’s biological action. The dietary selenium compoundscan be divided into those that are redox active (selenite andCysSeSeCys) and those that are not (SeMet and MeSeCys), toprovide a general explanation for their differing biological activities.When the metabolites of these compounds are considered,most can be classified into two main groups: the selenolates

(RSe�, and the hydrogen selenide ion HSe�) and the diselenides(R–Se–Se–R; in which the selenenylsulfides (R–Se–S–R) can beincluded, for convenience). Selenolates and diselenides are redoxactive, and as all of the dietary selenium compounds can potentiallygenerate HSe� and the selenolate MeSe�, describing the dietaryselenium compounds as redox active or inactive, without con-sideration of the properties of their metabolites, neglects theimportant role metabolism plays in selenium biology.

The properties of selenium itself are important to consider whendiscussing the element’s biological activity. The comparison ofselenium to sulfur serves to highlight some key properties ofselenium. The greater atomic radius of selenium compared to sulfurresults in weaker bonding to carbon and hydrogen in seleniumcompounds than in their sulfur analogues. The pKa of 5.2143 of theSeCys selenol results in free SeCys existing as a selenolate atphysiological pH, whereas the Cys thiol (pKa 8.3)144 is intact underthe same conditions. The pKa of other selenols is assumed to besimilar to that of the SeCys selenol. Selenolates are highly nucleo-philic and selenoxides are highly electrophilic – a combination ofproperties that are responsible for the GPx-like activity of seleniumcompounds.145 The redox potential of SeCys/CysSeSeCys is widelyreported to be �488 mV,144 although the validity of this measure-ment has been questioned by Nauser et al. who measured apotential of �380 mV.146 In either case, SeCys has a much lowerreduction potential than Cys (the Cys/cystine redox potential is�233 mV).144 These chemical properties of selenium are useful inconsidering both the functions of selenoproteins and the biologicalactivities of low molecular weight selenium compounds.

Most mechanisms by which selenium exerts its biologicalactivity can be split into antioxidant and prooxidant mechan-isms. The antioxidant mechanisms include increased expres-sion of antioxidant selenoproteins and the GPx-like, radicalscavenging and metal-binding activities of low molecularweight selenium metabolites. These mechanisms are generallyassociated with disease prevention by selenium supplementa-tion, due to the relationship between oxidative stress anddisease. Reactive oxygen and nitrogen species (ROS and RNS)are formed during endogenous metabolic processes, but exces-sive generation of ROS and RNS by endogenous and exogenoussources alters the redox balance. Oxidative damage to biologicalmolecules including DNA, lipids and proteins will follow.Damage to DNA affects transcription and signaling pathwaysand causes replication errors and genomic instability – all ofwhich are associated with carcinogenesis.147 Higher levels ofROS and/or reduced levels of antioxidants lead to oxidativestress and are implicated in cardiovascular diseases due toperoxidation and oxidation of protein and lipid thiols.148 Bothincreased superoxide generation by NADPH oxidase and oxida-tive damage to mitochondrial DNA has been linked to athero-sclerosis.149 Oxidative stress leads to dysfunction of endothelialcells, vascular smooth muscle cells and monocytes, and thisdamage is the initiating step in cardiovascular disease leadingto the formation of atherosclerotic lesions.149 The brain isparticularly susceptible to oxidative stress due to its highoxygen consumption and presence of redox active metals. Thereis evidence for high levels of oxidative stress in Alzheimer’s and


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Parkinson’s diseases, although the oxidative stress may beassociated with progression more so than the initiation ofthese diseases.148

The prooxidant activities of selenium include oxidation ofprotein thiols and ROS generation. The cytotoxicity and anti-canceractivity of selenium is attributed to its prooxidant action. Cancercells are more sensitive to selenium than normal cells as demon-strated in patient-matched pairs of malignant and normal prostatecells,150 in several cancer cells lines versus normal human fibroblastcells151 and malignant mesothelioma cells versus benign mesothe-lial cells.152 In each of the aforementioned cases, the sensitivity ofthe cancer cells to selenite and/or CysSeSeCys was attributed to thegreater sensitivity of the cancer cells to oxidative stress. Olm et al.showed that extracellular thiols are critical for the uptake of redoxactive selenium compounds by cancer cells, and that cells in whichthe xc

� cystine transporter was more highly expressed, and wherethe export of Cys was therefore greatest, were most susceptible toselenite toxicity.153 Both the intracellular and extracellular redoxstatus of cancer cells contributes to their sensitivity to selenium.Selenium compounds have also demonstrated a higher toxicitytowards drug-resistant cancer cells that may be related to theactivity of TrxR and the ability of the cells to cope with oxidativestress.153,154 The intriguing relationship between selenium toxicityand TrxR activity will be explored.

Finally, novel mechanisms of selenium compounds – thegeneration of HDAC inhibitors by the metabolism of theselenoamino acids and the potential of biologically activenanoparticulate elemental selenium – will be covered.

All of the dietary selenium compounds have demonstrated bothantioxidant and prooxidant activities. Selenolates, for example,undergo redox-cycling with cellular thiols and cellular oxidants.Via this redox-cycling, selenolates are capable of scavenging radicalsand may also generate ROS. The antioxidant and prooxidantactivities of dietary selenium compounds are two sides of the samecoin: metabolism, dose, the intracellular redox environment as wellas the activities of redox-sensitive proteins and proteins with whichthe redox cycling of selenium is coupled influence whether anti-oxidant or prooxidant activities predominate. When it comes tounderstanding the biological mechanisms of a particular seleniummetabolite, we would ideally have a good understanding of itsmechanism in vitro, in cell studies to prove its biological relevance,and in animal studies to demonstrate its efficacy in diseaseprevention and/or treatment. Few of the proposed mechanismsfor the biological activity of various selenium metabolites have all ofthese lines of evidence. We review the key mechanisms that havebeen described and actively researched in recent years, payingparticular attention to the species of selenium responsible forthe mechanism and the strength of the in vitro and in vivoevidence for the mechanism.

7 Upregulation of antioxidantselenoproteins

The focus of this review is on the disease prevention activitiesof low molecular weight selenium metabolites, and we will only

briefly discuss the antioxidant roles of the GPxs, TrxRs andSelP, which have been reviewed elsewhere.63,155,156 Seleniumsupplementation has the potential to increase selenoproteinexpression, as will be discussed below. However, it is difficult toknow at what level selenium supplementation is correctinga deficiency in selenoprotein expression, maximising or opti-mising selenoprotein levels or resulting in the overexpressionof selenoproteins with associated prophylactic, neutral orperhaps toxic effects.157 This uncertainty is due in part to theheterogeneous response of various biomarkers of seleniumstatus, which include serum selenium levels and the levelsof the selenoproteins SelP and GPx3.158,159 How differentbiomarkers reflect overall selenoprotein expression is not wellunderstood.61

The GPxs may inhibit the initiation of cancer by catalyzing thereduction by GSH of a range of hydroperoxides (and peroxy-nitrite) to water or their corresponding alcohols. Presumablythe SeCys selenolate at the active site of GPx is oxidised to aselenenic acid (yet to be detected) that is reduced back to theselenolate by GSH via a selenenylsulfide (Fig. 3a).160 Brigelius-Flohe and Kipp report that nutritional levels of seleniumare sufficient for the prevention of hydroperoxide-driven carcino-genesis.161 Selenium supplementation, by organic and inorganicselenium compounds, has been shown to increase selenoproteinexpression and activity. CysSeSeCys, SeMet and selenite wereequally effective in increasing the activity of GPx1 in rat tissuesand blood.162 GPx1 and GPx4 expression is enhanced byselenite supplementation of lung cancer cells.163 Selenite andMeSeCys, but not SeMet increase expression and activity ofGPx1 in human coronary artery endothelial cells, while selenitesupplementation increased GPx1 activity in patients withcoronary artery disease whose mean baseline selenium levels,at >90 ng mL�1, were already considered sufficient for maximalGPx expression.164 Increased GPx activity, induced by selenite

Fig. 3 The (a) GPx redox cycle and (b) GPx-like redox cycle of selenolates andmonoselenides. Adapted from Brigelius-Flohe and Maiorino160 and Rahmantoand Davies.181


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and SeMet, in rat cardiomyocytes that were subject to hypoxia/reperfusion damage resulted in reduced lipid peroxidation.165

TrxR, with Trx and NADPH, constitutes the major intra-cellular redox system: the Trx system. TrxR has control over theredox environment of the cell and cell growth via the regulationof Trx, which reduces many substrates including the disulfidescommonly found in transcription factors.166 In addition, theTrxR system is capable of reducing hydrogen peroxide, organichydroperoxides and lipid hydroperoxides.131 Due to its centralrole in maintaining the intracellular redox status, the TrxRsystem is potentially linked to many diseases.167 TrxR may beparticularly important in preventing cardiovascular disease, butmeta-analyses of clinical trials have found no benefit fromselenium supplementation for these diseases. TrxR may bedifficult to increase with selenium supplementation: TrxR1activity rapidly increased in rats fed high selenite diets, butgradually declined as supplementation continued;168,169 andMeSeCys and MeSeA supplementation had no effect on TrxR1activity in rats.170 Nonetheless, laboratory studies point tobenefits from selenium supplementation for models of cardio-vascular diseases. Low doses of selenite (5–40 nM) increasedboth TrxR and GPx activities and protected human coronary arteryand human umbilical vein endothelial cells from peroxide toxi-city, but TrxR appeared to be most important in exerting theantioxidant effects.171 In rats supplemented with 1 ppm seleniumper day as selenite for 5 weeks there was a modest increase inTrxR and GPx levels, with a corresponding modest improvementin cardiac function post ischaemia-reperfusion.172 Conversely,TrxR is overexpressed in many cancer cells and is associatedwith the inhibition of apoptosis173 – a factor that will bediscussed in due course.

Selenoprotein P, the major plasma selenoprotein that isinvolved in selenium transport, has some antioxidant activity.It is capable of reducing phospholipid hydroperoxides174 andhas been implicated in the prevention of oxidative damage inhuman astrocytes175 and in the complexation of heavy metalselenides.176 SelP has been found colocalised with amyloidplaques and neurofibrillary tangles in human brain tissue177

and, given this and the importance of SelP in the transport ofselenium to the brain, it may be involved in the aetiology ofneurodegenerative disease.166 Recent work has shown that thehistidine-rich motif of SelP binds Zn2+, preventing the Zn2+-mediated aggregation of the amyloid-b peptide, Ab42, in vitroand reducing the toxicity of Ab42-Zn2+ to cells.178

The potential role of selenium and selenoproteins in DNAdamage repair was recently reviewed179 and there is evidencethat both selenoproteins and low molecular weight seleniumcompounds may be involved in the redox regulation of signal-ing pathways and redox-sensitive proteins involved in DNAdamage repair. In the protection of prostate cancer cells fromUVA and H2O2 induced ROS, low doses of SeMet and seleniteaided the repair of oxidative DNA damage, potentially throughthe increased activity of GPx and TrxR.180 However, there is currentlylimited evidence of a role for selenium species or selenoproteinsin DNA damage repair, let alone any certainty about a potentialmechanism.

8 GPx-like and radical scavenging activity ofselenium compounds

Many low molecular weight organoselenium compounds exhibitGPx-like and radical scavenging activities that contribute to theirantioxidant properties. The direct antioxidant activities of organo-selenium compounds are attributed to the nucleophilicity ofselenolates and the ready oxidation of SeMet and SeCys.181 Inaddition, the selenoxides and selenenic (RSeOH) and seleninic(RSeO2H) acids produced by the oxidation of selenolatesare readily reduced by cellular thiols and NADPH-dependentreductase systems.181,182 Suryo Rahmanto and Davies haverecently published an excellent review on the direct scavengingof oxidants by the selenoamino acids with particular focus onSeMet and the kinetics of the reactions that we recommend forfurther reading.181

8.1 GPx-like activity of selenium compounds

Synthetic organoselenium compounds of the cyclic selenenylamide, diaryl diselenide, and monoselenide classes have alldemonstrated GPx-like activity.183 Ebselen is the best-knownGPx mimic. It catalyses the reduction of hydroperoxides in thepresence of thiols, although the mechanism by which this occurs isuncertain: the catalytic cycle being alternately described as differentfrom184 and similar to185 the GPx catalytic cycle that involvesselenol, selenenic acid and selenenylsulfide intermediates(Fig. 3a). A study of the GPx-like activity of synthetic mono-selenides, using a non-biological thiol, found that the selenideswere oxidised by hydrogen peroxide to selenoxide and finally toa hydroxy perhydroxy selenane (R–(HOO)–Se–(OH)–R), that inturn oxidises the thiol.186 That recently proposed scheme iscontrary to the established scheme that a selenoxide/selenenicacid is the most highly oxidised selenium compound in thecycle (Fig. 3b).181 Complete catalytic cycles of GPx and itsmimics are yet to be elucidated.

Nonetheless, selenite,187 SeMet, MeSeCys and CysSeSeCysall have demonstrated GPx-like activity in vitro, as measured bythe decay of NADPH in the presence of GSH, GR and cuminehydroperoxides.188 Of the four dietary selenium compounds,only selenite failed to protect red blood cells against peroxyl-radicalinduced haemolysis and lipid peroxidation. The presence ofGSH and GR in these experiments indicates that selenite andCysSeSeCys were reduced to their selenolate metabolites, akinto the reduction of the SeCys residue of GPx. Recently, SeMethas been reported to catalyse the reduction of protein-boundhydroperoxides in concert with GSH and/or the TrxR systemin vitro and in the lysates of SeMet-treated murine macrophage-like cell lines exposed to t-butylhydroperoxide.189 There was noincrease in cytosolic GPx1 levels in the cells and therefore theincreased peroxidase-like activity was attributed to redox-cyclingbetween SeMet and selenomethionine selenoxide.

8.2 Radical scavenging activities of selenium compounds

Selenium compounds have demonstrated radical scavengingactivity. Due to the inability of selanyl radicals (RSe�) to oxidiseproteins, their generation may terminate the propagation of


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radical damage.190 In a study of the catalysis of electrontransfer by SeCys, Nauser et al. surmise that the nucleophilicselenolates, which are better able to access their radical oxidationstate (RSe�) than their thiolate counterparts at pH 7, are capable ofreducing many oxidants.146 In the aforementioned study ofperoxyl-radical induced haemolysis, the monoselenides SeMetand MeSeCys and the diselenide CysSeSeCys showed peroxylradical scavenging activity as well as GPx-like activity.188 SeMetand CysSeSeCys reduced peroxynitrite-initiated oxidationand nitration reactions191 and protected plasmid DNA fromperoxynitrite-induced single-strand breaks.192 SeMet, MeSeCysand CysSeSeCys inhibited g-radiation induced lipid peroxida-tion, protein carbonylation and single-strand plasmid DNAbreaks in vitro.193 Both CysSeSeCys and GSSeH scavenge freetyrosyl radicals and tyrosyl radicals in proteins several orders ofmagnitude faster than their sulfur analogues, and are therebycapable of mitigating damage to protein structure and prevent-ing further oxidative damage to cells.194 Notably, these in vitrostudies have not tested the abilities of selenium metabolites,such as GSSeSG, to exert radical scavenging activity and so thesestudies need to be expanded to better resemble in vivo speciation.The in vivo radical scavenging activity of the selenolate MeSe�maybe linked to angiogenesis: the reduction in levels of ROS by MeSeAin hypoxic tumour cells was associated with, and presumablyled to, the inhibition of hypoxia-inducible factor 1a (HIF-1a).53

The same inhibition was also observed as a result of treatmentof mice with MeSeCys, which resulted in the inhibition oftumour xenograft growth.

8.3 Novel functions of SeMet in proteins

The free selenoamino acids act as GPx mimics and free radicalscavengers as described above, but there is precedent for aminoacid residues to act as antioxidants in proteins. Methionineresidues can be oxidised by hydrogen peroxide to methioninesulfoxide that is subsequently reduced by methionine sulfoxidereductases, and thus act as antioxidants that protect criticalresidues at the active sites of proteins from oxidation.195 SeMetcan be oxidised to its corresponding selenoxide by peroxynitriteat a rate 10- to 1000-fold higher than that of the oxidation ofMet.196 The fact that selenomethionine selenoxide is readilyreduced to SeMet by GSH,144 as opposed to the enzymatic reductionof methionine selenoxide, suggests that the adventitious incorpora-tion of SeMet in place of Met in proteins may confer protectionagainst radical species to susceptible nearby amino acidresidues.197 Similarly, SeMet incorporation in place of Met inamyloid proteins has been shown to modulate their aggrega-tion and neurotoxicity and the authors suggest that the abilityof SeMet incorporation to modulate fibrillogenesis may haveimplications for amyloid diseases.198 Whether fibrillogenesisand toxicity is increased, decreased or unchanged was depen-dent upon the location at which SeMet was substituted.

These novel antioxidant and anti-fibrillogenesis mechanismsare appealing, but the random incorporation of SeMet meansthat its antioxidant protection of other residues and effect onfibrillogenesis would be non-specific and therefore ineffective.Furthermore, the finding that SeMet supplementation increased

the proportion of selenium atoms in human plasma from 1 per8000 Met residues to 1 in 2800 Met residues without alteringthe ability of plasma proteins to quench peroxynitrite suggeststhat these in vitro mechanisms will not be relevant to seleniumsupplementation in vivo.199 There is, however, some evidencefor GPx-like and radical scavenging activities of the metabolitesof the dietary selenium compounds in vitro and, in the case ofradical scavenging activity, in vivo.

9 Metal binding

Metals, both endogenous and exogenous, have been implicatedin the aetiology of cancer, cardiovascular and neurodegenera-tive diseases, and their toxicity is associated with their ability togenerate oxidative stress, amongst other mechanisms such asthe binding of protein sulfhydryl groups.200 While copper, ironand other endogenous metals are usually tightly regulated,their mis-regulation may result in uncontrolled ROS generationvia the Fenton reaction.201 Cu(I) and Fe(II) react with hydrogenperoxide in the Fenton reaction to generate short-lived hydroxylradicals. The short-lived radicals spawn longer lasting radicalsas they react with biological molecules such as lipids and proteins,eventually leading to oxidative stress.

9.1 Metal binding in vitro

Brumaghim et al. have demonstrated, in vitro, that some organo-selenium compounds are capable of binding copper and iron andpreventing oxidative DNA damage induced by these metals.202–204

Many organoselenium compounds (including CysSeSeCys andSeMet) were found to bind one of the metals, but the ability ofthe compounds to prevent metal-mediated oxidative damagedepended on the selenium species: amino acids with both amineand carboxylate groups were required to prevent copper-mediatedoxidative damage and MeSeCys was the only one of 10 testedorganoselenium compounds to inhibit both copper- and iron-mediated damage.203 NMR, XAS and DFT studies were used toshow that SeMet and MeSeCys bind Cu(I) via the selenium atomsand, most likely, the amino groups.204 In contrast, the binding ofCu(II) by SeMet was found to occur via the nitrogen and oxygenatoms of the amino and carboxylate groups.205 The ability of theselenium compounds to inhibit Cu(I)-mediated oxidative DNAdamage was not related to their GPx-like hydrogen peroxidescavenging activity or radical scavenging activity, although radicalscavenging was considered a secondary mechanism for the inhibi-tion of Fe(II)-mediated oxidative damage by selenium compounds.203

The fact that all tested organoselenium compounds bound themetals, but only a few prevent metal-mediated oxidative DNAdamage suggests that the antioxidant mechanism is more complexthan metal-binding alone.203

More recently, the hypothesis that selenium may preventDNA oxidative damage via the binding of DNA-bound metalswas tested using electronic absorption spectroscopy.206 Theauthors found that oxidative DNA damage (due to the genera-tion of ROS by the Fenton reaction) was dramatically decreasedwhen selenite or selenium dioxide was incubated with Fe(II) orCr(III) in the presence of H2O2 prior to the addition of DNA.


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When the selenium compounds were added to a solution ofDNA and metal ions, there was a much smaller decrease inoxidative DNA damage. These results suggest that the bindingof free metal ions, rather than DNA-bound ions reduces oxida-tive damage. However, the authors also found that the metal–selenium species produced a similar amount of ROS to freemetals in solution with H2O2. On this basis, they propose thatthe metal–selenium species may protect DNA from oxidativedamage by binding at sites where ROS generation causes lessdamage, or prevents excess H2O2 from reaching more sensitiveareas of the DNA molecule. This hypothesis remains to betested. Of further note are the different results obtained in thisstudy with Cu(II): selenite and selenium dioxide were able toprevent oxidative damage to a far lesser extent than observedfor Fe(II) and Cr(III), yet only the Cu(II)–selenium species pro-duced fewer ROS than the free metal. The ability of selenium toprevent oxidative damage by the binding of metal ions in vitro isdependent on both the speciation of the selenium compoundand the identity of the free metal ion. Thus there are likelyseveral mechanisms by which selenium compounds can reducemetal-mediated oxidative damage: by direct interaction withthe free metals, GPx-like and radical scavenging in solution,and in association with DNA.

9.2 Metal-binding in vivo

The hypothesis that metal-binding properties of organoseleniumcompounds such as SeMet and MeSeCys contributes to theirantioxidant activities needs to be tested in cell and animal models.The antagonism between Cu and selenite has been studied in cellsand animals. Thus far, in testing the ability of copper to preventselenite-induced cytotoxicity in human colonic carcinoma cells, itappears that copper and selenite interact extracellularly to preventthe selenite-mediated generation of superoxide,207,208 which isin line with in vitro studies.209 Studies in rats also attest to theantagonism between copper and selenium: the toxicity of seleniteor selenocystamine to rats was ameliorated by dietary coppersupplementation.210 Both selenium and copper were found toaccrue in the liver and spleen of rats fed both minerals at levelsabove those observed fed either copper or selenium alone. How-ever, there has been no direct evidence of the formation of thepostulated CuSe or any other species containing a Cu–Se moietyin cells or animals treated with both copper and selenium.Furthermore, free metal concentrations in the cells are typicallyextremely low and cast doubt on the in vivo significance ofhydroxyl radical generation via Fenton chemistry.147 Until experi-ments have been conducted to determine the existence of suchmetal–selenium species, the relevance of the observed in vitrometal-binding activity to biological systems, where copper andiron are found tightly bound to proteins and ligands and DNA iscompartmentalised in the nucleus, remains uncertain.

Despite the lack of evidence for the formation of CuSe in theprevention of oxidative stress by selenium, there are precedentsfor metal–selenium binding mechanisms: Ag2Se has beendetected in the livers of marine mammals211 and HgSe hasbeen found in the brain tissue of humans exposed to acutemercury poisoning and with high fish consumption,212 both

complexes are inert forms of otherwise toxic metals that havebeen implicated in the aetiology of many diseases. The bindingof selenium to various endogenous and exogenous metals hasrecently come under systematic scrutiny. It was found that Ag+,Cd2+, Cu2+, Hg2+, Pb2+ and Zn2+ all form insoluble metal–selenide complexes in Saccharomyces Cerevisiae and protect yeastfrom sodium selenide toxicity, but metals that do not form metal–selenide complexes (Ca2+, Mg2+, Mn2+ and Fe2+) do not offerprotection.92 Interestingly, Co2+ and Ni2+ were found to offer protec-tion via both the formation of metal–selenide and by catalyzing theaerobic oxidation of selenide to elemental selenium. Despite theprotection against metal toxicity afforded by the formation of metalselenides, a consequence of metal–selenium binding may be thereduced availability of selenium for other biological functions,leading to problems associated with selenium deficiency.213 Eventhe apparently simple metal-binding antioxidant mechanism ofselenium has myriad consequences.

10 Generation of ROS

The generation of ROS by selenium compounds has been underinvestigation for two decades. Seko et al. detected ROS insolutions of GSH with selenite, GSSeSG and HSe� in the presenceof oxygen.214 In the presence of mammary tumour cells, seleniteand CysSeSeCys were also observed to generate large amounts ofROS, including superoxide and hydrogen peroxide, whereasSeMet and selenate generated ROS to a much lesser extent.215

Selenium compounds that can be reduced to selenolates byexcess GSH (e.g. selenite, CysSeSeCys and MeSeA) are capableof producing ROS in vitro and thus ROS generation is usuallyattributed to redox cycling of selenolates with GSH and oxygen.216–218

MeSeCys and SeMet, which do not generate superoxide in vivo,may be capable of generating ROS in cells where they can becleaved to MeSe�.217

10.1 ROS generation by metabolites of selenite

The ROS-generating capabilities of selenite and its metaboliteshave been studied extensively in cells. Selenite-mediated ROSgeneration occurs in concert with a reduction in GSH levels andincreased levels of GSSG and has been associated with oxidativestress leading to DNA strand breaks and apoptosis in cancercells.219 Superoxide, generated by selenolates,218 is generallyheld to induce selenite-mediated apoptosis, but other speciesmay be involved. Shen et al. found that the selenite-inducedapoptosis in human hepatoma cells was inhibited by super-oxide dismutase219 and Kim et al. observed that superoxidedismutase, but not catalase overexpression, prevented autophagiccell death in selenite-treated malignant glioma cells;220 but Parket al. recently reported that superoxide dismutase had no effect onselenite-induced apoptosis in human lung cancer cells.221 Hydroxylradicals have been implicated in the nicking of DNA in vitro bysodium selenide and a solution of sodium selenite and GSH, and inthe induction of double strand breaks by sodium selenide leadingto loss of viability in Saccharomyces Cerevisiae.222 The in vitro DNAdamage caused by sodium selenide was reduced by the hydroxylradical scavenger mannitol, but not by SOD or catalase.


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Further investigation into the ability of the highly reactive andtherefore short-lived hydroxyl radical to directly cause DNAdamage in cells is required. Rahmanto et al. have highlightedthe need for further studies into the mechanisms of reactionsof selenolates and thiols with oxygen by arguing that theformation of radicals from molecule–molecule interactions donot have favourable thermodynamics:189 improved understandingof the radical species that are generated by selenium compoundsin cells will improve our understanding of the cytotoxicmechanisms of selenium species.

Nonetheless, it has become apparent that selenite-inducedcell death is mediated via the mitochondrial pathway. Selenitehas been shown to result in the decrease of mitochondrialmembrane potential,223,224 and release cytochrome c into thecytosol,225,226 leading to apoptosis in a number of cancer celllines. In the case of prostate cancer cells, overexpression ofSOD2, but not SOD1 reduced superoxide generation, preventedthe decrease in mitochondrial membrane potential and caspaseactivation and inhibited apoptosis.224

10.2 ROS generation by CysSeSeCys and its metabolites

CysSeSeCys, given its reducibility to a selenolate by GSH, maybe expected to generate ROS in a similar manner to selenite.CysSeSeCys was shown to generate ROS in a breast cancer cellline227 and generate ROS, induce chromosomal DNA strandbreaks and initiate apoptosis in a number of different cancercell lines.151 The results of investigations into the inhibition ofROS and apoptosis by a variety of radical scavengers and thiol-reducing agents suggested that mitochondria, xanthine oxidaseand NADPH oxidase contribute to CysSeSeCys-induced ROSgeneration. Conversely, Wallenberg et al. reported that themitochondrial superoxide production by CysSeSeCys was negligiblein a large-cell lung carcinoma cell line, but was substantial forselenite and GSSeSG.105 All three selenium compounds werefound to be substrates for the Grx system, but CysSeSeCys wasreduced to SeCys in a stoichiometric manner, whereas seleniteand GSSeSG underwent a non-stoichiometric reaction. Thedifferent stoichiometry of the reactions with selenite andGSSeSG is due to the redox cycling of HSe� and oxygen withthe Grx system (and also the Trx system) that leads to thegeneration of ROS.

10.3 ROS generation by metabolites of SeMet and MeSeCys

There are limited reports of the ROS generating abilities of theselenoamino acids, yet a wide variety of ROS have been observedand a number of apoptotic mechanisms – both similar to anddifferent from those attributed to selenite – have been proposed.MeSeCys, presumably after metabolism to MeSe�, was reportedto induce ROS, DNA fragmentation and apoptosis in a leukemiacell line.228 MeSe� was generated in prostate cancer cells fromthe a,g-elimination of SeMet catalysed by methioninase.229 Themethylated metabolite caused superoxide-mediated DNA fragmen-tation and apoptosis via a mitochondrial pathway, as observedfor selenite-treated cells.229 Hydrogen peroxide, amongst otherROS, has been observed in both SeMet- and selenite-treatedhuman lung cancer cells.230 Rapamycin, the inhibitor of mTOR,

a protein involved in cell growth and proliferation, inhibitedSeMet-induced ROS generation and apoptosis, but did notinhibit selenite-induced apoptosis. The authors did not measurethe inhibition of ROS generation by rapamycin in selenite-treated cells, but the implication is that the apoptotic pathwaysinitiated by SeMet and selenite differ, despite the apparentability of both to generate ROS. In a direct comparison of theROS production and apoptotic pathways induced by selenite andMeSeA in prostate cancer cells lines, Li et al. reported thatselenite generated superoxide and hydrogen peroxide and whileneither of those species were detected in MeSeA-treated cells,both selenium compounds induced apoptosis.231 Conversely,hydrogen peroxide was detected and implicated in the apoptosisof MeSeA-treated lung cancer cell lines.227

10.4 Selenolate involvement in ROS production

A recent experiment further underscores the complexity of ROSgeneration and ROS-mediated apoptosis by different seleniumcompounds and the necessity of understanding the mechanismsby which ROS are produced and by which they exert theirprooxidant and apoptotic effects. Fernandes et al. comparedthe efficacy of MeSe� and selenide and found that while MeSe�

was more efficient at reducing levels of intracellular thiols andwas more toxic, it produced lower levels of superoxide thanselenide.232 The authors suggest that the superior nucleophili-city of MeSe� compared to selenide means that it will scavengeROS under reducing conditions, resulting in a net decrease inintracellular ROS. The potential dual role of selenolates in ROSgeneration and oxidant scavenging is summarised in Fig. 4.

There is sufficient evidence to conclude that the induction ofapoptosis in many cancer cell lines by selenium compoundscannot simply be attributed to the generation of superoxideanion radical (or any other single ROS) by the redox cycling ofselenolates and oxygen as has been implied by the results ofin vitro experiments. Treatment of cancer cells with seleniumcompounds, particularly selenite, appears to induce the genera-tion of ROS by endogenous systems. The sources and the speciesof ROS that make a greater contribution to selenium-mediatedapoptosis are yet to be determined. However, the weight ofevidence suggests that the production of selenolates is importantin selenium-mediated ROS production.

Fig. 4 Redox cycling of selenolates with oxidants, thiols and NADPH-dependentreductase systems (e.g. the TrxR system). Note that this is an expansion of Fig. 3bto include both the antioxidant and prooxidant capabilities of the selenolates.Adapted from Lu et al.233


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11 Oxidation of thiols

Selenium compounds are capable of altering the intracellularredox balance via the oxidation of thiols, not only through thegeneration of ROS, but by direct oxidation. Ganther describesfour bonds that result from the direct and indirect oxidationof protein thiols by selenium compounds: selenotrisulfide(S–Se–S), selenenylsulfide (S–Se) and disulfide (S–S), as well asthe formation of diselenide bonds with protein selenols.234

The ability of selenium to cross-link protein thiols to give aselenotrisulfide was first demonstrated in vitro with reducedpancreatic ribonuclease.235 The cross-linking of mitochondrialproteins by selenite and CysSeSeCys has been linked to theinduction of the mitochondrial permeability transition and celldeath.236 The oxidation of thiols by selenols and selenenylsulfidesin the presence of GSH (i.e. under overall reducing conditions) isremarkable as it occurs despite the lower reduction potential ofselenols relative to thiols.237 The ability of selenium compoundsto oxidise protein thiols may have ramifications for redox-sensitive proteins, which have diverse and important roles insignaling, transcription and DNA repair.

11.1 Oxidation of intracellular thiols

The metabolism of selenite by human lung cancer cells causesincreases in levels of GSSG and cystine, as well as increasing thelevels of protein-bound Cys and GSSeSG.105 HSe� and MeSe� havealso been found to decrease intracellular thiols.232 CysSeSeCysoxidised protein free thiols in human hepatoma cells whereasSeMet did not.236 SeMet was capable of increasing levels ofdiselenides and selenenylsulfides in Saccharomyces cerevisiae,238

which lacks the ability to synthesise selenoproteins (thuseliminating a major SeMet metabolic pathway), which suggeststhat some metabolites of SeMet are capable of oxidisingthiols.239 As we observed for ROS generation by seleniumcompounds, thiol oxidation by selenium compounds is notconsistent between cell lines, nor is thiol oxidation alwaysassociated with increased ROS levels: SeMet oxidised cellularfree thiols in A549 lung cancer cells, but not in BEAS-2B lungepithelial cells; MeSeA and CysSeSeCys oxidised free thiols inboth cell lines, but only CysSeSeCys generated ROS and only in theA549 cell line.240 Furthermore, the toxicity of selenium compoundsincreases with changes to intracellular thiol content: interestingly,both increases and decreases in GSH levels induce selenite-inducedoxidative stress in human hepatoma cells241 and sensitisedlung cancer cell lines to MeSeA.227 Clearly, the redox status ofthe cell likely influences the balance between the antioxidantand prooxidant activities, and therefore the cytotoxicities, ofselenium metabolites.232,240

11.2 Oxidation of proteins with zinc–thiolate coordinationsites

There is a significant body of work that demonstratesthe in vitro susceptibility of proteins containing zinc–thiolatecoordination sites to oxidation by selenium compounds.Selenium compounds, including the selenium drug ebselen,are known to release Zn from metallothioneins in vitro via the

formation of a selenenylsulfide.242–244 The GSH–GSSG redox paircouples reducible selenium compounds to the metallothionein–thionein–thionin cycle, as summarised in Fig. 5.245 Selenolates,via a glutathionyl selenenylsulfide (R–Se–SG), form selenenyl-sulfide (MT–S–Se–R) bonds with Cys at the zinc-binding site ofmetallothioneins to release zinc. The resulting unstable Se–Sbond is attacked by intramolecular thiols to form the disulfide-containing thionin (MT–S2). The thionin can react again with theselenolate to form another selenenylsulfide before its reductionby GSH back to thionein (MT–(SH)2), at which point a Zn atomcan bind.245 The ability of selenium compounds to catalysethe release of zinc in the presence of GSH suggests that thismechanism is possible in vivo.242

Metallothioneins are not alone in their oxidation by catalyticselenols. Selenium compounds in oxidation states from�I to +II

have been shown to be capable of inhibiting DNA-bindingactivity and releasing zinc from a Cys-rich zinc finger proteininvolved in nucleotide excision repair.246 Selenite has beenobserved to inhibit the DNA binding capability of the zinc fingerproteins transcription factor IIIA (TFIIIA) and Sp1, in the presenceof dithiothreitol.247,248 Before the elucidation of the selenium-mediated zinc release mechanism, selenite had been shown toinhibit the DNA-binding activity of the transcription factor NF-kB inhuman T cells and lung adenocarcinoma cells,249 presumably viaselenite-mediated oxidation of Cys residues. However, Larabee et al.reported that selenite did not inhibit the DNA binding domain ofNF-kB in vitro, whereas ebselen did, indicating differences in themechanisms by which ebselen and the metabolites of seleniteinhibit redox-active proteins.248 In an osteoblast model of meta-stasis, NF-kB activation was attenuated by MeSeA, but the con-tributions of selenoproteins and low molecular weight seleniumcompounds to the attenuation were not determined.250 The com-plexity and uncertainty surrounding the mechanisms of seleniumcompounds in the inhibition of NF-kB activation exemplifies theneed for in vitro and in vivo evidence that the oxidation andinhibition of redox-sensitive proteins by selenium compounds isa mechanism by which they exert their anti-cancer effects.

The selenoamino acids, which are in a fully reduced state(oxidation state � II), are incapable of releasing zinc in vitro,246

but selenolate-containing metabolites such as SeCys and MeSe�

would be expected to have catalytic Zn-releasing activity.Investigation into the activity of MeSe� towards zinc–thiolatecoordination sites is warranted, particularly in light of thereport that MeSe� is a better substrate to the Trx and Grxsystems than HSe�.232

Fig. 5 The metallothionein and GSH/GSSG redox systems coupled by seleno-lates. Adapted from Chen and Maret.245


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11.3 Oxidation of other redox sensitive proteins

Selenium compound-mediated oxidation does not just targetproteins with zinc–thiolate sites, but redox-sensitive proteins ingeneral. Selenite and GSSeSG inhibit the DNA binding activityof the redox-sensitive transcription factor AP-1 in nuclearextracts of 3B6 lymphocytes.251 MeSe� has been shown tocatalyse the oxidation of active site Cys thiols in protein kinaseC (PKC), which has a role in signaling and has been implicatedin both tumour progression and promotion.252 Treatment ofprostate cancer cells with MeSeA led to PKC inhibition, apop-tosis and cell death. The authors proposed the followingmechanism of inhibition of PKC based on in vitro experiments:(1) MeSeA, reduced to MeSe� in the cell, can be re-oxidised bylipid peroxides to MeSeA and (2) MeSeA oxidises PKC as it isitself reduced back to MeSe�. It is possible that this redox-cyclemay be maintained in close proximity to PKC, because PKCitself binds phospholipids and their peroxides. Thus theauthors have provided an explanation of how a small amountof MeSeA might be capable of intracellular inhibition of PKC,based on evidence from in vitro and cell studies. PKC can alsobe inhibited by selenite, GSSeSG and CysSeSeCys, but experi-ments with protein kinase A (PKA) revealed that only GSSeSGand CysSeSeCys were capable of its inhibition.253 Selenite’s lackof inhibitory action towards PKA suggested to the authors thatthe lower number of Cys residues in the catalytic domain ofPKA compared to PKC affected the activity of the seleniumcompounds. Interestingly, testing the inhibition of PKC byselenite, GSSeSG and selenocystine in cells revealed that theredox modulation of PKC was compartmentally independent ofglutathione: the addition of thiols reversed the redox modula-tion.254 In this case, the presence of GSH was not required tomake selenite biologically active, although a metabolite ofselenite (GSSeSG) also inhibited PKC.

Oxidation of protein thiols may be an important mechanism ofselenite and its metabolites, as well as the selenolate metabolitesof other dietary selenium compounds. However, the ability ofselenium compounds to oxidise protein thiols in vivo will dependon the activities of systems that maintain the cellular redox status,such as the Trx system. There is substantial in vitro evidence,and some evidence from cell culture studies, of the oxidation ofCys-rich proteins by the metabolites of selenium compounds.However, more studies where both in vitro mechanisms andactivity in cells are investigated, exemplified by the study ofGundimeda et al. of the inhibition of PKC by MeSeA,252 arerequired to better understand the targets and consequences ofselenium compound-mediated protein thiol oxidation.

12 TrxR, selenium and drug resistance incancer cells

As discussed earlier, TrxR is a selenoprotein that helps tomaintain the redox status of the cells and, as such, is involvedin the regulation of many cellular events that are linked todisease states.167 There is abundant evidence that increasedTrxR expression can protect against cardiovascular disease255

and so low level selenium supplementation, using an appropriateform of supplement to increase TrxR activity, may be useful in thiscontext. The relationship between selenium supplementation, theTrxR system and cancer is not straightforward. In normal cells,TrxR protects against selenite toxicity. Human embryo kidneycells overexpressing TrxR were less sensitive to selenite than cellswith native levels of TrxR.256 The protective role of TrxR in normalcells was highlighted by preincubation of the cells with low dosesof selenite (0.1 mM) prior to higher concentration selenite treat-ment (10 mM). The low doses of selenite significantly increasedTrxR in the cells already overexpressing TrxR and made then evenless sensitive to selenite. TrxR is overexpressed in many cancercells, a characteristic that is thought to help protect againstapoptosis and promote cell growth and makes it an anti-cancertarget.233,257 The redox regulatory functions and activation oftumour suppressors also indicate a role for TrxR in cancerprevention.62 The sensitivity of cancer cells in general, as well aschemotherapeutic drug-resistant cancer cells, to selenium, may belinked to TrxR levels and activities in those cells, although theexact nature of the relationship is yet to be elucidated.

12.1 Selenium and TrxR activity in cancer cells

In a study of the sensitivity of lung cancer cell lines towardsselenite, the cell line with the greatest TrxR expression, whichalso had the greatest resistance to doxorubicin, was mostsensitive.258 At low to moderate doses, selenite increased TrxRlevels in the cells, but high selenite concentrations reduced levelsof the protein; inhibition of TrxR by auranofin further enhancedselenite’s toxicity. The sensitivity of doxorubicin-resistant cells toselenite and similar responses of TrxR to high selenite doses hasbeen observed in other lung cancer cell lines.154,259 In malignantmesothelioma cells, selenite was more toxic to the therapy-resistant sarcomatoid cells than the responsive epithelioidcells.152 Both cell types experienced TrxR inhibition with sele-nite treatment, but the sarcomatoid cells experienced greaterloss of TrxR activity. The involvement of TrxR activity in selenitetoxicity is highlighted by the sensitivity of TrxR-deficient malignantmouse cells to the compound.260 These results are in accordancewith results from a study of human lung cancer cells in whichTrxR expression was knocked down. TrxR knock down cells weremore sensitive to CysSeSeCys than control cells (there was nodifference between knock down and control cells’ sensitivitiesto SeMet or MeSeA).261 Taken together, these studies show thatwhile high baseline TrxR can protect cells from selenite-mediatedoxidative stress, the response of TrxR levels and activities toselenite treatment is an important factor in the susceptibility ofcancer cells to selenite treatment. Selenite is more toxic to cellswhose concentration and/or activity is reduced with or has alimited response to selenite treatment.

12.2 Are dietary selenium compounds inhibitors of TrxRactivity?

How selenite reduces TrxR concentration and activity leading tocell death is uncertain. Nilsonne et al. suggest that oxidativestress and oxidation of protein thiols lead to the inhibition ofTrxR activity,152 but Tobe et al. found the toxicity of selenite


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toward TrxR-deficient cells was not caused by oxidative stressand was unrelated to levels of Trx, but associated withincreased GSH production. Given the demonstrated ability ofselenite metabolites to directly oxidise protein thiols, theformation of diselenide bonds with SeCys residues in TrxRhas been postulated as a mechanism by which selenium couldinhibit TrxR activity. Selenite, GSSeSG, CysSeSeCys and HSe�

and MeSe� are all substrates for the TrxR system117,118,131,232

and could compete with Trx.117,170,262 In a study in rat liverhomogenates, TrxR activity was inhibited by addition of MeSeA,implying that selenolates competitively inhibit TrxR,170 but theauthors question whether the levels of selenium that theyfound to inhibit TrxR in vitro could be achieved in vivo. Signifi-cantly, neither MeSeCys nor MeSeA significantly altered TrxRactivity in vivo. In testing the hypothesis that selenium com-pounds can inhibit TrxR, Gromer et al. reported that theirchosen selenium compound, methylseleninate (MeSeO2

�), isnot only a weak inhibitor of TrxR in vitro, but is a good substrateof the enzyme.262 Thus far, there is no evidence of the dietaryselenium compounds or their metabolites inhibiting TrxR activityby the formation of diselenide bonds, and, given the importantrole the Trx system plays in the metabolism of selenium, it seemsunlikely that TrxR inhibitors would be found amongst metabolitesof dietary selenium compounds. Although the effects of dietaryselenium compounds on TrxR activity may be indirectly mediated,a synthetic selenium compound, ethaselen, containing twoelectrophilic Se–N bonds, has been shown to bind to the Cys–SeCys redox pair at the active site of TrxR.263 The organo-selenium compound inhibits TrxR activity and reduces cellviability in A549 lung cancer cells.

A potential application of selenite, and other seleniumcompounds, in cancer treatment is its use as an adjunct toTrxR inhibitors. Treatment of cancer cells with selenite and thegold-based TrxR inhibitor auranofin has a synergistic effect, asobserved in lung cancer cells and ovarian cells.258,264 Recently,CysSeSeCys and auranofin were shown to have a synergisticeffect in inhibiting the growth of MCF-7 breast cancer cells,which overexpress TrxR.54 Co-treatment with CysSeSeCysincreased ROS generation and also had a synergistic effect inreducing TrxR activity, without any effect on TrxR expression.Alternatively, targeting selenium metabolism and selenoproteinsynthesis to prevent synthesis of TrxR has been proposed as ananti-cancer strategy.265

13 Selenium compounds as HDAC inhibitors

Histone deacetylases (HDAC) are involved in the regulation ofgene expression and due to their aberrant recruitment and over-expression in cancer cell lines, HDACs are anti-cancer targets.266

Tumour cells are more sensitive to HDAC inhibitors than normalcells: inhibition of HDAC selectively alters gene expression andthereby opens a number of anti-tumour pathways.267 There issome evidence that metabolites of selenium compoundsinhibit HDACs.

The enzyme-catalysed transamination reaction of SeMet andMeSeCys generates KMSB and MSP, respectively. KMSB and

MSP resemble short chain fatty acid inhibitors of histonedeacetylase (HDAC) and are competitive inhibitors of HDACin vitro.268 The inhibition probably occurs via coordination of acarboxylate moiety with the HDAC active site zinc atom andhydrogen bonding with amino acid residues in the bindingpocket. KMSB and MSP inhibit HDAC1 and HDAC8 in anumber of prostate142 and colon cancer cell lines268 leadingto apoptosis in the latter. Nian et al. found that MeSeCys andSeMet had limited HDAC-inhibitory effects, but in colon cancercells MeSeCys exhibited some ability to increase histoneacetylation.268 Similarly, MeSeCys, MSP and KMSB each hadan inhibitory effect on HDAC in prostate cancer cell lines, butonly MSP and KMSB had an inhibitory effect when the threecompounds were directly applied to nuclear fractions.142 SeMetdid not inhibit HDAC when incubated with prostate cancer celllines, but KMSB did: thus it is clear that the seleno a-ketoacid metabolites of SeMet and MeSeCys inhibit HDAC, butthese metabolites are generated only in systems in which thetransaminases are active.

MeSeA, which does not have an amino acid moiety and isthus not a substrate for transaminase reactions, increasedhistone acetylation without altering the expression of HDACproteins in diffuse large B-cell lymphoma cell lines.269 Thus theinhibition of HDAC may also be caused by MeSe� that, in theabsence of a zinc-binding moiety, has been suggested to oxidiseconserved Cys residues whose modification is known to disruptthe activity of class I HDACs.269,270 MeSeA also suppressed theinduction of a pro-angiogenic transcription factor, HIF-1a, andthe secretion of vascular endothelial growth factor (VEGF)under hypoxic conditions in the lymphoma cell line.269 Thereduction of HIF-1a and VEGF and was also observed in ratsinjected with prostate adenocarcinoma cells and treated withMeSeA and was associated with a significant reduction in meta-static lung foci.271 Although the authors did not establish a causallink between HDAC inhibition and HIF-1a suppression in thelymphoma cells, they hypothesise that this chain of events is oneexplanation of the anti-angiogenic mechanisms of MeSe�.

The evidence that MeSe�, in addition to seleno a-keto acids,may inhibit HDAC activity implies that selenols may be a classof HDAC inhibitors and thus selenium compounds have twomechanisms of HDAC inhibition. MeSeCys, which can be lysedto MeSe� and transaminated to MSP, may be capable of producingtwo different HDAC inhibitors in vivo. Further experiments arerequired to confirm that MeSe� does indeed directly inhibitHDAC activity and to make the causal link between MeSe�, HDACinhibition, HIF1-a suppression and anti-angiogenic activity. Theseleno keto amino acids require in vivo testing of their HDACactivity and potential anti-angiogenic activity.

14 Elemental selenium

Elemental selenium was long held to be biologically inert, butthere is evidence that nanoparticulate elemental selenium(a description that encompasses particles from 5 to 550 nmin size272,273) has biological activity. Elemental selenium hasbeen identified in human cells as a metabolite of selenite,


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where it appears to accumulate in the cytosol, but it is notknown whether elemental selenium, as a product of selenitemetabolism, is nano-sized or forms larger aggregates.121 However,nano-sized selenium can be prepared at neutral pH by the reductionof selenite by GSH in the presence of bovine serum albumin, whichstabilises the red elemental selenium, preventing its degradationand aggregation into grey and black elemental selenium: a similarprocess can be envisaged in biological systems.274 Selenium nano-particles are less toxic than selenite,275,276 but still induce increasesin the activities of GPx and TrxR1 in rat and mice tissues thatare comparable to those induced by selenite,274 SeMet277 andMeSeCys.278 The induction of selenoprotein activities appearsto be independent of nanoparticle size.272

There is some evidence of elemental selenium acting as bothan antioxidant and a prooxidant. Elemental selenium has beenshown to protect against paraquat toxicity in human hepatomacells274 and has demonstrated free radical scavenging activityin vitro.279 In the sperm of rats exposed to cisplatin, seleniumnanoparticles coadministered intraperitoneally reduced DNAdamage and oxidative stress.273 Against a number of cancer celllines, selenium nanoparticles exhibited a similar toxicity to selenite,but were less toxic to human fibroblasts.280 In melanoma cells,the selenium nanoparticles generated ROS and induced mito-chondria-mediated apoptosis. The apparently low toxicity ofnanoparticulate red elemental selenium compared to selenitemakes it an attractive candidate for selenium supplementation, butknowledge of its biological activity is limited and the mechanismsby which it exerts its prooxidant and antioxidant activities areunknown. The ability of elemental selenium to form conjugateswith proteins281 may present opportunities to develop novelselenium–protein conjugates for specific targets. The demon-strated biological activity of exogenous selenium nanoparticlesgives rise to two questions: is nanoparticulate selenium generatedduring the metabolism of selenite? If so, does this endogenousnanoparticulate selenium have biological activity?

15 Conclusion

With regards to the connection between selenium supplementa-tion and disease prevention and treatment, there is an astoundinggap between the efficacy observed in laboratory studies and themixed results of clinical trials. The choice of selenium supplementspeciation in laboratory and clinical studies may explain some ofthis efficacy gap. Selenite has been the preferred form of seleniumfor use in laboratory studies; but methylated selenium com-pounds, diselenides and synthetic organic selenium compoundsare becoming more commonly employed. Yet most humanstudies use SeMet or selenised yeast as supplements.

In this review, we have summarised the metabolic pathwaysof the dietary selenium compounds to show that the seleniummetabolites that would be expected in vivo depend upon theactivity of the metabolic pathways. Most of the metabolites can beclassified as either selenolates or diselenides (including selenenyl-sulfides), which are redox active. Regardless of whetherthe dietary selenium compound is redox active (selenite andCysSeSeCys) or inactive (SeMet and MeSeCys), they are all

capable of producing redox active metabolites, such as HSe�

and MeSe�, that can redox cycle with oxygen, thiols and the Trxsystem. The dual prooxidant and antioxidant mechanismsof the redox active metabolites contributes to selenium’s pro-mising biological activity.

Care must be taken in drawing conclusions about the efficacyof dietary selenium compounds from in vitro and cell studies,where the metabolites will differ from in vivo metabolites due tothe different intracellular redox environments of, and differentmetabolic pathways available in, each system. The distinct meta-bolic pathways of selenium compounds and the dependence oftheir metabolism on the activities of lyases and intracellular redoxsystems may confer an advantage in using selenium in diseaseprevention and treatment. Armed with knowledge of the diseaseand the tissues to target, it may be possible to chose an effectiveselenium supplement based on the metabolite that is likely to begenerated in the target tissues and perhaps minimising sideeffects. It is important to consider the dietary selenium com-pounds as prodrugs activated by enzymes138 and the redox statusof cells.153 Miki et al. employed this approach in transducingtumour cells with the g-lyase gene and treating mice intra-peritoneally with SeMet to generate MeSe� at the tumour site,resulting in inhibited tumour growth and prolonged survival.282

Many mechanisms by which selenium compounds mayexert their biological activity have been studied (as summarisedin Table 1), but in only a few cases have mechanisms observedin vitro been studied and observed in cells and in vivo. There isgood evidence for GPx-like, radical scavenging activities in vitroand in vivo, but the relevance of metal binding activities has notbeen explored in vivo. The generation of ROS by selenolates andother metabolites of selenite and CysSeSeCys is well-documented,but the ultimate source of ROS generation and the species respon-sible for exerting selenium’s proapoptotic effects is uncertain.Similarly, the oxidation of thiols by selenolates and metabolitesof selenite is well known, but whether the direct oxidation of keyredox sensitive proteins involved in, for example, transcription andcell signaling, is a major contributor to selenium’s anti-canceractivity is unknown. Understanding how selenium supplementa-tion affects TrxR activity is an important question.

Despite uncertainties over which mechanisms describedin vitro are most important in vivo, some general statementscan be made about the potential of each of the seleniumcompounds in disease prevention and treatment, based on theliterature reviewed above. The evidence that SeMet supplementa-tion improves selenium status in humans and the apparent lackof g-lyase activities in tissues, may make it a good choice forselenium supplementation to correct a deficiency: the SeMetinevitably stored in proteins will eventually be recycled as theproteins are degraded. The methylated forms of selenium, withlower toxicities compared to selenite and in the absence of aroute for adventitious incorporation into proteins, are theobvious choice for future trials of selenium supplementationin disease prevention. The activity of selenite against cancercells has been extensively studied. Its higher toxicity may be abarrier to its use in the long-term supplementation used fordisease prevention, but it may be useful in therapy, particularly


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given the sensitivity of drug resistant cells to its metabolitesand the synergistic effects between it and auranofin. CysSe-SeCys shows similar activity to selenite. These generalisationshowever, are not prescriptive. MeSeA, for example, has recentlybeen shown to have potential in combination therapy againstan aggressive form of breast cancer in vitro and in vivo283 andreduced the spontaneous metastasis of Lewis lung carcinomain mice (whereas SeMet did not).24

Current challenges in research into selenium as a supplementfor disease prevention and treatment include: understanding themetabolism and distribution of selenium species in vivo, deter-mining the amount of selenium required for optimal seleno-protein expression and the concentrations at which seleniummay prevent disease without side-effects (e.g. type 2 diabetes). Incontinuing laboratory and epidemiological research into seleniumsupplementation as a disease prevention or treatment strategy,

Table 1 Summary of critical metabolites of dietary selenium compounds and antioxidant, prooxidant and other mechanisms observed in vitro, in cells or in vivo

Criticalmetabolites Metabolite of:

Identification incells or tissuehomogenatesa Antioxidant mechanisms Prooxidant mechanisms

Othermechanisms

CysSeSeCys Increased GPx1 activity in rats162 ROS generation in cancercells151,227,240

Radical scavenging188,193,194 Oxidation of cellularthiols,236,240 presumablyexerted by its metabolites

Metal-binding and prevention ofmetal-mediated damage in vitro203

Inhibition of PKA and PKCin vitro253

Elementalselenium

Selenite Human lungcancer cells121

Induction of GPx and Trx1 inrats and mice274,277,278

ROS generation inmelanoma cells280

Free radical scavenging activityin vitro279

Reduced oxidative damage in ratsperm273

GSSeH Selenite — Radical scavenging in vitro194

GSSeSG Selenite Rat liver cytosol119 Superoxide generation in lungcancer cells105

Porcine intestinalhomogenate120

Inhibition of PKC and PKAin vitro253

H2Se/HSe� Selenite, SeMet,MeSeCys,CysSeSeCys

— GPx-like activity in vitro188 ROS generation in lung cancercells105

Radical scavenging in vitro194 Oxidation of cellular thiols232

KMSP SeMet In vitro only142 HDAC inhibitorin cancercells142,268

MeSeCys — Cancer cell lines122 Increased GPx activity inHCAEC164

ROS generation in cancer celllines,228 presumably exerted byits metabolitesRat hepatocytes284 GPx-like and radical-scavenging

activity in vitro188,193

Metal-binding in vitro204 andprevention of metal-mediateddamage in vitro203

MeSeH/MeSe�

Selenite, SeMet,MeSeCys,MeSeA,CysSeSeCys

— Radical scavenging in hypoxictumour cells53

ROS generation in cancer celllines227–231

Reduced HDACactivity inlymphomacells269

Radical scavenging activity in lungcancer cells232

Inhibits PKC in prostate cancercells252

Oxidation of cellularthiols232,240

MSP MeSeCys Prostate cancercells142

HDAC inhibitorin cancercells142,268

SeCys CysSeSeCys,SeMet, selenite

Rat tissuehomogenates285

GPx-like activity in vitro188 ROS generation in cancercells,151,230 presumably exertedby its metabolites

Selenite — Rat hepatocytes284 Increased GPx activity inhumans164

ROS generation in cancercells218,219

Increased TrxR activity in rats169 Oxidation of protein thiols,236

presumably exerted by itsmetabolitesInhibition of PKC in vitro253

SeMet — Cancer cell lines122 Increased GPx1 activity in rats162 Oxidation of proteins in cancercells lines,240 presumablyexerted by its metabolites

Incorporationalters propertiesof amyloidproteinsin vitro198

Rat hepatocytes284 GPx-like activity in cell lysates189

Radical scavenging activityin vitro193

Metal-binding in vitro203

a Identification is not necessarily unequivocal, refer to ref. 16 for a more comprehensive list of selenium metabolites identified in vivo.


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considerations of dose and the disease state and baseline seleniumstatus of the target population are important, but choosing themost effective selenium species is also essential. We must integrateknowledge of the metabolism, speciation and biological activity tohelp determine the most appropriate selenium compound(s) fordisease prevention and treatment.

Abbreviations

Cys CysteineCysSeSeCys SelenocystineCysSeSG Selenocysteine-glutathione selenenylsulfideCysSSeSG Cysteine-glutathione selenotrisulfideDMSe DimethylselenideGTK Glutamine transaminase KGTL Glutamine transaminase Lg-glutamyl-MeSeCys

g-Glutamyl-methylselenocysteineGR Glutathione reductaseGPx Glutathione peroxidaseGrx GlutaredoxinGSH GlutathioneGSSeGalNAc Glutathionylseleno-N-acetylgalactosamineGSSeH Glutathione selenenylsulfideGSSeSG SelenodiglutathioneH2Se/HSe� Hydrogen selenide/hydrogen selenide ionHDAC Histone deacetylaseHIF-1a Hypoxia-inducible factor 1aKMSB a-Keto-g-methylselenobutyrateMeSeA Methylseleninic acidMeSeCys MethylselenocysteineMeSeGalNH2 Se-methylselenogalactosamineMeSeGalNAc Se-methylseleno-N-acetylgalactosamineMeSeGluNAc Se-methylseleno-N-acetylglucosamineMeSeH/MeSe� Methylselenol/methylselenolateMet MethionineMSP b-MethylselenopyruvateNPC Nutritional prevention of cancer (trial)PKA Protein kinase APKC Protein kinase CPREADViSE Prevention of Alzheimer’s by vitamin E and

selenium (trial)RNS Reactive nitrogen speciesROS Reactive oxygen speciesSAM S-Adenosyl-L-methionineSeCys SelenocysteineSeCysta SelenocystathionineSeHCys SelenohomocysteineSELECT Selenium and vitamin E cancer prevention trialSelP SelenoproteinSeMet SelenomethionineTFIIIA Transcription factor IIIATMSe+ TrimethylselenoniumTrx ThioredoxinTrxR Thioredoxin reductaseVEGF Vascular endothelial growth factor

Acknowledgements

We thank Dr Jurgen Gailer for helpful comments on the manuscript.We gratefully acknowledge funding from the Australian ResearchCouncil (DP0985807 to HHH) and the Australian SynchrotronPostgraduate Award (CMW).

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