Post on 18-Jul-2020
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
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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.
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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.
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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 — — — —
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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.
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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.
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
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.
References
1 D. R. Lloyd, D. H. Philips and P. L. Carmichael, Chem. Res.Toxicol., 1997, 10, 393–400.
2 I. Fridovich, Science, 1978, 201, 8785–8880.3 S. De Flora and A. Izzotti, Mutat. Res., Fundam. Mol. Mech.Mutagen., 2007, 621, 5–17.
4 G. J. Brewer, Exp. Biol. Med., 2007, 232, 323–335.5 D. Bar-Or, G. W. Thomas, L. T. Rael, E. P. Lau and J. V. Winkler,Biochem. Biophys. Res. Commun., 2001, 282, 356–360.
6 S. Park and J. A. Imlay, J. Bacteriol., 2003, 185, 1942–1950.7 G. Perry, A. D. Cash, R. Srinivas and M. A. Smith, Drug Dev.Res., 2002, 56, 293–299.
8 I. Angel, A. Bar, T. Horovitz, G. Taler, M. Krakovsky,D. Resnitsky, G. Rosenberg, S. Striem, J. E. Friedman andA. Kozak, Drug Dev. Res., 2002, 56, 300–309.
9 K. Keyer and J. A. Imlay, Proc. Natl. Acad. Sci. USA, 1996, 93,13635–13640.
10 A. Ala, A. P. Walker, K. Ashkan, J. S. Dooley and M. L. Schilsky,Lancet, 2007, 369, 397–408.
11 E. Beutler, Blood Cells Mol. Dis., 2007, 39, 140–147.12 G. J. S. Cooper, Y.-K. Chan, A. M. Dissanayake, F. E. Leahy,
G. F. Koegh, C. M. Frampton, G. D. Gamble, D. H. Brunton,J. R. Baker and S. D. Poppitt, Diabetes, 2005, 54, 1468–1476.
13 N. Leone, D. Courbon, P. Ducimetiere andM. Zureik, Epidemiology,2006, 17, 308–314.
14 M. B. Reddy and L. Clark, Nutr. Rev., 2004, 62, 120–124.15 K. Shumann, H. G. Classen, H. H. Dieter, J. Konig, G. Multhaup,
M. Rukgauer, K. H. Summer, J. Bernhardt and H. K. Biesalski,Eur. J. Clin. Nutr., 2002, 56, 469–483.
16 S. Swaminathan, V. A. Fonseca, M. G. Alam and S. V. Shah,Diabetes Care, 2007, 30, 1926–1933.
17 R. F. Burk, Nutr. Clin. Care, 2002, 5, 75–79.18 R. Moreno-Reyes, F. Mathieu, M. Boelaert, F. Begaux,
C. Suetens, M. T. Rivera, J. Neve, N. Perlmutter andJ. Vanderpas, Am. J. Clin. Nutr., 2003, 78, 137–144.
19 K. Zou, G. Liu, T. Wu and L. Du, Osteoarthr. Cartilage, 2009, 17,144–151.
20 R. J. Coppinger and A. M. Diamond, Selenium: Its MolecularBiology and Role in Human Health, Kluwer Academic Publishers,1st edn, 2001, pp. 219–233.
21 M. de Lorgeril and P. Salen, Heart Failure Rev., 2006, 11,13–17.
22 J. Gromadzinska, E. Reszka, K. Bruzelius, W. Wasowicz andB. Akesson, Eur. J. Nutr., 2008, 47, 29–50.
23 C. Koriyama, F. I. Campos, M. Yamamoto, M. Serra,G. Carrasquilla, E. Carrascal and S. Akiba, J. Toxicol. Sci.,2008, 33, 227–235.
24 G. Pourmand, S. Salem, K. Moradi, M. R. Nikoobakht, P. Tajikand A. Mehrsai, Nutr. Cancer, 2008, 60, 171–176.
25 C.-L. Shen, W. Song and B. C. Pence,Cancer Epidemiol. BiomarkersPrev., 2001, 10, 385–390.
26 G. F. Combs Jr. and W. P. Gray, Pharmacol. Ther., 1998, 79,179–192.
27 F. Fernandex-Banares, E. Cabre, M. Esteve, M. D. Mingorance,A. Abad-Lacruz, M. Lachica, A. Gil and M. A. Gassull, Am. J.Gastroenterol., 2002, 97, 2103–2108.
28 P. Knekt, J. Marniemi, L. Teppo, M. Heliovaara and A. Aromaa,Am. J. Epidemiol., 1998, 189, 1343–1349.
29 G. N. Schrauzer, D. A. White and C. J. Schneider, Bioinorg.Chem., 1977, 7, 35–56.
30 R. J. Shamberger and D. V. Frost, Can. Med. Assoc. J., 1969, 104,82.
31 G. F. Combs, Jr., Brit. J. Cancer, 2004, 91, 195–199.32 M. E. Reid, A. J. Duffield-Lillico, E. Slate, N. Natarajan,
B. Turnbull, E. Jacobs, G. F. Combs, D. S. Alberts, L. C. Clarkand J. R. Marshall, Nutr. Cancer, 2008, 60, 155–163.
33 S. M. Lippman, E. A. Klein, P. J. Goodman, M. S. Lucia,I. M. Thompson, L. G. Ford, H. L. Parnes, L. M. Minasian,J. M. Gaziano, J. A. Hartline, J. K. Parsons, J. D. Bearden, III,E. D. Crawford, G. E. Goodman, J. Claudio, E. Winquist,E. D. Cook, D. D. Karp, P. Walther, M. M. Lieber,A. R. Kristal, A. K. Darke, K. B. Arnold, P. A. Ganz,R. M. Santella, D. Albanes, P. R. Taylor, J. L. Probstfield,T. J. Jagpal, J. J. Crowley, F. L. Meyskens, Jr., L. H. Baker andC. A. Coltman, Jr., J. Am. Med. Assoc., 2009, 301, 39–51.
34 L. Zhao, G. Zhao, B. Hui, Z. Zhao, J. Tong and X. Hu, J. Food Sci.,2004, 69, 184–188.
35 G. Mugesh and H. B. Singh, Chem. Soc. Rev., 2000, 29, 347–357.36 A. L. Choi, E. Budtz-Jørgensen, P. J. Jørgensen, U. Steuerwald,
F. Debes, P. Weihe and P. Grandjean, Environ. Res., 2008, 107,45–52.
37 N. V. C. Ralston, C. R. Ralston, J. L. Blackwell III andL. J. Raymond, NeuroToxicology, 2008, 29, 802–811.
38 L. J. Raymond and N. V. C. Ralston, Fuel Process. Technol., 2009,90, 1333–1338.
39 P. D. Whanger, Trace Elem. Electrolytes Health Dis. J., 1992, 6,209–221.
40 H. Gurer and N. Ercal, Free Radical Biol. Med., 2000, 29, 927–945.41 P.-C. Hsu and Y. L. Guo, Toxicology, 2002, 180, 33–44.42 L. G. Ljungdahl and J. R. Andreesen,Methods Enzymol., 1978, 53,
360–373.43 E. Garcin, X. Vernede, E. C. Hatchikian, A. Volbeda, M. Frey and
J. C. Fontecilla-Camps, Structure, 1999, 7, 557–566.44 E. E. Battin, N. R. Perron and J. L. Brumaghim, Inorg. Chem.,
2006, 45, 499–501.45 E. S. Henle, Y. Luo and S. Linn, Biochemistry, 1996, 35,
12212–12219.46 X. Liu and F. J. Millero, Geochim. Cosmochim. Acta, 1999, 63,
3487–3497.47 N. R. Perron, J. N. Hodges, M. Jenkins and J. L. Brumaghim,
Inorg. Chem., 2008, 47, 6153–6161.48 Z. Lu, W. Huang and J. J. Vittal, New J. Chem., 2002, 26,
1122–1129.49 T. Oikawa, N. Esaki, H. Tanaka and K. Soda, Proc. Natl. Acad.
Sci. USA, 1991, 88, 3057–3059.50 G. D. Christian, Analytical Chemistry, John Wiley & Sons,
New York, 1980, p. 616.51 S.-B. Yu, G. C. Papaefthymiou and R. H. Holm, Inorg. Chem.,
1991, 30, 3476–3485.52 J. L. Breton, J. A. Farrar, M. C. Kennedy, H. Beinert and
A. J. Thomson, Biochem. J., 1995, 311, 197–202.53 E. E. Battin and J. L. Brumaghim, Cell Biochem. Biophys., 2009,
55, 1–23.54 R. R. Ramoutar and J. L. Brumaghim, Cell Biochem. Biophys.,
2010, 58, 1–23.55 B. Mishra, K. I. Priyadarsini, H. Mohan and G. Mugesh, Bioorg.
Med. Chem. Lett., 2006, 16, 5334–5338.56 B. Mishra, A. Barik, A. Kunwar, L. B. Kumbhare,
K. I. Priyadarsini and V. K. Jain, Phosphorus, Sulfur Silicon Relat.Elem., 2008, 183, 1018–1025.
57 G. Mugesh and W.-W. du Mont, Chem.–Eur. J., 2001, 7,1365–1370.
58 K. Yasuda, H. Watanabe, S. Yamazaki and S. Toda, Biochem.Biophys. Res. Commun., 1980, 96, 243–249.
59 S. R. Wilson, P. A. Zucker, R.-R. C. Huang and A. Spector, J. Am.Chem. Soc., 1989, 111, 5936–5939.
60 G. Mugesh, A. Panda, H. B. Singh, N. S. Punekar andR. J. Butcher, J. Am. Chem. Soc., 2001, 123, 839–850.
61 T. Yamaguchi, K. Sano, K. Takakura, I. Saito, Y. Shinohara,T. Asano and H. Yasuhara, Stroke, 1998, 29, 12–17.
62 D. Blum, S. Torch, N. Lambeng, M. F. Nissou, A. L. Benabid andJ. M. Verna, Prog. Neurobiol., 2001, 65, 135–172.
63 S. Moussaoui, M. C. Obinu, N. Daniel, M. Reibaud, V. Blanchardand A. Imperato, Exp. Neurol., 2000, 166, 235–245.
64 M. Parnham and H. Sies, Expert Opin. Invest. Drugs, 2000, 9,607–609.
65 A. M.-M. Mareque, J. M. Faez, L. Chistiaens, S. Kohnen,C. Deby, M. Hoebeke, M. Lamy and G. Deby-Dupont, RedoxRep., 2004, 9, 81–87.
66 B. K. Sarma and G. Mugesh, J. Am. Chem. Soc., 2005, 127,11477–11485.
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online
512 Metallomics, 2011, 3, 503–512 This journal is c The Royal Society of Chemistry 2011
67 V. De Silva, M. M. Woznichak, K. L. Burns, K. B. Grank andS. W. May, J. Am. Chem. Soc., 2004, 126, 2409–2413.
68 B. Halliwell and J. M. Gutteride, Arch. Biochem. Biophys., 1986,246, 501–514.
69 B. P. Yu, Physiol. Rev., 1994, 74, 139–162.70 Y. Bai, T. Wang, Y. Liu and W. Zheng, Colloids Surf., B, 2009, 74,
150–153.71 C. A. Collins, F. H. Fry, A. L. Holme, A. Yiakouvaki, A. Al-Qenaei,
C. Pourzand and C. Jacob, Org. Biomol. Chem., 2005, 3, 1541–1546.72 K. T. Suzuki, C. Sasakura and S. Yoneda, Biochim. Biophys. Acta,
Protein Struct. Mol. Enzymol., 1998, 1429, 102–112.73 A. Molter and F. Mohr, Coord. Chem. Rev., 2010, 254, 19–45.74 J. L. Pierre, M. Fontecave and R. R. Crichton, BioMetals, 2002,
15, 341–346.75 E. E. Battin and J. L. Brumaghim, J. Inorg. Biochem., 2008, 102,
2036–2042.76 R. R. Ramoutar and J. L. Brumaghim, Main Group Chem., 2007,
6, 143–153.77 R. Breitkreutz, N. Pittack, C. T. Nebe, D. Schuster, J. Brust,
M. Beichert, V. Hack, V. Daniel, L. Edler and W. Droge, J. Mol.Med., 2000, 78, 55–62.
78 A. T. Fleischauer and L. Arab, J. Nutr., 2001, 131, 1032S–1040S.79 D. P. Jones, Antioxid. Redox Signaling, 2006, 8, 1865–1879.
80 E. A. Lutsenko, J. M. Carcamo and D. W. Golde, J. Biol. Chem.,2002, 277, 16895–16899.
81 C. Mytilineou, B. C. Kramer and J. A. Yabut, Parkinsonism Relat.Disord., 2002, 8, 385–387.
82 J. T. Pinto and R. S. Rivlin, J. Nutr., 2001, 131, 1058S–1060S.83 S. Tanaka, K. Haruma, M. Yoshihara, G. Kajiyama, K. Kira,
H. Amagase and K. Chayama, J. Nutr., 2006, 136, 821S–826S.84 K. M. Beeh, I. C. Beier, O. Kornmann, P. Micke and R. Buhl, Eur.
Respir. J., 2002, 19, 1119–1123.85 S. Kumar, H. B. Singh and G. Wolmershauser, Organometallics,
2006, 25, 382–393.86 C. Ip and H. E. Ganther, Carcinogenesis, 1992, 13, 1167–1170.87 R. S. Lloyd, C. W. Haidle and D. L. Robberson, Biochemistry,
1978, 17, 1890–1896.88 R. P. Hertzber and P. B. Dervan, J. Am. Chem. Soc., 1982, 104,
313–315.89 N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa
and R. M. M. Singh, Biochemistry, 1966, 5, 467–477.90 D. A. Perkowski and M. Perkowski, Data and Probability
Connections, Pearson Prentice Hall, New Jersey, 2007,pp. 318–337.
91 A. J. Bard and L. R. Faulkner, Electrochemical Methods,Fundamentals and Applications, 2nd edn, Wiley, New York, p. 3.
Dow
nloa
ded
by E
BSC
O o
n 08
Jun
e 20
11Pu
blis
hed
on 0
2 Fe
brua
ry 2
011
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C0M
T00
063A
View Online