Abstracts and Proceedings of the 12th International Symposium on

FeFeCuCu

ZnZnAuAu

SiSi HgHg CrCrRuRuPtPt PbPb

ISBN: 978-9974-0-0911-0

Punta del Este, Uruguay

11, 12 and 13 March 2013

Abstracts and Proceedings of the

12th International Symposium on

Metal Ions in Biology and Medicine

María H. Torre and Dinorah Gambino (Eds.)

March 2013, Montevideo, Uruguay

ISBN: 978-9974-0-0911-0

Editors:

María H. Torre and Dinorah Gambino

General Chairpersons

Dr. Dinorah Gambino and Dr. María H. Torre

Inorganic Chemistry, Faculty of Chemistry

Organizing committee

Dr. Alicia Cuevas Inorganic Chemistry, Faculty of Chemistry

Dr. Gianella Facchin Inorganic Chemistry, Faculty of Chemistry

Ing. Eduardo Kremer Inorganic Chemistry, Faculty of Chemistry

Dr. William Manzanares Intensive Care Unit, Hospital de Clínicas, Faculty of Medicine

Dr. Nelly Mañay Toxicology, Faculty of Chemistry

Dr. Lucía Otero Inorganic Chemistry, Faculty of Chemistry

Dr. Mariela Pistón Analytical Chemistry, Faculty of Chemistry

Dr. Ana Rey Radiochemistry, Faculty of Chemistry

Dr. Inés Viera Inorganic Chemistry, Faculty of Chemistry

Scientific Committee

Philippe Collery (France) Susana Etcheverry (Argentina)

Gérard Bastian (France) Antonio José Costa-Filho (Brazil)

Laetitia Pele (UK) Alzir Azevedo Batista (Brazil)

Alejandro Vila (Argentina) Ademir Neves (Brazil)

Etelka Farkas (Hungary) Enrique J. Baran (Argentina)

Lena Ruiz-Azuara (Mexico) Virtudes Moreno (Spain)

Victor Deflon (Brazil) Ana María da Costa Ferreira (Brazil)

Paul Tchounwou (USA)

Declared of interest by:

Ministerio de Turismo, Uruguay

Edited by:

Cristina Álvarez

Gold Sponsors:

Bronze Sponsor:

Other sponsors:

Plenary Lectures ...........................................................................................................................................................6

Keynotes .......................................................................................................................................................................9

Oral Presentations ......................................................................................................................................................28

Poster Presentations ..................................................................................................................................................70

Proceedings ..............................................................................................................................................................145

Areas

MBD Metal based drugs

AAMMIBBS Advanced analytical methods for metal ions in biochemical and biological systems

MIEH Metal ions in environmental health

TEMIBS Toxicological effects of metal ions in biological systems

NATME Nutritional aspects of trace and major elements

SBM Structural biology of metalloproteins and metal-based redox processes

Plenary Lectures

12th International Symposium on Metal Ions in Biology and Medicine

6

Abstract code: 149 OPENING LECTURE

Designing metalloproteins with hydrolytic, redox and electron transfer centers

Pecoraro, V.L.; Zastrow, M.; Cangelosi, V.; Tegoni, M.; Yu, F.; Tebo, A.; Plegaria, J. Department of Chemistry, University of Michigan, Ann Arbor, MI, USA 48109-1055

We will use de Novo Protein Design to provide synthetic constructs to develop metalloproteins capable of hydrolytic

and redox catalysis. Examples will include hydrolases containing mononuclear Zn centers and reductases

containing Cu. We will discuss the approach and advantages of this strategy to understand the functioning of

metals in biology. We will show a mimic of carbonic anhydrase that precisely mimics the first coordination sphere of

this enzyme and is capable of efficient, multiturnover hydrolysis of nitrophenylacetate or carbón dioxide in aqueous

solution over the pH range 7.5 to 9.5. We will examine how changing the location of the site impacts the catalytic

reaction and effects substrate recognition. We will then discuss using the same peptide, now substituted with

copper, which can reduce nitrie to NO mimicking the Cu Nitrite Reductase. We will show how surface modifications

can influence the copper reduction potential and modify the reaction rate. If time permits, we will also discuss

progress on preparing rubredoxin and cupredoxin centers within designed systems. These studies illustrate how

advances in solid phase peptide synthesis and high level gene expression allow chemists to probe fundamental

questions in metallobiochemistry.


7

Abstract code: 150 CLOSING LECTURE

Reactions of Mn-superoxide dismutase and manganese porphyrins with peroxynitrite: relevance to mitochondrial dysfunction and redox-based pharmacology

Radi, R.

Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

e-mail: [email protected]

Excess biological formation rates of free radicals and oxidants are associated to biomolecular oxidative damage

and disease development. Among the key endogenous antioxidant mechanisms is the mitochondrial, manganese-

containing, superoxide dismutase (MnSOD). MnSOD is an essential protein in mammals with its primary

functionbeing the detoxification of mitochondrial-derived superoxide radical (O2�−). The diffusion-controlled reaction

of O2�− with nitric oxide (�NO) leads to the formation of peroxynitrite (ONOO−), an oxidizing and nitrating cytotoxin,

known to cause mitochondrial dysfunction. Thus, in mitochondria, MnSOD is a key element controlling the

formation of peroxynitrite and therefore, provides protection from its mitochondrial-damaging effects. However,

peroxynitrite readily reacts with MnSOD (k ~ 105M-1s-1) and therefore, sustained mitochondrial levels of

peroxynitrite promote its inactivation via a Mn-catalyzed nitration reaction at the active site Tyr34 both in vitro and in

vivo. Then, peroxynitrite-dependent MnSOD nitration and inactivation triggers a pro-oxidant vicious cycle, that can

ultimately cause severe alteration in mitochondrial, and subsequently cellular, homeostasis. As a way to provide

further antioxidant protection to mitochondria under pathophysiologically-relevant conditions involvingperoxynitrite,

we have studied a series of Mn-based compounds, manganese-porphyrins (MnP), originally conceived to act as

“superoxide dismutase-mimics”. We have found that Mn2+P react fast with peroxynitrite (k ~ 106 -107 M-1s-1) and

reduce it to nitrite; in turn, the oxidized Mn species (Mn4+, Mn3+) can be reduced back to the Mn2+ redox state by

mitochondrial reductants and the electron transport chain to complete a catalytic cycle of peroxynitrite

detoxification. These redox interactions of MnP with peroxynitrite and mitochondrial reductants provide

pharmacological protection in vitro and in vivo, and have opened promising opportunities for mitochondrial- and

redox-based pharmacology aimed to cope with the toxic effects of peroxynitrite.

Keynotes


9

Abstract code: 016 AREA: AAMMIBBS KEYNOTE

Trace elemental and speciation analysis based on volatile species generation and on atomic absorption/fluorescence detectors - current state and perspectives

Dědina, J.1; Kratzer, J.1; Matoušek, T.1; Musil, S.1; Svoboda, M.1; Currier, J.M.2; Stýblo, M.2 1Department of Trace Element Analysis, Institute of Analytical Chemistry of the ASCR, v. v. i., Veveří 97, Brno, Czech Republic,

Prague, Czech Republic 2Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, USA [email protected]

Detection power of atomic spectrometry detectors can be substantially improved employing generation of volatile

species (VSG) of elements since separation from the sample matrix and high efficiency analyte transfer from

sample to spectrometer are involved. In addition, it makes analyte preconcentration easy. All these factors result in

a superior sensitivity. VSG is therefore ideally suited for trace and ultratrace elemental/speciation analysis.

This presentation will briefly overview the recent developments and perspectives of individual approaches to trace

and ultratrace elemental/speciation analysis based on atomic absorption (AAS) and atomic fluorescence (AFS)

detection coupled to VSG. It will be shown that the combination of VSG with AAS or AFS can bring substantial

benefits for elemental as well as speciation analysis of those elements that can be converted to analytically useful

volatile species. VSG with AAS or AFS can thus substitute or even surpass conventional approaches to elemental

and speciation analysis based on the liquid phase sampling with inductively coupled plasma mass spectrometry

which generally serves as a trademark of unparalleled sensitivity.

The applicability of the combination of VSG with AAS or AFS will be illustrated on our recent results:

- hydride trapping in quartz tube atomizers for AAS for ultratrace elemental determination of hydride forming

elements, namely in the presence of very high concentrations of other hydride forming elements;

- generation of substituted arsines followed by cryogenic trapping and atomization in the multiatomizer (AAS

detector) for arsenic speciation analysis in extremely difficult matrices and in liver homogenate slurry;

- generation of substituted arsines followed by cryogenic trapping and atomization in the flame-in-gas-shield

atomizer for AFS for ultratrace analysis of toxicologically important arsenic species.

Acknowledgment: The institutional support RVO:6808171 as well as support from ASCR (m200311202), MŠMT

program Kontakt II (LH12040) and UNC Gillings School of Global Public Health is gratefully acknowledged.


10

Abstract code: 132 AREA: AAMMIBBS KEYNOTE

Metalloproteomics and chemical speciation: towards to a perfect couple

Zezzi Arruda, M.A. Spectrometry, Sample preparation and Mechanization Group – GEPAM

Institute of Science and Technology for Bioanalytics, Institute of Chemistry, Department of Analytical Chemistry, UNICAMP Campinas, Brazil

[email protected]

Metalloproteomics encompasses the inorganic elements content and assemble of their complexes with proteins [1].

Although metalloenzymes and metalloproteins are responsible for nitrogen fixation, photosynthesis and respiration,

and for catalyzing molecular oxygen reduction [2], the information related to proteins and metals is frequently found

fragmented in the literature. This lack of information contributes to remain allusive some mechanisms involving

metal incorporated as a cofactor in a cell [3], metal-sensing related to metalloproteins [4], among others. In this

way, this presentation outcome, besides brief definitions and contextualization of the area, some studies which are

being carried out in our research group encompassing comparative metalloproteomics involving plant stress as well

as some preliminary experiments related to chemical speciation involving metalloproteins.

References

[1] Garcia, J. S., Magalhães, C. S., Arruda, M. A. Z., Talanta 69 (2006) 1-15

[2] Lu, Y., Yeung, N., Sierack, N., Marshall N. M., Nature 460 (2009) 855-862

[3] Szpunar, J., Anal. Bioanal. Chem. 378 (2004) 54-56

[4] Waldron, K. J., Rutherford, J. C., Ford, D., Robinson, N. J., Nature 460 (2009) 823-830

Acknowledgements: Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil),

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil) and Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) are greatly acknowledged for financial

support and fellowships.


11

Abstract code: 014 AREA: MBD KEYNOTE

Metal complexes based on oxindolimine ligands: antitumor and antiparasite activities

Da Costa Ferreira, A.M.1; de Paula, Q.A.1; Sabino, G.L.1; Dario, B.S.1; Ribeiro, G.A.2; Vieira, L.Q.2 1Department of Chemistry, University of Sao Paulo, Sao Paulo, Brasil

2Institute Biological Sciences, Federal University Minas Gerais, Belo Horizonte, Brasil [email protected]

Some oxindolimine compounds have been designed and prepared, based on oxindole derivatives already tested

clinically. These compounds were then metallated with essential ions (copper, zinc or vanadium) given rise to very

stable complexes, isolated and characterized by different spectroscopic techniques. Copper complexes showed

stability constants with log K = 15-17. Their reactivities towards fundamental biomolecules were verified in order to

investigate its possibilities as new therapeutical agents. In a process modulated by the ligand, monitored by CD

and EPR measurements, the corresponding copper(II) complexes interact with human serum albumin, inserting

selectively the metal ion at the N-terminal site, and changing remarkably the α-helix structure of the protein. They

can also effectively bind to DNA, with binding constants determined in the range 102 for plasmidial DNA, probably

at minor or major grooves, depending on the ligand. As cationic lipophilic species, these complexes can cross

membranes and enter into cells, where in the presence of common oxidant agents are able to generate reactive

oxygen species (ROS). The cytotoxicity of these complexes were tested against different tumor cells

(neuroblastoma, melanoma, and sarcoma), indicating that some of them are very effective on causing oxidative

damage to intracellular organelles, and on inducing apoptosis. The corresponding zinc(II) complexes also showed

interaction with HSA, in different metal binding sites, being less cytotoxic against tumor cells. Analogous vanadyl

complexes were also isolated and characterized. Furthermore, these metal complexes showed good activity

against T. cruzi, modulated both by the metal ion and the ligand.

Acknowledgements: FAPESP / INCT Redoxoma / Nanobiomed


12


Cell and tissue distribution of rhenium and selenium after a rhenium diselenate treatment

Collery, P.1; Santoni, F.2; Mohsen, A.3; Desmaele, D.4; D'Angelo, J.5; Wei, M.6; Collery, T.7; Bastian, G.8 1Corsica, Polyclinique Maymard, Bastia, France

2Office d'Equipement Hydraulique, Bastia, France

3Cairo, Egypt

4Université Paris Sud, Paris, France

5Université Paris Sud, Paris, France 6Cellvax Laboratory, Paris, France

7Polyclinique Maymard, Bastia, France

8Laboratoire de Pharmacologie, Paris, France

[email protected]

Rhenium diselenate is a new anticancer agent, combining an atom of rhenium (Re) and two atoms of selenium

(Se).

We studied the Re and Se cell uptake in different cancer cells, with several doses and after different times of

exposure and also after a period of wash-out.

The tissue distribution was studied after an oral daily administration (5 days a week for 4 weeks) of either 10 or 40

mg/kg of rhenium diselenate in mice bearing a MDA- MB 231 tumour, with a comparison with controls receiving a

vehicle. We also studied the interactions with essential metals, magnesium (Mg), zinc (Zn), iron (Fe).

We observed a correlation between the Re cell uptake and the inhibitory effects of rhenium diselenate in cancer

cells, depending on the type of cancer cells. We also noted an efflux from the cells after a period of 72 h after the

end of the exposure time.

In mice, the Re and Se concentrations increased from 10 to 40 mg/kg but much more in the healthy tissues than in

the tumour. The main concentrations were noted in the liver.

The results on the effects of the rhenium diselenate compound on the concentrations of essential metals will be

presented, but the data are not yet available and cannot be included in this abstract.

We conclude that pharmacological data are essential to determine the best schedule of treatment of metal

compounds.


13


Consequences of metal coordination to nucleobases: from alkali ions to platinum

Lippert, B. Prof. of Inorganic Chemistry, Faculty of Chemistry, Technische Universitaet Dortmund, Germany

[email protected]

Potassium, sodium, and magnesium ions are the dominant natural counter ions of the negatively charged nucleic

acids, binding usually in a purely electrostatic or outer-sphere fashion, with the capacity, however, to influence the

overall structure (e.g. DNA curvature) and/or to stabilize particular structures (e.g. nucleobase quartets). Inner-

shere binding, hence coordination of exogenous (transition) metal ions to nucleic acid constituents in general has

more profound consequences on the electronic properties of the nucleobases. These include effects on acid-base

properties of the nucleobases, on tautomerism and hydrogen bonding properties, on base pairing, and on overall

structure. Occasionally the metal can also cause irreversible modifications of the nucleobase or the oligonucleotide.

The lecture will provide an overview on metal binding patterns to nucleic acids in general and to nucleobases in

particular, and will focus especially on X-ray structural work as well as solution studies of Pt complexes of model

nucleobases carried out in the author's laboratory during the past three decades. The relationship and relevance to

biological findings (e.g. antitumor activity of Cisplatin, mutagenicity of heavy metals) will be pointed out and

discussed.


14


Mixed chelates copper(II) compounds Casiopeínas, first and second generation: Antitumor activity and mechanism of action

Ruiz-Azuara, L.1*; Mejía, C.2; Bravo Gómez, M.E.1; García Ramos, J.C.1; Cortés, F.3; Galindo, R.4; Moreno-Esparza, R.1; Hernández-Lemus, E.5; Alemón, R.6; Ballinas, G.1; Reina, M.1; Pessoa, J.7; Gambino, D.8; Moreno,

V.9 1Facultad de Química;

2Instituto de Investigaciones Biomédicas;

3Instituto de Química, UNAM, Mexico,

4University of Utah,

5INMEGEN Mexico,

6INP Mexico,

7Instituto Superior Tecnico, Lisboa, Portugal,

8Universidad de la República, Montevideo,

Uruguay, 9Universidad de Barcelona, España. *[email protected]

Metal complexes have gained a growing interest as pharmaceuticals for their use as diagnostic agents or as

chemotherapeutic drugs [1]. In our group some efforts have been done in the development of anticancer agents

employing essential metals such as the family of copper (II) compounds known as Casiopeínas®, with a general

formula [Cu (N-N) (X-Y) H2O] NO3 where N-N is a diimine (phen or bipy) and X-Y is a bidentate ligand (acac, salal,

aminoacidate, peptide, benzimidazol). These compounds have shown cytostatic, cytotoxic and antineoplastic

activity in vitro and in vivo [2]. Comparison between first and second generation of Casiopeina´s activity is

presented. Also albumin/Casiopeinas interaction is discussed.

The mechanism of action is still not completely elucidated. However, experimental evidence suggests the

interaction of coordination compounds with DNA (nuclear or mitochondrial) and their components and the

generation of reactive oxygen species (ROS) as the main action pathways. Induction of apoptosis has been proved

to be the main death tumoral cell pathway.

DNA interaction has been done by several methods such as comet assay, AFM, electrophoresis, CD [3] and the

nuclease activity studies of 4 Casiopeínas® employing plasmidic DNA in several reaction conditions is presented as

well as the microarray assay of isolated RNA to visualize the complete pathways implicated in the cell death.

Comparison of the results shown that compounds with 4,7-dimethyl phenanthroline in the copper (II) coordination

sphere have an extraordinary DNA cleavage capacity, compared with the 4, 4´-diimine analogs, then is suggested

an intercalation process as the first step for the DNA damage. Also calculations have been done in order to

modeling the interaction between Casiopeínas and DNA [4, 5].

References

[1] S. H. van Rijt, P. J. Sadler, Drug Discov. Today, 14 (2009) 1089-1097.

[2] L. Ruiz-Azuara, M.E. Bravo-Gómez, Curr. Med. Chem., 17 (2010) 3606-3615.

[3] L. Becco, A. Rodríguez, M. E. Bravo, M. J. Prieto, L. Ruiz-Azuara, B. Garat, V. Moreno, D. Gambino J. Inorg.

Biochem., 109 (2012) 49-56.

[4] R. Galindo-Murillo, J. Hernandez-Lima, M. González-Rendón, F. Cortés-Guzmán, L. Ruíz-Azuara, R. Moreno-

Esparza Phys. Chem. Chem. Phys., 13 (2011) 14511-14516.

[5] R. Galindo-Murillo, L. Ruíz-Azuara, R. Moreno-Esparza, F. Cortés-Guzmán Phys. Chem. Chem. Phys., 14

(2012) 15539-15546.


15


Second coordination-sphere effects increase the catalytic efficiency of hydrolases biomimetics

Neves, A.

LABINC – Laboratory of Bioinorganic and Crystallography, Department of Chemistry – UFSC, 88040-900, Florianópolis – SC, Brazil

[email protected]

Phosphatases, oxidases and a variety of other enzymes have been successfully modeled through metal complexes

that can reproduce their physico-chemical properties and even their catalytic activity, however with a much lower

efficiency1.

As has been suggested, these discrepancies in catalytic efficiency between model compounds and true enzymes

are, by a large extent, due to the lack of many important intramolecular interactions in the second-coordination

sphere of the model systems in comparison to the enzyme2.

In this work we propose a new extension of dinuclear hydrolases models by binding the FeIIIMII catalytic unit to a

small polyethyleneimine chain (PEI, 1200 Da) that can emulate the enzyme microenvironment around the active

site (Figure 1)2. In addition we also present the synthesis, characterization and hydrolase activity of conjugates in

which the well known DNA intercalator pyrene is anchored to the FeIIIMII catalytic center2.

Figure 1

Acknowledgment: CNPQ and INCT-catálise

References

1) Mitic´, N.; Smith, S. J.; Neves, A.; Guddat, L. W.; Gahan, L. R.; Schenk, G. Chem. Rev. 2006, 106, 3338. 2) Neves et al, unpublished results.


16


New metal ions in cancer research

Bastian, G. Oncopharmacology, CHU Pitié-Salpêtrière, 91 Bld de l’hôpital, 75013, Paris, France.

Metal compounds have been used in medicine for several hundreds of years. In oncology, the first, and still the

most used, metal ion is Platinum. More than three thousands platinum derivatives have been synthesized and

tested against cancer cells. In this talk, last development about this metal will be presented (attempt of oral

chemotherapy with Cis platinum, Micro and Nano particles containing various platinum derivativesa).

Rhodium belongs to the same group as Platinum and show interesting antitumor activity but with the same

nephrotoxicity. Clinical phase I will be discussed.

Gallium anticancer properties are known since 1971. The antiproliferative properties of Gallium could be related to

its competition with the iron metalGallium nitrate, gallium chloride have been tested successfully orally or

intravenously in different clinical trials. Gallium salicylate has been tested in preclinical trial. It was observed that

the inhibitory effects of gallium on malignant cells were dependent of the doses but also of the duration of

exposure.

Arsenic compounds are natural substances used in China for medical treatment. Arsenic trioxide is the most widely

used in cancer research for treating patients with acute promyelocytic leukemia

Copper complexes have been extensively investigated those past years as potential anticancer agents.

Cassiopeins have shown very promising activity and are ready to start clinical trials. Copper complexes associated

with ligands like peptides (Alanin and Phenylalanin) seem to be good candidates after in vitro testing against

various human tumor cell lines with very low level of toxicity against normal cell lines. Cell cycle modifications after

Copper treatment will be presented.

Rhenium complexes associated with Selenium ligands show also, at fairly high concentration, antitumor properties

which are potentialized with Cisplatin or Gallium salts. No toxicity is observed on normal cells or in animals. In vivo

biodistribution in nude mice of Rhenium, Gallium and Selenium will be presented.

Palladium is also a promising metal ion in cancer research. Compounds like Padeliporphin or Padoporphinare

palladium bacteriopheophorbide photosensitizers and are currently in phase II and phase III clinical trial.

Padeliporphin, which is water soluble, given in patients with low-risk prostate cancer yielded negative biopsies in

more than 80% of patients treated. Further clinical trial will be presented.

Gold complexes were used in Chinese and Arabic medicine. They bind to DNA and have more cytotoxicity than

Cisplatin. They tend also to be active against Cisplatin resistant cell lines. Organogold compound like Auranophin

is currently in clinical trial and results will be discussed.

Despite the tremendous amount of research done on metal chelating agents, very few data concerning the

molecular basis of the mechanisms underlying their antitumor activity are available and need to be investigated at

the time where cancer treatment is more and more targeted.


17

Abstract code: 019 AREA: MIEH KEYNOTE

Biomonitoring of lead exposure: Experience in Uruguay

Mañay, N.; Cousillas, A.; Heller, T.; Pereira Testa, L. Toxicology and Environmental Hygiene, Faculty of Chemistry, Montevideo, Uruguay

[email protected]

Lead (Pb) is a well known bioavailable toxic environmental chemical pollutant, thus it can be absorbed and cause

adverse health effects on susceptible living organisms. The biomonitoring for lead human exposure reflects an

individual’s current body burden, which is a function of recent and/or past exposure activities. Lead in whole blood

(BLL, B-Pb) showed to be the best biomarker for screening, biomonitoring and diagnostic purposes to assess lead

exposure.

In Uruguay, lead pollution problem became of public concern on 2001, and it has been multidisplinary approached

since then. This work highlights the importance of lead biomonitoring of human and animal populations illustrated

by the Uruguayan experience.

Uruguayan studies in human populations (infants, children, adults, workers) and animals (dogs) lead studies are

reviewed to show the incidence of different variables on BLL, and the fact that animals can be "sentinels" of

environmental lead human health hazards.

Results of thousands of analyses done in a 10 ys period, indicated that almost 50% children had BLL above

reference intervention limits (>10ug/dL) initially, but a significant decrease in BLL was found over time in the

studies results, demonstrating the importance of medical intervention, nutrition, and environmental education. The

phase-out of leaded gasoline have also contributed to this improvement.

The severity of lead pollution discovered, required official governmental actions, both to reduce sources of lead

contamination and to address the health implications for lead exposed children. Besides, dogs provided key

diagnostic elements to consider that children, living with them, may be contaminated by lead because these pet

species are susceptible to develop early severe intoxication symptoms to lead exposure.

In conclusion, there has been a significant change in preventing lead exposure in Uruguay due not only to

biomonitoring research work, but also to the public sensitization together with the integration of multidisciplinary

actions promoted.


18


Synthesis of resorcin[4]arenes as potential receptors for metal ions

Botta, B.; D’Acquarica, I.; Ghirga, F.; Santi, L. Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, Rome, Italy

Calixarenes and/or resorcinarenes are a suitable alternative to cyclodextrins for the ability of mimicking the

molecular recognition in the living systems.

Three main features play a fundamental role in the recognition process:

i) variability in the cavity size and shape

ii) chirality

iii) solubility in water, where the biological processes occur.

Two well separated main coordination regions are generally identified in the structure of these macrocycles when

they act as receptors of transition metal cations: the so-called upper rim, the π-basic cavity corresponding to the

wide side of the aromatic rings system, and the lower rim, localized in the region limited by the substituents.

As a part of our studies on modifications of lower rim substituents of resorcin[4]arenes octamethyl ethers, we

prepared tetramine- and the corresponding 4-(aminomethyl)pyridine-derivatives and we will discuss during this

lecture the interaction with CuII, Co+2 and Hg+2 of these compounds as host.

Moreover, we have shown also that resorcin[4]arenes, bearing carboalkyloxy groups in the side chains, are able to

interact with FeIII in organic media.1H-NMR studies, carried out using GaIII instead of FeIII, suggested that these

systems have two active sites for interaction, the first one located at the aromatic rim and the other in the vicinity of

the carbonyl groups. As a confirmation, resorcin[4]arenes without carbonyl groups in the side chains have been

found to exhibit only one active site. Notably, in the latter case the interaction resulted in configurational changes.

Finally we synthesized three resorcin[4]arene-capped porphyrins that differ in the porphyrin skeleton, in the linking

arms and in the cavity dimensions, as well as for their capability to inhibit the oxidation of Co(II) to Co(III).


19


Cognition and behavior in children: are they worse off when undernutrition and toxic metal exposures occur together?

Kordas, K. 3Nutritional Sciences, Pennsylvania State University, United States, University Park, PA

Dr. Kordas will discuss the potential interactions between undernutrition and toxic metal exposures in terms of their

effects on cognition and behavior in children. She will use iron deficiency/anemia and lead exposure as case

studies to outline the reasons why additive or multiplicative effects of both conditions are possible, to examine the

available research evidence and to make recommendations for future studies. She will also discuss her ongoing

research collaborations in Uruguay and the evidence they are generating to address the question posed in the

talk's title.


20

Abstract code: 131 AREA: NATME KEYNOTE

Correlations between trace metal ions (TEs) and the development of infectious diseases in home parenteral nutrition patients

Carver, P.

University of Michigan College of Pharmacy, Department of Clinical, Social, and Administrative Sciences, and University of Michigan Hospitals, Ann Arbor, MI, USA

[email protected]

Background and Objectives: Although patients with ↑ plasma concentrations of Fe have an ↑ risk of certain

bacterial and fungal infections, the effect of ↑ or ↓ plasma concentrations of other metal ions is poorly understood.

Metal ions are important components of nutrition, and are required for cellular growth and metabolism, and may

play a role patients' susceptibility to infection. We evaluated patients receiving long-term (≥1 year) home TPN, to

determine the relationship between bacterial and fungal infections and plasma concentrations of trace metal ions

(Zn, Cu, Se, Mn, and Cr).

Methodology: Data collection included all fungal and bacterial infections, doses and plasma concentrations of

TEs, and risk factors for infections. Descriptive statistics were used for quantitative data, and a recurrent events

survival analysis and a Glimmix procedure to analyze plasma concentrations of the trace metals, and their

correlation with infections.

Results: In long term home TPN patients, TE doses correlate significantly with plasma concentrations of Zn, Cr,

and Mn, but NOT Se, Cu, or Fe. Although % Fe saturation and IV dosages of Fe were predictive of an ↑ risk of

“first” infection, ↑ serum [Fe] and ferritin were not. Higher cumulative doses of Fe are highly predictive of an overall

↑ risk of gram negative or fungal infections; modest correlations were seen for Zn and Mn. A novel and significant

finding was that ↑ Mn doses correlated significantly with plasma [Mn] and with a ↓ rate of infections.

Conclusions: Correlations found between TE doses and plasma [TE]s, and with [TE]s and infections may help to

improve our dosing and monitoring of trace elements, and minimize the risk of inadequate nutrition or life-

threatening infections. The clinical significance of higher Mn doses should be explored further.


21

Abstract code: 137 AREA: RMNM KEYNOTE

Bioorganometallic technetium and rhenium chemistry for imaging and therapy: progresses and perspectives

Alberto, R.; Can, D.; Suleiman, S.; Shen, Y.

University of Zürich, Institute of Inorganic Chemistry, Zürich, Switzerland

Visualization of molecular events on the cellular and subcellular level requires a careful design of highly specific

compounds. Their design does not only concern the biological carrier of the imaging agent as often believed, but

the label in particular. Consequently, the label cannot be considered as “just a tag”, but its properties and

opportunities have to be integrated into the entire design. Following this approach, we have introduced the phenyl-

cyclopentadienyl analogy concept in which phenyl groups of a lead structure are replaced by a [(Cp-R)99mTc(CO)3 ]

moiety. Along a novel approach, we synthesized a wide variety of [(Cp-R)99mTc(CO)3] complexes in which “R”

represents a broad spectra of targeting functions.

The cyclopentadienyl complex may be an essential part of the overall structure. We have prepared 99mTc

analogues of carbonic anhydrase and histone deacetylase inhibitors but also “simple structures such as artificial

amino acids1. Thereby, the function “R” is not exclusively a carboxylate group but can combine different linkers and

coupling groups. For metal-labeled mimetics of pharmacophores, the 99mTc complex is used for imaging and the

homologous Re complex for therapy, a new form of theragnostics. Classical Werner type ligands are as important

as organometallic cyclopentadienyl complexes. We have synthesized bifunctional 2,3-diamino-propionic acid based

ligands which belong to the smallest tripods2. They have been coupled to peptides and to glucose. Synthetic

pathways and biological behaviour will be discussed in the context of Cp-chemistry. The presentation will

emphasize the importance of fundamental chemistry. A focus will be on Cp-complexes to corroborate the 3D-space

occupation concept. Aspects of the theragnostics will be presented as well as the integration of Tc and Re in very

small biological molecules.

(1) Can, D., Spingler, B., Schmutz, P., Mendes, F., Raposinho, P., Fernandes, C., Carta, F.,Innocenti, A., Santos,

I., Supuran, C. T., and Alberto, R. [(Cp-R)M(CO)3] (M=Re or 99mTc) Arylsulfonamide, Arylsulfamide, and

Arylsulfamate Conjugates for Selective Targeting of Human Carbonic Anhydrase IX. Angew. Chem. Int. Edit. 2012,

51, 3354-3357.

(2) Liu, Y., Oliveira, B. L., Correia, J. D. G., Santos, I. C., Santos, I., Spingler, B., and Alberto, R. Syntheses of

bifunctional 2,3-diamino propionic acid-based chelators as small and strong tripod ligands for the labelling of

biomolecules with 99mTc. Org. Biomol. Chem. 2010, 8, 2829-2839.


22

Abstract code: 010 AREA: SBMMBRP KEYNOTE

Catalytic mechanism and evolutionary traits of Zn-dependent beta-lactamases

Meini, M.R.; González, M.; González, L.; Barh, G.; Palacios, A.; Llarrull, L.; Vila, A. Biophysics Section, Instituto de Biología Molecular y Celular de Rosario (IBR), Rosario, Argentina

[email protected]

β-lactamases represent the prevalent resistance mechanism to β-lactam antibiotics. In the last decade, the

dissemination of genes coding for metallo-β-lactamases (MBL´s) has become an emergent clinical problem. MBL´s

are Zn(II)-dependent enzymes. The exponential growth of MBL sequences being characterized has revealed an

initially unforeseen structural diversity, that gives rise to the presence of mono- and dinuclear metal sites. MBL´s

have been recently subdivided into classes B1, B2 and B3, each of them displaying different zinc ligands and

coordination geometries (1).

We have studied the structural features of MBL´s from different subclasses with the aim of finding common

structural and catalytic features. By means of mutagenesis, functional and structural studies, we conclude that a Zn

site, previously regarded as non essential for catalysis, plays a major role in substrate binding and catalysis (2-4,

10-12). Non-steady state kinetic studies, aided by time-resolved electronic, EPR and Resonance Raman

spectroscopy have allowed us to trap a key intermediate in β-lactam hydrolysis, and to assess the role of each

metal binding site in the mechanism and stabilization of this intermediate (5,6).

Directed evolution was used as an evolutionary engineering tool to explore the effect of challenging MBLs towards

different antibiotics. BcII (the MBL from B.cereus) has been considered as a precursor of more efficient MBL’s

present in pathogenic bacteria (7,8). These results suggest that evolution of the chemical landscape can be

predicted by means of this approach. We have also observed that evolution acts by enhancing the Zn(II) binding

capability upon under metal-defficient conditions (9).

References

1. M. W. Crowder, J. Spencer and A.J.Vila Acc. Chem.Res, 39, 721-728 (2006).

2. Llarrull et al. J. Biol.Chem., 282, 18276-18285 (2007).

3. Morán-Barrio et al. J.Biol.Chem., 282, 18286-93 (2007).

4. Llarrull et al. J.Biol.Chem., 282, 30586-95 (2007).

5. Tioni et al. J.Am.Chem.Soc., 130, 15852-15863 (2008).

6. Llarrull et al. J.Am.Chem.Soc., 130, 15842-15851 (2008).

7. Tomatis et al. Proc.Natl.Acad.Sci.USA, 102, 13761-13766 (2005).

8. Tomatis et al. Proc.Natl.Acad.Sci.USA, 105, 20605-20610 (2008).

9. Gonzalez et al. Nature Chem. Biol., 8, 698-700 (2012).

10. Lisa et al. Antimicrobial Agents and Chem., 56, 1769-1773 (2012).

11. J.M.González, et al. Biochemistry, 49, 7930–7938 (2010).

12. Lisa et al. J. Biol.Chem., 285, 4570-7 (2010).


23


Electrostatically-driven switches in electron transfer reactions of metalloproteins

Murgida, D. INQUIMAE-DQIAyQF, CONICET y Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires,

Argentina [email protected]

Natural selection has evolved specialized redox proteins able to perform fast intra- and inter-molecular long range

electron transfer (ET) reactions, in the milliseconds time scale and below, in spite of involving nearly null

thermodynamic driving forces. Most of these nonadiabatic reactions are well described by the high temperature

limit expression of Marcus semiclassical theory. Accordingly, the ET rate is proportional to the square of the

electronic coupling matrix element, and to a Frank-Condon exponential term that accounts for the thermal

accessibility of such degeneracy. The latter is dominated by the reorganization energy (λ) that accounts for both

the energy required to distort the reactants towards the equilibrium configuration of the products and the

reorganization of the solvent around the redox center. Here we present a combined spectroscopic, electrochemical

and computational study showing that for heme and Cu proteins these parameters are strongly modulated by

electrostatic interactions which, in turn, activate either second-sphere ligand conformational switches or enable

alternative electronic ground states that provide optimized pathways for electron entry and exit, respectively. Based

on these results, we propose that electron transfer chains involved in electron-proton energy transduction are

strongly regulated by changes of the membrane potential, as well as by protein-protein interactions, which

eventually may lead to a change or gain of function.


24


Structural determinants of the reaction mechanism and stereoselectivity in heme containing dioxygenases

Marti, M.A.

Departamento de Química Biológica e INQUIMAE, Facultad de Ciencias Exactas y Naturales - CONICET, Universidad de Buenos Aires, Argentina [email protected]

Indoleamine 2,3 dioxygenase (IDO) and tryptophan dioxygenase (TDO) are two heme- proteins that catalyze the

oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). Human IDO (hIDO) has recently been

recognized as a potent anti-cancer drug target, a fact that triggered intense research on the reaction and inhibition

mechanisms of hIDO. In order to shed light into the IDO and TDO reaction mechanism we have used a

combination of theoretical QM/MM based methods with kinetic and spectroscopic studies. Our results show that the

dioxygenase reaction is initiated by addition of a ferric iron-bound superoxide to the C2=C3 bond of Trp to form a

ferryl and Trp-epoxide intermediate, whose presence was confirmed using Resonance Raman spectroscopy.

These data demonstrated that the two atoms of dioxygen are inserted into the substrate in a stepwise fashion,

challenging the paradigm of heme-based dioxygenase chemistry. For the second oxygen insertion step our results

show that the most energetically favored pathway involves proton transfer from Trp-NH3+ to the epoxide oxygen,

triggering epoxide ring-opening and a concerted nucleophilic attack of the ferryl oxygen to the C2 of Trp that leads

to a meta-stable reaction intermediate that subsequently converts to NFK, following C2-C3 bond cleavage and the

associated back proton transfer from the oxygen to the amino group of Trp. Last but not least, we show how the

different enzymes select L-Trp over D-Trp highlighting how the active site structure and dynamics achieve this

challenging task.

References

1. Capece L. et. al., J Phys Chem B. 2012;116(4):1401-13.

2. Capece, L. et. al., Proteins, struct. Func. And Bioinformatics. 2010, 78(14) 2961-72.

3. Lewis-Ballester et. al. Proc. Natl. Acad. Sci USA. 2009, 106(41) 17371- 17376.


25

Abstract code: 025 AREA: TEMIBSS KEYNOTE

Iron homeostatic mechanisms in normal and pathological retina: its implication in aging and retinal degeneration

Courtois, Y.1; Jeanny, J.C.1; Yefimova, M.2; Behar Cohen, F.1; Picard, E.1 1Centre de recherches des Cordeliers team17, INSERM University Paris V, Paris, France

2INSERM UMRS872 team17, Sechenov Institute of Evolutionary Physiology and Biochemistry Russian Academy of Sciences,

St-Petersburg, Russia - France [email protected]

Background: Iron is an essential metabolic component in the retina required for the vision mechanisms. All the

enzymes necessary for the control of its level and distribution have been recently described to be in place in the

retina. Iron accumulation has been reported in the retina as a function of aging and/or occurring in inflammatory

conditions, under oxidative stress or due to genetic defects in several proteins involved in iron metabolism. Since

excess iron is toxic for retina, it is postulated that in all the previous conditions described above, iron regulation

level by Transferrin (Tf) the main iron binding transporter will help to protect this organ.

Methodology: Localization of iron on retinal sections by Perl’s staining, analysis of the role of Tf on rod outer

segment phagocytosis by retinal epithelial pigmented cells (RPE). Addition of Tf on Muller cells loaded with iron. In

vivo injection of Tf in several genetic animal models of retinal degeneration: rd10 mice, RCS rats or in light-induced

retinal degeneration in mice. Comparison of retinal degeneration rate in transgenic mice carrying both the rd10 and

the hTf gene with rd10 mice.

Results: Iron accumulates during retinal degeneration in these models. Tf enhances phagocytosis process. It

protects Muller cells against iron excess. In animal models we observed that the delivery into the eyes of human Tf

by different routes (iv, ivt or gene transfection) at high dose protects or delays retinal degeneration, and in some

cases such as in light-induced degeneration maintains its function.

Conclusions: Iron has multiple functions in retinal physiology and its level is controlled by autonomous

homeostasis mechanisms. Many conditions disturb this equilibrium. Iron in excess induces further oxidative stress.

We show that increasing the level of Tf in the retina has a potent neuroprotective effect and could have potential

pharmacological applications for Age-related macular degeneration and other degenerative diseases

Acknowledgments: This work was funded by ANR and INSERM. We thank Mohamed El Sanharawi, Marianne

Berdugo for their expertise in ophthalmologic techniques.

Reference: Picard E, et al. Mol Vis. 2010 ; 16:2612-25.


26

Abstract code: 147 AREA: TEMIBSS KEYNOTE

Assessing the public health import of children’s exposures to metals

Bellinger, D.C.

The impact of environmental chemicals on children’s neurodevelopment is sometimes dismissed as unimportant

because the impairments observed among individual childrenare viewed as “clinically insignificant.” This reflects a

failure to distinguish between individual and population risk. The population impact of a risk factor depends both on

the effect size associated with it and its distribution (or incidence/prevalence). A set of analyses will be discussed in

which the total number of IQ points lost, among U.S. children under 5 years of age, for a variety of medical

conditions and events (e.g., congenital heart disease, preterm birth, traumatic brain injury, iron deficiency, ADHD)

and certain environmental chemical exposures (lead, methylmercury). The calculations show that the numbers of

IQ points lost as a result of chemical exposures exceed those associated with other neurodevelopmental risk

factors, largely due to the high prevalence of these exposures. The relative importance of different risk factors likely

differs by region and culture. The strategy described provides a rational basis for establishing priorities for reducing

neurodevelopmental morbidities in children, suggesting that a “population-oriented” approach to risk assessment

provides a better sense of public health impact of an exposure than does a “disease-oriented” approach.

Oral Presentations


28

Abstract code: 053 AREA: AAMMIBBS ORAL

Developing techniques for determining the spatial distribution, speciation and chemical environment of metallic elements in brain tissues affected by neurological disorders

Lankosz, M.1; Szczerbowska-Boruchowska, M.1; Wandzilak, A.1; Czyzycki, M.1; Adamek, D.2; Radwanska, E.2 1Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Krakow, Poland

2Chair of Pathomorphology, Faculty of Medicine, Jagiellonian University, Krakow, Poland

[email protected]

The minor and trace elements are playing an essential role in pathophysiology of neoplastic and

neurodegenerative disorders. Selected elements may contribute, directly or indirectly, on the cancerous and

neurodegenerative processes. Molecular medicine is in need of the application of structural methods which are

capable of monitoring biochemical processes and interactions within the tissues. The methods based on

synchrotron radiation i.e. synchrotron radiation X-ray fluorescence (SRXRF), X-ray absorption near edge structure

spectroscopy (XANES), and extended X-ray absorption fine structure spectroscopy (EXAFS) were utilized for

analysis of metals and other elements in human brain samples. The measurements for this study were carried out

at DLS on the beam line I18, on the bending magnet beam lines L and C at HASYLAB, and on beam line BM23 at

ESRF.

The SRXRF technique was applied to the quantitative evaluation of elemental changes in substantia nigra pars

compacta (SNc) in Parkinson’s disease (PD). Two-dimensional maps of elemental distribution show that the

locations of nerve cells in sections are precisely visualized by the high levels of most elements. It was found that Cl,

Fe, Ca and Zn are the most significant elements in the general discrimination between PD and control nerve cells.

The samples from brain gliomas (BG) of various grades of malignancy were collected intraoperatively. The SRXRF

and XANES micro-spectroscopies were performed in thin freeze-dried sections. The results of imaging of areas of

calcification in BG showed that a high level of Ca was accompanied by an increased level of Zn. The Fe, Zn and

Cu XANES spectra were collected at selected points of different regions of samples. Results of measurements

revealed that the Fe oxidation state in the studied samples was between 2+ and 3+, while that of Cu was between

1+ and 2+ and that of Zn was 2+. The cryo-EXAFS method was used for studies of frozen BG. The key findings from

these measurements relate to the oxidation state and the chemical environment of Fe, Cu and Zn in the samples. A

trend can be observed that the higher the tumor grade, the more Fe2+ as compared to Fe3+ the sample contains.

Analysis of Fe EXAFS spectra showed that the distance at which the atoms of the first coordination zone are

located slightly increases with the increase in the tumor malignancy grade. The Cu and Zn EXAFS functions are

indicating slight differences in the paths of scattering obtained for measured samples. The evident contribution of

chemical elements to the pathophysiology of PD and BG was shown.

Acknowledgements: The research leading to these results has received funding from: the European Community's

Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 226716, Diamond Light Source Ltd,

Didcot, Great Britain, European Synchrotron Radiation Facility, Grenoble, France, HASYLAB, Hamburg, Germany

and the Ministry of Science and Higher Education (Warsaw, Poland) grant no N N518 377 537


29


Protein and peptide labeling with metals in nano-HPLC-UV-ICP-MS detection

Lyrio Tenorio Correia, C.1; Holste, A.2; Tholey, A.3; Saint'Pierre, S.1; Schaumloffel, D.2 1Department of Chemistry, PUC Rio, Rio de Janeiro, Brazil 2Department of Analytical Chemistry, UPPA, Pau, France

3Division Systematic Proteom Sechearch, Christian-Albrechts-University, Kiel, Germany

[email protected]

The new challenge of analytical chemistry, to identify and understand the mechanisms of interaction between metal

ions and macromolecules, in addition to separation techniques, analytical techniques are required that can identify

and quantify elemental species, present in biological tissues at concentrations in the ng L-1. The principal approach

of this study is based on the use of metals detection in ICP-MS and derivatization of protein and peptide by metal

labels as a new approach to protein quantification to generate a method that allows higher sensitivity and accuracy.

Reduction for all peptides was carried out using a two-fold molar excess of TCEP per cysteine residue. After that,

the model peptides were derivatized with 100-fold molar excess of NHS-DOTA ester to each free amino group.

Subsequently, the complexation of peptides labelled with NHS-DOTA ester was carried out with lanthanide metal

ions. The end concentration of peptide was 5 pmol/mL. Moreover, a mixture of five peptides was prepared and the

same procedure for reduction, derivatization and complexation was applied. Every samples was analyzed by nano-

HPLC with UV detection and the metals detected by ICP-MS. Peptides were eluted onto a column separation C18

and performed with a flow rate of 0.3 mL/min and the following eluents: A (0.05% aqueous TFA) and B (80% ACN,

0.04% TFA, 20% deionized water. In order to study the MS and MS/MS behavior of NHS-DOTA-labelled peptides a

number of synthetic peptides containing lysine residues or primary amino groups were analyzed by MALDI-TOF-

MS. The data obtained demonstrates the suitability of MALDI-MS and -MS/MS for the characterization of metal-

labelled peptides. The benefits of the complementary application of molecular and elemental ICP-MS clearly show

that upon use of this approach a straightforward accurate and precise absolute quantification of the labelled

proteins is possible.


30


Post-mortem biochemistry: Determination of trace elements in vitreous humor by ICP-MS

Santos Júnior, J.C.1; Höehr Nelci, F.1; Mollo Filho, P.C.2; Felice Guidugli, R.B.2; Eberlin, M.N.3; Pessôa, G. de S.4; da Silva, E.G.5; Zezzi Arruda, M.A.5

1Department of Clinical Pathology, School of Medical Sciences - University of Campinas — UNICAMP, Campinas/SP, Brazil

2Team of Forensic Medicine West, Medico-Legal Institute - Police Technical Scientific Superintendence — SPTC, São

Paulo/SP, Brasil 3ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry - University of Campinas — UNICAMP, Campinas/SP, Brazil

4Campinas/SP, Institute of Chemistry - University of Campinas — UNICAMP, Group of Spectrometry, Sample Preparation and

Mechanization (GEPAM), Brasil 5Group of Spectrometry, Sample Preparation and Mechanization (GEPAM), Institute of Chemistry - University of Campinas —

UNICAMP, Campinas/SP, Brazil [email protected]

Background: Biochemistry is of great importance in forensic pathology [1]. The vitreous humor for its features and

sterility, was a good sample for our analyzes [2]. Mass spectrometry with inductively coupled plasma source allows

quantification multi-elemental metals and metalloids with a dynamic range of 6-7 orders of magnitude [3].

Objective: To investigate the pattern of metals (Fe, Sn, Mo and Mg) in vitreous humor bodies and the correlation

of decomposing and non-decomposing bodies for different periods.

Methodology: The vitreous humor was extracted using puncture site, packed in Vacutainer ® brand tubes BD

(REF 368521), separated and identified. For the decomposition system in mini- vials, 200 µL of samples were

added to 200 µL of HNO3 sub-distilled and 100 µL of H2O2. The concentrations were determined with an ICP-MS

(Elan DRC-e, PerkinElmer) under appropriate conditions. Methane was used to minimize interferences.

Results: There is an increase of iron concentration in the samples of decomposing bodies and there is no

correlation between time and concentration of Sn, Mo, and Mg ions.

Conclusions: The use of ICP / MS in biochemistry postmortem check is extremely valuable in correlating the

phenomena of decomposition, where the relationships can serve as indicative postmortem on body decomposing.

Acknowledgments: The FCM / UNICAMP (No1270/2010), the West IML (No 09/2011) and to CNPq for the

postgraduate scholarship.

References

[1] MAEDA H.; ISHIKAWA T.; MICHIUE T. Forensic biochemistry for functional investigation of death: Concept and practical application. Legal Medicine. 2011; 13:55-67.

[2] Sanches et al. Determination of opiates in whole blood and vitreous humor: a study of the matrix effect and an experimental design to optimize conditions for the enzymatic hydrolysis of glucuronides. Journal of analytical toxicology. 2012; 36:162-170.

[3] Sussulini A, Garcia JS, Arruda MA. Microwave- assisted decomposition of polyacrylamide gels containing metalloproteins using mini-vials: an auxiliary strategy for metallomics studies. Anal Biochem. 2007; 361(1):146-8.


31


Microwave induced combustion for metals determination in biological matrices

Flores, E.M.M.1; Smanioto Barin, J.2; Bizzi, C.A.1; Foster Mesko, M.3; Severo Fagundes Pereira, J.1; de Azevedo Mello, P.1

1Departamento de Química, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil

2Departamento de Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil 3Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, RS, Brazil

[email protected]

Determination of trace elements in biological and food samples has received increased concern due to nutritional

or toxic aspects. In this sense, the determination of metals in biological matrices as milk powder and bovine liver is

of great importance and an appropriate sample preparation method should be used in order to obtain an efficient

sample digestion. The use of concentrated reagents is hazardous, could require a dilution step of the digests

before analytes determination and generates high volumes of concentrated acids as effluents. Considering the

limitations of methods based on microwave-assisted wet digestion (MAWD), concentrated acids are generally

required, increasing the blank values or could cause interferences in the determination step by some analytical

techniques, such as ICP OES. Recently the microwave-induced combustion (MIC) method has attended several

premises related low reagent consumption, use of diluted acids and high digestion efficiency. In the present work

MIC method is proposed for biological digestion for further metals determination (Cd, Cr, Cu, Mn, Zn, etc) by ICP

OES and and ICP-MS. The concentration of absorbing solution for analytes retention was studied using a reflux

step after sample combustion.

For results comparison, milk powder digestion was also performed by MAWD and MIC in closed vessels. Accuracy

was evaluated using certified reference materials of milk powder and bovine liver and it was better than 95% for all

the analytes. Efficiency of digestion was evaluated by the residual carbon content in digests and values lower of

1% were obtained for all the samples. It was possible to use diluted nitric acid solution for analytes absorption and

digests were suitable for determination of metals by ICP OES or ICP-MS.

Acknowledgements: CNPq, INCT-Bioanalítica, FAPERGS, UFPel, UFSM


32

Abstract code: 002 AREA: MBD ORAL

Tungsten reacting with 5-hydroxymethylcytosine

Okamoto, A. Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan [email protected]

tokyo.ac.jp

5-Hydroxymethylcytosine (hmC) is a newly discovered natural nucleobase that may play an important intermediary

role in the active DNA demethylation pathway. An effective method to detect the presence and abundance of hmC

in DNA is required to elucidate the relationship between the generation of hmC and the mechanism of

demethylation. We have found that the oxidation using dinuclear peroxotungstate K2[{W(=O)(O2)2(H2O)}2(m-

O)]•2H2O is hmC selective, and is useful for the discrimination of hmC from its epigenetic precursors, unmethylated

cytosine (C) and 5-methylcytosine (mC) in DNA.1 A fluorescein-labeled model DNA containing CG, mCG, or hmCG

dinucleotides was prepared, and the dinuclear peroxotungstate was added to a DNA solution in a pH 7 sodium

phosphate buffer. The mixture was incubated at 50°C for 5 h. The reaction proceeded hmC-selectively. The mass

spectrum of the oxidation product obtained from the hmC-containing DNA indicated the formation of trihydroxylated

thymine in DNA. The tungsten oxidation products induced the incorporation of adenine into the complementary site

in a primer extension, and made it possible to detect hmC in a DNA sequence of interest in a conventional DNA

sequencing analysis. Tungsten oxidation worked as a simple chemical reaction for the effective detection of hmC,

and will provide beneficial information on the design of a powerful method for hmC scanning and typing to solve the

mystery of the initialization of gene function through demethylation.

References

Okamoto, A., et al. 5-Hydroxymethylcytosine-selective oxidation with peroxotungstate. Chem Commun, 2011, 47, 11231–11233.


33


Trisenox-based chemotherapy of acute promyelocytic leukemia: Molecular mechanisms of action

Tchounwou, P.B.; Yedjou, C.G.; Brown, E.; Rogers, C. 1Molecular Toxicology Research Laboratory, NIH-Center for Environmental Health, College of Science, Engineering and

Technology, Jackson State University, 1400 Lynch Street, Box 18540, Jackson, Mississippi, USA [email protected]

Background: Acute promyelocytic leukemia (APL) is a blood cancer characterized by a rapid accumulation of

abnormal white blood cells in the bone marrow and blood resulting in anemia, susceptibility to infections, bleeding,

and hemorrhage. Trisenox (TX)-based chemotherapy has recently been approved by the FDA based on its

effectiveness in providing for a complete remission in de novo and relapsed APL patients. Although TX has been

reported to induce degradation of PML-RAR alpha protein in these patients, its molecular mechanisms of action

against cancer cells remain to be elucidated. In this research, we hypothesize that TX pharmacology is mediated

through oxidative stress leading to p53 activation, genotoxicity, and apoptosis in cancer cells.

Methods: To test this hypothesis, we performed the MTT-assay for cell viability, thiobarbituric acid test for lipid

peroxidation, Western Blot analysis for p53 expression, microgel electrophoresis (Comet) assay for genotoxicity,

flow cytometry analysis of annexin-5 and caspase-3, and DNA laddering assay for apoptosis, using the human

leukemia (HL60) cell line as a test model.

Results: MTT assay indicated a strong dose-response relationship with regard to the cytotoxic property of TX. The

24 hr-LD50 was 6.35 ± 0.57µg/mL. Western Blot analysis also demonstrated a strong dose-response relationship

with regard to the expression of tumor suppression protein, p53. Similarly, strong dose-response relationships

(p<0.05) were obtained in connection with TX- induced MDA formation, and DNA damage as characterized by the

percentages of DNA fragmentation, and the lengths of the comet tails in leukemic cells. AT-induced apoptosis was

characterized by a significant increase in the percentages of annexin-5 and caspase-3 positive cells, as well as in

the degree of DNA fragmentation.

Conclusions: TX pharmacotherapy is associated with a cytotoxicity that is mediated by oxidative stress, up-

regulation of p53, DNA damage and morphological changes leading to apoptotic death in cancer cells.

Aknowledgments: Research supported by NIH Grant # 1G12RR13459 and DoD Grant # W911NF-11-1-0123 at

Jackson State University.


34


Synthesis, characterization and antitrypanosomal evaluation of ferrocenyl and cyrhetrenyl thiosemicarbazone complexes of platinium (II)

Arancibia, R.1; Lapier, M.2; Maya, J.D.2; Klahn, H.1 1Instituto de Quimica, PUCV, Valparaiso, Chile

2Facultad de Medicina, U. de Chile, Santiago, Chile

[email protected]

In the last few years, our research group has been involved on bioorganometallic compounds with potential

antiparasitic activity, particularly as antimalarial1 and antitrypanosomal2. By considering that Chagas’ disease or

American trypanosomiasis is still an important health problem that affects around 20 million people in Central and

South America3 and with the aim to find out new and more effective antichagasic agents, we decided to investigate

new complexes derivatives from organometallic thiosemicarbazones (TSCs). The very well known organic TSCs

complexes are currently being investigated as potential chemotherapeutics, including as antichagasic4, but

organometallic TSCs analogues have received considerably less attention, according to our acknowledge some

ferrocenic-TSCs complexes have been evaluated as antimalarial4 but their antitrypanosomal activity is still

unreported. In the present work, we would like to report the synthesis, characterization and anti-T. cruzi evaluation

of ferrocenyl and cyrhetrenyl thiosemicarbazone complexes of platinium (II) (Esqueme 1).

All compounds were characterized for IR and NMR. The 31P–{1H} NMR spectra showed a singlet, shifted to higher

frequency from the spectrum of the free phosphine, in agreement with phosphorus coordination to metal center.

The results of an in vitro antitrypanosomal assay of the compounds against strains of T. cruzi using MTT, indicate

that they have moderated antichagasic activity.

References

[1] R. Arancibia, F. Dubar, B. Pradines, I. Forfar, D. Dive, A. H. Klahn, C. Biot ; Bioorg. Med. Chem.; 2010, 18, 8085.

[2] R. Arancibia, A. H. Klahn, G. Buono-Core, E. Gutierrez-Puebla, A. Monge, M. Medina, C. Olea-Azar, J. Maya, F. Godoy; J. Organomet. Chem.; 2011, 696, 3238.

[3] http://www.who.int/healthinfo/statistics/healthinfoachmsreport2006.pdf

[4] (a) H. Cerecetto, M. Gonzalez; Mini Rev. Med. Chem.; 2008, 8, 1355; (b) C. Biot, B. Pradines, M. Sergeant, J. Gut, P. Rosenthal, K. Chibale; Bioorg. Med. Chem. Lett.; 2007, 17, 6434.

Acknowledgements: We thank the FONDECYT-Chile (Project 1110669; Project 3120091) and D.I.-PUCV.


35


Paramagnetic resonance studies on Ru(III) azole complexes of medicinal interest

Telser, J.1; Alamiri, A.1; Reisner, E.2; Arion, V.B.3; Büchel, G.3; Freitag, L.4; Tierney, D.L.5; Marts, A.R.5 1Biological, Chemical and Physical Sciences, Roosevelt University, Chicago, IL, USA

2Chemistry, Cambridge University, Cambridge, UK

3Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria 4Institute of Physical Chemistry, University of Vienna, Vienna, Austria

5Chemistry and Biochemistry, Miami University, Oxford, OH, USA [email protected]

Ruthenium complexes have recently emerged as candidates for anti-cancer drugs. These include organoruthenium

complexes (of diamagnetic Ru(II), 4d6) as well as coordination complexes of paramagnetic Ru(III) (4d5). The latter

class of Ru complexes, which are analogous to well-known Pt-based drugs, comprise chlorido ligands, in some

cases dmso (dimethylsulfoxide), and a variety of azole ligands such as imidazole (Him) and indazole (Hind). Two

such complexes are in clinical trials: H2im[trans-RuCl4(dmso)(Him)] (NAMI-A), and H2ind[trans-RuCl4(Hind)2],

(KP1019, also known as FFC14a). A spectroscopic advantage of Ru(III) is that its S = 1/2 ground state is suitable

for investigation by EPR, which provides information on electronic structure not available by other means. We

describe low temperature EPR studies at both X-band (~9 GHz) and Q-band (35 GHz) frequencies on a series of

Ru(III) tetrachlorido complexes in DMF, which solvent had already been used for electrochemical studies [1]. In

addition, paramagnetic 1H NMR spectra in DMF-d7 at 300 K were recorded, which provided fluid solution

information on these complexes for comparison with EPR. Electronic absorption spectra in DMF were also

recorded to provide data for computational determination of electronic energy levels and redox potentials. The

complexes studied were of three types: the anionic "A" series, [trans-RuCl4(dmso)L]–; the anionic "B" series, [trans-

RuCl4L2]–; and the neutral, "C" series: [mer-RuCl3L3], where L = a wide range of azole ligands. For the A and B

series, the azolium ion served as cation, although other cations were also investigated. The A and C series of

complexes each showed a distinct type of electronic structure, with little variation within each series. The B series

showed the presence of two forms in solution, which we associate with cis/trans isomerism, which has been

investigated computationally. The implications of these structures on reactivity of these complexes will be

discussed.

References

[1] Reisner, E; Arion, VB; Guedes da Silva, MFC; Lichtenecker, R; Eichinger, A; Keppler, BK; Kukushkin, VY; Pombeiro, AJL; Tuning of Redox Potentials for the Design of Ruthenium Anticancer Drugs - an Electrochemical Study of [trans- RuCl4L(DMSO)]- and [trans-RuCl4L2]

- Complexes, where L = Imidazole, 1,2,4-Triazole, Indazole. Inorg Chem; 2004; 43; 7083-7093.


36


Bioinduced investigation on Cobalt(II)/(III) – hydroxamate systems

Farkas, E.; Szabó, O. Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér, Debrecen, Hungary

[email protected]

Hydroxamic acids are among the most well studied compounds due to their significance in so many fields. For

example (i) due to their good metal binding capability, they are effective inhibitors of many metalloenzymes. Among

others, hydroxamic acids are well-known inhibitors of cobalt containing metallohydrolases. To get deeper insight

into the mechanism of inhibition, numerous dinuclear complexes (including Co(II) containing ones) as structural

and functional models for catalytic centers have been synthesized and characterized and their interaction with

different hydroxamic acids in the solid state has been investigated [1-2]. (ii) Remarkable results have recently been

published in the literature [3-4]. Namely, different kinetically inert Co(III) complexes have been synthesized as

carriers to deactivate hydroxamate-based MMP (Matrix Metalloproteinases) inhibitors by chelation through the

hydroxamate binding group. The goal in this study was the development of Co(III)-containing prodrugs for selective

delivering of MMP inhibitors to target tumor sites. Since the number of equilibrium results for the cobalt(II)- and

cobalt(III)-hydroxamate systems in the literature is very limited, the investigation of such kind of systems is in the

focus of interest nowadays in our lab in Debrecen.

Although many different hydroxamic acids have been involved in this study, the most interesting results have been

obtained on the complexes formed with the most well-known hydroxamate based siderophore desferrioaxamine B

(DFB), and also with desferricoprogen (DFC).

HO O N O

NH

HN

O N O O

O

NH

OH

HOO

OH

N O

HO

+H 3NN O

HN

O

HON

NH

OH O

O

N

O

HO

Desferricoprogen (DFC) Desferrioxamine B (DFB) B (DFB (DFB) (DFB)

Surprisingly, oxidation of Co(II)-siderophore complexes at high pH resulted the formation of extremely high stability

complexes, higher stability than the those of the corresponding Fe(III) species. Whether does it mean the possibility

of disruption of the microbial iron-uptake in the presence of this metal ion? This question and also the possibility of

the application of the extremely high stability and inert Co(III)-siderophore complexes as pro-drugs are planned to

discuss in a presentation on the conference.

Acknowledgments:

The work was supported by OTKA-NKTH CK77586 and TAMOP 4.2.1/B-09/1/KONV-2010-0007 National

Hungarian Fund

References

[1] D.A. Brown, W. Errington, W.K. Glass, W. Haase, T.J. Kemp, H. Nimir, S.M.Ostrovsky, R. Werner, Inorg. Chem. 40 (2001) 5962.


37

[2] Z. Tomkowicz, S. Ostrovsky, H. Müller-Bunz, A.J. Hussein Eltmimi, M. Rams, D.A. Brown, W. Haase, Inorg. Chem. 47 (2008) 6956.

[3] M.D. Hall, T.W. Failes, N. Yamamoto, T.W. Hambley, Dalton Trans. (2007) 3983.

[4] T.W. Failes, C. Cullinane, C.I. Diakos, N. Yamamoto, J.G. Lyons, T.W. Hambley, Chem. Eur. J. 13 (2007) 2974.


38


In vitro antitumor activities of complexes with the general formula: [Ru(N-S)(bipy)(P-P)]PF6, (N-S = thiolates ligands; bipy = 2, 2’-bipyridine; P-P = diphosphines)

Lima, B.A.V.1; Graminha, A.E.1; Ribeiro, A. de S.B.B.2; Pereira, F. de C.2; de Lima, A.P.2; Lacerda, E de P.S.*2; Batista, A.A.*1

1Department of Chemisty, Federal University of São Carlos, São Carlos(SP), Brazil

2Institute of Biologycal Ciences, Federal University of Goiás, Goiânia, Goiás, Brazil

*Corresponding author: [email protected] (A.A. Batista); [email protected], (E. de P. Silveira-Lacerda)

The reaction of the complexes cis-[RuCl2 (bipy)(P-P)], bipy = 2,2’-bipyridine and P-P = 1,3-

bis(diphenylphosphino)propane (dppp) and 1,2- bis(diphenylphosphino)etane (dppe), with the ligands pySH(2-

mercaptopyridine), Hprm (2-mercaptopyrimidine) and dmpm (4,6-dimethyl-2-mercaptopyrimidine) yielded

complexes of the type [Ru(N-S)(bipy)(P-P)]PF6, N-S = pyS, prm or dmpm. Preliminary in vitro tests for antitumour

activity against the MDA-MB-231 human breast tumor cell line, using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide], were carried out for the new complexes, for the precursors complexes and for the free

ligands. The complexes tested against MDA-MB-231tumour cells showed high activity, with IC50 values lower than

1 µmol L-1.

It was shown that the complexes are able to increase the population of cells in the G0/G1 phase. This G0/G1

population increase suggests that these complexes interfere in cell metabolism in a way that the cell cannot enter

the DNA duplication phase (S phase), thereby preventing cell proliferation. While S180 tumour cells already have a

larger cell population in the G0/G1 phase, which is characteristic of melanomas, the complexes enhance the

increase of this population, further reducing the efficiency of cell cycling and thus cell proliferation.

Acknowledgements

We thank FAPES, CNPq, CAPES, and CYTED for financial support.


39


Interaction of divalent cations with protein PARK9

Zoroddu, M.A.1; Peana, M.1; Medici, S.1; Solinas, C.1; Juliano, C.1; Remelli, M.2 1Chemistry and Pharmacy, University of Sassari, Sassari, Italy

2Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy

[email protected]

Metals have been shown to play a role in the genesis and development of many neurodegenerative diseases.

Park9 encoded protein can protect cells from manganese poisoning, an environmental risk factor for a Parkinson’s

disease- like syndrome1,2. Park9 belongs to a family of ATP-ases involved in metal coordination and transportation;

familial mutations of this gene may result in early development of PD. We tested two peptide sequences from

Park9, -P1D2E3K4H5E6L7- (1) and -F1C2G3D4G5A6N7D8C9G10- (2), for Mn(II), Zn(II) and Cu(II) binding. These

fragments are located from 1165 to 1171 and from 1184 to 1193 residues in Park9 sequence, and are highly

conserved in a number of organisms, from yeasts to humans. Experiments have been carried out at different pH

values and ligand/metal molar ratios with both potentiometric and spectroscopic (NMR, UV-vis) techniques,

showing that the three metals are able to effectively bind the examined peptides. Mn(II) and Zn(II) coordination with

peptide (1) involves imidazol of His5 and carboxyl γ-O of Asp2, Glu3 and Glu6 residues, in a distorted octahedral

geometry, possibly involving bidentate interaction of carboxyl groups; four donor atoms participate in Zn(II) binding,

resulting in a tetracoordinated geometry. Mn(II) and Zn(II) coordination involves the two cysteines in peptide (2);

Mn(II) accepts additional ligand bonds from D4 and D8 to complete the coordination sphere, together with some

water molecules. Details of Cu(II) coordination are under study.

References

[1] Gitler A.D., Chesi A., Geddie M.L., Strathearn K.E., Hamamichi S., Hill K.J., Caldwell K.A., Caldwell G.A., Cooper A.A., Rochet J.-C., Lindquist S., Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity, Nat Genet, 2009, 41, 308-315.

[2] Schmidt K., Wolfe D.M., Stiller B., Pearce D.A., Cd2+, Mn2+, Ni2+ and Se2+ toxicity to Saccharomyces cerevisiae lacking YPK9p the orthologue of human ATP13A2, Biochem Biophys Res Com, 2009, 383, 198-202.


40


A novel Cu(II) ternary complex with iminodiacetate and dimethyl-bipyridine. Synthesis, characterization and DNA interaction

Alvarez, N.1; Costa-Filho, A.2; Torre, M.H.1; Ellena, J.3; Facchin, G.1 1Cátedra de Química Inorgánica, Facultad de Química, UdelaR, Montevideo, Uruguay

2Departamento de Biofísica Molecular, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, USP, Ribeirão Preto, Brazil

3Grupo de Cristalografía, Instituto de Física de São Carlos, USP, São Carlos, Brazil [email protected]

The importance of metal complexes in medicine dates from the XVIth century with reports on the use of

metallodrugs in anticancer therapy. Inorganic Medicinal Chemistry has made great advances in search of new

metal compounds presenting antitumor activity, for instance, by use of essential metals. In this work, in the search

of new antitumor agents, the ternary complex [Cu(iminodiacetate)(4,4’-dimethyl-2,2’-bipyridine)]•4H2O was

prepared employing the reaction of copper acetate with iminodiacetic acid in presence of dimethyl-bipyridine. The

obtained complex was characterized by elemental analysis, single crystal X-ray diffraction, Fourier Transformed

Infrared spectroscopy (FT- IR), Electron Paramagnetic Resonance (EPR) and UV-visible spectroscopy (UV-vis).

The complex crystallizes in a triclinic unit cell, in the centrosymmetric space group P-1. The Cu(II) center is bonded

to a bypiridine ligand -through its N atoms- and to the iminodiacetate ion -through the carboxylate O atoms and the

imine N atom-. The molecular structure was confirmed by FT-IR where bands involving the ligand and donor atom-

Copper bonds are observed. Knowing the atom array in solid state is considered fundamental for further aqueous

solution analysis and comprehension of the complexes behavior in biological media. UV-vis absorption bands in

aqueous solution are consistent with a square pyramidal Cu(II) environment, which suggests the maintenance of

the coordination sphere observed in solid state. The ability of the complex to interact with deoxyribonucleic acid

(DNA) was studied by Circular Dichroism (CD) in constant concentration of DNA with increasing concentration of

complex. The profile observed in the CD spectra evidences complex-DNA interaction which is consistent with an

intercalative interaction, as observed in other complexes containing dimethyl-bipyridine.


41


Targeting human topoisomerase I with different vanadyl compounds

Vieira, S.1; Katkar, P.1; Castelli, S.1; Benítez, J.2; Costa Ferreira, A.M.3; Gambino, D.2; Desideri, A.1 1Department of Biology, University of Roma Tor Vergata, Rome, Italy

2Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay.

3Department of Chemistry, University of Sao Paulo, Sao Paulo, Brasil [email protected]

DNA topoisomerases are essential and ubiquitous enzymes, belonging to two classes (type Iand type II), both

characterized by a catalytic mechanism whichinvolves a nucleophilic attack of a DNA phosphodiester bondby a

tyrosyl residue from the enzyme, but type I cleaves only oneDNA strand, whereas type II cleaves both strands.

These enzymeshave been shown to be essential in nearly all processes of DNAmetabolism such as replication,

transcription, recombination andchromosomal segregation. The human topoisomerase I enzyme is composed of

765 amino acids and has four distinct domains: the N-terminal domain (1–214), the core domain (215–635), the

linker domain (636–712) and the C-terminal domain (713–765). The three-dimensional structure of the

reconstituted N-terminal truncated version of human topoisomerase in complex with a 22 bp DNA molecule shows

the enzyme organized in multiple domains which ‘clamp on’ the DNA molecule. Human topoisomerase I is of

significant medical interest since it is the unique cellular target of camptothecin a plant alkaloid with strong

anticancer activity, that rapidly blocks both DNA and RNA synthesis. Here we present some recent results

concerning the interaction of the enzyme with different ligands coordinated to the vanadyl cation. The efficiency of

the metal compounds in inhibiting the enzyme is different depending on t the ligand dimension and polarity and

such differences are discussed in the attempt to derive a structure-function correlation and select the best ligand to

target the enzyme.


42


The studies of the interactions between metal complexes and DNA

Mao, Z. MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen

University, Guangzhou, China [email protected]

DNA, the carrier of the genetic information and the substantial basis of the expression of genes, plays a significant

role in the metabolic processes of lives like growth and reproduction, and is involved in the excitation of the silent

genes of cancers. Due to the diversity of the structures of the DNA and the fact that the DNA can be easily targeted

in the therapy of cancers, the studies of the interactions between the small molecular metal complexes and the

DNA, has a great impact on both the discovery of the structures or the functions of nucleic acids and the

development of potential anti-cancer drugs. Thus, there are two aspects. The effective exploration of the mimic of

metallonucleases that cleave dsDNA under physiological conditions provides us not only a solid theoretical

foundation for the chemical process of lives but the estimated applications in the fields of diagnosis and therapy of

cancers in the future. And the synthesis of the metal complexes targeting and stabilizing the G-quadruplex DNA

brings about the hope of conquering cancers. Considering the structural features of the duplex DNA and the G-

quadruplex DNA, we have designed and synthesized a series of Cu and Pt complexes with functional groups for

the research of the interactions in recent years, which is as follows:

1) By exploring the thermodynamics and the kinetics mechanism of the interactions between model compounds of

nuclease enzyme and the DNA systematically, we made a conclusion of the different abilities to shear the DNA

from many angles, including the structures of ligands and the properties of the electrons of substituents of main

ligands.

2) We found a fact that some Pt complexes composed of pyridines are able to induce the G-quadruplex DNA and

promote the stability of the specific structures, which is successfully confirmed by the data of PCR-stop, FRET and

CD. Based on the research above, in addition, we focus on the synthesis of some Pt complexes with unique

configurations via supermolecular self-assembly. And the very complexes can stabilize the G-quadruplex DNA

efficiently and may be used as an inhibitor of telomere. We tried to explain the mechanism of the interactions at the

level of cells or molecules to provide theoretical and experimental proofs for the further development of therapeutic

reagents with higher selectivities and lower toxic side effects.

Acknowledgements: Financial support from the National Natural Science Foundation of China, the Guangdong

Provincial Natural Science Foundation and National Basic Research Program of China.

References

1. An, Y.; Tong, M. L.; Ji, L. N; Mao, Z. W. Dalton Trans., 2006, 35, 2066.

2. An Y.; Lin, Y. Y.; Wang, H.; Sun, H. Z.; Tong, M. L.; Ji, L. N.; Mao, Z. W. Dalton Trans., 2007, 36, 1250.

3. He, J.; Hu, P.; Wang, Y. J.; Tong, M. L.; Sun, H. Z.; Mao, Z. W.; Ji, L. N. Dalton Trans., 2008, 38, 3207.

4. Wang, J. T.; Xia, Q.; Zheng, X. H.; Chen, H. Y.; Chao, H.; Mao, Z. W. Dalton Trans., 2010, 39, 2128.

5. Wang, J. T.; Zheng, X. H.; Xia, Q.; Mao, Z. W. Ji, L. N.; Wang, K. Dalton Trans., 2010, 39, 7214.

6. Wang, J. T.; Li, Y.; Tan, J. H.; Ji, L. N.; Mao, Z. W. Dalton Trans., 2011, 40, 564.

7. Zheng, X. H.; Zhong, Y. F.; Tan, C. P.; Ji, L. N.; Mao, Z. W. Dalton Trans., 2012, 41, 11807.

8. Zheng, X. H.; Chen, H. Y.; Tong, M. L.; Ji, L. N.; Mao, Z. W. Chem. Commun., 2012, 48, 7607.


43


Dual mode binding of ruthenium arene anticancer complexes to DNA: Coordination and intercalation

Liu, H.

Jiangsu Province Key Laboratory of Biofunctional Material, School of Chemistry and Environmental Science, Nanjing Normal University, Nanjing, China [email protected]

Organometallic Ru(II) arene complexes of the type [(η6-arene)Ru(en)Cl]+ [1], are toxic to cancer cells, including

cisplatin-resistant cell lines. We have investigated the kinetics and thermodynamics of binding to DNA. We find that

Ru-arene complexes with extended rings can bind to DNA duplex by bi-functional binding modes (coordinative and

intercalative) and further work has provided evidence that such bifunctional interactions of Ru-bip can affect the

basepairing of duplex DNA [2-3]. Two-dimensional NMR experiments [1,4] show the presence of a novel

penetrating intercalation of Ru-tha into a DNA hexamer. The results presented here provide and may help to

explain why ruthenium arene complexes have a different mechanism of antitumour activity compared to cisplatin

[4]. We also relate these interactions to themechanism of anticancer activity and to structure-activity relationships.

In addition, we find that the interactions between these complexes and DNA show close similarities to those of

covalent polycyclic aromatic carcinogens [5]. It is the first time that the anticancer mechanism and carcinogenesis

mechanism are tied together based on the above researches..

References

1. Liu, H. -K., Sadler, P. J., (2011). "Interactions of Metallodrugs with DNA in NMR of Biomolecules: Towards Mechanistic Systems Biology". I. Bertini, K. McGreevy (Ed), Wiley VCH., 16, 283.

2. Liu, H. -K., Burners-Price, S, Sadler, P. J.,* Angew. Chem., Int. Ed., 2006, 45, 8153.

3. Liu, H. -K., Sadler, P. J.,* Chem. Eur. J., 2006, 12, 6151.

4. Liu, H. -K.*, Sadler, P. J.,* Chem. Sci. 1 (2010), 258.

5. Liu, H. -K.,* Sadler, P. J.*, Acc. Chem. Res., 2011, 44, 349.


44


Towards the development of new copper compounds for the treatment of cancer: Study of the cytotoxic activity of [Cu(L-dipeptide)(1, 10-o-phenantroline)] complexes

Iglesias, S.1; Noble, C.1; Kramer, G.2; González, R.2; Torre, M.H.1; Kremer, E.1; Facchin, G.1 1Cátedra de Química Inorgánica, Facultad de Química, UdelaR, Montevideo, Uruguay

2Departamento de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, Montevideo, Uruguay

[email protected]

Coordination compounds of essential metals are being explored as new antitumor species, in the search of drugs

with expanded activity and/or reduced side effects. To date, several Cu(II) compounds have shown promising

antitumor activities in cellular lines, some of them are being studied in vivo. Their mechanism of action is not

completely understood; however, copper and its complexes, present the ability to participate in redox reactions.

This event generates reactive oxygen species (ROS) that are toxic to cancerous cells. In addition, many of the

complexes present the ability to intercalate DNA, interfering with DNA replication of dividing cells, which may also

be part of their mechanism of antitumor action.

We have synthesized several new [Cu(L-dipeptide)(1,10-phenanthroline)] compounds. In solid state they present a

pyramidal geometry, where the Cu(II) ion is coordinated both to the phenanthroline, through its nitrogen atoms, and

to the dipeptide, through the amine and the amide nitrogen atoms and the carboxylic oxygen (Figure). They are

stable in aqueous solution, where its coordination is maintained. This work focuses on the study of the lipophilicity

and antitumor activity of [Cu(L- dipeptide)(1,10-phenanthroline)] (were L-dipeptide= phe-phe, phe-val, phe-ala, ala-

phe, gly-val, ala- gly). The cancer cell lines utilized were: HeLa (human cervical cancer), NMU (rat breast cancer)

and 4T1 (mouse breast cancer). The lipophilicity, depends on the dipeptide, being the [Cu(L-Phe- Phe)(1,10-

phenanthroline)] complex the most lipophilic. The estimated IC50 varied from 1 to 30 µM in these tumor cells, being

the complexes containing the dipeptides phe-val, phe-ala and ala-phe the ones that showed best antitumor activity

in vitro. Therefore, complexes [Cu(L-dipeptide)(1,10- phenanthroline)] with dipeptides phe-val, phe-ala and ala-phe,

may be the candidates to test its antitumor activity in animal models of breast cancer.


45


Synthesis, characterization and anti-malarial activity of a binuclear copper(II) acetate-quinine complex

Pastrana Alta, R.Y.1; Aguilar, V.H.1; Katzin, A.M.2; Espósito, B.P.1; Valderrama Negron, A.C.3 1Instituto de

Química, Universidade de São Paulo, São Paulo, Brasil

2Instituto de Biociencias, Universidade de São Paulo, São Paulo, Brasil

3Facultad de Ciencias, Universidad Nacional de Ingeniería, Lima, Perú

[email protected]

Malaria is one of the tropical neglected disease whose therapy may benefit from a bioinorganic chemistry

approach.

In this work, we prepared a binuclear copper(II) derivative of the type [Cu(O2CCH3)(µ-Qn)]2 (Qn = quinine). Its

structure was resolved by X-ray diffraction and showed two copper metal centers each surrounded by three oxygen

atoms and one nitrogen atom (CuNO3). The complex has been further characterized by thermogravimetric and

spectroscopic (UV- Vis, IR and EPR) analysis, as well as magnetic measurements. The complex present geometric

and electronic similarities with Type 3 copper centers of the multicopper oxidases and hemocyanin/tyrosinase.

Quinine and acetate coordinate to Cu as a zwitterionic bidentade ligand (O,N) and monodentade ligand (O),

respectively.

The antimalarial activity of the complex and the starting ligand was evaluated in vitro against bloodstream schizonts

of Plasmodium falciparum. The complex (IC50 = 14.6 nM) was more active than quinine (IC50 = 60.15 nM). Both

present superior activity in comparison with other reported antimalarial complexes [1].

References

[1] Nikhil H. Gokhale a, Subhash B. Padhye, David C. Billington,Daniel L. Rathbone, Simon L. Croft, Howard D. Kendrick, Christopher E. Anson, Annie K. Powell. Synthesis and characterization of copper(II) complexes of pyridine-2-carboxamidrazones as potent antimalarial agents. Inorganica Chimica Acta 2003;349:23-/29.


46


Rhenium and technetium complexes with a dithiocarbazate ligand as potential radiopharmaceuticals

de Araujo Fernandes, A.G.1; Lafratta, A.E.2; Portela Luz, C.2; Navarro Marques, F.L.2; Ulrich, A.3; Deflon, V.M.4 1Departamento de Ciências Exatas e Tecnológicas - DCET, Universidade Estadual de Santa Cruz - UESC, Ilhéus, Brazil

2Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brasil

3Institute of Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany

4Instituto de Química de São Carlos, Universidade de São Paulo, São Paulo, Brazil

[email protected]

Introduction: 99mTc is a leading radionuclide in nuclear medicine, being used in diagnostic, while 186Re and 188Re

have a potential usage in radiotherapy. The compound 5-hydroxy-3-methyl-5-phenyl-pyrazoline-1-(S-

benzyldithiocarbazate) (H2-BDTC) consist on a versatile free ligand, being able to coordinate mono-, bi- or

tridentate as well as mono- or dianionically. This work presents rhenium and technetium complexes with H2-BDTC

mono- and dianions as ligands, in different metal:ligand stoichiometric proportions (1:1 and 1:2) and coordination

modes (bi- and tridentate).

Material and methods: The distorted octahedral complexes [ReO(BDTC)(H-BDTC)] and [99TcOCl(BDTC)] were

prepared by reacting [ReOCl3(PPh3)2] and (NBu4)[99TcOCl4] with H2-BDTC, respectively, in

dichloromethane/methanol. The complexes were fully characterized by standard methods and crystal structure

determinations.

The 99mTc-BDTC complex was obtained by addition of 0.3 mL (1mg/mL – H2-BDTC/DMSO) to 0.3 mL of

Na99mTcO4 solution (370 MBq) followed by 20 µL of acid solution of Sn2+ (0.25 mg/mL), and 0.640 mL of 0.1 N

phosphate buffer (pH=7.4). Labeling efficiency was assessed by W3MM paper chromatography using acetone or

saline, and by HPLC [C18 (4,6x250 mm) acetone 1 mL/min] using [ReO(BDTC)(H-BDTC)] as standard.

Electrophoresis was evaluated using W3MM paper and PBS (50 mM, pH 7.4):methanol (1:1), at 275V, partition

coefficient (LogP) was assessed by standard procedure.

Results: Labeling efficiency was over 95 %. The compound showed charge zero and logP = 1.36. HPLC analysis

gave a compound retained at 2.1 min (100%), but was being converted to another compound with retention time of

12.8 min (88% at 12h), closer to 11.7 min to[ReO(BDTC)(H-BDTC)2].

Conclusion: Labeling process of H2-BDTC with technetium is simple and in high yield. The compounds, analyzed

just after preparation, showed neutral and lipophilic characteristics suggesting its use for radiopharmaceutical brain

studies.

Acknowledgements: The authors thank to FAPESP, CNPq and CAPES for the financial support.


47


PTA (1, 3, 5-triaza-7-phosphaadamantane) as co-ligand for metal complexes bearing anti T. cruzi activity

Cipriani, M.1; Toloza, J.2; Curbelo, E.1; Olea Azar, C.2; Maya, J.D.3; Gambino, D.1; Otero, L.1 1Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay

2Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Santiago, Chile

3Programa de Farmacología Molecular y Clínica, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). Being originally an

autochthonous Latin American endemic disease, it is becoming a global health problem because of immigration. In

the search of an effective and safe chemotherapy for this incurable illness, we have been working in the rational

design of metal complexes with active ligands against T. cruzi in order to improve their activity and pharmacological

properties. Different co-ligands have also been included in the new species with the aim of modulating their

physicochemical properties. In particular, PTA (1,3,5-triaza-7-phosphaadamantane) had been previously included

in metal complexes bearing antitumor activity leading to an improvement in their aqueous solubility and to a pH

dependent DNA interaction.

In this work, we report the development of palladium and platinum complexes with bioactive 5-nitrofuryl containing

thiosemicarbazones (L) and PTA as ligands. Complexes of the formula [MCl(PTA)L] with M = Pd or Pt were

synthesized and characterized by elemental analysis, conductivity measurements and 1H NMR and infrared

spectroscopies. Their activity in vitro against the trypomastigote form of T. cruzi. was evaluated. All complexes

resulted more active than the reference drug nifurtimox and than the previously obtained Ru-PTA-L complexes.

Lipophilicity of the obtained complexes was studied by reverse phase TLC and its relation to obtained biological

activity was stated. In addition, being DNA a potential target for PTA containing metal complexes, the ability of the

obtained compound to interact with this macromolecule was studied. The inclusion of co-ligands that confer desired

properties to metal complexes seems to be a valid strategy to rationally design potential antiparasitic metal

compounds.


48


Redox non-innocent thioether ligands and their copper compexes

Castillo, I.1; Martínez-Alanis, P.1; Sánchez Eguía, B.1; Ugalde-Saldívar, V.2; Aullón, G.3 1Instituto de Química, Universidad Nacional Autónoma de México, México DFMéxico

2Facultad de Química, División de Estudios de Posgrado, Universidad Nacional Autónoma de México, México DF, México

3Departament de Quimica Inorgànica, Universitat de Barcelona, Barcelona, Spain

[email protected]

The presence of thioether ligands in the active sites of copper-containing metalloenzymes has resulted in a

growing number of reports involving these sulfur-based donors. Intriguingly, methionine residues have also been

identified in several types of copper monooxygenases such as Dopamine-β-monooxygenase (DβM), although their

role has not been firmly established. This raises the need for Cu+ complexes with thioether-rich coordination

environments; the known examples usually get oxidized to Cu2+ analogues, but the coordination environment

provided by tripodal benzimidazolylamino-thioether ligands results in a HOMO level with predominant arylthioether

character. A combined experimental and DFT study demonstrates that oxidation of such complexes occurs at the

thioether groups, giving rise to cascade ligand decomposition reactions initiated by sulfur-based radical cations.

The implications of these results for bioinorganic systems will be discussed.


49

Abstract code: 064 AREA: MIEH ORAL

Determination of Cd in urine: validation of a routine methodology

Alvarez, C.; Pizzorno, P.; Pereira, N.; Mañay, N. Cátedra de Toxicología - DEC, Facultad de Química, Montevideo, Uruguay

[email protected]

Cadmium toxicity is well known and one of its main sources of exposure for general population is tobacco smoke.

According to the International Agency for Research on Cancer (IARC) cadmium and cadmium compounds are

carcinogenic to humans (Group 1). Therefore standardized protocols, validated and reliable for monitoring this

metal in humans are needed.

Urinary cadmium (U-Cd) has been shown to accurately reflect the amount of cadmium in the body, whereas blood

cadmium (B-Cd) shows recent exposure to the metal [1]. Reference values for U-Cd in general population are 0-

1.4 µg L-1 [2].

A routine methodology for Cd determination in urine is proposed and validated.

The method is based in the extraction of Cd from urine with diethylammonium diethyldithiocarbamate (DDDC) /

Triton X-100 / 4-methylpentan-2-one (methyl isobutyl ketone, MIBK), and its subsequent determination by

electrothermal atomic absorption spectrometry (ET-AAS) at 228,8 nm.

Urine samples were stored at -20ºC until analysis. For the extraction, 1 mL of DDDC 0.2% (w/v), Triton X-100 1%

(w/v) in MIBK were added to 2.5 mL of urine. The mixture was shaked for 5 minutes and then centrifuged for 5

minutes at 3500 rpm. The supernatant was introduced into the graphite furnace to perform the Cd determination.

The figures of merit were: LOD: 0.06 µg L-1 (3s), LOQ: 0.19 µg L-1 (10s), linearity: up to 5 µg L-1 (r2>0.99), precision:

RSD%>5 (n=5), recovery: 108% (spiked samples, n=5).

The proposed methodology was adequate to the purpose and can be implemented as a routine analysis of Cd in

urine.

References

1) Agency for Toxic Substances and Disease Registry (ATSDR) ToxFAQs™ for Cadmium. http://www.atsdr.cdc.gov/toxfaqs/tf.asp?id=47&tid=15. Accessed November, 20, 2012.

2) Repetto, M.R. y Repetto, M. Tabla de concentraciones de xenobióticos en fluidos biológicos humanos como referencia para el diagnóstico toxicológico (actualización 2007). http://www.esmed.com.ar/download/postgrado/medicina_legal/criminalistica/toxicologia_de_laboratorio_17-10-2009/modulo_1_tabla_valores_ref.pdf. Accessed November 20, 2012


50


Lung oxidative metabolism after an acute exposure to transition metals present in ambient particulate matter

Magnani, N.1; Marchini, T.1; Mebert, A.1; Tasat, D.2; Desimone, M.1; Diaz, L.1; Alvarez, S.1; Evelson, P.1 1Universidad de Buenos Aires, Buenos Aires, Argentina

2Universidad Nacional de San Martín, Buenos Aires, Argentina

[email protected]

Transition metals present in particulate matter (PM) could alter lung oxidative metabolism due to their ability to

participate in Fenton reactions thus increasing reactive oxygen species production. The aim of this work was to

study lung oxidative metabolism after an acute exposure to Residual Oil Fly Ashes (ROFA) and transition metals

present in PM. Mice were intranasally instilled with a ROFA suspension (1.0 mg/kg body weight) or silica

nanoparticles (NP) containing Ni or Cr adsorbed to its surface (0.01-1.00 mg Ni/kg body weight). Lung samples

were analysed 1 h after instillation. Tissue O2 consumption, NADPH oxidase (Nox) activity and TBARS content

were evaluated after PM and NP exposure. Mitochondrial respiration, H2O2 and ATP production rates,

mitochondrial membrane potential and oxidative damage markers were assessed in isolated mitochondria after

ROFA treatment.ROFA exposure was found to be associated with 61% increased tissue O2 consumption, a 30%

increase in Nox activity, and a 33% increased mitochondrial active respiration (state 3). During state 3, decreased

mitochondrial membrane potential and a 53% decreased ATP production rate were observed. No changes were

found in either H2O2 production rate or oxidative damage markers in isolated mitochondria. Regarding NP

exposure, tissue O2 consumption, Nox activity and TBARS were found significantly increased in every Ni

concentration assessed, while NP coated with Cr only showed significantly increased O2 consumption and TBARS

content at 0.05 mg/kg. After ROFA exposure, increased tissue O2 consumption may account for an augmented Nox

activity and mitochondrial respiration. The mitochondrial function modifications observed may prevent oxidative

damage within the organelle. Transition metals like Ni or Cr present in PM seem to be important components

involved in the altered lung oxidative metabolism after ROFA exposure. These findings provide new insights to the

understanding of lung oxidative damage mechanisms triggered by metals present in environmental PM.


51


The exposure to an environmental particulate matter surrogate rich in transition metals impairs cardiac mitochondrial function and contractile reserve

Marchini, T.1; Magnani, N.1; D’Annunzio, V.1; Tasat, D.2; Gelpi, R.J.1; Alvarez, S.1; Evelson, P.1 1Universidad de Buenos Aires, Buenos Aires, Argentina

2Universidad Nacional de San Martín, Buenos Aires, Argentina

[email protected]

The exposure to environmental particulate matter (PM) is associated with increased cardiovascular morbidity and

mortality. Transition metal content of PM is suggested to play a predominant role, owing to their ability to drive

Fenton- like reactions leading to oxidative metabolism alterations. A byproduct of fossil fuel combustion known as

residual oil fly ashes (ROFA) is particularly rich in iron, nickel and vanadium, and is frequently used as a PM

surrogate to evaluate the contribution of transition metals on PM biological effects. To address this hypothesis, with

focus on heart mitochondrial and cardiac function, an in vivo animal model of acute exposure to ROFA was used.

Female Swiss mice weighting 25 g were intranasally instilled with a ROFA suspension (1 mg/kg) and hearts were

removed after 3 hours. Tissue and isolated mitochondrial oxygen consumption was followed polarographically with

a Clark-type oxygen electrode. Mitochondrial membrane potential was evaluated by flow cytometry. Mitochondrial

ATP production rate was determined by a chemiluminescent assay. Cardiac contractile reserve was evaluated in

isolated perfused hearts at constant flow, as left ventricular developed pressure before and after a β-adrenergic

stimulus with isoproterenol.

Tissue oxygen consumption was significantly decreased by 38% in ROFA-exposed mice (control: 1180±70 ng-at

O/min g tissue, p 0.001). Likewise, mitochondrial respiration was decreased by 30% in state 4 (control: 87±5 ng-at

O/min mg prot, p 0.05) and by 24% in state 3 (control: 240±20 ng-at O/min mg prot, p 0.01). These findings were

associated with significant mitochondrial depolarization and deficient ATP production. When contractile reserve

was evaluated, basal contractility was not modified (control: 75±5 mmHg). However, isolated perfused hearts failed

to properly respond to isoproterenol in ROFA-exposed mice. The present results show an impaired mitochondrial

function associated with deficient cardiac contractile reserve, and may provide new insights to the mechanisms of

the adverse cardiovascular effects triggered by PM exposure.


52


Exposure to copper in a community chronically exposed to mining waste, Chile

Cortes, S.; Molina, L. Departamento de Salud Pública, Pontificia Universidad Catolica de Chile, Santiago, Chile

[email protected]

Background: North of Chile has some waste mining’s deposits. Chañaral is one the most important due their high

environmental impact in the bay. Environmental studies show the principal metal associated is copper. We had

measured urinary copper in 2009 by ICP-MS at INTA (Nutrition and Food Technology Institute, University of Chile).

The urinary levels varied between 4,4 – 107,4 µg/L, assuming this group was exposed chronically to copper. We

have made measurement of serum copper and ceruloplasmin to establish the real exposure to copper in this

population.

Methodology: we interviewed 167 adults volunteers from our first study on exposure to metals (2009). In 2011

they gave blood sample in fast to measure in serum copper and ceruloplasmin. Ceruloplasmin was made by

enzymatic methods while copper by atomic spectrophotometry at INTA.

Results: our preliminary results shows:

Urinary copper µg/L1 Serum copper µg/L Serum ceruloplasmin (%)

N 204 129 129

Medium ± DE 20,2 ± 11,5 85,2 ± 24,9 37,8 ± 13,1

Mínimum 4,4 38,4 14,7

Máximum 107,4 206,9 97,0

Percentile 95 41,9 115,7 52,5

References value 13,02 70 – 1503 26 - 33 %4

It is showed that exist urinary excretion of copper, according high levels in the waste. Medium levels of biomarkers

of internal function were inside normal values. Serum level of copper and ceruloplasmin showed maximum level

above the normality.

Conclusions: It is not possible to establish this population has a normal excretory pattern. Could be necessary to

collect more data to define exposure (in air, water and food). This information together with this will estimate about

deleterious effects on health.

Acknowledgements: Thank to Profesor Miguel Arredondo.

References

1) Universidad de Chile. Cortes S. Thesis Doctoral 2009.

2) J Trace Elem Med Biol. 2006;20(4):253-62.

3) Biol Trace Elem Res. 2008; 123(1-3): 261-269.

4) Anal Biochem. 1960; 3: 452-456.


53

Abstract code: 055 AREA: NATME ORAL

Chemical speciation of phytate at gastrointestinal level: nutritional quality of vegetable-based diets

Veiga, N.; Torres, J.; Kremer, C.

Departamento Estrella Campos, Facultad de Química, Universidad de la República, Montevideo, Uruguay [email protected]

Myo-inositol phosphates are essential biomolecules in all eukaryotic cells. Among them, phytate (Figure 1, L12-) is

largely abundant in vegetable-based products and it possesses various beneficial effects in health. Conversely, it

has been categorized as an antinutrient, a precipitating agent which reduces the absorption of Fe, Zn and Ca,

causing a global nutritional problem. This dichotomy has raised a debate1, and chemical quantitative data are still

not available to give an answer to this problem.

The interaction of phytate with metal ions was studied by potentiometry. Besides, the solid metal phytates were

synthesized and fully characterized. Solubility measurements were also carried out. These data and all the

nutritional and physiological information2,3 were included in a mathematical model, allowing the calculation of the

gastrointestinal speciation of phytate for each tested intake.

Soluble mononuclear ([Mn+(HxL)](12-n-x)-) and polynuclear ([Mx(HyL)]) metal complexes were detected. Under metal

ion excess conditions, scarcely soluble polynuclear species (Zn5(H2L)•14H2O and Fe4L•22H2O) were obtained. The

resulting model showed nutritional trends that are in agreement with those already reported. In the duodenum,

phytate is mostly precipitated with magnesium and calcium. Phytate reduces zinc and iron bioavailability by

disturbing their adsorption and solution equilibria. Among all, the citrate and glucosamine are the most important

solubilizant agents at gastrointestinal level.

In sum, a large gastrointestinal model was generated. It was successfully applied to predict nutritional tendencies in

the bioavailability of essential metal ions. The chemical information can be used to improve the nutritional quality of

vegetable-based diets.

Aknowledgments: The authors wish to thank ANII for financial support.

References

1. Harland, B. F., Morris, E. R. Phytate: A good or a bad food component? Nutrition Research; 1995; 15; 733-754.

2. Diem, K., Lentner, C. Tablas Científicas Documenta Geigy. Barcelona, España: Ciba-Geigy, 1975.

3. Tablas de composición de alimentos. El pequeño "Souci-Fachmann-Kraut". Zaragoza, España: Acribia S.A., 1999.


54

Abstract code: 143 AREA: NATME ORAL

Pharmaconutrition with selenium in the critically ill

Manzanares,W.; Galusso, F.; Tacchini, R.; Facchin, G.; Torre, M.H.; Biestro, A.; Hardy, G. [email protected]

In critically ill patients, low plasma selenium (Se) levels are associated with systemic inflammatory response

syndrome (SIRS) and multiple organ failure. In Uruguay, we have found that Se and glutathione peroxidase-3

(GPX-3) are significantly decreased in SIRS patients. Furthermore, we demonstrated that plasma Se has a

relatively good predictive value for intensive care unit (ICU) mortality (P= 0.034). The Se cut-off value of 60 µg/L

and the GPx-3 cut-off value of 0.5 U/mL have high specificity for severity of critical illness.

The optimum safe dose and method of administration remains controversial but the best clinical outcomes in SIRS

have been reported from very high Se doses, administered first by bolus followed by continuous infusion. Selenite

is postulated to have a biphasic action; initially as a pro-oxidant and then as an antioxidant. A loading dose given

as a bolus could have a direct reversible inhibition of NF-κB binding to DNA controlling gene expression and thus

down-regulating the synthesis of pro-inflammatory cytokines. In 20 critically ill SIRS patients we performed a dosing

study comparing the pharmacokinetic profile of two high doses of selenite (as selenious acid). Data analysis

showed that the concentration/time curves for Se overlapped. However the pharmacodynamic profile of GPx-3

showed that optimal GPx-3 levels were only achieved with the higher dose of selenite (1600µg/d Se). In both

groups, GPx –3 decreased after day 7 of supplementation. This suggests that GPx-3 inhibition or saturation occurs

as a result of insufficient synthesis of precursors such as hydrogen selenide or selenocysteine, and/or limitation of

glutathione synthesis.

Finally, in a phase II randomized we have investigated high-dose Se, administered first as a bolus (2,000 µg over 2

h), and thereafter 1,600 µg/day Se. After 10 days, ICU patients exhibited less organ dysfunction (P = 0.0001) and

the incidence of hospital-acquired pneumonia was lower (P= 0.03). Additionally, Se monotherapy was not

associated with a higher incidence of adverse events during 28 days follow-up.


55

Abstract code: 008 AREA: RMNM ORAL

Influence of pretreatment with caspofungin in the early diagnosis of fungal infections with 99mTc- tricarbonyl-caspofungin complex

Fernández, L.1; Vilche, M.2; Reyes, L.3; Terán, M.4; Rey, A.5

Departamento Estrella Campos, Facultad de Química, Montevideo, Uruguay [email protected]

Opportunistic fungal infections occur primarily in immunesupressed hosts, the development of fast and accurate

diagnostic techniques is necessary for appropriate treatment. The aim of this work was the synthesis,

physicochemical and biological evaluation of 99mTc-tricarbonyl-Caspofungin as potential diagnostic agent. The

complex was synthesized using Tc-tricarbonyl precursor and substitution with ecaspofungin (Merck).

Physicochemical studies included stability over time, lipophilicity of the complex (coefficient octanol/water), % of

binding to yeast, challenge against cysteine and histidine, and plasmatic protein binding. Biodistributions and

scintigraphic images were performed in CD1 female mice 20 ± 5 g (n = 3/group). Before the administration of the

complex, animals were immunosuppressed with cyclophosphamide (6mg inicial/2mg maintainment) and divided

into 3 groups: Group 1 was inoculated into the right hind leg with Candida albicans (9x108UFC). Group 2 was

inoculated with sterile water. Group 3 control was immunosuppressed without infection. Groups 1 and 3 received

pre-treatment with caspofungin at therapeutic concentration (1mg/kg) and Group 2 only saline to match stress

conditions. To verify the efficacy of immunosuppression, total count of viable white cells in the spleens was

determined in 3 groups of animals (normal as control and immunosuppressed with and without infection). The

complex was obtained with radiochemical purity higher than 95%, was stable until 6 hours post labelling. The Log P

was 0.56, suitable for passage through biological membranes. Binding to yeast was 11%. Stability in plasma was 4

hours post incubation. The complex retained over 90% radiochemical purity when challenged against cysteine and

histidine and plasma protein binding ranged from 65% to 75%. The biological evaluation showed that there is clear

differentiation of tissue target/nontarget = 3.4 and that pretreatment with caspofungin does not compete with the

radiolabeled compound at the infection site, so it could be used for detection mycosis in these conditions.

Acknowledgements: Merck, PEDECIBA, ANII.


56


Development and evaluation of transition metal complexes with nitroimidazole ligands with potential application in Nuclear Medicine

Fernández, S.1; Giglio, J.2; Dematteis, S.3; Cerecetto, H.4; Rey, A.1 1Cátedra de Radioquímica, Facultad de Química, UdelaR, Montevideo, Uruguay

2Centro Uruguayo de Imagenología Molecular, Montevideo, Uruguay

3Cátedra de Inmunología, Facultad de Química, UdelaR, Montevideo, Uruguay

4Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, UdelaR,

Montevideo, Uruguay [email protected]

Tumour perfusion and oxygenation status are important factors for poor treatment outcome after radio and

chemotherapy. Herein, we present the development of 99mTc and 68Ga complexes bearing the 5-nitroimidazol-1-yl

moiety as potential radiopharmaceutical for imaging tumour hypoxia in Nuclear Medicine. 5-Nitroimidazol-1-yl

moiety has bioreductive capacity since it is irreversibly reduced in hypoxic tissue to metabolites that are entrapped

within the cells.

All ligands were synthesized using Metronidazole®, a pharmaceutical containing the pharmacophore, as starting

reagent, and introducing an adequate chelator. Identity of final products was confirmed by IR, NMR spectroscopy

and mass spectrometry.

Labelling with 99mTc was successfully performed through the formation of 99mTc(I)-tricarbonyl, 99mTc(V)-nitride and 99mTc-“4+1” complexes. 68Ga-labelling was achieved by the formation of the 68Ga(III)-complex with the chelator

DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) previously coupled to the 5-nitroimidazol-1-yl

moiety. Radiochemical purity was above 90% and compounds were stable in labelling milieu and human plasma

for up to 2-4 hours. Binding to plasma protein (studied by molecular exclusion) was between 2 and 85% and

lipophilicity, determined as Log of the partition coefficient octanol/phosphate buffer pH 7,4 ranged between -3,3 to

1,5.

Uptake in HCT-15 cells both in normoxia and hypoxia was assessed. Biodistribution in animals bearing induced

3LL Lewis murine lung carcinoma was studied. 68Ga-complexes and some of 99mTc-complexes showed high uptake

in hypoxic cells in vitro and a very favourable biodistribution profile in mice bearing induced tumours. The highest

hypoxia/normoxia ratios were achieved for 68Ga-complexes: 3,27±0,03 and 2,03±0,02. In vivo, tumour uptake was

similar for all complexes (1.0% per gram) but biodistribution profile showed significant differences. Selective uptake

and retention in tumour together with favourable tumour/muscle ratio (6,8±1,8 for the best complex at 2 h) make

some of these compounds promising candidates for further evaluation as potential hypoxia imaging agents.

Acknowledgements: ANII, Pedeciba-Química, Gramón-Bagó S.A., Covidien, M. Moreno, L. Reyes.


57


Development of a potential 99mTc-radiopharmaceutical for estrogen receptors imaging

Tejería, E.1; Cerecetto, H.2; Fernández, S.1; Giglio, J.1; Rey, A.1 1Cátedra de Radioquímica, Facultad de Química - Universidad de la República, Montevideo, Uruguay

2Grupo de Química Medicinal, Facultad de Química- Facultad de Ciencias - Universidad de la República, Montevideo, Uruguay

[email protected]

Breast cancer is the main type of neoplasia in women and adequate treatment requires assessment of the

presence of estrogen receptors. With the objective to develop a potential radiopharmaceutical for estrogen

receptors imaging we have prepared and evaluated an ethinylestradiol derivative labelled with 99mTc.

Ethinylestradiol was derivatized by the so-called “click chemistry”, by reaction (50 µL of a 1x10-2 M solution) with N-

Boc-protected azidoalanine (65µL of a 1x10-2 M solution) for 60 minutes at 75ºC. Deprotection was achieved by

adding 1 µL of trifluoroacetic acid for 60 minutes at 75ºC.

99mTc- labelling was performed in 2 steps: preparation of fac[99mTc(CO)3(H2O)3] by reduction of 99mTc- pertechnetate

(40-50 mCi, 1.0 mL) with sodium borohydride (7.0 mg) in CO(g) atmosphere at 70 °C for 20 minutes; substitution

by mixing 0.5 mL of neutralized precursor with the derivatized ligand and incubation for 30 minutes at 75ºC.

Fig. 1 - Proposed structure of 99mTc-complex

Radiochemical purity (RP) was controlled by reverse phase HPLC. The precursor showed a retention time (rt) of 4

min and a RP > 90 %. The final complex had a rt of 20.3 min and a RP > 72 % which remained unchanged for at

least 6 hours. The HPLC purified complex was stable in human plasma at 37°C and when incubated with histidine

(100M excess) for at least 2 hours. Lipofilicity expressed as logP (partition coefficient between octanol and

phosphate buffer pH=7.4) was 1.43 ± 0.04. Plasma protein binding was 76.4 ± 0.6 %.

Biodistribution in normal rats at 30, 60 and 120 minutes post-injection showed high liver uptake (40.8 ± 2.4 %), as

expected from a lipophilic compound with high protein binding. Excretion occurred mainly by the hepatobiliary tract.

Uptake in other organs was negligible.

These results are promising for a potential oncological radiopharmaceutical.

Acknowledgements: ANII, Pedeciba-Química, Bayer Schering Pharma AG, Inés Sanz, Sylvia Dematteis.


58


68Gallium and 177Lutetium for diagnosis and therapy of tumors expressing CCK-2 receptors

Trindade, V.1; Reyes, L.2; Vasilskis, E.2; Kreimerman, I.1; Oliver, P.2; Engler, H.3; Balter, H.1 1Radiofarmacia, CUDIM, Montevideo, Uruguay

2Preclínico, CUDIM, Montevideo, Uruguay

3CUDIM, Dirección, Uruguay [email protected]

Cholecystokinin Subtype 2 (CCK-2) receptors are present at high expression levels in medullary thyroid carcinoma

(MTC), as well as neuroendocrine gut tumors, stromal tumors, small cell lung cancer and others. Thus, there is a

high interest in finding CCK-2 analogues suitable for imaging (68Ga, β+, Eγ:511 keV, T1/2:68min) and treatment

(177Lu, Eβ-:497 keV, T1/2:6.7days) of these tumors. Both radiometals form very stable complexes with DOTA

(1,4,7,10- Tetraazacyclododecane-1,4,7,10-tetraacetic acid) which can be conjugated to biomolecules.

Minigastrin (His2-MG11)His-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 is a CCK-2 analog with high uptake in this

kind of tumor.

We optimized radiolabelling with 68Ga and 177Lu of DOTA-His2-MG11, quality control methods and studied in vitro

and in vivo stability as well as biological behavior.

Parameters evaluated included peptide to activity ratio and incubation time. For 177Lu labelling also was evaluated

the addition of gentisic acid to avoid radiolysis.

Yield for 68Ga-DOTA-His2-MG11 was ˃90% for pH 4.0, 4MBq/µg of peptide, in sodium acetate 1.14M, incubation at

90ºC-9min, while for 177Lu-DOTA-His2-MG11 was >94% at pH 4.5, 6.5MBq/µg of peptide, in sodium acetate 0.4M

with gentisic acid 0.24M, incubation at 80ºC-20min.

Higher temperatures or longer incubation times increased the amount of the Met-oxidized species in the

peptidewhich results in loss of receptor affinity.

Biodistribution of 177Lu-DOTA-His2-MG11 was done in normal and in nude mice with tumors induced with AR42J

cells. Normal mice showed a rapid blood clearance, high urinary excretion (92% at 24h), very low renal uptake

(0.4% at 24h) and neglectable uptake by other organs and tissues, as expected for peptides.

Images obtained in a SPECT/PET/CT camera up to 9 days, showed high tumor uptake (tumor/blood:25), confirmed

by autoradiography.

These results are very promising to apply the theragnostic approach in tumors expressing CCK-2 receptors, due to

the possibility to reach a molecularly specific target, both with positrons and beta emitters.


59


Clinical applications of radiometals in diagnostic and treatment

Alonso, O. Centro de Medicina Nuclear, Hospital de Clínicas, Montevideo, Uruguay

Nuclear medicine is a discipline that, applying the tracer principle, acts in the diagnosis and therapy of patients.

Radiopharmaceuticals are then administered and external devices, which generate a planar or tomographic

functional image, detect the emitted radiation. Thus, according to the nature of the radioactive sources and the

equipment involved we can acquire "SPECT" (single photon emission tomography) or "PET" (positron emission

tomography) images.

Currently, the Molecular Imaging (MI) concept has emerged, allowing the evaluation of the body’s biological activity

at the molecular level of organization that underlies the development of different diseases. For this purpose it is

necessary to develop labeled target-specific ligands that can be detected externally either by radioisotopes or by

other means. Although 90% of clinical MI images correspond to PET and SPECT, optical imaging and other

modalities have promising potential. Ligands should be developed taking into account the identification of

molecular targets of clinical relevance.

It is well known that molecular changes occur early in disease development. Genomic alterations are responsible

for changes in protein synthesis determining, for example, an over-expression of protein molecular targets such as

cell membrane receptors. Most neuroendocrine tumors express somatostatin receptors (integrated membrane

proteins). Somatostatin analogues (regulator peptide) may be labeled with either 99mTc (99mTc -HYNIC-Octreotide)

or 68Ga (68Ga- DOTATATE), resulting in PET or SPECT imaging, respectively. Moreover, these molecules can also

be labeled with 90Y or 177Lu, which due to their physical characteristics (high-energy electron emission) lead to a

selective molecular-based therapy. Accordingly, this approach allows diagnosis and therapy based on a single

molecular target.


60


68Ga an emergent radionuclide for molecular imaging

Balter, H. Centro Uruguayo de Imagenologia Molecular (CUDIM), Montevideo - Uruguay

68Ga is a positron emitter with 68 min half life and available from 68Ge/68Ga radionuclide generator system. The

long half life (T1/2 271 days) of 68Ge is attractive to operate the generator over a long period of time, ensuring daily

availability with high yield and radionuclidic purity.

The coordination chemistry of Ga3+ is dominated by its hard acid character. A variety of mono and bifunctional

chelators have been developed which allow the formation of stable 68Ga3+ complexes and convenient coupling to

biomolecules. 68Ga coupling to small biomolecules has the potential to facilitate development of clinically practical

PET based on molecularly specific recognition. In particular, peptides targeting G-protein coupled receptors

overexpressed on human tumour cells have shown preclinically and clinically high and specific tumour uptake.

Radiopharmaceuticals based on 68Ga based somatostatin analogues are attracting interest because of their

significant proven potential for the management of patients suffering from neuroendocrine tumours. Other 68Ga

labelled peptides like 68Ga-DOTA-Minigastin, 68Ga-DOTA-RGD, 68Ga-NOTA-UBI 29-41are promising for the

diagnosis of some cancer types, such as lung, breast and prostate, as well as for infection detection. Small non-

peptide molecules labelled with 68Ga have potential in PET imaging of other indications such as bone disease ie 68Ga-BPPEN, 68Ga-BPPED and 68Ga-BPAMD. Carbon nanoparticles labeled with 68Ga (Gallgas) are also an

interesting approach for lung ventilation studies. In order to ensure safe and efficacious use of 68Ga based PET

agents, standardization of radiopharmaceutical preparation, quality assurance and preclinical trails are of utmost

priority.

68Ga-based peptide tracers offer a superior vehicle for tumor imaging/diagnosis, chemo- and radiotherapy planning

and monitoring as well as pretherapeutical dosimetry for radiotherapy. Furthermore, after the diagnosis and

dosimetry 68Ga might be substituted in the same vector with a therapeutical radionuclide like 177Lu or 90Y.


61


Development of therapeutic radiopharmaceuticals based on 177Lu

Cabral, P. Centro de Investigaciones Nucleares, Facultad de Ciencias, UdelaR, Uruguay

Lutetium-177 (177Lu) belongs to the same chemical group (3A) of the periodic table as yttrium (90Y). Lutetium

chemistry is comparable to that of other trivalent metals such as 111In or 90Y and has been widely investigated, used

in nuclear medicine therapy of different disorders.

177Lu has favorable characteristics that makes it a very good candidate to label peptides and monoclonal antibodies

to use in radionuclide therapy.

Its physical characteristics are: semi-decay period of 6.71 days, negative beta emission with a maximum energy of

497 keV (78% abundance), negative beta energy of 0.134 MeV average with an average range of 2 mm in soft

tissue, gamma photon energy of 208 keV (11% abundance) suitable for in vivo scintigraphic imaging and for

custom dosimetric studies.

Chemically, 177Lu is obtained with a specific activity of 20 Ci / mg or 45 Ci / mg depending on its method of

production. It is produced by direction (n, γ) thermal neutron using two methods: a. natural white or enriched 176Lu

oxide or b. by irradiating a target of 175Yb.

The main radionuclide contaminant is 177mLu and its concentration is about 95 ppm. As trace metal contamination,

commercial products have less than 20µg of Fe / Ci of 177Lu. This impurity (Fe) is the most problematic, it interferes

significantly decreasing yields, as many researchers have experimentally verified.


62

Abstract code: 032 AREA: SBMMBRP ORAL

Biochemical and structural basis of iron-sulfur cluster coordination by trypanosomes glutaredoxins: Implications in protein function

Manta, B.1; Fleitas, L.1; Sturlese, M.2; Pavan, C.2; Bellanda, M.2; Comini, M.A.1 1Lab. Redox Biology of Trypanosomes, Institut Pasteur Montevideo, Montevideo, Uruguay

2Dep. of Chemical Sciences, University of Padova, Padova, Italy

[email protected]

Background: Iron-sulfur clusters (ISCs) are versatile inorganic structures that plays regulatory and/or catalytic

roles as cofactors of several proteins. Glutaredoxins (Grxs) are small proteins belonging to the thioredoxin-fold

family with multiple functions provided by their capacity to act as thiol/disulfide oxidoreductases and as mediators

of ISC biogenesis. African trypanosomes possess five Grxs, two with a dithiol CXXC (2-C-Grxs) and three with a

monothiol CXXS (1-C-Grxs) active site. In this work, we have investigated the ability of trypanosomal Grxs to bind

an ISC as well as the biochemical and structural components implicated.

Methodology: ISC reconstitution assays were performed on recombinant wild-type and mutant versions of three

Trypanosoma brucei and one T. cruzi Grx using a chemo-enzymatic method and different low molecular mass

thiols (LMM-RSH). The apo- and holo-proteins were studied by chromatographic, spectroscopic (absorption and

circular dichroism) and structural approaches (NMR), while the interaction of ISC and LMM-RSH with the apo-

proteins was analyzed by spectroscopic and calorimetric techniques.

Results: All the Grxs studied are able to bind ISC but with different outputs in terms of oligomeric conformation and

stoichiometry. Structural analysis of ISC/LMM-RSH interaction with apo-proteins revealed different binding

mechanisms. Point and deletion mutants contributed to dissect the role of specific aminoacids and regions in

ISC/LMM-RSH coordination/binding. Interestingly, trypanothione (bis-glutathyonylspermidine, the main LMM-RSH

in trypanosomes) forms stable protein-free ISC complexes that could be incorporated to 2-C-Grx1 but not to 1-C-

Grx1. Gene function studies supported different roles for the ISC in these proteins and highlighted the in vivo

indispensability of the holo-1-C-Grx1.

Conclusion: Several structural features of 2-C-Grx1 and 1-C-Grx1 explain the functional specificity of these

proteins in the thiol- and iron-metabolism of trypanosomatids, and led us to propose a new mode of ISC

coordination.


63


Hemin promotes tyrosine nitration in membranes

Bartesaghi, S.1,2,3; Radi, R.1,3 1Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

2Departamento de Educación Médica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

3Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

[email protected]

Hemin is a redox-active bioinorganic complex, normally present as a prosthetic group in heme-proteins. However,

under oxidative stress and inflammatory conditions, it can be released and participate in uncontrolled oxidation

reactions to biomolecules. Indeed, in the presence of oxidants hemin promotes, for example, lipid peroxidation and

protein tyrosine oxidation reactions. In this work, we have assessed the role of hemin in nitration of tyrosine

residues present in lipid bilayers. For this, we have used a model system of membranes consisting in

phosphatidylcholine (PC) liposomes of different fatty-acid composition, in which we have incorporated a

hydrophobic tyrosine analog, N-t-BOC L-tyrosine tert-butyl ester (BTBE).We have observed that in saturated fatty-

acid containing liposomes hemin promoted peroxynitrite-mediated tyrosine nitration and the same was observed

when the peroxyl radical-donor 2, 2´-azobis (2-amidinopropane) hydrochloride (ABAP), was used in the presence

of nitrite (NO2-). These results are consistent with transition metal-catalyzed processes favoring the oxidation of

tyrosine (TyrH) to tyrosyl radical (�Tyr) and the formation of nitrogen dioxide (�NO2) by reactions of hemin as

follows:

hemin Fe3+ + ONOOH →hemin Fe4+ + �NO2 + OH- [1]

hemin Fe4+ + TyrH →hemin Fe3+ + �Tyr + H+ [2]

�Tyr + �NO2 →NO2 Tyr [3]

In unsaturated fatty-acid containing liposomes (e.g.1-palmitoyl, 2-linoleyl-PC) exposed to the same oxidizing

systems, hemin primarily induced lipid peroxidation and through the formation of lipid peroxyl (LOO�) radicals,

secondarily promoted tyrosine oxidation and nitration:

LOO� + TyrH →LOOH + �Tyr [4]

In summary, hemin hydrophobicity favors its penetration into membranes and facilitates tyrosine nitration via two

possible mechanisms, one through a direct metal- catalyzed nitration reaction (Eqs. 1-3) and another that is

connected to the lipid peroxidation process (Eq. 4). Further studies are being carried out in 23 amino acid

transmembrane tyrosine-containing peptides.


64


Reactivity and cytoprotective capacity of the synthetic catalytic antioxidants MnPorphyrins towards peroxynitrite

Carballal, S.1; Valez, V.1; Batinic-Haberle, I.2; Ferrer-Sueta, G.1; Radi, R.1 1Universidad de la República, Facultad de Medicina and Center for Free Radical and Biomedical Research, Montevideo,

Uruguay 2Duke University Medical Center, Department of Radiation Oncology, USA

[email protected]

Peroxynitrite (ONOO–) is a cytotoxic oxidant formed in vivo from the diffusion-controlled reaction between

superoxide (O2–) and nitric oxide (NO) radicals. Peroxynitrite- dependent oxidative damage promotes cell

dysfunction and/or death. In several of these conditions, excess mitochondrial formation of oxidants, including

peroxynitrite, has been related to the pathogenesis of multiple diseases. Thus, a major interest has been generated

in the search for potential synthetic catalytic antioxidants. Metalloporphyrins catalyze numerous redox reactions, in

particular manganese porphyrins (MnPorphyrins) can reach mitochondria and as reported previously can act as

efficient catalytic antioxidants accelerating the reduction of oxygen- and nitrogen reactive species. In adddition,

MnPorphyrins have been shown remarkably protective in different animal models of disease. The reactivity of

MnPorphyrins can be modulated by changes in charge and lipophilicity providing opportunities to adapt redox

activity and subcellular distribution. In this work we have studied a new generation of MnPorphyrins compounds

differing in their substituents where the enhanced property resides in its bioavailability.

We have characterized the reactivity of Mn(III)Porphyrins with ONOO– using stopped- flow, with rate constants

ranging from 1.35 x 105 to 3.5 x 107 M-1 s-1 (pH 7.4, 37 °C). We also extended the kinetic characterization of

Mn(III)Porphyrins reaction with another relevant biological oxidant, the myeloperoxidase-produced hypochlorite,

with similar rate constants. In an effort to provide the links between chemical reactivity and the obtained kinetic

information with action mechanisms in vivo, we performed cytoprotection experiments by exposing vascular

endothelial cells, in the presence of MnPorphyrins (5-50 M), to a nitro-oxidative stress conditions generated by the

peroxynitrite donor SIN-1. We observed an inhibition of cell damage as evaluated by a decrease in protein tyrosine

nitration and a protection in the apoptotic cell death. Thus, these MnPorphyrins serve to attenuate the peroxynitrite-

mediated injury and represent a detoxification mechanism which can also be expanded to another oxidants in

biological systems.


65

Abstract code: 020 AREA: TEMIBSS ORAL

Histopathological effects of silver nanoparticle exposure to chick embryos

Gagnon, Z.E.1; Pavel-Sizemore, I.2; Trivedi, P.3; Isaac, L.4 1School of Science, Marist College, Poughkeepsie, NY, United States

2Chemistry, Wright State University, Dayton, OH, United States

3University Hospital, Cincinnati, OH, United States

4Pathology, St.Francis Hospital, Poughkeepsie, NY, United States

[email protected]

The presence of silver nanoparticles (AgNPs) in industrial and household products has become increasingly

prevalent in recent years. Six-day old fertile-specific pathogen free white leghorn strain chick embryos were

exposed to AgNP concentrations of 15, 30, 60, and 100 ppm via injections into the egg air sac on the 7th and 14th

days of incubation. A modified Creighton method was employed, using AgNO3 and NaBH4 to manufacture AgNPs.

In addition to no injection and deionized water injection (DI) controls, AgNO3 and NaBH4 control treatments were

established having the same concentrations as those used for AgNP synthesis. On the 20th day of incubation,

embryos were sacrificed and the brains, hearts, livers, and tibiotarsi were harvested. Histological analysis and

graphite furnace atomic absorption spectroscopy analysis (GFAAS) were conducted on the collected tissue

samples. Statistical analyses were performed to determine variation in silver absorption levels. Mortality was

experienced in all treatments except for the DI and no injection controls. Analysis revealed correlation between Ag

concentrations in the tissues and pathological changes observed in the histological preparations. No pathological

changes were observed in the liver tissues of embryos exposed to 15 or 30 ppm AgNP concentrations. At the 60

ppm exposure level, fatty vacuoles in the liver cells were evident. Proliferation of hepatocytes, hyperplasia and

increased cellular mitotic activity was also observed in liver samples. Additionally, Von Kossa staining revealed cell

calcification, especially in the brain tissues. At the 100 ppm exposure, apoptotic bodies with nuclear fragmentation

were evident, and silver precipitation was observed in the intercellular spaces of liver tissue. Pathological changes

in AgNP-exposed chick embryonic tissue, especially accelerated cell death, support further experimentation on

possible human health effects. In light of these findings, increasing AgNP exposure to the human population

suggests that environmental risk assessment of this emerging factor should be considered.


66

Abstract code: 063 AREA: TEMIBS ORAL

Blood lead, iron deficiency, IQ and executive functions in Uruguayan first-graders

Barg, G.1; Queirolo, E.1; Mañay, N.2; Roy, A.3; Kordas, K.3 1Facultad de Psicología, Universidad Católica del Uruguay, Montevideo, Uruguay

2Facultad de Química, UdelaR, Montevideo, Uruguay

3Nutritional Sciences, Pennsylvania State University, United States, University Park, PA

[email protected]

Cognition in children with both iron deficiency (ID) and elevated blood lead levels (BLL) is not well studied. In 1st-

grade children (n=150, age 6.4±0.6 y, 56.7% boys) from Montevideo, Uruguay we investigated associations among

BLL≥5µg/dL, serum ferritin (SF) 12µg/L), IQ and two executive function (EF) tasks (Internal/External Dimensional

Shift—theIED shift—and Stockings of Cambridge—the SOC—boh part of the CANTAB testing battery). Mean ± SD

IQ was 92.9±17.0 points. Two of the EF task outcomes were 1) the IED shift errors (mean ± SD 19.2 ±10.5, range

0-36) and 2) initial thinking time on the SOC (mean ± SD 3.1 ± 5.8 sec, 0 – 46.4 sec). Mean SF and BLL were

15.0±14.1 µg/L, 4.7±2.2µg/dL, respectively, with 48.8 and 30.2% of study children having BLL ≥5µg/dL and ID,

respectively, and 13.4% having both conditions. BLL and SF were not correlated. In unadjusted linear regressions,

BLL and SF were not independently associated with IQ or the two EF tasks; neither were BLL ≥5µg/dL nor ID.

However, children with both elevated BLL and ID had lower IQ 11.7 points lower than their ID-only peers (p=0.07).

Preliminary analyses suggest that elevated BLL and ID may result in poor general cognitive function in school

children.


67


Selenium-induced oxidative stress in the model organism zebrafish

Hauser-Davis, R.A.1,2; Ziolli, R.L.3; Zezzi Arruda, M.A.1,2 1Instituto de Química/UNICAMP, Departamento de Química Analítica, Grupo de espectrometria, Preparo de amostras e

Mecanização-GEPAM, Campinas, SP, Brazil 2Instituto Nacional de Ciência e Tecnologia de Bioanalítica/ UNICAMP, Grupo de espectrometria, Preparo de amostras e

Mecanização-GEPAM, Campinas, SP, Brazil 3Universidade Federal do Estado do Rio de Janeiro – UNIRIO, Rio de Janeiro – RJ, Brazil.

[email protected]

Selenium toxicity has been suggested as a result of oxidative damage [1]. The zebrafish is a potentially useful

model organism for the study of selenium metabolism and its role in biology and medicine. Zebrafish were exposed

to Se to analyze effects on fish metabolism. A control and a Se-exposed group (selenium selenite, 1 mg L-1) were

maintained in a static system for 96hs. Brains, livers and gallbladders were removed. Pooled samples were used

for metallothionein and GSH determination: 10 brains, 10 livers and 4 gallbladders from the controls and 10 brains,

7 livers and 8 gallbladders from Se-exposed fish. MT extraction was conducted by heat treatment. Samples were

homogeneized in Tris containing PMSF and β-mercaptoethanol, centrifuged at 20.000 x g for 1 h at 4º C, heated at

70ºC for 10 min and centrifuged again at 20.000 x g for 30 min. Supernatants were treated with HCl/EDTA and

NaCl/ buffered DTNB, and incubated for 30 min. The samples were then centrifuged at 3000 x g for 5 min and the

absorbance was evaluated at 412 nm. MT content was estimated by assuming mol MT = 20 mols GSH. Samples

for GSH determination were homogenized in Na-phosphate buffer, pH 7.0 followed by centrifugation at 11.000 x g.

Absorbance was evaluated at 412 nm. MT in bile is lower than liver, as expected, and MT in Se-exposed bile was

higher than in controls, indicating MT induction and efficient excretion through the bile leading to less MT

accumulation in liver. The brain, with no detoxification route as quick as the liver-gallbladder route, accumulated

significantly higher MT in the Se-exposed group. Se-exposed fish showed significant GSH induction compared to

the controls. Thus, selenium exposure induced significant oxidative stress in this model organism, being of interest

in the understanding and further study of Se metabolism.

References

[1] Misra, S., Niyogi, S. Selenite causes cytotoxicity in rainbow trout (Oncorhynchus mykiss) hepatocytes by inducing oxidative stress. Toxicol In Vitro; 2009; 23; 1249–1258.


68


Evaluation of the intellectual capacity of children in areas which are exposed and unexposed to environmental pollution in the extreme south of Brazil

Dupont-Soares, M.; Muccillo-Baisch, A.L.; Garcia, E.; Baisch, P.R.; Soares, M.C.F.

Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio Grande - FURG, Rio Grande, Brazil [email protected]

The study area, located in the extreme south of Brazil, has been the focus of studies due to problems with

environmental pollution. This study evaluated the intellectual capacity of children living in areas considered

exposed (E) or not exposed (NE) to environmental pollution. 246 students between the ages of 8 years and 11

years 8 months were evaluated. Data was collected between May and June 2010 utilizing a semi-structured

questionnaire to investigate factors associated with compromised intellectual capacity. Intellectual capacity was

evaluated by Raven’s Progressive Matrices Scale. Study areas that were exposed or not exposed were compared

by the chi-squared test and raw and adjusted analyses were carried out using Poisson regression with a robust

error estimate. General occurrence of intellectual impairment was 28.9%, with 22.0% in the non-exposed area and

36.1% in the exposed area (p=0.01). The outcome showed association with: lack of companionship (RP= 2.77;

p<0.00); maternal education less than five years (RP=2.13; p<0.00); mother not Caucasian (RP=1.57; p<0.00);

residence in an exposed area (RP=1.88; p<0.00); nutritional risk/malnutrition (RP=2.83; p<0.00). Results showed a

high occurrence of intellectual impairment and in this study there was a significant difference between the areas.

Further study is needed in order to improve understanding of some results. The town’s priorities should include

measures related to environmental health.

Poster Presentations


70

Abstract code: 036 AREA: AAMMIBBS POSTER

Determination of Cd in soybean and wheat seeds using TS-FF-AAS and preconcentration with cloud point extraction (CPE)

Morales, G.; Knochen, M.; Pistón, M.*

Cátedra de Química Analítica, Facultad de Química, Montevideo, Uruguay [email protected], [email protected]

Antecedents: Cadmium is a toxic element associated with the environment. Seeds can accumulate it, so it is

important to determine the concentration of this element.

Thermospray Flame Furnace (TS-FF) coupled to a flow-injection preconcentration system can be used to enhance

detectability in flame atomic absorption spectrometry (FAAS) providing an alternative to the use of ET-AAS or ICP-

MS.

Methodology: The method is based on the adsorption of a complex of Cd and methyl green, potassium iodide and

Triton X-114 in a minicolumn filled with cotton. The elution was carried out with HNO3. The system consists of a

peristaltic pump, an injection valve and, a nickel tube 10 cm long with 7 holes. The analytical determinations were

carried out by FAAS at 228.8 nm.

The seeds were milled, and 1.8 g of the obtained flour was digested by means of an acidic treatment with a mixture

of HNO3/H2O2. The resultant solution was neutralized with solid NaOH.

Results: The figures of merit were: LD (3s) and LQ (10s): 0.6 and 2.1 µg L-1 respectively, linearity: up to 10 µg L-1

(r2= 0.999), precision: sr(%) = 2.8 (n = 5), sampling frequency: 30 h-1 with a preconcentration factor of 30 compared

with FAAS.

Accuracy was evaluated with the CRM Wheat Flour 1567a (NIST), the recoveries were between 98% and 105 %

(n = 10). There was no evidence of the influence of potential interferences.

Conclusions: This method can be implemented for the control of Cd levels in soybean and wheat seeds.

Acknowledgements: Agencia Nacional de Investigación e Innovación (ANII-2841). Dr. Harald Berndt for providing

the TS-FF-AAS system.


71


Determination of copper in honey by on-line flow-injection cloud-point extraction with atomic absorption spectrometry (AAS) detection

Sixto, A.1; Knochen, M.1; Heinzen, H.2 1Departamento Estrella Campos (DEC), Facultad de Química, Montevideo, Uruguay 2Departamento de Química Orgánica, Facultad de Química, Montevideo, Uruguay

[email protected]

Background: the content of metals in honey is highly variable depending on the botanical and geographical origins

of the floral resources available for bees. Bioaccessibility of Cu from honey depends on different factors, including

physicochemical forms of Cu species and other dietary components present. It has been reported that Cu in honey

is highly bioaccesible. Considering the daily consumption of honey for adults, it can provide approximately 10% of

the recommended daily intake of Cu. Cu content is also an indicator of contamination during handling, processing

and storage. Determination of metals in honey usually involves tedious procedures such as ashing or wet

digestion. Methods based on flow analysis can be advantageous in terms of speed, ease, and possibility of

automation. A system based on the concept of multicommutated flow analysis (MCFA)1 with on-line cloud-point

extraction and AAS detection was developed for the automated determination of Cu in honey.

Methodology: the system scheme was as follows:

The operation of the system was carried out by means of a PC and software compiled in Quick Basic. The sample

was dissolved in water and analysed without further processing.

Results: the calibration curve was linear up to 0.9 mg L-1 (A = 0.0834 C + 0.0137), repeatability (sr (%)) was ≤ 5,

LOD (3σ) was 0.07 mg L-1, LOQ (10σ) was 0.14 mg L-1, the sampling frequency was 20 samples h-1, recovery was

over 80% (3 samples). The content of Cu of the samples analysed was <0.78 mg kg-1.

Conclusions: regarding sampling preparation the method is much simpler than the traditional methods.

References

1- Rocha, F.R.P.; Reis, B.F.; Zagatto, E.A.G.; Lima, J.L.F.C.; Lapa, R.A.S.; Santos, J.L.M. Multiconmutation in flow analysis: concepts, applications and trends. Anal Chim Acta (2002) 468 119-131.


72


Determination of selenium in antioxidant softgels by Continuous Flow-Hydride Generation-Atomic Absorption Spectrometry (CF-HG-AAS)

Barbato, S.1; Mollo, A.1; Knochen, M.1; Viera, S.2; Huertas, R.2 1Analytical Chemistry, School of Chemistry, Montevideo, Uruguay

2Atomic Spectrometry Department of Food and Environment, Technological Laboratory of Uruguay, Montevideo, Uruguay

[email protected]

Background: Selenium is an essential trace element for human health recognized as antioxidant and related with

the reduction of certain types of cancer. As frequently the dietary intake is not sufficient there is a great interest into

develop Se- enriched nutritional supplements prepared based on selenised enriched yeast Saccharomyces

cerevisiae, in which selenium is present mostly as selenomethionine.

Hydride Generation Atomic Absorption Spectrometry (HG-AAS) is a widespread technique for the determination of

selenium. New trends in quality control laboratories aim to automated processes offering a number of advantages

such as low cost, lower detection limits, better precision and high sample throughput. Continuous Flow (CF)

techniques enable to generate the hydride on line by continuous pumping of both reactant and sample through the

manifold.

Methodology: The aim of this work was to develop an automated system for the determination of selenium in

antioxidant softgel. Sample preparation was achieved by acidic-oxidizing wet digestion, both by means of

microwave oven and hot plate. The prepared sample was introduced in the automated manifold and selenium

determined by Atomic Absorption Spectrometry at 196.03 nm.

Results: Both digestion strategies exhibited results differing less than 1%.

Accuracy was assessed by comparison with a reference method (microwave-assisted acid digestion followed by

Inductively-Coupled Plasma Optical-Emission Spectrometry). Results from both methods differed 6 % and the

precision sr(%) ≤ 10, n=10 at 6 levels, fitting the purpose of the analysis.

Conclusion: The CF-HG-AAS system proposed provides an accurate determination of selenium by means of a

simple and low cost manifold suitable for implementing in quality control laboratories.

Acknowledgements: The authors are grateful to Mariela Pistón for useful discussion and Germán Morales for his

collaboration in the early stages of this work.


73


Differential metal binding behavior determined by LC(SEC)-ICP-MS of brain metallothioneins in high- and low- freezing rats used as a genetic model of anxiety

Hauser-Davis, R.A.1; Gomes, V. de C.2; Rocha, R.3; Ziolli, R.4; Saint'Pierre, T.3; Zezzi Arruda, M.A.1; Landeira-Fernandez, J.5

1UNICAMP, Brazil

2Uniformed Services University of the Health Sciences, Department of Psychiatry, USA

3Chemistry Department, PUC-Rio, Brazil

4UNIRIO, Brazil

5PUC-Rio, Brazil

[email protected]

Carioca high- and low- conditioned freezing (CHF and CLF) Wistar rat selected lines represent the most recent

genetic model in the study of anxiety disorders. Metal-binding behavior of brain metallothioneins (MT) by size

exclusion chromatography (SEC) coupled to liquid chromatography (LC) separation and ICP-MS detection was

investigated to verify differences between these groups. Seven CHF, CLF and randomly selected animals, males

and females (n=42), were sacrificed and whole brains removed. Samples were homogeneized in a Tris buffer

containing PMSF and β - mercaptoethanol, centrifuged at 20.000 g for 1 h, heated at 70ºC for 10 min and

centrifuged again at 20.000 g for 30 min. Elemental detection was performed on-line by a NexION 300X Perkin

Elmer ICP-MS coupled to a Shimadzu UV HPLC with a Superdex 75 SEC column. Results showed no differential

metal-binding behavior in males. Differential binding in females was observed, where a Zn and Cd peak presented

retention time between 10 and 15 min, and between 15 and 20 min, respectively, present in the control group but

absent from CHF and CLF females. Comparing males and females, females presented several differential metal-

bound peaks: CHF presented Cd and Pb peaks at 10 min and another Pb peak at ca. 12.5 min, absent in male

CHF; female CLF also presented these same peaks, and an extra Zn peak at 15 min, which was absent in male

CLF. Comparing male CHF and female CLF, and male CLF compared to female CHF both females presented an

extra Cd peak at 10 min, absent from the males. This indicates that female CHF and CLF animals may show

alterations in basic homeostasis, since Zn and Cd are essential trace elements, and that inter-gender differences

are present, with females presenting more metal-binding proteins than males in the central nervous system.


74


Microwave-assisted digestion of milk powder with diluted nitric acid using oxygen as auxiliary reagent

Bizzi, C.A.1; Smanioto Barin, J.2; Nóbrega, J. de A.3; Foster Mesko, M.4; de Azevedo Mello, P.1; Flores, E.M.M.1 1Departamento de Química, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil

2Departamento de Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil

3Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil

4Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, RS, Brazil

[email protected]

The feasibility of using diluted HNO3 solutions under oxygen pressure for decomposition of whole and non-fat milk

powders and whey powder samples has been evaluated. Digestion efficiency was evaluated by determining the

carbon content in solution (digests) and the determination of Ca, Cd, Cu, Fe, K, Mg, Mn, Mo, Na, Pb and Zn was

performed by inductively coupled plasma optical emission spectrometry and Hg by chemical vapor generation

coupled to inductively coupled plasma mass spectrometry. Samples (up to 500 mg) were digested using HNO3

solutions (1 to 14 mol L-1) and the effect of oxygen pressure was evaluated between 2.5 and 20 bar. It was possible

to perform the digestion of 500 mg of milk powder using 2 mol L-1 HNO3 with oxygen pressure ranging from 7.5 to

20 bar with resultant carbon content in digests lower than 1700 mg L-1. Using optimized conditions, less than 0.86

mL of concentrated nitric acid (14 mol L-1) was enough to digest 500 mg of sample. The accuracy was evaluated by

determination of metal concentrations in certified reference materials, which presented an agreement better than

95% (Student t test, P < 0.05) for all the analytes.

Acknowledgements: CNPq, INCT-Analítica, INCT-Bioanalítica, FAPESP, FAPERGS, UFSM


75

Abstract code: 012 AREA: MBD POSTER

Bioactivity of a new polyoxometalate with copper. Anti-tumoral activity in a human osteosarcoma model

Leon, I.E.1; Di Virgilio, A.L.1; Porro, V.2; Egusquiza, G.3; Cabello, C.3; Bollati- Fogolin, M.2; Etcheverry, S.B.1 1Cátedra de Bioquimica Patológica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina

2Unidad de Biologia Celular, Instituto Pasteur, Montevideo, Uruguay

3Centro de Investigación y Desarrollo en Ciencias Aplicadas CINDECA, Universidad Nacional de La Plata, La Plata, Argentina

[email protected]

The polyoxometalates are transition metals clusters of oxide anions, usually d0 species (V(V), Mo(VI) y W (VI))

formed by a self assembly process. In addition, the heteropolyoxometalates (HPOMs) contain one or more

heteroatoms with electrons of valency p-, d - and f. The structural and electronic properties of this type of

compounds that are easy to modify and their characteristics makes them attractive for several applications in

different fields, especially as pharmaceutical drugs. The biomedical importance of the POMs is based principally on

its interaction with proteins, including enzymes and constituents of the cellular systems. The selected

heteropolytungstate belongs to an isomorfic serie [(PW9O34)2M4]10- with M=Cu(II), was synthesized and completely

characterized in our laboratories.

In the frame of a research project devoted to study the potential pharmacological effects of heteropolytungstates

with copper ([(PW9O34)2Cu4]10-), we present herein the results of its biological effects in an osteosarcoma human

model.

The effects on cell proliferation, mitochondria and lysosomal activity and morphology were investigated. Besides,

the mechanisms of action involved in the cytotoxicity were also studied (oxidative stress, level of GSH, GSH/GSSG

ratio and Cell cycle arrest).

[(PW9O34)2Cu4]10- caused inhibition of cell proliferation in the range of 25-100 µM (p<0.01). Besides, similar results

were obtained by MTT and Neutral Red uptake cytotoxicity assays. Complementary, morphological features were

studied by DAPI/ Cell Mask staining and epifluorescence microscopy. These results were in parallel with those of

the viability studies. The compound increased the level of Reactive Oxigen Species (ROS) 350% over basal and

decreased the GSH/GSSG ratio in the same range (p<0.01). The complex arrested the cell cycle in G2 phase at 24

h and 48 h (p<0.05)

Taken together, these results suggest that this compound is potentially a good candidate for future use in

alternative cancer treatments.


76


Vibrational spectra of three maltolato complexes with interesting pharmacological properties

Baran, E.J.; Parajón-Costa, B.S.

Centro de Química Inorgánica (CEQUINOR, CONICET/UNLP), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina

[email protected]

Maltol, 3-hydroxy-3-methyl-4-pyrone, is one of several hydroxypyrones long known for high bioavailability and

favourable toxicity profiles. It has a high affinity for different metal cations and is, therefore, an excellent choice for

the formulation of therapeutic and/or diagnostic metallopharmaceuticals1.

We have prepared three metallic complexes of this ligand, namely with VO2+, Zn(II), and Ga(III) respectively. The

first two show insulino mimetic activity1-3 and the other one is a very promising antitumoral drug4.

The FTIR and Raman spectra of polycrystalline samples of the three complexes were recorded and briefly

discussed on the basis of their crystal structures and by comparison with the spectroscopic behavior of free maltol

and of other, previously investigated, maltolato complexes.

References:

1 Thompson, K.H.; Barta, Ch.A.; Orvig, Ch. Metal complexes of maltol and close analogues in medicinal inorganic chemistry. Chem Soc Rev; 2006; 35; 545-556.

2 Thompson, K.H.; Orvig, Ch. Vanadium: in diabetes: 100 years from phase to phase I. J Inorg Biochem; 2006; 100; 1925-1935.

3 Yoshikawa, Y., Ueda, E., Kawabe, K., Miyake, H., Sakurai, H., Kojima, Y. New insulin-mimetic zinc(II) complexes; bis-maltolato zinc(II) and bis-2- hydroxypyridine-N-oxido zinc(II) with Zn(O4) coordination mode. Chem Lett; 2000; 874-875.

4 Jakupec, M.A., Keppler, B.K. Gallium in cancer treatment. Curr Top Med Chem; 2004; 4; 1575-1583


77


Apoptosis and autophagy in neuroblastoma by copper compounds

Gutiérrez, A.G.1; Vázquez-Aguirre, A.1; Palma-Tirado, L.2; Ruiz-Azuara, L.3; Hernández-Lemus, E.4; Mejía, C.1 1Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, UNAM, Ciudad de México, México

2Unidad de Microscopía, Instituto de Neurobiología, Campus Juriquilla, UNAM, Qurétaro,

3Química Inorgánica y Nuclear, Facultad de Química, UNAM, Ciudad de México, México

4Departamento de Genómica Computacional, Instituto Nacional de Medicina Genómica, Ciudad de México, México

[email protected]

Background: Neuroblastoma is a childhood solid cancer refractory to several anti-carcinogenic agents1.

Casiopeínas® are a group of coordination complexes with a central Copper atom2, designed to be an alternative to

cancer therapy by means of apoptosis activation3. Controversy exists about autophagy may be a caspase- and

apoptosis-independent cell death, or rather, is a self-limited process that protect cells from death4.

Methodology: We determined apoptotic and autophagic molecules by Western blot and electronic microscopy,

and ROS expression by flow cytometry. Finally, all results were analyzed with an integrative computational model.

Results: We found that Cas produce early mitochondrial apoptosis with an increase of cytoplasmic apoptotic

molecules Bcl-2, cytochrome C, and caspase-3, followed by a decrease of Bax and a disruption of the

mitochondrial transmembrane potential; and later autophagic molecules Beclin-1 and LC3-II were expresed. Both

processes appear in ROS presence, concomitantly with a decrease in glutathione levels. The protein regulatory

network for mitochondrial apoptosis and autophagy in NB by effect of Casiopeínas, showed that most molecules

are highly connected, especially as a result of a pro-oxidant environment.


78


Spectroscopical studies of the peroxidase activity of cytoglobin

Nascimento, O.R.1; Ferreira, J.C.2; Icimoto, M.Y.2; Marcondes, M.Y.2; Oliveira, V.2; Nantes, I.L.3 1Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil

2Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, SP, Brazil

3Santo André, SP, Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Brasil

[email protected]

This paper reports spectroscopical analysis by UV-visible absorption, magnetic circular dichroism (MCD) and

electron paramagnetic resonance (EPR) of the structure of a recombinant Cygb and its reactivity with hydrogen

peroxide (HOOH), tert-butylhydroperoxide (t-BuOOH) and cumene hydroperoxide (CuOOH). UV-visible direct EPR

of heme iron. Cygb was converted to the high valence species by peroxides, promoted homolytic scission of the O-

O bond and generated alkoxyl radicals. The secondary methyl and peroxyl radicals resulted respectively from the

β-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen. From kinetic studies

of Cygb with different peroxide concentrations they were determined the catalytic constants kcat/KM for CuOOH

(0.0028 ± 0.0006), t- BuOOH (0.0032 ± 0,0006) and HOOH (0.0061± 0,0010 s-1.µM-1). EPR spectra obtained in the

course of the reactions corroborated the conversion of the hexacoordinated heme iron to compound II. These

results are consistent with the biological role of Cygb grounded on pro-oxidant signaling activity.


79


On the thermal oxidation and denaturation of oxy-HbGp, in presence of SDS, at pH 7.0, monitoring by optical absorption and CD

Carvalho, J.W.P.; Tabak, M.

Departamento de Físico-Química, Instituto de Química de São Carlos-IQSC/Universidade de São Paulo-USP, São Carlos-SP, Brazil

[email protected]

HbGp is an oligomeric protein with a molecular mass of 3.6 MDa, presenting a high oligomeric stability, at pH 7, in

the presence of both surfactant and high temperature. At 20ºC, the oxy-HbGp, in the absence and presence of

SDS, is characterized by absorption spectra with an intense Soret band, at 415 nm, and two Q-bands, at 535 and

575 nm. Increase of temperature promotes the decrease of intensity and shifting of λmax from 415 nm (20ºC) to

411-409 nm (70ºC). The Q-bands also present a decrease in intensity. The appearance of two bands, at 500 and

540 nm, at high temperatures, is observed. SDS concentration, in the range from 0.2 to 0.6 mmol/L, induces

changes on the Soret and Q-bands, already at lower temperatures. Our data indicate the presence in solution of a

mixture of oxy-, met-HbGp and hemichrome, at higher temperatures. Met-HbGp shows a Soret band at 405 nm,

and Q-bands at 500 and 540 nm, while the hemichrome displays a Soret band at 413 nm, and Q-bands at 534 and

564 nm. CD data show similar results, suggesting the oxy-HbGp oligomeric dissociation, in the presence of SDS.

These changes on the iron oxidation and coordination promote a decrease in the unfolding temperature of HbGp

from 56±2 (absence of surfactant) to 33±3ºC (with 0.6 mmol/L of SDS). Thus, our data show clearly that the iron

oxidation state is very important for the oligomeric stability of HbGp.

Aknowledgments: The authors thank FAPESP and CNPq for financial support.


80


Ni(II) binding to 429-460 peptide fragment from human toll-like receptor (hTLR4)

Zoroddu, M.A.1; Peana, M.1; Medici, S.1; Solinas, C.1; Potocki, S.2; Kozlowski, H.2 1Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy

2Department of Chemistry, University of Wroclaw, Wroclaw, Poland

[email protected]

Contact allergy, commonly induced by nickel, is the most frequent cause of contact hypersensitivity in industrialized

countries, with 30% of population being affected. Ni(II) seems to trigger an inflammatory response by activating

human Tool-like-Receptor 4 (hTLR4). Species-specific activation, as in this case, required distinct sequence motifs

that are present in human but not in mouse, a species not sensitive to nickel-induced allergies. The specific region

of human TLR4 responsible for nickel responses could be a sequence containing three histidine residues, H431, and

the non- conserved H456 and H458, localized in the C-terminus. It has been proposed that the imidazole side chains

of the histidine residues H456 and H458 provide a potential binding site for nickel because they were located at an

optimal distance to interact with Ni(II) ions, whereas H431 was further apart. We decided to verify the possibility of

metal binding to FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR sequence, containing the three histidines

supposedly involved in nickel response, in order to study the binding properties of the peptide fragment and on the

thermodynamic stability of its metal complexes. Formation equilibria of Ni(II) complexes have been investigated in

aqueous solution and in a wide pH range. Protonation and complex-formation constants have been

potentiometrically determined; complex-formation models and species stoichiometry have been checked by means

of UV-Vis absorption and CD spectroscopy and investigation through NMR is currently being carried out. The

predominant species for a 1:1 peptide/Ni(II) molar ratio was obtained at physiological pH and showed an effective

binding of the metal to the target sequence.


81


Synthesis, characterization and antibacterial activities of a novel Ag(I) complex with sulfadoxin

Abbehausen, C.; Zanvettor, N.T.; Bergamini, F.R.G.; Lancellotti, M.; Corbi, P.P.

Inorganic Chemistry Department, University of Campinas, Campinas, Brazil [email protected]

The antibacterial action of silver is known since ancient times. Later, the use of silver compounds in therapy was

significantly reduced by the discovery of more efficient and less toxic antibiotics. However, the development of

resistant bacterial strains brought the resurgence of silver based drugs. The most used is silver sulfadiazine, which

was approved for clinical treatment of burns and skin infections[1].

Sulfadoxin (C12H13N4SO4, SFX), as sulfadiazine, is a sulfonamide used in the treatment of malaria in combinatory

therapies. Sulfadoxin increases the activity of pyrimethamine against the malaria protozoa Plasmodium

falciparum[2]. The coordination of sulfadoxin to silver, similarly to the coordination of sulfadiazine to silver, could

generate a new and potent antibiotic. Studies of metal coordination to sulfadoxin were not described previously.

Here, we describe the synthesis, characterization and antibacterial studies of a silver complex with sulfadoxin.

The silver-sulfadoxin (Ag-SFX) complex was prepared by the reaction of an alkaline solution of sulfadoxin with an

aqueous solution of AgNO3. A white solid was formed within one hour of reaction. Elemental analysis led to the

composition AgC12H13N4SO4. Anal. Calc. for AgC12H13N4SO4 (%): C 34.5; H 2.64; N 13.4. Found (%): C 34.3; H

2.52; N 13.0.

The absence of the ν(NH) band at 3240 cm-1 in the infrared spectrum of Ag-SFX in comparison to the free ligand

suggests the coordination of the nitrogen atom of the sulfonamide group to silver. Nitrogen coordination was

confirmed by 13C NMR in the solid state and by DFT studies. An antibiogram assay showed the significant

antibacterial activity of the silver complex over six pathogenic Gram-positive and Gram-negative strains.

References

[1] Bakhtiar, R; Ochiai, Ei-I Pharmacological applications of inorganic complexes Gen Pharmacol 1999, 32, 525-540.

[2] Chulay, JD; Watkins, WM; Sixsmith, DG, Synergistic antimalarial activity of pyrimethamine and sulfadoxine against Plasmodium Falciparumin vitro. Am J Trop Med Hyg 1984, 33, 325-330.


82


A new nytrosil ruthenium compound with some special features

Mondelli, M.1; Carnizello, A.1; Tavares, D.2; Corrêa, R.1; Ellena, J.3; Batista, A.1 1Departamento de Química, Universidade Federal de São Carlos (UFSCar), São Carlos, Brazil

2Laboratório de Mutagênese, Universidade de Franca (UNIFRAN), Franca, Brazil

3Departamento de Física e Informática, Instituto de Física, Universidade de São Paulo (USP), São Carlos, Brazil

[email protected]

NO is a little molecule that in the last twenty five years has extensively been studied for their beneficial effects in

the body. In 1992 was chosen by Science as "molecule of the year" and in 1998 R. Furchgott, F. Murad and L.

Ignarro won the Medicine Nobel Prize "for their discoveries concerning nitric oxide as a signalling molecule in the

cardiovascular system". 4,6-dimethyl-2-mercaptopyrimidine (spymm) has antibacterial, antithyroideal, antitumor,

antiviral and fungicide activity1. A new compound, [RuCl(spymm)2(NO)] (1), was synthesized and characterized by

cyclic voltammetry, molar conductivity, 1H NMR and IR/UV-vis spectroscopies, and its structure was determined by

X-ray analysis. The complex showed an octahedral distorted structure where the nitrogen atoms were trans

between each other, and also the sulfur atoms, in an equatorial plane; whereas the nitrosonium (NO+) was trans

with respect to chloride in the axial plane (Figure 1). 1H NMR spectrum showed two singlets in 7,3 ppm and in 6,7

ppm attributed to the hydrogen of the aromatic ring, and also in 2,5 ppm and 2,3 ppm attributed to hydrogens of

methyl groups; this differences could be explained by evaluating how the intermolecular hydrogen bonds are

affecting the other atoms present in the molecule and in the nearest neighbourhood. In the infrared spectrum was

observed an intense peak at 1856 cm-1, corresponding to νNO. An electrolytic process demonstrated the liberation

of NO+ from the molecule, property that can be useful for medical applications, as for example in photodynamic

therapy2. Besides, IC50 was evaluated in three cell lines V79 (chinese hamster fibroblast), HeLa (human epithelial

carcinoma) and U251 (glioblastoma), showing a promisor result in HeLa (18,70 µM).

References

1) H. Parveen; F. Hayat; A. Salahuddin and A. Azam, “Syntheses, characterization and biological evaluation of novel 6- ferrocenyl-4-aryl-2-substituted pyrimidine derivatives”. Eur J Med Chem, 2010; 45;3497-3503.

2) G. Stochel; A. Wanat; E. Kulís and Z. Stasicka, “Light and metal complexes in medicine”. Coord Chem Rev, 1998; 171; 203-220.

Acknowledgments: CAPES, CNPq, FAPESP.


83


Synthesis, characterization and in vitro cytotoxic activity of the palladium(II) complexes with the naphthaldehyde thiosemicarbazone ligands against various human tumor cell

lines

Hernández Gorritti, W.R.1; Paz Castillo, J.1; Vaisberg, A.2; Manzur, J.3; Spitridonova, E.4; Beyer, L.5 1Facultad de Ingeniería Industrial, Universidad de Lima, Lima, Perú

2Laboratorio de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima,

Perú 3Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, CEDENNA, Santiago, Chile

4Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, CEDENNA, Santiago, Chile

5Fakultät für Chemie und Mineralogie, Universität Leipzig, Leipzig, Germany

[email protected]

It has been reported that the ligands derived from α–(N)-heterocyclic carbaldehyde, benzaldehyde and furaldehyde

thiosemicarbazone, and their metal complexes inhibit tumor cell growth [1-2].

The present work describes the preparation, characterization and antitumor activity of palladium(II) bis-chelates

Pd(L1-3)2 of naphthaldehyde thiosemicarbazone derivatives. The ligands and their palladium complexes have been

synthetized and characterized by elemental analysis and IR and NMR (1H, 13C) spectroscopy.

Analytical and spectroscopy data are consistent with the proposed structural formulas for the ligands and their

complexes (Fig. 1). The spectroscopy data revealed that the desprotonated naphthaldehyde thiosemicarbazone

derivatives are coordinated to the Pd(II) ion through the nitrogen and sulphur atoms.

N

NPd

S

N

N S

N

NH

H

H

H

X

X

(X = H, NO2, OCH3)

Figure 1. Structural formulas of the synthetized palladium(II) complexes.

The in vitro antitumor activity of the ligands and their complexes was determined against six human tumor cell

lines: H460, DU145, M14, HT29, K562 and MCF-7. The antitumor studies revealed that the palladium(II)

complexes are more cytotoxic than their ligands with IC50 values at the range of 0.65–2.39 µM. The complex

Pd(L2)2, with the 4-phenyl-1-(1´-nitro-2´-naphthaldehyde) thiosemicarbazone ligand, showed higher antiproliferative

activity (CI50 = 0.65- 0.68 µM) than the other tested palladium(II) complexes against H460 and K562 tumor cell

lines.

Acknowledgements: W. H. thanks the Universidad de Lima Scientific Research Institute for financial support to

carry out the research work. We also thank the Research Laboratory of the Faculty of Sciences and Philosophy,

Universidad Peruana Cayetano Heredia, for the cytotoxic studies of the compounds. E. S. and J. M. thank

Financiamiento Basal FB0807 Project.


84

References

1.-J. Patole, S. Padhye, M.S. Moodbidri and N. Shirsat. “Antiproliferative activities of iron and platinum conjugates of salicylaldehyde semi-/thiosemicarbazones against C6 glioma cells”.Eur J Med Chem; 2005; 40; 1052–1055.

2.-W. Hernández, J. Paz, J. Vaisberg, E. Spodine, R. Richter y L. Beyer.“Synthesis and characterization of new palladium(II) complexes with ligands derived from furan-2-carbaldehyde and benzaldehyde thiosemicarbazone and their in vitro cytotoxic activities against various human tumor cell lines”. Z Naturforsch; 2010; 65b; 1271-1278.


85


Effects of copper-dipeptide complexes on lipid model membranes

Pianca Barroso, R.1; Noble, C.2; Facchin, G.2; Torre, M.H.2; Costa-Filho, A.1 1Instituto de Física, Universidade de São Paulo, Ribeirão Preto, Brasil

2Cátedra de Química Inorgánica, Universidad de la República, Montevideo, Uruguay

[email protected]

Several copper-dipeptide complexes have demonstrated pharmacological activity, such as antiproliferative agents

on cell cultures, interaction with DNA and superoxide dismutase activity. As part of the mechanism underlying their

pharmacological activity is certainly the interaction with membranes that regulate the flux of molecules in and out of

the cell. Surprisingly this is an issue that has received little attention over the years. Studies about the possible

interactions of copper-dipetides with lipid membranes can then significantly contribute to the understanding of basic

aspects of complex activity. In this context, we use differential scanning calorimetry (DSC) and electron spin

resonance (ESR) to investigate the effect of copper-dipeptide complexes on the lipid phase transition and acyl

chain dynamics of lipid model membranes. Different membrane regions were monitored by spin probes located at

different positions along the lipid chain. The spin labels used were 1-palmitoyl-2-stearoyl-(n-doxyl)-sn-glycero-3-

phosphocholine, where n = 5, 10 and 16. The lipid membranes were made from 1,2-dipalmitoyl-sn-glycero-3-

phosphocholine (DPPC) and from 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG). The results

showed interactions between the complexes and the lipid model membranes. The presence of copper-dipeptide

complexes caused changes on the mobility (fluidity) of the lipid membranes, thus indicating that non-specific

complex-membrane interactions could help the entrance of the molecules.

Aknowledgments: Financial support: FAPESP and CNPQ.


86


Development of new coordination complexes of Co(III) with antitumoral activity

Noble, C.1; Iglesias, S.1; Pianca Barroso, R.2; Torre, M.H.1; Costa-Filho, A.2; Facchin, G.1 1Departamento Estrella Campos, Facultad de Química, UdelaR, Montevideo, Uruguay

2Departamento de Biofísica Molecular, Faculdade de Filosofía, Ciências y Letras de Ribeirão Preto, Universidade de São Paulo,

Ribeirão Preto, Brasil [email protected]

Nowadays society is in need of new antitumor drugs with less collateral damage to the human body and with

different spectra of action. The development of new compounds with essential elements, aims to find new

compounds with less severe side effects. There is a background in coordination compounds of essential metals

such as Mn, Co, Cu and Zn, among others, which present pharmacologic activity. For instance, different mixed

ligand Co(III) complexes including phenanthroline inhibit cancer cell proliferation. There are numerous records of

antineoplasic drugs whose action mechanisms involves interaction with DNA, causing damage that interfere with

the cellular proliferation process, and eventually resolve in cellular death. Because of this, the development of new

metallic complexes, capable of interacting with DNA is a potential path in finding new antineoplastic compounds.

Our research group has synthesized and characterized several Cu(II) coordination compounds with L-dipeptides

and with mixed ligands, including L-dipeptides and 1,10-phenathroline (phen) which showed very good antitumor

activity in vitro studies in HeLa cells. Other studies have yielded that in vitro the aforementioned complexes are

capable of interacting with DNA. To continue advancing on the development of better antitumor drugs with

essential elements, this work focused on the synthesis of new complexes of Co(III), analogues to [Cu(II)(L-

dipeptide)(phen)]. The syntheses of these new complexes, [Co(III)(L-dipeptide)(phen)(H2O)], were made by

substitution in aqueous solution of Co(II) and further oxidation of the complexes. The structural elucidation in solid-

state was made by infrared spectroscopy, elemental analysis, and in aqueous solution by EPR, UV-Vis and DC.

Their lipophilicity, DNA interaction and antiproliferative activity was assessed. These studies have demonstrated

that these new complexes present one L-dipeptide and one phen coordinated to the Co(III) ion, and that they

interact with DNA. Furthermore, they exhibit antitumor activity in HeLa cells, possible due to its ability to interact

with DNA.


87


New heteroleptic copper complexes with saccharinate: synthesis, characterization and cytotoxicity against osteoblast and osteosarcome cell lines

Santi, E.1; Viera, I.1; León, I.2; Baran, E.J.2; Etcheverry, S.2; Torre, M.H.1* 1 Cátedra de Química Inorgánica, DEC, Facultad de Química (UdelaR), Montevideo, Uruguay

2Centro de Química Inorgánica (CEQUINOR/CONICET, UNLP), Facultad de Ciencias Exactas (UNLP), La Plata, Argentina

[email protected]

With the aim of providing efficacious and safe products, the pharmaceutical industry needs to incorporate different

substances to maintain their attributes and stability, to aid in the administration and to impede the contamination.

Usually, these compounds can form complexes with metals coming from the pharmaceutical formulations or food.

Saccharin (sac) is an important sweetening agent used in pharmaceutical vehicles, foods, beverages and in diets

for diabetics. Besides, it has three potential coordinating centers: the heterocyclic nitrogen and the carbonyl and

sulphonyl oxygen atoms. On the other hand, aminoacids (aa) are ligands that usually transport metals in blood. For

this reason it is interesting to study the interaction of essential cations with saccharin and aminoacids.

As a part of our work in the development of metal complexes with pharmacological activities or pharmacotechnical

interest1, in this work we report the synthesis and characterization (elemental analysis, UV-Vis, IR and Raman

spectroscopies) of new mixed Cu(II) complexes with saccharin and aminoacids (gly, ser, gln, asp) with general

formula Na2[Cu(sac)2(aa)2]•nH2O. The aminoacids coordinated as bidentate ligands through the amino and

carboxylate groups and the sacharinate as monodentate through the heterocyclic nitrogen.

Besides, due to the antecedents that several coadjuvants showed biological adverse effect, the antiproliferative

activity against osteoblastic cells (MC3T3-E1) and human osteosarcoma cells (MG-63) for [Cu(sac)2(H2O)4]•2H2O

(Cu-sac) and Na2[Cu(sac)2(gln)2]•H2O (Cu-sac-gln) were tested. The results presented in the Figure showed that

the first complex is less cytotoxic than the second one in both cell lines, showing the importance of these mixed

complexes. On the other hand the heteroleptic complexes presented different superoxide dismutase-like activity

than the homoleptic one.

References

1 Santi E., Viera I., Mombrú A., Castiglioni J., Baran E.J., Torre M.H., Synthesis and characterization of heteroleptic copper and zinc complexes with saccharinate and aminoacids. Evaluation of SOD-like activity of the copper complexes, Biol. Trace. Elem., 2011, 143, 3, 1843-1855.


88


Physicochemical study, crystal structure and antitumoral activity of a novel vanadium(V) complex with an isoniazid-derived hydrazone ligand

González Baró, A.C.1; Ferraesi Curotto, V.2; Parajón-Costa, B.S.1; Pis Diez, R.1; Resende, J.A.L.C.3; Paula, F.C.S.4; Pereira, M.E.C.4; Rey, N.A.5

1CEQUINOR- Departamento de Química, CONICET-UNLP (Facultad de Ciencias Exactas), La Plata, Argentina

2Departamento de Química, Facultad de Ciencias Exactas y Naturales UNCa, Catamarca, Argentina

3Departamento de Quimica, Universdidade Federal Fluminense, Nitrói, RJ, Brazil

4Departamento de Quimica, Universidade Federal de Minas Gerais, Brazil, Belo Horizonte

5LABSO-BIO, Departamento de Química, Rio de Janeiro, Brazil

[email protected]

Condensation reaction of the antituberculous agent isoniazid with o-vanillin leads to the formation of a stable and

active hydrazone, INHOVA1. The interaction of this compound with vanadium was studied in an attempt to obtain

metal complexes of therapeutic interest. The complex was prepared from ethanolic solutions of INHOVA and VOCl2

in a 1:1 molar ratio and obtained in the hydrochloride form, with protonation of the pyridine N atom. It crystallizes in

the monoclinic system, space group P21/c. INHOVA acts as a tridentate ligand and the distorted coordination

environment is completed by an oxo ligand, a disordered ethoxide and a water molecule.

The optimized geometry of the complex, the vibrational analysis and the vertical electronic transitions were

obtained within the context of the DFT. Basis sets of triple-z quality were used. Solvent effects were included

through the IEF- PCM.

The IR spectrum of the solid and the UV-Vis spectra in DMSO and ethanol were compared with those of the free

ligand. The assignments were accomplished with the help of theoretical calculations. Electronic spectra were

similar in both solvents, in accordance with theoretical predictions. The bands could be assigned to intraligand and

charge transfer transitions. The redox behavior of the complex and the ligand was studied by cyclic voltammetry in

the same solvents. Several irreversible oxidation and reduction processes involving the metal center and the

coordinated ligand were observed.

Both INHOVA and the complex showed activity against the chronic myelogenous leukemia K562 cell line in a

concentration-dependent manner. The IC50 (concentration required to inhibit 50% of cellular growth) values

obtained were, respectively, 36.46 ± 4.13 and 28.80 ± 3.48 µmol.L-1.


89

References

1 Ana C. González-Baró, Reinaldo Pis-Diez, Beatriz S. Parajón-Costa, Nicolás A. Rey; Spectroscopic and theoretical study of the o-vanillin hydrazone of the mycobactericidal drug isoniazid. J. Mol. Struct. 2012, 1007, 95–101.


90


Synthesis, spectroscopic characterization and DFT studies of a novel Pd(II) complex with rimantadine

Sucena, S.; de Paiva, R.E.F.; Formiga, A.L.B.; Corbi, P.P.

Department of Inorganic Chemistry, University of Campinas, Campinas, Brazil [email protected]

Since the discovery of cisplatin, reports concerning the use of metal complexes in cancer treatment have

experimented an extraordinary increasing. Platinum(II) and palladium(II) complexes were shown to present a wide

range of pharmacological activities, such as antitumoral and antibacterial activities [1]. Despite the widespread use

of cisplatin, this compound is also a toxic substance. In order to reduce the toxic side effects of cisplatin, new

compounds based on the cisplatin structure have been synthesized. Among the non-platinum compounds with

potential for the clinical treatment of human malignancies, palladium derivatives have received considerable

interest, because of the structural and electronic analogy between the Pt(II) and Pd(II) complexes [2].

Rimantadine (C12H21N, rtd) is a diamantoid obtained by the functionalization of adamantine with an ethanamine

group. It is a bioactive compound used as an antiviral agent in the treatment of influenza A, depression and some

cases of parkinsonism [3]. No studies of coordination compounds with rimantadine was previously reported in the

literature, excepted in the case of the gold(I) rimantadine complex published by us [4]. Here, we describe the

synthesis, characterization and antibacterial studies of a new palladium complex with rimantadine.

The palladium(II) complex with rimantadine (Pd-rtd) was prepared by the reaction of an alkaline methanolic solution

of rimantadine with a methanolic solution of Li2[PdCl4], in a 2:1 ligand/metal ratio. A yellow solid was formed within

one hour of reaction. Elemental analysis led to the composition PdCl2C24H42N2. Anal. Calc. for PdCl2C24H42N2 (%):

C 53.79; H 7.37; N 5.23. Found (%): C 53.79; H 7.89; N 5.23.

The shifted of the (NH2) deformation band in the IR spectra of the complex, which appears at 1728 cm-1 for the free

rimantadine indicates nitrogen coordination of the ligand to Pd(II). Nitrogen coordination was confirmed by 1H and 13C and 1H- 15N HMBC NMR. The second one shows a ∆δ 13C (complex – free ligand) = 3.5 ppm. Studies based on

DFT are in progress in order to optimize the structure of the compound.

References

[1] Iyidogan, A. K.; Tasdemir, D.; Emre, E. E. O.; Balzarini, Novel platinum(II) and palladium(II) complexes of thiosemicarbazones derived from 5-substitutedthiophene-2-carboxaldehydes and their antiviral and cytotoxic activities. J. Eur. J. Med. Chem.2011, 46, 5616-5624.

[2] Ghani, N. T. A.; Mansour, A. M. Novel palladium(II) and platinum(II) complexes with 1H-benzimidazol-2-ylmethyl-N-(4- bromo-phenyl)-amine: Structural studies and anticancer activity. Eur. J. Med. Chem. 2012, 47, 399-411.

[3] Garcia, J. C.; Justo, J. F.; Machado, W. V. M.; Assali, L. V. C. J. Structural, Electronic, and Vibrational Properties of Amino-adamantane and Rimantadine Isomers Phys. Chem. A 2010, 114, 11977-11983.

[4] Sucena, S. F.; Paiva, R. E. F.; Abbehausen, C.; Mattos, I. B.; Lancellotti, M.; Formiga, A. L. B.; Corbi, P. P. Chemical, spectroscopic characterization, DFT studies and antibacterial activities in vitro of a new gold(I) complex with rimantadine. Spectrochim. Acta A. 2012, 89, 114-118.


91


Interaction with proteins and antitumoral activity of a ruthenium semicarbazone complex

Otero, L.1; Demoro, B.1; De Almeida, R.2; Marques, F.3; Costa Pessoa, J.4; Tomaz, I.5; Gambino, D.1 1DEC, Facultad de Química, UdelaR, Montevideo, Uruguay

2CQB/DQB, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal

3UCQR, Instituto Superior Técnico/Instituto Tecnológico e Nuclear, Lisboa, Portugal

4CQE, Instituto Superior Técnico, UT Lisboa, Lisboa, Portugal

5CCMM/DQB, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal

[email protected]

Ruthenium complexes hold great potential in cancer treatment as non-platinum alternatives. Some 5-

nitrofurylsemicarbazone derivatives have shown activity against Trypanosoma cruzi, the parasite responsible for

Chagas’ disease. A panel of [RuIICl2(DMSO)2L] complexes with these ligands was previously developed by some of

us and screened for the activity against T. cruzi [1]. Some metal-based anti-trypanosomal compounds have

exhibited antitumor properties as well, probably due to metabolic similarities between tumor cells and pathogenic

trypanosomes.

In this work, we evaluate the antiproliferative activity of [RuIICl2(DMSO)2(5-nitrofurylsemicarbazone)], RuNTF, in

three human cancer cell lines with different sensitivity to cisplatin (A2780 ovarian adenocarcinoma, MCF7 breast

adenocarcinoma and PC3 grade IV prostate carcinoma) and the effect of serum protein binding on the cytotoxic

activity in the A2780 cell line. Human serum albumin (HSA), the most abundant protein in human plasma, is the

major nonspecific transporter in the circulatory system and is known to play an important role in drug

pharmacokinetics. As a first approach to the pharmacokinetics of RuNTF, we assessed its interaction with HSA at

37ºC by Circular Dichroism (CD), UV-visible absorption and both steady-state and time-resolved Fluorescence in

simulated blood plasma conditions. The great quenching extent of the Fluorescence emission from the HSA

Trp214 aminoacid residue observed upon binding of RuNTF to HSA, indicates a strong complex-protein affinity,

which is supported by CD data, with an Induced CD broad band at ~450 nm observed after short incubation times.

Acknowledgements: Authors thank CYTED network RIIDFCM and Portuguese Foundation for Science and

Technology (PTDC/QuiQui/101187/2008, PEst-OE/QUI/UI0100/2011, PEst-OE/QUI/UI0612/2011, PEst-

OE/QUI/UI0536/2011, Ciência2007 and 2008 Initiatives). B.D. thanks ANII Uruguay for a postgraduate fellowship.

References

[1] L. Otero, P. Noblia, D. Gambino, H. Cerecetto, M. González, J. A. Ellena, O. E. Piro, Inorg. Chim. Acta 344 (2003) 85.


92


[Ru(tpy)(adtpy)]2+ and [Ru(dtp)(adtpy)]2+ as dual topoisomerase I/II Inhibitors

Chao, H. School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, China

[email protected]

DNA topoisomerases are essential enzymes that control and modify the topological states of DNA. In accordance,

topoisomerase activities are activated in cancer cell growths, and thus are important cellular targets for

antineoplastic drugs.As modern therapeutics, ruthenium complexes have specifically received considerable interest

as potential anticancer drugs in recent years. Here two novel ruthenium(II) anthraquinone complexes

[Ru(tpy)(adtpy)]2+ (1) and [Ru(dtp)(adtpy)]2+ (2) have been synthesized and characterized. Spectroscopic and

viscosity measurementssuggested that two Ru(II) complexes bind to DNA in an intercalative mode. Topoisomerase

inhibition and DNA strand passage assay confirmed that two Ru(II) complexes acted as efficient dual inhibitors of

topoisomerases I and II by interference with the DNA religation. In MTT cytotoxicity studies, two Ru(II) complexes

exhibited antitumor activity against HeLa, BEL-7402, MCF-7 and HepG2 tumor cells. The AO/EB staining assay,

Alexa Fluor® 488 annexin V/PI double staining assay and alkaline single-cell gel electrophoresis (comet assay)

indicated that Ru(II) complexes could induce the apoptosis of HeLa cells.

Acknowledgements: This work was supported by NSFC(No. 21071155, 21172273, 21171177), GDNSFC

(9351027501000003), the National High Technology Research and Development Program of China (863 Program,

2012AA020305), StateKey Laboratory of Optoelectronic Materials and Technologies (2010-ZY-4-5) and Sun Yat-

Sen University.

References

[1] K. J. Du, J. Q. Wang, J. F. Kou, G. Y. Li, L. L. Wang, H. Chao, L. N. Ji, Synthesis, DNA binding and topoisomerase inhibitory activity of ruthenium(II) polypyridyl complexes, Eur. J. Med. Chem., 2011, 46, 1056-1065.

[2] J. F. Kou, C. Qian, J. Q. Wang, X. Chen, L. L. Wang, H. Chao, L. N. Ji,Chiral Ruthenium(II) Anthraquinone Complexes as Dual Inhibitors of Topoisomerases I and II, J. Biol. Inorg. Chem., 2012, 17, 81-96.


93


A novel silver(I) complex with Ibuprofen: synthesis, spectroscopic characterization in the solid-state and preliminary antibacterial assays

Silva, I.M.P.1; de Paiva, R.E.F.1; Profirio, D.M.1; Lancellotti, M.2; Corbi, P.P.1; Formiga, A.L.B.1 1Department of Inorganic Chemistry, University of Campinas, Campinas, Brazil

2 Department of Biochemistry, University of Campinas, Campinas, Brazil

[email protected]

The wide-ranging biological properties associated with metal ions have recently stimulated the development of new

metal-based drugs. Silver complexes have been extensively studied due to the antibacterial properties exhibited by

this metal, and the combination of silver with biologically active molecules can enhance this effect even further [1].

Ibuprofen (C13H18O2, Ibu) is a non-steroidal anti-inflammatory drug used to treat headaches, rheumatoid arthritis,

fever and acute or chronic pain related with the inflammatory processes. The present work describes the synthesis,

spectroscopic characterization and preliminary antibacterial assays of a novel silver(I) complex with ibuprofen (Ag-

Ibu).

The Ag-Ibu complex was synthesized by the reaction of equimolar quantities of silver nitrate and sodium

ibuprofenate in aqueous solution. Elemental analysis indicates the composition AgC13H17O2. Anal. Calc for

AgC13H17O2(%): C, 49.9; H 5.47. Found(%): C, 49.6; H, 5.01. Thermal decomposition starts at 180°C and ends at

400°C, with a mass loss of 64,6%, leading to the formation of Ag0 as the final residue (calcd 34.5%; found 36.4%).

The CP/MAS 13C NMR spectrum of the complex shows that, upon coordination, the carbon atom of the carboxylic

group is shifted upfield by 3.97 ppm. The IR spectrum shows that the value of separation Dn = νasym(COO) -

νsym(COO) for the complex (175 cm-1) is higher than the value observed for sodium ibuprofenate (138 cm-1),

suggesting a monodentate carboxylate coordination. However, according to the literature, a dimeric bidentate

bridging coordination cannot be discarded [2]. The DFT studies are being performed in order to further evaluate the

coordination mode.

The antibacterial activities of the complex were evaluated in aqueous suspension, against Gram-negative (E. coli

ATCC 25922, P. aeruginosa ATCC 27853 and P. aeruginosa PA IMI) and Gram-positive (S. aureus ATCC25923,

Rib 1 and BEC9393) bacterial strains. The minimum inhibitory concentration values (MIC) were of ~25 mg mL-1 for

all tested bacterial strains, confirming the antibacterial activity of the compound in the considered experimental

conditions.

References

[1] J. L. Clement, P. Ss. Jarret. Antibacterial Silver. Metal Bases Drugs 1994, 1, 467-482.

[2] V. Zelenak, Z. Vargová, K. Gyoryová. Correlation of infrared spectra of zinc(II) carboxylates with their structures.Spectrochim Acta A 2007, 66, 262-272


94


Synthesis, characterization and DNA interactions of two new ruthenium(II) compounds

Silva, P.P.; Pereira-Maia, E.C. Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

[email protected]

Recently, ruthenium compounds have received considerable attention because the complexes imidazolium-trans-

dimethylsulfoxide-imidazole-tetrachlororuthenate(III) and indazolium-trans-bis(1H-indazole)-tetrachlororuthenate(III)

entered human clinical trials. This fact has stimulated us to search for new ruthenium compounds as antitumoral

agents. This work reports on the synthesis, characterization and DNA interactions of two new ligands and mixed-

ligand complexes of ruthenium(II). Their general formula is [Ru(L)(phen)(dmso)(Cl)](PF6), in which L= pqdS [N'-(6-

oxo-1,10- phenanthrolin-5(6H)-ylidene)thiophene-2-carbohydrazide] for complex 1 and pqdO [N'-(6-oxo-1,10-

phenanthrolin-5(6H)- ylidene)furan-2-carbohydrazide] for complex 2 (Figure 1). The ligands pqdS and pqdO were

synthesized by reacting 1,10-phenanthroline-5,6-dione with thiophene-2-carbohydrazide (ligand pqdS) or furan-2-

carbohydrazide (ligand pqdO) in ethanol with 10% acetic acid. The ligands were characterized by elemental

analyses, vibrational, electronic, and 1H NMR spectrometries. The complexes were prepared by refluxing cis-

[RuCl2(dmso)4] with pqdS (1) or pqdO (2) in methanol for 1 hour. Afterwards, 1,10- phenanthroline and NH4PF6

were added and the mixture kept in reflux for more 3 hours. The complexes were characterized by elemental and

conductivity analyses, vibrational, electronic, and ESI-MS spectrometries. The presence of the complexes in

solution was confirmed by ESI-MS spectrometry in positive mode. Complex 1 give a main peak at m/z 728.6

assigned to [Ru(pqdS)(phen)(dmso)(Cl)]+ (calculated mass 729.2) and complex 2 at 712.6 due to

[Ru(pqdO)(phen)(dmso)(Cl)]+ (calculated mass 713.2). The calculated isotopic distribution for the proposed species

agrees with the experimental spectra. The molar conductivity values of 10-3 mol L-1 solutions of 1 and 2 in

nitromethane, at 25ºC, are ΛM= 89.6 and 77.9 µS·cm-1, respectively, indicating that they are 1:1 electrolytes. The

addition of calf thymus DNA to a solution of the complexes induces a hypochromic effect in their UV-Vis spectra

indicating the formation of a ternary complex with DNA. From the spectrophotometric data, the following binding

constants were calculated 6.1 × 104 for 1 and 1.4 × 104 for 2.

Ru

S

Cl

N

NN

N O

N NH

C

SOCH3H3C

O

Complex 1

+

Acknowledgments: CNPq, FAPEMIG, CAPES and INCT-Catálise.


95


Antitumoral potential of platinum(II) compounds with 4-aminoquinoline derivatives

de Paula, F.C.S.1; Carmo, A.M.L.2; Fontes, A.P.S.2; Pereira-Maia, E.C.1 1Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil 2 Department of Chemistry, Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil

[email protected]

Quinoline is extensively used as a starting compound to synthesize molecules with pharmacological properties. In

this work, we examined the antitumoral potential of five platinum(II) compounds with a series of 4-amino-7-

chloroquinoline analogues (Figure 1). The synthesis and characterization of the complexes were performed as

previously described1. The sensitivity to drug was evaluated in a chronic myelogenous leukemia cell line from the

drug concentration needed to inhibit cell growth by 50%, the IC50. Measurements of cellular accumulation were

made by incubating cells in the culture medium with various drug concentrations for 72 h. Partition coefficients of

the complexes were determined in an n-octanol/water system. Platinum content was determined by graphite

furnace atomic absorption spectrometry. All complexes inhibited cellular growth with the following IC50 values:

47.16 ± 0.8 for complex 1; 21.02 ± 0.4 for 2; 8.94 ± 0.4 for 3; 10.45 ± 1.1 for 4; and 2.02 ± 0.3 for 5. There is a

general trend of enhancing cytotoxic activity with the increase in the carbon chain length. Comparing compounds

carrying the same R and R1 groups, one observes that increasing “n” from 2 to 3, it doubles the cytotoxic activity in

the case of 1 and 2 (R=R1=H) and it leads to a 5-fold increase in the cytotoxic activity in the case of 4 and 5

(R=R1= CH2CCH). Incubation of cells with equitoxic complex doses leads to the same intracellular platinum

concentrations. Increasing lipophilicity enhances cellular uptake and, consequently, the cytotoxic activity.

Figure 1. Structures of complexes 1-5

Acknowledgments: CNPq, FAPEMIG, CAPES and INCT-Catálise.

References

[1] Carmo AML, Silva FMC, Machado PA, Fontes APS, Pavan FR, Leite CQF, Leite SRA, Coimbra ES, Silva AD.

Synthesis of 4-aminoquinoline analogues and their platinum(II) complexes as new antileishmanial and

antitubercular agents. Biomed Pharmacother; 2011; 65; 204–209.


96


Novel ortho-substituted binucleating ligands and the mononuclear copper(II) complexes of their partially hydrolyzed derivatives display potent antimicrobial activity

Cruz, W.S.1; Resende, J.A.L.C.2; Siqueira, S.A.3; Beraldo, H. de O.3; Rey, N.A.1 1Department of Chemistry, PUC-Rio, Rio de Janeiro, Brazil

2Department of Chemistry, UFF, Niterói, Brazil

3Department of Chemistry, UFMG, Belo Horizonte, Brazil

[email protected]

The utility of copper ions, either alone or in combination with bioactive ligands, as a biocidal tool is well

documented in literature1. On the other hand, metal complexes of Schiff bases have shown antibacterial and

antifungal activities2. In the present work, we report the synthesis and characterization of a series of new o-

substituted binucleating ligands, as well as the preparation and study of copper(II) complexes concerning their

partially hydrolyzed Schiff base products. The antimicrobial potential of all compounds was assayed against

bacteria (P. aerouginosa, S. aureus) and fungus (C. albicans).

Crystal structures of complexes containing methyl (1) and iodo (2) substituents were resolved by X-ray diffraction.

The compounds are mononuclear, with two partially hydrolyzed ligands coordinated through their phenolate oxygen

and Schiff base nitrogen atoms. Geometry is essentially square-planar in 2, while a heavily distorted tetrahedral

coordination sphere is observed for 1 since both methyl groups are on the same side of the ligands plane, causing

relevant steric repulsion.

When tested against bacteria, all ligands showed some activity, being the iodo-substituted compound the best of

them (MIC = 165 and 83 mmol L-1 for P. aerouginosa - ATCC: 9027 and S. aureus - ATCC: 6538, respectively).

Complexation only improved the activity profile against Staphylococcus, with complexes 1 and 3 (nitro-substituted)

presenting MIC values equal to 87 and 76 mmol L-1, correspondingly.

In the case of C. albicans, methyl and nitro-substituted ligands were as active as the reference drug fluconazole,

showing MIC around 25 mmol L-1.


97

References

1) G. Borkow, J. Gabbay; Copper as a biocidal tool. Curr. Med. Chem. 2005, 12, 2163–2175.

2) S. Kumar, D. N. Dhar, P. N. Saxena; Applications of metal complexes of Schiff bases – A review. J. Sci. & Ind. Res. 2009, 68, 181–187.

Aknowledgments: CNPq, PUC-Rio


98


Synthesis, characterization and antimycobaterial activity of Ag(I) complexes with N, N´-bis[thiophenil-2-methylidene]-1, 2-diamine

da Silva, S.A.1; Cuin, A.1; Bergamini, F.2; Corbi, P.P. 2; Leite, C.Q.3; Pavan, F.3 1Department of Chemistry, UFJF, Juiz de Fora, Brazil

2Department of Chemistry, UNICAMP, Campinas, Brazil

3Department of Biological Sciences, UNESP, Araraquara, Brazil

[email protected]

Tuberculosis, an infectious disease, occurs mainly in Africa, Asia and America, and is caused by the bacillus

Mycobacterium tuberculosis (MTB) [1]. It causes nearly 3 million deaths annually worldwide, and estimated 8.8

million of new cases every year [2]. The long period of treatment and co-infection with HIV, are one of the factors

responsible for the emergence of multidrug-resistant strains [2]. The WHO estimates some 500,000 cases of

multiresistant tuberculosis (MDR-TB) in 2007 [1]. Therefore, the discovery of new drugs to combat the MDR-TB is

necessary. In 2007, A. Cuin et al. synthesized Ag(I) complexes with alpha-hydroxy acids, with MIC values lower

than 8.0mg mL-1 against MTB [2]. Recently, Ag(I) complex with 2-(2-thienyl) benzothiazole, that showed effective

antituberculosis activity, was synthesized in our laboratory [3]. The present work aims the synthesis and

characterization of the ligand (BNH) and its Ag(I) complexes, AgBNH, Ag(BNH)2 and AgBNH(PPh3)2.

Methodology: The ligand was synthesized mixing 2-thiophenecarboxaldehyde and ethylenediamine in methanol

and recrystallized from n-hexane (yield: 97%). The complexes, AgBNH and Ag(BNH)2 were synthesized in

isopropyl alcohol solution (yield: 47% and 41% respectively). The complex AgBNH(PPh3)2 was synthesized adding

AgNO3 in BNH and PPh3 methanolic solutions, in proportion 1:1:2. The complex was precipitated in water (yield:

70%).

Results: IR bands of C=N group were assigned at 1634, 1632, 1629 and 1630 cm-1 for AgBNH, Ag(BNH)2,

AgBNH(PPh3)2 and BNH, respectively. The 1H- and 13C-NMR of the compounds showed chemical shifts (ppm) for

N=C-H group at: BNH (8.40; 155.6), AgBNH (8.85, 159.0), Ag(BNH)2 (8.85, 158.7) and AgBNH(PPh3)2 (8.48,

155.9). The minimal formulas of Ag(I) complexes were obtained by elemental and thermal analyses. For AgBNH

(34.3%C, 2.68%H, 10.7%N, 25.8%Ag - C24H24Ag2N6O6S4), Ag(BNH)2 (42.2%C, 3.51%H, 10.4%N, 16.2%Ag -

C24H24AgN5O3S4) and AgBNH(PPh3)2 (65.2%C, 4.25%H, 2.38%N, 11.3%Ag - C48H42AgN3O3P2S2). Tests against

Mycobacterium tuberculosis are underway for the four compounds.

Conclusions: Three promising compounds antituberculosis and a ligand with excellent yield were synthesized.

Acknowledgements: The authors are thankful to FAPEMIG and FAPESP/Brazil process–CEXAPQ-00256/11 and

2012/08230-2 and UFJF/Proquali for financial supports.

References

[1] SouzaW. Doenças Negligenciadas. Rio de Janeiro: Academia Brasileira de Ciências, 2010, 43.

[2] Cuin A, Massabni AC, Leite CQF, Sato DN, Neves A, Szpoganicz B, Silva MS, Bortoluzzi AJ. Synthesis, X-ray structure and antimycobacterial activity of silver complexes with a-hydroxycarboxylic acids. J Inorg Biochem; 2007; 101; 291-296.

[3] Pereira GA, Massabni AC, Castellano EE, Costa LAS, Leite CQF, Pavan FR, Cuin A. A broad study of two new promising antimycobacterial drugs: Ag(I) and Au(I) complexes with 2-(2-thienyl)benzothiazole. Polyhedron; 2012; 38; 291-296.


99


DNA structural changes on binding and photoreaction with water-soluble tricarbonyl Re(I) complexes

Ragone, F.1; Yañuk, J.2; Cabrerizo, F.2; Wolcan, E.1; Ruiz, G.1 1INIFTA-CONICET, UNLP, Universidad Nacional de La Plata, UNLP, La Plata, Argentina

2IIB–INTECH–CONICET, Universidad Nacional de San Martín, CHASCOMUS, ARGENTINA

[email protected]

Metal complexes containing the fac-{ReI(CO)3} core derivatized with different diimine ligands have been known to

be efficient emitters for many years and, therefore, their photophysical properties have been widely studied1. In

particular, fac-Re(CO)3L1L2 complexes, with L1 = dipyridil[3,2-a:2’3’-c]phenazine (dppz) and L2 = pyridine

derivatives, have been recognized as good intercalators in double helix of DNA2. Intercalative binding can disrupt

the helical nature of DNA, causing profound effects on DNA integrity. Because of their photophysical and redox

properties, many of those complexes have the capacity to cause DNA damage by photoinduced oxidative strand

breakage3. Furthermore, the binding modes to DNA for Re(I)-Pterin complexes (L1 = 2-amine-4-pteridinone) have

been scarcely studied.

In this work we used UV-vis absorption and fluorescence spectroscopy to investigate the binding properties of a

new Re(I)-Pterin complex4 with DNA (plasmid DNA, double-stranded calf thymus DNA (CT-DNA) and synthetic

polynucleotides Poly[dAdT]2 and Poly[dGdC]2), in aqueous solutions. In addition, the consequences on the DNA

chemical structure and the excited state reactions of these complex and super-coiled plasmid DNA were

investigated by means of electrophoresis. Briefly, when solutions of the complex were incubated (30 min.) with

plasmid DNA, agarose gels showed morphological changes on circular DNA changing its conformation from

supercoiled to relaxed. Thermal processes show a progression of the conversion with increasing [Re(I)

complex]/[DNA] ratio. Irradiation with 350 nm light reverted the changes observed in darkness and no

photocleavage was observed.

On the other hand, recently we have demonstrated that a Re(I)-dppz complex, (4,4'-bpy)ReI(CO)3(dppz)+,

intercalates to Poly[dGdC]2 and with some selectivity to Poly[dAdT]2, Kb = 1.8 x 105 M-1 5. These studies are now

complemented with binding to CT-DNA and photocleavage to plasmid DNA after intercalation.

All the experimental data will be discussed in relation to the potential applications of those complexes as both

luminescent and conformational probes to DNA and molecular scissors to the double helix.

Acknowledgements: This work was supported in part by ANPCyT (PICT 1435), CONICET (PIP 0389),

Universidad Nacional de La Plata (UNLP 11/X533 y 11/X611) of Argentina. G.T.R., F.M.C. and E.W. are Research

Members of CONICET (Argentina).

References

1- A. Kumar, S. Sun and A. J. Lees.Photophysics and Photochemistry of Organometallic Rhenium Diimine Complexes. Top OrganometChem (2010) 29: 1–35

2- K. K. Lo. Exploitation of Luminescent Organometallic Rhenium(I) and Iridium(III) Complexes in Biological Studies. Top OrganometChem (2010) 29: 115–158

3- J. A. Smith, M. W. George and J. M. Kelly.Transient spectroscopy of dipyridophenazine metal complexes which undergo photo-induced electron transfer with DNA.CoordChem Rev 255 (2011) 2666– 2675


100

4- F. Ragone, G. T. Ruiz, O. E. Piro, G. A. Echeverría, F. M. Cabrerizo, G. Petroselli, R. Erra-Balsells, K. Hiraoka, F. S. García Einschlag and E. Wolcan. Water-Soluble (Pterin)rhenium(I) Complex: Synthesis, Structural Characterization, and Two Reversible Protonation–Deprotonation Behavior in Aqueous Solutions. Eur J InorgChem, 2012, 4801–4810.

5- G.T. Ruiz, M.P. Juliarena, R.O. Lezna, E.Wolcan, M.R. Féliz and G. Ferraudi. Intercalation of fac-(4,4’- bpy)ReI(CO)3(dppz)+, dppz = dipyridil[3,2-a:2’3’-c]phenazine, in polynucleotides. On the UV-visphotophysics of the Re(I) intercalate and the redox reactions with pulse radiolysis-generated radicals. J ChemSoc, Dalton Trans, 2007, 20, 2020–2029.


101


On the reaction between trans-[Ru(NH3)4(4- pic)(H2O)]3+ and cysteine

Franco, D.W.1; Souza, M.L.1; Castellano, E.E.2 1Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, Brazil 2Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil

[email protected]

Cysteine and glutathione (RSH) are the non protein thiols of higher concentration in the human body (0.1 – 1.0

mM) being part of an important redox buffer involving the oxidized form (RSSR)1. Furthermore they are very good

candidates to act as in vivo reductant of ruthenium nitrosyls2 trough a series of reactions in which is conceivable

the formation of the trans-[Ru(NH3)4(4-pic)(H2O)]3+/2+species3. Aiming to better understand this system the complex

trans-[Ru(NH3)4(4-pic)(H2O)](CF3SO3)3.H2O has been isolated and studied through elemental analysis, UV-vis, X-

ray and EPR spectroscopies and cyclic voltammetry. The water molecule in trans-[Ru(NH3)4(4-pic)(H2O)]3+ is

substituted by Br- and Cl- ions at the rate constants of 1.5×10-4 M-1s-1 and 2.5×10-4 M-1s-1 (55 ± 0.5ºC, [H+] = 1.0×10-

2M), respectively. The trans-[RuIII(NH3)4(4-pic)(OH2)]3+ did not reacts with cysteine while trans-[RuIII(NH3)4(4-

pic)(OH)]2+ does at the specific rate constant of 7.4 M-1s-1 (pH = 4.8 µ = 0.2 M, 25ºC) yielding trans-[RuII(NH3)4(4-

pic)(OH2)]2+ and cystine, through an outer sphere mechanism. The calculated equilibrium constant for this reaction,

Keq= 2,6×102 M-1, allows to calculate, in the above experimental conditions, Eº’RSSR/RSH equal to +0.088 vs.

NHE.

Acknowledgements: The authors are thankful to the Brazilian agencies FAPESP, CNPq and CAPES for financial

support.

References

[1] Schafer, F.Q.; Buettner. G.R.; Redox environment of the cell as viewed through the redox state of the

glutathione disulfide/glutathione couple. Free Radical Biology & Medicine; 2001; 30; 1191–1212.

[2] Tfouni, E; Truzzi, D.R.; Tavares, A.; Gomes, A.J.; Figueiredo, L.E.; Franco, D. W. Biological activity of ruthenium

nitrosyl complexes. Nitric Oxide; 2012; 26; 38-53.

[3] Roncaroli, F.; Olabe. J.A.; The Reactions of Nitrosyl Complexes with Cysteine. Inorganic Chemistry; 2005; 44;

4719 – 4727.


102


New ruthenium(II) cyclopentadienyl thiosemicarbazone complexes with antitrypanosomal activity

Sarniguet, C.1; Morais, T.S.2; Tomaz, A.I.2; Medeiros, A.3; Comini, M.4; Otero, L.1; Garcia, M.H.2; Gambino, D.1 1Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay

2Faculdade de Ciências da Universidade de Lisboa, Centro de Ciências Moleculares e Materiais, Lisboa, Portugal

3Departamento de Bioquímica, Facultad de Medicina, Universidad de la República / Group Redox Biology of Trypanosomes,

Institut Pasteur de Montevideo, Montevideo, Uruguay 4Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay

Both classical octahedral ruthenium complexes and organoruthenium compounds have been widely explored for

their potential use in medicine as chemotherapeutics against different diseases. In particular, they have

demonstrated high potentiality as drug candidates for cancer therapy being promising alternatives to platinum

complexes. Indeed, two ruthenium compounds have already reached clinical trials for cancer therapy. The interest

on ruthenium stems on the fact that it shows chemical and biological properties that make its compounds

particularly suitable for the development of drug candidates. These properties make interesting the design of

antiparasitic ruthenium drugs1.

Among the infectious illnesses designated by the World Health Organization as neglected tropical diseases, those

caused by genetically related parasites from the Trypanosomatidae family (genus Trypanosoma and Leishmania)

constitute major health concerns in the developing world. Our group has been working on the development of

prospective antitrypanosomal ruthenium complexes with bioactive 5-nitrofuryl containing semicarbazones and

thiosemicarbazones as ligands. Metal-drug combined effects could lead to enhance ligands´ activity and to

generate new mechanisms of action.

In this work, we describe the synthesis, characterization and evaluation on Trypanosoma brucei brucei (strain 427)

of a novel organoruthenium (II) cyclopentadienyl compound with 5-nitrofuryl thiosemicarbazone (TN) as co-ligand.

The compound [RuCp(PPh3)(TN)] was synthesized by ligand substitution from a suitable Ru-Cp precursor and

characterized by elemental analysis, infrared and 1H, 13C and 31P NMR spectroscopies. It shows to be a highly

selective anti-trypanosomal compound with parasiticidal effect (IC50 T. brucei brucei 3.2 < M; IC50 J774

macrophages < 100 < M; selectivity index < 31.2).

Acknowledgements: Authors thank CYTED network RIIDFCM and Portuguese national funds through FCT, the

Portuguese Foundation for Science and Technology (PTDC/QUI-QUI/101187/2008, PTDC/QUI-QUI/118077/2010

PEst- OE/QUI/UI536/2011, Ciência2008 Initiative and grant SFRH/BD/45871/2008).

References

1. D. Gambino, L. Otero, Perspectives on what ruthenium-based compounds could offer in the development of potential antiparasitic drugs. Inorg Chim Acta 2012, 393, 103-114.


103


Structure activity relationships of new prospective antiparasitic compounds based on oxidovanadium(IV) complexes

Benítez, J.1; Fernández, M.1; Becco, L.2; Varela, J.3; Birriel, E.3; Correia, I.4; Lavaggi, M.L.3; González, M.3; Cerecetto, H.3; Moreno, V.5; Garat, B.2; Costa Pessoa, J.4; Gambino, D.1

1Cátedra de Química Inorgánica, Facultad de Química, UDELAR, Montevideo, Uruguay.

2Laboratorio de Interacciones Moleculares, Facultad de Ciencias, UDELAR, Montevideo, Uruguay.

3Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la

República, Montevideo, Uruguay. 4Centro de Química Estrutural, Instituto Superior Técnico, TU-Lisbon, Lisbon, Portugal.

5Departamento de Química Inorgánica, Universitat Barcelona, Spain.

American trypanosomiasis, caused by Trypanosoma cruzi, is endemic throughout Latin America and causes more

deaths in this region than any other parasitic disease. The available chemotherapy is based on old non specific

drugs that give rise to undesirable toxic effects, show limited efficacy in the chronic phase of the disease and suffer

from development of resistance.

In the search for new therapeutic tools against this disease twenty five structurally related oxidovanadium(IV)

mixed- ligand complexes, [VIVO(L-2H)(NN)], including five tridentate salicylaldehyde semicarbazone derivatives (L)

and five polypyridyl NN derivatives (NN) as ligands were synthesized and characterized in the solid state and in

solution. The compounds were evaluated in vitro on Trypanosoma cruzi. Most of the complexes showed

significantly higher in vitro activities than the reference drug Nifurtimox and than the free semicarbazone and NN

ligands. In addition, most of the complexes showed high selectivity indexes towards the parasite in respect to

mammalian cells (J774 murine macrophages). The interaction of the complexes with DNA was demonstrated by

gel electrophoresis, atomic force microscopy and viscosity measurements, suggesting that this biomolecule may be

a parasite target.

Having a large number of structurally related VIVO-compounds structure-activity relationships may be established.

The lipophilicity was determined for the whole series of compounds. A nearly parabolic relationship between

biological response and lipophilicity was observed and an optimal lipophilicity value could be established as a guide

for further drug design. The nature of the NN coligand was determinant of the biological activity. The substitution on

the phenol moiety of the semicarbazone ligand seemed to have only a low incidence on the antitrypanosomal

activity.

Acknowledgements: Authors thank CYTED networks RIIDFCM and RIDIMEDCHAG and Portuguese Foundation

for Science and Technology (FCT): (PTDC/QuiQui/101187/2008, PEst-OE/QUI/UI0100/2011, PEst-

OE/QUI/UI0612/2011, PEst-OE/QUI/UI0536/2011, Ciência2007 and 2008). M.F. thanks ANII Uruguay for a

fellowship.


104


Synthesis, single crystal structure analysis and evaluation of apoptosis in vitro of the trans-bis(3-aminoflavone)dichloroplatinum(II)

Ochocki, J.1; Fabijanska, M.1; Raber, G.2; Raab, A.3; Cebula-Obrzut, B.4; Smolewski, P.4; Krupp, E.M.5 1Department of Bioinorganic Chemistry, Medical University of Lodz, Lodz, Poland

2Institute of Analytical Chemistry, Karl-Franzens-University, Graz, Austria

3Department of Chemistry, University of Aberdeen, United Kingdom

4Department of Experimental Hematology, Medical University of Lodz, Lodz, Poland

5Department of Chemistry, University of Aberdeen / ACES Aberdeen Centre for Environmental Sustainability, Aberdeen,

Scotland [email protected]

Background: Currently, cisplatin (cis-DDP) is widely used in chemotherapy in many types of cancer.

Unfortunately, its application is limited because of serious side effects such as nephrotoxicity, myelotoxicity,

ototoxicity, allergy and the development of resistance in tumor cells. Therefore, studies are concentrated to search

for other platinum compounds, with at least equally effectiveness in chemotherapy and lower toxicity. Because of

the biological relevance of flavanone system, the derivatives of flavanone were used as the ligands in the synthesis

of novel cis Pt(II) and trans Pt(II) complexes, for which a possible application as antitumor agents is conceived.

Methodology: The compound was characterized using reversed HPLC (High Performance Liquid

Chromathography), Mass Spectrometry (MS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS),

Results: Our previous studies revealed that cis-bis(3-aminoflavone) dichloroplatinum(II) exhibited higher toxicity,

induced a higher degree of apoptosis and necrosis and a higher percentage of P53 and BAX expression in A549

human non small cell lung cancer in comparison with the classical antitumor agent cis-DDP. This complex

structurally related to cis-DDP, possesses flavones derivatives as non leaving ligands instead of amine with two

cis- bound labile chloride ligands. In this study, the crystal and molecular structure of trans-Pt[(af)2Cl2], (af = 3-

aminoflavone) shows a square planar geometry of PtL2Cl2, with two organic molecules and two chloride leaving

ligands in a trans configuration. The proapoptotic effect of platinum(II) complex in comparison with cisplatin in

leukemia cell lines (L1210, L1210R) and the mechanisms of apoptosis induction was examined using flow

cytometry analysis (detection of caspase-3 activity and Annexin V/PI assay).

Conclusions: Results suggested that trans-Pt[(af)2 Cl2] effectively induced programmed cell death in a dose- and

time- depended manner. Furthermore, the new compound revealed stronger apoptotic effect in contrast to cis-

DDP.

Acknowledgements: This work supported by Polish Ministry of Science and Higher Education grant No. N N405

674040


105


Bis-thiazole copper (II) complex in the research of new cytotoxic drugs

Velluti, F.1; Guidali, F.2; Alvarez, N.1; Serra, G.2; Medeiros, A.3; Comini, M.3; Ellena, J.4; Scarone, L.2*; Torre, M.H.1* 1Química Inorgánica (DEC), Facultad de Química, Universidad de la República, Montevido, Uruguay

2Química Farmacéutica (DQO), Facultad de Química, Universidad de la República, Montevideo, Uruguay

3Group of Redox Biology of Trypanosomes, Institut Pasteur, Montevideo, Uruguay

4Laboratorio de Cristalografía, Instituto de Física de São Carlos, Universidade de São Paulo, Sao Paulo, Brasil

Small natural macrocyclic peptides have attracted significant attention due to their interesting biological activities

[1]. Their geometry and the set of possible donor atoms suggest that their biological function might involve the

binding of metal ions inside the organisms. Taking into account previous studies based on 2-amino-thiazole copper

(II) and manganese (II) complexes [2], as part of our research for new candidates with anticancer activity employing

molecular simplification, the bis-thiazole type BT (Figure a) and their copper(II) complex (Figure b), were

synthesized.

The characterization of the ligand was performed by NMR, HRMS and IR and for the complex by elemental

analysis, UV- Vis, IR and X-ray diffraction spectroscopies.

The stoichiometry of this new compound was [Cu2(BT)2(Cl)2]•H2O. It presented a dimeric structure where two BT

molecules acted as bridges between both Cu(II) ions (Figure b). Each BT molecule linked with one Cu(II) through

the thiazole and amide nitrogen atoms and with the other Cu(II) through the other thiazole N atom and the

carboxylic oxygen. The distorted square pyramidal geometry of each Cu(II) is completed by Cl- ions. The

spectroscopic measurements agreed with the structure found.

The biological activity of BT and the copper complex against tumor cell lines (MCF-7 and HT29) and the cytotoxicity

in Murine macrophage J774 were determined.

References

[1] Comba, P.; Gahan, L.R.; Haberhauer, G.; Hanson, G.R.; Noble, C. J.; Seibold, B.; van den Brenk, A.L. ”Copper(II) Coordination Chemistry of Westiellamide and Its Imidazole, Oxazole, and Thiazole Analogues”. Chem. Eur. J. 2008, 14, 4393 – 4403.

[2] Alexandru, M.G.; Velickovic, T.C.; Jitaru, I.; Grguric-Sipka, S.; Draghici, C. “Synthesis, characterization and antitumor activity of Cu(II), Co(II), Zn(II) and Mn(II) complex compounds with aminothiazole acetate derivative”. Cent. Eur. J. Chem. 2010, 8(3), 639–645.


106


Antitumor activity of novel trans-platinum(II) complexes based on intercalating and sulfonamide ligands

Navarro-Ranninger, C.1; Del Solar, V.1; Martín-Santos, C.1; Alemán, J.2 1Inorganic Chemistry Department, Universidad Autónoma Madrid, Madrid, Spain 2Organic Chemistry Department, Universidad Autónoma Madrid, Madrid, Spain

[email protected]

Cisplatin (CDDP) is one of the most successful and traditional antitumor metal compoundswhich has been used in

nearly 50% of all tumor therapies1. However, different adverse effects, such as dose-limiting, nephrotoxicity,

peripheral neuropathy, tinnitus and hearing loss are known.Thousands of platinum complexes have been tested

during the last half-century;therefore, the continuous search for new platinum complexes (and especially with trans

geometry in our group) is an interesting area which have directed to hundred of chemists to do huge efforts in this

topic.

At this moment, only few platinum complexes with sulfonamide ligands and cis-geometry have been synthesized

and none of them have been evaluated in biological assays. Therefore, we will present the synthesis and in vitro

evaluation of new trans-monosulfonamide platinum complexes will be presented (right, Scheme). In addition, we

will focus our attention in simple intercalating compounds2, which can be incorporated into the platinum complex.

We will show how to prepare these compounds in one step from commercial available compounds in high yields

(50-95%), and their citoxicity studies (left, Scheme).

Acknowledgements: We acknowledge grants from the Spanish MICINN (SAF2009-09431, and CTQ2008-06806-

C02-01/BQU).J. A. thanks the Spanish MICINN for Ramón y Cajal contract. C. M. And V. S. thank Autónoma

University andSpanish government for pre-doctoral fellowships, respectively.

References

[1] For selected reviews in platinum anticancer complexes, see: (a) Bruyninx, M., Sadler, P. J., Curr Opin Chem Biol, 2008, 12, 197-206; (b) L. Kelland Expert Opin Investig Drugs, 2007, 16, 1009-1021.

[2] S. Neidle, Principles of Nucleic Acid Structure. Elsevier. Chapter 5, page 132, 2008.


107


Iodido complexes “the second generation”: trans-[PtI2(amine)(amine´)] with different aliphatic amines

Álvarez-Valdés, A.1; Parro, T.1; Medrano, M.A.1; Cubo, L.1; Carnero, A.2; Quiroga, A.G.1 1Inorganic Chemistry, Universidad Autónoma Madrid, Madrid, Spain

2IBIS. Hospital Universitario Virgen del Rocío, Sevilla, Spain

[email protected]

The research developed in our laboratories has demonstrated that the use of chemical modifications in metallic

based drugs structures (in particular trans platinum drugs) can result in improved anticancer activity [1]. Our work

regarding the importance of the leaving group has afforded very interesting results in the evaluation of iodido

complexes [2]. The potential of cis iodide complexes as antitumoral agent was firstly proven and the studies of the

trans parent complexes have not only afforded a higher cytotoxicity values, but also proven to have a peculiar

different reactivity (compared to cisplatin) versus sulfur donors and Cyt C [3].

Our experience on the design of non-conventional metallodrugs manifested in most of the cases, a higher

cytotoxicity using different aliphatic amines in the same structure [1].

Following these results, we are now exploring the activity of the iodido complexes bearing different aliphatic amines

in trans configuration. In this contribution, we will present the synthesis, characterization and cytotoxicity values in a

selected panel of cell lines of complexes with general formulae trans-[PtI2(amine)(amine´)]. The interaction with

biomolecules such as 5-GMP, supercoiled pBR322, N-AcCys and N-AcMet (sulphur donor models) will be also

presented, discussed and compared with the interaction. The interaction with sulphur donors is also investigated.

Acknowledgements: Financial support of Spanish MICINN: SAF 2009-09431 and SAF-2012-34424 and COST

CM1105 are acknowledged.

References

[1] Quiroga, A. G. Understanding trans platinum complexes as potential antitumor drugs beyond targeting DNA. J Inorg Biochem2012 114. 106-112.

[2] Messori, L., Casini, A., Gabbiani, C., Micheluci, E., Cubo, L., Rios-Luci, C., Padrón, J. M., Navarro-Ranninger, C., Quiroga, A. G. Cytotoxic Profile and Peculiar Reactivity with biomolecules of a Novel “Rule-Breaker” Iodidoplatinum(II) Complex.ACS Med Chem Lett 2010;1, 381-385.

[3] Messori, L., Cubo, L., Gabbiani, C., Álvarez-Valdés, A., Michelucci, E., Pieraccini, G., Ríos-Luci,C., León, L.G., Padrón, J.M., Navarro-Ranninger, C., Casini, A., Quiroga, A.G. Reactivity and Biological Properties of a Series of Cytotoxic PtI2(amine)2 Complexes, Either cis or trans Configured.Inorg Chem 2012; 51, 1717-1726.


108


Searching for gallium bioactive compounds: a new gallium(III) complex as prospective antitumoral and anti-Trypanosoma cruzi compound

Machado, I.1; Fernández, M.1; Becco, L.2; Brissos, R.F.3; Zabarska, N.3; Gamez, P.3,4; Marques, F.5; Garat, B.2; Gambino, D.1

1Cátedra de Química Inorgánica, Facultad de Química, UDELAR, Montevideo, Uruguay.

2Laboratorio de Interacciones Moleculares, Facultad de Ciencias, UDELAR, Montevideo, Uruguay.

3Departament de Química Inorgànica, QBI, Universitat de Barcelona, Barcelona, España.

4Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, España.

5Unidade de Ciências Químicas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Sacavém, Portugal

Cancer is the second largest cause of death in developed countries. The need to overcome the drawbacks exerted

by cisplatin, oxaliplatin and carboplatin, the worldwide approved metal-based anti-tumoral compounds, has lead to

the search for other metallodrugs. Chagas disease (American Trypanosomiasis), caused by the protozoa

Trypanosoma cruzi, constitutes a major health problem concentrated in the poorest regions of Latin America.

The anti-neoplastic activity of simple Ga(III) salts was discovered in the 1970s and much work has been performed

since then to elucidate their mechanism of action. To prevent hydrolysis processes and to improve cell membrane

permeation and oral bioavailability, coordination compounds of this metal ion are currently being developed. So far,

tris(8-quinolato) gallium(III) and tris(maltolato) gallium(III) have entered the clinical trials phase as prospective oral

antitumor agents.

In the search for gallium bioactive compounds we have been developing Ga(III) complexes with tridentate ligands

having an N, N, O donor set. In this work we report the synthesis and characterization of the complex [GaIII(L-

H)2](NO3) with 2-methoxy-6-(pyridin-2-ylcarbohydrazonoyl) phenol as ligand. Its biological potential has been

explored. Highly- proliferative cells such as Trypanosoma parasites and tumor cells show metabolic similarities that

have lead in many cases to a correlation between antitrypanosomal and antitumor activities. Therefore, both

activities are evaluated in this work. Three human tumor cell lines were selected that show different degrees of

resistance to metallodrugs: ovarian A2780 (low resistance), breast MCF7 (medium resistance) and prostate PC3

(high resistance) cells. The complex shows IC50 values in the low micromolar range on T. cruzi epimastigotes

(Dm28c strain) and on A280 cells. Complexation with gallium leads to the enhancement of the cytotoxic potency of

the organic ligand.

Acknowledgements: I.M. and M.F. thank ANII-Uruguay for a research grant. Authors thank ICREA (Institució

Catalana de Recerca i Estudis Avançats) and Ministerio de Economía y Competitividad de España (Proyecto

CTQ2011-27929- C02-01).


109


Antibacterial activity of silver(I) complexes with ligands having anti- inflammatory properties

Azócar, M.I.*; Muñoz, H.; Levin, P.; Dinamarca, N.; Gómez, G.; Paez, M.A.

Laboratory of Bioinorganic Chemistry, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile.

During recent decades silver complexes have been extensively studied for their excellent antibacterial properties,

which have proven to be even more effective than silver salts. This metal is active at low concentrations and has a

low toxicity [1]. Antibacterial experiments have shown broader antimicrobial activity spectra for silver complexes

with Ag-O and Ag-N bonds than with Ag-P and Ag-S bonds [1]. Investigations on silver complexes to date have

attributed their enhanced antimicrobial properties to distinctive weak Ag-O and Ag-N bonds in their structure [3,4].

Figure 1: Ligands used in the synthesis of silver compounds.

Herein, we describe the preparation, characterization (by FTIR, NMR, UV-C radiation and X-ray diffraction) and

antibacterial activity (MIC and Viability test) of five silver (I) complexes: AgIbu, AgNap, AgMef, AgAsp and AgSal,

where Ibu= iso-butyl-propanoic- phenolate, Nap=(+)-(S)-2-(6-methoxynaphthalen-2-yl) propanoate, Mef=2-(2,3-

dimethylphenyl)aminobenzoate, Asp=2-acetoxybenzoate and Sal= 2- hydroxybenzoate(See figure 1) against E.

coli and S. aureus.

The results show a tendency between the nature of Ag-X (X = Oxygen and Nitrogen) bonds and the rate or

efficiency of antibacterial behavior.

Acknowledgements

This work was supported by FONDECYT (Grants No 11080133 and 1100537), MINCYT- CONICYT(2010-188) and

CONICYT (Grant: 79090024). M.A. Paez and M. I. Azócar are also grateful to VRID USACH.

References

[1] Ahmad, S.; Isab, A.A.; Ali, S. and Al-Arfaj, A.R. Perspectives in bioinorganic chemistry of some metal based therapeutic agents. Polyhedron, 2006, 25, 1633 and references therein.

[2] Kasuga, N. C.; Sugie, A. and Nomiya, K. Syntheses, structures and antimicrobial activities of watersoluble silver(I)–oxygen bonding complexes with chiral and racemic camphanic acid (Hca) ligands. Dalton Trans., 2004, 3732.

[3] Abarca, R.; Gomez, G.; Velasquez, C.; Paez, M.A.; Gulppi, M.; Arrieta, A.; Azocar, M.I. Antibacterial behavior of pyridinecarboxylate silver(I) complexes. Chin J Chem. 2012, 30, 1631.


110


Antitumor activity of [Ru(O-O)(bipy)(dppb)]PF6 complexes and their interactions with bovine serum albumin

[O-O=cinnamic acid derivatives; dppb = 1,4-bis(diphenylphosphino)butane; bipy = 2,2´- bipyridine]

Graminha, A.E.*1; Honorato, J.2, Menezes, A.C.S.2; Cominetti, M.R.3, Batista, A.A.1 1Department of Chemistry, Federal University of São Carlos, São Carlos(SP), Brazil

2Department of Chemistry, Federal University of Goiás, Goiânia, Goiás, Brazil

3Department of Gerontology, Federal University of São Carlos, São Carlos(SP), Brazil

Phenolic compounds are secondary metabolites that are capable of inhibiting DNA damage1 and protecting the skin

cells from damage caused by UV radiation2. Among the phenolic acids, cinammic acid found to be an important

bioactive substance. Moreover, ruthenium complexes have been showing antitumor activity, in different cancer

cells, including metastatic cancer3. Therefore, in this work we aim to combine the antitumor properties of the

cinnamic acid derivatives, using it as ligand, with the ruthenium complex core, containing the ruthenium/dppb/bipy.

Hence, five new complexes of ruthenium (II) were synthesized from the precursor, cis- [RuCl2(bipy)(dppb)], with

ligands derivative of the cinnamic acid. The reaction with α- methylcinnamic acid (α-metcn); 4-methoxycinnamic

acid (4-metoxcn); 3-Hydroxy-4-methoxycinnamic acid (3,4-metoxocn) yielded complexes of the type [Ru(O-

O)(bipy)(dppb)]PF6, O-O = coordination by carboxylic group (Fig. 1), as products, by displacement of the chloride

ions from the precursor complex. The complexes were characterized by elemental analysis, spectroscopic and

electrochemical techniques. Furthermore, albumin binding studies with complexes showed good binding affinity to

BSA proteins giving relatively high binding constants. In vitro tests for antitumor activity against the MCF-7 human

breast tumor cell line were carried out for the new complexes, for the precursor complex (cis-[RuCl2(bipy)(dppb)]),

and for the free ligands as well. The complexes tested showed higher activities than the free ligands, and than the

reference drugs, cisplatin and doxorubicin.

Figure 1. Representative scheme of reaction between cis-[RuCl2(dppb)(bipy)] and cinnamic acid.

Acknowledgements

We thank CNPq, CAPES and FAPESP, for financial support.

References

1. Rice-Evans C, Spencer JP, Schroeter H, Rechner AR., Drug Metabol Drug Interact. 2000,17: 291-310.

2. Saija A, Tomaino A, Trombetta D, De Pasquale A, Uccella N, Barbuzzi T, Paolino D, Bonina F. Int J Pharm.,2000, 199: 39-47.

3. C.G. Hartinger, S. Zorbas-Seifried, M.A. Jakupec, B. Kynast, H. Zorbas, B.K. Keppler, J. Inorg. Biochem., 2006, 100: 891-904.


111


Synthesis of semi-sandwich complexes η6-C10H14-Ru(II) with nitrogen chelating ligands and interaction with DNA

Colina-Vegas, L.*1; Villarreal-Peña, W.1; Navarro, M.2; Batista, A.A.1 1Departamento de Química, Universidade Federal de São Carlos – UFSCar, São Carlos – SP, Brazil

2School of Chemical and Mathematical Sciences, Murdoch University, Australia

The development of half-sandwich η6arene-Ru (II) has attracted particular attention and extensively studied over

the past 20 years because these complexes show applications in different areas of scientific and industrial interest

[1]. The synthesis, spectroscopic characterization, crystal structures and electrochemistry of π-arene piano-stool

ruthenium (II) complexes with bipyridine and derivatives as nitrogen donor ligands are described. A series of six

organometallic compounds of formula [RuCl(η6-C10H14)(N∩N)]PF6 (N∩N = 1,10’-phenantroline, 4,7’-diphenyl-1,10’-

phenantroline, 2,2’-bipyridine, 5,5’-dimethyl-2,2’- bipyridine, 4,4’-dimethoxi-2,2’-bipyridine and 4,4’-di-t-buthyl-2,2’-

bipyridine) were synthesized and characterized. The solid state structures of the six complexes were determined by

X-ray crystallography (Figure 1) and their characterization were completed by IR spectroscopy, molar conductivity

and NMR studies (1H and 13C). Additionally, were done spectroscopic titration with DNA, to estimate interaction

constants between the metal complex and this macromolecule and in vitro tests for antitumor activity against the

MDA- MB-231 human breast tumor cell line were carried out for the new complexes. The electrochemical

measurements in dichloromethane compounds do not exhibit a reversible process, is only observed after addition

of acetonitrile, which showed the exchange between the ligand Cl- for CH3CN [2]. The crystallographic structures of

the compounds show a distorted octahedral geometry, the semi-sandwich, in all lengths and bond angles do not

vary significantly. In the study of metal complex and DNA interaction by spectroscopic titrations allowed us to

calculate constant interaction using two calculation methods well reported, the values of the interaction constants

are between 105 and 103 M-1 indicate a reversible interaction with DNA [3]. The complexes tested showed higher

activities.

Figure 1. ORTEP view of the complexes 1-6 with the thermal ellipsoids at the 50 % probability level.

Acknowledgements: We thank CAPES, CNPq and FAPESP for financial support.

References:

[1] Dutta, B., Scolaro, C., Scopelliti, R., Dyson, P. J., Severin, K., Importance of the π-Ligand: Remarkable Effect of the Cyclopentadienyl Ring on the Cytotoxicity of Ruthenium PTA Compounds, Organometallics, 2008, 27, 1355-1357.

[2] Gollas, B., Speiser, B., Zagos, I., Maichle-Mössmer, C., Electrochemistry of ruthenium metallocenes: Part 3. Synthesis and properties of ruthenium [22]paracyclophane complexes with methacrylic acid and methacrylate ester substitutents, J. Organomet. Chem. 2000, 602, 75-90.

[3] Navarro, M., Castro, W., Martínez, A., Sánchez-Delgado, R. A., The mechanism of antimalarial action of [Au(CQ)(PPh3)]PF6: Structural effects and increased drug lipophilicity enhance heme aggregation inhibition at lipid/water interfaces, J. Inorg. Biochem., 2011, 105, 276–282.


112


Study of the mechanism of action of Pt(CQ)2(Cl)2 through its interaction with nitrogenous bases

Colina-Vegas, L.*1,2, Castro, W.2, Navarro, M.2 1Universidad del Zulia, Maracaibo, Venezuela

2Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela

Transition metals in medicine is a field of growing chemical development which has enormous potential [1]. One

drug design strategy used in our laboratory was based on the binding of an organic compound with known

therapeutic value to a transition metal looking for a synergistic metal-drug effect [2]. Recently, we have found that

Pt(CQ)2(Cl)2 displayed good activity against various cancer cell lines and its mechanism of action seems to be

related with its interaction with DNA, through covalent binding [3]. In the present work, we show the synthesis of

Pt(CQ)2(Cl)2 and the study of the hydrolysis of this complex as a funtion of time using conductivity and UV-vis

spectroscopy was carried out. Additionally, we have studied the interaction of title complex with guanosine,

adenosine, guanosine 5'- monophosphate (5'-GMP) and adenosine 5'-monophosphate (5'-AMP) by NMR-1H and

NMR- 31P spectroscopic. The results indicated that both chloride ligands of the complex exchanges with water

molecules, these exchanges showed dependency with the time, temperature and solvent. Also, we have observed

that the interaction between this complex and guanosine and adenosine produced different changes in the

absorption spectrum of these nucleosides such as bathochromic shift and hyperchromic. The absorption spectra of

5'-GMP and 5'-AMP showed hypochromic (at 251 and 260 nm respectively), hyperchromic at 268, 330 and 345 nm,

and several isobestic points at 264 and 310 nm when they was tritrated with Pt(CQ)2(Cl)2. The study by NMR-1H

and NMR-31P indicated that the compound interacts with the guanosine and adenosine via primary amine; the

reaction was warmed in the case of adenosine. 5'-GMP in order to promote the interaction with the oxygen of the

phosphate group.

Figure 1. From left to right: Molecular structure of complex Pt(CQ)2(Cl)2 and spectroscopic titration with 5'-GMP and

5' –AMP

Ackowledge: This work was funded by IVIC.

References:

[1] Farrel, N. Uses of inorganic chemistry in medicine, The Royal Society of Chemistry, Bodmic, 1999.

[2] Gill, M.R., Thomas, J.A,. Ruthenium(II) polypyridyl complexes and DNA—from structural probes to cellular imaging and therapeutics. Chem. Soc. Rev. 2012, 41, 3179-3192.

[3] Navarro, M., Castro, W., Higuera-Padilla, A., Sierraalta, A., Abad, M., Taylor P., Sánchez-Delgado, R., Synthesis, characterization and biological activity of trans-platinum(II) complexes with chloroquine. J. Inorg. Biochem. 2011, 105, 1684-1691.


113


Adaptation of a novel class of proteasome inhibitors for treatment of multiple myeloma

Szenkier-Garcia, N.1; Navon, A.2; Sterchook, R.3, Gumienna-Kontecka, E.4; Szebesczyk, A.4; Kozlowski, H.4; Shanzer, A.1

1Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

2Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

3Biological Services, Weizmann Institute of Science, Rehovot, Israel

4Faculty of Chemistry, University of Wroclaw.

The proteasome is a large complex protein present in eukaryotes and Archaea, responsible for the ubiquitine-

dependent degradation of intracelular proteins, envolved in the regulation of multiple physiological functions, and

an established target of anticancer drugs.

In this work we describe the proteasome inhibition of a thiosemicarbazate-Cu complex, which, in contrast to all

other known proteasme inhibitors, lacks an electrophillic moiety capable of covalently binding the active sites.

We explored the possible mechanism of inhibition, and present experimental evidence and in silico calculations

showing that catalysis of the formation of a disulfide bond in the proximity of the β-2 active site may be envolved.

A series of derivatives possesing different substituents was prepared, as well as complexes of other transition

metals, and their activities were compared to the original compound. Among them, for one derivative, we suceded

to diminish proteasome activity in one order of magnitude.

Physicochemical characterization, including species distribution curves is also presented.

In conclusion, we have explored the chemical, biochemical and biological aspects of proteasome inhibitory activity

of thiosemicatbazate-Copper complexes.


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Abstract code: 021 AREA: MIEH POSTER

Arsenic environmental and health issues in Uruguay: a multidisciplinary approach

Mañay, N.1*; Pistón, M.2; Goso, C.3 1Cátedra de Toxicología, Facultad de Química, Montevideo, Uruguay

2Cátedra de Química Analítica, Facultad de Química, Montevideo, Uruguay

3Departamento de Evolución de Cuencas, Facultad de Ciencias, Montevideo, Uruguay

[email protected]

Scientific research has been recently conducted in Uruguay to study the presence of geogenic arsenic (As) in

groundwater and preliminary results, showed As levels above those recommended by WHO for drinking water

(10 µgL-1) in different aquifers of the country. For example, in Raigon aquifer, water samples showed arsenic

concentrations ranged from 1,4 to 39,7 µgL-1 with a median of 24,6 µgL-1.

Several laws and decrees regulate water sources quality and the drinking water is supplied only by a state

company that is controlled by a state regulation institute, being As new recommended levels < 20 µgL-1. In a 6

years period study of those data assessment, As concentration in drinking water samples taken from different

areas of the country, showed an increase from not detectable to quantifiable levels but within local regulation limits.

This environmental health issue is now becoming a matter of concern with a multidisciplinary approach, as similar

aims research teams and experts from geosciences and biosciences, joined to face those arsenic exposure risks

problems. The aim of this work is to review those environmental and health arsenic issues in Uruguay to show the

recent advances in our country.

As preliminary results, several multidisciplinary studies have been developed focusing on arsenic geological and

toxicological aspects. For this purpose, analytical and speciation methodologies were optimized and validated, for

arsenic analysis in water and urine as available tools in Uruguay. Arsenic biomonitoring and determination of urine

metabolites on general population and exposed workers, are currently ongoing research studies.

To assess population’s health impacts from chronic exposure to inorganic arsenic through drinking water and in the

workplace, it is important to continue developing those systematic studies and analytical tools because the risk of

the Uruguayan population has not yet been estimated.


115


Occupational exposure evaluation of workers of portland cement industry in Uruguay

Cousillas, A.; Pereira Testa, L.; Clavijo, G.; Mañay, N. Cátedra de Toxicología, Departamento Estrella Campos, Facultad de Química, Montevideo, Uruguay

[email protected]

Portland cement is a light gray or white powder used as a building material in the production of concrete with strong

adhesive properties when mixed with water. Employees who work with Portland cement are at risk of developing

skin problems, ranging from mild and brief to severe and chronic. Portland cement becomes highly caustic (pH>12)

when it isin contact with moisture in eyes or on skin, or when mixed with water, and will damage or burn the eyes or

skin. Inhalation may cause irritation to the moist mucous membranes of the nose, throat and upper respiratory

system or may cause or may aggravate certain lung diseases or conditions.

The aim of this work is to evaluate occupational exposure of workers of three uruguayan Portland cement plants.

46 personal and zone airborne samples were collected in plants 1, 2 and 3 and analyzed to determine occupational

exposure. Samples of total and respirable dust were collected according to NIOSH methods 0500 and 0600.

2010 ACGIH TLVs were considered as reference values being 1 mg/m3 for respirable Portland cement dust and

10 mg/m3 for total dust (PNOS).

We found that in Plant 1: 50 % of the samples were above the TLV value and 25 % exceeded del action level. In

Plant 2: 43 % > TLV and 21% > action level. In Plant 3: 38 % > TLV and 8 % > action level.

We discuss the reasons we observed that could lead to this results and conclude that there is a need of improving

working conditions. Important measures should be taken to prevent occupational exposure.


116


Portland Cement and health impacts

Pereira Testa, L.; Cousillas, A.; Mañay, N. Cátedra de Toxicología, Departamento Estrella Campos, Facultad de Química, Montevideo, Uruguay

[email protected]

Portland Cement is basically a finely ground cement clinker mixed with a small amount of calcium sulfate

dehydrate. It is obtained by heating to a high temperature a mixture of substances such as limestone, sand, clay

and shale. It is a light gray or white powder. Cement dusts are produced during blasting of raw materials, grinding

of cement clinker and packaging or loading of finished cement.

Although this material is widely used in building and construction works as an important ingredient in concrete

products, very scarce information on health effects of the particulate air pollution from cement factories, is

available. However, most cement dusts chemical elements are potentially harmful to the environmental biota in

general and children are the most susceptible affected human populations.

Some of the dangerous elements of cement dust include: Particulate Matter (PM2.5 & PM10), lead, arsenic,

chromium VI and chromium III, mercury, manganese, cadmium, crystalline silica among others.

It is well known that when Portland Cement, contacts with moisture in eyes or on skin, or when mixed with water, it

becomes highly caustic and will damage or burn the eyes or skin.

Uruguay has several Portland Cement kilns in different parts of the country and there are only few studies on

occupational exposure assessment.

This work describes the main health risks of populations living near a cement kiln and of occupationally exposed

workers. Risk assessment and safety conditions are explained with special emphasis on minimizing toxic effects of

cement pollutants.

We conclude that there must be a concern on risk management not only for health prevention but also for the

environment protection in cement quarrying sites and around cement factories.


117


Iron analysis from Octopus hubbsorum (Cephalopoda: Octopodidae) for human health

Palacios-Abrantes, J.1; Melo, V.1; Urbano, B.2 1Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Xochimilco, Ciudad de México, México

2Malacología, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Ciudad de México,

México [email protected]

The Food and Agriculture Organization of the United Nations declared that 12% of the world’s population is

undernourishand and for Mexico around 5% of the populations is included. Minerals are important components of

the daily diet. Hence, the important task of the scientific community is to augment information about the chemical

composition of edible organisms that human population consume on regular basis, this information will help to

improve world’s people health, nutrition, food security and ecological sustainability. Minerals are essential in

humans, the lack of them have been related to many degenerative diseases. Iron (Fe) is an important mineral on

human diet, and on the role of their function of the immune and central nervous systems, among others. Adult man

contains of iron on his body with a relation of 60 mg/kg, because the highly conservation rate (except menstruating

and pregnant woman), the daily need is considerably low for man. Despite this, iron anemia is the most common

nutritional deficiency worldwide, that lead governments to fight undernutrition, hunger and promote health. Typically

Mexican population gets the iron needs from vegetables (wich have poor absorption rates) and costal population

include fish every once in a while in their diet. Simpson et al. (1981) mention the impact of fiver on iron absorption It

is mention that that addition of 12g/day of bran to a meal decreases iron absorption by 51% to 74%, a considerable

fact for Mexican population who's basic food is rich in cereals. The aim of this work was to analyze the iron

contents of Octopus hubbsorum (Cephalopoda: Octopodidae) an animal reported at all mexican Pacific coast. Tree

specimens where analyzed, two from the north and one from central Pacific Ocean. Data obtained for organism of

central Pacific Octopus 0.008% and data for North Pacific was 0.00% and 0.011%. In conclusion, the aport of Iron

to the mexican diet provided by Octopus hubbsorum is significant, therefore is recommended to consume more of

this resource, to diminish iron deficiency, a world-wide problem and to improve human health.


118


Interactions of Mo(VI) oxyanions with environmentally relevant metal cations in natural ground waters

Gonzatto, L.1; Tissot, F.2; Torres, J.1; Kremer, C.1; Kremer, E.1 1Cátedra de Química Inorgánica, Facultad de Química, Montevideo, Uruguay 2Cátedra de Química Analítica, Facultad de Química, Montevideo, Uruguay

[email protected]

Molybdenum is an essential trace element, whose mobility in the upper crust is highly dependant on the

hydrological cycle. In natural waters, speciation and concentration of the main anionic components are important in

determining its bioavailability and environmental impact. Its chemical speciation in natural aquatic scenarios has

been yet poorly studied. This work reports the potentiometric study of oxyanions of Mo(VI) and their interaction with

some divalent metal ions of special environmental relevance.

Experimental conditions were chosen to simulate the natural ground waters media of low depth (20.0 C, low ionic

strength). The influence of the supporting electrolyte was also evaluated. In the absence of metal ions, MoO42- is

the predominant species for pH values above 4.4, HMoO4- predominates from pH 3.6 to pH 4.4and H2MoO4 is the

most abundant species below pH 3.6. Polymeric species such as [Mo8O26]4- and [Mo7O-]6- appear when the total

molybdenum concentration exceeds 1 mM.

The M2+ cations are bound to molybdenum anions to a considerable extent. For total molybdenum concentration

below 1 mM, the predominant form of molybdenum (VI) is still MoO42- above pH 4.4, with a variable percentage of

[M(MoO4)]+, depending on the metal ion concentration. As the pH value becomes lower than 4.4, the most

abundant species is [M(HMoO4)]. For example, for 10-7 M Mo(VI) in the presence of mean natural ground waters

concentrations of Ca2+ (16.3 mM) shows the predominance of [Ca(MoO4)]for pH values above 4.4, and

[Ca(HMoO4)]+ for more acidic media. For some transition metal ions, the low solubility of the MMoO4 salt prevents

the accurate detection of the soluble species [M(MoO4)] above pH 4.4.

Results show that in conditions simulating natural ground waters, the availability of Mo(VI) is strongly influenced by

pH media and the presence of +2 metal ions.

Aknowledgments: This work was partially supported by CSIC.


119


Metal ions binding and extraction using functionalized magnetic nanoparticles

Mendoza, C.1; Pericàs, M.2; Vilar, R.3; Jansat, S.2 1DEC, Facultad de Química, Universidad de la República, Montevideo, Uruguay

2Institute of Chemical Research of Catalonia (ICIQ), Tarragona, España

3Department of Chemistry, Imperial College London, United Kingdom, London

[email protected]

Magnetic nanoparticles are being widely applied in catalysis, cellular delivery systems, MRI and ion recognition and

sensing1. They offer the advantage that their movement can be controlled as required by the application of an

external magnetic field.

These nanoparticles, functionalized with ligands that have the ability to selectively bind metal cations could be

applied in metal recovery process and decontamination of water samples or biological fluids. The ligand

coordinates the cations and the nanoparticles are then removed from the solution by the application of an external

magnetic field2-3.

Copper (I) catalyzed 1,3-dipolar cycloaddition of an azide and an alkyne (click chemistry) has received broad

attention for its convenient characteristics as high yields and no by products, and it has been employed in the

functionalization of a variety of materials: polymers, silica, or nanoparticles.

In this work click chemistry was used to anchor crown ether- or monoaza-crown ether- type macrocycles onto

azide-magnetite nanoparticles. The obtained nanoparticles (1) were characterized by infrared spectroscopy,

thermogravimetric analysis, TEM microscopy and microanalysis. They were used in the extraction of heavy metals

from aqueous solutions. Metal concentrations were determined by flame atomic absorption spectroscopy,

achieving high extractions percentages. The same binding moieties were attached to a variety of polystyrene (PS)

resins, and the resulting functional polymers have also been used for the selective removal of cations from

aqueous and organic solvents solutions (2). The merits of these two extraction systems for particular situations are

discussed.

References

1. Lu, A.-H.; Salabas, E. L.; Schüth, F., Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew Chem Int Ed; 46; 2007; 1222-1244.


120

2. Wang, L.; Yang, Z.; Gao, J.; Xu, K.; Gu, H.; Zhang, B.; Zhang, X.; Xu, B., A biocompatible method of decorporation: biophosphonate-modified magnetite nanoparticles to remove uranyl ions from blood. J Am Chem Soc; 128; 2006; 13358-13359.

3. Koehler, F. M.; Rossier, M.; Waelle, M.; Athanassiou, E. K.; Limbach, L.K.; Grass, R N.; Günther, D.; Stark, W.J., Magnetic EDTA: coupling heavy metal chelators to metal nanomagnets for rapid removal of cadmium, lead and copper from contaminated water. Chem Commun; 2009; 4862-4864.


121


Manipulation of metalloproteins for heavy metal bioremediation and detection

Zhao, J.1; Wei, W.2 1School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

2School of Life Sciences, Nanjing University, Nanjing, China

[email protected]

A gold-specific sensory protein GolS from Samonella gol regulon was incorporated into E. coli, which in conjunction

with an engineered downstream red fluorescence protein allowed the highly sensitive and selective whole-cell

detection of gold(III) ions by naked eyes. The putative gold-chaperone, GolB, in the gol regulon was next verified to

be specific to gold(I) ions over other metal ions including copper(I). The subsequent display of GolB on E. coli cell

surface permitted selective enrichment of gold ions from media containing various thiophilic metal ions. The cell

surface- enriched gold(I) was further shown to be easily recovered and the gold-deprived bacteria were capable for

re-usage. E. coli bacteria harboring these gold-specific elements from the gol regulon could be a valuable tool for

visual detection and facile recycling of gold ions from environmental resources. A similar approach to lead ions will

also be discussed.


122


Mechanisms of cell death induced by cytochrome c metal-substituted species

Prieto, T.1; Smaili, S.2; Nascimento, O.3; Nantes, I.1 1Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, Brazil

2Farmacologia, Universidade Federal de São Paulo, São Paulo, Brazil

3Instituto de Física, Universidade de São Paulo, São Carlos, Brazil

[email protected]

Cytochrome c is a mitochondrial respiratory hemeprotein that participates of respiratory chain, elimination of

superoxide ion and apoptosis. The latter role is played by the protein in the cytosol as resulting of endogenous

mobilization or exogenous deliver to cells via microinjection or cell carriers. In the present study, apoptosis was

induced by the delivery of exogenous metal-substituted cytochrome c species and microperoxidases (hemepeptide

product of cytochrome c proteolytic digestion) to rabbit aortic smooth muscle cells. The delivery vehicles were

microinjection and dioctadecyl dimethylammonium bromide (DODAB) liposomes. It was observed that only zinc-

substituted cytochrome c that retains the native cytochrome c structural features was efficient to activate caspase-

3. Although unable to active caspase 3, free base-, Fe3+- and Mn3+ -microperoxidases-11 as well as free base-

cytochrome c led to the occurrence of typical events of the apoptosis program, such as: nuclear fragmentation,

membrane blebbing and extracellular exposure of phosphatidylserine. In addition, it was also observed that the

delivery of free base and native iron cytochrome c led to lose of cellular adhesion simultaneously to the induction of

cell death. DODAB liposomes loaded with heme-peptides and proteins were less efficient than microinjection to

induce cell death. These results suggest that protein structure and surface rather than the redox center determine

the efficiency of cytochrome c to activate caspases, however the peroxidase activity of the redox centers promote

oxidative stress that could induce apoptosis via endogenous cytochrome c mobilization.

Aknowledgments: Supported by FAPESP and CNPq


123


Correlations among water, blood and hair lead concentrations in first-grade children from Uruguay

Martínez Savio, G.1; Mañay, N.1; Guido, M.2; Kordas, K.3; Queirolo, E.4 1Catedra de Toxicología e Higiene Ambiental, Facultad de Química (UdelaR), Montevideo, Uruguay

2Catedra de Toxicología e Higiene Ambiental, Facultad de Química(UDELAR), UCUDAL, Montevideo, Uruguay

3UCUDAL, Pensilvania, Estados Unidos

4UCUDAL, Montevideo, Uruguay

[email protected]

Over the last decade, environmental lead exposure and its impact on public health have been one of the biggest

concerns in Uruguay, mainly within the low income populations. Lead does not play any physiological role in

humans. However, often significant body lead burdens are present in humans, especially children, in relation to the

recommended limit values. Children’s exposure to lead, depending on the concentration, can be related to

physiological disorders, intellectual impairment or behavior problems. Therefore, it is important to monitor lead

concentrations in children’s environment to understand its relationship to the accummulation of the metal in

chidlren’s bodies.

The aim of this study was to measure and correlate lead levels in biological and environmental samples in first-

grade Montevideo children (N=134, ages 6-7 years). Lead children’s whole bood, in household water used for daily

consumption, and children’s hair was measured using atomic absorption spectrometry and ICP-MS, respectively.

Mean blood lead concentrations (BLL) in the study sample were: (4,9 ±2.2) µg/dL (range 1,80-13,2), Pb-hair (3,5 ±

3.9) µg/g (range 0,17-23,8) and Pb-Water (4,6 ± 10.6) µg/L (range 0,10-57,2). 31.3 % of the children have BLL > 5

µg/L, 10.4 % of the household water was > 10 µg/L, and 25.4 % children had > 3.9 µg/g of Pb in hair. SPSS was

used for this Exploratory Data Analysis (EDA). None of the three variables studied were normally distributed,

therefore bivariate correlations for pairs of variables performed using Spearman Rank Correlation Test.

Hair and water Pb levels were significantly correlated (p =0.04), p<005. However there was no significative

correlation between Pb in water and Pb in blood (p > 0,05), and neither between Pb in hair and BLL.

Lead in blood represents environmental exposure over the short term while lead in hair may represent longer-term

exposure. Thus, the contribution of lead in water as an environmental health risk should be taken into account for

further studies.


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Assessment of environmental exposure to methyl mercury in polish women of childbearing age

Trzcinka-Ochocka, M.; Brodzka, R.

Biological Monitoring Laboratory, Nofer Institute of Occupational Medicine, Lodz, Poland [email protected]

Background: Methyl mercury (MeHg) is well–documented neurotoxicant and primarily affects the central nervous

system. The main exposure pathway of Hg to humans is through the consumption of marine fishery products. The

MeHg exposure of 306 occupationally non-exposed women of childbearing age, inhabitants of three Polish regions,

with different levels of fish consumption: Lodz (n=100), Wladyslawowo (n=107) and Gizycko (n=99) was assessed.

Methodology: Total mercury concentrations in hair (THg-H) was determined by flow-injection cold vapor atomic

absorption spectrometry technique using Mercury Analizer after mineralization of hair samples in microwave

digestion system. Detection limit (DL 3s) of method is 0.052 µg/g and quantification limit (QL) 0.104 µg/g. Accuracy

of the method was verified by the using certified reference materials: BCR CRM 397 (accuracy = 0.8%) and NCS

DC 73347 (accuracy = 13.89%).

Results: The results showed that the geometric mean concentrations of THg-H among women of Lodz were

statistically significantly lower 0.114 ± 2.02 µg/g (2.6 fish/month) than in the other two regions (p<0.05). The

concentrations of THg- H of women in Wladyslawowo and Gizycko increased in proportion to the consumption of

fish per month and amounted 0.177 µg/g ± 2.89 (6.5 fish/month) and 0.235 µg/g ± 2.17 (10.4 fish/month)

respectively. We found a statistically significant positive correlation (p<0.05) between THg-H concentration and the

number of fish meals/month in all examined regions: for Lodz, y = 0.0087 x + 0.1223, r = 0.226; for Wladyslawowo,

y = 0.0215 x + 0.1456, r = 0.464, and for Gizycko, y = 0.0151 x + 0.1727, r = 0.433.

Conclusions: The obtained results indicated that THg-H were low but in accordance with data quoted in the

current literature. The hair mercury concentration increased with increasing of fish consumption.


125

Abstract code: 040 AREA: NATME POSTER

Trace elements in soybean seeds and their correlation with the in vitro quality parameters

Machado, I.1; Cardoso, J.2; Irigoyen, J.O.3; Viera, I.2; Pistón, M.1*; Torre, M.H.2 1Cátedra de Química Analítica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay

2Cátedra de Química Inorgánica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay

3Agropecuaria Valdense S.R.L., Colonia, Uruguay

*[email protected]

Soybean is an important food source in the world. To ensure a good harvest, country governments recommend the

evaluation of seed quality using several in vitro tests for viability and vigor, properties related to the potential for

rapid, uniform emergence, and development of normal seedlings under a wide range of field conditions. However,

the results of these in vitro tests do not always fully correlate with the actual development of plants in the crops. On

the other hand, it is well known that mineral content in plants and seeds are fundamental for plant growth and

health.

As a part of our studies, Fe, Cu, Zn, Mn and Ni levels in soybean seeds were assessed and correlated with vigor

and viability tests done on the same seeds, with the aim of understanding the poor performance observed for some

of them.

Sample treatment was performed according to AOAC 975.03. The trace elements were determined by Flame

Atomic Absorption Spectrometry in 10 representative lots of seeds with good and bad viability.

To ensure the accuracy a reference material was also analyzed (NIST 1567a). Analytical precision was better than

6%. Soybean seeds with viability higher than 95% presented ranges of: 47.0 - 90.6 mgkg-1, 10.7 - 15.0 mgkg-1, 33.9

- 51.5 mgkg-1, 19.5 - 24.1 mgkg-1, 3.9 - 5.8mgkg-1 for Fe, Cu, Zn, Mn and Ni respectively.

These results are in accordance with the reported ranges in the literature for soybean seeds. No significant

difference in metal level was observed between seeds with viability less than 95% and those with higher viability,

except for Zn levels that were higher in the former. On the other hand, a negative correlation between vigor results

and Fe levels was observed.


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Escamoles ant eggs Liometopum apiculatum M source of metal ions for human health

Melo Ruiz, V.1; Quirino, T.1; Macín, S.1; Calvo, C.2; Muñiz, I.1 1Division de Ciencias Biologicas y de la Salud, Universidad Autónoma Metropolitana Unidad Xochimilco, Ciudad de México,

México 2Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Ciudad de México, México

[email protected]

The earth crust is believed to be made of a mass of minerals and the participation of these elements in the physical

world is matched by their importance in human life. The human body like other leaving organisms depend on

several minerals as essential constituents of its existence. Metal ions in foodstuff are in different chemical forms, as

inorganic salts or organic molecules or complexes with other compounds such as proteins, amino acids, enzymes

and some vitamins, among others, that play an important role in human health. Entomophagy, insect consumption

by several ethnic groups in Mexico as cultural tradition since prehispanic era represent an option for population to

obtain the minerals needed by the body to keep a good health. Escamoles ant eggs of the Liometopum apiculatum

M genus, much appreciated either at rural communities as well as in urban cities, contain minerals with a favorable

effect in human health. The aim of this study was to investigate the mineral composition of Escamoles and the

benefits they can provide to the human body. Sampling was perform at an arid region of the Hidalgo state on April

2012. Minerals in dry basis were determined by Atomic Absorption Spectrophotometry, with the exception of

phosphorus, content was obtained from a triple acid digested extract and determined colorimetrically. Data of

mineral analysis in dry basis of Escamoles was: total minerals 5.92%; Na 0.079%; K 0.075%; Ca 0.097%; P

0.701%; Fe 0.021%; Zn 0.035%; Cu 0.009%; Mg 0.998%; Mn 0.002%. Minerals quantify are not equal to total ash

contained because not all of them were determined. Elements concentration depends not only of the total mineral

composition of foodstuff but also on their availability and lability on it. Minerals in Escamoles ant eggs have a

considerable influence in the condition of human health.


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Effect of zinc supplementation on the production of IL-17 in elderly individuals

Aguilar Cardenas, A.E.1; Sanchez, M.T.C.1; Muñoz, B.1; Espejel, G.2; Saldivar, L.2; Pastelin, R.1 1Department of Biology, University of Mexico, Mexico DF, Mexico

2Department of Analytical Chemistry, University of Mexico, Mexico DF, México

[email protected]

The suboptimal contribution of micronutrients may damage the immune response. In the particular case of zinc, this

microelement has shown their participation in various immunological processes around perinatal stages. In the

elderly, eating lesser amounts of food can cause a negative balance in the concentration of zinc and its possible

effects on immunity. In this regard, it is interesting to know the impact of zinc may have on proinflammatory

cytokines such as IL-17 for its role in delayed hypersensitivity and its possible effect in the induction of

autoimmunity. This study aimed to evaluate the effects of zinc supplementation on the production of IL-17 over

elderly stages. For this, we used an experimental model of zinc supplemented mice (500 microgram/mL zinc

acetate, BALB/c inbred mice). We measured the microelement concentration in lymphoid organs by Atomic

Absorption Spectrometry and also the cytokine production in serum by double antibody immunoenzime assay.

Results showed that female group of supplemented mice diminished the cytokine production 34.35 % compared

with control group whereas male group of supplemented animals showed a noticeable reduction of 54.34 %.

Further, zinc concentration increased in the spleen of female supplemented mice (80%). We concluded that

supplementation of zinc in mice has the effect of causing decreased production of IL-17 by Th17 cells, which far to

produce a problem of autoimmunity this means a regulatory response to supplemented individuals.


128


Star fish source of calcium to prevent this metal ion deficiendy

Palacios, J.; Melo, V.; Quirino, T.; Dominguez, O.; Schettino, B. Universidad Autónoma Metropolitana, Xochimilco, México

[email protected]

Calcium is the most abundant mineral in the body. It forms a functional reserve during mineralization of bone and is

required by essentially all body metabolic processes1. Therefore finely harmony homeostatic control mechanisms

to maintain constant blood levels of this mineral should be studied, since, it has complex cellular mechanisms to

control movement of intracellular calcium. Inadequate calcium consumption might cause on increased risk of

several diseases. Scientific community think Star fish (Echinodermata: Asteroidea) is high in minerals mainly in

calcium due to the calcareous osicles that forms the animal's body. Available all year, sometimes is consumed by

residents along the Atlantic coast. The objective of this research is to assess calcium content to inform people the

benefits of an adequate intake can provide in human health. Starfish obtained in Acapulco Bay last August was

analyzed by tirtation with EDTA method. Data obtained was 1.3%. In conclusion starfish is a good calcium source

to reduce the risk to several diseases such as osteoporosis, hypertension, cardiovascular dysfunction, cancer and

kidney stones in human health, it can also be an alternative food source of calcium for lactose intolerant

people2,3,4,5,6,7. Consumption is highly recommended suggesting no excessive calcium intake to avoid adverse

effect for human health and it's consumption is highly recommended. It can also be an alternative food source of

calcium for lactose intolerant people.

References

1. Crabb E. & Moore E (Ed.) Metals and life. RscPublishing 2010

2. Gebhardt L. & Silva E. Dairy augumentation of total and central fat loss in obese subjects. Int Jobes Relat Metab Disord. 2005 Vol 29 pp 391-397

3. Dawson-Hughes B. Interaction of dietary calcium and protein in bone health in humans. J Nutr. 2003 Vol 133 pp 8525-8545

4. Wu K., Willett W.C., Fuchs C., GOlditz G., Giovanuchi E. Calcium intake and risk of colon cancer in women and men. J Natl. Cancer Inst. 2002 Vol 94 pp 437-446

5. Heaney R. Calcium diary products and osteoporosis. J am Coll Nutri, 2000 Vol. 19 pp 835-995

6. Heller J. The role of calcium in the preention of kidney stones. J Am Coll Nutr. 1999 Vol 19 pp 3375-3785

7. Bucher H., Cook R., Guyatt G., Lang J., Hatala R., Hunt D. Effects of dietary calcium supplementation on blood pressure: a metaanalysis of randomized controlled trials. JAMA 1996 Vol 275 pp 1016-1022


129


Preliminary study of superoxide dismutase activity in soybean seeds with good and poor in vitro viability used in Uruguay

Cardoso, J.1; Machado, I.2; Irigoyen, J.O.3; Pistón, M.2; Viera, I.1; Torre, M.H.1* 1Química Inorgánica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay 2Química Analítica, DEC, Facultad de Química, UdelaR, Montevideo, Uruguay

3Agropecuaria Valdense (J.J.Ferreira), Colonia, Uruguay

[email protected]

Reactive oxygen species (ROS) such as superoxide and hydroxyl radicals are present in seeds, plants and animals

as a result of normal aerobic metabolism. If the organisms are not able to control the intracellular ROS level, they

may suffer damage in membrane lipids, proteins and nucleic acids leading to the death of the cells. For

detoxification of ROS, efficient antioxidant systems both enzymatic and non-enzymatic ones are present in all cells.

Superoxide dismutases (SOD) can react with O2- radical to prevent the damage. They are metalloproteins where

Cu, Mn, Zn or Ni are linked to the peptide residues. Therefore when the metal levels are low the activity of these

enzymes decrease and different disorders can appear.

The SOD extraction from seeds was optimized by means of multivariate experiments (best conditions: stirring time

60 min using 10 mL of phosphate buffer pH=7).

The SOD activity was determined by the method based on the inhibitory effect of SOD over the reduction of NBT

by the O2- generated by xanthine/xanthine oxidase system.

SOD activity of soybean seeds used in our country was in the range 20-63 %, similar in all the analyzed seeds. The

results showed that no significant difference was observed in seeds with viability less than 95%. In these

conditions, the poor viability of soybean seeds used in our country is not explained by oxidative stress.


130

Abstract code: 024 AREA: RMNM POSTER

α-MSH analog cyclized through rhenium coordination as new potential agent for melanoma imaging

Teixeira, V.1; Fernadez, M.1; Moreno, M.2; Chabalgoity, J.A.2; Gambini, J.P.3; Porcal, W.4; Quinn, T.5; Cabral, P.1 1Radiofarmacia, Facultad de Ciencias, UdelaR, Montevideo, Uruguay

2Biotecnología, Facultad de Medicina, UdelaR, Montevideo, Uruguay

3Medicina Nuclear, Facultad de Medicina, UdelaR, Montevideo, Uruguay

4Química Orgánica, Facultad de Química, UdelaR, Montevideo, Uruguay

5Biochemistry, University of Missouri, Columbia, USA

[email protected]

Background: α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium metal

coordination (ReCCMSH) present high chemical stability and affinity for the MC1R receptors of melanoma cells. In

this way the radiolabelled ReCCMSH is a potential radiopharmaceutical for melanoma diagnosis and treatment. 99mTc is the radioisotope most used in nuclear medicine diagnostic imaging owing to its optimal nuclear properties,

ready availability, low cost and favourable dosimetry. The bifunctional chelators tetraamine (N4) forms octahedral

complexes with 99mTc as [Tc(V)O2](tetraamine)+. 99mTc-tetraamine (N4)]ReCCMSH, presented herein, and can be

used as a potential MC1R-targeting radiotracer based on 99mTc for non invasive in vivo imaging of melanoma.

Methodology: the synthesis of the peptide N4-ReCCMSH was performed in an automated peptide synthesizer. N4-

ReCCMSH was radiolabeled with pertechnetate in alkaline conditions (pH 11.5) in presence of citrate and stannous

chloride as reduceding agent. Radiochemical evaluation of conjugates, as well as studies of stability and partition

coefficient was performed. Biological studies included binding, internalization, blocking studies at 30, 60, 90 and

120 minutes in B16-F1 mouse melanoma cells, biodistribution at 1, 2 and 24 hours in normal mice and in tumor-

bearing mice (C57 BK with B16-F1 mouse melanoma cells).

Results: The radiolabeling was efficiently performed at 37ºC in 30 minutes. 99mTc-N4ReCCMSH was obtained with

a purity >99% and was stable up to 24 hours post incubation. The cell studies exhibit high binding, fast

internalization and inhibition of 70% against unlabeled peptide. Biodistribution studies in mice demonstrated a

favourable pharmacokinetics with rapid urinary excretion, very low hepatobiliary elimination, combined with a high

and specific uptake in the tumor (1H, 8% ID/g)

Conclusions: these promising preclinical results of 99mTc-N4ReCCMSH warrant its potential candidature for clinical

translation as a novel 99mTc based in vivo non invasive diagnostic melanoma agent.

Acknowledgements: ANII, PEDECIBA QUIMICA and CHLCC.


131


Development and optimization of 177Lu-DOTA-Tocilizumab: a potential Multiple Myeloma theragnostic agent

Camacho, X.1; Calzada, V.1; Gutiérrez, E.1; Fernández, M.1; Moreno, M.2; Chabalgoity, J.A.2; Riva, E.3; Cabral, P.1 1Nuclear Research Centre, University of the Republic, Faculty of Sciences, Montevideo, Uruguay

2Institute of Hygiene, University of the Republic, Faculty of Medicine, Montevideo, Uruguay

3Clinical Hematology, University of the Republic Faculty of Medicine, Montevideo, Uruguay

[email protected]

Antecedents: Interleukin 6 (IL-6) is one of the key molecules related to growth, survival and proliferation of Multiple

Myeloma (MM) cells. Tocilizumab is a humanized monoclonal antibody directed against the receptor both soluble

and membrane of IL-6. Lutetium is a lanthanide element, in the f-block of the periodic table. Its only common

oxidation state is +3. 177Lu is a ß-particle emitter (497 keV, 90%), γ-rays (113 and 208 keV, 6% and 11%) and half-

life of 6.7 days. Tocilizumab labeled with 177Lu could be a potential MM theragnostic agent.

Methodology: Tocilizumab was purified by PD-10 column equilibrated and eluted with NaHCO3 0.1 M, pH=8.5 and

was derivatized with DOTA-NHS at 4ºC for 18 h. DOTA-Tocilizumab was radiolabeled with 177LuCl3 (15 MBq/mg) at

37ºC for 1 h. MALDI TOF/TOF was used to determine the level of DOTA conjugation to Tocilizumab.

Radiochemical purity was determined by ITLC-SG and by HPLC. In-vitro stability of 177Lu-DOTA-Tocilizumab was

studied in solution, serum, DTPA and EDTA. In-vitro studies binding and competition were performed with U266 MM

cells up to 120 minutes. Biodistribution studies were performed in normal Balb/C mice at 24 and 48 h (n = 5).

Results: DOTA-Tocilizumab was efficiently labeled with 177LuCl3. The in-vitro stability of labeled product was

optimal over 72 h in solution and serum, being stable and don’t showing significant transchelation. In-vitro

experiments binding and displacement confirm the specificity of recognition of 177Lu-DOTA-Tocilizumab by IL6R.

Biodistribution studies showed hepatic uptake and renal elimination.

Conclusions: 177Lu-DOTA-Tocilizumab was easily and rapidly labeled. This is a potential new agent for MM

theragnostic agent.

Acknowledgements: ANII, Roche Laboratories, Pro.In.Bio, PEDECIBA Química.


132


Validation of an atomic absorption spectroscopic method for the quantification of copper in tetrakis (2-methoxy isobutyl isonitrile) copper (I) tetrafluorborate

Leonardi, N.1; Fuda, J.2; Torti, H.2; Molinari, M.3; Zubata, P.4; Salgueiro, J.1; Zubillaga, M.1 1Radioisotopes Laboratory, Physics Department, School of Pharmacy and Biochemistry, Buenos Aires, Argentina

2Physics Department, School of Pharmacy and Biochemistry, Buenos Aires, Argentina

3Quality Assurance, Bacon Laboratories, Buenos Aires, Argentina

4Technical Direction, Bacon Laboratories, Buenos Aires, Argentina

[email protected]

Antecedents: The quantification of tetrakis (2-methoxy isobutyl isonitrile) copper (I) tetrafluorborate (CuTFB) is an

important issue when measuring samples of Sestamibi, a common radiopharmaceutical for cardiac imaging. The

aim of this work was to validate the copper determination in CuTFB by means of an atomic absorption

spectroscopic (AAS) methodology to be use as an indirect quantitative method for CuTFB.

Material and method: Validation characteristics assayed were linearity, precision, accuracy, specificity and range

according to the ICH Q2. All CuTFB and copper standard solutions were prepared using distilled water and

processed in order to be measure by AAS.

Results: The method proved to be linear showing a correlation coefficient (R2) for linearity of 0,9962. In precision

studies the results obtained for repeatability were 0% and for intermediate precision were 0,2%. There was no

significant difference for both analysts working under different conditions (different days). For accuracy, the

recovery percentages were between 99,5 % and 101,1 %. In specificity studies there were no noticeable

interferences of tin in copper determination. In robustness studies there was a lack of influence of operative

variables studied compared to results obtained without variables. For the purpose of this methodology, the

specified range is between 50 µg and 1,5 mg of CuTFB.

Conclusion: The results obtained showed that the proposed method for the quantification of CuTFB, by means of

copper detection in AAS, proved to be linear, precise, accurate and specific.

References: International Conference on Harmonization (ICH Q2), version 4, 2005.

Acknowledgments: This work was financial supported by project UBACYT 20020100100489 from the University

of Buenos Aires.


133


99mTc labelling of phenazine dioxides with recognized DNA interaction capacity. In vitro and in vivo studies

Tejería, E.1; Berchesi, A.2; Fernández, S.1; Sanz, I.1; Cerecetto, H.2; González, M.2; Lavaggi, M.L.2; Rey, A.1 1Cátedra de Radioquímica, Facultad de Química, UdelaR, Montevideo, Uruguay

2Grupo de Química Medicinal, Facultad de Ciencias, UdelaR, Montevideo, Uruguay

[email protected]

Phenazine dioxides are drugs used for antitumor therapy that exhibit DNA-interaction and produce DNA-damage.

We have selected two of them that have demonstrated good in vitro DNA interaction, namely ML44 (2-

aminophenazine N,N’-dioxide) and ML84 (2- amino-7-fluorophenazine N,N’-dioxide) and we herein present their

labeling with 99mTc- and their evaluation as potential radiopharmaceuticals. Labeling through formation of 99mTc(I)-

tricarbonyl complexes, was performed in two stages: 1) preparation of the precursor fac[99mTc(CO)3 (H2O)3]+ by

sodium pertechnetate reduction (40-50 mCi, 1.0 mL) with sodium borohydride (7.0mg) under CO (g) atmosphere, in

alkaline medium at 70°C, 20 minutes, 2) substitution with phenazine: ML44 or ML84 (2-3 mg in 0.5 mL of DMSO,

30 minutes at 70°C). The radiochemical purity (RP) of precursor and the final compounds were controlled by

reverse phase HPLC. The precursor showed a retention time (rt) of 4 min and a RP > 90 %. The labelled

phenazines had a rt of 21 and 22 min., respectively and a RP > 95 % and were stable for at least 4 h. Both

complexes were 100 % stable in plasma at 37°C. The 99mTc(CO)3 -ML44 complex showed 100% stability against

histidine solution (100-fold excess), while the 99mTc(CO)3 -ML84 complex suffered a 15 % transchelation. The

lipophilicity (partition coefficient octanol:phosphate buffer pH = 7.4) was log P = 0.12 ± 0.07 and 0.50 ± 0.06,

respectively. The plasma protein binding (PPB) was 35.2 ± 4.5% and 54.0 ± 0.9%, respectively. Attempts to reduce

the PPB through formation of more stable 2+1 complexes using acetylacetone as bidentate coligand were

unsuccessful. Biodistribution at 30 and 120 minutes in normal mice (females, CD1, 3 months, 3 animals per group)

showed high liver uptake: 28.1 ± 2.9% and 35.9 ± 3.6% at 2 h, respectively, consistent with the high PPB. Excretion

occurs mainly via the hepatobiliary tract and uptake in other organs is low. Studies in tumor bearing mice will

determine its potentiality as radiopharmaceuticals for Nuclear Oncology.

Acknowledgements: CSIC (Proy. 622), ANII, Pedeciba-Química


134


Value of 99mTc in the characterization of an anti-Tn chimeric antibody

Vasilskis, E.1; Trindade, V.1; Giglio, J.1; Reyes, L.1; Oliver, P.1; Balter, H.1; Osinaga, E.2; Engler, H.1 1CUDIM, Montevideo, Uruguay

2Medicine Faculty, Montevideo, Uruguay

[email protected]

Tc (I)-Tricarbonyl complexes synthesis leads to stable precursors in aqueous solution and wide pH range, useful in

the labeling of biomolecules by ligand exchange. Carboboranes as reducers and CO groups source in situ, allow

synthesize [99mTc(CO)3(H2O)3]+, as intermediary complex. H2O molecules in carbonyl complex have adequate Kd

for the exchange and transference to protein and peptides through histidine residues.

We report here the labeling optimization of the monoclonal chimeric anti-Tn with 99mTc through [99mTc(CO)3(H2O)3]+

complex, and its characterization by HPLC and ITLC-SG as well as biodistribution studies.

[99mTc(CO)3(H2O)3]+ was obtained by addition of 1mL of 99mTcO4-(0.24-3.98 GBq) to the Isolink kit (given by

Mallinckrodt), 20m incubation at 100°C and controlled by RP-HPLC C18 column. Retention times (RT) were 3.96 ±

0.70 min for [99mTc(CO)3(H2O)3]+ and 12.70 ± 0.60 min for 99mTcO- 4. Then 0.3- 2.0 nmol antiTn was added to

[99mTc (CO)3(H2O)3]+, incubated 1 to 3 h at 37ºC and 45ºC to synthetize 99mTc(CO)3-anti-Tn (99mTcAb) and

controlled by HPLC molecular exclusion, RT [99mTc (CO)3(H2O)3]+: 5.6 min; 99mTc(CO)3-anti-Tn: 3.3 min; 99mTcO4-

:11.8 min.

99mTc red-hidr was determined by ITLC-SG, pretreated with 5% BSA, in EtOH: NH4OH: H2O (2:1:5).

The radiolabeled antibody was purified by PD-10 gel filtration. Stability was assessed up to 48h post-labeling.

Biodistribution studies were done in Balb-C and NudehealthySPF at 4, 16 and 24h postintravenous injection of1 to

12MBq.

[99mTc(CO)3(H2O)3]+ yield was above 99%. Anti-Tn antibody was successfully labeled reaching 100% radiochemical

purity post purification. The antibody was stable up to 48h post labeling for up to 600 MBq/nmol.

Biexponential pharmacokinetics, with urinary (35.0±6.73; 50.8±3.28) and hepato-biliary (24.4±4.07; 28.9±4.93)

elimination was found for Balb-C and Nude mice, respectively.

Labeling through tricarbonyl complex formation is useful for anti-Tn labeling showing the typical profile of the

antibodies biological behavior.


135


Biological evaluation of two glucose derivates radiolabeled with 99m-Tc as potential imaging agents for melanoma

Dapueto, R.1; Fernández, M.1; Gambini, J.P.2; Aguiar, R.3; Marques, F.4; Chammas, R.3; Cabral, P.1; Porcal, W.5 1Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

2Centro de Medicina Nuclear, Facultad de Medicina, Hospital de Clínicas, Montevideo, Uruguay

3Radiologia e Oncologia, Facultade de Medicina, Universidade do Sao Paulo, Sao Paulo, Brazil

4Centro da Medicina Nuclear, Facultade de Medicina, Universidade do Sao Paulo, Sao Paulo, Brazil

5Departamento de Química Orgánica, Facultad de Química-Facultad de Ciencias, Universidad de la República, Montevideo,

Uruguay [email protected]

Background: Malignant tumors can be detected with high sensibility by imaging the increased metabolism of

glucose, aminoacids and lipids. GLUT´s over- expression in tumor cells allows the detection of several cancer

types, using 18FDG with PET images, worldwide. Glucose derivates radiolabeled with 99mTc had been widely

studied to obtain a 18FDG analogue that substitute or complement 18FDG-PET images with SPECT.

Methodology: Two glucose derivatives, 11 and 2 (Figure 1) were synthesized. Radiolabeling was achieved by

adding 5-20 mCi of Na99mTcO4- to the corresponding compound and SnCl2 as reducing agent. After agitation, the

radiochemical purity was controlled using paper chromatography and HPLC. In vitro internalization was

accomplished incubating the radiolabeled compounds with B16F10 melanoma murine cells for 30, 60 and 120

minutes at 37ºC and 4ºC. Competition studies in vitro with 18FDG and the unlabeled compounds were done.

Images with C57BL6 mice bearing B16F1 melanoma were acquired at 60 and 120 minutes post- injection of the

radiolabeled compound, using a gamma camera.

Figure 1. Glucose derivates 1 and 2 used for radiolabeling with 99m-Tc and biological study

Results and conclusions: 99mTc radiolabeling of 1 and 2 was accomplished with high radiochemical purity

(>98%). In vitro studies of the radiolabeled compounds showed low cell accumulation (0,2- 0,5 %) at both

incubation temperatures and both unlabeled compounds did not alter 18FDG internalization. In vivo studies with

B16F1 melanoma bearing mice showed high accumulation in the tumor and renal bioelimination. These results

suggests that different mechanisms could be occurring in the live animal: in vivo accumulation could be nonspecific

due to an interaction with biomolecules in the tumor micro-environment.

Acknowledgements: PEDECIBA - Química, ANII, Espacio Interdisciplinario-UdelaR, CMN-FMUSP.


136

References

1. Dapueto, R; Castelli, R; Fernández, M; Chabalgoity, J;Moreno, M; Gambini, J.P; Cabral, P; Porcal,W. Biological evaluation of Glucose and Deoxyglucose derivatives radiolabeled with [99mTc(CO)3(H2O)3]

+ core as potential melanoma imaging agents. Bioorg Med Chem Lett. 23, 7102-7106.


137


Preparation and biologic evaluation of glucose derivates bearing a thiol group radiolabeled with 99mTc as potential diagnostic agents for cancer

Castelli, R.1; Fernández, M.1; Gambini, J.P.2; Aguiar, R.3; Marques, F.4; Chammas, R.3; Cabral, P.1; Porcal, W.5 1Laboratorio de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Montevideo, Uruguay

2Centro de Medicina Nuclear, Hospital de Clínicas, Facultad de Medicina, Montevideo, Uruguay

3Departamento de Radiologia e Oncologia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil

4Centro de Medicina Nuclear, Hospital das Clínicas da Facultade de Medicina da Universidade de São Paulo, São Paulo, Brasil

5Laboratorio de Química Orgánica, Instituto de Química Biológica, Facultad de Ciencias-Facultad de Química, Montevideo,

Uruguay [email protected]

Antecedents: PET-CT with FDG is a very important technique among the several existing of imaging melanoma

and non-Hodking lymphoma (LNH)1-3. Many research groups worldwide are working on the development of imaging

agents based on the expression of glucose transporters (GLUT `s) that are over-expressed in most tumor cells.

Therefore, the development of novel analogs of FDG for use in SPECT imaging, adapted to non-invasively and in

vivo evaluation of patients with melanoma and non-Hodgkin lymphoma is very desirable.

Methodology: We performed organic synthesis, purification and characterization of two new glucose derivates

which incorporated a thiol group for coordination with 99mTc (Figure) through an ethylene linker in the anomeric

carbon of the glucose. Labeling was performed using 99mTcO4- in presence of SnCl2 as reductive agent. In vitro

internalization was carried out using murine cellular lines of LNH (A20) and melanoma (B16F10 and B16F1). In

vivo images were performed in mice C57BL6 carrying of melanoma and LNH, at different times, using a gamma

camera.

O

OAc

AcOAcO

OAc

OSH

O

OH

HOHO

OH

OSNa

1 2

Figure.Structureoftwonew glucosederivateswhichincorporated a thiol group for coordination with 99mTc

Results and conclusions: Labeling of the compounds 1 and 2 with 99mTc was obtained with high purity. In vitro

studies showed a moderate percentage of cellular cellular internalization for both compounds. In vivo studies

showed high tumoral uptake and renal elimination in both tumoral models.

References

1. Friedman, KP.; Wahl, RL. Clinical use of positron emission tomography in the management of cutaneous melanoma. Semin Nucl Med. 2004, 34, 242-253.

2. Karantanisa, D.; Durskia, J.; Lowea, V.; Nathana, M.; Mullana, B.; Georgioub, E.; Johnstonc, P.; Wisemana, G. 18F- FDG PET and PET/CT in Burkitt’s lymphoma. Europ J of Radiol. 2010, 75, e68–e73.

3. Ho Shon, IA.; Chung, DK.; Saw, RP.; Thompson, JF. Imaging in cutaneous melanoma. Nucl Med Commun. 2008, 29, 847-876.

Acknowledgements: CSIC-UdelaR, AUGM, Espacio Interdisciplinario-UdelaR, CMN-FMUSP.


138


Clinical application of two neutral 99mTc radiopharmaceuticals in diagnosis of brain pathologies

Crócamo, N.

Servicio de Medicina Nuclear Dr. Touya y Dr. Gaudiano, Montevideo, Uruguay [email protected]

The aim of this paper is to present the experience in the preparation and clinical application of two neutral 99mTc

radiopharmaceuticals, 99mTc-etilendicisteína (ECD) and 99mTc-TRODAT. These radiopharmaceuticals cross the

intact blood-brain barrier and are used to image cerebral perfusion and dopamine transporters in

neurodegenerative lesions (such as Parkinson's), respectively.

The preparation of 99mTc-ECD requires the use of a kit formulation: one vial containing dihidroclorhidric ester of N,

N'- (1,2 ethylendiethyl) bis-L-cysteine (ligand), stannous chloride dihydrate (reductant), disodium

ethylendiaminotetraacetic acid dihidrated (stabilizer) and mannitol (inert filler); vial 2 containing phosphate buffer

and water to reach the proper pH. Labelling is performed by addition of 99mTcO4-(A 50mCi) at room temperature for

45 minutes. The radiochemical purity (PR) determined by paper chromatography on Whatman 1 paper with ethyl

acetate is above 90%.

The 99mTc-TRODAT is obtained by adding sodium pertechnetate (A 50mCi) to a single vial containing TRODAT

(the ligand), stannous chloride dihydrate and calcium glucoheptonate, as intermediate ligand and heated at 100ºC

for 30 minutes. The PR is also greater than 90%.

Both radiopharmaceuticals are administered intravenously at a dose between 27 and 30mCi. The images are

obtained using a SPECT camera The acquisition time should be less than 30 minutes and 360 ° rotation following

the contour of the body.

For 99mTc-ECD the normal biodistribution pattern is increased uptake in cerebellum and occipital lobe and a nearly

uniform and symmetrical distribution in the rest of the brain. Hypoperfusion or hyperperfusion in some of these

regions are considered pathological.

For 99mTc-TRODAT, images are acquired 4 hours post-injection, Areas of interest are drawn around caudate

nucleus, putamens, striatum and occipital cortex. Decreased uptake of the radiotracer in any of the previous zones

may denote a neurodegenerative disorder.

Acknowledgements: Dr. A. Rey


139

Abstract code: 004 AREA: SBMMBRP POSTER

pH effects on the iron oxidation states of the extracellular hemoglobin of Glossoscolex paulistus (HbGp)

Carvalho, F.A.O.; Tabak, M.

Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Paulo, SP, Brazil, São Carlos, Brazil

[email protected]

HbGp is characterized by a molecular mass (MM) of 3.6MDa, a high resistance to oxidation and high oligomeric

stability. The present work has as the main focus to study the pH effect on the HbGp iron oxidation states and the

characterization of the species in alkaline medium, by optical absorption and analytical ultracentrifugation (AUC). At

pH 7.0, oxy-HbGp displays an intense Soret band at 415nm, and other two bands (Q bands) at 576 and 540nm,

while met-HbGp presents a Soret band at 403nm, and Q bands at 500 and 540nm. At pH 8.0, for oxy-HbGp, Soret

and Q bands λabs values have no significant changes, suggesting that in this case the heme group remains intact.

For met-HbGp, pH 8.0 induces significant changes in the absorption spectrum due to the formation of the

hemichrome species. The hemichrome is characterized by a Soret band centered at 413nm, and Q bands at 534

and 565nm. The changes in the metal coordination sphere induce other phenomena in the HbGp structure. Thus,

oxy-HbGp, at pH 8.0 and 9.0, is partially dissociated, with 88 and 56% of native protein contribution, respectively.

The sedimentation coefficient c(S) curves for the oxy- form at these pH values show three species in the

equilibrium: tetramer with sedimentation coefficient ( 0w,20s ) and MM of 4.9S and 68.4kDa, dodecamer with 0

w,20s

and MM of 9.3S and 204kDa, and whole protein with 0w,20s and MM of 58.4S and 3600kDa. Met-HbGp, at pH

values 5.0 and 7.0, has a single species in the c(S) distribution, attributed to the native protein, while, at pH 8.0,

only smaller species, of MM between 16 and 70kDa, are observed in equilibrium. Our results show that the HbGp

stability depends on the iron oxidation state and on the ligand coordinated in heme group.

Support: Brazilian agency FAPESP.


140

Abstract code: 078 AREA: SBMMBRP POSTER

Effect of cationic surfactant (DTAB) on the HbGp iron oxidation at neutral and acidic pH values

Alves, F.R.; Carvalho, J.W.; Oliveira Carvalho, F.A.; Tabak, M.

Química e Física Molecular, USP, São Carlos, Brasil [email protected]

HbGp has a hexagonal bilayer structure, with a high molecular mass of 3.6MDa, and a high oligomeric stability. In

the absence of DTAB, oxy-HbGp presents characteristic absorbances at 415, 540 and 575nm. At pH 5.0, the

absorption spectrum remains unchanged up to 0.5mmol/L of surfactant, indicating the stability of the heme group

iron. The increase of DTAB concentrations, above 0.5mmol/L, induces a blue-shift of the Soret band from 413 to

405nm, as well as of Q bands from 540 and 575nm to 533 and 568nm, suggesting the formation of met-HbGp and

hemichrome species. The appearance of the LMCT band at 618nm is also observed above 1.0mmol/L DTAB,

indicative of aquomet-HbGp species. However, at pH 7.0, the presence of low DTAB concentration (in the range

0.1-0.5mmol/L) alters the absorption spectrum of oxy-HbGp drastically. DTAB concentration required for the

formation of aquomet-HbGp and hemichrome species is smaller at pH 7.0, as compared to pH 5.0, despite the

similarity of absorption spectra. The HbGp fluorescence emission, at pH 5.0, changes slightly less, in the presence

of DTAB, when compared with pH 7.0, increasing, roughly, 30-fold. The protein structural changes and oxidation of

the heme groups occur at smaller DTAB concentrations, at pH 7.0 as compared to pH 5.0, due to the acidic pI of

protein (5.5) that favors the oxy-HbGp-cationic surfactant interaction at pH 7.0. In conclusion, our present data

show that differences and similarities are noticed for DTAB and CTAC (Santiago et al. BBA 2007) effects upon

HbGp spectroscopic features.

Aknowledgments: Financial support: FAPESP and CNPq.


141

Abstract code: 009 AREA: TEMIBS POSTER

A particular approach of the correlation between metals and health: arsenic removal from groundwater by Fe-rich geo-materials

Botto, I.L.1; González, M.J.2; Gazzoli, D.3; Soto, E.L.4 1Quimica Universidad Nacional de La Plata, CEQUINOR Facultad de Ciencias Exactas, La Plata, Argentina

2Facultad de Ciencias Exactas, Univ. Nacional de La Plata, CEQUINOR-INREMI, La Plata, Argentina

3Dipartimento di Chimica, Universitá La Sapienza di Roma, Roma, Italia 4Facultad de Ciencias Exactas, UNLP, PLAPIMU, La Plata, Argentina

[email protected]

Arsenic is a very toxic element, observed in some groundwaters at levels above those suggested by the WHO (10

µgL-1). The geo-genetic origin is responsible of serious health problems, particularly the pathology known as

Chronic Endemic Regional Hydroarsenicism (CERHA). In Argentina the arsenical grounwaters are consumed in the

big Chaco- pampean area, affecting more than 4 million people. In general, several technological removing

strategies were used, but the adsorption arsenate/metal-oxide seem to be the most appropriate treatment for areas

of low population density and/or low income. In our case, the method was supported by the abundance of mineral

species naturally rich in iron or possible to modify with this metal. Chemical modification treatment was carried out

by using solutions of Fe (III) salts, through a hydrolytic process and adjustment of pH, promoting the formation of

Fe-O-H2O active clusters (precursors of nanoscopic ferrihydrite). Alumino-silicates such as clinoptilolite,

pyrophylite, pyroclastic material and kaolinite (Fe2O3 content lower or close to 5%) and Fe-rich clay minerals (~40%

Fe2O3), have been analyzed. The performance of the process was studied by means of Jar tests and

physicochemical techniques (micro-Raman, XR diffraction, chemical analysis (ICP-AAS), scanning electron

microscopy (SEM-EDS) and surface (BET) assays). It was noted that a small increase in the iron content (2-10%)

correlates with an increase in mesoporosity and a marked increase in the arsenate(V) adsorption, measured from

the number of treatment cycles. During the process, scorodite precursor is formed, whose stability in the clay matrix

ensures the As isolation. The results revealed that the trigger alternative, dependent on structural and surface

mineral properties, constitute a low-cost, effective, accessible and eco-friendly technology. Hence, several

prototypes were successfully installed in rural and/or vulnerable localities of the affected region, producing water

with As values lower than 10µgL-1.


142


Line of research: Effects of multiple metals on cognitive ability and behavior in children from Montevideo Uruguay

Queirolo, E.1; Barg, G.1; Mañay, N.2; Kordas, K.3 1Facultad de Psicología, Universidad Católica del Uruguay, Montevideo, Uruguay

2Facultad de Química, UdelaR, Montevideo, Uruguay

3Nutritional Sciences, Pennsylvania State University, United States, University Park, PA

[email protected]

Since 2006 an interdisciplinary and collaborative group formed by the Catholic University of Uruguay, Pennsylvania

State University and the Republic University of Uruguay is studying the effects of nutrition and heavy metals on

development, behavior and learning of children. We present a review of studies: In 2007 the authors screened lead

and hemoglobin levels in capillary blood of 222 preschool children from Montevideo, Uruguay. In 2008 the authors

examined metal exposures in 109 young children from Montevideo, Uruguay and their mothers participating in a

community-based study. In 2009, 150 children from first grade were enrolled. Underwent psychological and clinical

assessments (blood, urine and hair was collected for measuring metals), information was requested from teachers

and parents. The mothers were interviewed nutritional and psychological. There was also a home visit and

sampled water, soil and household dust. This year we are investigating the relationship between exposure to low

levels of arsenic and neurobehavioral disorders in children.

RESULTS

Study 1

Older child age, hemoglobin 10.5g/dL, and putting finges/toys in the mouth were associated with higher blood lead

level (BLLs). Young maternal age, less education, father's job with potential risk of lead exposure, and fewer family

possessions were also associated with higher BLLs.

Study 2

BLLs ≥ 5 µg/dL in mother or child were associated with lower maternal perceptions of being skilled at discipline

(panemia was associated with lower likelihood that mothers would let their children explore and play (panemia was

associated with maternal perception of lower emotional support (p<0.01).

Study 3

Mean IQ, SF and BLL were 92.9±17.0 points, 15.0±14.1 µg/L, 4.7±2.2 µg/dL, respectively. 48.8% and 30.2% of

children had BLL ≥5µg/dL and iron deficiency respectively, 13.4% had both. 31% of children had IQ ≤ 85 points.

Conclusions

The consolidation of an interdisciplinary group specializing in environmental pollution and the experience of a

collaborative work between different universities and institutes is a significant step in understanding the problem

and finding solutions to the most vulnerable populations: children and reproductive-age women.


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The use of biomarkers in evaluation of mutagenicity risk in offspring of rats exposed to contaminated soils - Contribution to the study of environmental contaminants

Garcia, E.M.1; Rodrigues da Silva Júnior, F.M.2; Dupont-Soares, M.1; Baisch, P.R.1; Muccillo-Baisch, A.L.1 1Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio Grande - FURG, Rio Grande, Brazil

2Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Rio Grande - FURG, Rio Grande, Brazil

[email protected]

Industrial advances have significantly increased the release of contaminants to various environmental

compartments, such as soil, which is static and accumulates substances. Frequent human exposure to

contaminated soil has led to more risks of mutagenic effects since environmental contaminants can be transferred

from mother to child. In order to measure irreversible DNA damage, micronucleus test was performed in the bone

marrow of rat offspring - in pre-pregnancy, pregnancy and lactation - exposed to soil affected by atmospheric

dispersion from the industrial complex in Rio Grande, a city located in the south of Brazil. Results showed an

increased micronucleus frequency in all periods, by comparison with the control animals. In addition, chemicals

released to the soil can induce mutagenicity in rat offspring whose mothers were exposed to it at different stages of

the reproductive period, leaving an alert so that fewer pollutants are emitted in order to avoid serious harm to

human health.

Proceedings


145

Tungsten reacting with 5-hydroxymethylcytosine

Okamoto, A.

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan

[email protected]

Abstract

5-Hydroxymethylcytosine (hmC) is a newly discovered natural nucleobase that may play an important intermediary

role in the active DNA demethylation pathway. An effective method to detect the presence and abundance of hmC

in DNA is required to elucidate the relationship between the generation of hmC and the mechanism of

demethylation. We have found that the oxidation using dinuclear peroxotungstate K2[{W(=O)(O2)2(H2O)}2(µ-

O)]•2H2O is hmC selective, and is useful for the discrimination of hmC from its epigenetic precursors, unmethylated

cytosine (C) and 5-methylcytosine (mC) in DNA. A fluorescein-labeled model DNA containing CG, mCG, or hmCG

dinucleotides was prepared, and the dinuclear peroxotungstate was added to a DNA solution in a pH 7 sodium

phosphate buffer. The mixture was incubated at 50°C for 5 h. The reaction proceeded hmC-selectively. The mass

spectrum of the oxidation product obtained from the hmC-containing DNA indicated the formation of trihydroxylated

thymine in DNA. The tungsten oxidation products induced the incorporation of adenine into the complementary site

in a primer extension, and made it possible to detect hmC in a DNA sequence of interest in a conventional DNA

sequencing analysis. Tungsten oxidation worked as a simple chemical reaction for the effective detection of hmC,

and will provide beneficial information on the design of a powerful method for hmC scanning and typing to solve the

mystery of the initialization of gene function through demethylation.

Keywords: tungsten, osmium, epigenetics, 5-hydroxymethylcytosine

Introduction

5-Hydroxymethylcytosine (hmC) is a newly discovered natural nucleobase that is induced by modification of 5-

methylcytosine (mC) with TET proteins, which have potential roles in epigenetic regulation (Scheme 1).1,2 Given the

critical role of mC in epigenetic regulation, hmC may play an important biological role in vivo, such as an

intermediary role in the pathway of active DNA demethylation.

Scheme 1. hmC in epigenetic modification.

An effective method to detect the presence and abundance of hmC in DNA is required to elucidate the relationship

between the generation of hmC and the mechanism of demethylation. Several assays have been attempted for

hmC detection.1–5 Although these methods can confirm whether or not hmC is present, including within a DNA

sample, it is difficult to carry out a scan that can detect which mC in a sequence is hydroxylated and whether or not

a specific mC is hydroxylated. An effective chemical reaction is required for the easy detection of hmC, such as the

chemical methods used to detect mC. If such a reaction is available, then the reaction product could be detected as


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an hmC-positive signal using a conventional detection method, such as site-selective strand cleavage assay,

sequencing analysis, and mass spectroscopy. In this paper, we report on an hmC-positive chemical reaction that is

effective for the discrimination of hmC from C and mC in a DNA of interest.6

Materials and Methods

Metal oxidation and hot piperidine treatment. The fluorescein-labelled DNA (5 µM) to be examined was

incubated in a solution of 5 mM of the metal oxidant (plus 50 mM hydrogen peroxide as an option) and 100 mM of

sodium chloride in 50 mM of sodium phosphate (pH 7.0) at 50°C for 5 h. The reaction solution was filtered to

deionize it using Micro Bio-Spin Columns with Bio-Gel P-6 (BioRad). After drying in vacuo, the precipitated DNA

was redissolved in 50 µL of 10% piperidine (v/v), heated at 90°C for 2 h, and then evaporated to dryness using a

vacuum rotary evaporator.

Sequencing. The DNA (3 ng) to be examined was incubated in a reaction mixture, and then it was deionized by

filtration. The reaction sample was analysed using capillary electrophoresis-based DNA sequencing technology

using BigDye® Terminator v1.1.

Results and Discussion

A fluorescein-labeled model sequence containing CG, mCG, or hmCG dinucleotides, DNA1(X) 5′-fluorescein-

AAAAAAG- XGAAAAAA-3′ (X = C, mC, or hmC), was prepared,7 and several metal oxidants have been tested as

oxidation agents of DNA1(X), considering their reactivity against nucleobases, reagent availability, and solubility in

water. The metal oxidant was added to a solution of DNA1(X) in a pH 7 sodium phosphate buffer, and the mixture

was incubated at 50 °C for 5 h, as described in Experimental section. After the solution was desalted through a

filter, the sample was treated with hot piperidine and analyzed using polyacrylamide gel electrophoresis (PAGE) to

find the oxidized nucleotides in DNA1(X) through the detection of DNA cleavage bands. The dinuclear

peroxotungstate K2[{W(=O)(O2)2(H2O)}2(µ-O)]•2H2O (1)8 was the most effective hmC-selective oxidant among

metal oxidants tested in this experiment (Fig. 1).

Fig. 1. Structure of dinuclear peroxotungstate.

PAGE analysis of the strand cleavage product after the reaction of DNA1(X) with 1 and hot piperidine treatment

showed a band at the hmC site of DNA1(hmC). The cleavage bands observed at the mC and C sites in DNA1(mC)

and DNA1(C), respectively, were negligible. The mass spectrum of the oxidation product of DNA1(hmC) indicated

the formation of trihydroxylated T in DNA, and the product increased in molecular mass by four when the oxidation

of DNA1(hmC) with 1 was carried out in a buffer solution prepared with H218O (Scheme 2).


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Scheme 2. Oxidation of hmC with 1.

The reactions of DNA1(X) with other metal oxidants, e.g., MeReO3,9 OsO4,

10 and K2[{Mo(=O)(O2)2(H2O)}2(µ-

O)]•2H2O dinuclear peroxomolybdate,8 which are known to oxidize a carbon–carbon double bond, were much less

hmC-selective (Fig. 2). Methylrhenium trioxide was weakly reactive, and osmium tetroxide and molybdenum

peroxocomplex exhibited an hmC selectivity that was much lower than that of tungstic acid.

hmC in a single-stranded DNA was efficiently oxidized with 1, whereas the oxidation efficiency for hmC in a DNA

duplex was lower. Oxidative cleavage of the fluorescence-labeled DNA2, 5′-fluorescein-

GCAGGGCCCACTAChmCGCTTCCTCCAGATGA-3′, was observed at hmC (79% conversion). In contrast, the

oxidation of bases in a hybrid with the fully matched complementary DNA was suppressed (7% conversion).

The protection of the C5−C6 double bond by the stacking of the flanking base pairs inhibits the attack of 1.

Fig. 2. hmC selectivity of metal oxidations.


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Tungsten oxidation was applicable to detection of hmC in fluorescence-labeled human genome sequences. The

DNA fragments containing hmCG dinucleotides in the promoter regions of human STAT3, BCL2, OCT4, NANOG,

HOX5A and TNF-β genes were prepared and oxidized with 1. They exhibited cleavage bands at hmC after hot

piperidine treatment, suggesting that tungsten oxidation is effective for hmC-selective reaction and detection of

hmC in DNA.

The deamination of cytosines makes it possible to incorporate adenines at the complementary position in a strand

extension process. Therefore, the deaminated product from the oxidation of hmC may help the efficient detection of

hmC through PCR amplification and sequencing. A DNA fragment from the human TNF-β putative promoter

region11 including CG, mCG, and hmCG dinucleotides was prepared and incubated with 1 at 50 °C for 5 h, and

then the sequences were analysed using capillary electrophoresis-based DNA sequencing technology. The

sequencing profiles of the tungsten oxidation products indicated that adenine was incorporated into the position

opposite the original hmC group (Fig. 3), whereas only guanine was incorporated into the position opposite mC and

C. The degree of the oxidative damage of guanine, which was observed on reaction with 1 in the PAGE analysis,

seemed to have little effect on the sequencing efficiency and fidelity in the present sequence analysis. The

selective base conversion of hmC in the sequencing process facilitated the ability to distinguish hmC from C and

mC in the DNA sequencing analysis.

Fig. 3. Sequence analysis of hmC- containing DNA using 1 oxidation. The sequencing profiles of human TNF-β

promoter fragments containing CCChmC-GGGC.

In the present study, the tungsten oxidation system was effective for discrimination of hmC from C and mC. The

key point of the discrimination was the use of the oxidation of a carbon-carbon double bond. The structural

difference between hmC and mC is the presence of a hydroxyl group (O–H). However, there is a large number of

chemically active O–H, N–H, and S–H bonds in nucleic acids and proteins. Therefore, it may be difficult to make a

chemical reagent react only the hydroxyl group of hmC. On the other hand, considering the structure of hmC in a

wider view, hmC sites possess a characteristic allyl alcohol group (C6 = C5–CH2OH), which is rare in biomolecules.

Tungsten-based oxidation systems are known to be effective for the oxidation of the carbon-carbon double bond of

allyl alcohol groups.8

Therefore, the allyl alcohol group of hmC worked as a key feature in the hmC-selective reaction of tungsten

oxidants.


149

Conclusions

Oxidation using tungsten oxidants, such as tungstic acid activated by hydrogen peroxide and dinuclear

peroxotungstate 1, is hmC selective, and is useful for the discrimination of hmC from C and mC in DNA. Tungsten

is a key element that can selectively access hmC, and is different from the other metallic elements that are close to

tungsten in the Periodic Table, such as osmium, molybdenum, and rhenium. Although the data shown here are

preliminary, and further optimization of the reaction conditions is required for improved reaction yield and

sequence-independent reactivity, the above reaction will provide beneficial information on the design of a powerful

method for hmC scanning and typing, and is undoubtedly a promising way to solve the mystery of the initialization

of gene function through demethylation.

References

1. Kriaucionis, S. and Heintz, N. The nuclear DNA base 5-hydroxymethylacytosine is present in Purkinje

neurons and the brain. Science, 2009, 324, 929–930.

2. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL

partner TET1. Science, 2009, 324, 930–935.

3. Münzel, M. et al. Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew Chem Int

Ed, 2010, 49, 5375–5377.

4. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass

specification. Nature, 2010, 466, 1129–1133.

5. Song, C.-X. et al. Selective chemical labeling reveals the genome-wide distribution of 5-

hydroxymathylcytosine. Nature Biotech, 2011, 29, 68–72.

6. Okamoto, A., et al. 5-Hydroxymethylcytosine-selective oxidation with peroxotungstate. Chem Commun,

2011, 47, 11231–11233.

7. Sugizaki, K., Ikeda, S., Yanagisawa, H. and Okamoto, A. Facile synthesis of hydroxymethylcytosine-containing

oligonucleotides and their reactivity upon osmium oxidation. Org Biomol Chem, 2011, 9, 4176–4181.

8. Kamata, K., Yamaguchi, K. and Mizuno, N. Highly selective, recyclable epoxidation of allylic alcohols with

hydrogen peroxide in water catalyzed by dinuclear peroxotungstate. Chem Eur J, 2004, 10, 4728–4734.

9. Tetzlaff, H. R. and Espenson, J. H. Kinetics and mechanism of the epoxidation of allylic alcohols by hydrogen

peroxide with methyltrioxorhenium as catalyst. Inorg Chem, 1999, 38, 881–885.

10. Dizdaroglu, M., Holwitt, E., Hagan, M. P. and Blakely, W. F. Formation of cytosine glycol and 5, 6-

dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide. Biochem J, 1986, 235, 531–536.

11. Nedwin, G. E. et al. Human lymphotoxin and tumor necrosis factor geges: structure, homology and

chromosomal localization. Nucleic Acids Res, 1985, 13, 6361–6373.


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Acknowledgments

We wish to thank Ms Kaori Sugizaki and Ms Akiko Nakamura (RIKEN) for the PAGE analysis. We are also grateful

to the Support Unit for Bio-material Analysis, RIKEN BSI Research Resources Center, for help with DNA

sequencing analysis.


151

Silver nanoparticle exposure: Pathological changes in developing chick embryos

Gagnon, Z.E.1*; Pavel Sizemore, I.E.2*; Trivedi, P.3; Dagher, J.M.2; Monahan, J.L.2; Lam, T.2; Isaac; L.A.C.4 1Marist College, School of Science, Department of Environmental Sciences, 3399 North Road, Poughkeepsie, NY 12601, USA;

e-mail: [email protected]; 2Department of Chemistry, Wright State University, Dayton, OH 45435: [email protected], [email protected], and

[email protected] 3University Hospital, Cincinnati, OH 45291: [email protected]

4St. Francis Hospital, Pathology Department, Poughkeepsie, NY 12601: [email protected]

Abstract

The presence of silver nanoparticles (AgNPs) in industrial and household products has become increasingly

prevalent in recent years. Six-day old chick embryos were exposed to AgNPs in concentrations of 15, 30, 60, and

100 µg mL-1 via injections into the egg air sac on the 7th and 14th days of incubation. The colloidal AgNPs were

synthesized according to the widely-used Creighton method through the aqueous reduction of silver nitrate

(AgNO3) with sodium borohydride (NaBH4). The control groups included no injection, highly purified water, AgNO3,

and NaBH4 injections (10 embryos per each control and treatment group). On the 20th day of incubation, embryos

were sacrificed, and the brains and livers were harvested. Histological analysis and flame atomic absorption

spectroscopy (FAAS) measurements were conducted on the collected tissue samples. The SPSS statistical

package was used to determine variation in serotonin and Ag bioaccumulation levels. Mortality occurred in all

treatments except for the water and no injection controls. A direct correlation was found between Ag

bioaccumulation levels in tissues and the pathological changes observed in chick embryos exposed to 60 and 100

µg mL-1. Fatty vacuoles were present in the liver cells for the 60 µg mL-1 of AgNP treatment. Proliferation of

hepatocytes, hyperplasia, and increased cellular mitotic activity was also observed in these samples. Additionally,

Von Kossa staining revealed cell calcification, especially in brain tissues. At the 100 µg mL-1 AgNP exposure level,

apoptotic bodies with nuclear fragmentation were evident, and Ag precipitation was detected in the intercellular

spaces of liver tissue. These pathological findings raise major health concerns about the increasing exposure of the

human population to AgNPs, and suggest that environmental risk assessment of this emerging factor should be

considered.

Key words: Creighton silver nanoparticles, chick embryos, liver and brain pathology, serotonin, flame atomic

absorption spectroscopy

Introduction

Although nanoparticles (NPs) are part of our natural environment, engineered NPs have recently become a

dominant factor in modern technology. Because of their unique physical, chemical and antimicrobial properties,

AgNPs are the most commonly used NPs for industrial and medical purposes, in particular in consumer products.

The Nanotechnology Consumer Product Inventory prepared in 2010 by the Woodrow Wilson Center’s Project on

Emerging Nanotechnologies [1] indicated that over 50% of the nanomaterial-based consumer products contain

nanosilver. Approximately 5% or over 1,200 tons of the total silver (Ag) that is produced worldwide is represented

by AgNPs. In spite of their numerous applications, AgNPs also have drawbacks. Questions regarding the

environmental fate of AgNPs in terrestrial and aquatic environments remain unanswered [2]. The potential effect of

AgNPs on human health is of even greater concern. Because there are currently no efficient methods for the


152

removal of AgNPs from wastewater effluents [2], it is imperative to learn more about AgNP uptake into the food

chain and their long term effects on human health and animals.

Recent nanotoxicology studies on AgNPs, ranging in size between 6 and 25 nm diameter and exposure levels

between 5 to 400 µg mL-1, were found to increase production of reactive oxygen species (ROS) in human lung

fibroblast cells [3], increase cell membrane leakage in mouse embryonic stem cells and mouse embryonic

fibroblasts [4], diminish mitochondrial function in Buffalo rat liver cells [5], and cause chronic alveolar inflammation

in rats [6]. The main goal of this study was to quantify the amount of Ag accumulated in the liver and brain tissues

of developing chick embryos, and to determine the histopathological effects associated with the exposure to AgNPs

in these organs.


Experimental Organisms. One hundred forty fertilized specific pathogen free white leghorn strain chick embryos

(Gallus domesticus) were obtained from Charles River Laboratories, Inc., and were divided into 14 experimental

groups of 10 chicks each. The experimental setup, embryo acclimation, and embryo preparation followed the

procedure described by Gagnon et al. [7]. Embryos were incubated for 20 days in a forced air chamber with

automatic turner at 38.5ºC ± 1ºC and 50-55% relative humidity. Hamburger and Hamilton (HH) [8] nomenclature

was used to estimate embryo developmental stages. Embryological age was established based on a full 24 hours

from the time of day the eggs were first placed in the incubator.

Synthesis and Characterization of AgNPs. Colloidal AgNPs were synthesized according to the widely-used

Creighton approach through the reduction of 1 mM of AgNO3 solution (50 mL) with 2.0 mM of NaBH4 solution (300

mL) in water. The reaction mechanism and the characterization of the Creighton colloid were presented in our

previous work [9, 10, 11]. This method was shown to lead to the formation of round AgNPs with an average

diameter of approximately 11 nm and a moderate size distribution in the 1-100 nm size range. The UV-VIS

absorption spectrum of the yellow colloid exhibited a typical sharp surface plasmon peak at 390 nm. These AgNPs

have a negative surface charge and a shelf life time of up to 3-6 months at 10ºC. Literature shows that aqueous

suspensions of AgNPs release Ag+ ions over time in the presence of dissolved O2 and H+ through an oxidation

process. However, this oxidation process is slow [12] (e.g., 6-125 days for citrate-capped AgNPs) and temperature

dependent. In our study, the Creighton colloidal AgNPs were used immediately after fabrication.

In Ova Injections. The following experimental treatments were established: a) control with no injection; b) 0.0 µg

mL-1 control treatment with deionized water only; c) four AgNP treatment aqueous suspensions of 15.0, 30.0, 60.0

and 100 µg mL-1 each; d) four AgNO3 treatment solutions of 23.6, 47.2, 94.4 and 157.4 µg mL-1 each; e) four

NaBH4 treatment solutions of 10.5, 21.0, 42.0 and 70.0 µg mL-1 each. There is some evidence that AgNPs in

colloidal solution can deaggregate to form their original compounds [13]. For this reason, AgNO3 and NaBH4

control treatments were established with the same concentrations as those for AgNP synthesis. These

concentrations were based on the equivalent amounts of AgNO3 and NaBH4 that used in the AgNP synthesis.

Stock solutions of AgNPs, AgNO3, and NaBH4 were prepared in sterile deionized water for subsequent 1 mL

injections. The pH of all solutions was adjusted to 7.2 with 0.1 N NaOH or HCl to compensate for minor treatment

variations (pH close to that of the stable, Creighton colloid). All solutions were filtered using a 0.22 µm filter (Fisher


153

Scientific). All treatments were injected directly into the air sac on the 7th and 14th days of incubation. The internal

membrane surrounding the embryo was not punctured, allowing for slow diffusion of liquid through the membrane.

Tissue Sampling. The experiment was terminated on the 20th day of incubation. Embryos were sacrificed through

cervical decapitation within the first 5-10 seconds after removal from the egg shell to avoid any major stresses. A

gross visual examination was performed to identify potential external and internal abnormalities. Chick brains,

livers, and tibiotarsi were extracted by dissection. Collected samples were separated for histology, chemical

analysis by FAAS, and serotonin ELISA immunoassay.

Histological Analysis. Tissue samples for histological analysis were rinsed in saline solution and fixed in 10%

phosphate-buffered formalin solution. Tissue cassettes were processed in a Vacuum Infiltration Processor (VIP),

and dehydrated with formalin, ethanol, and americlear. Paraffin molds were sectioned at 3 µm using a Spencer

microtome. Prepared slides were stained using a hematoxylin-eosin (H&E) dye (LEICA Autostainer XL), and

examined for pathological changes via light microscopy. Following these analyses, a Von Kossa Stain Kit

(American Master Tech Scientific, Inc.) was used to identify tissue calcification.

Serotonin Analysis. Analyses were performed on homogenized tissue samples using modified ELISA

Immunoassay (LDN Labor Diagnostica Nord GmbH & Co. KG). Chick brain tissue samples (0.1 g) were

homogenized in 2 mL of freshly prepared buffer (1mg mL-1 of ascorbic acid added to 50 mM of Tris, pH of 7.5). In

the assay, acetylated serotonin was bound to the solid phase of the microtiter plate. Substrate TMB (3,3′,5,5′-

tetramethylbenzidine)/peroxidase reaction was determined by recording the difference in absorbance using a

BIOLOG Reader at 450 nm. A calibration curve was developed using six LDN Standard Controls of different,

known serotonin concentrations. Concentration of serotonin was obtained using the Datalogger program and curve

fitting techniques.

FAAS Analysis. Tissues were dehydrated at 80°C for 72 h. Samples (0.5 g) were then homogenized, and digested

in 5 mL of high purity nitric acid (HNO3) (Fisher Scientific, Optima Grade). Ag content analyses were conducted via

FAAS. An Ag stock standard solution of 1.0 x 103 µg mL-1 (Fisher Scientific, Lot Number: CL4-132AG, certified by

SPEX CertiPrep) was used for the preparation of seven Ag standards (0.01, 0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 µg mL-

1). The pH of each sample solution was adjusted to 2.0 using high purity nitric acid. Samples corresponding to

higher Ag-exposure levels were diluted 2x or 4x using a blank solution before each measurement to fit within the

concentration range of the Ag calibration curve. A fast sequential spectrometer (model AA240 FS, Varian Inc.)

equipped with an air-acetylene burner and a Ag hollow cathode lamp (5 mA) was used to determine Ag content

within samples. The acquisition parameters were as follows: wavelength of 328.1 nm, slit width of 0.5 nm, air flow

rate of 13.50 L min-1, and acetylene flow rate of 2.00 L min-1. Ag amounts were determined through standard

external calibration using least-squares fit of regression curves.

Statistical Analysis. The effect of applied treatments on Ag accumulation and serotonin concentration in brain

tissues was analyzed using SPSS. One-way analysis of variance (ANOVA) by multi-comparison Student-Newman-

Keuls procedure was used to examine sample variation at the probability level α ≤ 0.05.


154


Mortality. One hundred percent mortality was observed in NaBH4 exposures at the 30, 60 and 100 µg mL-1 levels

and in AgNO3 exposures at the 94.4 and 157.4 µg mL-1 levels. The lower AgNO3 and NaBH4 treatment levels

exhibited mortality proportional to increasing compound concentrations, as recorded in Gagnon et al. [7]. The

chicks surviving the lower AgNO3 and NaBH4 exposure levels were smaller, unhealthy in appearance, and severely

underdeveloped in comparison to the no injection and water controls. Additionally, tissue damage was so extensive

that the structural integrity of the organs of interest rendered them unusable for analysis. Chick mortality for AgNP

treatments occurred at all exposure levels, but was lower in comparison to the paternal compound exposures. At

60 µg mL-1 of AgNP, 7 of 10 specimens died before harvest. However, at the highest AgNP concentration, only 2

specimens were dead at harvest. No mortality occurred in the no injection or water control groups.

FAAS Analysis. The results of FAAS analysis are presented in Table 1. Each sample was measured three times

and the absorbance values were averaged before interpolating the Ag concentration from the calibration curve (r2 >

0.998). Calibrations were performed every 20 samples, using acid-matched standard solutions [14]. Statistically

significant differences in Ag concentration were found in liver tissue samples. Ag bioaccumulation was found to

increase with the increase in the AgNP concentration of the injected solutions. The results demonstrate a

correlation between Ag concentrations in the tissues and the pathological changes observed in the histological

preparations. Additionally, there was higher Ag accumulation from AgNO3 sources than from AgNPs.

Table 1. Total amount of Ag (in µg mL-1) accumulated in chick embryo liver tissue as estimated by FAAS. Each

value is the mean of three measurements ± standard deviation.

Treatment Total amount of Ag accumulated

in 0.5 g of liver tissue (µg mL-1

)

Control (no injection) 0.0

Control (water injection) 0.0

23.6 µg mL-1 of AgNO3 1.6 ± 0.3

47.2 µg mL-1 of AgNO3 3.0 ± 0.1

15 µg mL-1 of AgNPs 1.2 ± 0.4

30 µg mL-1 of AgNPs 2.4 ± 0.6

60 µg mL-1 of AgNPs 5.5 ± 1.1

100 µg mL-1 of AgNPs 6.2 ± 1.2

All NaBH4 samples 0.0

Liver Toxicity. AgNP exposures of 15 and 30 µg mL-1 resulted in no pathological changes in liver tissue. The 60

µg mL-1 treatment of AgNPs, however, had a dramatic effect on liver tissue integrity and health. Fatty vacuoles in

the liver cells were evident. Fatty vacuoles of individual cells coalesced and caused cell membrane to disintegrate.

There is a significant body of research demonstrating formation and accumulation of fat in the liver of animals,

including avian species, caused by exposure to heavy metals, mostly mercury and lead [15]. Identification of fatty

liver in the embryonic liver tissue of mammalian and avian models is not uncommon in many pathological

conditions, including human population.


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H&E stained liver tissue showed black pebbly structures (Figure 1a). Von Kossa staining was conducted confirming

cell calcification. Proliferation of hepatocytes, hyperplasia, and increased cellular mitotic activity was observed in

liver tissue. Proliferation of hepatocytes with undifferentiated hepatocytes was not uniformly dispersed but rather in

clusters spread out among normally differentiated hepatocytes.

Deposition of ultra-fine AgNPs in hepatocytes could cause inhibition of cell differentiation. It is known that liver

proliferation in experimental animals can be induced by a variety of external conditions such as hepatectomy or

heavy metals [16]. The liver is known to be a main target for heavy metal exposure. In this study, regenerative

hyperplasia was accompanied by liver enlargement. Therefore, increased mitotic activity and DNA synthesis could

be an adaptive response to AgNP toxicity.

Another possibly adaptive response to liver toxicity from AgNPs was demonstrated by vascular proliferation. It

seems that developing hypoxia was caused by two AgNP injections during embryonic development, and stimulated

pathological angiogenesis. The 7th day correlates with HH stages 30 and 31, when limb bud is developing. The 14th

day corresponds to HH stage 40, when structural development is completed and only growth of existing structures

is taking place [17]. Widespread hypoxia, which induced vascular proliferation, was observed in the Ag-exposed

chick liver. In healthy tissue, the occurrence of vascular proliferation is extremely low [18]. Our study also revealed

angiogenesis, thick vessels in the proliferation area, which can be induced in response to several physiological and

pathological stimuli.

Ag deposits were observed within the liver cells and in the extracellular space. As indicated in the previous section,

the FAAS analysis confirmed the Ag bioaccumulation and quantified the total Ag amounts in tissue. Ham and

Tange [19] reported Ag deposition in liver of rats receiving a 0.25% solution of AgNO3 in distilled water as their

drinking water for several weeks. There is a large body of knowledge on Ag affinity to proteins, especially to the SH

groups. However, the composition of the Ag aggregates can be more diverse and requires further investigations.

Ag aggregates formed in cells and tissues can be composed of metallic Ag, silver oxide, or silver sulphide [19].

In this study, clusters of undifferentiated liver cells were observed. At the same time, however, cells undergoing

pyknosis and apoptotic bodies with nuclear fragmentation were also observed. Dramatic pathological changes

were noted at AgNP exposure level of 100 µg mL-1 with a ghost like appearance of cytoplasm. This type of

piecemeal necrosis in the liver also occurs in viral hepatitis and other autoimmune conditions.

Brain Toxicity. At an AgNP exposure level of 15 µg mL-1, no changes were observed in brain tissue. However, at a

concentration level of 30 µg mL-1, black deposits of Ag were clearly identified in the subcortical space of the pia

matter under light microscopy at 400x (Figure 1b). The pia matter is a delicate membrane that covers the brain

surface and is impermeable to fluids. In addition, patchy loss of structural integrity was demonstrated by localized

dense nuclei and highly diffused cellular arrangement. Von Kossa staining revealed cell calcification most

prominent in brain tissue. The authors’ previous research on chick embryo exposed to platinum group metals

identified widespread brain tissue calcification [20]. In contrast, this experiment’s patchy spatial tissue calcification

could be correlated with target-like calcium distribution and is believed to indicate the onset of neuron cell death.


156

a)

b)

Figure 1. a). Cross section of chick embryo liver exposed to 100 µg mL-1 of AgNPs. Visible pyknosis (arrow) shows

the irreversible condensation of chromatin in the nucleus of an apoptotic cell (100x). b) Brain tissue of a chick

embryo exposed to 60 µg mL-1 of AgNPs. The Ag appears in the tissues as small dark granules. Ag deposits form a

patchy pattern. Note the presence of very few neurons outside the area of Ag deposition (indicated by arrow).

Serotonin Content. There was a statistically significant difference in the level of serotonin in the brains of AgNP-

exposed chick embryos with respect to the control groups. The significant serotonin increase at the 60 µg mL-1

AgNP exposure level was followed by a decrease at the highest AgNP exposure level of 100 µg mL-1, possibly

indicative of an unconventional dose response. In comparison with no injection group, serotonin levels in the 10.5

µg mL-1 NaBH4 group experienced a significant decrease. Due to low survival rate, no AgNO3 exposed chicks were

available for serotonin examination.

In principal, the blood brain barrier (BBB) protects the brain from blood-borne exposure to heavy metals. During

embryonic development, however, the BBB is not fully developed. In humans, the placenta provides additional

protection to the embryo. Ag deposition in brain tissue indicates that the BBB is not an effective barrier for NPs

(including AgNPs). It has been demonstrated that serotonin plays a decisive role in cell motility initiation coupled

with the process of gastrulation. According to Wallace [21], serotonin is a common neurotransmitter in most animal

embryos during this early period of development. In his experiments, chick embryos deprived of normal access to

serotonin by either serotonin synthesis inhibitors or by serotonin receptor blockers showed typical morphological

disturbance of gastrulation, neurulation, and semitogenesis. Our results suggest that decreased levels of serotonin

in the chick brain tissue could have a significant effect on embryo development.

Conclusions

The exposure to Creighton AgNPs had multiple effects on developing chick embryos. Our results demonstrate that

there was significant bioaccumulation of Ag in the chick liver tissue and reduction of serotonin level in the brain

tissue for the highest AgNP exposure. In addition, our previous AgNP study [7] documented an effect on the

immune system. In our controlled experimental environment, bioaccumulation of AgNPs in tissues and repeatable


157

results in 10 replicates suggest that the pathological changes in the immune system and serotonin level are the

effect of exposure to AgNPs. As part of a comprehensive investigation on AgNP exposure, our study presents

multiple pathological outcomes using the chick embryo model that could suggest similar responses in humans.

DNA damage, cell cycle arrest, morphological changes of cells, Ag tissue deposits, and brain calcification raise

concerns about the safety associated with widespread use of AgNPs.

References

1. Maynard A, Michelson E. 2009. Woodrow Wilson International Center for Scholars: Analysis-Consumer

Products-Nanotechnology Project. [http://www.nanotechproject.org/inventories/consumer/analysis_draft/]

webcite1.

2. Hornyak GL, Tibbals HF, Dutta J, Moor JJ. 2009. Introduction to Nanoscience and Nanotechnology. Boca

Raton, FL: CRC Press.

3. AshaRani PV, Low Kah Mun G, Hande MP, Valiyaveettil, S. Cytotoxicity and genotoxicity of silver

nanoparticles in human cells. ACS Nano 2008 3(2):279-290.

4. Braydich-Stolle L, Hussain S, Schlager JJ, Hofmann M-C. In vitro cytotoxicity of nanoparticles in mammalian

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5. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat

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exposure to silver nanoparticles. Inhal Toxicol 2008 20(6):567-574.

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response in an avian model. In: Pele L, Powell JJ, Kinrade S, Jugdaohsingh R, Collery P, Maymard I, Badawi

A, eds. Metal Ions in Biology and Medicine, Volume 11. Paris: John Libby Eurotext, 2011:172-177.

8. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol

1951 88:49-92.

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DA. Estimating the analytical and surface enhancement factors in surface-enhanced Raman scattering

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biosensors. J Am Chem Soc 2010 132:10970-10972.

11. Anders CB, Baker JD, Stahler AC, Williams A, Sisco JN, Trefry JC, Wooley DP, Pavel Sizemore IE.

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2010 44:2169-2175.

13. Jayabalan J, Singh A, Chari C, Srivastava H, Mukhopadhyay PK, Srivastava AK, Oak SM. Aggregated

nanoplatelets: Optical properties and optically induced disaggregation. J Phys-Condens Mat 2008

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14. Creighton JA, Eadon DG. Ultraviolet-visible absorption spectra of the colloidal spectra elements. J Chem Soc

Faraday T 1991 87(24):3881-3992.

15. Gomez-Munoz A, Rodriguez-Fernandez C. Fatty acid composition of liver lipids during ontogeny of the chick

embryo: effect of a single dose of triiodothyronine. Exp Clin Endocrinol Diabetes 1988 92(3):323-327.

16. Columbano A, Ledda GM, Sirigu P, Perra T, Pani P. Liver cell proliferation induced by a single dose of lead

nitrate. Am J Pathol 1983 110(1):83-88.

17. Bellaris R, Osmond M. The Atlas of Chick Development. New York: Academic Press, 1998.

18. Humar R, Kiefer FN, Berns H, Resnik TJ, Battegay EJ. Hypoxia enhances vascular cell proliferation and

angiogenesis in vitro via rapamycin (mTOR) - dependent signaling. FASEB J. 2002 16(8):771-80

19. Ham KN, Tange JD. Silver deposition in rat glomerular basement membrane. Immunol Cell Biol 1972

50:423–434.

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metals. In: Collery P, Maymard I, Theophanides T, Khassanova L, Collery T, Eds. Metal Ions in Biology and

Medicine, Volume 10. Paris: John Libbey Eurotext, 2008:82-87.

21. Wallace JA. Monoamines in the early chick embryo: Demonstration of serotonin synthesis and the regional

distribution of serotonin-concentrating cells during morphogenesis. Am J Anat 1982 165(3):261-276.


159

Determination of Cd in soybean and wheat seeds using TS-FF-AAS and preconcentration with cloud point extraction (CPE)

Morales, G.; Knochen, M.; Pistón, M.* Cátedra de Química Analítica, Facultad de Química, Universidad de la República

Montevideo, Uruguay [email protected]

Abstract

Cadmium is a toxic element associated with the environment. Seeds can accumulate it, so it is important to

determine the concentration of this element.


detectability in flame atomic absorption spectrometry (FAAS) providing an alternative to the use of ET-AAS or ICP-

MS.

The method is based on the adsorption of a complex of Cd and methyl green, potassium iodide and Triton X-114 in

a minicolumn filled with cotton. The elution was carried out with HNO3. The system consists of a peristaltic pump,

an injection valve and a nickel tube 10 cm long with 7 holes. The analytical determinations were carried out by

FAAS at 228.8 nm.

The seeds were milled, and 1.8 g of the obtained flour was digested by means of an acidic treatment with a mixture

of HNO3/H2O2. The resultant solution was neutralized with solid NaOH.

The figures of merit were: LD (3s) and LQ (10s): 0.6 and 2.1 µg L-1 respectively, linearity: up to 10 µgL-1

(r2 = 0.999), precision: sr(%) = 2.8 (n = 5), sampling frequency: 30 h-1 with a preconcentration factor of 30

compared with FAAS.

Accuracy was evaluated with the CRM Wheat Flour 1567a (NIST), the recoveries were between 98% and 105 %

(n = 10). There was no evidence of the influence of potential interferences.

This method can be implemented for the control of Cd levels in soybean and wheat seeds.

Keywords: cadmium, thermospray, soybean, wheat

Introduction

Cadmium is one of the most toxic elements associated with environmental and industrial pollution, either as a

secondary product of metallurgic industry, where it reaches the soil by aerial deposition, or through the use of

phosphate fertilizers and the use of compost. Soybean and wheat seeds can accumulate this element from the

environment, and then it is very important to determine the concentration of Cd in the seeds because wheat is the

raw material of many foods [1].

Analytical determinations of Cd can be performed using different detection systems. Once the pretreatment of the

sample is performed (digestion, extraction, preconcentration), this element can be quantified by flame atomic

absorption spectrometry (FAAS), by atomic absorption spectrometry with electrothermal atomization (ET-AAS) or

by atomic emission inductively coupled plasma (ICP-OES, ICP-MS), among others [2].


160


detectability and sensitivity in flame atomic absorption spectrometry (AAS) of some volatile elements providing an

alternative to the use of ET-AAS or ICP-MS [3].

To further improve detectability of trace elements, strategies of on-line preconcentration can be used. Cloud point

extraction (CPE) is a separation/preconcentration procedure which has proved to be very efficient for determination

of trace elements in varied matrices and also in good agreement with the principles of Green Chemistry [4-7].

CPE is based on the principle that a surfactant containing aqueous solution becomes cloudy and separates into

two phases, if certain conditions are adjusted appropriately as temperature, pressure or if an adequate substance

is added. When the surfactant solution becomes cloudy it has reached the "cloud point". At this point, the original

layer of surfactant phase is separated into a small volume of solution which will be rich in the analyte of interest

(linked to organic or inorganic species by having high affinity), trapped by micellar type structures [4].

Flow injection systems with micelle mediated preconcentracion are made up with minicolumns filled with cotton,

this material is commonly used for the retention of micellar aggregates [5].

In Uruguay the local regulation establishes that the maximum concentration of cadmium admitted in solid foods is

0.2 mg kg-1 [8]. Thus analytical methods used for the determination of this element in foods should provide high

sensitivity.

The aim of this work is to propose an automated method for routinely Cd determination in soybean and wheat

seeds using a flow injection system (FI) with on-line CPE preconcentraton coupled to Thermospray Furnace (TS-

FF) and detection by flame atomic absorption spectrometry (FAAS).


All reagents were of analytical reagent grade. Purified water (ASTM Type I) was obtained from a Millipore (São

Paulo, Brazil) Simplicity 185 purifier fed with glass-distilled water.

All glassware was soaked overnight in 10% (v/v) nitric acid and then rinsed exhaustively with distilled water. For

calibration, adequate dilutions in purified water were prepared from a standard stock solution of Cd 1000 mg L-1 in

HNO3 4% (v/v) (SCP SCIENCE traceable to NIST).

The proposed method is based on micelle formation by the reaction between the reagent methyl green, and

potassium iodide (KI) using Triton X-114 surfactant. These micelles are retained in a Teflon PFA minicolumn (60 x

2.5) mm packed with 40 mg of cotton. Elution is carried out with HNO3 1 mol L-1.

The system consists of a peristaltic pump (Rainin Dynamax) fitted with Tygon tubing, a 6-ports injection valve

(Valco Cheminert), connections coils were made from 0.8 mm internal diameter Teflon PFA tubing. The detection

system used consisted of an atomic absorption spectrometer Perkin Elmer AAnalyst 200, equipped with a

thermospray flame furnace system (TS-FF-AAS). A nickel tube of 10 cm long by 10 mm of diameter with 7 holes

was used above an adapted burner with ceramic supports. The measurements were carried out at a wavelength of

228.8 nm using a deuterium lamp for background correction.

During the preconcentration step, the reaction occurs between the Cd present in the sample, KI 0.5 mol L-1, methyl

green 0.005 mol L-1 and Triton X-114 0.5% (v/v), to form micelles that are retained on the cotton minicolumn. After


161

one minute of preconcentration (charge position), micelles are dissolved in HNO3 1 mol L-1 in the elution step

(elution position).

Figure 1 ilustrates the system.

Figure 1. Flow injection system used for the preconcentration of Cd. P: peristaltic pump; V: 6-port injection valve;

C: Column, W: waste, S: sample, T: Triton X-114, MG: methyl green, KI: potassium iodide, HNO3: nitric acid. Solid

lines represent the valve position during the preconcentration step, dotted lines represents the valve position during

the elution step.

The soybean and wheat seeds were obtained from a local distributor (Agropecuaria Valdense S.R.L). The seeds

were milled, and 1.8 g of the obtained flour was digested by means of an acidic treatment with a mixture of 30 mL

of HNO3 and 4 mL of H2O2 (30% (v/v) heated in a hot plate for 30 minutes in a vessel with reflux and afterwards the

evaporated volume was completed to 25 mL with purified water. The resultant solution was neutralized with solid

NaOH.

Certificate reference material of wheat flour purchased from NIST (1567a) was also prepared as described above.

Reagent blanks were also run.

Results and discussion

The optimum concentrations of the reagents were obtained by means of a three-level central composite design [9].

It was decided to work with 1 minute as preconcentration time, because it is a reasonable time in agreement with

good results in terms of the best net signal. The operative parameters of the system were optimized using

multivariate experiments, where the net signal of a standard solution was monitored.

Once the operative conditions were optimized, the figures of merit were evaluated to complete the validation of the

method. The results obtained are show in Table 1.

Accuracy was evaluated by means of trueness and precision. Trueness was studied using a reference material of

wheat flour (NIST-1567a) by means of the recoveries of Cd. The precision of the method was evaluated taking into

account the intermediate precision (relative standard deviation between 3 days).


162

Table 1. Figures of merit

Parameter Result

Limit of detection (3s) 0.6 µg L-1

Limit of Quantification (10s) 2.1 µg L-1

Linearity up to 10 µg L-1

Precision (sr, n=5) 2.8 %

Accuracy (n=10) 98% - 105%

Sampling frequency 30 h-1

Enrichment factor 30 respect to FAAS

Preconcentration factor 6 respect to TS-FF-AAS

Sample consumption (mL) 4

The validated method was applied to analyze 24 samples of wheat and 14 of soybean seeds, the obtained values

were in the range of 0.03 and 0.10 mg kg-1 and 0.02 and 0.11 mg kg-1 of Cd respectively.

Conclusions

All the analyzed samples met the requirements of the regulations and can be used as raw material for food.

The proposed method was successful for the application and can be implemented for the control of Cd levels in

wheat and soybean seeds.

References

[1] Da Matta Chasin, A.A., Cardoso, L. M. N. Cádmio. In De Azeredo, F.A. and Da Matta Chasin, A.A. (Eds.).

Metais, Geranciamento da Toxicidade. San Pablo, Editora Atheneu, 2003, San Pablo, 187-298.

[2] Ferreira, S. L.C., de Andrade J. B., María das Graças A. Korn, M., Pereira, M. G., Lemosc, V. A., dos Santos,

Walter N.L., de Medeiros Rodrigues, F. Frederico, Souza, A. S., Ferreira, H. S., da Silva E. G.P. Review of

procedures involving separation and preconcentration for the determination of cadmium using spectrometric

techniques. J Hazard Mater; 2007; 145; 358-367.

[3] Tarley, C.R.T., Arruda, M.A.Z. A sensitive method for cadmium determination using an on-line polyurethane

foam preconcentration system and thermospray flame furnace atomic absorption spectrometry. Anal Sci; 2004; 20;

961-966.

[4] de Almeida Bezerra, M., Zezzi Arruda, M.A., Ferreira, S. L. C. Cloud Point Extraction as a Procedure of

Separation and Pre-Concentration for Metal Determination Using Spectroanalytical Techniques: A Review. Appl

Spectrosc Rev; 2005; 40; 269-299.


163

[5] Silva, E. L., dos Santos Roldan P. Simultaneous flow injection preconcentration of lead and cadmium using

cloud point extraction and determination by atomic absorption spectrometry. J Hazard Mater, 2009; 161; 142-147.

[6] Xiang G., Wen, S., Wu, X., Jiang, X. Lijun He, L., Liu, Y. Selective cloud point extraction for the determination of

cadmium in food samples by flame atomic absorption spectrometry. Food Chem, 2012; 132; 532-536.

[7] Silva, E. L., dos Santos Roldan P., Giné, M. F. Simultaneous preconcentration of copper, zinc, cadmium, and

nickel in water samples by cloud point extraction using 4-(2-pyridylazo)- resorcinol and their determination by

inductively coupled plasma optic emission spectrometry. J Hazard Mater, 2009; 171; 1133-1138.

[8] Reglamento Bromatológico Nacional. Características de los alimentos. Disposiciones Generales. Decreto

315/94; 1994.

[9] Massart, D. L., Vandeginste, B. G. M., Buydens, L. M. C., De Jong, S., Lewi, P.J., Smeyers-Verbeke, J.,

Handbook of Chemometrics and Qualimetrics: Part A, Amsterdam, Elsevier Science, 1997, 711-716.

Acknowledgements

Agencia Nacional de Investigación e Innovación (ANII) for Scientific Initiation Grant – ANII-2841). Dr. Harald Berndt

for providing the TS-FF-AAS system.

Dra. María H. Torre and Agropecuaria Valdense S.R.L for the seeds samples.


164

Escamoles ant eggs of Liometopum apiculatum M source of metal ions for human health

Melo, V.1; Calvo, C.2; Quirino, T.1; Macín, S.1; Muñiz, I.1 1Universidad Autónoma Metropolitana-X. Calzada del Hueso 1100, Edif. Central, 1er. Piso, Coyoacán, 04960, D.F. México.

[email protected] 2Instituto Nacional de Ciencias Médicas y Nutrición SZ. Vasco de Quiroga 14. Tlalpan, 14000, D.F., México.

Abstract

The earth crust is believed to be made of a mass of minerals and the participation of these elements in the physical

world is matched by their importance in human life. The human body like other leaving organisms depend on

several minerals as essential constituents of its existence. Metal ions in foodstuff are in different chemical forms, as

inorganic salts or organic molecules or complexes with other compounds such as proteins, amino acids, enzymes

and some vitamins, among others, that play an important role in human health. Entomophagy, insect consumption

by several ethnic groups in México as cultural tradition since prehispanic era, represents an option for population to

obtain the minerals needed by the body to keep a good health. Escamoles ant eggs of the Liometopum apiculatum

M genus, much appreciated either at rural communities as well as in urban cities, contain minerals with a favorable

effect in human health. The aim of this study was to investigate the mineral composition of Escamoles and the

benefits they can provide to the human body. Sampling was perform at an arid region of the Hidalgo state on April

2012. Minerals in dry basis were determined by Atomic Absorption Spectrophotometry, with the exception of

phosphorus, content was obtained from a triple acid digested extract and determined colorimetrically. Data of

mineral analysis in dry basis of Escamoles was: total minerals 5.92%; Na 0.079%; K 0.075%; Ca 0.097%; P

0.701%; Fe 0.021%; Zn 0.035%; Cu 0.009%; Mg 0.998%; Mn 0.002%. Minerals quantify are not equal to total ash

contained because not all of them were determined. Elements concentration depends not only of the total mineral

composition of foodstuff but also on their availability and avail of it. Minerals in Escamoles ant eggs have a

considerable influence in the condition of human health.

Introduction

Entomophagy is traditional in México since ancient times, escamoles ant eggs of the Formicidae family are part of

the cultural diet in rural communities and urban restaurants, consider as delicacies in México. Some other coutries

of Latin America, Asia and Australia ant eggs are much appreciated as well by their delicate flavor. [1]. Ant eggs

provide a good amount of macronutrients and micronutrients such as minerals, these insects reproduced at

desertic regions at an altitud range from 1800 to 3000 mts. above sea level in underground nests depth 1 to 1.5

mts, to keep low humidity and warm environment for natural develop of eggs. Nests are at xerophit ticket, near by

maguey agave sp or nopal opuntia sp cactus, as source of food sugars, amino acids and minerals. Ants mantain a

relationship with their habitat diversity and preserve an equilibrium of the ecology where they leave. [2]

Poor nutrition is a problem worldwide and is getting worse every day [3]. The natural available sources of food are

often less considered, whereas research of new technologies for synthesis and conservation, raises prices and

make the products inaccesible to the majority of population, particularly social groups from rural communities and

urban cities. Poor nutrition affects an individual´s performance and makes him/her vulnerable to different chronic

degenerative diseases [4,5,6]. Mineral deficiency represents a serious problem in the development and behavior of

the human being [7,8,9]. Absence of knowledge of both the edible species and their nutritional value, determine


165

lack of consumption and decreases potential food benefits from a large variety of organisms existing in the

environment.

The escamoles ant eggs of the Liometopum apiculatum M found at an arid regions of México and several other

countries worldwide, are consider as a food source with a good nutritional value of metal ions that could contribute

to improve human health of vulnerable groups. The aim of this is to assess mineral composition of the Liometopum

apiculatum M ant eggs and the benefits they can provide to the human body [10]

Material and Methods

Seasonal escamoles ant eggs reproduced from the second week of February to the end of May at Cardonal,

Hidalgo State, arid zone situated at 2100 masl, in a xerophit ticket environment of about 2,5 km, with a BS kw

climate, they were harvested early in the morning, with special techniques, by escamoleros, local country men. The

samples (450 g of eggs and few adults each time) were collected at February 11, March 30 and May 4, of 2012.

Samples captured from nests near by maguey cactus, were washed and transported by land in plastic containers

to the laboratory of Universidad Autónoma Metropolitana–X, to determin the taxonomy in adult insects and the

mineral analysis in eggs. Samples were analysed separately and by triplicate each one.

Material obtained was divided, into: adult ants and ant eggs. Adult ants were used to determine taxonomy and ant

eggs to perform analysis for chemical quantification of metal ions [11,12] The moisture content was measured by

simple drying in an oven at 80º C for 24 h. The dry product was powdered in a Willey Mill to 60 mesh size. The fine

powder so obtained was used for further analysis. Total minerals content were determined by incineration in a cold

muffle furnance set a 550º C by 2 h or until whitish/greyish ash was obtained. Individual ions, Na, K, Ca, Fe, Zn,

Cu, Mg and Mn, were analysed by atomic absorption spectrophotometry, and P content in acid digested extracts

was determined colorimetrically.


The Escamoles ant eggs are classified as an invertebrate, class insecta, order Hymenoptera, family Formicidae,

genus Liometopum, specie apiculatum M (Table 1) the moisture content is high (Table 2). The total minerals in

escamoles was 5.92 g/100g dry basis. There is a difference between the value of the total inorganic matter

obtained and the individual ions reported because not all elements content were assessed (Table 3, 4). The

availability of Escamoles ant eggs are seasonal (Table 5). The data obtained might change accorging biotic and

abiotic conditions of environment.

Table 1. Taxonomy determination of Escamoles

Class Insecta

Order Hymenoptera

Family Formicidae

Genus Liometopum

Specie apiculatum M

Common name Escamoles

Morón, M.A., Terrón, R. 1980. [22]


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Table 2. Moisture content of Escamoles %

Moisture 65.93

Dry matter 34.07

Table 3. Metal ions content in Escamoles ant eggs g/100g dry basis

Sodium 0.079

Potasium 0.075

Calcium 0.097

Phosphorus 0.701

Iron 0.021

Zinc 0.035

Copper 0.009

Magnesium 0.998

Manganesum 0.002

Tabla 4. Escamoles availability on year at Cardonal, Hidalgo State.

Season Winter Spring Summer Autumm

Months Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

X X X X

The magnitude of the under-nutrition problem, due to lack of metal ions, has reached significant proportions in the

world population, mainly in developing countries like México. The inadequate consumption of foods containing

adequate amounts of metal ions is closely related to phisical and mental development and the good health of

population [7]. Sodium and potasium are important ions in the functioning of the sodium-potasium pump [13].

Copper fulfills key metabolic functions in different organs and systems [14,15]. Iron is important in avoiding different

degrees of anemia which reduce the capacity of the individual work, another characteristic is the incapacity to

mantain body temperatura in a cold environment. Low consumption of iron produces a deficiency in psychomotor

development and intelectual activity, changes in the behavior of breast-fed infants up to two years of age and a

lower resistence to infections. Iron deficiency induces a substantial increase in risk of greater absorption of lead,

however, excessive administration of iron competes with absorption of zinc and copper that fulfills key metabolic

functions in different organs and systems [16]. Zinc a cell component is important for regulatory capacity of cells

since its intracelular concentration can be homeostatically controlled in a specific way for each tissue. It also has

functional and structural actions for many metalloenzymes and macromolecules [17,18,19]. Calcium and

phosphorus are indispensable for the formation of bone. Although phosphorus also acts as a structural part of high

energy compounds, the amounts of calcium and phosphorus necessary to mantain metabolic equilibrium depend

on the physiological needs of these metal ions and the capacity of the intestine to absorb them and the possibility


167

of the kidneys conserving them [20]. Magnesium is an enzyme cofactor that intervenes in the metabolism of

carbohydrates chalesterol and proteins [21].

Conclusion

The Escamoles ant eggs are seasonal but for a further consumption can be storaged refrigerated douring one

month. After that period of time they should be frozen. Most people intake Escamoles by their sensory

characteristics not for their nutritional value. It is very well accepted by population and can be intake either raw or in

different preparations. Consumption of this foodstuff is recomended for all population groups in low and high

density population regions, to void degenerative diseases and improve health.

References

1. Hopkins, J. 2004. Extreme Cusine. Periplus Editions Ltd. Singapore.

2. Speight, M.R., Hunter, M.D. and Wat, A. 1999. Ecology of Insects. Blacwell Science Ltd. London, UK.

3. Pelletier, D.L., Olson, C.M., and Fronguillo, EA. 2006. Food Insecurity. Hunger and undernutrition in: B.A.

Bowman and R.M. Russell (eds) Present Knowledge in Nutrition. ILSI Press, Washington DC. USA.

4. Chavez, A. and Chavez M. M. 2009. Nutrición su impacto en la salud humana y en la capacidad funcional.

Instituto Nacional de Ciencias Médicas y Nutricion Salvador Zubiran. México.

5. Chavez M. M. et al, 2010. Tablas de valor nutritivo de los alimentos en México. International Mc Graw Hill.

México.

6. FAO 1995. Conferencia Mundial Cumbre 50 Aniversario. FAO World Conference Report, Quebec, Canada.

7. Reilly, C. 2004. The Nutritional Trace Metals. Blackwell Publishing Ltd. Oxford, UK.

8. Conor, R 2006. The nutritional trace metals. Blackwell Publishing Ltd. Oxford, UK.

9. Crabb, E and More, E. 2010. Metals and Life. RSC Publishing. Cambridge, UK

10. Melo, V. Reyes, J, Castejon, E., Nogueda, N. 2006. Metal in three species of edible insects in México. Metal

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Park9 interaction with Manganese and other divalent cations

Zoroddu, M.A.a*; Remelli, M.b; Peana, M.a; Medici, S.a; Solinas, C.a aDepartment of Chemistry and Pharmacy, University of Sassari, Sassari (Italy),

bDepartment of Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara (Italy),

*Email: [email protected]

Abstract

Two peptide sequences from Park9 Parkinson’s disease gene, P1D2E3K4H5E6L7 (1) and F1C2G3D4G5A6N7D8C9G10

(2) have been studied in their interaction with Mn(II) and Zn(II) ions. These fragments lie from residue 1165 to 1171

and from 1184 to 1193 in the Park9 encoded protein, that can protect cells from manganese poisoning, an

environmental risk factor for a Parkinson’s disease-like syndrome called Manganism. The study was carried out

through potentiometric and spectroscopic (UV-Vis, EPR, mono- and multidimensional NMR) techniques, to cast

light on the details of metal binding at different pH values and different ligand to metal molar ratios.

Keywords: Parkinson, Manganism, divalent cations

Introduction

Recently, a connection between genetic and environmental causes of Parkinson’s disease (PD) has been reported

[1,2]. It was known that environmental and occupational exposure to Mn(II) [3] can lead to symptoms that resemble

Parkinson’s disease, called Parkinsonism or Manganism [4], but two articles appearing almost at the same time in

the literature showed that a human PD gene, PARK9 (a member of the P5-type ATPase family, perhaps a metal

transporter, considering its richness in coordinating residues), and its homologue in yeast, YPK9, can prevent

manganese- induced PD and protect neurons and cells from manganese poisoning [1]. In fact, deletion of the

YPK9 gene, which is 58% similar and 38% identical in its amino acid sequence to human PARK9, confers

sensitivity to growth defects in the presence of cadmium, nickel, selenium, and manganese, suggesting that YPK9

protein may play a role in the sequestration of divalent heavy metal ions. In the same way, a mutation on PARK9

may expose humans to these cations, especially to manganese. We have thus decided to verify the possibility of

metal binding to some simplified portions of PARK9 and YPK9, by choosing promising sites for metal interaction

due to the presence of coordinating residues, far from the membrane-spanning and the alpha-helical rich domains

not easily accessible by metals, and taking into account highly conserved binding motifs on both PARK9 and

YPK9. We identified two interesting fragments and tested them for metal binding with manganese and zinc [5,6].

Materials and methods

Peptides were synthesized using solid-phase Fmoc chemistry in an Applied Biosystems Synthesizer. Peptides

were N-terminally acetylated and C-terminally amidated in order to mimic this region of YPK9 within the full-length

protein.

NMR experiments were performed on a Bruker AscendTM 400 MHz spectrometer equipped with a 5 mm automated

tuning and matching broad band probe (BBFO) with z-gradients. Samples were 2.5 or 5 mM in concentration and

dissolved in 90/10 (v/v) H2O/D2O solutions, at 298 K in 5 mm NMR tubes. NMR data were processed with TopSpin

(Bruker Instruments) software and analyzed by Sparky 3.11 MestRe Nova 6.0.2 (Mestrelab Research S.L.)

programs.


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Potentiometric titrations were performed with Orion EA 940 and Orion 720A pH-meters equipped with a combined

glass electrode (Metrohm EA125), Hamilton MicroLab M and MicroLab300 motor burettes, and Hamilton syringes.

The temperature was kept at 298.2 ± 0.1 K by use of a thermostat. All measurements were carried out under

nitrogen atmosphere.

EPR spectra were recorded with an X-band (9.4 GHz) Bruker EMX spectrometer, 100 kHz field modulation, at 120

K on neat aqueous solutions or mixed with few drops of ethylene glycol, at different ligand to metal molar ratios,

ranging from 1:0.2 to 1:1, pH 7 and 1 mM peptide concentration.


Peptide 1, P1D2E3K4H5E6L7

Only mono-nuclear complexes have been potentiometrically detected in the systems containing Mn(II) and the

investigated peptides, and the speciation diagrams are in agreement with the results found with NMR, UV-vis and

EPR measurements.

Mn(II) is a paramagnetic ion, thus the effect of its addition to peptide 1 and 2 was monitored through the NMR line

broadening and/or disappearance it causes on the resonances of the free ligand (Fig.1).

Fig. 1 Selection of aliphatic regions in 1H-13C HSQC NMR spectra for the Ac-P1D2E3K4H5E6L7- Am peptide, 2.5 mM,

pH 6.2, T 298 K in the absence (orange) and in the presence (blue) of 0.1 equivalents of Mn(II).

As expected whenever a histidine residue is present on a peptide fragment coordinating a metal, this was the

favoured binding site for both Mn(II) and Zn(II) ions with peptide 1.

From the different sets of experiments performed, it was clear that once His5 has anchored the peptide at slightly

acidic pH, other residues are involved in coordination as the pH is raised. Mn(II) progressively affected the

relaxation rates of the amidic HN protons from the backbone together with the complete disappearance of Asp2,


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Glu3 and Glu6 aliphatic protons signals in the 2D TOCSY spectra, and a decrease or total loss of their proton–

carbon correlations in the 2D 1H-13C HSQCs.

Moreover, the analysis of the aliphatic HSQC region revealed that Qγ protons of Glu3 and Glu6 vanished upon

Mn(II) addition, indicating that glutamic carboxylic groups are involved in the coordination, a fate shared also by the

aspartate residue. All the other signals remained unaffected upon manganese addition. From the analysis of the

data thus collected, the donor groups involved in metal coordination might be the Nε nitrogen from the imidazole

ring of His5, and the γ-O from three carboxylic moieties of Asp2, Glu3 and Asp6 side-chains (Fig 1).

EPR spectra of the Mn(II) complexes reflect the symmetry of the electronic environment of the ion which is imposed

by the ligands in the primary coordination sphere. Changes in the composition or geometric arrangement of the

ligands in the coordination sphere of the metal ion lead to changes in the position of the EPR spectral lines. The

Mn(II)–peptide 1 spectra are almost indistinguishable from those of free Mn(II). The spectral identity indicated that

the interaction, in aqueous solution, between the ligand and Mn(II) ions does not affect the electronic structure of

the metal ion itself. Thus, it is possible that the peptide does coordinate to Mn(II) by displacement of aqua ligands;

however, the six donor set and coordination geometry is almost maintained so that the zfs parameters remain

nearly unchanged. The spectra are in agreement with an octahedral or distorted octahedral geometry, probably

involving bidentate interaction of carboxyl groups carboxyl groups (Fig. 2b).

Fig. 2 a) The model of the YPK9 protein built with ESyPred3D using the 3D crystal structure of the sodium-

potassium pump (PDB 3B8E chain A) as the template, and the 3D structural models proposed for b) the Mn(II) ion

complexed with Ac-P1D2E3K4H5E6L7-Am and c) with the Ac- F1C2G3D4G5A6N7D8C9G10-Am peptide.

Diamagnetic zinc ion was studied in order to have more information about the coordination behaviour of both

investigated peptides. In fact, we were able to observe that the signals affected by diamagnetic shift under zinc

interaction were the same which experienced paramagnetic broadening under manganese interaction, suggesting

that the donor atoms involved in the coordination with peptide 1 are the same for both metal ions. In addition, a


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change in the resonances for HN and Hγ2 of Lys4, and for HN of Leu7 shows that these two residues, although not

directly involved in the complex formation, experience a new electronic environment after metal coordination.

Peptide 2, F1C2G3D4G5A6N7D8C9G10

Coordination of manganese to peptide 2 starts with the binding to a cysteine residue, as evidenced by the

broadening of its signals already at very low metal concentration. So, in the absence of a histidine residue, cysteine

acts as the anchoring site for the metal ion. In fact, Mn(II) progressively affected the relaxation rates in the

resonances of both Cys2 and Cys9 with a complete disappearance of their signals in the 2D TOCSY spectra and

the total loss of their proton–carbon correlations in the 2D 1H-13C HSQCs (Fig. 3).

Fig. 3 Aliphatic regions of 1H-13C HSQC NMR spectra of the Ac-F1C2G3D4G5A6N7D8C9G10-Am peptide, 2.5 mM, pH

6.3, T 298 K, in the absence (orange) and in the presence (blue) of 0.1 equivalents of Mn(II).

Asp4 and Asp8 residues experience the main difference in the observed Mn(II)-induced effects for the carbon and

proton frequencies, indicating that they take part, together with the cysteine residues, in the complex formation,

most likely through their carboxyl groups (Fig. 2c). The presence of the paramagnetic metal provides a signal

relaxation also for HN protons of Gly5, Ala6 and Asn7 residues. This behaviour could be due to an indirect

paramagnetic line broadening effect caused by a through-space relaxation of these residues along the peptide

chain which happen to be close to the Mn(II) centre.

The EPR spectra of Mn(II)–peptide 2 complex and of free Mn(II) ion in aqueous (glass) solution show that they are

qualitatively the same in the resolved sextet region, but the overall breadth of the pattern is a little different,

indicating that the axial zfs parameter is different for the Mn–peptide species. This can be explained by a decrease

in the number of water ligands around Mn(II) in the presence of the peptide fragment, suggesting their replacement


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by oxygen and sulphur donor atoms. Diamagnetic NMR with Zn(II) ions evidenced that the residues involved in

metal coordination are those experiencing the strongest chemical shift differences: Cys2 and Cys9. Other residues

appear to be indirectly interested in the complex formation by slight perturbations: Phe1, Gly3, Asp8 and Gly10.

Gathering the results from the NMR experiments we are able to infer that zinc coordination to peptide 2 involved

the sulphur donors from the two cysteine side chains, forming a macrochelate complex. It is interesting to point out

that the involvement of the two carboxylic groups from Asp4 and Asp8, in contrast to manganese coordination, has

not been evidenced for Zn(II) coordination, thus indicating that, in this case, the behaviour of peptide 2 is different

as a function of the different metal ion.

Conclusions

The preliminary results of the study on these two Park9 protein fragments show that both metals are able to bind

them in an effective way, although with different coordination features depending on the fragment chosen,

evidencing that portions of the same protein can interact in different ways with different ions. Although the

fragments used are small and only represent a part of the entire protein, nevertheless this kind of study may

provide insight in the divalent cations trafficking (transportation, detoxification) within the cell, at the base of the

connection between genetic and environmental Parkinson’s disease causes. Further investigations, involving

bigger domains of PARK9/YPK9 protein, are planned to shed light on the role of the entire protein in sequestering

manganese and in the involved detoxification mechanism.

References

1) Gitler AD, Chesi A, Geddie ML, Strathearn KE, Hamamichi S, Hill KJ, Caldwell KA, Caldwell GA, Cooper AA,

Rochet JC, Lindquist S. Alpha-synuclein is part of a diverse and highly conserved interaction network that includes

PARK9 and manganese toxicity. Nat Genet. 2009; 41; 308-15

2) Schmidt K, Wolfe DM, Stiller B, Pearce DA. Cd2+, Mn2+, Ni2+ and Se2+ toxicity to Saccharomyces cerevisiae

lacking YPK9p the orthologue of human ATP13A2. Biochem Biophys Res Commun. 2009 ; 383; 198-202

3) Covy JP, Giasson BI. α-Synuclein, leucine-rich repeat kinase-2, and manganese in the pathogenesis of

Parkinson disease. Neurotoxicology. 2011; 32; 622-9

4) Perl DP, Olanow CW. The neuropathology of manganese-induced Parkinsonism. J Neuropathol Exp Neurol.

2007; 66; 675-82

5) Medici S, Peana M, Delogu LG, Zoroddu MA. Mn(II) and Zn(II) interactions with peptide fragments from

Parkinson's disease genes. Dalton Trans. 2012 ;41; 4378-88

6) Remelli M, Peana M, Medici S, Delogu LG, Zoroddu MA. Interaction of divalent cations with peptide fragments

from Parkinson's disease genes. Dalton Trans. 2012; DOI: 10.1039/C2DT32222F

Acknowledgments

This work was also supported by the Regione Autonoma Sardegna FSE Sardegna 2007–2013 L.R.7/2007

“Promozione della ricerca scientifica e dell’innovazione tecnologica in Sardegna” programme, the L.R.7/2010

programme and the Fondazione Banco di Sardegna.


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Effect of zinc supplementation on the production of IL-17 in elderly individuals

Aguilar, A.E.1; Sanchez, M.T.C.1; Muñoz, B.1; Espejel, G.2; Saldivar, L.2; Pastelin, R.1

School of Chemistry, 1Biology Department,

2Analytical Chemistry Department, University of Mexico, UNAM, Mexico DF 04510,

Mexico

Abstract

The suboptimal contribution of micronutrients may damage the immune response. In the particular case of zinc, this

microelement has shown their participation in various immunological processes around perinatal stages. In the

elderly, eating lesser amounts of food can cause a negative balance in the concentration of zinc and its possible



autoimmunity. This study aimed to evaluate the effects of zinc supplementation on the production of IL-17 over

elderly stages. For this, we used an experimental model of zinc supplemented mice (500 microgram/mL zinc

acetate, BALB/c inbred mice). We measured the microelement concentration in lymphoid organs by Atomic

Absorption Spectrometry and also the cytokine production in serum by double antibody enzyme-immuno assay.

Results showed that female group of supplemented mice diminished the cytokine production 34.35 % compared

with control group whereas male group of supplemented animals showed a noticeable reduction of 54.34 %.

Further, zinc concentration increased in the spleen of female supplemented mice (80%). We concluded that

supplementation of zinc in mice has the effect of causing decreased production of IL-17 by Th17 cells, which far to

produce a problem of autoimmunity this means a regulatory response to supplemented individuals.

Keywords: zinc, IL-17, elderly

Introduction

Zinc has various effects on the immune system, acting on the maturation of CD4+ and CD8+ T lymphocytes and

serum concentrations of this element are modified in autoimmune diseases. In case of deficiency causes an

imbalance in the function of the Th1 and Th2 cells in the periphery due to the reduced production of IFN-γ and IL-2

while the production of Th2 cytokines such as IL-4, IL-6 and IL-10, is not affected.[1]

Interleukins are a group of proteins that act as chemical messengers because these are the main intracellular

communication in a microbial attack. Cytokines serve to initiate the inflammatory response, and to define the extent

and nature of the specific immune response. Its main function is to regulate the events belonging to the functions of

the different populations of immune system cells, such as activation, differentiation or proliferation of various cell

types, antibody secretion, chemotaxis and regulation of other cytokines and factors. [2]

Some IL-17 studies have shown that IL-17/1L-17RA system is essential for host defense in various extracellular

bacterial infections. About the fungi infections, the Th cell activation occurs by 17 polysaccharides constituting the

cell wall. [3] Another important point is that in the absence of the signaling of IL-17, the influx and activation of

neutrophils in the infected organs undergoes serious delay, which means that there is not properly control of the

infections.[4] However, this cytokine overproduction can trigger autoimmune disorders like rheumatoid arthritis,

autoimmune encephalomyelitis, multiple sclerosis and psoriasis. [5]


175

In the elderly, nutritional deficiency can cause a negative balance in the concentration of zinc and its possible



autoimmunity. [6, 7]

This study aimed to evaluate the effects of zinc supplementation on the production of IL-17 over elderly stages.

Material and Methods

We used an experimental model in inbred mice. Balb/cAnN, male and female animals, received zinc acetate (500

microgram/mL, Mallinckrodt-Baker, Mexico, Cat 8740)) in drinking water for two weeks. Controls did not drink

supplement. The animals were divided into groups according to the received supplementation (Zn 0, Zn500), and

considering the gender of the animal. All mice were kept 7 months (28 weeks) in plastic boxes with stainless steel

covers. For water intake were used glass bottles and glass drinking tubes. The animals were fed ad libitum with

commercial diet 2018S HarlanTeklad Global Diets (USA). This study was performed according to the Guide for the

Care and Use of Laboratory Animals.

The Zn concentrations in thymus and spleen were obtained by AAS technique. In order to validated the method

and results we utilized the NIST (Standard Reference Material, 1577b, bovine liver).

To study the effect of zinc supplementation on the production of IL-17 and its detection in serum was assayed a

double antibody ELISA (PeproTech 900-M392), where antibodies anti-IL-17 were produced in rabbits and the

detection antibodies were biotinylated; as developing agent was employed the avidine-peroxidase system plus

ABTS (Sigma Cat A3219) and absorbance were measured to 405 nm in a Behring EL 301 microreader, with

correction absorbance at 630 nm.

Finally, statistical analysis was performed using GraphPad Prism software version 6.01


Aging is associated with damaged immune response which is linked with several immune related diseases in the

elderly. Nutritional intervention could be a promise for modulating immunity. [7]

Our studies showed that in vivo supplementation increases zinc concentration in the spleen and remains constant

the absorption in the thymus, and gender differences were observed because the trace element is localized mainly

in the spleen of elderly supplemented female group, with an increase of 80%, whereas in male individuals remains

mostly in the thymus, with an increase of 70%. Figure 1

This difference can be explained by the observation that the effect of zinc concentration is hormonal dependent

and the age of animal is critical to the analysis of results.


176

We observed the typical proportional relationship between the absorbance and the concentration of murine IL-17,

in pg / mL. Figure 2

Figure 2. Murine IL-17 standard curve

The experimental groups were supplemented with zinc acetate (2H2O), in drinking water at 500 microgram/mL for

two weeks, showing a decline of IL-17 in serum (Figure 3A) being the diminish of 34.35% in the group of elderly

females and (Figure 3B) and 54.34% for elderly male mice. These data have some coincidence with the tendency

reported by Kitabayashi et al [1]. However our results indicated biological aspects which we need to investigate in

depth eg cytokine cellular concentration.


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Figure 3A, B. Murine IL 17 in pg/mL obtained in serum from supplemented groups compared with the controls

Conclusion

We concluded that supplementation of zinc in mice has the effect of causing decreased production of IL-17 by

Th17 cells, which far to produce a problem of autoimmunity this means a regulatory response to supplemented

individuals.

References

1. Kitabayashi Ch, Fukada T, Kanamoto M, Ohashi W, Hojyo Sh, Atsumi T, Ueda N, Azuma I, Hirota H, Murakami

M, Hirano T. Zinc suppresses Th17 development via inhibition of STAT3 activation. Int Immunol. 2010, 22: 375–386

2. Kindt TJ, Osborne BA, Goldsby RA, Kuby J. Immunology. Freedman WH Publisher, USA, 2007. pp 236-238

3. Louten J, Boniface K, de Waal Malefyt R. Development and function of TH17 cells in health and disease. J

Allergy Clin Immunol. 2009, 123:1004-1011

4. Korn T, Oukka M, Kuchroo V, Bettelli E. Th17 cells: Effector T cells with inflammatory properties. Seminars in

Immunology. 2007,19:362–371


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5. Hemdan NYA, Birkenmeier G, Wichmann G, Abu El-Saad AM, Krieger T, Conrad K, Sack U. Interleukin-17-

producing T helper cells in autoimmunity. Autoimmunity Rev. 2010, 9:785–792

6. Haase H and Rink L. The immune system and the impact of zinc during aging. Immunity and Ageing. 2009, 6:1-

17

7. Pae M, Meydani S N, Wu D. The role of nutrition in enhancing immunity in aging. Aging and disease. 2012, 3:91-

119

8. National Research Council Guide for the Care and Use of Laboratory Animals. National Academy Press. Mexico

2002


179

A novel Cu(II) ternary complex with iminodiacetate and dimethyl-bipyridine. Synthesis, characterization and DNA interaction

Alvarez, N.a; Costa-Filho, A.c; Torre, M.H.a; Ellena, J.b; Facchin, G.a. aCátedra de Química Inorgánica, Facultad de Química, Universidad de la República, Montevideo, Uruguay.

bInstituto de Física de São Carlos, Universidad de São Paulo, São Carlos, Brasil.

cFaculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brasil.

[email protected]

Abstract

The importance of metal complexes in medicine dates from the XVIth century with reports on the use of

metallodrugs in anticancer therapy. Inorganic Medicinal Chemistry has made great advances in search of new

metal compounds presenting antitumor activity, for instance, by use of essential metals. In this work, in the search

of new antitumor agents, the ternary complex [Cu(iminodiacetate)(4,4’-dimethyl-2,2’-bipyridine)]�4H2O was

prepared employing the reaction of copper acetate with iminodiacetic acid in presence of dimethyl-bipyridine. The

obtained complex was characterized by elemental analysis, single crystal X-ray diffraction, Fourier Transform

Infrared spectroscopy (FT-IR), Electron Paramagnetic Resonance (EPR) and UV-visible spectroscopy (UV-vis).

The complex crystallizes in a triclinic unit cell, in the centrosymmetric space group P-1. The Cu(II) center is bonded

to the bypiridine ligand -through its N atoms- and to the iminodiacetate ion -through the carboxylate O atoms and

the imine N atom-. The molecular structure was confirmed by FT-IR where bands involving the ligand and donor

atom-Copper bonds are observed. Knowing the atom array in solid state is considered fundamental for further

aqueous solution analysis and comprehension of the complex behavior in biological media. UV-vis absorption

bands in aqueous solution are consistent with a square pyramidal Cu(II) environment, which suggests the

maintenance of the coordination sphere observed in solid state. The ability of the complex to interact with

deoxyribonucleic acid (DNA) was studied by Circular Dichroism (CD) in constant concentration of DNA with

increasing concentration of complex. The profile observed in the CD spectra evidences complex-DNA interaction

which is consistent with an intercalative interaction, as observed in other complexes containing dimethyl-bipyridine.

Keywords: copper complex, crystal structure, DNA interaction.

Introduction

One of the target biomolecules for therapeutics with metallodrugs in cancer is the deoxyribonucleic acid (DNA). If

the complex binds to the DNA or participates in any form of interaction, it could alter its structure, impeding the

cellular system's ability to correctly read the genetic code, which can result in the interruption of protein synthesis or

synthesis of non-functional proteins. Any of these results leads eventually to cell death.

In the search of metallodrugs with fewer side effects, one of the strategies in Inorganic Medicinal Chemistry is to

synthesize coordination compounds with essential metals. The main advantage in the use of essential metals is

due to pre-existing biodistribution and excretion pathways. However, their therapeutic use should be controlled

since in excess they become toxic. In particular, different series of Cu(II) complexes have shown promising

antiproliferative activity in different tumor cell lines. Among them, the heteroleptic complexes with di-imine ligands

denominated Casiopeínas® have completed preclinical requirements and complexes CasII-gly [(Cu(II))(4,7-

dimethyl-1,10-phenanthroline)(glycinate)(NO3)(H2O)] and CasI-II-ia [(Cu(II))(4,4’-dimethyl-2,2’-


180

bipyridine)(acetilacetonate)(NO3)(H2O)] entered phase I clinical trials [1; 2]. The proposed mechanism for the latter

is DNA oxidative damage, as well as damage to other biomolecules and organelles, through production of reactive

oxygen species (ROS) in the presence of a reducing agent [3; 4].

The research group where the investigation takes place has experience in synthesis and characterization both in

solid state and aqueous solution of Cu(II) coordination complexes of pharmacological interest. The ability of these

complexes to interact with DNA has also been assessed. Antiproliferative studies of Cu-dipeptide and Cu-

dipeptide-phenanthroline complexes in different tumor cell lines show promising results [5; 6].

In this work the study of a new ternary Cu(II) complex with iminodiacetic acid (ida) and dimethyl-bipyridine (dmb) is

presented.


Materials

All reactants, 4,4’-dimethyl-2,2’-bipyridine (Sigma, 99%), iminodiacetic acid (Sigma, 98%), Copper (II) acetate

(Sigma, 98%), as well as organic solvents and distilled water were used without further purification.

Synthesis and analytical characterization

To 10 mL of a 25 mM aqueous solution of Copper (II) acetate with constant stirring at 50°C, were added 10 mL of a

25 mM ethanolic solution of dimethyl-bipyridine and 10 mL of a 25 mM solution of iminodiacetic acid deprotonated

by NaOH (pH=7). Single crystals were grown by slow evaporation of solvent from the resulting solution at 4°C and

its crystal structure determined by X-ray diffraction. Anal. Calc. for C16H25CuN3O8: N: 9,32; C: 42,62; H: 5,59.

Found: N: 9,06; C: 42,93; H: 5,09.

Crystal structure determination

Data from suitable single crystals was collected at 298(2) K in a Enraf-Nonius FR590 Kappa-CCD diffractometer

using graphite monochromated MoKα radiation (0,71073 Å). The structure was solved by direct methods and the

model refined with SHELXL-97. Gaussian absorption correction was applied. All non-hydrogen atoms were refined

on F2 by full-matrix least-squares procedure using anisotropic displacement parameters. Hydrogen atoms for C-H

groups were stereochemically positioned, whereas imino hydrogen and water hydrogen atoms were found in the

difference Fourier map and positionally fixed. All hydrogen atoms were refined using the riding-model. The values

of the refinement indicators are: Goodness of fit: 1,070 (1 restrain), R1: 0,038 (I>2σ(I)) and wR2: 0,102 (I>2σ(I)).

Spectroscopic measurements

FT-IR spectra were measured on a Bomen MB 102 instrument in the range 4000-400 cm-1. Electronic absorption

spectra on aqueous solution were recorded on a Shimadzu UV-1800 in the range 200-900 nm using 1 cm quartz

cells. X-band (9.5 GHz) EPR measurements were carried out on polycrystalline samples and frozen aqueous

solution using a Bruker ELEXSYS E580 system with 100 kHz field modulation. Measurements were performed at

N2 liquid temperature (77 K).


181

DNA-complex interaction

DNA-complex interaction was characterized by circular dichroism in a JASCO J-815 spectrometer in the 200-300

nm range. Dichroism spectra of 1.5 mM DNA aqueous solution with increasing concentration of complex were

measured at 20°C.


Crystallographic data analysis

The coordination compound studied crystallizes in the spatial group P-1. Cell parameters are: a=8.0498(11),

b=10.8189(17), c=12.5674(14), α=67.215(7), β=88.141(5) and γ=84.128(8). The coordination sphere surrounding

the Cu(II) ion has in the equatorial positions three nitrogen atoms (two from the bipyridine ligand and one from the

iminodiacetate ion) and one oxygen (from the terminal carboxylate of the iminodiacetate ion), whereas the apical

position is occupied by the remaining oxygen from the iminodiacetate ion.


182

a) b)

Figure 1. (a) Representation of the complex [Cu(ida)(dmb)]�4H2O. (b) Crystal packing.

The crystalline arrangement is sustained by intermolecular hydrogen bonding (N-H•••O y H-O•••H) between water

molecules, carboxylate and imine groups as well as π-π interactions between the aromatic rings in the bypiridine

ligand (Figure 2).

Spectroscopic measurements

FT-IR spectroscopy confirms the coordination scheme observed in the crystal. Some of the most representative

stretching and rocking modes are: ν(C-H), ν(O-H) in 2900-3500 cm-1, ν(COO-)as in 1620 cm-1, ν(C-C), ν(C-N) in

1450, 1493 cm-1, δ(C-C-C), δ(C-C-N) in 921, 957 cm-1, aromatic δoop(C-H) in 839 cm-1, ν(Cu-N) in 514 cm-1 and

ν(Cu-N) in 425 cm-1.

Electronic spectrum of aqueous solution of the complex shows intraligand charge transferences in the ultraviolet

region and an asymmetric band at 656 nm (ε656=63 cm-1M-1) due to d-d electron transfer consistent with

pentacoordinated Cu(II) centers, suggesting that the coordination scheme observed in solid state is preserved in

solution.

EPR spectra both in solid state and frozen aqueous solution do not show any hyperfine structure. This is probably

a consequence of exchange interactions between the paramagnetic copper ions. In these cases, exchange

interaction averages out other interactions such as hyperfine.

DNA-complex interaction

The CD curves corresponding to DNA and DNA plus complex are shown in Figure 2. DNA concentration during the

whole experiment is fixed and the concentration of the complex is increased in each measurement. Even though

the general profile of the curve is similar it can be observed that after addition of the complex intensities around the

bands at 215, 244 and 270 nm vary due to an induced CD spectrum. In the case of the band at 244 nm the

intensities increase and have a shift to lower wavelengths, whereas in the case of the band at 270 there is no


183

wavelength shift but the intensities decrease. This behavior is consistent with an intercalative mode of binding,

which has been previously observed for other complexes with dimethyl-bipyridine [7].

200 220 240 260 280 300

-12

-8

-4

0

4

8

12

DNA

9 mM

25 mM

38 mM

49 mM

θ (m

deg

)

λ (nm)

Figure 2. Circular Dichroism spectra of DNA with increasing concentrations of complex.

Conclusions

The crystalline structure of a new heteroleptic complex [Cu(II)(ida)(dmb)]�4H2O with square-based pyramidal

geometry was determined. The molecular motif of the complex is maintained in aqueous solution. The complex

interacts with DNA, probably by intercalative binding.

Acknowledgements

The authors thank the Agencia Nacional de Investigación e Innovación (INI_X_2011_1_3940, N. Alvarez), Instituto

de Física de São Carlos (Winter internship, N. Alvarez) and PEDECIBA Química for financial support.

References

[1] J. Serment-Guerrero, P. Cano-Sanchez, E. Reyes-Perez, F. Velazquez-Garcia, M.E. Bravo-Gomez, L. Ruiz-

Azuara, Genotoxicity of the copper antineoplastic coordination complexes casiopeinas®. Toxicol In Vitro 25 (2011)

1376-1384.

[2] M.E. Bravo-Gómez, J.C. García-Ramos, I. Gracia-Mora, L. Ruiz-Azuara, Antiproliferative activity and QSAR

study of copper(II) mixed chelate [Cu(N–N)(acetylacetonato)]NO3 and [Cu(N–N)(glycinato)]NO3 complexes,

(Casiopeínas®). J Inorg Biochem 103 (2009) 299-309.

[3] A. Marı́n-Hernández, I. Gracia-Mora, L. Ruiz-Ramı́rez, R. Moreno-Sánchez, Toxic effects of copper-based

antineoplastic drugs (Casiopeinas®) on mitochondrial functions. Biochem Pharmacol 65 (2003) 1979-1989.

[4] X. Zhong, J. Yi, J. Sun, H.L. Wei, W.S. Liu, K.B. Yu, Synthesis and crystal structure of some transition metal

complexes with a novel bis-Schiff base ligand and their antitumor activities. Eur J Med Chem 41 (2006) 1090-1092.


184

[5] G. Facchin, E. Kremer, D.A. Barrio, S.B. Etcheverry, A.J. Costa-Filho, M.H. Torre, Interaction of Cu-dipeptide

complexes with Calf Thymus DNA and antiproliferative activity of [Cu(ala-phe)] in osteosarcoma-derived cells.

Polyhedron 28 (2009) 2329-2334.

[6] N. Alvarez, S. Iglesias, R. Sapiro, M.H. Torre, G. Facchin, Antioxidant and pro-oxidant properties and

antiproliferative activity of homoleptic and heteroleptic copper complexes, potential antitumoral species, Badawi,

(Ed.), Metal Ions in Biology and Medicine, John Libbey Eurotext, Cambridge, United Kingdom, 2011, pp. 117-122.

[7] Y.S. Wu, K.R. Koch, V.R. Abratt, H.H. Klump, Intercalation into the DNA double helix and in vivo biological

activity of water-soluble planar [Pt(diimine)(N,N-dihydroxyethyl-N'-benzoylthioureato)]+Cl- complexes: a study of

their thermal stability, their CD spectra and their gel mobility. Arch Biochem Biophys 440 (2005) 28-37.


185

Towards the development of new copper compounds for the treatment of cancer: Study

of the cytotoxic activity of [[[[Cu(L-dipeptide)(1,10-phenanthroline)]]]] complexes

Iglesias, S.a; Noble, C.a; González, R.b; Torre, M.H.a; Kremer, E.a; Kramer, G.b; Facchin, G.a aCátedra de Química Inorgánica, Facultad de Química, Universidad de la República, Montevideo, Uruguay.

bDepartamento de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, Universidad de la República,

Montevideo, Uruguay. [email protected], [email protected]

Abstract

Coordination compounds of essential metals are being explored as new antitumor species, in the search of drugs

with expanded activity and/or reduced side effects. To date, several Cu(II) compounds have shown promising

antitumor activities in cellular lines, some of them are being studied in vivo. Their mechanisms of action are not

completely understood; however, it is well known that copper and its complexes, present the ability to participate in

redox reactions. This event generates reactive oxygen species (ROS) that are toxic to cancerous cells. In addition,

many of these complexes present the ability to intercalate DNA, interfering with DNA replication of dividing cells,

which may also be part of their mechanism of antitumor action. We have synthesized several new [Cu(L-

dipeptide)(1,10-phenanthroline)] compounds. In solid state they present a pyramidal geometry, where the Cu(II) ion

is coordinated both to the phenanthroline, through its nitrogen atoms, and to the dipeptide, through the amine and

the amide nitrogen atoms and the carboxylic oxygen. These compounds are stable in aqueous solution, where its

coordination is maintained. In this work we have focused on the study of the lipophilicity and antitumor activity of

[Cu(L-dipeptide)(1,10-phenanthroline)] (were L-dipeptide= phe-phe, phe-val, phe-ala, ala-phe, gly-val, ala-gly). The

cancer cell lines utilized were HeLa (human cervical cancer), NMU (rat breast cancer) and 4T1 (mouse breast

cancer). The lipophilicity, depends on the dipeptide, being the [Cu(L-Phe-Phe)(1,10-phenanthroline)] complex the

most lipophilic. The estimated IC50 varied from 1 to 30 µM in these tumor cells, being the complexes containing the

dipeptides phe-val, phe-ala and ala-phe the ones that showed best cytotoxic activity in vitro. Therefore, complexes

[Cu(L-dipeptide)(1,10-phenanthroline)] with dipeptides phe-val, phe-ala and ala-phe, may be good candidates to

test its antitumor activity in animal models of cancer.

Keywords: copper phenanthroline dipeptide complex, lipophilicity, antiproliferative activity.

Introduction

Cisplatin, a coordination compound of Pt(II), is a widely used antitumor drug [1]. In search for new antitumor active

metal coordination complexes with expanded activity and/or reduced side effects, several copper (II) complexes

have been studied and reported to be promising as anticancer drugs. Copper is an essential metal therefore there

are specific metabolic routes of detoxification which may lead to complexes with less severe side effects. The

mechanisms of action of copper complexes are not completely understood; however, two early molecular events

are commonly accepted. One of them is the ability of copper and its complexes to participate in redox reactions

producing reactive oxygen species (ROS) that are toxic to cancerous cells, which are especially sensitive to

oxidative stress. The other one is the ability to intercalate or coordinate with the DNA, interfering with DNA

replication of dividing cells, which may also be part of their mechanism of antitumor action.


186

Cu(I) complexes with the metal chelator bis-1,10-phenanthroline (phen) were synthesized and evaluated by

Sigman et al.. Especially, [Cu(phen)2]+ was reported to oxidatively degrade DNA and RNA by attacking the sugar

groups. The presence of large quantities of an external reductant, mediated the generation of freely diffusible OH

radical through the redox cycling between the Cu(II)/Cu(I) couple [2].

In spite of their large study, the biological application of bis phen copper complexes presents a restriction: their

stability is rather unfavorable under physiological conditions, because of the small association constant of the

second phen. One way to overcome this problem is to prepare mixed ligand complexes including only one phen

and additional ligands that form a stable complex [2]. An outstanding example of this approach are the complexes

denominated Casiopeinas, developed by L. Ruiz-Azuara et al., which are entering phase I human clinical studies

[3].

Our research group has developed different series of new mixed copper complexes, including phen and derivatives

as ligands. In particular, a series of new [Cu(L-dipeptide)(phen)] compounds has been synthesized and

characterized. They present a pyramidal geometry, where the Cu ion is coordinated both to one phen, through its

both nitrogen atoms, and to the dipeptide, through the amine and the amide nitrogen atoms and the carboxylic

oxygen. Its coordination is maintained in aqueous solution, where they are stable [4]. As the complexes present a

bulky lipophilic ligand, they may access lipophilic sites, such as solid tumors.

In this frame, the present work describes the study of the lipophilicity and evaluation of antitumor activity of [Cu(L-

dipeptide)(1,10-phenanthroline)] (were L-dipeptide= phe-phe, phe-val, phe-ala, ala-phe, gly-val, ala-gly) in breast

cancer and cervical carcinoma cell lines.


Synthesis and analytical characterization

Copper complexes with the L-dipeptides (Sigma): phe-phe, phe-val, phe-ala, ala-phe, gly-val, ala-gly, and 1,10-o-

phenantroline (Sigma) were synthesized and characterized as reported previously [4]. The obtained stoichiometries

[Cu(L-dipeptide)(1,10-phenanthroline)] were determined by Elemental Analysis (Fig.1).

Figure 1. Representation of the solid state structure of [Cu(phe-phe)(1,10-phenanthroline)]


187

Lipophilicity

Lipophilicity tests were performed determining the partition coefficient of the complexes in n-octanol/ physiological

solution. The copper concentration was measured by atomic absorption spectroscopy using a Perkin-Elmer 5000

instrument, with a Photron lamp for copper analysis.

Antiproliferative studies

Cell lines were obtained from the American Type Culture Collection (ATCC): HeLa (CCL-2™, human cervical

adenocarcinoma), NMU (CRL-1743™, mammary adenocarcinoma induced in Sprague Dawley rat), 4T1 (CRL-

2539™ mammary adenocarcinoma from BALB/c mouse). Cells were grown in Dulbecco's Modified Eagle Medium

(DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS) and removed enzymatically from T25 flasks

using 0.01% trypsin. Incubations of cultured cells were at 37°C on 5% CO2 atmosphere. Cell viability in response to

the Cu complexes was determined by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) and absorbance of converted dye was measured at 570 nm as specified by manufactures (Sigma-

Aldrich).


Lipophilicity

Table 1 presents the partition coefficients (Kp) between n-octanol and physiologic solution. The general trend

observed in the tabulated Kp-values for [Cu(dipeptide)(phen)] complexes suggests that lipophilicity increases with

ligand complexity. This trend follows a well known behavior, according to which in a homologous series the

partition coefficients increase by added CH2 or phenyl groups. In spite of this, [Cu(phe-ala)(phen)] is more lipophilic

than [Cu(ala-phe)(phen)], showing the importance of the overall arrangement of the ligands in the complex in

aqueous solution.

Table 1. Partition coefficients (Kp) between n-octanol and physiologic solution

Compound Compound code Kp

[Cu(phe-phe)(phen)] 1 4.12

[Cu(phe-val)(phen)] 2 0.59

[Cu(phe-ala)(phen)] 3 0.30

[Cu(ala-phe)(phen)] 4 0.18

[Cu(gly-val)(phen)] 5 ND

[Cu(ala-gly)(phen)] 6 ND

ND: not detected

Antiproliferative studies

Cell viability was determined by MTT Cell Proliferation Assay. MTT is a yellow substrate that can be reduced by

dehydrogenase enzymes in the mitochondria of living cells, giving a purple compound (MTT formazan).The amount


188

of solubilized MTT formazan produced is directly proportional to the number of living cells [5]. An estimation of the

IC50 at 24 h was calculated using closer to 50% cell viability data and considering a linear correlation.

Table 2, 3 and 4 presents the mean percentage value of cell viability obtained for 4T1, NMU and HeLa cancer

cells, respectively, incubated for 24 and 48 hours in the presence of the indicated concentrations of the studied

complexes. Experiments were done in duplicate and standard deviation was less than 10 % of each value.

Table 2. Percentage of cell viability measured on 4T1 breast cancer cell line and IC50 estimated at 24 h.

5 µM 20 µM 100 µM IC50 µM

Compound 24 h 48 h 24 h 48 h 24 h 48 h 24 h

1 40.13 2.08 0.64 0.44 0.73 0.73 4.2

2 46.26 2.53 1.23 0.82 0.36 0.36 4.7

3 28.81 1.45 5.00 0.19 0.45 0.45 3.5

4 17.50 1.45 0.68 0.63 0.55 0.55 3.0

5 105.63 82.23 30.68 2.31 1.17 1.17 14.4

6 59.32 8.10 1.75 0.84 0.68 0.68 6.1

7 113.4 124.31 108.93 106.13 23.37 23.37 65.3

Table 3. Percentage of cell viability measured on NMU breast cancer cell line and IC50 estimated at 24 h.

5 µM 20 µM 100 µM IC50 µM


1 1.87 4.85 3.20 0.37 50.13 3.48 < 5

2 2.40 0.87 2.67 0.50 57.07 0.99 < 5

3 3.20 0.50 2.13 0.50 9.33 1.12 < 5

4 4.00 1.37 3.20 0.37 20.00 1.49 < 5

5 2.68 1.01 16.96 0.78 141.07 253.27 < 5

6 2.38 0.89 3.27 0.34 100.89 15.86 < 5

7 116.96 65.66 128.87 178.78 31.85 79.51 73.4


189

Table 4. Percentage of cell viability measured on HeLa cervical cancer cell line and IC50 estimated at 24 h.

5 µM 20 µM 100 µM IC50


1 4.49 4.47 4.33 3.35 13.98 6.70 < 5

2 5.66 4.47 6.32 9.50 15.97 6.33 < 5

3 5.49 4.47 6.99 6.70 7.32 9.31 < 5

4 5.99 5.21 5.66 5.59 19.30 4.84 < 5

5 7.16 2.70 12.08 2.43 94.41 101.08 < 5

6 6.71 3.24 4.03 1.89 16.11 7.30 < 5

7 13.42 10.81 33.56 27.84 9.40 4.05 -

We found that all Cu complexes present antitumor activity in the three studied cancer cell lines. Among them, the

complexes containing the dipeptides phe-val, phe-ala and ala-phe showed best antitumor activity in vitro. However,

we did not found a clear relationship between lipophilicity and antiproliferative activity. Possibly other factors as the

redox potential or the ability to interact with DNA influence the antiprolifferative activity.

Conclusions

The dipeptide present in the [Cu(L-dipeptide)(1,10-phenanthroline)] compounds determine lipophilicity, being

[Cu(L-Phe-Phe)(1,10-phenanthroline)] the most lipophilic. Among all the complexes studied, the ones containing

dipeptides phe-val, phe-ala and ala-phe, showed best antitumor activity in cancer cell lines and therefore, these

compounds may be good candidates to test its antitumor activity in vivo.

Acknowledgements

The authors thank PEDECIBA Química for financial support. We also thank members of both laboratories for

helpful technical advices.

References

1. Florea, A.-M. and D. Büsselberg, Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects. Cancers, 2011. 3: p. 1351-1371.

2. Tisato, F., et al., Copper in Diseases and Treatments, and Copper-Based Anticancer Strategies. Medicinal Research Reviews, 2010. 30(4): p. 708-749.

3. Alemón-Medina, R., et al., Induction of oxidative damage by copper-based antineoplastic drugs (Casiopeínas). Cancer Chemother Pharmacol, 2007. 60: p. 219-228.

4. Alvarez, N., G. Facchin, and M. Torre, Antioxidant and pro-oxidant properties and antiproliferative activity of homoleptic and heteroleptic copper complexes, potential antitumoral species. Metal Ions in Biology and Medicine, 2011: p. 117-122.

5. Cole, S.P., Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother Pharmacol, 1986. 17: p. 259 -263.


190

Preliminary study of superoxide dismutase activity in soybean seeds with good and poor in vitro viability used in Uruguay

Cardoso, J.1; Machado, I.2; Irigoyen, J.O.3; Pistón, M.2; Viera, I.1; Torre, M.H.1* 1

Química Inorgánica and 2 Química Analítica, DEC, Facultad de Química, UdelaR, Gral. Flores 2124, CC1157, Montevideo,

Uruguay 3 Agropecuaria Valdense (J.J. Ferreira), Colonia, Uruguay

*e-mail: [email protected]

Abstract

Reactive oxygen species (ROS) such as superoxide and hydroxyl radicals are present in seeds, plants and animals

as a result of normal aerobic metabolism. If the organisms are not able to control the intracellular ROS level, they

may suffer damage in membrane lipids, proteins and nucleic acids leading to the death of the cells. For

detoxification of ROS, efficient antioxidant systems both enzymatic and non-enzymatic ones are present in all cells.

Superoxide dismutases (SOD) can react with O2- radical to prevent the damage. They are metalloproteins where

Cu, Mn, Zn or Ni are linked to the peptide residues. Therefore when the metal levels are low the activity of these

enzymes decrease and different disorders can appear.

The SOD extraction from seeds was optimized by means of multivariate experiments (best conditions: stirring time

60 min using 10 mL of phosphate buffer pH=7).


by the O2- generated by xanthine/xanthine oxidase system.

SOD activity of soybean seeds used in our country was in the range 20-63 %, similar in all the analyzed seeds. The

results showed that no significant difference was observed in seeds with viability less than 95%. In these

conditions, the poor viability of soybean seeds used in our country is not explained by oxidative stress.

I) Introduction

Loss of viability during plant seeds storage is an important problem in agriculture that produces great economic

losses. There are different factors producing changes in seed viability. One of them is the presence of reactive

oxygen species (ROS) including the superoxide radical (O2-), hydroxyl radical (OH-) and hydrogen peroxide (H2O2),

in high levels [1]. These bioproducts are generated as a result of normal aerobic metabolism. If the organisms are

not able to control the intracellular ROS level, these species can produce damage in membrane lipids, proteins and

nucleic acids leading to cell death [2].

It is now known that the stress produced in seeds by drought, high temperatures, salinity, toxic metals, among

others, can cause molecular damage directly or indirectly through the formation of ROS [1,3-5].

To scavenge ROS and to avoid oxidative damage, efficient antioxidant systems both enzymatic and non-enzymatic

ones are present in all cells. One of them is the superoxide dismutases (SOD) that catalyze the reaction 2O2- +

2H+→ H2O2 + O2. These enzymes are Cu and Zn, Mn, Fe or Ni dependant and it is well known that if the metal

levels are low the activity of these enzymes decreases and different disorders can appear [2]. Besides there are

differences in the SOD levels depending on seed genotype, location, climatic and storage conditions and presence

of toxic metals, among others [4,5].


191

With these antecedents, our work was to measure the SOD activity on soybean seeds with good and poor in vitro

viability previously determined with the aim to observe a possible correlation between SOD activity and viability and

provide new information about regional soybean seeds.

II) Experimental

Materials

Reagents and solvents were commercially available research-grade chemicals and were used without further

purification.

Seed samples

The soybean seeds were provided by Agropecuaria Valdense (Juan Jorge Ferreira), a seed importing company,

from Colonia (Uruguay).

Samples from 5 different batches of seed were analyzed: 3 of them with good viability and 2 presenting poor

viability. All tests were run in duplicate.

Enzyme extraction method

The SOD method of extraction from seeds was optimized by means of multivariate experiments (central composite

design) [7]. The variables were stirring time and volume of extraction and were analyzed in three levels. The effect

of Polyvinylpyrrolidone (PVP) concentration was also studied.

After the development of the method of extraction, the following technique was performed:

For each sample, 1 g of ground and dried (80ºC, 24 hours) soybean seeds was weighed and mixed with 0,2 g of

PVP. After that, 10 mL of extraction phosphate buffer, pH 7, was added. The homogenate was stirred for 1 hour,

centrifuged at 13,000 rpm for 15 min and finally extracted using a Sephadex G-25 column. The eluate was

collected in 10 tubes of two mL each one. These solutions were used to estimate protein concentration

(spectrophotometric method, A280) and initial superoxide anion level (as reduction of NBT, A 560) (data not shown),

and to evaluate the SOD activity for the most concentrated tube.

SOD activity determination


by the O2- generated by xanthine/xanthine oxidase system, adapted from Beauchamp and Fridovich’s method [8].

The test tubes contained 0.1 mL of the sample, 0.2 mL of 4.6 mM xantine, 0.1 mL of 0.75 mM NBT, 0.1 mL of 3

mM EDTA, 0.1 mL of 1.50 mg mL-1 bovine albumin, 0.1 mL of 10 mg prot. mL-1 and buffer phosphate (pH=7.4) until

2 mL.

After the incubation at 28ºC, the reaction was stopped with 0.1 mL of 6 mM CuCl2 and the absorbance of the blank

solution and reaction solutions were measured with a spectrophotometer Thermo Scientific mEvolution60 at 560

nm.


192

SOD activities were calculated with the equation: SOD activity (%) = (1 − A/B) × 100, being A= absorbance of

sample and B= absorbance of blank, taking into account the initial O2- level.

III) Results

III-1) Enzyme extraction method

The different experimental conditions of the extraction method and the result of the eluate in tube 4 are presented

in Table 1.

Table 1. Absorbance in different experimental conditions

Stirring Time (min) Volume of extraction (mL) Concentration (AU*) Tube 4

15 10 6,028

30 10 6,050

60 10 9,966

60 20 3,003

30 20 3,388

30 40 1,131

15 40 1,133

* AU = A280 x f(including dilution factor and absortivity)

As expected, more concentrated solutions are obtained when the volume is reduced and time of stirring is

increased.

The effect of changing the volume beyond 10 mL is more important than changing the time.

The analysis of the multivariate experiment is shown in Figure 1.

Figure 1. 3D Surface Plot, central composite design, two variables, three levels


193

According to the 3D surface plot the conditions tested did not include the maxima of the curve for any variable. In

spite of this no more changes in experimental conditions were tried due to several reasons. First of all, any of the

protein concentrations obtained in these experiments proved adequate to perform both superoxide anion level and

SOD activity determinations by the method used. An increase in time would result in having to dilute the solution for

afore mentioned determinations.

On the other hand working below 10 mL, extraction mixture forms a semi-solid paste, very difficult to stir

homogenously.

Another variable was PVP quantity. The experiments showed that more than 1g meddled with the extraction

mixture and made it very difficult to separate from the solution in the column.

After the development of the technique, the elution profile was performed in all the experiments. Absorbance at 280

nm showed that the eluate in the fourth tube was the most concentrated in protein content for all samples.

III-2) SOD activity determinations

The results obtained in the SOD activity tests for seed sample 003 are presented in Figure 2.

Figure 2. Protein concentration and SOD activity for seed sample 003.

As the graph shows, tube 4 presented both the maximum protein concentration and maximum SOD activity. Similar

elution profile and SOD activity was observed in the other samples.

The preliminary SOD activity results for all the studied samples are shown in Table 3.

Table 3. Mean inhibition (%) of NBT reduction by the O2- generated by xanthine/xanthine oxidase system, in tube 4

Seed 001 002 003 V6,2 009

% 20 23 63 55 62

As Table 3 shows, the SOD activity of soybean seeds used in our country was in the range 20-63 %. To our

knowledge, there is no information about SOD activity in soybean seeds in the region but it is known that SOD

activity changes with latitude and genotype. For this reason, this preliminary study can be an interesting


194

antecedent. Studies performed on other seeds than those used in Uruguay showed SOD activities in the range of

our results[5].

No differences were found between seeds with good or poor viability. Consequently, in these conditions, the poor

viability of soybean seeds used in our country is not explained by oxidative stress.

Further studies are necessary to investigate this aspect.

Biobliography

[1] Somone García J., Lupino Gratao P., Antunes Azevedo R., Zezzi Arruda M.A. Metal contamination effects on

sunflowers (Helianthus annuus L.) growth and protein expression in leaves during development. J Agric Food

Chem; 2006; 54; 8623-8630.

[2] Kaim W., Schwederski B. Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life. Chichester:

J.Wiley & Sons, 1994.

[3] Cabrini L., Barzanti V., Cipollone M., Fiorentini D., Grossi G., Tolomelli B., Zamboni L., Landi L. Antioxidants

and total peroxyl radical-trapping ability of olive and seed oils. J Agric Food Chem; 2001; 49; 6026-6032.

[4] Wang G., Huang J., Gao W., Lu J., Li J., Liao R., Jaleel C. A. The effect of high-voltage electrostatic field

(HVEF) on aged rice (Oryza sativa L.) seeds vigor and lipid peroxidation of seedlings. J Electrostatic; 2009; 67;

759-764.

[5] Bao X., Bing-chang Z., Qin-hua L., Jing-liang. Study on the superoxide dismutase of soybean seeds from

different species in subgenus soja. Acta Botanica Sinica; 1989; 31(7); 517-522.

[6] Kranner I., Colville L. Metals and seeds: Biochemical and molecular implications and their significance for seed

germination. Environ Exp Botany; 2011; 72; 93-105.

[7] Massart D. L., Vandeginste B. G. M., Buydens L. M. C., De Jong S., Lewi P.J., and Smeyers-Verbeke J.

Handbook of Chemometrics and Qualimetrics: Part A. Amsterdam: Elsevier Science, 1997.

[8] Beauchamp C., Fridovich J. Superoxide Dismutase: Improved assays and an assay applicable to acrylamide

gels. Anal Biochem; 1971; 44; 276-287.


195

Metal ions and human diseases

Theophanides, T.1; Anastassopoulou, J.1; Dritsa, V.1; Pissaridi, K.2; Michalis, E.3 1National Technical University of Athens, Chemical Engineering Department, Radiation Chemistry &Biospectroscopy, Zografou

Campus, 15780 Athens 2Athens Institute for Education and Research, 8 Valaoritou Street, 10671 Athens

3General Hospital, Messogion Av. Athens, Greece

Introduction

The metals are natural chemical elements and they were needed for life since the origin of life. Another aspect of

metals is the metals in everyday life.Without metals the computers, mobile phones and batteries could not exist.

The metals can be divided in three categories: 1) The life metals that are necessary for our health and life, for

example, the metal ions that are found in every human cell, i.e. Na+, K+,Mg2+ and Ca2+. In this category there are

also all the metal ions that are the life metals, e.g. iron, in haemoglobin, copper in copper-proteins,vanadium,

chrome, manganese, zinc, cobalt, molybdenum, etc. Iron makes our blood blue. 2) There are the very toxic metals,

such as arsenic, mercury, lead and cadmium and others and 3) There are the metals that have been used in

pharmaceuticals for several diseases, such as the antitumor coordination complex, cis-platinum, i.e. cis-

Pt(NH2)2Cl2, discovered in 1965 by Barnett Rosenberg1 and second and third generation platinum complexes and

other metal complexes or metal salts used as pharmaceuticals such as the gold complexes for arthritis, etc.The

metals constitute almost 80% of all the elements of the Periodic Table.

The life metals and their usefulness in health and life are known for a long time4,5. The metals sodium (Na),

potassium (K), magnesium (Mg) and calcium (Ca) are essential for all cells6,7. It is well known that the metal

sodium is very toxic and could kill a person immediately, if he tries to eat the metal, whereas when it is a cation,

Na+ as in sodium chloride (NaCl) is not harmful at all. It is even very tasty. We use it in our food very often as

mineral nutrient, likewise for K, Mg and Ca. The metal, Na when is becoming an ion, is fundamental for our health,

both in cells and in our food (see equation [1]):

−+ +→ eNa Na [1]

This sodium example is good evidence that a natural element can be toxic or good for our health, if it is

transformed chemically or energetically7. The metals of the Periodical Table can be used in biology and medicine,

as nutrients or as pharmaceuticals or as toxic materials when they surpass the optimum concentration (Fig.1). In all

these categories the dose will determine their properties towards humans8.

Figure1. The double-bell function for the activity of all elements in our life.


196

Patients and Methods

Materials: Blood serum and marrow were taken from 100 patients who had plasmacell dyscrasias (46-76 years).

They were characterized as IgG:4, IgA:1, IgM:1 and λ-light chains.The carotid and coronary arteries were taken

from 150 patients, who underwent endarterectomy, while 30 aortic valves were taken from patients, who underwent

surgical aortic valve substitution due to mineralization (45-85 years).

Methods: The spectra were recorded with a Nicolet 6700 thermoscientific spectrometer, equipped with an ATR-

FTIR technology. Each plot consisted of 120 co-added spectra at a spectral resolution of 4 cm−1 and the OMNIC

7.1 software was used for data analysis.

For the fine detection of aortic valve surfaces it was used the Scanning Electron Microscopy (SEM).The instrument

was from Fei Co, The Netherlands. It must be noted that there was not any coating of the samples with carbon or

gold. XRD was performed with a Simens D-500 X-Ray diffractometer based on an automatic adjustment and

analysis system. The diffraction interval was of 2θ 5o-80o and the scan rate was of 0.030o/s.


Metal ions found in cardiovascular systems

SEM-EDAX analyses showed that the atheromatic plaques were rich in foam cells, which were the preferential

sites of mineralization. In some atheromatic plaques were detected toxic metal ions, such as lead, silver, aluminum

and copper.Figures2A and C shows the morphology of atheromatic plaque of carotid artery as it was described by

SEM. From the electron density distribution, as it is plotted by pixel value (Fig. 2 B&D), in the mineral salts it

became clear that copper ions must bind to oxygen and nitrogen atoms of proteins most likely through chelation,

while calcium ions produced salts with the oxygen anions of phospholipids. This is indicated from the fact that in

the calcium salt the electron density is evenly distributed, as in an ionic salt, whereas in the case of copper there is

a hole in the middle showing a covalent bonding in the copper complex (see Fig.2, B and D).

The non-life metals, such as lead and silver, which were found at the arteries, were due to patient’s occupational

environment. It is well known from literature that copper induces oxidative stress, leading to atherosclerosis9,10. The

oxidized organic products that were formed during oxidative stress could be detected easily using the characteristic

absorption FT-IR bands and SEM analysis.


197

Figure 2.A and C are SEM images of the carotid artery at different sites of the tissue. The squares show the

presence of Ca2+ and Cu2+ binding. B and D are the corresponding electron density environments at the square

surfaces using ImageJ software for the pixel analysis.

Figure 3.FT-IR spectra of 1: carotid artery, 2: coronary artery and 3: aortic valve

The FT-IR spectra of the samples in the region 3000-2800 cm-1 are shown in Fig. 3.The spectra showed significant

increase of the intensity of the bands,due to stretching vibrations of CH3 and CH2, suggesting that the permeability

of the membranes changed significantly (Fig.3). We propose that these changes could be related to free radical

reactions with lipid macromolecules, leading finally to a shortening of the chains of proteins. These findings are in


198

accord with the appearance of the band at 3080 cm-1, assigned to C-H stretching vibration of the double bonds

related to LDL of patients. The band at 1735 cm-1 is assigned to aldehyde groups, which confirms the peroxidation

of LDL10. The intensity of this band is also related to LDL of patients and could be used as a “marker band”.From

the changes in the region of the infrared spectra, 1700-1500 cm-1, where the amide I and amide II of the proteins

absorb it was suggested that atherogenesis arises with breaking of intramolecular and intermolecular hydrogen

bonds of proteins and membranes11.

Finally, considerable changes were observed in the region 900-400 cm-1, which suggest that glutathioles (GRS), R-

S-and R-SH were transformed into sulfones (S-S). In all patients’ spectra of aortic artery there were found bands in

the region, 1150-900 cm-1, which corresponded to the stretching vibrations of phosphate groups (vPO4), most likely

from hydroxyapatite12. It was observed by XRD that in sites rich in magnesium concentration the hydroxyapatite

was amorphous. Magnesium cations (Mg2+) are known to regulate ATP metabolism and is cofactor in more than

300 intracellular enzymes13,14. It was described that Mg2+ inhibits the release of Ca2+ from the sarcoplasmic

reticulum, blocks the influx of Ca2+ into the cell by inactivating the Ca2+ channels in the cell membrane, and

competes with Ca2+ at binding sites on troponin C and myosin, thereby inhibiting the ability of Ca2+cations to

stimulate myocardial tension15.

Moreover, in the carotid and coronary arteries were detected molybdenum ions by the method of SEM-EDAX,

which indicates that the enzyme of xanthine (molybdenum-enzyme) is inert and that it does not reverse the

xanthine molecule into hypoxanthine16,17.

The influence of copper concentration in patients with plasma dyscrasias

Plasma dyscrasias is a malignant disease, which affects people usually after 50 years old. It refers in a series of

abnormalities of monoclonal immunoglobulin and includes the multiple myeloma and Waldenström’s

macroglobulinemia. In Figure 4 are shown the FT-IR spectra of a patient with plasma dyscrasias (1) and healthy

donor (2). It is important that the ratio of the intensities of Amide I/Amide II in the spectra of the patient is <1, due to

the influence of malignancy. In healthy people the ratio of the intensities Amide I/Amide II is found to be >1.


199

Figure 4.FT-IR spectra of blood plasma. 1: patient with plasma dyscrasias and 2: healthy donor.

Considerable changes were observed in the region, where the sugar moieties of DNA absorb. The intensity of the

band at 830 cm-1 increases in spectra of the patients and shifts to higher frequencies. These spectral changes in

the spectra of DNA are most likely due to DNA structural changes, maybe related to the high concentration of Cu2+

ions, which is known that strongly perturbs the sugar-phosphate group vibrations as well as the base vibrations18,19.

SEM-EDAX analyses showed that in the patients with plasma dyscrasias the relative concentration of copper

increases and varies between 7%-14%, while in the healthy donors the relative concentration was found to be

between 1% and 4,5%.

Summary

From all our research data it was shown that toxic metals could induce oxidative stress leading to atherogenesis.

The FT-IR spectra, in particular showed that the mechanism of plaque formation mustbe different in carotid and

coronary arteries from those of aortic valve mineral deposition.

In patients with blood diseases the influence of copper concentration compared to that of healthy subjects was

increased to double and even SEM-EDAX analysis showed that in the patients with plasma dyscrasias the copper

concentration varies between 7%-14%, while in the healthy donors the concentration is found between 1% and

4,5%, although no one of them had Wilson’s disease.

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201

Authors Index

Abbehausen, C., 82

Adamek, D., 29

Aguiar, R., 136, 138

Aguilar Cardenas, A.E., 128

Aguilar, A.E., 175

Aguilar, V.H., 46

Alamiri, A., 36

Alberto, R., 22

Alemán, J., 107

Alemón, R., 15

Alonso, O., 60

Alvarez, C., 50

Alvarez, N., 41, 106, 180

Alvarez, S., 51, 52

Álvarez-Valdés, A., 108

Alves, F.R., 141

Anastassopoulou, J., 196

Arancibia, R., 35

Arion, V.B., 36

Aullón, G., 49

Azócar, M.I., 110

Baisch, P.R., 69, 144

Ballinas, G., 15

Balter, H., 59, 61, 135

Baran, E.J., 77, 88

Barbato, S., 73

Barg, G., 67, 143

Barh, G., 23

Bartesaghi, S., 64

Bastian, G., 13, 17

Batinic-Haberle, I., 65

Batista, A., 83

Batista, A.A., 39, 111, 112

Becco, L., 104, 109

Behar Cohen, F., 26

Bellanda, M., 63

Bellinger, D.C., 27

Benítez, J., 42, 104

Beraldo, H. de O., 97

Berchesi, A., 134

Bergamini, F., 99

Bergamini, F.R.G., 82

Beyer, L., 84

Biestro, A., 55

Birriel, E., 104

Bizzi, C.A., 32, 75

Bollati- Fogolin, M., 76

Botta, B., 19

Botto, I.L., 142

Bravo Gómez, M.E., 15

Brissos, R.F., 109

Brodzka, R., 125

Brown, E., 34

Büchel, G., 36

Cabello, C., 76

Cabral, P., 62, 131, 132, 136, 138

Cabrerizo, F., 100

Calvo, C., 127, 165

Calzada, V., 132

Camacho, X., 132

Can, D., 22

Cangelosi, V., 7

Carballal, S., 65

Cardoso, J., 126, 130, 191

Carmo, A.M.L., 96

Carnero, A., 108

Carnizello, A., 83

Carvalho, F.A.O., 140

202

Carvalho, J.W., 141

Carvalho, J.W.P., 80

Carver, P., 21

Castellano, E.E., 102

Castelli, R., 138

Castelli, S., 42

Castillo, I., 49

Castro, W., 113

Cebula-Obrzut, B., 105

Cerecetto, H., 57, 58, 104, 134

Chabalgoity, J.A., 131, 132

Chammas, R., 136, 138

Chao, H., 93

Cipriani, M., 48

Clavijo, G., 116

Colina-Vegas, L., 112, 113

Collery, P., 13

Collery, T., 13

Cominetti, M.R., 111

Comini, M., 103, 106

Comini, M.A., 63

Corbi, P.P., 82, 91, 94, 99

Corrêa, R., 83

Correia, I., 104

Cortés, F., 15

Cortes, S., 53

Costa Ferreira, A.M., 42

Costa Pessoa, J., 92, 104

Costa-Filho, A., 41, 86, 87, 180

Courtois, Y., 26

Cousillas, A., 18, 116, 117

Crócamo, N., 139

Cruz, W.S., 97

Cubo, L., 108

Cuin, A., 99

Curbelo, E., 48

Currier, J.M., 10

Czyzycki, M., 29

Da Costa Ferreira, A.M., 12

da Silva, E.G., 31

da Silva, S.A., 99

Dagher, J.M., 152

Dapueto, R., 136

Dario, B.S., 12

De Almeida, R., 92

de Araujo Fernandes, A.G., 47

de Azevedo Mello, P., 32, 75

de Lima, A.P., 39

de Paiva, R.E.F., 91, 94

de Paula, F.C.S., 96

de Paula, Q.A., 12

Dědina, J., 10

Deflon, V.M., 47

Del Solar, V., 107

Dematteis, S., 57

Demoro, B., 92

Desideri, A., 42

Desimone, M., 51

Desmaele, D., 13

Di Virgilio, A.L., 76

Diaz, L., 51

Dinamarca, N., 110

Dominguez, O., 129

Dritsa, V., 196

Dupont-Soares, M., 69, 144

Eberlin, M.N., 31

Egusquiza, G., 76

Ellena, J., 41, 83, 106, 180

Engler, H., 59, 135

Espejel, G., 128, 175

Espósito, B.P., 46

Etcheverry, S., 88

203

Etcheverry, S.B., 76

Evelson, P., 51, 52

Fabijanska, M., 105

Facchin, G., 41, 45, 55, 86, 87, 180, 186

Farkas, E., 37

Felice Guidugli, R.B., 31

Fernadez, M., 131

Fernández, L., 56

Fernández, M., 104, 109, 132, 136, 138

Fernández, S., 57, 58, 134

Ferraesi Curotto, V., 89

Ferreira, J.C., 79

Ferrer-Sueta, G., 65

Fleitas, L., 63

Flores, E.M.M., 32, 75

Fontes, A.P.S., 96

Formiga, A.L.B., 91, 94

Foster Mesko, M., 32, 75

Franco, D.W., 102

Freitag, L., 36

Fuda, J., 133

Gagnon, Z.E., 66, 152

Galindo, R., 15

Galusso, F., 55

Gambini, J.P., 131, 136, 138

Gambino, D., 15, 42, 48, 92, 103, 104, 109

Gamez, P., 109

Garat, B., 104, 109

García Ramos, J.C., 15

Garcia, E., 69

Garcia, E.M., 144

Garcia, M.H., 103

Gazzoli, D., 142

Gelpi, R.J., 52

Ghirga, F., 19

Giglio, J., 57, 58, 135

Gomes, V. de C., 74

Gómez, G., 110

González Baró, A.C., 89

González, L., 23

González, M., 23, 104, 134

González, M.J., 142

González, R., 45, 186

Gonzatto, L., 119

Goso, C., 115

Graminha, A.E., 39, 111

Guidali, F., 106

Guido, M., 124

Gumienna-Kontecka, E., 114

Gutiérrez, A.G., 78

Gutiérrez, E., 132

Hardy, G., 55

Hauser-Davis, R.A., 68, 74

Heinzen, H., 72

Heller, T., 18

Hernández Gorritti, W.R., 84

Hernández-Lemus, E., 15, 78

Höehr Nelci, F., 31

Holste, A., 30

Honorato, J., 111

Huertas, R., 73

Icimoto, M.Y., 79

Iglesias, S., 45, 87, 186

Irigoyen, J.O., 126, 130, 191

Isaac, L., 66

Isaac; L.A.C., 152

Jansat, S., 120

Jeanny, J.C., 26

Juliano, C., 40

Katkar, P., 42

Katzin, A.M., 46

Klahn, H., 35

204

Knochen, M., 71, 72, 73, 160

Kordas, K., 20, 67, 124, 143

Kozlowski, H., 81, 114

Kramer, G., 45, 186

Kratzer, J., 10

Kreimerman, I., 59

Kremer, C., 54, 119

Kremer, E., 45, 119, 186

Krupp, E.M., 105

Lacerda, E de P.S., 39

Lafratta, A.E., 47

Lam, T., 152

Lancellotti, M., 82, 94

Landeira-Fernandez, J., 74

Lankosz, M., 29

Lapier, M., 35

Lavaggi, M.L., 104, 134

Leite, C.Q., 99

León, I., 88

Leon, I.E., 76

Leonardi, N., 133

Levin, P., 110

Lima, B.A.V., 39

Lippert, B., 14

Liu, H., 44

Llarrull, L., 23

Lyrio Tenorio Correia, C., 30

Machado, I., 109, 126, 130, 191

Macín, S., 127, 165

Magnani, N., 51, 52

Mañay, N., 18, 50, 67, 115, 116, 117, 124, 143

Manta, B., 63

Manzanares,W., 55

Manzur, J., 84

Mao, Z., 43

Marchini, T., 51, 52

Marcondes, M.Y., 79

Marques, F., 92, 109, 136, 138

Marti, M.A., 25

Martínez Savio, G., 124

Martínez-Alanis, P., 49

Martín-Santos, C., 107

Marts, A.R., 36

Matoušek, T., 10

Maya, J.D., 35, 48

Mebert, A., 51

Medeiros, A., 103, 106

Medici, S., 40, 81, 170

Medrano, M.A., 108

Meini, M.R., 23

Mejía, C., 15, 78

Melo Ruiz, V., 127

Melo, V., 118, 129, 165

Mendoza, C., 120

Menezes, A.C.S., 111

Michalis, E., 196

Mohsen, A., 13

Molina, L., 53

Molinari, M., 133

Mollo Filho, P.C., 31

Mollo, A., 73

Monahan, J.L., 152

Mondelli, M., 83

Morais, T.S., 103

Morales, G., 71, 160

Moreno, M., 131, 132

Moreno, V., 15, 104

Moreno-Esparza, R., 15

Muccillo-Baisch, A.L., 69, 144

Muñiz, I., 127, 165

Muñoz, B., 128, 175

Muñoz, H., 110

205

Murgida, D., 24

Musil, S., 10

Nantes, I., 123

Nantes, I.L., 79

Nascimento, O., 123

Nascimento, O.R., 79

Navarro Marques, F.L., 47

Navarro, M., 112, 113

Navarro-Ranninger, C., 107

Navon, A., 114

Neves, A., 16

Noble, C., 45, 86, 87, 186

Nóbrega, J. de A., 75

Ochocki, J., 105

Okamoto, A., 33, 146

Olea Azar, C., 48

Oliveira Carvalho, F.A., 141

Oliveira, V., 79

Oliver, P., 59, 135

Osinaga, E., 135

Otero, L., 48, 92, 103

Paez, M.A., 110

Palacios, A., 23

Palacios, J., 129

Palacios-Abrantes, J., 118

Palma-Tirado, L., 78

Parajón-Costa, B.S., 77, 89

Parro, T., 108

Pastelin, R., 128, 175

Pastrana Alta, R.Y., 46

Paula, F.C.S., 89

Pavan, C., 63

Pavan, F., 99

Pavel Sizemore, I.E., 152

Pavel-Sizemore, I., 66

Paz Castillo, J., 84

Peana, M., 40, 81, 170

Pecoraro, V.L., 7

Pereira Testa, L., 18, 116, 117

Pereira, F. de C., 39

Pereira, M.E.C., 89

Pereira, N., 50

Pereira-Maia, E.C., 95, 96

Pericàs, M., 120

Pessôa, G. de S., 31

Pessoa, J., 15

Pianca Barroso, R., 86, 87

Picard, E., 26

Pis Diez, R., 89

Pissaridi, K., 196

Pistón, M., 71, 115, 126, 130, 160, 191

Pizzorno, P., 50

Plegaria, J., 7

Porcal, W., 131, 136, 138

Porro, V., 76

Portela Luz, C., 47

Potocki, S., 81

Prieto, T., 123

Profirio, D.M., 94

Queirolo, E., 67, 124, 143

Quinn, T., 131

Quirino, T., 127, 129, 165

Quiroga, A.G., 108

Raab, A., 105

Raber, G., 105

Radi, R., 8, 64, 65

Radwanska, E., 29

Ragone, F., 100

Reina, M., 15

Reisner, E., 36

Remelli, M., 40, 170

Resende, J.A.L.C., 89, 97

206

Rey, A., 56, 57, 58, 134

Rey, N.A., 89, 97

Reyes, L., 56, 59, 135

Ribeiro, A. de S.B.B., 39

Ribeiro, G.A., 12

Riva, E., 132

Rocha, R., 74

Rodrigues da Silva Júnior, F.M., 144

Rogers, C., 34

Roy, A., 67

Ruiz, G., 100

Ruiz-Azuara, L., 15, 78

Sabino, G.L., 12

Saldivar, L., 128, 175

Salgueiro, J., 133

Sánchez Eguía, B., 49

Sanchez, M.T.C., 128, 175

Santi, E., 88

Santi, L., 19

Santoni, F., 13

Santos Júnior, J.C., 31

Sanz, I., 134

Sarniguet, C., 103

Scarone, L., 106

Schaumloffel, D., 30

Schettino, B., 129

Serra, G., 106

Severo Fagundes Pereira, J., 32

Shanzer, A., 114

Shen, Y., 22

Silva, I.M.P., 94

Silva, P.P., 95

Siqueira, S.A., 97

Sixto, A., 72

Smaili, S., 123

Smanioto Barin, J., 32, 75

Smolewski, P., 105

Soares, M.C.F., 69

Solinas, C., 40, 81, 170

Soto, E.L., 142

Souza, M.L., 102

Spitridonova, E., 84

Sterchook, R., 114

Sturlese, M., 63

Stýblo, M., 10

Sucena, S., 91

Suleiman, S., 22

Svoboda, M., 10

Szabó, O., 37

Szczerbowska-Boruchowska, M., 29

Szebesczyk, A., 114

Szenkier-Garcia, N., 114

Tabak, M., 80, 140, 141

Tacchini, R., 55

Tasat, D., 51, 52

Tavares, D., 83

Tchounwou, P.B., 34

Tebo, A., 7

Tegoni, M., 7

Teixeira, V., 131

Tejería, E., 58, 134

Telser, J., 36

Terán, M., 56

Theophanides, T., 196

Tholey, A., 30

Tierney, D.L., 36

Tissot, F., 119

Toloza, J., 48

Tomaz, A.I., 103

Tomaz, I., 92

Torre, M.H., 41, 45, 55, 86, 87, 88, 106, 126, 130,

180, 186, 191

207

Torres, J., 54, 119

Torti, H., 133

Trindade, V., 59, 135

Trivedi, P., 66, 152

Trzcinka-Ochocka, M., 125

Ugalde- Saldívar, V., 49

Ulrich, A., 47

Urbano, B., 118

Vaisberg, A., 84

Valderrama Negron, A.C., 46

Valez, V., 65

Varela, J., 104

Vasilskis, E., 59, 135

Vázquez-Aguirre, A., 78

Veiga, N., 54

Velluti, F., 106

Vieira, L.Q., 12

Vieira, S., 42

Viera, I., 88, 126, 130, 191

Viera, S., 73

Vila, A., 23

Vilar, R., 120

Vilche, M., 56

Villarreal-Peña, W., 112

Wandzilak, A., 29

Wei, M., 13

Wei, W., 122

Wolcan, E., 100

Yañuk, J., 100

Yedjou, C.G., 34

Yefimova, M., 26

Yu, F., 7

Zabarska, N., 109

Zanvettor, N.T., 82

Zastrow, M., 7

Zezzi Arruda, M.A., 11, 31, 68, 74

Zhao, J., 122

Ziolli, R., 74

Ziolli, R.L., 68

Zoroddu, M.A., 40, 81, 170

Zubata, P., 133

Zubillaga, M., 133

Documents

Abstracts and Proceedings of the 12th International Symposium on