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Research Article
Effects of Diphenyl Diselenide on Methylmercury
Toxicity in Rats
Cristiane L. Dalla Corte,1Caroline Wagner,2Jéssie H. Sudati,2
Bruna Comparsi,3Gerlania O. Leite,4Alcindo Busanello,1Félix A. A. Soares,1
Michael Aschner,5,6 and João B. T. Rocha1
1Biochemistry and Molecular Biology Department, Graduation Program in Biological Sciences: Toxicological Biochemistry,
Natural and Exact Sciences Center, Federal University of Santa Maria, 97105-900 Santa Maria, RS, Brazil
2Federal University of Pampa—Cac¸apava do Sul Campus, Avenida Pedro Anunciac¸˜
ao, Vila Batista,
96570-000 Cac¸apava do Sul, RS, Brazil
3Higher Education Cenecista Institute of Santo ˆ
Angelo—IESA, Rua Dr. Jo˜
ao Augusto Rodrigues 471,
98801-015 Santo ˆ
Angelo, RS, Brazil
4Regional University of Cariri, Pharmacology and Molecular Chemistry Laboratory, Rua Cel. Antˆ
onio Lu´
ıs 1161,
63100-000 Crato, CE, Brazil
5Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
6Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, USA
Correspondence should be addressed to Cristiane L. Dalla Corte; crisbioq@yahoo.com.br
and Jo˜
ao B. T. Rocha; jbtrocha@yahoo.com.br
Received September ; Revised November ; Accepted November
Academic Editor: Fernando Barbosa Jr.
Copyright © Cristiane L. Dalla Corte et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
is study investigates the ecacy of diphenyl diselenide [(PhSe)2] in attenuating methylmercury- (MeHg-)induced toxicity in
rats. Adult rats were treated with MeHg [ mg/kg/day, intragastrically (i.g.)] and/ or (PhSe)2[ mg/kg/day, intraperitoneally (i.p.)]
for days. Body weight gain and motor decits were evaluated prior to treatment, on treatment days and . In addition,
hepatic and cerebral mitochondrial function (reactive oxygen species (ROS) formation, total and nonprotein thiol levels, membrane
potential (ΔΨm), metabolic function, and swelling), hepatic, cerebral, and muscular mercury levels, and hepatic, cerebral, and
renal thioredoxin reductase (TrxR) activity were evaluated. MeHg caused hepatic and cerebral mitochondrial dysfunction and
inhibited TrxR activity in liver (,%), brain (,%), and kidney (,%). Cotreatment with (PhSe)2protected hepatic and cerebral
mitochondrial thiols from depletion by MeHg but failed to completely reverse MeHg’s eect on hepatic and cerebral mitochondrial
dysfunction or hepatic, cerebral, and renal inhibition of TrxR activity. Additionally, the cotreatment with (PhSe)2increased Hg
accumulation in the liver (,%) and brain (,%) and increased the MeHg-induced motor decits and body-weight loss. In
conclusion, these results indicate that (PhSe)2can increase Hg body burden as well as the neurotoxic eects induced by MeHg
exposure in rats.
1. Introduction
MeHg is one of the most poisonous environmental contam-
inants, causing toxic eects in humans and experimental
animals [,]. Environmental MeHg is largely derived from
inorganic mercury biomethylation carried out primarily by
aquatic microorganisms []withsubsequentaccumulationin
the aquatic food chain and human consumption []. MeHg
causes acute and chronic damage to multiple organs, most
profoundly to the central nervous system (CNS), in partic-
ular when exposures occur during the neurodevelopmental
period [,,].
e events that mediate MeHg toxicity are largely depen-
dent upon its electrophilic properties, which allow for its
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interaction with so nucleophilic groups (mainly thiols
and selenols) from either low- or high-molecular-weight
biomolecules []. e interaction of MeHg with so nucle-
ophilic groups from biomolecules is responsible, at least in
part, for decreased antioxidant capacity and increased ROS
generation [,]. Notably, MeHg can disrupt the activity of
thiol- and selenol-containing proteins, such as glutathione
peroxidase (GPx), thioredoxin (Trx), and TrxR [,–].
ese proteins are important components of the cellular
antioxidant system, and their inhibition contributes to the
disruption of the normal redox balance of cells [].
In addition, MeHg can disrupt mitochondrial function
by targeting specic thiol-containing proteins, including
respiratory chain complexes [,]. e inhibition of these
complexes or enzymes can contribute to mitochondrial depo-
larization and swelling upon MeHg exposure. Mitochondrial
targeting by MeHg has also been associated with increased
mitochondrial ROS generation, which can further exacerbate
thetoxicityofMeHgbyattackingadditionalnucleophiliccen-
ters in mitochondria and in other subcellular compartments
[,,–], leading to a vicious cycle of cell demise.
Several studies demonstrated that organic and inorganic
selenium (Se) compounds inuence the deposition and
toxicity of MeHg [,,]. Se is an essential trace element
for a wide range of living organisms, including humans [].
Se is necessary for the expression of approximately Se-
dependent proteins, including GPx, TrxR, and several other
enzymes and proteins, which can modulate the cellular redox
and antioxidant status [].
In addition to inorganic and naturally occurring organo-
selenium compounds, synthetic organoselenium compounds
can also exhibit protective eects against MeHg. For example,
ebselen and (PhSe)2have been shown to exert benecial
eects against in vitro and in vivo MeHg-induced toxicity [–
]. (PhSe)2(which is the simplest of the diaryl diselenides
[]) protected against an array of toxic eects of MeHg and
lowered the Hg burden in the brain, liver, and kidneys of adult
mice []. e molecular mechanism(s) which underlie(s) the
protective eects of (PhSe)2in mice likely reect the direct
interaction of MeHg with “selenol intermediate” of (PhSe)2
aer its reaction with thiols, or indirectly, by modulating
oxidative stress levels [,]. In short, the protective eects
of (PhSe)2against MeHg-induced toxicity are likely related
to its antioxidant properties and its ability to form stable
complexes with MeHg, which can increase Hg excretion and
decrease the MeHg body burden.
Of particular pharmacological signicance, the toxicity
and pharmacokinetics of MeHg [] are dierent in mice
and rat which can be explained by the higher binding anity
of rat hemoglobin, which contains more cysteinyl residues
than mice protein, for MeHg when compared to the mice
hemoglobin []. (PhSe)2toxicity and pharmacokinetics
dierences between mice and rat also exist and could be
explained by a faster metabolization of (PhSe)2in mice [–
].
erefore, the aim of the present study was to investigate
the potential protective eects of (PhSe)2against MeHg-
induced toxicity and mitochondrial dysfunction in rats. To
accomplish this goal, the eects of (PhSe)2on Hg deposition
in liver and brain and on behavioral and biochemical param-
eters were studied in rats.
2. Materials and Methods
2.1. Chemicals. Chemicals, including ethylene glycol-bis(𝛽-
aminoethylether)-N,N,N,N -tetraacetic acid (EGTA), -
(-hydroxyethyl)--piperazineethanesulfonic acid (HEPES),
, dinitrophenol (, DNP), -(,-dimethylthiazol--yl)-
,-diphenyltetrazolium bromide (MTT), glutamic acid,
safranin O, ,-dichlorouorescin diacetate (H2-DCFDA),
and methylmercury chloride were obtained from Sigma
Aldrich (St. Louis, MO, USA). (PhSe)2was synthesized
according to the method by Paulmier []. All other chemi-
cals were of analytical reagent grade and purchased from local
commercial suppliers.
2.2. Animals. Male Wistar rats, weighing –g and with
age from to . months, from our own breeding colony
were kept in cages (four animals in each). Rats were placed
in a room with controlled temperature (22 ± 3∘C) on a h
light/dark cycle (lights on at : a.m.) and had continuous
access to food and water. All experiments were conducted
in accordance with the Committee on Care and Use of
Experimental Animal Resources of the Federal University of
Santa Maria, Brazil.
2.3. Treatment. Sixteen rats were equally divided into four
experimental groups as follows: () control ( mL/Kg of
water i.g. and mL/Kg of soybean oil i.p.); () (PhSe)2
( mL/Kg of water i.g. and mg/Kg of (PhSe)2i.p.); () MeHg
( mg/Kg of MeHg i.g. and mL/Kg of soybean oil i.p.); and
() (PhSe)2+ MeHg (mg/Kg of MeHg i.g. and mg/Kg
of (PhSe)2i.p.). Based on previous studies, exposures were
performed daily over a -day period [,,]. Twenty-four
hours aer the last exposure, the animals were sacriced and
the livers, brains, kidneys, and skeletal muscle were quickly
removed, placed on ice and homogenized.
2.4. Determination of Hg Levels. Tissue levels of total Hg
were measured in liver, brain, and skeletal muscle collected
at the time of euthanasia []. Approximately . g (wet
weight) of the tissues was weighed and digested with mL
of HNO3acid (%). Digested samples were diluted to mL
with ultrapure water before analysis using a Multitype ICP
Emission Spectrometer (ICPE-, Shimadzu). Calibration
standard curve was prepared freshly using mercury stock
standard solution.
2.5. Motor Coordination Tests
2.5.1. Open Field Test. General locomotor activity was eval-
uated as previously described []. e number of line
crossings (number of segments crossed with the four paws)
and rearings was measured over min and taken as an
indicator of locomotor activity. e test was carried out at
time points: hours prior to treatment (basal), and on
treatment days and .
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2.5.2. Rotarod Test. Motor coordination was tested on the
rotarod apparatus as described previously [,]. e
latency to fall and the number of falls from the apparatus were
recorded for s. e tests were conducted times: hours
prior to treatment (basal), and on treatment days and .
2.6. TrxR
2.6.1. TrxR Purication. TrxR was partially puried by a
modication of the method by Holmgren and Bjornstedt
[]. Tissues were homogenized in buered saline ( mM
NaCl, . mM KCl, . mM Na2HPO4,and.mMKH
2PO4,
pH .). Livers, brains, and kidneys (. g) were homogenized
in,,andvolumesofbueredsaline,respectively.
Homogenates were centrifuged at , g for min. e
protein concentration in the supernatant was measured and
adjusted to mg/mL. e supernatant was dialyzed against
buered saline for h to remove endogenous glutathione
(GSH) and Trx. e dialysate was heated at ∘Cformin,
cooled, and centrifuged at , g for min to remove
denatured protein.
2.6.2. TrxR Activity. TrxR activity was measured by the
method of Holmgren and Bjornstedt []. e reaction
mixture consisted of the following: . mM NADPH,
mM EDTA, mM potassium phosphate buer (pH .),
mg/mL ,dithiobis--nitrobenzoic acid (DTNB), and
. mg/mL of BSA. e partially puried TrxR was added
(to nal concentration of – 𝜇gofprotein)tothecuvette
containing the reaction mixture, and the absorbance was
followed at nm for a maximum of min.
2.7. Isolation of Rat Brain and Liver Mitochondria. Rat brain
and liver mitochondria were isolated as previously described
by Brustovetsky and Dubinsky [], with some modications.
Brain and liver were rapidly weighed and homogenized in :
(w/v) ice-cold “isolation buer I” containing mM man-
nitol, mM sucrose, mM K+-EGTA, .% bovine serum
albumin (BSA), and mM K+-HEPES,pH..etissuewas
then manually homogenized with a potter glass. e result-
ing suspension was centrifuged for min at , g. Aer
centrifugation the supernatant was recentrifuged for min
at , g. e pellet was resuspended in “isolation buer
II” containing mM mannitol, mM sucrose, mM K+-
EGTA, and mM K+-HEPES pH . and recentrifuged
at , g for min. e supernatant was discarded and
the nal pellet gently washed and resuspended in “isolation
buer II” without EGTA.
2.8. Mitochondrial Nonprotein and Total iol Content. Mito-
chondrial nonprotein and total thiol content were measured
according to the method of Ellman []. To determine total
thiol groups, mitochondria (.mg protein) were added to
the reaction medium containing mM Tris-HCl pH ., %
SDS, and mM DTNB. Nonprotein thiol content was mea-
sured by adding 𝜇L%TCAto𝜇Lofthemitochondria
(. mg protein). Aer centrifugation (, ×gat
∘Cfor
min), the protein pellet was discarded and an aliquot of
the clear supernatant, neutralized with .M NaOH, was
added to the medium containing mM Tris-HCl pH .
and mM DTNB. e samples absorbance was measured
spectrophotometrically at a wavelength of nm.
2.9. Measurements of Mitochondrial ΔΨm. Mitochondrial
ΔΨm was estimated by uorescence changes in safranin
O ( mM) recorded by RF- Shimadzu spectrouorom-
eter (Kyoto, Japan) operating at excitation and emission
wavelengths of and nm, with slit widths of . nm
[]. Data on ΔΨm in the gures is presented in Arbitrary
Fluorescence Units (AFU).
2.10. Estimation of ROS Production. e mitochondrial
generation of ROS was determined spectrouorimetrically,
using the membrane permeable uorescent dye H2-DCFDA
recorded by RF- Shimadzu spectrouorometer (Kyoto,
Japan) operating at excitation and emission wavelengths of
and nm, with slit widths of nm []. Data of
ROS production in the gures is presented as Arbitrary
Fluorescence Units (AFU).
2.11. Assessment of Mitochondrial Metabolic Function. e
mitochondrial metabolic function was assessed by the con-
version of MTT to a dark violet formazan product by mito-
chondrial dehydrogenases []. e rate of MTT reduction
was measured spectrophotometrically at a wavelength of
nm. Results were expressed as the percentage of MTT
reductionrelativetocontrolvalues.
2.12. Assessment of Mitochondrial Swelling. Measurement of
mitochondrial swelling was performed in a RF- Shi-
madzu spectrouorometer at nm (slit . nm for exci-
tation and emission) []. Data for mitochondrial swelling
are expressed as Arbitrary Absorbance Units (AAU). e
dierence (ΔA) between the initial absorbance reading and
thenalabsorbancereadingwasusedforstatisticalanalysis.
2.13. Protein Measurement. Protein was assayed by the
method of Bradford []withbovineserumalbuminas
standard.
2.14. Statistical Analysis. Normality assumption was tested
with Kolmogorov-Smirnov test and the distribution of the
majority of results is not normal. Data were analyzed sta-
tistically by Mann-Whitney or Kruskal-Wallis, followed by
Dunn’s post-hoc tests when appropriate. e results were
considered statistically signicant at 𝑃 < 0.05.Allstatistical
analyses were conducted using GraphPad Prism (Version
., GraphPad Soware, Inc., USA).
3. Results
3.1. Eects of (PhSe)2and MeHg on Body Weight. Treat me n t
with MeHg led to body-weight loss from the second week
until the end of the treatment compared to controls (𝑃<
0.05,Figure ). Rats cotreated with (PhSe)2and MeHg also
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0 7 14 21
160
210
260
310
360
Control MeHg
Days
Weight (g)
(PhSe)2(PhSe)2+ MeHg
∗
∗
∗
F : Eect of MeHg and/or (PhSe)2on the body weight gain in
adult rats. Data are expressed as mean ±S.D., 𝑛=4.(∗)represents
𝑃 < 0.05 as compared to controls by Mann-Whitney test.
showed a decrease in the body weight when compared to
the control group (𝑃 < 0.05,Figure ). Rats treated with
(PhSe)2lost weight aer the rst week of treatment (𝑃<
0.05) but showed a trend towards a recovery and were
statistically indistinguishable from the controls at the end of
the treatment (Figure ).
3.2. Eects of (PhSe)2andMeHgonHgDeposition. Tre at m e n t
with MeHg increased the levels of Hg in liver, brain, and
skeletal muscle compared with controls (𝑃 < 0.05,Figure ).
e cotreatment with (PhSe)2caused a greater increase
in brain Hg deposition when compared to MeHg alone
treatment, both in brain (Figure (b))andliver(Figure (a)),
and showed a trend towards increased deposition in skeletal
muscle (Figure (c)).
3.3. Eects of (PhSe)2and MeHg on Motor Coordination
and Spontaneous Locomotor Activity. e eects of MeHg
and/or (PhSe)2on locomotion and motor coordination were
assessed by the open-eld and rotarod tests, respectively.
Aer days, rats treated with MeHg showed increased
number of falls on the rotarod and decreased latency to the
rst fall when compared to controls (𝑃 < 0.05,Figures(a)
and (b)). Rats treated with (PhSe)2did not show statistically
signicant dierences on the rotarod test when compared
to controls; however, rats cotreated with (PhSe)2and MeHg
showed increased loss of motor coordination as evidenced by
increased number of falls and reduced latency to the rst fall
(𝑃 < 0.05,Figures(a) and (b)). e rotarod test could not
be performed at the end of the treatment in rats receiving
MeHgsincetheywereunabletoremainintheapparatusdue
to severe motor impairment caused by MeHg.
Rats treated with MeHg showed a decrease in the number
of crossings and rearings in the open-eld at the end of the
treatment compared to the control rats (𝑃 < 0.05,Figures
(c) and (d)). Rats cotreated with MeHg and (PhSe)2also
showed a signicant decrease in the number of crossings
aer days of treatment and a decrease in the number of
rearings at the end of the treatment (𝑃 < 0.05,Figure (d)).
Tre at m e n t w it h ( PhS e ) 2didnotaecttherats’performancein
the open-eld. e decrease in the number of crossings and
rearings observed in all groups on treatment days and
was expected given that the animals habituate to the open-
eld arena [].
3.4. Eects of (PhSe)2and MeHg on
Mitochondrial Dysfunction
3.4.1. Mitochondrial Metabolic Function. e hepatic mito-
chondrialmetabolicintegrity(MTTreduction)wasnotaec-
ted by MeHg and/or (PhSe)2(Figure (a)). Treatment with
MeHg or cotreatment with MeHg and (PhSe)2decreased the
capacity of brain mitochondrial dehydrogenases to reduce
MTT compared to controls (𝑃 < 0.05,Figure (b)). Treat-
ment with (PhSe)2alonedidnotaectthecerebralmito-
chondrial metabolic function.
3.4.2. Mitochondrial Total and Nonprotein iols. MeHg
treatment decreased the total mitochondrial thiol levels in
brainandliverwhencomparedtocontrols(𝑃 < 0.05,
Figure ). Treatment with (PhSe)2alone did not alter the
mitochondrial total thiol levels in liver and brain (Figure ).
e cotreatment with (PhSe)2blunted the MeHg-induced
mitochondrial total thiol level depletion in rats’ liver and
brain (𝑃 < 0.05,Figure ). Rats treated with MeHg showed
decreased mitochondrial nonprotein thiol levels in the liver
compared to controls, and coadministration of (PhSe)2
blunted the MeHg-induced decrease in hepatic nonprotein
thiol content (𝑃 < 0.05,Figure (a)). Brain mitochondrial
nonprotein thiol levels were not aected by any of the
treatments (Figure (b)).
3.4.3. Mitochondrial Swelling. Treatment with MeHg sig-
nicantly increased hepatic mitochondrial swelling when
compared to controls (𝑃 < 0.05,Figure (a)). Cotreatment
with (PhSe)2partially prevented the MeHg-induced mito-
chondrial swelling in liver (Figure (a)). Similarly, treatment
with MeHg showed a trend towards increased mitochondrial
swelling in brain (Figure (b)). e cotreatment with MeHg
and (PhSe)2signicantly increased cerebral mitochondrial
swelling when compared to controls (𝑃 < 0.05,Figure (b)).
Tre at m e n t w it h ( PhS e ) 2alone did not alter the mitochondrial
swelling in brain or liver compared to the controls (Figures
(a) and (b)).
3.4.4. Mitochondrial ROS Production. Mitochondrial ROS
production (DCFH oxidation) was signicantly increased in
livers of rats treated with MeHg or cotreated with MeHg and
(PhSe)2(𝑃 < 0.05,Figure (a)). Rats treated with (PhSe)2
showed hepatic mitochondrial ROS levels indistinguishable
from controls. ROS production in cerebral mitochondria was
not aected by any of the treatments (Figure (b)).
3.4.5. Mitochondrial ΔΨm. Polarization (ΔΨm) of mitochon-
driafromliverofratscotreatedwithMeHgand(PhSe)
2
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0
50
100
150
200
250
(𝜇g Hg/g tissue)
Control (PhSe)2MeHg (PhSe)2+ MeH
g
∗#
∗
(a)
0
10
20
30
40
50
Control (PhSe)2MeHg (PhSe)2+ MeH
g
∗#
∗
(𝜇g Hg/g tissue)
(b)
0
20
40
60
80
∗
∗
Control (PhSe)2MeHg (PhSe)2+ MeHg
(𝜇g Hg/g tissue)
(c)
F : Hg content in liver (a), brain (b), and muscle (c) of rats exposed to MeHg and/or (PhSe)2. Data are expressed as mean ±S.D., 𝑛=4.
(∗)represents𝑃 < 0.05 as compared to controls by Mann-Whitney test. () represents 𝑃 < 0.05 as compared to MeHg by Mann-Whitney
test.
showed only a trend towards decreased (Figures (a) and
(c)). Treatment with (PhSe)2and MeHg alone did not cause
mitochondrial depolarization in liver of rats (Figures (a) and
(c)). Treatment with (PhSe)2and/or MeHg had no eect on
mitochondrial ΔΨm in brain of rats (Figures (b) and (d)).
3.5. Eects of (PhSe)2andMeHgonTrxRActivity.MeHg
is known to inhibit TrxR activity both in vitro and in vivo
[,,]. (PhSe)2treatment signicantly increased renal TrxR
activities when compared to controls (𝑃 < 0.05,Figures(a)
and (b)). Hepatic and cerebral TrxR activity showed a trend
towards increased in rats treated with (PhSe)2(Figure (c)).
MeHg treatment also led to signicant inhibition of TrxR
in liver, kidney, and brain compared to controls (𝑃 < 0.05,
Figure ). Cotreatment with (PhSe)2failed to signicantly
attenuate the MeHg-induced inhibition of TrxR activity in the
liver, kidney, or brain (Figure ).
4. Discussion
e present study investigated the ecacy of (PhSe)2,an
organoselenium compound, in attenuating MeHg-induced
toxicity in rats. Our results established that MeHg decreased
body weight (Figure ) and induced motor decits (Figure )
as well hepatic and cerebral mitochondrial dysfunction (Fig-
ures (b),,(a),and(a))andinhibitedTrxRactivityin
liver, brain, and kidney (Figure ) in the rat. e cotreatment
with (PhSe)2and MeHg increased Hg accumulation in the
liver and brain (Figure ). Furthermore, the cotreatment with
(PhSe)2protected hepatic and cerebral mitochondrial thiols
from depletion by MeHg (Figure ) but did not prevent
hepatic and cerebral mitochondrial dysfunction (Figures
(b),(b),and(a)) nor did it reverse the MeHg-induced
motor decits (Figure ), body-weight loss (Figure ), and the
MeHg-induced inhibition of TrxR activity in liver, brain, and
kidney (Figure ).
Cotreatment with (PhSe)2and MeHg increased Hg depo-
sition in the brain and liver of exposed rats (Figure ).
ese results dier from those of de Freitas et al. []where
(PhSe)2led to a signicant reduction in Hg concentrations
in brain, liver, and kidney of MeHg-exposed mice. e
discrepancies between the studies may be attributed to
metabolic dierences between the species and the route of
administration. e toxicity and pharmacokinetics of MeHg
[] are dierent in mice and rat which can be explained
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0
2
4
6
8
10
Basal
Day 11
Number of falls
Control (PhSe)2MeHg (PhSe)2+ MeH
g
∗
∗
(a)
Basal
Day 11
0
25
50
75
100
125
150
Latency to rst fall (s)
Control (PhSe)2MeHg (PhSe)2+ MeHg
∗
∗
(b)
0
20
40
60
80
100
Basal
Day 11
Day 21
Control (PhSe)2MeHg (PhSe)2+ MeH
g
∗
∗
∗
Ambulation (number of crossings/5 min)
(c)
Basal
Day 11
Day 21
0
10
20
30
40
50
Control (PhSe)2MeHg (PhSe)2+ MeHg
Rearing (number of times/5 min)
∗#
∗
(d)
F : Rotarod and open eld tests in rats exposed to MeHg and/or (PhSe)2. e number of falls (a) and latency for the rst fall (b)
ambulation (crossing) (a) and rearing (b) were recorded. Data are expressed as mean ±S.D., 𝑛=4.(∗)represents𝑃 < 0.05 as compared to
controls by Kruskal-Wallis test followed by multiple comparison test. () represents 𝑃 < 0.05 as compared to (PhSe)2by Kruskal-Wallis test
followed by multiple comparison test.
by the higher binding anity of rat hemoglobin, containing
more cysteinyl residues, for MeHg when compared to the
mice hemoglobin []. (PhSe)2toxicity and pharmacokinet-
ics dierences between mice and rat also exist and could
be explained by a faster metabolization of (PhSe)2in mice
[–]. Notably, herein rats were administered (PhSe)2i.p.,
whilst in the study by de Freitas et al. [](PhSe)
2was
subcutaneously (s.c.) administered to the mice. Another
dierence between the two works is in relation to the dose
of MeHg: in our study we used a dose . times higher than
in the study of de Freitas et al. (mg/Kg). However, the
duration of the treatment was shorter in our study, versus
days. On the other hand, the dose of (PhSe)2was similar
betweenthetwostudies.ehigherdoseofMeHgusedinour
study may have contributed to the discrepancies since it could
generate a more severe toxicity which could not be prevented
by (PhSe)2. However, we realize that the dierences in the
pharmacokinetics between rats and mice for the (PhSe)2is
the major factor involved in the discrepancies found here
[].
In the study by de Freitas et al. []theproposed
mechanism for the reduction Hg’s organ burden by (PhSe)2
was the formation of a selenol/selenolate (PhSeH/PhSe−)
intermediate, which could interact with MeHg, generating
the readily excretable PhSeHgMe complex. One possible
explanation for the increase in hepatic and cerebral Hg depo-
sition (Figures (a) and (b), resp.) by the cotreatment with
(PhSe)2observed herein may be the conversion of (PhSe)2
to inorganic selenium, which is subsequently metabolized
to selenhidric acid (HSe−). HSe−couldbindtoMeHgto
form a less soluble complex [], which can be degraded to
HgSe [,]. In addition, Palmer and Parkin []showed
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Control (PhSe)2MeHg (PhSe)2+ MeHg
0
50
100
150
MTT reduction (% of control)
(a)
Control (PhSe)2MeHg (PhSe)2+ MeHg
∗
∗
0
50
100
150
MTT reduction (% of control)
(b)
F : MTT reduction in liver (a) and brain (b) mitochondria of rats exposed to MeHg and/or (PhSe)2. Data are expressed as mean ±
S.D., 𝑛=4.(∗)represents𝑃 < 0.05 as compared to controls by Mann-Whitney test.
∗
Control (PhSe)2MeHg (PhSe)2+ MeHg
0
2
4
6
8
10
12
Total thiols (nmol/mg protein)
(a)
∗
Control (PhSe)2MeHg (PhSe)2+ MeHg
0
2
4
6
8
10
12
Total thiols (nmol/mg protein)
(b)
∗
Control (PhSe)2MeHg (PhSe)2+ MeHg
0.0
0.2
0.4
0.6
0.8
Nonprotein thiols (nmol/mg protein)
(c)
Control (PhSe)2MeHg (PhSe)2+ MeH
g
0.0
0.2
0.4
0.6
Nonprotein thiols (nmol/mg protein)
(d)
F : Total and nonprotein thiol content in liver (a), (c) and brain (b), (d) mitochondria of rats exposed to MeHg and/or (PhSe)2.Data
are expressed as mean ±S.D., 𝑛=4.(∗)represents𝑃 < 0.05 as compared to controls by Mann-Whitney test.
that organoselenium can also form a complex with mercury.
us the increase in hepatic and cerebral Hg deposition
by the cotreatment with (PhSe)2possibly involves Hg:Se
interactions and the formation of a less excretable compound
that accumulates in these organs []. ese results are in
agreement with other studies that showed elevated deposition
of Hg in key brain regions upon oral Se administration [,
].Ithasbeenspeculatedthattheformationofinsoluble
HgSe salt could reduce the toxicity of MeHg. However, exper-
imental evidence supporting this assumption has yet to be
BioMed Research International
Control (PhSe)2MeHg (PhSe)2+ MeHg
∗
0
10
20
30
40
50
Swelling (ΔA)
(a)
Control (PhSe)2MeHg (PhSe)2+ MeH
g
∗
0
10
20
30
40
Swelling (ΔA)
(b)
F : Mitochondrial swelling in liver (a) and brain (b) of rats exposed to MeHg and/or (PhSe)2. Data are expressed as mean ±S.D., 𝑛=4.
(∗)represents𝑃 < 0.05 as compared to controls by Mann-Whitney test.
Control (PhSe)2MeHg (PhSe)2+ MeHg
∗∗
0
50
100
150
H2-DCFH oxidation (AFU)
(a)
Control (PhSe)2MeHg (PhSe)2+ MeH
g
0
50
100
150
200
250
H2-DCFH oxidation (AFU)
(b)
F : ROS production (H2-DCFH oxidation) in liver (a) and brain (b) mitochondria of rats exposed to MeHg and/or (PhSe)2.Dataare
expressed as mean ±S.D., 𝑛=4.(∗)represents𝑃 < 0.05 as compared to controls by Mann-Whitney test.
generated. Although the cotreatment with (PhSe)2increased
Hg levels in brain and liver, these were accompanied by
a partial protection against MeHg-induced mitochondrial
dysfunction. We suggest that the formation of an insoluble
and inert complex between Hg and Se could decrease the
availability of MeHg that could react with important cellular
components decreasing its toxicity.
Decreased weight gain and weight loss are prominent
and readily observed features of severe MeHg toxicity. In
this study, rats treated with MeHg showed body-weight
loss (Figure ). Notably, the most severe eect on weight
loss occurred in rats cotreated with (PhSe)2and MeHg
(Figure ). In addition, rats treated with MeHg showed
decreased locomotor activity (Figure ). Cotreatment with
(PhSe)2andMeHgincreasedtheseverityofmotordysfunc-
tion (rotarod test) (Figure ), likely as a result of increased
Hg deposition in the brain (Figure (b)). Motor decits
are the most apparent neurological eects following MeHg
exposure []. In vivo studies in rodents point to impairment
in intracellular calcium homeostasis, alteration in glutamate
homeostasis, and oxidative stress as critical mediators of
MeHg-induced neurotoxicity []. e overactivation of
N-methyl-D-aspartate- (NMDA-) type glutamate receptors
increases Ca2+ inux into neurons, thereby leading to cell
death []. Alternatively, Ca2+ taken up by mitochondria may
stimulate the generation of ROS [].
Several studies corroborate MeHg’s ability to induce
mitochondrial dysfunction and ROS generation [,,].
e high anity binding of MeHg to thiol groups inactivates
enzymes, including respiratory chain complexes [,,],
decreasing mitochondrial dehydrogenases activity. Inhibi-
tion of these complexes may contribute to mitochondrial
swelling and ROS production aer MeHg exposure (Figures
and ). However, in brain, the MeHg-induced decrease in
mitochondrial dehydrogenases activity (MTT reduction) was
not accompanied by an increase in ROS production. ese
BioMed Research International
0 50 100 150 200 250 300
0
100
200
300 Mitochondria 2,4-DNP
Time (s)
Membrane potential (AFU)
Control
(PhSe)2
MeHg
(PhSe)2+ MeHg
(a)
0 50 100 150 200 250 300
0
100
200
300
Mitochondria 2,4-DNP
Time (s)
Membrane potential (AFU)
Control
(PhSe)2
MeHg
(PhSe)2+ MeHg
(b)
0
50
100
150
200
250
Control (PhSe)2MeHg (PhSe)2+ MeHg
Membrane potential (ΔΨm)
ΔΨm1
ΔΨm2
(c)
0
50
100
150
200
250
Control (PhSe)2MeHg (PhSe)2+ MeH
g
Membrane potential (ΔΨm)
ΔΨm1
ΔΨm2
(d)
F : Mitochondrial depolarization in liver (a), (c) and brain (b), (d) of rats exposed to MeHg and/or (PhSe)2. Figures (a) and (b) show
mitochondrial membrane potential (AFU). Figures (c) and (d) show mitochondrial ΔΨm. ΔΨm = delta of uorescence before (time ) and
aer addition of mitochondria (time seconds) and ΔΨm = delta of uorescence before (time seconds) and aer addition of , DNP
(time seconds). Data are expressed as mean ±S.D., 𝑛=4.
results are corroborated by the fact that MeHg aected total
thiols but not nonprotein thiol levels in brain mitochondria.
MeHg caused a decrease in the total mitochondrial thiol
levels in brain, which is related mainly with protein thiols,
andisinagreementwiththeinhibitionofmitochondrial
dehydrogenases activity in this tissue. On the other hand,
MeHg did not aect nonprotein thiol levels (mainly GSH)
in brain mitochondria, which can explain the normal ROS
production, since GSH is the main antioxidant in brain.
e cotreatment with (PhSe)2prevented the MeHg-
induced mitochondrial total and nonprotein thiol groups
depletion in the brain and liver (Figure ). e ecacy of
(PhSe)2in preventing thiol depletion may reside in its ability
to form a complex with MeHg, thus eectively reducing
MeHg binding to protein and free thiols. Treatment with
(PhSe)2also partially protected the liver from mitochondrial
MeHg-induced swelling (Figure (a)). However, the cotreat-
ment with (PhSe)2failed to reverse the MeHg-induced mito-
chondrial swelling (Figure (b))anddecreasedmitochon-
drial metabolic function (Figure (b))inthebrainaswellas
increased mitochondrial ROS production (Figure (a))inthe
liver. ese results indicate that mechanisms other than the
interaction with important free and protein thiols are likely
involved in the MeHg-induced mitochondrial dysfunction.
us, the preferential anity of MeHg for specic, and as
of yet unidentied, mitochondrial protein targets may have
acriticalroleinMeHg’stoxicity.
Previous studies have demonstrated that MeHg can
directly inhibit TrxR activity both in vitro and in vivo [,
,]. Mammalian TrxR is a selenoenzyme containing a
BioMed Research International
Control (PhSe)2MeHg (PhSe)2+ MeH
g
∗
∗
0
3
6
9
12
(nmol of TNB/min/mg protein)
(a)
Control (PhSe)2MeHg (PhSe)2+ MeH
g
∗
∗
#
0
2
4
6
8
10
(nmol of TNB/min/mg protein)
(b)
Control (PhSe)2MeHg (PhSe)2+ MeHg
∗
∗
0
1
2
3
4
(nmol of TNB/min/mg protein)
(c)
F : Tr xR activity in liver (a), kidney (b), and brain (c) of rats exposed to MeHg and/or (PhSe)2. Data are expressed as mean ±S.D., 𝑛=4.
(∗)represents𝑃 < 0.05 as compared to controls by Mann-Whitney test. () represents 𝑃 < 0.05 as compared to controls by Mann-Whitney
test.
unique, catalytically active selenolthiol/selenenylsulde in
the conserved C-terminal sequence (-Gly-Cys-Sec-Gly) [].
ree mammalian TrxR selenoenzymes have been identi-
ed, the cytosolic enzyme TrxR, the mitochondrial enzyme
TrxR, and a testis-specic enzyme thioredoxin-glutathione
reductase (TGR/TrxR) []. Here, we show that MeHg
treatment inhibited rat TrxR activity in brain, liver, and
kidney (Figure ). MeHg forms covalent bonds between its
Hg moiety and the Se of the selenocysteine of the enzyme,
thus directly inhibiting the activity of TrxR []. Since TrxR is
critical for cellular antioxidant defense system the inhibition
ofthisenzymelikelyhasacentralroleinmediatingthe
toxicity of MeHg.
Recently, diphenyl diselenide was demonstrated to be
a substrate for cerebral and hepatic rat TrxR, which could
account, at least in part, for the antioxidant properties of
(PhSe)2[]. Herein, rats treated solely with (PhSe) showed
an increase in the activity of renal TrxR (Figures (a) and
(b), resp.). e formation of selenhidric acid from (PhSe)2
couldalsoexplaintheincreaseinTrxRactivity,sincethis
inorganic form of Se can be converted to selenocysteine
and incorporated to selenoenzymes, such as TrxR [,
]. Accordingly, Zhang et al. [] have demonstrated that
organoselenium compounds (including diselenide) increase
theexpressionofTrxRinwhitebloodcellslinesinculture.
e cotreatment with (PhSe)2and MeHg was ineective in
attenuating the inhibition of MeHg-induced TrxR in liver,
kidney, and brain (Figure ). Similarly, studies in vitro and
in vivo have previously corroborated that selenite was able
to recover the activity of HgCl2-induced TrxR inhibition but
not in response to MeHg. e eect of Se (as selenide) was
attributed to the displacement of Hg from the active site,
giving rise to mercury selenide and regenerating the TrxR
selenol [,].
5. Conclusions
In conclusion, the results of this study established that
(PhSe)2can increase Hg body burden (likely associated with
release of inorganic Se from (PhSe)2) and MeHg neurotoxic-
ityinratsdespitethefactthat(PhSe)
2blunted the deleterious
eects of MeHg on thiol levels. e results presented herein
also reinforce the central role of mitochondrial dysfunction in
mediating the aberrant eects of MeHg in vivo,aswellasthe
role of TrxR as a molecular target for MeHg in the rat. Further
research into MeHg-(PhSe)2interactions will be helpful in
characterizing the consequences concomitant exposures to
theseandrelatedcompounds.
BioMed Research International
Conflict of Interests
e authors declare that there is no conict of interests regar-
ding the publication of this paper.
Acknowledgments
is work was supported by Fundac¸˜
ao de Amparo a Pesquisa
do Estado do Rio Grande do Sul (FAPERGS), CAPES
(Coordenac¸˜
ao de Aperfeic¸oamento de Pessoal de N´
ıvel
Superior), CNPq (Conselho Nacional de Desenvolvimento
Cient´
ıco e Tecnol ´
ogico), FINEP (Rede Instituto Brasileiro
de Neurociˆ
encia (IBN-Net) no. ..-), FAPERGS-
PRONEX-CNPQ, and INCT-EN (Instituto Nacional de
Ciˆ
encia e Tecnologia em Excitotoxicidade e Neuroprotec¸˜
ao).
Cristiane L. Dalla Corte is recipient of CAPES fellowship.
F´
elix A. A. Soares and Jo˜
ao B. T. Rocha are recipients of
CNPq fellowships. Michael Aschner was partially supported
by grants from the US PHS, R ES and R ES.
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