This journal is c The Royal Society of Chemistry 2011 Metallomics, 2011, 3, 503–512 503

Cite this: Metallomics, 2011, 3, 503–512

Preventing metal-mediated oxidative DNA damage with selenium

compoundsw

Erin E. Battin, Matthew T. Zimmerman, Ria R. Ramoutar, Carolyn E. Quarles

and Julia L. Brumaghim*

Received 22nd October 2010, Accepted 18th January 2011

DOI: 10.1039/c0mt00063a

Copper and iron are two widely studied transition metals associated with hydroxyl radical (�OH)

generation, oxidative damage, and disease development. Because antioxidants ameliorate

metal-mediated DNA damage, DNA gel electrophoresis assays were used to quantify the ability

of ten selenium-containing compounds to inhibit metal-mediated DNA damage by hydroxyl

radical. In the CuI/H2O2 system, selenocystine, selenomethionine, and methyl-selenocysteine

inhibit DNA damage with IC50 values ranging from 3.34 to 25.1 mM. Four selenium compounds

also prevent DNA damage from FeII and H2O2. Additional gel electrophoresis experiments

indicate that CuI or FeII coordination is responsible for the selenium antioxidant activity. Mass

spectrometry studies show that a 1 : 1 stoichiometry is the most common for iron and copper

complexes of the tested compounds, even if no antioxidant activity is observed, suggesting that

metal coordination is necessary but not sufficient for selenium antioxidant activity. A majority

of the selenium compounds are electroactive, regardless of antioxidant activity, and the glutathione

peroxidase activities of the selenium compounds show no correlation to DNA damage inhibition.

Thus, metal binding is a primary mechanism of selenium antioxidant activity, and both the

chemical functionality of the selenium compound and the metal ion generating damaging hydroxyl

radical significantly affect selenium antioxidant behavior.

Introduction

During the course of cellular metabolism, reactive oxygen

species (ROS) such as hydroxyl radical (�OH) cause oxidative

damage to living organisms.1 This highly reactive species

oxidizes lipids, proteins, and DNA, and contributes to develop-

ment of cancer, chronic inflammation, and neurodegenerative

and cardiovascular diseases.1–4 In cells, transition metal ions

such as CuI and FeII generate �OH from endogenous hydrogen

peroxide (H2O2) in the Fenton and Fenton-like reactions

(reaction (1)). This metal-mediated �OH formation is the

primary cause of cellular DNA damage and death.5,6

FeII/CuI + H2O2 - FeIII/CuII + �OH + OH� (1)

Copper and iron are essential redox-active metal ions

required for normal physiological function. Cells employ

multiple pathways to maintain metal homeostasis, but under

conditions of oxidative stress, mis-regulation can lead to excessive

concentrations of non-protein-bound (labile) metal ions asso-

ciated with oxidative stress and DNA damage due to �OH

generation.7–9 Specifically, elevated levels of labile copper and iron

have been linked to hemochromatosis, anemia, diabetes, cancer,

and amyotrophic lateral sclerosis (ALS), as well as cardiovascular,

Wilson’s, Menke’s, and Alzheimer’s diseases.4,10–16 Because anti-

oxidants can ameliorate oxidative damage caused by ROS, there

has been an increasing focus on selenium antioxidants to prevent

or treat diseases caused by oxidative stress.

Selenium deficiency is associated with many diseases. In areas of

China with low soil selenium levels, Keshan disease caused

cardiomyopathy in children in the 1970s; incidence of this disease

has decreased considerably with selenium supplementation.17–19

Selenium deficiency has also been associated with other diseases,

including Kashin-Beck, HIV infection, arteriosclerosis, mis-

carriage, neurological disorders, and cancer.18–24

Selenium in dietary supplements is primarily found as

organic selenomethionine and inorganic selenite.25 Interest

regarding selenium supplementation has increased considerably

in recent years due to numerous clinical studies indicating

the benefits of selenium in disease prevention, especially

chemoprevention.26–30 The Nutritional Prevention of Cancer

(NPC) trial reported that daily selenium supplementation

(200 mg day�1) resulted in a 37% decrease in cancer incidence,

including prostate cancer.31,32 In contrast, the selenium and

vitamin E cancer prevention trial (SELECT) found that selenium

supplementation (200 mg day�1) did not uniformly protect against

Clemson University South Carolina, USAw Electronic supplementary information (ESI) available: Gel electro-phoresis results, UV-vis experiments, tabular data of gel electrophoresisexperiments, and CV and DPV plots. See DOI: 10.1039/c0mt00063a

Metallomics Dynamic Article Links

www.rsc.org/metallomics PAPER

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504 Metallomics, 2011, 3, 503–512 This journal is c The Royal Society of Chemistry 2011

prostate cancer.33 The conflicting results of these studies highlight

the need for a greater understanding of selenium antioxidant

mechanisms.

In proteins, selenium is incorporated into selenocysteine, an

amino acid required for the synthesis of several selenoenzymes,

including glutathione peroxidase, iodothyronine deiodinases,

and thioredoxin reductases.34 Glutathione peroxidase (GPx) is

an antioxidant enzyme that protects intracellular components

from oxidative stress by catalyzing the reduction of H2O2 to

water via the selenocysteine-containing active site.35 Because

of the biological importance of this process, traditional

quantification of selenium antioxidant activity has been deter-

mined by spectroscopic measurements of H2O2 reduction,

similar to the GPx mechanism.35

Selenium complexes have been observed with mercury, and

this complexation may prevent ROS damage caused by heavy

metal toxicity.36–38 It has also been proposed that selenium

binding protects against lead toxicity.39–41 Selenocysteine

also coordinates metal ions in enzymes: molybdenum and

tungsten in formate dehydrogenases,42 and nickel in [NiFeSe]

hydrogenase.43 Thus, given the role of copper and iron in �OH

generation, it is important to explore the role of metal

coordination in selenium antioxidant behavior.

We previously reported the ability of selenomethionine, seleno-

cystine, and 2-aminophenyl diselenide to inhibit CuI-mediated

DNA damage.44 In the present work, we report the quantifica-

tion of copper-mediated DNA damage inhibition for seven

additional selenium compounds. In addition, we have quantified

and compared the ability of all ten selenium compounds to

prevent FeII-mediated DNA damage. Mass spectrometry and

cyclic voltammetry studies were also performed to investigate

the metal binding and electrochemical activity of selenium

antioxidants. Metal coordination is compared with GPx-like

activity for prevention of metal-mediated DNA damage to

determine the primary mechanism for DNA damage inhibition.

Determining the chemical properties and mechanims that

affect antioxidant activity of selenium compounds will aid

researchers in identifying more potent selenium antioxidants

to treat and prevent disease.

Results and discussion

Determining antioxidant activity for selenium compounds

The selenium compounds in Fig. 1 were examined using gel

electrophoresis to determine their ability to inhibit oxidative

DNA damage caused by CuI/H2O2 (pH 7) or FeII/H2O2 (pH 6).

This DNA damage assay enables quantification and direct

comparison of metal-mediated oxidative DNA damage preven-

tion to determine biologically-relevant antioxidant potency for

a variety of selenium compounds.

Fig. 2 shows the results of a gel electrophoresis experiment

testing methyl-selenocysteine (MeSeCys) for its ability to inhibit

oxidative DNA damage caused by CuI/H2O2. Hydrogen

peroxide alone does not damage DNA (lane 2); however,

combining CuI with H2O2 results in 98% DNA damage

(lane 4). Adding methyl-selenocysteine in increasing concen-

trations results in significant reduction of oxidative DNA

damage, with greater than 95% DNA damage inhibition at

concentrations Z 100 mM. A best-fit dose-response curve for

methyl-selenocysteine is shown in Fig. 3, and the concentra-

tion of methyl-selenocysteine required to inhibit 50% of

copper-mediated oxidative DNA damage (IC50 value) was

calculated to be 8.64 � 0.02 mM. The remaining selenium

compounds (Fig. 1) were similarly examined for their ability to

inhibit copper-mediated DNA damage using the same method

(see ESIw for gel images, IC50 plots, and data tables).

Selenomethionine and selenocystine have been previously

studied, and inhibit >90% of copper-mediated DNA damage

at 100 mM with IC50 values (Table 1) of 25.10 � 0.01 mM and

3.34 � 0.08 mM, respectively.44 In contrast, selenocystamine

had significant prooxidant activity at low concentrations

(1–500 mM), causing a maximum of 22 � 5% DNA damage

(p= 0.02), but preventing 59 � 3% DNA damage at 1000 mM.

Under these conditions, 3,30-diselenobispropionic acid, 3,30-seleno-

bispropionic acid, 2-aminophenyl diselenide, 2-carboxyphenyl

diselenide, 2-carboxyphenyl selenide, and 4-carboxyphenyl

diselenide do not inhibit CuI-mediated DNA damage.

Of the ten selenium compounds tested, those containing both

amine and carboxylate groups (selenocystine, selenomethionine,

and methyl-selenocysteine) significantly inhibit copper-mediated

DNA damage. In contrast, selenocystamine, a selenium

compound that has amine functionality but lacks carboxylate

groups, shows prooxidant activity at low concentrations

despite its structural similarities to the three amino acids.

Similarly, selenium compounds with only carboxylate groups

(3,30-diselenobispropionic acid and 3,30-selenobispropionic

acid) or aromatic groups (2-aminophenyl diselenide, 2-carboxy-

phenyl diselenide, and 2-carboxyphenyl selenide) also show no

ability to inhibit copper-mediated DNA damage. Thus, the

antioxidant ability of selenium compounds is strongly influenced

by the functional groups present in the species.

Inhibition of iron-mediated DNA damage by selenium

compounds

Using a similar plasmid nicking assay, the ability of selenium

compounds to prevent DNA damage by FeII/H2O2 was

also determined. These gel electrophoresis experiments with

iron were conducted at pH 6 rather than pH 7 due to iron

insolubility at pH> 6.45–47 Four of the ten selenium compounds

tested inhibit iron-mediated oxidative DNA damage. Seleno-

cystamine inhibits 79 � 1% of iron-mediated DNA damage

at 250 mM (Fig. 4). Methyl-selenocysteine, 3,30-diseleno-

bispropionic acid, and 3,30-selenobispropionic acid inhibit

60 � 2%, 76 � 1%, and 42 � 8% of oxidative DNA damage

at 1000 mM, respectively. The remaining selenium compounds

have no effect on iron-mediated oxidative DNA damage.

IC50 values for inhibition of iron-mediated DNA damage

were calculated for selenocystamine (121.4 � 0.3 mM) and

methyl-selenocysteine (378.4 � 0.1 mM; Table 2). An IC50

value of B75 mM is estimated for 3,30-diselenobispropionic

acid based on averaging DNA damage inhibition at 50 mM(42 � 7%) and 100 mM (59 � 6%), since a dose-response

curve did not accurately fit the gel data for this compound.

In all cases, inhibition of iron-mediated DNA damage

occurred at higher concentrations of selenium compound than

inhibition of copper-mediated damage. Surprisingly, only

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This journal is c The Royal Society of Chemistry 2011 Metallomics, 2011, 3, 503–512 505

methyl-selenocysteine inhibited DNA damage from both CuI

and FeII; selenocystamine, 3,30-diselenobispropionic acid, and

3,30-selenobispropionic acid inhibited only iron-mediated

damage.

The striking differences observed for selenium antioxidant

prevention of metal-mediated damage by CuI and FeII high-

light the critical role of both the metal ion and selenium

functionality in selenium antioxidant behavior. Compounds

containing both amine and carboxylate substituents

demonstrate the greatest antioxidant activity with CuI-

mediated damage; however, for inhibition of FeII-mediated

damage, both substituents are not required. Selenocystamine,

3,30-diselenobispropionic acid, and 3,30-selenobispropionic

acid, compounds that inhibit iron-mediated DNA damage,

contain either amine or carboxylate substituents. Interestingly,

selenocystamine, with only amine substituents, has a lower

IC50 value for prevention of iron-mediated damage than

methyl-selenocysteine, with both amine and carboxylate sub-

stituents. Other selenium compounds with both amine and

carboxylate substituents, such as selenocystine and seleno-

methionine, do not inhibit iron-meditated oxidative damage.

Studies with CuI/H2O2 suggest that the presence of two

selenium atoms instead of one may increase antioxidant

activity, and a similar trend is observed in the FeII/H2O2

system. The diselenide compounds selenocystamine and 3,30-

diselenobispropionic acid inhibit significantly more DNA

damage at lower concentrations than the selenide compounds

methyl-selenocysteine and 3,30-selenobispropionic acid. Thus,

functionality of the selenium compounds also plays an im-

portant role in selenium antioxidant activity with iron, but

determination of antioxidant efficacy based on chemical fea-

tures of these compounds is not straightforward and requires

further investigation.

The role of metal coordination in antioxidant activity

Previous gel electrophoresis experiments with [Cu(bipy)2]+

(bipy = 2,20-bipyridine) instead of CuI as the copper source,

established that prevention of CuI-mediated DNA damage by

selenocystine and selenomethionine is due to CuI coordina-

tion.44 The bipyridyl ligands fully coordinate copper in

[Cu(bipy)2]+; thus, DNA damage inhibition should not be

observed if copper coordination is responsible for selenium

antioxidant activity. In DNA damage assays with [Cu(bipy)2]+/

H2O2, selenocystine, selenomethionine, and methyl-

selenocysteine show no inhibition of oxidative DNA damage

even at high concentrations (Supplementary Information).

Because prior coordination of the copper eliminates

antioxidant activity for these selenium compounds, our gel

studies clearly indicate that their antioxidant behavior is due

to copper coordination.

UV-vis spectroscopy was also performed to determine

whether CuI coordination occurs upon addition of the sele-

nium compounds. Most of the selenium compounds that

prevent DNA damage in the gel studies with CuI show

absorption bands around 226 nm in the UV-vis spectra

(Fig. S7, ESIw), whereas these absorbances are not observed

for the selenium compounds that had no antioxidant activity

with CuI/H2O2. Selenocystamine showed no UV absorption

band in the presence of CuI, although both prooxidant and

antioxidant activities were observed. Copper-selenium charge

transfer bands commonly occur between lmax = 241–260 nm.48,49

Similar UV-vis bands were not observed for selenocystine,

selenomethionine and methyl-selenocysteine in the presence of

[Cu(bipy)2]+.

Gel electrophoresis experiments with [Fe(EDTA)]2� were also

conducted to determine whether the antioxidant activity of

methyl-selenocysteine, selenocystamine, 3,30-diselenobis-propionic

Fig. 1 Selenium compounds tested for DNA damage inhibition.

Fig. 2 Agarose gel showing a reduction in oxidative DNA damage

with increasing methyl-selenocysteine concentration. Lanes: MW) 1 kb

ladder; (1) plasmid DNA; (2) DNA+ H2O2; (3) DNA+ Se + H2O2;

(4) DNA + CuII/ascorbate + H2O2; (5–11) same as lane 4 with

increasing [MeSeCys]: 0.1, 1, 5, 10, 50, 100, and 1000 mM, respectively.

Fig. 3 A best-fit dose-response curve for methyl-selenocysteine

prevention of DNA damage by CuI; the concentration of methyl-

selenocysteine (MeSeCys) required to inhibit 50% of copper-mediated

DNA damage (IC50) is 8.64 � 0.02 mM.

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506 Metallomics, 2011, 3, 503–512 This journal is c The Royal Society of Chemistry 2011

acid, and 3,30-selenobispropionic acid is due to iron coordination.

Similarly to bipyridyl in the CuI system, EDTA fully coordinates

FeII and prevents antioxidant binding, but [Fe(EDTA)]2� still

reacts with H2O2 to cause DNA damage.

Under these conditions, methyl-selenocysteine inhibits

27 � 3% of oxidative DNA damage at 1000 mM with

[Fe(EDTA)]2�/H2O2 compared to 60� 2% inhibition observed

for the FeII/H2O2 system. Similarly, selenocystamine inhibits

60 � 2% of DNA damage at 1000 mM with [Fe(EDTA)]2�,

significantly less than the 100% inhibition observed at 1000 mMwith FeII. Both 3,30-diselenobispropionic acid and 3,30-seleno-

bispropionic acid also demonstrate DNA damage inhibition

with [Fe(EDTA)]2� (17 � 3 and 12 � 6%, respectively, at

1000 mM) but to a significantly lesser extent than with FeII/H2O2

(76 � 1 and 42 � 8%, repectively, at 1000 mM). Due to the

high FeII-EDTA binding affinity (2 � 1014 50), competition

for iron binding sites from selenium compounds is not likely.

Thus, the DNA damage inhibition observed at higher concen-

trations in the presence of [Fe(EDTA)]2� as the iron source is

likely a result of radical scavenging.

UV-vis experiments were also conducted with selenium

compounds and FeII/H2O2 to examine possible iron-selenium

binding. Iron-selenium coordination has been observed spectro-

scopically at lmax = 316–340 nm for iron-selenium-thiolate

clusters51 and at lmax = 311–389 nm for iron-selenium

clusters.52 In contrast to similar experiments with CuI, UV-vis

spectra of the tested selenium compounds showed no significant

changes upon addition of FeII. Based on these results, either

iron coordination is very weak, or no direct iron-selenium

binding is present. Because iron binding is a significant factor

in DNA damage inhibition, as established by electrophoresis

experiments with [Fe(EDTA]2�, this suggests that coordinaton

of iron to the selenium compounds may occur through the

amine and/or carboxylate groups as well as the selenium atom.

Of the ten selenium compounds, only methyl-selenocysteine

prevents both copper- and iron-mediated DNA damage. Two

selenium compounds (3,30-diselenobispropionic acid, and

3,30-selenobispropionic acid) demonstrate antioxidant activity

with FeII but do not inhibit CuI-mediated DNA damage, and

two compounds (selenocystine and selenomethionine) prevent

only CuI-mediated damage. Surprisingly, selenocystamine is a

prooxidant at low concentrations with copper (1–500 mM), but

acts only as an antioxidant with iron. Because biological

selenium concentrations are typically in the low micromolar

Table 1 IC50 values, lmax, and mass spectrometry data for the tested selenium compounds with CuI, as well as GPx activity measurements

Compound CuI IC50 (mM)a CuI lmax (nm) m/z (Da) CuI : Se compoundRelativeGPx Activityb

Selenocystine 3.34 � 0.08c 240c 397.8, 731.6 1 : 1, 1 : 2 4.5Methyl-selenocysteine 8.64 � 0.02 241 247.8, 427.8, 598.1 1 : 1, 1 : 2, 1 : 3 0.7Selenomethionine 25.10 � 0.01c 226c 261.9, 456.9, 650.9 1 : 1, 1 : 2, 1 : 3 0.6Selenocystamine 59 � 3% (1000 mM)c — — — 8.82-Aminophenyl diselenide — — — — 6.42-Carboxyphenyl diselenide — — 466.9 1 : 1 B02-Carboxyphenyl selenide — — 383.9 1 : 1[d] B04-Carboxyphenyl diselenide — — 466.9 1 : 1 B03,30-Diselenobispropionic acid — — 370.6, 673.4 1 : 1, 1 : 2 B03,30-Selenobispropionic acid — — 290.7, 515.6 1 : 1, 1 : 2 B0

a IC50 is defined as the concentration at which the compound inhibited 50% of DNA damage. b GPx relative activity values are reported relative to

ebselen in methanol. c Values from ref. 44. d ESI mass spectrometry voltage of 5500 V.

Fig. 4 Agarose gel showing reduction in iron-mediated oxidative

DNA damage with selenocystamine. Lanes: MW) 1 kb ladder;

(1) plasmid DNA; (2) DNA + H2O2; (3) DNA + Se + H2O2;

(4) DNA + FeII + H2O2; (5–9) same as lane 4 with increasing

[SeCysta]: 1, 10, 100, 500, and 1000 mM, respectively.

Table 2 IC50 values, and mass spectrometry data for the tested selenium compounds with FeII

Compound FeII IC50 (mM)a m/z (Da) FeII : Se compound

3,30-Diselenobispropionic acid B75b 360.6, 664.4 1 : 1, 1 : 2Selenocystamine 121.4 � 0.3 415.1 1 : 3Methyl-selenocysteine 378.4 � 0.1 239.1, 420.8, 601.8 1 : 1, 1 : 2, 1 : 33,30-Selenobispropionic acid 42 � 8% inhibition (1000 mM) 288.7, 506.6 1 : 1, 1 : 2Selenocystine — 390.8, 724.7, 1058.2 1 : 1, 1 : 2, 1 : 32-Aminophenyl diselenide — 404.8 1 : 1c

2-Carboxyphenyl diselenide — 465.7 1 : 1c

2-Carboxyphenyl selenide — 376.9, 696.7 1 : 1, 1 : 2c

4-Carboxyphenyl diselenide — 455.1 1 : 1Selenomethionine — 448.9, 643.9 1 : 2, 1 : 3

a IC50 is defined as the concentration at which the compound inhibited 50% of DNA damage. b Estimated value. c ESI mass spectrometry voltage

of 5500 V.

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This journal is c The Royal Society of Chemistry 2011 Metallomics, 2011, 3, 503–512 507

range (B1–10 mM),53,54 selenium antioxidant activity may be

much more pronounced for copper-mediated oxidative damage

than for iron in cells. In addition, the antioxidant activity of

these compounds is more complex than previously reported,

since the activity is affected by the properties of the selenium

compounds and the functional groups present. It is clear that

the precise chemical mechanism(s) for the observed selenium

activity requires further study. However, this work suggests

that it may be possible to select selenium antioxidants for

prevention of oxidative damage by specific metal ions, since

the observed antioxidant activity of the selenium com-

pounds is greatly affected by the metal ion causing the DNA

damage.

Glutathione peroxidase activity of selenium compounds

GPx activity measurement is a typical method used to determine

selenium antioxidant activity, and a great deal of research has

focused on the development of more potent GPx mimics,

including small organoselenium compounds.55–60 The selenium

drug ebselen (2-phenyl-1,2-benzisoselazol-3(2H)-one) protects

against ischemic brain injury in human clinical trials,61 and

prevents neurotoxicity in Parkinson’s disease, a disease where

ROS are considered the primary cause of pathogenesis.61–64

The mechanism for this protective ability is thought to be

reducing oxidative damage by H2O2 in a manner similar to

that of the GPx enzyme, and ebselen is typically used as the

standard relative to which all selenium GPx measurements are

compared.60,65,66

Although GPx activity is commonly used to assess antioxidant

behavior of selenium compounds, these measurements are not

necessarily biologically relevant.67 GPx activity measurements

are known to be greatly affected by the experimental method

used,56,58,65,66 and Sarma et al. have demonstrated that an

undesired thiol exchange reaction can artificially decrease GPx

activity measurements for selenium compounds.66 In addition,

H2O2 is a relatively non-reactive oxygen species in the absence

of metal ions. Hydroxyl radical, in contrast, is one of the most

highly reactive and damaging radical species.6,68 Therefore,

directly preventing hydroxyl radical formation or release at the

metal center would directly prevent more oxidative damage

than scavenging H2O2.69

To determine how this metal-binding mechanism for selenium

antioxidant activity compares to GPx activity of selenium

compounds, we measured their ability to reduce H2O2 in the

presence of benzenethiol according to the method reported by

Mareque et al.65 Selenomethionine, 2-aminophenyl diselenide,

and selenocystine have been previously reported to have GPx

activities of 0.6, 6.4, and 4.5 compared to ebselen in methanol,

respectively.44 Selenocystamine and methyl-selenocysteine

have GPx activities of 8.8 and 0.7 compared to ebselen,

whereas the remaining selenium compounds do not demonstrate

significant GPx activity under these conditions. Mishra and

coworkers56 reported that selenocystamine has a much higher

GPx activity (B22 times higher) than 3,30-diselenobispropionic

acid, a trend that we also observed (Table 1). In addition,

Yasuda et al. reported that selenocystine and selenocystamine

have similar GPx activity for decomposition of t-butyl

hydroperoxide.57

If GPx activity were the primary mechanism for DNA

damage inhibition, these results suggest that selenocystamine,

2-aminophenyl diselenide, and selenocystine should have the

highest antioxidant activity, whereas the remaining selenium

compounds should be poor antioxidants due to their lower

GPx activities. Our DNA damage assays directly contradict

this hypothesis. Methyl-selenocysteine has a low GPx activity

and prevents both copper- and iron-mediated DNA damage.

Selenocystamine has a high GPx activity, but inhibits only

iron-mediated DNA damage. Other selenium compounds,

including 3,30-diselenobispropionic acid and 3,30-seleno-

bispropionic acid show very little or no GPx activity; however,

these compounds demonstrate significant antioxidant activity

with FeII. Thus, it is apparent that the majority of metal-

mediated DNA damage inhibition is due to a mechanism

distinct from GPx activity. Based on our DNA damage assay

results, metal-antioxidant binding is the primary mechanism

for the observed selenium antioxidant activity, although radical

scavenging is likely a secondary mechanism for FeII-mediated

damage.

Electrochemistry of selenium compounds

To determine whether there is a correlation between selenium

antioxidant activity and electrochemistry of the selenium com-

pounds (Fig. 2), we conducted cyclic voltammetry (CV) and

differential pulse voltammetry (DPV) experiments in aqueous

buffer at pH 7. Only three of the ten compounds have measureable

E1/2 values using a glassy carbon working electrode: seleno-

cystamine (�329 mV), 2-carboxyphenyl diselenide (535 mV),

and 2-aminophenyl diselenide (with two redox potentials at

308 mV and �429 mV; Table 3). In contrast, selenomethionine,

2-carboxyphenyl selenide, 3,30-diselenobispropionic acid, and

selenocystine show only a single oxidation wave, whereas

3,30-selenobispropionic acid, 4-carboxyphenyl diselenide, and

Table 3 Electrochemical properties of selenium compounds versus normal hydrogen electrode (NHE)

Compound Epa (mV) Epc (mV) DE (mV) E1/2 (mV)

Selenomethionine 693 — — —Selenocystine �115 — — —Methyl-selenocysteine — — — —Selenocystamine �131 �526 394 �3292-Aminophenyl diselenide 593, 120 24, �978 569, 1099 308, �4292-Carboxyphenyl diselenide 501 569 67 5352-Carboxyphenyl selenide 832 — — —4-Carboxyphenyl diselenide — — — —3,30-Diselenobispropionic acid 907 — — —3,30-Selenobispropionic acid — — — —

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508 Metallomics, 2011, 3, 503–512 This journal is c The Royal Society of Chemistry 2011

methyl-selenocysteine exhibit no electrochemical activity

between �1000 mV and 1000 mV (CV data are provided in

the Supplemetary Information).

Bai et al. observed two anodic waves for selenocystine at

810 mV and B1200 mV; as well as two anodic waves

for selenomethionine at B640 mV and 1100 mV.70 This dis-

crepancy is likely due to the difference in both the pH and choice

of working electrode; Bai and coworkers reported these poten-

tials at pH 3.9 using a gold working electrode. Many electro-

chemical studies of selenium antioxidants have been performed

with either mercury or gold electrodes;70,71 however, both

mercury72 and gold73 can form complexes with selenium com-

pounds. As a result, observed electrochemical behavior may be a

result of the selenium compound interacting with the metal

electrode. Our electrochemical measurements using a glassy

carbon electrode are reflective of the electrochemical behavior

of the uncoordinated selenium compounds. Because �OH

generation and neutralization are one-electron redox processes,

and because antioxidant activity may be correlated to the redox

activity of the selenium compound alone, it is vital to determine

the electrochemistry of the uncoordinated selenium compounds.

Selenocystine, an antioxidant with both amine and

carboxylate functionalities, has a single irreversible anodic

wave at �115 mV, whereas the amine-functionalized seleno-

cystamine exhibits quasi-reversible behavior with a similar

redox potential of �329 mV. In contrast, the similar amino

acids selenomethionine and methyl-selenocysteine show very

different electrochemical behavior: selenocystine has a single

anodic wave at 693 mV, whereas methyl-selenocysteine

exhibited no electrochemical behavior. These electrochemical

differences are somewhat surprising, since selenomethionine

and methyl-selenocysteine differ only by one methylene group,

and one might expect more similar electrochemistry.

The results of our DNA damage prevention assays do not

correlate with the observed electrochemistry of the selenium

compounds. Methyl-selenocysteine inhibits both CuI- and

FeII-mediated DNA damage, but shows no electrochemical

activity, whereas selenomethionine inhibits CuI-mediated DNA

damage and has only an oxidation potential. Similarly,

3,30-diselenobispropionic acid undergoes only oxidation and

inhibits FeII-mediated DNA damage, whereas 3,30-seleno-

bispropionic acid also inhibits FeII-mediated DNA damage, but

has no observable electrochemical behavior. Both 2-carboxy-

phenyl selenide and 2-carboxyphenyl diselenide also differ by a

single selenium atom, but 2-carboxyphenyl diselenide undergoes

both oxidation and reduction, whereas 2-carboxyphenyl selenide

exhibits only an oxidation potential. Despite their high oxidation

potentials, neither 2-carboxyphenyl diselenide or 2-carboxyphenyl

selenide inhibit CuI- or FeII-mediated DNA damage. Since

no correlation is observed between oxidation potential of the

selenium compound and DNA damage prevention, radical

scavenging is likely not a major mechanism for the observed

antioxidant activity.

Mass spectrometry evidence for metal coordination to selenium

antioxidants

Electrospray ionization mass spectrometry (ESI-MS) was used

to examine the stoichiometry of CuI or FeII coordination to

the selenium compounds in Fig. 2. Most selenium compounds

form 1 : 1 and 1 : 2 (Cu : ligand) complexes with CuI, except for

2-aminophenyl diselenide (forms only a 1 : 1 complex) and

selenocystamine (shows no coordination). Interestingly

both selenomethionine and methyl-selenocysteine also form

complexes with CuI in a 1 : 3 ratio.

Because CuI typically adopts a tetrahedral coordination

geometry, it is probable that many of these selenium compounds

bind CuI through the selenium atom, as well as through the

amine nitrogen and/or carboxylate oxygen atom(s). Since the

majority of selenium compounds show metal coordination

regardless of antioxidant activity, these results suggest that

metal coordination is necessary but not sufficient for preven-

tion of copper-mediated DNA damage.

Similar mass spectrometry studies with FeII reveal iron

binding to all of tested selenium compounds. Both 1 : 1 and

1 : 2 stoichiometry (Fe : ligand) is observed for all compounds

except 4-carboxyphenyl diselenide (only a 1 : 1 ratio) and

selenocystamine (only a 1 : 3 ratio). A 1 : 3 coordination ratio

was also observed for selenomethionine, selenocystine, and

methyl-selenocysteine. Again, all the selenium compounds

coordinate FeII, regardless of antioxidant activity, suggesting

that iron coordination is necessary but not sufficient for

antioxidant activity. Metal-antioxidant binding may prevent

DNA damage by altering the redox potential of the metal ion

and preventing H2O2 reduction to �OH or CuII and FeIII

reduction by cellular reductants.74 Alternatively, the metal-

bound selenium antioxidant may be poised to efficiently

scavenge �OH immediately upon formation at the metal center

before it can be released.

A comparison of selenium and sulfur antioxidant activity

The antioxidant properties of sulfur compounds have also

been extensively investigated and are commonly compared to

those of selenium antioxidants.35,75–83 Sulfur antioxidants

have chemopreventitive effects against certain types of cancer

and are of great interest for the treatment and prevention

of diseases.77–78,82,83 Sulfur compounds also protect cells

against oxidative damage.84 However, selenium compounds are

generally more effective antioxidants than sulfur compounds.

Kumar et al. showed that selenium compounds protected cells

and DNA more effectively from peroxynitrite damage than

analogous sulfur compounds.85 Similarly, Ip et al. compared

the efficacy of selenium and sulfur compounds for chemo-

prevention in rats and reported that the concentration of

selenium compound (selenocystamine, methyl-selenocysteine,

and selenobetaine) required for comparable antiocarcinogenic

efficacy was 500- to 750-fold lower than that of analogous sulfur

compounds (cystamine, S-methylcysteine, and sulfobetaine).86

We have reported the ability of sulfur compounds to inhibit

iron- and copper-mediated DNA damage using the same

DNA damage assays.75 Of the ten sulfur compounds tested,

oxidized and reduced glutathione, cystine, cysteine, methyl-

cysteine, and methionine prevented copper-mediated DNA

damage with IC50 values between 3–12 mM. 2-Aminophenyl

disulfide, 3-carboxypropyl disulfide, and cystamine showed no

antioxidant activity with CuI/H2O2, whereas 2-carboxyphenyl

disulfide showed prooxidant activity at 25 mM.75

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This journal is c The Royal Society of Chemistry 2011 Metallomics, 2011, 3, 503–512 509

Comparing selenium and sulfur antioxidant activity using

CuI/H2O2 mediated DNA damage inhibition, selenocystine

and cystine have the lowest IC50 values (3.34 � 0.08 mM and

3.34 � 0.07 mM, respectively). Similarly, selenomethionine

and methionine have IC50 values of 25.10 � 0.01 mM and

11.20 � 0.01 mM respectively, and methyl-selenocysteine

and methyl-cysteine have IC50 values of 8.64 � 0.02 mM and

8.90 � 0.01 mM, respectively.53,75 Both 2,20-dithiosalicylic acid

and 2-carboxyphenyl diselenide have no effect on CuI-mediated

DNA damage; similar results were also observed for 2-amino-

phenyl disulfide and 2-aminophenyl diselenide.53,75 Our results

indicate that selenium compounds prevent CuI-mediated DNA

damage similarly to their sulfur analogs.

With FeII and H2O2, however, sulfur compounds do not

demonstrate similar antioxidant activity compared to their

selenium counterparts. Only 3-carboxypropyl disulfide and

glutathione inhibit iron-mediated DNA damage at very high

concentrations; methyl-cysteine, and cystamine do not.75 Thus,

selenium compounds are significantly more potent antioxidants

in preventing iron-mediated DNA damage compared to their

sulfur analogs.

Based on our results, neither glutathione peroxidase measure-

ments nor electrochemical activity show a correlation to

prevention of metal-mediated DNA damage by selenium

compounds, although reactive oxygen species scavenging

may occur at high concentrations. Instead, our work demon-

strates that the biochemical mechanisms for selenium anti-

oxidant activity are much more complex than previously

reported. Prevention of metal-mediated DNA damage by

selenium compounds occurs primarily through a metal-binding

mechanism, but metal binding alone is not sufficient for anti-

oxidant activity. Both the functional groups on the selenium

compound and the identity of the metal ion also play significant

roles in selenium antioxidant activity.

Experimental section

Materials

Water was deionized using a Nano Pure DIamond Ultrapure

H2O system (Barnstead International). H2O2 (30%) and

CuSO4 were purchased from Fisher. NaCl (99.999% to

avoid trace metal contamination), selenocystamine and seleno-

cystine were purchased from Sigma-Aldrich; ethanol (99.5%),

3-(N-morpholino)propanesulfonic acid (MOPS), seleno-

methionine, 2-aminophenyl diselenide, andmethyl-selenocysteine

were obtained from Acros. 2,2-Bipyridine and FeSO4

were purchased from Alfa Aesar. 2-Carboxyphenyl selenide,

2-carboxyphenyl diselenide, and 4-carboxyphenyl diselenide

were purchased from Focus. EDTA (disodium salt) and

ascorbic acid were obtained from J. T. Baker; 2-(N-morpholino)-

ethanesulfonic acid (MES) buffer was purchased from

Calbiochem. All UV-vis spectra and GPx measurements were

obtained using a Shimadzu UV-3101 PC spectrophotometer.

Electrochemical experiments were conducted on CH Electro-

chemical Analyzer (CH Instruments, Inc.). Prior to use,

all selenium compounds were stored under nitrogen in a

desiccator at 15 1C to prevent oxidation.

Purification of plasmid DNA

Plasmid DNA (pBluescript SK) was purified from E. coliDH1

using a Qiaprep Spin Miniprep kit (Qiagen, Chatsworth, CA).

Plasmid DNA was eluted in Tris-EDTA buffer and dialyzed

for 24 h at 4 1C against 130 mM NaCl. Absorbance ratios

of A250/A260 r 0.95 and A260/A280 Z 1.8 were determined for

DNA used in all experiments.

DNA damage experiments

CuSO4 (6 mM), the indicated concentrations of selenium

compound, ethanol (10 mM), deionized H2O, MOPS (10 mM),

NaCl (130 mM), and ascorbic acid (1.25 equiv; 7.5 mM, to

reduce CuII to CuI) were combined at pH 7 and allowed to

stand for 5 min at room temperature. Plasmid (pBSSK, 0.1 pmol

in 130 mM NaCl) was added to the reaction mixture and the

solution was again allowed to stand for an additional 5 min.

The �OH formation was then initiated by the addition of H2O2

(50 mM). After 30 min, EDTA (50mM) was added to quench

the reaction. For Cu(bipy)2+ gel electrophoresis studies,

Cu(bipy)22+ (50 mM) was used in place of CuSO4, and the

concentration of ascorbic acid was increased to 62.5 mM. For

FeSO4 DNA damage experiments, FeSO4 (2 mM) and MES

(10 mM, pH 6) were substituted for the CuSO4, ascorbic acid,

and MOPS. For [Fe(EDTA)]2� DNA damage experiments,

[Fe(EDTA)]2� (400 mM) was substituted for FeSO4. All con-

centrations indicated are the final concentrations in a 10 mLvolume.

Damaged and undamaged forms of plasmid DNA were

separated using 1% agarose gel electrophoresis in TAE buffer

(140 V for 30 min). The gels were stained with ethidium

bromide and imaged under UV light, where the amount of

damaged (nicked) DNA versus undamaged (supercoiled) DNA

was determined using UViProMW (Jencons Scientific Inc., 2003).

Due to less efficient staining by ethidium bromide, supercoiled

DNA band intensities were multiplied by 1.24 prior to

comparison.87,88 Additionally, DNA band intensities in each

lane were normalized so that addition of supercoiled and

nicked DNA equaled 100%. At the concentration used in

the copper gels, ascorbic acid shows no effect on DNA

damage. Studies with only CuII/ascorbate or FeII, DNA, and

the selenium compounds also showed no DNA damage. MES

and MOPS buffers were treated with chelating resin before use

and have no significant interactions with metal ions.89

Calculation of DNA damage inhibition

DNA damage inhibition was calculated using the 1-[% N/%

B]*100, where % N is the percentage of nicked DNA in the

selenium-containing lanes (lanes 6–9) and% B is the percentage

of nicked DNA in the Cu+/H2O2 or Fe2+/H2O2 lane (lane 5).

Both % N and % B were normalized for residual nicking

(lane 2) prior to calculation, and results are the average of at

least three trials with standard deviations indicated by error

bars. Additionally, for all selenium DNA damage inhibition

studies, no linear DNA was observed. Statistical significance

was determined by calculating p values at 95% confidence

(p o 0.05 indicates significance) as described by Perkowski

et al.;90 tables listing these values can be found in the ESIw.

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510 Metallomics, 2011, 3, 503–512 This journal is c The Royal Society of Chemistry 2011

IC50 determination

The plots of percent inhibition of DNA damage versus

log concentration of selenium compound (mM) were fit to a

variable-slope sigmoidal dose-response curve using SigmaPlot

(v. 9.01, Systat Software, Inc.). IC50 value errors were calculated

from error propagation of the gel electrophoresis measurements.

UV-vis measurements

CuSO4 (58 mM), ascorbic acid (72.5 mM) and selenium compound

(116 mM) were combined; the pH was adjusted to 7 using

MOPS buffer (10 mM). Similarly, compounds demonstrating

antioxidant activity with CuI were tested with Cu(bipy)2+,

where Cu(bipy)22+ (29 mM), ascorbic acid (36.25 mM), and the

selenium compound (58 mM) were combined at pH 7 using

MOPS (10 mM). For FeII experiments, FeSO4 (300 mM) and

selenium compound (600 mM) were combined in MES buffer

(10 mM; pH 6). All concentrations indicated are the final

concentrations in a 3 mL reaction volume.

Glutathione peroxidase measurements (GPx)

Glutathione peroxidase measurements were conducted according

to the reported procedure byMareque et al.65 The tested selenium

compound in methanol (selenomethionine, selenocystamine,

methyl-selenocysteine, 2-aminophenyl diselenide, 2-carboxy-

phenyl diselenide, 2-carboxyphenyl selenide, 4-carboxyphenyl

diselenide, 3,30-diselenobispropionic acid, 3,30-selenobispropio-

nic acid), water (selenomethionine, selenocystine) or DMSO

(ebselen, 2-aminophenyl diselenide) (100 mM) was added to

H2O2 (3.75 mM) in methanol and the formation of PhSSPh

from PhSH (10 mM) was monitored spectroscopically at 305 nm

for 25 min at 25 1C (300 scans). This catalytic reaction was

initiated by the addition of the H2O2 to the solution of PhSH.

Initial rates were determined by fitting the linear portion of the

curve; rates were determined from the slope of best-fit line. All

rates were determined as the average of at least three trials and

are reported relative to ebselen in methanol. For compounds

that were not soluble in methanol, GPx measurements were

compared to those of another compound in the same solvent

and methanol. The relative GPx measurement in methanol

was then calculated based on this comparison.

Mass spectroscopy measurements

Electrospray ionization (ESI) mass spectra were obtained using

the QSTARXLHybrid MS/MS System (Applied Biosystems),

with direct injection of the sample (flow rate = 0.05 mL min�1)

into the Turbo Ionspray ionization source. Samples were run

under positive mode, with an ionspray voltage of 3000 V

(except as otherwise indicated) in time-of-flight scan mode

(error � 2 m/z). Mass spectrometry samples for a 1 : 3 metal to

ligand ratio were prepared by combining CuSO4 (75 mM) and

ascorbic acid (94 mM) in methanol–water, and allowed to

stand for 3 min at room temperature. The selenium compound

(225 mM) was added to the solution to obtain a final volume of

1 mL, and allowed to stand for 5 min at room temperature. A

similar procedure was followed for studies with FeII, combining

FeSO4 (75 mM) and the selenium compound (225 mM) for a

1 : 3 metal to ligand ratio. All concentrations indicated are the

final concentrations in a 1 mL volume. All reported m/z peak

envelopes matched theoretical peak envelopes for the assigned

complexes.

Electrochemical measurements

Cyclic voltammetry samples were prepared by dissolving the

selenium compounds (300 mM) in MOPS buffer (10 mM, pH 7)

with KNO3 (10 mM) as a supporting electrolyte. Solutions

were degassed for 5 min with N2 before each experiment. All

CV experiments were conducted at 100 mV s�1. For differential

pulse voltammetry experiments, a pulse amplitude of 0.080 V,

a pulse width of 0.100, a sample width of 0.045, and a pulse

period of 0.200 were used. Samples of each selenium compound

were cycled between �1000 mV and 1000 mV (2-aminophenyl

diselenide was cycled between �1200 mV and 800 mV) using a

glassy carbon working electrode, a Pt counter electrode, and a

Ag/AgCl (197 mV vs. NHE)91 reference electrode. Electro-

chemical data for all compounds are given in the ESIw.

Conclusions

The selenium compounds selenocystine, selenomethionine and

methyl-selenocysteine effectively prevent copper-mediated DNA

damage with IC50 values of 3-26 mM. 3,30-Diselenobispropionic

acid, 3,30-selenobispropionic acid, selenocystamine, 2-amino-

phenyl diselenide, 2-carboxyphenyl diselenide, 2-carboxyphenyl

selenide, and 4-carboxyphenyl diselenide show no antioxidant

activity under these conditions. For these selenium compounds,

antioxidant activity primarily occurs by a copper-binding

mechanism, not by GPx-like activity or radical scavenging.

The observed antioxidant activity of selenium compounds

with CuI occurs at biological concentrations (B1–10 mM) of

selenium.53,54

In contrast, inhibition of iron-mediated DNA damage was

observed for four selenium compounds (methyl-selenocysteine,

selenocystamine, 3,30-diselenobispropionic acid, and 3,30-seleno-

bispropionic acid) at high concentrations; methyl-selenocysteine

was the only compound found to inhibit both copper- and

iron-mediated DNA damage. Metal binding also plays a

significant role in the observed antioxidant activity with FeII,

but a radical scavenging mechanism is also likely at high con-

centrations. Overall, selenium compounds were more effective

at inhibiting iron-mediated DNA damage than their sulfur

analogs.

Electrochemical studies show no clear correlation between

selenium antioxidant activity and oxidation potentials of the

selenium compounds. Additionally, mass spectrometry studies

of CuI and FeII coordination to selenium compounds indicate

that while metal coordination is necessary for antioxidant

activity, it is not sufficient. Both the identity of the metal ion

and the functional groups on the selenium compounds play a

very significant role in antioxidant behavior. Because reactive

oxygen species are implicated in cellular damage and disease,

further investigating this novel metal-binding antioxidant

mechanism and the effects of selenium speciation on anti-

oxidant activity are vital to understanding the biological

antioxidant activity of selenium.

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This journal is c The Royal Society of Chemistry 2011 Metallomics, 2011, 3, 503–512 511

Acknowledgements

We thank the American Heart Association (0665344U) and

the Clemson University Research Grant Committee for funding

this work.

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