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April 9, 2015
FFULisboa Auditurium
08:45-09:15 Registration
09:15-09:30 Opening Session
09:30-10:30 The Thioredoxin System in Health and Disease………………
Arne Holmgren (Karolinska Institutet)
10:30-11:00 Coffee-break
11:00-11:50 Cancer, Antitumorals and Selenoproteins ……………………….
Jun Lu (Karolinska Institutet)
11:50-12:30 Selenium and Selenoproteins: Role on Mercury Toxicity
Cristina Carvalho (FFUL/iMed.ULisboa)
12:30-14:00 Lunch
14:00-14:30
The Mitochondrial Thioredoxin System and its Importance for
Neurotoxicity Development - The Case Study of Mercury
Compounds
Vasco Branco (FFUL/iMed.ULisboa)
14:30 -15:15
Current Selenium Research in Portugal: Oral Presentations of
Selected Posters………………………………………………………
(to be announced)
15:15-16:00 Posters View / Coffee-break
16:00-16:30 Food intake of Selenium and Health Benefits: a study in a population
of Portuguese pregnant women……………………….
……………………..
Luis Carvalho (FFUL/iMed.ULisboa)
16:30-16:40 Best Communication Prize announcement
16:40 Closing Session
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The Thioredoxin System in Health and Disease
Arne Holmgren
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm,
Sweden
Email: [email protected]
Thioredoxin (Trx), NADPH, thioredoxin reductase (TrxR) comprise a thioredoxin
system and is present universally in living organisms. Most organisms also have a
glutaredoxin system comprising glutathione (GSH), NADPH, glutathione reductase (GR)
and glutaredoxin (Grx). Both systems were discovered in studies of how
deoxyribonucleotides for DNA synthesis are formed de novo (1).The essential enzyme
catalyzing this reaction is ribonucleotide reductase (RNR) and for each catalytic cycle
Trx1 or Grx1 is required to reduce a disulfide in the active site of the enzyme. Recent
results on the mechanism of the thioredoxin system for mammalian and bacterial RNR
enzymes will be reviewed. Mammalian TrxR is radically different from the corresponding
enzyme in bacteria, yeast and plants by having a higher molecular weight and an essential
selenocysteine (Sec) residue in the active site. A new way of killing bacteria via inhibition
of thioredoxin reductase in bacteria by ebselen, a selenium-containing antioxidant
selenazole drug will be described. This new antibiotic mechanism will also target defense
against reactive oxygen species (2) like hydrogen peroxide via peroxiredoxins (thioredoxin
peroxidases). Mammalian Trx and TrxR are present in many isoforms with a large number
of functions as disulfide reductases in control of cellular redox potential. TrxR and Trx are
required for reduction of selenite to make selenide required for synthesis of Sec which is
inserted in selenoproteins via a cotranslational mechanism.The thioredoxin system is
central in redox regulation via H2O2 or NO (3). Recent data on the autoregulation of human
Trx1 in signal transduction by H2O2 and peroxiredoxins will be presented. The thioredoxin
system is of particular importance for carcinogensis and treatment of cancer. Many drugs
target the Sec residue in TrxR1 in the cytosol or TrxR2 in the mitochondria causing
oxidative stress by gain of function in TrxR to an NADPH oxidase while inactivating its
function as a Trx reductase. Of particular interest for this meeting will be that mercury
compounds target both human Trx1 and TrxR1 (4).
References
1. Lillig, C.H. and Holmgren, A.: Thioredoxin and related molecules: from biology to
health and disease. Antioxid. Redox Signal. , 9, 25-47, 2007.
2. Lu, J. and Holmgren, A.: The thioredoxin antioxidant system. Free Radic. Biol.
Med. 66, 75-87, 2014.
3. Sengupta, R. and Holmgren, A.: Thioredoxin and thioredoxin reductase in relation
to reversible S-nitrosylation. Antioxid. Redox Signal. , 18, 259-269, 2013.
4. Carvalho, C.M.L., Chew, E.-H., Hashemy, S.I., Lu, J. and Holmgren, A.: Inhibition
of human thioredoxin system: a molecular mechanism of mercury toxicity. J. Biol.
Chem., 283, 11913-11923, 2008.
5
Cancer, Antitumorals and Selenoproteins
Jun Lu
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm,
Sweden
Email: [email protected]
Selenium is an essential micronutrient for human. A major role of Se is attributed
to its presence in selenoproteins as selenocysteine (Sec). Sec has similar chemical
properties as cysteine, but possesses a lower pKa value, thus a stronger nucleophilicity
and higher reactivity. Twenty-five selenoprotein genes have been identified in the human
genome. Thioredoxin reductases (TrxR) and glutathione peroxidases are the best
characterized selenoproteins and play critical roles in disulfide reduction, redox signaling,
and antioxidant defense. TrxR is the highly efficient enzyme to reduce oxidized thioredoxin
(Trx), and glutathione system, the other thiol dependent electron transfer system, can
serve as a backup to reduce oxidized Trx. Cytosolic TrxR1 and Trx1 are overexpressed
in many cancer cells to maintain cancer cell redox environment and participate in
carcinogenesis, cancer progression and drug resistance. The cancer cell survival is more
reliable on the Trx system compared to normal cells. Trx redox state and TrxR activity are
determining factors for cell’s fate, Trx oxidation results in the cell death. Inhibition of TrxR
can block Trx-mediated activity in DNA synthesis and defense against oxidative stress.
Therefore, Trx system emerges as an interesting anticancer drug target. There are
several chemotherapeutic strategies to cause the blockage of Trx-mediated pathway,
resulting in cancer cell death: 1) Inhibition of TrxR1 activity and converting the reductant
TrxR1 into a pro-oxidant to kill the cancer cells or enhance cancer cell radiosensitization;
2) Blockage of both Trx1 system and glutathione system; 3) Disruption of mitochondrial
Trx2 system to cause the elevation of reactive oxygen species production in mitochondria
and mitochondrial dependent cancer cell death; 4) Induction of overexpression of Trx
endogenous inhibitor, thioredoxin interacting protein; 5) Inhibition of TrxR1 together with
the reaction with Trx1 structural cysteines.
References
1. Lu, J., and Holmgren, A. (2009) Selenoproteins. J Biol Chem 284, 723-727
2. Lu, J., Chew, E. H., and Holmgren, A. (2007) Targeting thioredoxin reductase is a basis
for cancer therapy by arsenic trioxide. Proc Natl Acad Sci U S A 104, 12288-12293
3. Lu, J., and Holmgren, A. (2012) Thioredoxin system in cell death progression. Antioxid
Redox Signal 17, 1738-1747
4. Zhang, X., Zheng, Y., Fried, L. E., Du, Y., Montano, S. J., Sohn, A., Lefkove, B.,
Holmgren, L., Arbiser, J. L., Holmgren, A., and Lu, J. (2011) Disruption of the mitochondrial
thioredoxin system as a cell death mechanism of cationic triphenylmethanes. Free Radic
Biol Med 50, 811-820
6
Selenium and Selenoproteins and their role in Mercury
Toxicity Development
Cristina M.L. Carvalho
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of
Lisbon, Av. Prof. Gama Pinto 1649-003 Lisbon, Portugal
Email: [email protected]
Mercury (Hg) toxicity mediated by different forms of mercury is a major health
problem; however, the molecular mechanisms underlying toxicity still remain elusive.
The inhibition of the thioredoxin system by mercury has been shown in vitro and in
vivo being the selenoenzyme thioredoxin reductase (TrxR) particularly sensitive to
mercury. Given the importance of the thioredoxin system for several cellular functions it is
assumed that this inhibition is a key step in the development of Hg toxicity in vivo.
Fishes were used as an animal model and exposure to methylmercury (MeHg),
showed a significant reduction in the activity of the thioredoxin system in the liver and in
brain and, a correlation between TrxR activity and histopathological changes in liver and
kidney was observed, with Hg2+
being the strongest inhibitor and causing the more severe
lesions. Co-exposure to Hg2+
and Se prevented TrxR inhibition in the liver and reduced
the severity of lesions.
TrxR was shown to be more sensitive to mercurials than GPx due to the higher
accessibility of the Sec in the active site.
In HepG2 cells, important differences were noted between the toxic behavior of
MeHg and Hg2+
. MeHg was more cytotoxic and increased the activity of caspase-3
indicating the onset of apoptosis, while Hg2+
did not. Co-exposure to selenium prevented
the inhibition of TrxR by mercurial, increasing its activity.
Selenite has been described as an antagonist of mercury toxicity. We have
confirmed that upon treatment with selenite and NADPH, TrxR inactivated by HgCl2
displayed almost full recovery of activity. Structural analysis indicated that mercury was
complexed with TrxR, but enzyme generated selenide removed mercury as mercury
selenide regenerating the active site selenocysteine and cysteine residues required for
activity. The antagonistic effects on TrxR inhibition were extended to endogenous
antioxidants such as GSH and clinically used exogenous chelating agents. Antidotes used
in clinics such as BAL, DMPS, DMSA and α-lipoic acid, were essentially effective towards
the inorganic form of mercury, HgCl2, and had minor effects on MeHg inhibitory effects,
which is consistent with the results obtained in patients treatment.
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BAL, DMPS and α-lipoic acid recovered 85% TrxR activity after HgCl2 inhibition
whereas the maximum recovery activity for MeHg inhibition was 45% with BAL. Selenite
was as effective as the antidotes tested in regenerating TrxR. Upon the treatment with
higher than 5µM selenite, TrxR displayed full activity recovery in selenite reduction assay
and 70-80% activity recovery in insulin reduction assay. Overall, the results strength the
clinical effects of mercury antidotes on TrxR and its role on the molecular mechanisms of
mercury toxicity. These results stress the role of TrxR as a target of mercurials and provide
the mechanism of selenite as a detoxification agent for mercury poisoning.
KEYWORDS: mercury; methylmercury; thioredoxin; thioredoxin reductase; selenium.
8
The Mitochondrial Thioredoxin System and its Importance in
Neurotoxicity Development – the Case Study of Mercury Compounds
Vasco Branco
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de
Lisboa
In the mitochondria redox-balance is tightly regulated and fully functional
antioxidant enzymes are essential to prevent excessive production of reactive oxygen
species (ROS) and, consequently, in protecting mitochondria from dysfunction-causing
oxidative stress. One of the most abundant ROS species in the mitochondria is hydrogen
peroxide (H2O2) which has important signaling functions at low levels, but in higher
concentrations generates the highly reactive hydroxyl radical (OH·). Several enzymes (e.g.
glutathione peroxidases –GPx; catalase - CAT) can metabolize H2O2 at the mitochondria
but, due to its abundance and reactivity towards peroxides, peroxiredoxin 3 (Prx3) is the
main scavenger (Brown et al., 2008). The activity of Prx3 is regulated via the mitochondrial
thioredoxin system that comprises thioredoxin 2 (Trx2) and the selenoprotein thioredoxin
reductase 2 (TrxR2). This pathway is particularly relevant in brain mitochondria where
CAT activity is absent. In fact, it has been shown that deletion of TrxR2 in neuronal cells,
increases sensitivity to Parkinson disease causing agents such as paraquat and 6-
hydroxydopamine (6OHDA), due to an unregulated build-up in ROS levels (Lopert et al.,
2012).
Our group has previously shown that, TrxR2 is particularly sensitive to exposure to
mercury compounds (MeHg and Hg2+
) since its expression is not regulated by redox
sensitive mechanisms involved in cellular response to stress such as, the Nrf-2 pathway
(Branco et al., 2014). Curiously, Trx2 is kept relatively protected from oxidation by
mercurials keeping Prx3 in a reduced active state. Knock-down of glutathione (GSH) levels
with BSO considerably increases Trx2 oxidation which suggests that GSH, possibly via
mitochondrial glutaredoxin 2 (Grx2) activity, acts as a backup for TrxR2.The possible
implications of these findings for the development of cell death following exposure to
mercury compounds will be discussed.
References:
- Branco et al. 2014. Mitochondrial thioredoxin reductase inhibition, selenium status,
and Nrf-2activation are determinant factors modulating the toxicity of mercury compounds.
Free Radic. Biol .Med. 73, 95-105.
- Brown, K.K. et al. 2008. Mitochondrial peroxiredoxin 3 is rapidly oxidized in cells treated
with isothiocyanates. Free Radic. Biol .Med. 45, 494-502.
- Lopert, P., Day, B. J., Patel, M. 2012.Thioredoxin reductase deficiency potentiates
oxidative stress, mitochondrial dysfunction and cell death in dopaminergic cells. PLoS
One. 7: e50683.
9
Food intake of Selenium and Health Benefits: a study in a
population of Portuguese pregnant women
Carvalho, Luis1
, Vasco Branco1
, and Cristina Carvalho1
.
1
Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia da
Universidade de Lisboa, Av. Prof. Gama Pinto 1649-003 Lisbon, Portugal.
Selenium is an essential trace mineral in human biology. The physiological
functions of selenium are achieved by the selenoproteins, which possess
selenocysteine in their active site and perform indispensable antioxidant functions
in the brain and neuroendocrine system. Foods like fish, meat, eggs and nuts are
important sources of this element in the diet. The selenium in soil is the primary
determinant for the selenium amounts in food, which results in a very variable
intake worldwide. An adequate selenium intake is associated with benefits like
reduced mortality, increased immunity and reduction of mercury toxicity. This
relationship with mercury is especially important in pregnant women since the
developing brain of the fetus is very susceptible to mercury, resulting in several
forms of brain damage. Given the possible protective effect of selenium during
development and the lack of studies concerning the portuguese selenium status in
this work we studied the selenium status on a population of 540 pregnant women
from Madeira, in Portugal. Selenium concentrations were determined in whole
blood and plasma by atomic absorption spectrometry. The average selenium found
in whole blood was 62.5 µg/L ± 13.2 µg/L, with a minimum and maximum of 37.8
µg/L and 151.8 µg/L, respectively. Plasma samples contained an average of 74%
of the concentration for whole blood. Our results were comparable to the ones
obtained in similar studies in other European countries. While the results weren’t
as high as initially expected, this might be explained by the sub-population we
studied. This project is ongoing and the dietary habits of the population in study
are currently being analyzed.
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11
Poster 1
The influence of bioaccessibility of selenium and methylmercury on
the evaluation of risk assessment associated to raw and cooked
tuna and blue shark fish consumption
Afonso, C. ab; Cardoso, C a; Matos, J. C; Costa S. a; Lourenço, H.M. ab; Brito, P. a;
Coelho, I. d; Batista, I. a; Bandarra, N. ab; Nunes, M.L. b
a Division of Aquaculture and Upgrading, Portuguese Institute of the Sea and Atmosphere,
IPMA, Avenida de Brasília, 1449-006 Lisboa, Portugal
b Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR),
University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal
c Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017
Lisbon, Portugal
d National Health Institute of Dr. Ricardo Jorge (INSA), Avenida Padre Cruz, 1649-016
Lisboa, Portugal.
The health benefits of a seafood diet are known. However, this consumption is subject to
a nutritional-toxicological conflict since they are the main route of exposure to some
contaminants, particularly methylmercury (MeHg). However, micronutrients normally
found in high levels in seafood, such as selenium (Se), have been shown to modify the
toxic effects of MeHg exposure. Furthermore, whenever there is quantification of a
compound exposure in the risk/benefit assessment calculations it is usually assumed that
its bioavailability is 100 %.
Thus, the current study aims to clarify the effect of different cooking/processing procedures
on the bioaccessibility of Se, total Hg and MeHg to contribute for a better assessment of
benefits/risks associated with tuna and blue shark consumption. For this purpose, an
optimized in vitro digestion method, that simulated the human digestive processes, was
used for acquire information about the bioaccessibility of Se, total Hg and MeHg from raw,
cooked (boiled and grilled) or processed tuna (canned in olive oil or water) and blue shark
(steamed and grilled). The results showed that Se bioaccessibility was high. Hg
bioaccessibility was lower in cooked or processed fish compared to raw. For the
bioaccessible fraction, Se:MeHg molar ratios were higher than one and all Se-HBV were
positive in tuna fish samples but in the case of blue shark the Se:MeHg ratios were lower
than one and all Se-HBV were negative. The risk assessment analyses showed that a
weekly meal of canned tuna and a year meal of blue shark present very low risk
12
Poster 2
Updated data and anatomical region differences on selenium levels
in the human brain
Patrícia Ramos1, Nair Pinto2, Ricardo Mendes2, Agostinho Santos2-5, Agostinho
Almeida1
1REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, Portugal
2National Institute of Legal Medicine and Forensic Sciences, North Branch, Portugal
3Faculty of Medicine, Porto University, Portugal
4School of Health Sciences, University of Minho, Portugal
5CENCIFOR – Forensic Science Center, Portugal
Disturbances in brain selenium (Se) homeostasis have been identified as potentially
responsible for the cognitive decline associated with normal ageing and development of
some neurodegenerative diseases but the evidence is still fragmentary and its definite role
remains unclear.
The main goals of this work were to establish the “normal” (reference) levels for Se in the
human brain and to evaluate the anatomical region differences and the age-related
changes, a prior and essential step in order to enlighten its role in human brain physiology
and its involvement in ageing and neurodegenerative processes.
From neurologically and psychiatrically healthy individuals submitted to autopsy (n=42;
71±12, range: 50–101 years old) the following 14 brain areas were sampled: frontal cortex,
superior and middle temporal, caudate nucleus, putamen, globus pallidus, cingulated
gyrus, hippocampus, inferior parietal lobule, occipital lobe, midbrain, pons, medulla and
cerebellum. After samples microwave-assisted acid digestion, Se levels were determined
by inductively coupled plasma-mass spectrometry.
Considering the whole data set (n=588; 42 individuals x 14 brain areas), Se levels (on a
dry weight basis) ranged from 552–1435 ng/g, with a mean±SD content of 959±178 ng/g.
Selenium distribution across the different brain areas was heterogeneous, with highest
levels in the putamen, parietal inferior lobule, and occipital cortex, and lowest in the
medulla and cerebellum. Selenium levels were unchanged with aging. Compared with the
age-matched control group, significantly increased levels of Se were found in the globus
pallidus, superior temporal gyrus, and frontal cortex of Parkinson’s disease (n=1) and
Alzheimer’s disease (n=2) patients.
13
Poster 3
Evaluation of selenium chemical species in foodstuffs by HPLC-ICP-
MS
Sandra Gueifão1, Inês Coelho1, Marta Ventura1, Isabel Castanheira1, Petru Jitaru2,
Paola Fisicaro3, Véronique Vacchina4, Fabienne Seby4
1- Department of Food and Nutrition, National Health Institute Doutor Ricardo Jorge, I.P.
(INSA), Avenida Padre Cruz 1649-016 Lisboa – Portugal.
2- Département Sciences et Techniques Agro-Industrielles (STAI), Institut Polytechnique
LaSalle Beauvais, Beauvais – France.
3- Department of Biomedical and Inorganic Chemistry. Laboratoire national de métrologie
et d’essais (LNE),1 rue Gaston Boissier 75015 Paris – France.
4- Ultra Traces Analyses Aquitaine (UT2A), Technopole Hélioparc, 2 avenue Pierre Angot,
PAU Cedex 9, 64053 – France.
Evaluation of Se levels in diet requires information on total Se and its species. The aim of
this work was to evaluate the concentration of total Se and Se species as
Selenomethionine (SeMet), Selenite (SeIV) and Selenate (SeVI) in milk and cabbage
consumed in Portugal.
Determination of total Se was carried out by Inductively Coupled Plasma Mass
Spectrometry (ICP-MS) and Se species by High Performance Liquid Chromatography
coupled with ICP-MS and equipped with a dynamic reaction cell (HPLC-DRC-ICP-MS).
For determination of total Se, samples were digested by closed-vessel microwave. To
optimize microwave procedure, several mineralization conditions, HNO3:H2O2 ratios (4/2;
4/3; 7/1) at different time and temperature, were studied. For Se species, enzymatic, acid
hydrolysis and water were applied as extraction procedures.
Performance of the method was assessed obtaining the main analytical figures of merit
using standard chemical solutions, CRMs and matrix spiked samples. The results
obtained with CRMs were in a good agreement with certified values and values for
recovery were under the acceptance criteria (> 80%).
Total Selenium content in analysed samples ranged from 67 ± 3 ug.kg-1 (cabbage) to 161
± 16 ug.kg-1 (milk). SeMet was major selenium specie in milk accounting for 85 -90 % of
total Se content. In cabbage inorganic specie selenate and selenoamino-acid (SeMet)
were found.
The results showed the suitability of the methods to determine total Se in both samples.
The conditions used in this work seemed effective to achieve accurate determination of
Se species in these difficult matrices.
14
Poster 4
S-Adenosylhomocysteine alters the Selenoproteome through RNA
hypomethylation
Madalena M Barroso1,2
; Anton A Turanov1
; Bradley A Carlson3
; Vadim N Gladyshev1
;
Dolph L Hatfield3
; Isabel Tavares de Almeida2
; Rita Castro2,4
; Joseph Loscalzo1
; Diane E
Handy1
1 Medicine, Brigham and Women’s Hosp, and Harvard Med Sch, Boston, MA, USA; 2
Met&Gen, iMed.ULisboa, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; 3
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 4Department
of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon,
Portugal.
The mechanisms by which hyperhomocysteinemia (HHcy) contributes to cardiovascular
disease are unclear. The accumulation of homocysteine precursor, S-
adenosylhomocysteine (SAH), which occurs during HHcy, induces DNA and protein
hypomethylation. Previous studies link HHcy-induced suppression of the antioxidant
selenoprotein GPx-1 (glutathione peroxidase-1) with endothelial dysfunction. Translation
of a subset of selenocysteine containing proteins, including GPx-1, is dependent on
methylation of the tRNASec to the Um34 form. We hypothesized that SAH accumulation
may impair tRNASec methylation, affecting the selenoproteome and altering cell redox
balance. To increase SAH in endothelial cells, we decreased SAH hydrolase activity with
a targeted siRNA or a pharmacological inhibitor. The selenoproteome was studied after
[75Se]-labeling. GPx-1 and thioredoxin reductase (TR1, TR2) expression was confirmed
by western-blotting. tRNASec methylation status was evaluated by RPC-5
chromatography. H2O2 release was measured with Amplex-Red and the recombinant
Hyper2 biosensor. An increase in intracellular SAH decreased the expression of
selenoproteins dependent on the Um34-containing tRNASec, with a 37±2.7% and
39.3±3.5% suppression of GPx-1 protein expression and activity, respectively (p<0.005).
Under excess SAH conditions, Um34-tRNASec was undetectable, although total tRNASec
was increased by 1.64±0.03-fold. In contrast, TR protein expression, which is independent
of Um34 modification, was upregulated 36.7% (TR1) and 23.1% (TR2) by excess SAH
(p<0.05). The SAH-induced reduction in stress-related selenoproteins, such as GPx-1,
may contribute to the 2.9±0.3-fold (p<0.001) increase in cellular H2O2. These results
suggest that SAH accumulation can induce tRNASec hypomethylation, which alters the
selenoproteome to contribute to a pro-atherogenic phenotype.
15
Poster 5
Selenium Role In an Human Biomonitoring Study Applied to
Occupational Health
Carina Ladeira 1,2, Susana Viegas 1,3, Elisabete Carolino 1,2, Manuel C. Gomes 4,
Miguel Brito 2
1 – Environmental Health Research Group, Escola Superior de Tecnologia da Saúde de
Lisboa – IPL, Portugal.
2 - Genetic and Metabolism Research Group, Escola Superior de Tecnologia da Saúde
de Lisboa – IPL, Portugal.
3 – CMDT – Universidade Nova de Lisboa, Portugal.
4 – Faculty of Sciences, University of Lisbon, Portugal.
Selenium functions as a co-factor for the reduction of antioxidant enzymes and is an
important component of antioxidant enzymes.
Dietary selenium significantly inhibits the induction of skin, liver, colon, and mammary
tumours in experimental animals by a number of different carcinogens, as well as the
induction of mammary tumours by viruses.
Selenium shows a “U” shaped curve for functionality, whereby too little is as damaging as
too much. At optimal levels, selenium may protect against the formation of DNA adducts,
DNA or chromosome breakage, chromosome gain or loss, mitochondrial DNA, and
telomere length and function.
The aim of the study was to investigate the relation between selenium and genotoxic
effects in a human biomonitoring study applied to occupational health. Therefore, it was
constituted two groups - occupationally exposed to cytostatics (n=46) and controls (n=46).
Blood samples were taken and used to measure genetic instability biomarkers
represented by DNA damage breaks and oxidative stress; also a food-frequency
questionnaire was fulfilled.
The mean of selenium intake obtained was 131.51±9.34 and 138.67±8.58 in exposed and
controls, respectively, measured in µg/d. It was not found any statistical significant
association between the genotoxicity biomarkers studied and selenium in each group. For
both groups, the increase of selenium intake was related with the decrease of genomic
instability, however without reaching statistical significance.
Current dietary recommendations do not consider the concept of genome stability which
is of concern because damage to the genome has been linked to the origin and
progression of many diseases and is the most fundamental pathology.
16
Poster 6
Selenium counteracts mercury inhibition of thioredoxin reductase in
neuroblastoma cells
(Eurotox)
Protective effects of selenium compounds in the toxicity of mercury over
the thioredoxin reductase in neuroblastoma cells
Inês Guerra1
, Vasco Branco1
, Juan Rodrigues1
, Cristina Carvalho1
.
1
Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia da
Universidade de Lisboa, Av. Prof. Gama Pinto 1649-003 Lisbon, Portugal.
Mercury (Hg) is a global contaminant without a known biological function.
Methylmercury (MeHg) is the most dangerous form of mercury to human health due to
neurotoxicity and the most susceptible are infants and developing embryos. The
thioredoxin system is the major system responsible for maintaining the redox state of cells
and this function involves thiol reduction mediated by selenol groups in thioredoxin
reductase (TrxR). In fact, it has been shown that Se supplementation can reactivate the
mercury-inhibited TrxR and recover the enzyme activity in vitro.
In this work, the effects of mercurials and different Se compounds on the growth of
human neuroblastoma SH-SY5Y cells were assessed by the MTT cell viability assay.
MeHg was more toxic than Hg2+
, having a rather lower (> 15 fold) GI50 after 72 hours of
exposure. Sodium selenite (Na2SeO3), sodium selenate (Na2SeO4) and selenomethionine
(SeMet) did not cause a reduction in cell growth by 50% even at concentrations as high
as 100 μM and the same was seen when cells were treated with selenocystine (SeC-SeC)
in concentrations up to 50 μM. MeHg is also a stronger inhibitor of TrxR activity in cell
lysates than Hg2+
(70 vs. 50% inhibition at 5 μM of MeHg and Hg2+
, respectively).
Therefore, these results show important differences between the toxic behaviour of MeHg
and Hg2+
in human neuroblastoma SH-SY5Y cells. Furthermore, the Se compounds
showed a significant protective effect when co-exposed to mercurials although they were
not able to fully prevent the inhibition of TrxR caused by the previous.
This study has been financed by MERTOX project (PTDC/QUI-BIQ/117281/2010)
and by iMED.UL through FCT’s strategic project: PEst-OE/SAU/UI4013/2011.
17
Poster 7
Mitochondrial thioredoxin system as a target for mercury
compounds in HepG2 cells
Vasco Branco1
, Ana Godinho-Santos1
, João Gonçalves1
, Jun Lu2
, Arne Holmgren2
,
Cristina Carvalho1
.
1
Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia da
Universidade de Lisboa, Av. Prof. Gama Pinto 1649-003 Lisbon, Portugal.
2
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177
Stockholm, Sweden
ABSTRACT
The thioredoxin system is essential for the maintenance of key cellular functions.
The activity of the cytosolic thioredoxin (Trx1) and thioredoxin reductase (TrxR1) is
inhibited by mercury compounds, but the effect over mitochondrial isoforms (TrxR2 and
Trx2) is unknown. Thus, the goal of this work was to understand how mercurials affected
the activity of mitochondrial and cytosolic thioredoxin systems and the transcription and
expression of TrxR1 and TrxR2 in HepG2 cells (human hepatoma). The effects of co-
exposure to selenite and mercurials on the thioredoxin systems were also addressed as
well as the role of Nrf-2 on TrxR transcription.
Methylmercury (MeHg) affected the activity of the mitochondrial and cytosolic
thioredoxin systems to the same extent, whereas mercuric mercury (Hg2+
) inhibited mostly
the activity of the mitochondrial enzymes. TrxR1 expression was upregulated by Hg2+
and
downregulated by MeHg. Co-exposure to selenite and Hg2+
led to a very significant
increase in TrxR1 expression but no such effect was observed for MeHg. Analysis of
nuclear translocation of Nrf-2 and TrxR1 mRNA levels indicate a slower induction of TrxR1
transcription by MeHg in comparison to Hg2+
which helps to explain the increased toxicity
of this organomercurial. Interestingly, TrxR2’s transcription and expression was not up-
regulated by any treatment.
These results show important differences in the mechanisms of toxicity of Hg2+
and
MeHg and stress the distinct effect of selenite co-exposure in counteracting the toxicity of
the two mercurials. Most notably, the mitochondrial thioredoxin system and in particular
TrxR2 seem to be a primary target in the development of mercury toxicity.
This study has been financed by MERTOX project (PTDC/QUI-BIQ/117281/2010)
and by iMED.UL through FCT’s strategic project: PEst-OE/SAU/UI4013/2011.
18
Poster 8
Assessment of selenium concentration in the plasma of the
pregnant women from Madeira Island (Portugal) and correlation with
blood levels
Cássia Barbosa, Luis Carvalho, Vasco Branco, Cristina Carvalho
1
Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia da
Universidade de Lisboa, Av. Prof. Gama Pinto 1649-003 Lisbon, Portugal
Selenium (Se) is an essential micronutrient and multiples studies have shown its
importance in the human body due to its antioxidant properties. Selenium is also known
for its neuro, cardioprotective and anti-inflammatory properties as well as its role in the
regulation of the immune system and thyroid gland and in cancer prevention. Selenium is
present in several foods and it can be found in higher concentration in fish, seafood and
Brazilian nuts.
The aim of this study was to determine the Se levels in 175 pregnant women from
Madeira Island (Portugal) due to insufficient information in this area. Plasma samples were
mineralized with hydrochloric acid at 95ºC and following reduction, volatile hydrides of the
element were analyzed with Atomic Absorption Spectrometry (HG-AAS).
The mean concentration found was 46 ± 6.3 µg/L. The minimum and the maximum
values observed were 25 µg/L and 102 µg/L, respectively. Similar results have been
reported observed in other countries such as New Zealand, Italy and Poland. The Se
levels in plasma were compared with the corresponding levels in blood and a good
correlation was observed.
