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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
AVALIAÇÃO DE EFEITOS BIOLÓGICOS DE ANTIINFLAMATÓRIOS DERIVADOS
DO ÁCIDO PROPIÔNICO ATRAVÉS DE MODELOS EXPERIMENTAIS EM NÍVEL
MOLECULAR E CELULAR.
Natal, RN
2010
ii
MARCIA DE OLIVEIRA PEREIRA
AVALIAÇÃO DE EFEITOS BIOLÓGICOS DE ANTIINFLAMATÓRIOS DERIVADOS
DO ÁCIDO PROPIÔNICO ATRAVÉS DE MODELOS EXPERIMENTAIS EM NÍVEL
MOLECULAR E CELULAR.
Dissertação apresentada à Universidade Federal do Rio
Grande do Norte- UFRN, para a obtenção do título de
Mestre em Ciências da Saúde pelo programa de Pós-
graduação em Ciências da Saúde.
Orientador: PROF. DR. MARIO BERNARDO-FILHO
Natal, RN
2010
iii
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenadora do Programa de Pós Graduação em Ciências da Saúde
Profa. Dra. Técia Maria de Oliveira Maranhão
Natal, RN
2010
iv
Dados Internacionais de Catalogação-na-Publicação (CIP)
P436 Pereira, Márcia de Oliveira.
Avaliação de efeitos biológicos de antiinflamatórios derivados do ácido propiônico através de modelos experimentais em nível molecular e celular / Márcia de Oliveira Pereira – Rio Grande do Norte, Natal 2010.
70 f; 30cm.
Dissertação (Mestrado em Ciências da Saúde) - Universidade Federal
do Rio Grande do Norte, 2010. Orientador: Prof. Dr. Mario Bernardo Filho
1. Ciência da saúde 2. Antiinflamatórios 3. Constituintes sanguíneos 4. DNA 5. Escherichia coli I.Título.
CDD 616.1
v
MARCIA DE OLIVEIRA PEREIRA
AVALIAÇÃO DE EFEITOS BIOLÓGICOS DE ANTIINFLAMATÓRIOS DERIVADOS
DO ÁCIDO PROPIÔNICO ATRAVÉS DE MODELOS EXPERIMENTAIS EM NÍVEL
MOLECULAR E CELULAR.
PRESIDENTE DA BANCA: Prof. Dr. Mario Bernardo-Filho (UERJ)
BANCA EXAMINADORA
Prof. Dr. Irami Araújo Filho
Prof. Dra. Patrícia Froes Meyer
SUPLENTES
Prof. Dra. Cecília Maria de Carvalho Xavier Holanda
Prof. Dra. Maria Teresa Jansem Catanho
vi
DEDICATÓRIA
Dedico este trabalho:
A meu esposo Elcio que me ensinou a ter paciência, a aceitar as minhas
limitações e me deu um incrível desejo de crescer como ser humano, além de que me
ensinar que o limite entre o possível e o impossível é diretamente proporcional ao
nosso nível de esforço.
A minha filha Jéssica que compreendeu minha ausência, me apoiou e incentivou
em todos os momentos.
A Deus por me acompanhar em todos os momentos dessa jornada.
vii
AGRADECIMENTOS
Agradeço ao meu orientador Mario Bernardo-Filho pelo profissionalismo e ética,
virtudes que a cada dia fui tendo a oportunidade de vivenciá-las.
A amiga Gabrielle, que me despertou o interesse pela pesquisa e sempre esteve
ao meu lado.
Ao professor Adenilson Fonseca, no que diz respeito ao desempenho, ao
compromisso, ao esforço, e a dedicação ele que foi fundamental para a existência
desse trabalho.
Ao Programa de Pós-graduação em Ciências da Saúde pela oportunidade de
fazer parte de um processo que hoje me faz ser uma nova profissional. Com uma visão
crítica da saúde e de todas as suas manifestações no indivíduo e no mundo.
As funcionárias da Secretaria do Programa de Pós-graduação em Ciências da
Saúde, que me auxiliaram e estiveram sempre solícitas.
A Deus por estar sempre presente na minha vida, e tornar tudo possível e me
fazer sentir sempre que toda dificuldade é um novo desafio para um novo começo.
Ao professor Sebastião Santos Davi que foi muito solícito e disponível em todos
os momentos.
Aos amigos do Laboratório de Radiofarmácia Experimental do Departamento de
Biofísica e Biometria e no Departamento de Histologia do Instituto de Biologia Roberto
Alcântara Gomes da Universidade do Estado do Rio de Janeiro por estarem sempre
solícitos e pacientes diante de todas as minhas dificuldades.
viii
SUMÁRIO
Sumário..................................................................................................................viii
Lista de abreviações siglas e símbolos...................................................................ix
Resumo………………...…………………………………........……………...................x
1. Introdução.............................................................................................................1
2. Revisão de literatura.............................................................................................3
3. Artigos anexados...................................................................................................7
3.1. Artigo publicado..................................................................................................7
3.2. Artigo submetido I..............................................................................................12
3.3. Artigo submetidoII..............................................................................................27
4. Comentários, críticas e conclusões......................................................................44
5. Anexos..................................................................................................................46
6. Referências...........................................................................................................52
7. Abstract.................................................................................................................61
ix
LISTA DE ABREVIAÇÕES, SIGLAS E SÍMBOLOS.
BC Blood Cell (célula sanguínea)
CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CNPQ Conselho Nacional de Desenvolvimento Científico e Tecnológico
FAPERJ Fundação de Amparo a Pesquisa do Rio de Janeiro
FI-C fração insolúvel da célula
FS-P fração solúvel da célula
FI-P fração insolúvel do plasma
FS-P fração solúvel do plasma
IBRAG Instituto de Biologia Roberto Alcantara Gomes
LRE Laboratório de Radiofarmácia Experimental
Mo molibdênio
P plasma
% ATI porcentagem de radioatividade incorporada
rpm rotações por minuto
SnCl2 cloreto estanoso
99mTc tecnécio-99m
Na99mTcO4 pertecnetato de sódio
TCA ácido tricloroacético
UERJ Universidade do Estado do Rio de Janeiro
UFRN Universidade Federal do Rio Grande do Norte
x
RESUMO
Os derivados do acido propiônico são antiinflamatórios não esteroidais inibidores
irreversíveis da enzima cicloxigenase amplamente utilizados. O objetivo deste trabalho
foi avaliar, através de diferentes modelos experimentais, efeitos biológicos de derivados
do ácido propiônico (fenoprofeno, naproxeno, ibuprofeno e cetoprofeno) em nível
celular e molecular. A marcação de constituintes sanguíneos com tecnécio–99m
(99mTc) e a análise morfológica de hemácias de sangue de ratos Wistar, bem como, o
crescimento, sobrevivência de culturas de Escherichia coli (E. coli) e a avaliação do
perfil eletroforético plasmídios bacterianos, foram modelos experimentais utilizados para
avaliação de possíveis efeitos biológicos dos antiinflamatórios. Os resultados obtidos
demonstram que, de modo geral, os antiinflamatórios avaliados não foram capazes de
alterar a marcação de constituintes sanguíneos com 99mTc, a morfologia de hemácias
de sangue de ratos Wistar, assim como, o crescimento de culturas de E. coli e o perfil
eletroforético de plasmídios. Entretanto, o naproxeno parece apresentar efeito citotóxico
em culturas bacterianas, efeito genotóxico em plasmídios e diminuição da ação do
cloreto estanoso em culturas de E. coli. A utilização de modelos experimentais de
rápida realização e baixo custo se mostrou importante para avaliação de efeitos
biológicos, contribuindo para uma melhor compreensão das propriedades dos derivados
do ácido propiônico estudados. Esse trabalho teve caráter multidisciplinar e na vigência
dos auxílios concedidos pela CAPES, FAPERJ e CNPq.
Palavras-chave: antinflamatórios; constituintes sanguíneos; tecnécio-99m; cloreto
estanoso; Escherichia coli; DNA.
1. INTRODUÇÃO
Os antiinflamatórios não-esteroidais são fármacos de diferentes classes
químicas, mas que apresentam propriedades terapêuticas similares, com atividade
inibitória da enzima ciclooxigenase 1.
Os derivados do acido propiônico são antiinflamatórios não-esteroidais prescritos
no mundo inteiro, sendo muito utilizados para doenças músculo-esqueléticas e
reumatológicas 2. Apesar de alguns possíveis efeitos biológicos não terem ainda sido
descritos, estes fármacos são muito utilizados para tratamento da dor e inflamação 3.
Fármacos inibidores das enzimas cicloxigenase são amplamente utilizados no
mundo inteiro, e devido a ações atribuídas ao uso desses fármacos, alguns já foram
retirados de circulação devido a identificação de efeitos biológicos deletérios 4,5.
Modelos experimentais têm sido utilizados para avaliar efeitos biológicos de
produtos naturais ou sintéticos. A marcação de constituintes sanguíneos com tecnécio–
99m (99mTc), a morfologia de hemácias 6-9, o crescimento 10 e a sobrevivência de
culturas bacterianas de Escherichia coli 11, tem sido usados para avaliar estes efeitos
em nível celular e, o perfil eletroforético de plasmídios bacterianos, para avaliação
destes efeitos em nível molecular 12.
O objetivo deste estudo foi avaliar efeitos biológicos de antiinflamatórios
derivados do ácido propiônico (ibuprofeno, naproxeno, cetoprofeno e fenoprofeno)
através de modelos experimentais em nível molecular e celular.
Esse estudo foi realizado no Laboratório de Radiofarmácia Experimental do
Departamento de Biofísica e Biometria, e no Departamento de Ciências Fisiológicas do
Instituto de Biologia Roberto Alcantara Gomes da Universidade do Estado do Rio de
2
Janeiro. Os experimentos foram possíveis através de convênio firmado entre a
Universidade do Estado do Rio de Janeiro e a Universidade Federal do Rio Grande do
Norte, sob a orientação do Professor Doutor Mario Bernardo Filho e na vigência dos
auxílios concedidos pela CAPES, FAPERJ e CNPq.
3
2. REVISÃO DE LITERATURA
Drogas antiinflamatórias não esteroidais derivadas do ácido propiônico, como o
ibuprofeno, naproxeno, cetoprofeno e fenoprofeno, estão entre as drogas mais
amplamente prescritas para o tratamento de dor, febre e inflamação 1,11. Estes
fármacos são extensamente utilizados para o tratamento de doenças músculo-
esqueléticas, tais como traumatismos, artrite reumática, espondilite anquilosante e
artrite gotosa, bem como outras enfermidades, com a dismenorréia 12.
As propriedades farmacodinâmicas dos derivados do ácido propiônico são
semelhantes às propriedades de outros antiinflamatórios não esteroidais. Estes
fármacos são inibidores eficazes da ciclooxigenase, embora exista variação
considerável em sua potência 3,11,12. O naproxeno, por exemplo, é vinte vezes mais
potente do que a aspirina. Enquanto o ibubrofeno, o fenoprofeno, e a aspirina são
eqüipotentes como inibidores da ciclooxigenase alterando a função plaquetária e
prolongando o tempo de sangramento 3.
Estudos demonstraram que efeitos biológicos de produtos naturais e sintéticos
podem ser avaliados através de modelos experimentais simples, de rápida realização e
baixo custo 4,6,8.
O 99mTc é o radionuclídeo mais utilizado para obtenção de imagens cintilograficas
do tipo SPECT (single photon emission computed tomography) devido às propriedades
de emissão de um fóton de 140 keV, de possuir meia vida física de 6 horas, impacto
ambiental desprezível e ser facilmente obtido em gerador13. Esse radionuclídeo também
tem sido utilizado em pesquisa 4-6,8.
4
A marcação de constituintes com tecnécio-99m (99mTc) tem sido utilizada como
um modelo experimental para avaliação de transporte de íons (pertecnetato e estanoso)
estrutura e função da membrana plasmática 14-17. Essa técnica é de grande relevância
na medicina nuclear 18-20.
Constituintes sanguíneos marcados com 99mTc são radiobiocomplexos que
podem ser utilizados na detecção de hemorragias gastrintestinais 21, hemangiomas 22
na avaliação da função cardíaca 23, bem como na medida do fluxo sanguíneo em
artérias periféricas 24.
Tem sido proposto que no processo de marcação de constituintes sanguíneos, o íon
pertecnetato atravessaria a membrana celular por troca com o íon cloreto e/ou
bicarbonato através do canal de ânions (banda-3) 25, e o íon estanoso, através dos
canais de cálcio 26.
Autores têm descrito que produtos naturais e fármacos sintéticos podem interferir na
radiomarcação dos constituintes sanguíneos com 99mTc 27,4,7 comprometendo a
interpretação de resultados clínicos ou a repetição de exame em Medicina Nuclear 28.
A alteração da marcação de constituintes sanguíneos com Tc-99m por fármacos
poderia ocorreria por: (i) formação de complexos com os íons estanoso e pertecnetato;
(ii) alterações morfológicas (qualitativas) e morfométricas (quantitativa) membrana
eritrocitária; (iii) competição com os referidos íons pelos mesmos sítios de ligação nas
proteínas plasmáticas e celulares e/ou (iv) oxidação direta do íon estanoso ou através
da (v) geração de radicais livres 27,29.
Estudos com células do sangue também podem ser realizados através de
distensões preparadas pelo espalhamento de uma gota de sangue sobre uma lâmina
5
para microscopia de luz 30. As hemácias são células que sofreram o processo de
extrusão de seu núcleo durante a diferenciação celular, o que facilita sua visualização
na microscopia óptica (análise qualitativa), visto que não apresenta sistemas
intracelulares de membranas 31. Da mesma forma, as análises de parâmetros, como a
relação perímetro/área, são de grande utilidade para avaliação de efeitos de fármacos
na morfologia de hemácias 6,7.
O crescimento de uma cultura bacteriana pode ser avaliado através da medida
da variação do número de células viáveis presentes na cultura com o tempo. O
aumento do número de células na cultura pode ser acompanhado através da titulação
da cultura em um meio não nutritivo (solução salina 0,9%, por exemplo) ou através da
densidade óptica da cultura, obtida em espectrofotômetro, utilizando comprimento de
onda de 600 nm 8. Pode-se, então, utilizar o crescimento de uma cultura bacteriana
como modelo experimental para avaliar a citotoxicidade de uma substância química
através da avaliação do crescimento da cultura incubada com esta substância 8,32.
A marcação de estruturas celulares ou moleculares de interesse biológico com
99mTc envolve, de modo geral, a utilização de um agente redutor 13,33. O cloreto
estanoso (SnCl2) tem sido largamente utilizado com essa finalidade 25,26. Entretanto,
têm sido descritas as ações citotóxica e mesmo genotóxica associada com essa
substância química 34,38. A citotoxicidade e genotoxicidade do SnCl2 parecem estar
associadas com a geração de radicais livres 35, embora uma ação direta sobre o
sistema biológico também tenha sido sugerida 36,38-40. Além disso, foi demonstrado que
a presença de aceptores de radicais livres (como a tiouréia, benzoato de sódio e
dipiridil) ou de fitoterápicos (Cymbopogon citratus, Baccharis genistelloides, Maytenus
6
ilicifolia e Peumus boldus) pode aumentar a sobrevivência de culturas bacterianas,
proficientes e deficientes nos mecanismos de reparo de lesões no DNA, ao tratamento
com SnCl2 40-43.
A avaliação do perfil eletroforético de plasmídios bacterianos pode ser utilizada
para o estudo do potencial genotóxico de substâncias químicas 6,8,10. A incubação de
plasmídios bacterianos com o SnCl2 pode alterar banda referente à forma circular
aberta, devido a quebras simples no DNA, que seriam induzidas pelas espécies ativas
de oxigênio 35. Entretanto, se esta incubação for realizada na presença de uma
substância que apresenta potencial antioxidante a produção de radicais livres será
reduzida e, em consequência, haverá uma menor alteração do perfil eletroforético dos
plasmídios 44,45.
7
3- Indexação de Artigos
3.1- Artigo Publicado
FENOPROFEN EFFECTS ON THE LABELING OF BLOOD CONSTITUENTS WITH
TECHNETIUM-99m, ON THE MORPHOLOGY OF RED BLOOD CELLS AND ON
THE PLASMID DNA
Marcia de Oliveira Pereira1, 2
, Gabrielle de Souza Rocha
1, 2, Simone dos Santos Lombardi
2,
Mauro Geller4, Mário José Pereira
4, Sebastião David Santos-Filho
2, Adenilson de Souza da
Fonseca2,4,*
and Mario Bernardo-Filho2,5
. 1Programa de Pós-Graduação em Ciências da Saúde, Centro de Ciências da Saúde, Universidade Federal do Rio
Grande do Norte, Avenida General Gustavo Cordeiro de Farias, s/n, 59010180, Natal, Rio Grande do Norte,
Brasil; 2Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do
Estado do Rio de Janeiro, [email protected]; 3Departamento de Fisiologia, Avenida 28 de setembro, 87, 20551030,
Rio de Janeiro, Brasil; 4
Centro de Ciências da Saúde, Centro Universitário Serra dos Órgãos, Avenida Alberto
Torres, 111, 25964004, Teresópolis, Rio de Janeiro, Brasil; 5Instituto Nacional do Câncer, Coordenadoria de
Pesquisa Básica, Praça Cruz Vermelha, 23, 20230-130, Rio de Janeiro, Brasil.
ABSTRACT
The aim of this work was to evaluate the effect of fenoprofen on the labeling of blood constituents with
technetium-99m, on the morphology of red blood cells and on the plasmid DNA. Blood samples from
Wistar rats were incubated with fenoprofen and the assay of labeling of blood constituents with
technetium-99m (
99mTc) was performed. Blood cells, plasma, soluble and insoluble fractions of blood cells
and plasma were separated. The radioactivity in each fraction was counted and percentage of
incorporated radioactivity (%ATI) was determined. Blood smears were prepared, fixed, stained and the
qualitative and quantitative morphology of the red blood cells (RBC) was evaluated. Plasmid (pBSK) was
incubated with fenoprofen with stannous chloride, and agarose gel electrophoresis procedure was
carried out to evaluate genotoxic and the protection of this drug against stannous chloride effect on DNA.
In conclusion, under the conditions used in this work, our data suggest that fenoprofen would not (i)
affect the fixation of the 99m
Tc on the blood constituents, (ii) alter the RBC membrane and (iii) present
genotoxic and redox effects.
Key words: technetium-99m, blood, morphology, plasmid, fenoprofen.
INTRODUCTION
Nonsteroidal antiinflammatory drugs are used for
treatment of rheumatic and other inflammatory,
degenerative, and articulate diseases (Insel, 2001).
The action mechanism of these drugs results from
the inhibition of cyclooxygenase activity, with a
consequent reduction of the synthesis of
prostaglandin, one of the main mediators of the
inflammatory process (Poggi et al., 2006).
Fenoprofen is a nonselective cyclooxygenase
inhibitor commonly used for the treatment of acute
and chronic pain (Insel, 2001).
In vitro red blood cells (RBC) labeled with
technetium-99m (99m
Tc) has been proposed as an
assay to assess biological effects of natural and
synthetic drugs (Fonseca et al., 2007; Benarroz et
al., 2008). Morphogical analysis of RBC has been
utilized as another method to evaluate effects of
drugs (Frydman et al., 2008). Electrophoretic
profile of bacterial plasmids has also been used as
a reliable assay to evaluate genotoxic effect of
drugs (Ferreira-Machado et al., 2004).
The aim of this work was to evaluate the effect of
fenoprofen on the labeling of blood constituents
with 99m
Tc, on the morphology of RBC and on the
plasmid DNA.
MATERIALS AND METHODS
Drugs
Fenoprofen used in this study was purchased from
Biolab Sanus Farmacêutica Ltda (São Paulo,
Brazil, lot 601034) and stannous chloride (SnCl2)
was purchased from Sigma Chemicals Co (St
Louis, USA).
Animals Adult male Wistar rats (3-4 months, 250-300 g)
were maintained in a controlled environment:
normal light/dark cycle conditions (12-h light/12-h
dark; lights on at 6 am), free access to water and
food and room temperature was kept at 25 2 ºC.
8
Experimental protocols were approved by the
Ethical Committee of the Instituto de Biologia
Roberto Alcantara Gomes, Universidade do
Estado do Rio de Janeiro (protocol number
CEA/203/2007).
In vitro radiolabeling of blood constituents
Samples of whole blood (n=7, for each fenoprofen
concentration) were incubated with this drug at
different concentrations (0.0, 0.1, 1.0, 10, 100,
1000 μg/mL; 1 hour). After that, SnCl2 (1.2
µg/mL, 1 hour) was added and, in sequence, 99m
Tc
(3.7 MBq, 10 minutes) as sodium pertechnetate
(Na99m
TcO4), recently milked from a 99
Mo/99m
Tc
generator (Instituto de Pesquisas Energéticas e
Nucleares, Comissão Nacional de Energia
Nuclear, São Paulo, Brazil). These samples were centrifuged (1500 rpm, 5 minutes) and plasma (P)
and blood cells (BC) were separated. Aliquots of P
and BC were also precipitated with trichloroacetic
acid (5 %) and soluble (SF) and insoluble (IF)
fractions were obtained. The radioactivity (% ATI)
in P, BC, IF-P, SF-P, IF-BC and SF-BC was
determined in a well gamma counter (Packard,
model C5002, Illinois, USA). The %ATI was
calculated as described previously (Bernardo-Filho
et al., 1983).
Morphological evaluation
Smears were prepared from blood samples
incubated with fenoprofen at different
concentration (0.0, 0.1, 1.0, 10, 100, 1000 μg/mL;
5 slides for each concentration) and stained by
May-Grünwald-Giemsa (Barcia, 2007). The slices
were analyzed by optical microscopy and for
morphometric measurements a total of five fields
per each slide were evaluated. A spherical shape
and normal size distribution were assumed to RBC
on control samples. Area and perimeter of RBC
were measured (Software image pro plus, media
Cibernetics, USA) and perimeter/area ratio was
calculated.
Plasmid DNA
Plasmid (pBSK) was obtained by alkaline cell
lysis method (Sambrook et al,. 1989) from
Escherichia coli DH5aF’Iq (rec-) strain hosting
this plasmid.
Plasmid treatment with fenoprofen
Plasmids were incubated with fenoprofen at
different concentrations (3.0, 30, 300 µg/mL). To
assess the action of fenoprofen on effects of SnCl2,
plasmids were incubated with fenoprofen, at the
same concentrations, in the presence of SnCl2 (200
g/mL). Plasmid incubated only with SnCl2 was
used as positive control and, as negative control,
plasmid incubated at 10 mM Tris buffer (vehicle,
pH 7.4). The incubations were carried out at room
temperature for 40 minutes. After that, each
sample was mixed with loading buffer (0.25%
xylene cyanol, 0.25% bromophenol blue and
glycerol in water) and applied in 0.8% agarose
horizontal gel electrophoresis chamber in Tris-
acetate-EDTA buffer (pH 8.0, 7 V/cm). The gel
was stained with ethidium bromide (0.5 g/mL)
and the DNA bands were visualized by
fluorescence under an ultraviolet transilumination
system. The assay was repeated at least four times,
the results were digitalized (Kodak Digital Science
1d, EDAS 120) and the bands semiquantified
using the computer program Image J for Windows.
Statistical analysis
Data are reported as (means ± SD) of the %ATI,
the perimeter/area ratio and the percentual of
plasmid forms. The One-way analysis of variance–
ANOVA test was performed to verify possible
statistical differences p<0.05 as less significant
level.
RESULTS
Table 1 presents the effects of fenoprofen on the
radioactivity distribution between cellular and
plasma compartments. This data indicates no
alteration (p<0.05) of 99m
Tc distribution in these
compartments.
Table 2 presents the effect of fenoprofen on the
fixation of 99m
Tc on insoluble and soluble fractions
plasma proteins. This data indicates that the
fenoprofen was not capable to interfere on the
fixation of the radioactivity on the insoluble and
soluble fractions of plasma.
No alteration on fixation of radioactivity on
proteins of blood cells from blood samples
incubated with fenoprofen (Table 3) was found.
9
Table 1 Effect of fenoprofen on the radioactivity distribution on
the cells and plasma compartments labeled with 99m
Tc.
Fenoprofen
( g/mL) %ATI
P BC
0.0 2.82 ± 0.54 97.18 ± 0.54
0.1 1.99 ± 0.59 98.01 ± 0.59
1.0 2.49 ± 1.21 97.51 ± 1.21
10 1.92 ± 0.55 98.08 ± 0.55
100 3.95 ± 1.60 96.05 ± 1.60
1000 4.39 ± 4.02 95.61 ± 4.02
Table 2 Effect of fenoprofen on the fixation of
99mTc on soluble
and insoluble fractions of plasma.
Fenoprofen
( g/mL) %ATI
SF-P IF-P
0.0 24.19 ± 2.67 75.81 ± 2.67
0.1 31.90 ± 3.87 68.10 ± 3.87
1.0 26.00 ± 4.00 74.00 ± 4.00
10 25.99 ± 6.18 74.01 ± 6.18
100 25.31 ± 5.55 74.69 ± 5.55
1000 25.87 ± 7.78 74.13 ± 7.78
Table 3 Effect of fenoprofen on the fixation of
99mTc on soluble
and insoluble fraction of blood cells.
Fenoprofen
( g/mL) %ATI
SF-BC IF-BC
0.0 19.56 ± 2.77 80.44 ± 2.77
0.1 19.81 ± 2.76 80.19 ± 2.76
1.0 18.34 ± 3.96 81.66 ± 3.96
10 18.84 ± 2.30 81.16 ± 2.30
100 20.63 ± 2.66 79.37 ± 2.66
1000 18.58 ± 3.05 81.42 ± 3.05
Photomicrographs of RBC from blood incubated
with 0.9% NaCl or fenoprofen (1000 g/mL)
under optical microscopy is shown in the figures 1
and 2. Qualitative evaluation of these figures
indicates no alterations on the shape of the RBC
incubated with fenoprofen.
Table 4 presents the perimeter/area ratio of RBC
from blood samples incubated with fenoprofen.
The results indicate that the perimeter/area ratio of
RBC was not significantly (p>0.05) altered by
fenoprofen at the concentrations used.
(a) (b)
Figure 1 - Photomicrography of blood smear from
blood incubated with 0.9% NaCl (control) (a) and
Photomicrography of blood smear from blood
incubated with fenoprofen (1000 g/mL) (b).
Table 4 Effect of fenoprofen the perimeter/area ratio of RBC.
Fenoprofen
( g/mL)
Perimeter/area
ratio (1/ m)
0.0 0.62 ± 0.01
0.1 0.64 ± 0.01
1.0 0.65 ± 0.02
10 0.66 ± 0.02
100 0.64 ± 0.01
1000 0.63 ± 0.01
The Figure 2 shows the photograph of agarose gel
electrophoresis of pBSK plasmid treated with
fenoprofen in presence and absence of SnCl2. This
figure indicates that fenoprofen is not capable to
induce alterations on the electrophoretic profile of
plasmids (lanes 3, 4 and 5) when compared with
negative control (lane 1). Also, figure 2 indicates
that the effect of SnCl2 (lane 2) is not altered by
fenoprofen at concentrations used (lanes 6, 7 and
8). These results were confirmed by
semiquantitative analyses of the percentages of
supercoiled (SC) and open circle (OC) plasmid
forms (Figure 3) indicating no alteration on the
electrophoretic profile.
DISCUSSION
Blood constituents labeled with 99m
Tc have been
used in several clinical examinations (Saha, 2004)
and also as an experimental assay to verify the
effect of drugs on radiopharmaceuticals (Fonseca
et al., 2007). This experimental model has
permitted the obtaining of relevant information
about properties of various chemical compounds
(synthetic and natural) (Benarroz et al., 2008). The
data obtained in this work indicates that there was
no alteration on the labeling of the blood
constituents with 99m
Tc when the blood was
10
incubated with fenoprofen (tables 1, 2 and 3).
Despite the absence of effects of the fenoprofen on
radiolabeling of blood constituents, it has
described drug-related immune hemolytic
anemia after use of fenoprofen in human beings
(Shirey et al., 1988). Other data has indicated
that fenoprofen is almost completely bond to
plasma proteins (Insel, 2001).
0
20
40
60
80
100
1 2 3 4 5 6 7 8
LANE
PE
RC
EN
TU
AL
FO
RM
(a)
1 2 3 4 5 6 7 8
(b) Figure 2: Percentage of topological forms (a) and
photograph (b) of agarose gel electrophoresis of
plasmid pBSK treated with fenoprofen in presence and
absence of SnCl2. Lanes: (1) pBSK + buffer (negative
control); (2) pBSK + SnCl2 (positive control); (3) pBSK
+ fenoprofen (300 µg/mL); (4) pBSK + fenoprofen (30
µg/mL); (5) pBSK + fenoprofen (0.3 µg/mL); (6) pBSK
+ fenoprofen (300 µg/mL) + SnCl2; (7) pBSK +
fenoprofen (30 µg/mL)+ SnCl2; (8) pBSK + fenoprofen
(3.0 µg/mL) + SnCl2. (■) OC (open circle); (□) SC
(supercoiled).
Morphological analysis has been used to
demonstrate effects of salicylic acid
derivatives on membrane of RBC (Li et al.,
1999). On the other hand, our data indicates that
fenoprofen would not alter the morphology of
RBC (Figure 1 and table 4). As morphological
analysis of RBC has been used as complementary
technique, these results could confirm the data
obtained with fenoprofen on the labeling of blood
constituents with 99m
Tc.
The genotoxic effect of stannous chloride on DNA
has been demonstrated by different experimental
models and the mechanism action has been so far
related to free radical generation (Melo et al. 2001,
Dantas et al. 2002). In fact, the presence of free
radicals scavengers could reduce the changes of
electrophoretic profile of plasmid DNA induced
by stannous chloride decreasing the DNA strand
breaks (Dantas et al. 1999, de Mattos et al., 2000).
Fenoprofen has been suggested to be scavenger of
free radicals (Costa, et al., 2006). However, at
conditions used in this work, fenoprofen did not
seem to protect plasmid DNA against the effects
of stannous chloride. In addition, fenoprofen could
not present genotoxic effect because no alteration
on the electrophoretic profile of plasmids was
observed (Figure 2).
In conclusion, under the conditions used in this
work, our data suggest that fenoprofen would not
(i) affect the fixation of the 99m
Tc on the blood
constituents, (ii) alter the RBC membrane and (iii)
present genotoxic and redox effects.
ACKNOWLEDGEMENTS
This study was supported by grants and financial
support from CAPES, CNPq and FAPERJ.
RESUMO
O objetivo deste trabalho foi avaliar o efeito do
fenoprofeno na marcação de constuintes
sanguíneos com tecnécio-99m (99m
Tc), na
morfologia de hemácias e no DNA plasmidial.
Amostras de sangue de ratos Wistar foram
incubadas com fenoprofeno e a marcação de
constituintes sangüíneos com 99m
Tc foi realizada.
Células sangüíneas (CS) e plasma (P) foram
isolados. Alíquotas de CS e P foram precipitadas,
frações insolúvel e solúvel foram separadas. A
radioatividade em cada fração foi contada e o
percentual de radioatividade incorporada (%ATI),
determinada. Distensões sangüíneas foram
preparadas, fixadas, coradas e análise morfológica,
qualitativa e quantitativa, de hemácias foi
realizada sob microscopia óptica. Plasmídios
pBSK foram incubados com fenoprofeno na
presença e ausência de cloreto estanoso, e o
procedimento de eletroforese em gel de agarose
realizado para avaliar o efeito genotóxico deste
fármaco e seu efeito sobre a ação do cloreto
estanoso no DNA. Os resultados obtidos sugerem
que, nas condições utilizadas nesse estudo, o
fenoprofeno não poderia: (i) afetar a fixação do 99m
Tc nos constituintes sanguíneos, (ii) alterar a
membrana de hemácias e (iii) apresentar efeitos
genotóxicos e redox.
11
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Lage, C. A. S.; Cabral-Neto, J. B.; Leitão, A. C.;
Bernardo-Filho, M.; Bezerra; R. J. A. C.; Carvalho; J.
J.; Caldeira-de-Araújo, A. (2002), Genotoxic effects
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Chem Toxicol., 40: 1493-1498.
Dantas, J. S. F.; Morais, O.; Mattos, C. P. J.; Bezerra, J.
A. C. R.; Carvalho, F. E.; Bernardo-Filho, M.;
Araújo, C. A. (1999), Stannous chloride mediates
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Ferreira-Machado, S. C.; Rodrigues, M. P.; Nunes, A.
P.; Dantas, F. J.; De Mattos, J. C.; Silva, C. R.;
Moura, E. G.; Bezerra, R. J.; Caldeira-de-Araujo, A.
(2004), Genotoxic potentiality of aqueous extract
prepared from Chrysobalanus icaco L. leaves.
Toxicol Lett., 151, 481-488.
Fonseca, A. S.; Frydman, J. N.; Rocha, V. C.;
Bernardo-Filho, M. (2007), Acetylsalicylic acid
decreases the labeling of blood constituents with
technetium-99M. Acta Biol Hung., 2, 187-198.
Freitas, R. S.; Moreno, S. R. F.; Lima-Filho, G. L.;
Fonseca, A. S.; Bernardo-Filho, M. (2007), Effect of
a comercial extract of Paulinia cupana (guaraná) on
the biding of 99mTc-DMSA on blood constituents:
An in vivo study. Appl Radiat Isot., 65,528-533.
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Essentials of nuclear medicine science. Williams and
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Gilman, A.G. (eds) The Pharmacological Baisis of
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617-657.
Li, A.; Seipelt, H.; Muller, C.; Artmann, M.; (1999),
Effects of salicylic acid derivatives on red blood cell
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R.; de Oliveira, M. B.; Bezerra, R. J.; Caldeira-de-
Araujo, A.; Bernardo-Filho, M. (2001), Effect of the
Cymbopogon citratus, Maytenus ilicifolia and
Baccharis genistelloides extracts against the stannous
chloride oxidative damage in Escherichia coli. Mutat
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Moreno, S. R. F.; Rocha, E. K.; Pereira, M.; Mandarim-
Lacerda, C.; Freitas, R. S.; Nascimento, A. L. R.
Carvalho, J. J.; Lima-Filho, G. L.; Diré, G.; Lima, E.
A. C.; Bernardo-Filho, M. (2004), Ginkgo biloba
extract: experimental model to evaluate its action on
the labeling of blood elements with Technetium-99m
and on the morphometry of red blood cells. Pak J
Nutr., 3, 68-71.
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Santos-Filho, S. D.; Bernardo-Filho, M. (2005), An
aqueous extract of Pfaffia sp. does not alter the
labeling of blood constituents with technetium-99m
and the morphology of the red blood cells. Braz J
Pharmacol., 15, 126-132.
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Cunha, F. Q.; Lanchote, V. L.; Reis, M. L. (2006),
Pharmacodynamics, Chiral Pharmacokinetics, and
Pharmacokinetic-Pharmacodynamic Modeling of
Fenoprofen in Patients With Diabetes Mellitus. J Clin
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Santos-Filho, S. D.; Bernardo-Filho, M. (2005), Effect
of Hypericum perforatum extract in vitro labeling of
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biovailability of sodium pertechentate in Wistar rats.
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Pharmacy. Springer-Verlag, New York.
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Shirey, R.S.;Morton; S. J.; Lawton, K. B., Lowell,
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Received: August 28, 2008;
Revised: September 16, 2008;
Accepted: September 18, 2008.
12
3.2- Artigo Submetido I
Evaluation of biological effects of the naproxen
Marcia de Oliveira Pereira1,2,Gabrielle de Souza Rocha1,2, Aldo Cunha Medeiros1,
Adenilson de Souza da Fonseca2,3,*, Mario Bernardo-Filho2,4.
1Programa de Pós-Graduação em Ciências da Saúde, Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Avenida General Gustavo Cordeiro de Farias, s/n, 59010180, Natal, Brasil 2Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, Vila Isabel, 20551030, Rio de Janeiro, Brasil 3Departamento de Ciências Fisiológicas, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro, Rua Frei Caneca, 94, 20211040, Rio de Janeiro, Brasil 4Instituto Nacional do Câncer; Praça Cruz Vermelha, 23, 20230130, Rio de Janeiro, Brasil Abstract The aim of this work was to evaluate biological effects of the naproxen through of the labeling of blood constituents with technetium-99m, survival of bacterial cultures and electrophoresis profile of plasmid DNA. Blood samples from Wistar rats were incubated with naproxen or with saline (0.9% NaCl), as control, and radiolabeling of blood constituents was performed. Influence of naproxen on the E. coli AB1157 culture growth and survival in presence or absence of SnCl2 was used to assess cytotoxic effect. Electrophoretic profile in agarose gels of bacterial plasmids in presence or absence of SnCl2 was used to evaluate antioxidant and genotoxic potential of naproxen. Results obtained suggest that naproxen could not interfere on the labeling of blood constituents with 99mTc but it could present cytotoxic effect and protect E. coli cultures of the lethal effect of stannous chloride at low concentrations. Moreover, naproxen could present genotoxic effect in isolated plasmid DNA. Keywords: Blood Constituents, DNA, Escherichia coli, Naproxen, Stannous Chloride.
13
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are a heterogeneous group of
compounds that exhibit anti-inflammatory, analgesic, and antipyretic properties. These
drugs can be separated into three groups: salicylates, represented by aspirin; propionic
acid derivatives, including ibuprofen and naproxen sodium; and the para-aminophenols,
represented by acetaminophen (Abramson et al., 2001). NSAIDs are the most
commonly prescribed analgesic medications worldwide, and their efficacy for treating
acute pain has been well demonstrated (Van Tunder et al., 2000; Warner et al., 2006).
They reversibly inhibit cyclooxygenase (prostaglandin endoperoxide synthase), the
enzyme mediating production of prostaglandins and thromboxane A2 (Fitzgerald and
Patrono, 2001).
Naproxen is a NSAID used for treatment of rheumatic as osteoarthritis (Fendrick
and Greenberg, 2009), other inflammatory degenerative and articulate diseases (Costa
et al., 2006; Insel, 2001). Clinicians prescribe NSAIDs on a routine basis to treat of mild-
to-moderate pain using doses ranging 440 up to 660 mg day (Schiff and Minic, 2004).
However nor all their biological effects have been well established and the use of
different experimental models could be worthwhile.
Blood constituents are labeled with technetium-99m (99mTc) using stannous
chloride (SnCl2) as reducing agent and they are utilized in nuclear medicine to aid in the
diagnostic image procedures (Saha, 2004). Red blood cells (RBC) labeled with 99mTc
are used in procedures of nuclear medicine, including studies for function evaluation of
spleen, gastrointestinal bleeding sites, blood cells mass, cardiovascular system (Saha,
2004). Sequential steps of the intracellular labeling process of the RBC include: (i)
14
transmembrane transport of stannous and pertechnetate ions into internal compartment
of RBC; (ii) reduction of 99mTc (99mTcO4) by the SnCl2 and (iv) binding of the reduced
99mTc to hemoglobin (Callahan and Rabito, 1990). Based on these sequential steps,
blood constituents from Wistar rats have been proposed as experimental model to
evaluate redox properties and possible interactions of drugs on cellular membrane
(Abreu et al., 2006; Fonseca et al., 2007; Benarroz et al., 2008; Frydman et al.,2008).
Some experimental models to evaluate genotoxic, citotoxic and potential redox,
involving SnCl2, have been suggested (Pungartnik et al., 2007 and Almeida et al., 2005).
Escherichia coli (E. coli) cultures have been used to evaluate the citotoxic effect of
extract of medicinal plants (Almeida et al., 2005; Mello et al., 2001). Assays based on
these cultures are fast, easy and very cheap to perform. Electrophoretic profile of
bacterial plasmids have been used to assess the ability of natural drugs to induce single
strand breaks in DNA through generation of free radicals in vitro (Ferreira-Machado et
al., 2004; Presta et al., 2007). Moreover, bacterial cultures and plasmid DNA samples
treated with SnCl2 has been also proposed as models to evaluate redox of drugs
(Almeida et al., 2005; Pereira et al., 2008).
The aim of this work was to evaluate biological effects of the naproxen through of
the labeling of blood constituents with 99mTc, of the survival of E. coli cultures and of
the electrophoresis profile of bacterial plasmid.
15
Materials and Methods
Drugs
Naproxen used in this study was purchased from Laboratory Teuto Brasileiro S/A.
(Goiás, Brazil, lot 601034) and SnCl2 was purchased from Sigma Chemicals Co (St
Louis, USA).
Animals
Adult male Wistar rats (3-4 months, 250-300g) were maintained in a controlled
environment: normal light/dark cycle conditions (12-h light/12-h dark; lights at 6 am), free
access to water and food, room temperature was kept at 25±2 ºC. Experimental
protocols were approved by the Ethical Committee of the Instituto de Biologia Roberto
Alcantara Gomes, Universidade do Estado do Rio de Janeiro (protocol number
CEA/203/2007).
In vitro radiolabeling of blood constituents
Samples of whole blood (n=7, for each naproxen concentration) were incubated
with this drug at different concentrations (0.1, 1.0, 10, 100, 1000 μg/mL; 1 hour). Blood
samples incubated with saline solution (0.9% NaCl). After that, SnCl2 (1.2 μg/mL, 1
hour) was added and, in sequence, 99mTc (3.7 MBq, 10 minutes), as sodium
pertechnetate (Na99mTcO4), recently milked from a 99Mo/99mTc generator (Instituto
de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, São
Paulo, Brazil). These samples were centrifuged (1500 rpm in clinical centrifuge, 5
minutes, room temperature) and plasma (P) and blood cells (BC) were separated.
Aliquots of P and BC were also precipitated with trichloroacetic acid (5 %) and soluble
(SF) and insoluble (IF) fractions were obtained. Radioactivity in P, BC, IF-P, SF-P, IF-BC
16
and SF-BC was counted in a well gamma counter (Packard, model C5002, Illinois, USA)
and the percentage of radioactivity incorporated (%ATI) on each fraction was calculated
as described previously (Bernardo-Filho et al., 1983). Briefly, %ATI for each fraction was
obtained by ratio between the radiation counting for a fraction and the sum of the
radiation counting for this fraction and the complementary fraction multiplied by 100.
Bacterial growth assay
From a stock (in glycerol 50% v/v) of E. coli AB1157, a wild-type strain proficient
in repairing DNA damage, an aliquot was grown in liquid LB (Luria and Burrous, 1957)
medium at 37 °C overnight up to stationary growth phase. Samples of these cultures
were grown in presence of naproxen at 3 and 30 μg/mL. After that, these samples were
allowed to grow for up to 4 hours. The growth of bacterial cultures was evaluated by the
optic density at 600 nm. As controls, bacterial cultures were grown in presence of saline
solution.
Bacterial survival assay
From cultures of E. coli AB1157, in stationary growth phase, aliquots were taken
and further incubated under the same conditions to reach exponential growth (108
cells/mL). The cells were collected by centrifugation, washed twice in saline and
suspended again in saline. After that, bacterial suspensions (108 cells/mL) were treated
with naproxen (0.3 and 30 μg/mL) in the presence or absence of stannous chloride (25
g/mL) (Almeida et al., 2005) for 60 minutes. Bacterial suspensions treated with saline
or stannous chloride alone were used as controls. Aliquots from these treatments were
diluted in saline, spread onto Petri dishes containing solidified LB medium (1.5% agar).
Colonies formed after overnight incubation at 37 oC were counted and the survival
17
fraction was calculated as described before (Almeida et al., 2005). Experiments were
carried out in triplicate and the results presented are the average mean of three
independent assays.
Plasmid treatment with naproxen and electrophoretic profile assay
Bacterial plasmids (pBSK) were obtained by alkaline cell lysis method (Sambrook
et al., 1989) from E. coli DH5aF’Iq (rec-) strain hosting this plasmid. Plasmid samples
were incubated with naproxen at different concentrations (3.0, 30, 300 g/mL). To
assess the action of naproxen on effects of SnCl2, plasmids were incubated with
naproxen, at the same concentrations, in the presence of SnCl2 (200 g/mL) (Sambrook
et al., 1989). Plasmids incubated with 10 mM Tris buffer (vehicle, pH 7.4) or SnCl2 alone
were used as positive control. The incubations were carried out at room temperature for
40 minutes. After that, each sample was mixed with loading buffer (0.25% xylene cyanol,
0.25% bromophenol blue and glycerol in water) and applied in 0.8% agarose horizontal
gel electrophoresis chamber in Tris-acetate-EDTA buffer (pH 8.0, 7 V/cm). The gel was
stained with ethidium bromide (0.5 g/mL) and the plasmids forms (supercoiled and
open circle) were visualized by fluorescence under an ultraviolet transilumination
system. The assay was repeated at least three times, the results were digitalized (Kodak
Digital Science 1d, EDAS 120) and the plasmid forms semiquantified using the computer
program Image J for Windows.
Statistical analysis
Data are reported as (means ± SD) of the %ATI, optic density, survival fraction
and percentage of plasmid forms. The One-way analysis of variance–ANOVA test was
performed to verify possible statistical differences with p<0.05 as less significant level.
18
Results
In vitro radiolabeling of blood constituents
Table 1 presents the effects of naproxen on the radioactivity distribution between
cellular and plasma compartments. These data indicate no alteration (p>0.05) of 99mTc
distribution into these compartments.
Table 2 presents the effect of naproxen on the fixation of 99mTc on insoluble and
soluble fractions plasma proteins. Similarly to present in Table 1, naproxen was not
capable to interfere significantly (p>0.05) on the fixation of the radioactivity on the
insoluble and soluble fractions of plasma.
No significant (p>0.05) alteration on the fixation of radioactivity on proteins of
blood cells from blood samples incubated with naproxen (Table 3) was also found.
Table 1. Effect of naproxen on the radioactivity distribution between cells and plasma compartments.
Naproxen
( g/mL)
%ATI
P BC
0.0 3.44 ± 2.04 96.56 ± 2.04 0.1 1.73 ± 1.29 98.27 ± 1.29 1.0 2.22 ± 1.20 97.78 ± 1.20
10.0 4.48 ± 2.69 95.52 ± 2.69 100.0 2.51 ± 1.72 97.49 ± 1.72
1000.0 3.53 ± 3.09 96.47± 3.09
Samples of whole blood from Wistar rats were treated with naproxen at different concentrations for 60 minutes. After that, labeling of blood constituents with 99mTc was carried out. Plasma (P) and blood cells (BC) were separated by centrifugation. Radioactivity in BC and P was counted and the %ATI was calculated. P <0.05, compared to control group of BC.
19
Table 2. Effect of naproxen on the fixation of 99mTc on soluble and insoluble fractions of plasma.
Naproxen
( g/kg)
%ATI
SF-P IF-P
0.0 31.72 ± 4.96 68.28 ± 4.96 0.1 29.83 ± 4.22 70.17 ± 4.22 1.0 34.89 ± 8.17 65.11 ± 8.17
10.0 27.61 ± 4.01 72.39 ± 4.01 100.0 27.86 ± 3.95 72.14 ± 3.95
1000.0 28.62 ± 2.80 71.38 ± 2.80
Samples of whole blood from Wistar rats were treated with naproxen at different concentrations. After that, labeling of blood constituents with 99mTc was carried out. Plasma (P) was separated from blood cells by centrifugation and soluble (SF) and insoluble (IF) fractions of plasma were isolated by precipitation in trichloroacetic acid and centrifugation. Radioactivity in BC and P was counted and the %ATI was calculated. P <0.05, compared to control group of IF-P.
Table 3. Effect of naproxen on the fixation of 99mTc on soluble and insoluble fraction of blood cells.
Naproxen
( g/kg)
%ATI
SF-BC IF-BC
0.0 19.71 ± 1.75 80.29 ± 1.75 0.1 18.48 ± 2.50 81.52 ± 2.50 1.0 19.03 ± 2.90 80.97 ± 2.90
10.0 20.09 ± 2.57 79.91 ± 2.57 100.0 17.96 ± 0.92 82.04 ± 0.92
1000.0 17.85 ± 2.17 82.15 ± 2.17
Samples of whole blood from Wistar rats were treated with naproxen at different concentrations. After that, labeling of blood constituents with 99mTc was carried out. Blood cells (BC) were separated from plasma by centrifugation and soluble (SF) and insoluble (IF) fractions of BC were isolated by precipitation in trichloroacetic acid and centrifugation. Radioactivity in each fraction was counted and the %ATI was calculated. P <0.05, compared to control group of IF-BC.
Bacterial growth assay
Figure 1 presents the optic density of E. coli AB1157 cultures grown in presence
and absence of naproxen. Data in this figure suggest that the treatment with naproxen,
at concentrations used (3 and 30 g/mL) would not present effects on the growth of E.
coli AB1157 cultures.
20
Figure 1: Growth curves of E. coli AB1157 in presence and absence of naproxen. From a culture in stationary growth phase, aliquots of E. coli AB1157 cultures were put in rich medium at 37 °C in presence an absence of naproxen at different concentrations (3, 30 μg/mL) and allowed to grow for up to 4 hours. Growth of bacterial cultures was evaluated by the optic density at 600 nm. Cultures grown in presence of saline solution (0.9% NaCl) were used as controls. (■)
control, (▲) naproxen 3 g/mL ( ), naproxen 30 g/mL.
Bacterial survival assay
Figure 2 represents the survival graphic of E. coli AB1157 in presence and
absence of SnCl2. Data in this figure suggest that the treatment with naproxen could
present cytotoxic effects on E. coli AB1157 cultures at concentrations used (3 and 30
g/mL). Other data in figure indicate that the lethal effect of SnCl2 on E. coli cultures
could be reduced in presence of naproxen at the lower dose used (3 g/mL).
21
Figure 2: Bacterial survival curves of E. coli AB1157 treated with naproxen in presence and absence of SnCl2. E. coli AB1157 cultures, in exponential growth phase, were centrifuged (3000 rpm, 20 minutes) and suspended in saline solution (0.9% NaCl). Samples of these bacterial suspensions were incubated (0, 30 and 60 minutes, 37 oC) with naproxen at different concentrations (3, 30 μg/mL) in the presence and absence of SnCl2 (25 μg/mL). Aliquots were diluted in saline and spread onto Petri dishes. After overnight incubation (37 oC), colony forming units were counted to determine survival fractions. As controls, bacterial samples incubated with
saline (negative control) or SnCl2 (positive control) alone. ( ) saline; (■) SnCl2; (□) naproxen 3μg/mL; (▲) naproxen 30 μg/mL; (∆) SnCl2 + naproxen 3 μg/mL; (●) SnCl2 + naproxen 30 μg/mL.
Plasmid treatment with naproxen and electrophoretic profile assay
The Figure 3b shows the photograph of agarose gel electrophoresis of bacterial
plasmids treated with naproxen in presence and absence of SnCl2. The results shown in
this figure indicate that naproxen is capable to induce alterations on the electrophoretic
profile of plasmids (lanes 3, 4 and 5) when compared with negative control (lane 1).
Also, the results of the figure 3b indicate that the effect of SnCl2 (lane 2) is increased by
naproxen at concentrations used (lanes 6, 7 and 8). These results were confirmed by
semiquantitative analyses of the percentages of supercoiled (SC) and open circle (OC)
plasmid forms (Figure 3a) indicating alteration on the electrophoretic profile.
22
Figure 3: Percentage of bacterial plasmid forms (a) and photograph (b) of agarose gel after electrophoresis of plasmid pBSK treated with naproxen in presence and absence of SnCl2. Samples of bacterial plasmids were incubated with naproxen (0.3, 30 and 300 μg/mL) in presence or absence of SnCl2 (200 μg/mL). After that, agarose gel electrophoresis procedure (0.8%, 7 V/cm) was performed, gels were stained with ethidium bromide (0.5 μg/mL), plasmid forms were visualized by fluorescence and digitalized to obtain the percentage of each plasmid forms. As controls, plasmids samples incubated with buffer (negative control) or SnCl2 (positive control) alone. Lanes: (1) pBSK + buffer; (2) pBSK + SnCl2; (3) pBSK + naproxen (300 μg/mL); (4) pBSK + naproxen (30 μg/mL); (5) pBSK + naproxen (0.3 μg/mL); (6) pBSK + naproxen (300 μg/mL) + SnCl2; (7) pBSK + naproxen (30 μg/mL) + SnCl2; (8) pBSK + naproxen (3.0 μg/mL) + SnCl2. (■) OC (open circle); (□) SC (supercoiled).
Discussion
Data obtained in this work indicate that there was not alteration on the labeling of
the blood constituents with 99mTc when the blood was incubated with naproxen (tables
1, 2 and 3). Despite the absence of effects of the naproxen on radiolabeling of blood
constituents, it has been reported hemolysis after use of naproxen in human beings
(Orhan and Sahin, 2001). Other data has indicated that naproxen is almost completely
bond to plasma proteins (Insel, 2001) and suffers reduction from its effectiveness when
it has reduction of the concentration of plasma proteins (Warner et al., 2006). These
findings could aid to understand the reason to the naproxen was not capable to interfere
on the labeling of the blood constituents with 99mTc. The importance of these founds is
relate to comprehension of possible interactions of medicines with radiopharmaceuticals.
b
(a)
23
Cytotoxic effect of some drugs has been demonstrated by different experimental
models (Hassan et al., 1999). Anti-inflammatory drugs have been reported to decrease
the survival of cancer cells (Ricchi et al., 2002). In our study, naproxen decreases the
survival of E. coli AB1157 cultures suggesting a cytotoxic effect (Figure 2). This cytotoxic
effect of naproxen is in agreement with data of other authors that have demonstrated
cytotoxic effect of this drug on intestinal mucosa cells (Oh et al., 2005). These
considerations are very important due to the level of biological organization is different in
E. coli and intestinal mucosa cells.
Stannous chloride has been suggested to decrease the survival of bacterial
cultures by free radical generation (Bernardo-Filho et al., 1994; Agostinho et al., 2008)
Natural products could abolish the effects of SnCl2 decreasing the free radical
production or as scavengers of these species (Almeida et al., 2007). Naproxen could
protect bacterial cultures of the lethal effect of SnCl2, probably, decreasing the free
radical production (figure 2). Although the data obtained with association of SnCl2 and
naproxen could be paradoxical, these results are in agreement with data that have
suggested that naproxen presents antioxidant property in non cellular and cellular
experimental models (Costa et al., 2006).
The genotoxic effect of SnCl2 on DNA has been suggested to occur by
mechanisms related to free radical generation (Pereira et al., 2008; Dantas et al., 1996;
Assis et al., 2002). These effects could, at last in part, to be attributed to the reactive
oxygen species, generated during the SnCl2 treatment (Dantas et al., 2002; Mattos et
al., 2000). In fact, the presence of free radicals scavengers could reduce the changes of
electrophoretic profile of bacterial plasmids induced by SnCl2 decreasing the DNA strand
24
breaks (Dantas et al., 1999; de Mattos et al., 2000). Naproxen has been suggested to
be scavenger of free radicals (Costa et al., 2006), in agreement with the results gotten in
this work, but this was not obtained with another NSAIDs (Pereira et al., 2008).
However, under the experimental conditions used in this work, naproxen did not seem to
protect plasmid DNA against the effects of SnCl2. On the other hand, naproxen seems to
present genotoxic effect in consequence of the alterations in the electrophoretic profile
of plasmids were found (Figure 3). This finding is very relevant, due to the importance of
this NSAID and the results were obtained wit isolated plasmid DNA. However, further
studies about this effect would be stimulated in specialized laboratories.
In conclusion, results obtained in this study suggest that naproxen could not
interfere on the labeling of blood constituents with 99mTc but it could present cytotoxic
effect and protect E. coli cultures of the lethal effect of stannous chloride at low
concentrations suggesting antioxidant effect. Moreover, naproxen could present
genotoxic effect in isolated plasmid DNA.
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Abreu PRC, Almeida MC, Bernardo RM, Bernardo LC, Brito LC, Garcia EAC, Fonseca AS, Bernardo-Filho M (2006) Guava extract (Psidium gaujava) alters the labeling of blood constituents with technetium-99m. Journal of Zheijiang University Science B 7:429-435. Agostinho RT, Santos-Filho SD, Fonseca AS, Missailidis S, Bernardo-Filho M (2008) The effect of an extract from ganoderma lucidum (reishi) on the labeling of blood constituents with technetium-99m and on the survival of Escherichia coli. Brazilian Archives of Biology and Techonology 51:157-162. Almeida MC, Soares SF, Abreu PR, Jesus LM, Brito LC, Bernardo-Filho M (2007) Protective effect of an aqueous extract of Harpagophytum upon Escherichia coli strains
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submitted to the lethal action of stannous chloride. Cellular and Molecular Biology (Noisy-le-grand-France) 53: 923-92. Assis ML, De Mattos JC, Caceres MR, Dantas FJ, Asad LM, Asad NR, Bezerra RJ, Caldeira-de-Araújo A, Bernardo-Filho M (2002) Adaptive response to H2O2 protects against SnCl2 damage: the OxyR system involvement. Biochimie 84:291-294. Benarroz MO, Fonseca AS, Rocha GS, Frydman JN, Rocha VC, Pereira M O, Bernardo-Filho M (2008) Cinnamomum zeylanicum extract on the radiolabelling of blood constituents and the morphometry of red blood cells: In vitro assay. Applied Radiation and Isotopes 66:139-146. Bernardo-Filho M, Moura INS, Boasquevisque M (1983) 99m technetium – labeled red blood “in vitro”. Brazilian Archives of Biology and Techonology 4:455-461. Bernardo-Filho M, Cunha MC, Valsa JO, Araujo AC, Silva FC, Fonseca AS (1994) Evaluation of potential genotoxicity of stannous chloride: inactivation, filamentation and lysogenic induction of Escherichia coli. Food Chemistry and Toxicology 32:477-479. Callahan RJ, Rabito CA (1990) Radiolabeling of erythrocytes with technetium-99m: role of band-3 protein in the transport of pertechnetate across the cell membrane. Journal of Nuclear Medicine 31: 2004-2008. Costa D, Moutinho L, Lima JLF, Fernandes E (2006) Antioxidant activity and inhibithion of human neutrophil oxidative burst mediated by arylpropionic acid non-steroidal anti-inflamatory drugs. Biological & Phamaceutical Bulletin 29:1659-1670. Dantas FJS, De Mattos JCP, Viana ME, Lage CAS, Cabral-Neto JB, Leitão AC, Bernardo-Filho M, Bezerra RJAC, Carvalho JJ, Caldeira-de-Araújo A (2002) Genotoxic effects of stannous chloride (SnCl2) in K562 cell line. Food Chemistry Toxicology 40:1493-1498. Dantas FJS, Moraes MO, Carvalho EF, Valsa JO, Bernardo-Filho M, Caldeira-de-Araújo A (1996) Lethality induced by stannous chloride on Escherichia coli AB1157: participation of reactive oxygen species. Food Chemistry and Toxicology 34:959-962. Dantas FJS, Moraes O, Mattos CPJ, Bezerra JACR, Carvalho FE, Bernardo-Filho M, Araújo CA (1999) Stannous chloride mediates single strand breaks in plasmid DNA through reactive oxygen species formation. Toxicology Letters 110:129-136. de Mattos JC, Dantas FJ, Bezerra RJ, Bernardo-Filho M, Cabral-Neto JB, Lage C, Leitão AC, Caldeira-de-Araújo A (2000) Damage induced by stannous chloride in plasmid DNA. Toxicology Letters 116:159-163.
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Fendrick AM, Greenberg BP (2009) A review of the benefits and risks of nonsteroidal anti-inflammatory drugs in the management of mild-to-moderate osteoarthritis. Osteopathic Medicine and Primary Care 6:3.
Ferreira-Machado SC, Rodrigues MP, Nunes AP, Dantas FJ, De Mattos JC, Silva CR, Moura EG, Bezerra RJ, Caldeira-de-Araujo A (2004). Genotoxic potentiality of aqueous extract prepared from Chrysobalanus icaco L. leaves. Toxicology Letters 151:481-488. Fitzgerald GA, Patrono C (2001). The coxibs, selective inhibitor of cyclooxygenase-2. New England Journal of Medicine 345:433-444. Fonseca AS, Frydman JN, Rocha VC, Bernardo-Filho M (2007) Acetylsalicylic acid decreases the labeling of blood constituents with technetium-99M. Acta Biologica Hungarica 2: 187-198. Frydman JNG, Rocha VC, Benarroz MO, Rocha GS, Pereira MO, Fonseca AS, Bernardo-Filho M (2008) Assessment of effects of a Cordia salicifolia extract on the radiolabeling of blood constituents and on the morphology of red blood cells. Journal of Medicinal Food 11:767–772. Hassan HN, Barsoum BN, Habid IH (1999) Simultaneous spectrophotometric determination of rutin, quercetin and ascorbic acid in drugs using a Kalman Filter approach. Journal of Pharmaceutical and Biomedical Analysis 20:315-320. Insel PA (2001) Analgesic-antipiryretic and antiinflamatory agentes and drugs employed in the treatment of gout. In: Harman, J.G., Limbird, L. E., Gilman, A.G. (eds) The Pharmacological Baisis of Therapeutics. 10th McGraw/Hill, New York, 617-657. Luria SE, Burrous JW (1957) Hybridization between E.coli and Shigella. Journal of Bacteriology 74:461-476. Mattos JPC, Dantas FJS, Bezerra RJA, Bernardo-Filho M, Cabral-Neto JB, Lage C, Leitão AC, Caldeira-de-Araújo A (2000) Damage induced by stannous chloride in plasmidial DNA. Toxicology Letters 116:159-163. Melo SF, Soares SF, da Costa RF, da Silva CR, de Oliveira MB, Bezerra RJ, Caldeira-de- Araujo A, Bernardo-Filho M (2001) Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli. Mutation Research 496:33-38. Oh TY, Ahn GJ, Choi SM, Ahn BO, Kim WB (2005) Increased susceptibility of ethanol-treated gastric mucosa to naproxen and its inhibition by DA-9601, an Artemisia asiatica extract. World Journal of Gastroenterology 11:7450–7456.
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Orhan H, Sahin G (2001) In vitro effects of NSAIDS and paracetamol on oxidative stress-related parameters of human erythrocytes. Experimental Toxicology and Pathology 53:133-140. Pereira MO, Rocha GS, Lombardi SS, Geller M, Pereira MJ, Santos-Filho SD, Fonseca AS, Bernardo-Filho M (2008) Effects of fenoprofen on the labeling of blood constituents with technetium-99m, the morphology of red blood cells and the plasmid. Braz Arch Biol Technol 51:135-141. Pungartnik C, Viau C, Picada J, Caldeira-de-Araujo A, Henriques JA, Brendel M (2005) Genotoxicity of stannous chloride in yeast and bacteria. Mutation Research 583:146-157. Presta GA, Fonseca AS, Bernardo-Filho M (2007)A Chrysobalanus icaco extract alters the plasmid topology and the effects of stannous chloride on the DNA of plasmids. Brazilian Journal of Pharmacognosy 17:331-335. Ricchi P, Di Matola T, Ruggiero G (2002) Effect of non-steroidal anti-inflammatory drugs on colon carcinoma Caco-2 cell responsiveness to topoisomerase inhibitor drugs. British Journal of Cancer 86:1501–1509. Saha GB (2004) Fundamentals in Nuclear Pharmacy. Springer-Verlag, New York. Sambrook J, Fritsch EF Maniatis T (1989) Extraction and purification of plasmid DNA. In: Molecular cloning. A laboratory manual. New York: Cold Spring Harbour Laboratory Press. Schiff M, Minic M (2004) Comparison of the analgesic efficacy and safety of nonprescription doses of naproxen sodium and ibrupofen in the treatment of osteoarthritis the knee. Journal of Rheumatology 31:1373-1383. Van Tunder MW, Sholten RJ, Koes BW, Deyo RA (2000) Nonsteroidal anti-inflamatory drugs for low back pain: a systematic review within the framework of the Cochrane Collaboration Back Review Group. Spine 25:2501-2513. Warner TD, Vojnovic I, Bishop-Bailey D, Mitchell JA (2006) Influence of plasma protein on the potencies of inhibitors of cyclooxygenase-1 and-2. The Faseb Journal 20:542-544.
28
3.3- Artigo submetido II
EVALUATION OF BIOLOGICAL EFFECTS OF THE IBUPROFEN AND KETOPROFEN
Marcia de Oliveira Pereira 1, 2,Gabrielle de Souza Rocha 1, 2, Adenilson de Souza da Fonseca 2,3 ,* and Mario Bernardo-Filho 2,4.
1Programa de Pós-Graduação em Ciências da Saúde, Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Avenida General Gustavo Cordeiro de
Farias, s/n, 59010180, Natal, Brasil. 2Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida
28 de Setembro, 87, Vila Isabel, 20551030, Rio de Janeiro, Brasil. 3Departamento de Ciências Fisiológicas, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro Rua Frei Caneca, 94, 20211040, Rio de Janeiro, Brasil. 4Instituto Nacional do
Câncer; Praça Cruz Vermelha, 23, 20230130, Rio de Janeiro, Brasil.
Abstract
The aim of this work was to evaluate biological effects of the ibuprofen and ketoprofen
through experimental models at cellular and molecular level. Blood samples from Wistar
rats were incubated with ibuprofen or ketoprofen and the assay of labeling of blood
constituents with technetium-99m (99mTc) was performed. Blood cells, plasma, soluble
and insoluble fractions of blood cells and plasma were separated. The radioactivity in
each fraction was counted and percentage of incorporated radioactivity (%ATI) was
determined. Blood smears were prepared, fixed, stained and the qualitative and
quantitative morphology of the red blood cells (RBC) was evaluated. Plasmid (pBSK)
was incubated with ibuprofen or ketoprofen with stannous chloride, and agarose gel
electrophoresis procedure was carried out to evaluate genotoxic and the protection of
this drug against stannous chloride effect on DNA. In conclusion, under the conditions
used in this work, our data suggest that ibuprofen and ketoprofen would not (i) affect
29
the fixation of the 99mTc on the blood constituents, (ii) alter the RBC membrane and (iii)
present genotoxic and redox effects.
Key words: ibuprofen, ketoprofen, technetium-99m, morphology, plasmid.
Introduction
Non-steroidal anti-inflammatory drugs widely used for the treatment of pain and
inflammation and represent the drugs of choice commonly used in the management of
musculoskeletal traumatisms, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis,
acute gouty arthritis and dysmenorrhoea (Costa, 2006).
The arylpropionic acid derivatives constitute an important group of non-steroidal
anti-inflammatory drugs widely used and with action similarly and very effective for
treatment of pain and inflammation as irreversible cyclooxygenase enzyme inhibitors
(Insel, 2001). Ibuprofen and ketoprofen are a peripherally acting non-steroidal anti-
inflammatory drug indicated for analgesia, antipyresis, and various arthritic conditions
(Olson, 2007). Clinicians prescribe NSAIDs on a routine basis to treat of mild-to-
moderate pain using doses ranging 200mg up to 800mg of ibuprofen and 150mg up to
300mg doses of ketoprofen (Korolkovas, 1999, Insel, 2001).
Nor all biological effects were evaluated to these anti-inflammatory drugs
justifying that experimental models can be used for evaluation of their effects.
Antioxidant action has been described for a number of chemical substances and
drugs (Pereira et al, 2008) and interest on them is explained because some chronic
diseases could be prevented when antioxidants are used (Valko, et al, 2006).
Oxidative stress induces a cellular redox imbalance (Valko, et al, 2006), at high
concentrations, reactive oxygen species (ROS) can be important mediators of damage
30
to cellular structures, including lipids and membranes, proteins and nucleic acids (G.
Poli,1996). There is compelling evidence that ROS mediated oxidative stress is involved
in a vast number of biological responses causing DNA modification, lipid peroxidation,
and production of inflammatory cytokines (Brigantini, S. 2003). This could contribute to
the pathogenesis of many inflammatory diseases (Zhou et al., 2009).
The aim of this work was to evaluate biological effects of the ibuprofen and
ketoprofen through experimental models at cellular and molecular level.
MATERIALS AND METHODS
Drugs
Ibuprofen and ketoprofen used in this study was purchased from EMS
Farmacêutica Ltda (São Paulo, Brazil, lot 12678) and Medley Farmacêutica (São Paulo,
Brazil, lot 06110713), respectively, and SnCl2 were purchased from Sigma Chemicals
Co (St Louis, USA).
Animals
Adult male Wistar rats (3-4 months, 250-300g) were maintained in a controlled
environment: normal light/dark cycle conditions (12-h light/12-h dark; lights at 6 am), free
access to water and food, room temperature was kept at 25±2 ºC. Experimental
protocols were approved by the Ethical Committee of the Instituto de Biologia Roberto
Alcantara Gomes, Universidade do Estado do Rio de Janeiro (protocol number
CEA/203/2007).
31
In vitro radiolabeling of blood constituents
Samples of whole blood (n=7, for each ibuprofen and ketoprofen concentration)
were incubated with this drug at different concentrations (0.1, 1.0, 10.0, 100.0, 1000.0
μg/mL; 1 hour). Blood samples incubated with saline solution (0.9% NaCl). After that,
SnCl2 (1.2 μg/mL, 1 hour) was added and, in sequence, 99mTc (3.7 MBq, 10 minutes)
as sodium pertechnetate (Na99mTcO4), recently milked from a 99Mo/99mTc generator
(Instituto de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia
Nuclear, São Paulo, Brazil). These samples were centrifuged (1500 rpm, 5 minutes) and
plasma (P) and blood cells (BC) were separated. Aliquots of P and BC were also
precipitated with trichloroacetic acid (5 %) and soluble (SF) and insoluble (IF) fractions
were obtained. The radioactivity (% ATI) in P, BC, IF-P, SF-P, IF-BC and SF-BC was
counted in a well gamma counter (Packard, model C5002, Illinois, USA). The %ATI was
calculated as described previously (Bernardo-Filho et al., 1983).
Morphological evaluation
Smears were prepared from blood samples incubated with ibuprofen and
ketoprofen at different concentration (0.0, 0.1, 1.0, 10.0, 100.0, 1000.0 μg/mL; 5 slides
for each concentration) and stained by May-Grünwald-Giemsa (Barcia, 2007). The slices
were analyzed by optical microscopy and for morphometric measurements a total of five
fields per each slide were evaluated. A spherical shape and normal size distribution
were assumed to RBC on control samples. Area and perimeter of RBC were measured
(Software image pro plus, media Cibernetics, USA) and perimeter/area ratio was
calculated.
32
Plasmid DNA
Plasmid (pBSK) was obtained by alkaline cell lysis method (Sambrook et al,.
1989) from E. coli DH5aF’Iq (rec-) strain hosting this plasmid.
Plasmid treatment with ibuprofen and ketoprofen and electrophoretic profile
assay
Plasmids were incubated with ibuprofen and ketoprofen at different
concentrations (3.0, 30.0, 300.0 g/mL). To assess the action of ibuprofen and
ketoprofen on effects of SnCl2, plasmids were incubated with ibuprofen and ketoprofen,
at the same concentrations, in the presence of SnCl2 (200.0 µg/mL). Plasmid incubated
only with SnCl2 was used as positive control and, as negative control, plasmid incubated
at 10 mM Tris buffer (vehicle, pH 7.4). The incubations were carried out at room
temperature for 40 minutes. After that, each sample was mixed with loading buffer
(0.25% xylene cyanol, 0.25% bromophenol blue and glycerol in water) and applied in
0.8% agarose horizontal gel electrophoresis chamber in Tris-acetate-EDTA buffer (pH
8.0, 7 V/cm). The gel was stained with ethidium bromide (0.5 µg/mL) and the DNA
bands were visualized by fluorescence under an ultraviolet transilumination system. The
assay was repeated at least four times, the results were digitalized (Kodak Digital
Science 1d, EDAS 120) and the bands semiquantified using the computer program
Image J for Windows.
33
Statistical analysis
Data are reported as (means ± SD) of the %ATI, morphological analysis, growth
bacterial of E. coli cultures and optic density and percentual of plasmid forms. The One-
way analysis of variance (ANOVA) test was performed to verify possible statistical
differences p<0.05 as less significant level.
Results
In vitro radiolabeling of blood constituents
Table 1 presents the effects of ibuprofen and ketoprofen on the radioactivity
distribution between cellular and plasma compartments. These data indicate no
alteration (p<0.05) of 99mTc distribution in these compartments.
Table 1- Effect of ibuprofen and ketoprofen on the radioactivity distribution on the cells and plasma compartments labeled with 99mTc.
%ATI Concentration Ibuprofen Ketoprofen
(µg/mL) P BC P BC
0.0 2.08 ± 1.10 97.92 ± 1.10 1.88 ± 0.74 98.12 ± 0.74 0.1 1.16 ± 0.76 98.84 ± 0.76 2.57 ± 2.98 97.43 ± 2.98 1 1.65 ± 1.23 98.35 ± 1.23 1.20 ± 0.69 98.80 ± 0.69 10 3.15 ± 2.73 96.85 ± 2.73 1.91 ± 0.98 98.09 ± 0.98 100 1.75 ± 1.37 98.25 ± 1.37 1.51 ± 1.34 98.49 ± 1.34 1.000 1.85 ± 0.82 98.15 ± 0.82 1.92 ± 0.83 98.08 ± 0.83
Samples of whole blood from Wistar rats were treated with naproxen at different concentrations for 60 minutes. After that, labeling of blood constituents with 99mTc was carried out. Plasma (P) and blood cells (BC) were separated by centrifugation. The radioactivity in BC and P was counted, and the %ATI was calculated. P <0.05, compared to control group BCs.
Table 2 presents the effect of ibuprofen and ketoprofen on the fixation of 99mTc
on insoluble and soluble fractions plasma proteins. Similarly to present in table 1,
ibuprofen and ketoprofen are not capable to interfere on the fixation of the radioactivity
on the insoluble and soluble fractions of plasma.
34
Table 2 - Effect of ibuprofen and ketoprofen on the fixation of 99mTc on soluble and
insoluble fractions of plasma.
%ATI Concentration Ibuprofen Ketoprofen
(µg/mL) FI- P FS-P FI-P FS-P
0.0 68.36 ± 5.73 31.64 ± 5.73 70.75 ± 5.45 29.24 ± 5.45 0.1 71.94 ± 3.33 28.06 ± 3.33 72.73 ± 7.02 27.26 ± 7.02 1 72.62 ± 4.58 27.38 ± 4.58 69.33 ± 6.83 30.66 ± 6.83 10 72.89 ± 4.42 27.11 ± 4.42 72.80 ± 5.75 27.19 ± 5.75 100 71.64 ± 3.61 28.36 ± 3.61 67.39 ± 7.74 32.60 ± 7.74 1.000 71.46 ± 4.89 28.54 ± 4.89 70.81 ± 5.18 29.18 ± 5.18
Samples of whole blood from Wistar rats were treated with naproxen at different concentrations. After that, labeling of blood constituents with 99mTc was carried out. Plasma and blood cells was separated by centrifugation, and fractions soluble plasma (SF-P) and insoluble (IF-P), were isolated by precipitation in trichloroacetic acid and centrifugation. The radioactivity P<0.05, compared to control group of IF-P.
No alteration on the fixation of radioactivity on proteins of blood cells from blood
samples incubated with ibuprofen and ketoprofen (Table 3) were found.
Table 3 - Effect of ibuprofen and ketoprofen on the fixation of 99mTc on soluble and insoluble fractions of blood cells.
%ATI Concentration Ibuprofen Ketoprofen
(µg/mL) FI- BC FS-BC FI-BC FS-BC
79.80 ± 2.88 20.20 ± 2.88 81.58 ± 2.24 18.41 ± 2.24 0.1 78.84 ± 2.78 21.16 ± 2.78 85.67 ± 3.53 14.32 ± 3.53 1 76.70 ± 2.02 23.28 ± 2.02 82.37 ± 2.76 17.62 ± 2.76 10 76.02 ± 2.02 27.11 ± 2.02 84.65 ± 4.11 15.34 ± 4.11 100 79.09 ± 3.37 20.90 ± 3.37 82.45 ± 1.97 17.57 ± 1.97 1.000 73.21 ± 6.28 23.68 ± 6.28 82.55 ± 1.76 17.44 ± 1.76
Samples of whole blood from Wistar rats were treated with naproxen at different concentrations. After that, labeling of blood constituents with 99mTc was carried out. BCs were separated by centrifugation, and fractions soluble at blood cells SF-BC and insolube IF-BC were isolated by precipitation in trichloroacetic acid and centrifugation. The radioactivity in each fraction was counted, and the %ATI was calculated. P<0.05, compared to control group of IF-BC.
Morphological evaluation
Photomicrographs of RBC from blood incubated with 0.9% NaCl (figure1a) or
ibuprofen or ketoprofen (1000 mg/mL) under optical microscopy is shown in the figures
35
1b and 1c. Qualitative evaluation of these figures indicates no alterations on the shape
of the RBC incubated with ibuprofen or ketoprofen.
Figure 1: Photomicrography of blood smear from blood incubated with 0.9% NaCl (control) (a) and
photomicrography of blood smear from blood incubated with ibuprofen (1000 g/mL) (b) and
incubated with ketoprofen (1000 g/mL) (c).
Table 4 presents the perimeter/area ratios of RBC from blood samples incubated
with ibuprofen or ketoprofen. The results indicate that the perimeter/area ratios of RBC
was not significantly (p>0.05) altered by at ibuprofen or ketoprofen the concentrations
used when compared with the control group.
Table 4 - Effect of treatment with ibuprofen or ketoprofen on the perimeter/area ratio of red blood cells.
Ibuprofen
ketoprofen
Concentration (µg/mL)
Perimeter/area ratio (1/ m)
0.0 0.62 ± 0.01 0.66 ± 0.01
0.1 0.69 ± 0.02 0.62 ± 0.02
1.0 0.68 ± 0.02 0.64 ± 0.02
10 0.67 ± 0.02 0.66 ± 0.02
100 0.66 ± 0.01 0.65 ± 0.02
1000 0.64 ± 0.02 0.67 ± 0.01 Morphometric measurements were performed to red blood cells from blood smears from Wistar rats treated with ibuprofen or ketoprofen for 60 minutes. A total of five fields per each slide and five slides to each extract dose were evaluated.
b
a c
36
Plasmid treatment with ibuprofen and ketoprofen and electrophoretic profile
assay
Figures 2b and 3b show the photograph of agarose gel electrophoresis of pBSK
plasmid treated with ibuprofen and ketoprofen, respectively, in presence and absence of
SnCl2. Data in these figures suggest that ibuprofen and ketoprofen are not capable to
induce alterations on the electrophoretic profile of plasmids (lanes 3, 4 and 5) when
compared with negative control (lane 1). Also, figures 2b and 3b indicate that the effect
of SnCl2 (lane 2) is not altered by ibuprofen and ketoprofen at concentrations used
(lanes 6, 7 and 8). These results are confirmed by semiquantitative analyses of the
percentages of supercoiled (SC) and open circle (OC) plasmid forms (Figure 2a and 3a )
indicating no alteration on the electrophoretic profile.
Figure 2: Percentage of bacterial plasmid forms (a) and photograph (b) of agarose gel after electrophoresis of plasmid pBSK treated with ibuprofen in presence and absence of SnCl2. Samples of bacterial plasmids were incubated with ibuprofen (0.3, 30 and 300 μg/mL) in
presence or absence of SnCl2 (200 g/mL). After that, agarose gel electrophoresis procedure
(0.8%, 7 V/cm) was performed, gels were stained with ethidium bromide (0.5 g/mL), plasmid forms were visualized by fluorescence and digitalized to obtain the percentage of each plasmid
(b)
(a)
37
forms. As controls, plasmids samples incubated with buffer (negative control) or SnCl2 (positive control) alone. Lanes: (1) pBSK + buffer; (2) pBSK + SnCl2; (3) pBSK + ibuprofen (300 μg/mL); (4) pBSK + ibuprofen (30 μg/mL); (5) pBSK + ibuprofen (0.3 μg/mL); (6) pBSK + ibuprofen (300 μg/mL) + SnCl2; (7) pBSK + ibuprofen (30μg/mL) + SnCl2; (8) pBSK + ibuprofen (3.0 μg/mL) + SnCl2. (■) OC (open circle); (□) SC (supercoiled).
Figure 3: Percentage of bacterial plasmid forms (a) and photograph (b) of agarose gel after electrophoresis of plasmid pBSK treated with ketoprofen in presence and absence of SnCl2. Samples of bacterial plasmids were incubated with ketoprofen (0.3, 30 and 300 μg/mL) in
presence or absence of SnCl2 (200 g/mL). After that, agarose gel electrophoresis procedure
(0.8%, 7 V/cm) was performed, gels were stained with ethidium bromide (0.5 g/mL), plasmid forms were visualized by fluorescence and digitalized to obtain the percentage of each plasmid forms. As controls, plasmids samples incubated with buffer (negative control) or SnCl2 (positive control) alone. Lanes: (1) pBSK + buffer; (2) pBSK + SnCl2; (3) pBSK + ketoprofen (300 μg/mL); (4) pBSK + ketoprofen (30 μg/mL); (5) pBSK + ketoprofen (0.3 μg/mL); (6) pBSK + ketoprofen (300 μg/mL) + SnCl2; (7) pBSK + ketoprofen (30μg/mL) + SnCl2; (8) pBSK + ketoprofen (3.0 μg/mL) + SnCl2. (■) OC (open circle); (□) SC (supercoiled).
DISCUSSION
In vitro blood constituents labeled with technetium-99m (99mTc) has been
proposed as an assay to assess biological effects, redox properties and interactions of
natural and synthetic drugs with cellular membrane (Fonseca et al., 2007; Benarroz et
(b)
(a)
38
al., 2008; Frydman et al., 2008). To this radiolabeling procedure, stannous chloride
(SnCl2) has been mostly used as reducing agent (Saha, 2004).
It has been reported that a number of natural or synthetic substances (Fonseca,
2005; Abreu et al., 2006; Fonseca, 2007; Benarroz, 2008; Frydman et al., 2008) could
alter the labeling of blood constituents with 99mTc. Other products are not capable to
interfere with this labeling process (Frydman et al 2004; Pereira, 2008). This procedure
has been proposed as an in vitro assay to verify some important properties, as
chelating/redox activities or interactions on cellular membrane, of products used daily by
humans (Fonseca et al., 2007; Benarroz, 2008). Our data in this work suggest no
alteration on the radiolabeling of blood constituents when blood was incubated with
ibuprofen as well as with ketoprofen (Table 1, 2 and 3).
The morphology of RBC is influenced by function of ion transport systems in
membrane (Lew and Bookchin, 2005). Morphological analysis of red blood cells (RBC)
has been utilized as another method to evaluate effects of drugs and products naturals
at membrane cellular (Li et al., 1999; Benarroz et al., 2008; Frydman et al., 2008;
Pereira et al., 2008). Our data indicates that ibuprofen and ketoprofen would not alter
the morphology of RBC (Figures 1a, 1b and 1c).
The morphometric analysis (area, shape and volume measurements) has been
used to evaluate the alterations induced by natural products and synthetic drugs on
membrane of red blood cells (Oliveira et al., 2002; Moreno et al., 2004, Frydman et al.,
2008). Non-steroidal anti-inflammatory drugs can change the morphometric parameters
of red blood cells (Frydman et al., 2008). Our data indicate that ibuprofen and
ketoprofen could not alter the perimeter/area ratio (table 4) indicating no modifications
39
on the shape of RBC membrane. These data are similar to another study carried out
with fenoprofen (Pereira et al, 2008).
Some experimental models to evaluate genotoxic and potential redox, involving
SnCl2, have been suggested (Melo et al. 2001, Dantas et al. 2002, Pungartnik et al,
2005; Almeida et al, 2007, Presta et al., 2007). Electrophoretic profile of bacterial
plasmids has also been used as a reliable assay to evaluate genotoxic effect of drugs
(Ferreira-Machado et al., 2004, Pereira et al, 2008). In fact, the presence of free radicals
scavengers could reduce the changes of electrophoretic profile of plasmid DNA induced
by stannous chloride decreasing the DNA strand breaks (Dantas et al. 1999, de Mattos
et al., 2000; Presta et al., 2007). Ibuprofen and ketoprofen has been suggested to be
scavenger of free radicals (Costa, et al., 2006). However, at conditions used in this work,
ibuprofen and ketoprofen did not seem to protect plasmid DNA against the effects of
stannous chloride. In addition, these drugs could not present genotoxic effect because
no alteration on the electrophoretic profile of plasmids was observed (Figures 2a, 2b, 3a
and 3b).
In conclusion, under the conditions used in this work, our data suggest that
ibuprofen and ketoprofen would not (i) affect the fixation of the 99mTc on the blood
constituents, (ii) alter the RBC membrane and (iii) present genotoxic and redox effects.
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Human Neutrophil Oxidative Burst Mediated by Arylpropionic Acid Non-steroidal Anti-
inflammatory Drugs, Biol Pharm Bull. 2006; 29:1569 - 1659.
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2- Insel PA, Analgesic-antipiryretic and antiinflamatory agentes and drugs employed in
the treatment of gout In: Harman JG, Limbird LE, Gilman AG (eds) The
Pharmacological Baisis of Therapeutics. 10th McGraw/Hill, New York 2006; p. 617-
657.
3- Chahade WH, Giorgi R D N, Szajubok J C M, Nonsteroidal anti-inflammatory drugs.
Einstein 2008; 6: S166-S74.
4- Korolkovas A, Dicionário Terapêutico Guanabara 2006/2007. Rio de Janeiro:
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5- Pereira MO, Rocha GS, Lombardi SS, Geller M, Pereira MJ, Santos-Filho SD,
Fonseca AS, Bernardo-Filho M. Effects of fenoprofen on the labeling of blood
constituents with technetium-99m, the morphology of red blood cells and the
plasmid. Braz Arch Biol Technol. 2008; 55:135-141.
6- Valko M, Rhodes CJ, Moncol J, Zakovic MI, Mazur M. Free radicals, metals and
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inflammation and chronic lung diseases. J Biochem Mol Biol. 2003; 36: 95-109.
10- Bernardo-Filho M, Moura INS, Boasquevisque M. 99m technetium – labeled red
blood “in vitro”. Arq Biol Technol. 1983; 4: 455-461.
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11- Barcia JJ. The Giemsa stain: its history and applications. Int J Surg Pathol 2007; 15:
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12- Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratorial manual. Cold
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decreases the labeling of blood constituents with technetium-99M. Acta Biol Hung.
2007; 58:187-198.
14- Benarroz MO, Fonseca AS, Rocha GS, Frydman JN, Rocha VC, Pereira MO,
Bernardo-Filho M. Cinnamomum zeylanicum extract on the radiolabelling of blood
constituents and the morphometry of red blood cells: In vitro assay. Appl Radiat Isot.
2008; 66: 139-146.
15- Frydman JN, Rocha VC, Benarroz MO, Rocha GS, Pereira MO, de Souza, da
Fonseca A, Bernardo-Filho M. Assessment of effects of a Cordia salicifolia extract on
the radiolabeling of blood constituents and on the morphology of red blood cells. J
Med Food. 2008; 11: 767-772.
16- Frydman JNG, Fonseca A, Rocha VC, Benarroz MO, Rocha GS, Pereira MO,
Pereira MJ, Medeiros AC, Bernardo-Filho M. Acetylsalicylic Acid and Morphology of
Red Blood Cells. Braz Arch Biol Technol. 2010; 53: 575-582.
17- Saha GB. Fundamentals of nuclear pharmacy. 5th ed. New York: Springer-Verlag;
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18- Abreu PR, Almeida MC, Bernardo RM, Bernardo LC, Brito LC, Garcia EA, Fonseca
AS, Bernardo-Filho M. Guava extract (Psidium guajava) alters the labelling of blood
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the plasmid topology and the effects of stannous chloride on the DNA of plasmids.
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M. Stannous chloride mediates single strand breaks in plasmid DNA. Toxicol Lett.
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44
4- COMENTÁRIOS, CRÍTICAS E CONCLUSÕES
A convivência inicial com um grupo de pesquisa com mestres e doutores, alunos
de iniciação científica, mestrandos e doutorandos foi um grande desafio. Um ambiente
restrito, cujo crivo não é apenas o saber, mas o aprimoramento do saber, o
compromisso com a ciência e a dedicação no estudo em desenvolvimento. Entretanto,
o mais desafiador foi realizar um projeto utilizando modelos experimentais, avaliando
efeitos biológicos e interações entre drogas naturais e sintéticas com radionuclídeo
(99mTc), linha de pesquisa do Laboratório de Radiofarmácia Experimental do
Departamento de Biofísica e Biometria (UERJ), local onde desenvolvi minha pesquisa.
O curso de pós-graduação strictu sensu possibilita ao aluno participar das
mudanças no contexto social decorrentes dos avanços tecnológicos, a partir do
aperfeiçoamento, capacitação e qualificação técnica, bem como agregar valores
inerentes ao cientista: investigação, crítica e criatividade
O Ingresso no Programa de Pós-graduação em Ciências da Saúde da
Universidade Federal do Rio Grande do Norte foi uma experiência muito gratificante,
bem como cursar as disciplinas em diferentes departamentos e compartilhar
experiências e o conhecimento com profissionais das áreas mais diversas foi muito
enriquecedor.
Este estudo, apesar de ser uma pesquisa experimental, tem caráter
multidisciplinar. Foi realizado em colaboração com o Setor de Medicina Nuclear e o
Laboratório de Endocrinologia do Hospital Universitário Pedro Ernesto – UERJ e
através do convênio entre duas universidades públicas, a UERJ e a UFRN. Essas
parcerias possibilitaram novas idéias e perspectivas, além de agregar conhecimentos
45
de diferentes áreas.
Os resultados deste estudo viabilizaram a publicação de um artigo original
intitulado: “Fenoprofen effects on the labeling of blood constituents with technetium-
99m, on the morphology of red blood cells and on the plasmid DNA” em revista
internacional de impacto relevante, Brazilian Archives of Biology and Technology, com
índice de impacto 0,353 (referente ao ano de 2008) e Qualis C Internacional
Um artigo que está submetido, intitulado: “Evaluation of biological effects of the
naproxen” em revista internacional Medicinal Chemistry Research Qualis internacional
C, bem como a co-autoria de outros artigos: “Assessment of effects of a Cordia
salicifolia extract on the radiolabeling of blood constituents and on the morphology of red
blood cells” (Journal of medicinal food - 2008), “Effects of a Chronic Sucralose
Sweetener on the Labeling of Blood Constituents with Technetium-99m, Morphology of
Red Blood Cells and the Biodistribution of Sodium Pertechnetate in Rats” (Brazilian
Archives of Biology and Technology - 2008), “Acetylsalicylic acid decreases the labeling
of blood constituents with technetium-99m” (Brazilian Archives of Biology and
Technology - 2010), “Acetylsalicylic acid and morphology of red blood cells” (Brazilian
Archives of Biology and Technology - 2010).
Como também a submissão do manuscrito mestrado intitulado: “Evaluation of
biological effects of the Ibuprofen and Ketoprofen”, para a revista Clinics
O estudo de efeitos biológicos de antiiflamatórios produziu resultados relevantes,
além de me proporcionar conhecimento em diferentes modelos experimentais em nível
celular e molecular.
46
5-Trabalhos apresentados em congressos
- Santos-Filho, S.D.; Pereira, M. O.; Carmo, F.S.; Diniz, C.L; Rocha, G. S. ; Fonseca,
A.S.; Bernardo-Filho, M. INFLUENCIA DE UM EXTRATO AQUOSO DE JUGLANS
REGIA NA MARCAÇÃO DE CONSTITUINTES SANGUINEOS COM TECNECIO-99M.
Publicado nos Anais da XXIV Reunião Anual da Federação de Sociedades de Biologia
Experimental, 2009, ÁGUAS DE LINDÓIA, SP.
- Rocha, G. S.; Pereira, M. O.; Pinto, N.S.; Santos-Filho, S.D.; Fonseca, A.S.; Bernardo-
Filho, M. AVALIAÇÃO DE PARAMETROS BIOQUIMICOS DO SANGUE DE RATOS
WISTAR TRATADOS COM SUCRALOSE. Publicado nos Anais da XXIV Reunião Anual
da Federação de Sociedades de Biologia Experimental, 2009, ÁGUAS DE LINDOIA,
SP.
- Rocha, G.S.; Pereira, M.O.; Pinto, N.S.; Santos-Filho, S.D.; Fonseca, A.S.; Bernardo-
Filho, M. TRATAMENTO AGUDO COM SUCRALOSE NÃO ALTERA PARAMETROS
HEMATOLOGICOS DE RATOS WISTAR. Publicado nos Anais da XXIV Reunião Anual
da Federação de Sociedades de Biologia Experimental, 2009, ÁGUAS DE LINDOIA,
SP.
- Rocha, G.S.; Lombardi, S.S.; Diniz, C.L.; Carmo, F.S.; Pereira, M.O.; Souza, R.S.S.;
Manoel, C.V.; Santos-Filho, S.D.; Fonseca, A.S.; Bernardo-Filho, M. EFEITO DO
ADOÇANTE COMERCIAL COM SUCRALOSE NA BIODISPONIBILIDADE DO
RADIOFÁRMACO ÁCIDO DIETILENOTIAMINOPENTACÉTICO (DTPA) EM RATOS
47
WISTAR. Publicado nos Anais da XXIII Reunião Anual da Federação de Sociedades de
Biologia Experimental, 2008, ÁGUAS DE LINDÓIA, SP.
- Santos-Filho, S.D.; Bernardo, R.M.; Lombardi, S.S.; Pereira, M.O.; Carmo, F.S.; Diniz,
C.L.; Rocha, G. S.; Fonseca, A.S.; Bernardo-Filho, M. INFLUÊNCIA DE UM EXTRATO
AQUOSO DE HYPERICUM PERFORATUM NA SOBREVIVÊNCIA DE ESCHERICHIA
COLI AB1157 E NA MOBILIDADE ELETROFORÉTICA DE DNA DE PLASMÍDEO
pBSK. Publicado nos Anais da XXIII Reunião Anual da Federação de Sociedades de
Biologia Experimental, 2008, ÁGUAS DE LINDÓIA, SP.
- Agostinho, R.T; Diniz, C.L.; Carmo, F.S.; Rocha, G.S.; Pereira, M.O.; Almeida, D.S.;
Santos-Filho, S.D.; Fonseca, A.S.; Bernardo-Filho, M. AVALIAÇÃO DE EFEITOS DO
EXTRATO AQUOSO DE GANODERMA LUCIDUM NA MARCAÇÃO DE
CONSTITUINTES SANGUÍNEOS COM 99M-TC E NA SOBREVIVÊNCIA DE
CULTURAS DE E.COLI AB1157. Publicado nos Anais do VI CONGRESSO DA
SOCIEDADE BRASILEIRA DE BIOCIÊNCIAS NUCLEARES, 2008, CABO FRIO - RJ.
MN METABÓLICA - SUPLEMENTO SBBN 2008.
- Lombardi, S.S; Pereira, M.O.; Carmo, F.S.; Diniz, C.L.; Rocha, G.S.; Santos-Filho,
S.D.; Fonseca, A.S.; Bernardo-Filho, M. EXTRATO AQUOSO DE SÁLVIA ALTERA A
BIODISPONIBILIDADE DO RADIOFÁRMACO PERTECNETATO DE SÓDIO EM
RATOS WISTAR. Publicado nos Anais da XXIII Reunião Anual da Federação de
Sociedades de Biologia Experimental, 2008, ÁGUAS DE LINDÓIA, SP.
48
- Rocha, G.S.; Lombardi, S.S.; Carmo, F.S.; Diniz, C.L.; Pereira, M.O.; Santos-Filho,
S.D.; Fonseca, A.S.; Bernardo-Filho, M. AVALIAÇÃO DO EFEITO CITOTÓXICO E
GENOTÓXICO DO ADOÇANTE COMERCIAL COM SUCRALOSE EM CULTURAS DE
E. COLI. Publicado nos Anais da XXIII Reunião Anual da Federação de Sociedades de
Biologia Experimental, 2008, ÁGUAS DE LINDÓIA, SP.
- Pereira, M.O.; Diniz, C.L.; Carmo, F.S.; Lombardi, S.S.; Rocha, G.S.; Fonseca, A.S.;
Santos-Filho, S.D.; Bernardo-Filho, M. AVALIAÇÃO DOS EFEITOS GENOTÓXICO E
CITOTÓXICO DO ANTIINFLAMATÓRIO CETOPROFENO EM PLASMÍDEOS E
CULTURAS DE E. COLI. Publicado nos Anais da XXIII Reunião Anual da Federação de
Sociedades de Biologia Experimental, 2008, ÁGUAS DE LINDÓIA, SP.
- Lombardi, S.S.; Pereira, M.O.; Carmo, F.S.; Diniz, C.L.; Rocha, G.S.; Santos-Filho,
S.D.; Fonseca, A.S.; Bernardo-Filho, M. AVALIAÇÃO DO EFEITO CITOTÓXICO DE
EXTRATO AQUOSO DE SALVIA OFFICINALIS EM CULTURAS DE E. COLI. Publicado
nos Anais da XXIII Reunião Anual da Federação de Sociedades de Biologia
Experimental, 2008, ÁGUAS DE LINDÓIA, SP.
- Pereira, M.O.; Lombardi, S.S.; Carmo, F.S.; Diniz, C.L.; Rocha, G.S.; Santos-Filho,
S.D.; Fonseca, A.S.; Bernardo-Filho, M. AVALIAÇÃO DOS EFEITOS GENOTÓXICO E
CITOTÓXICO DO ANTIINFLAMATÓRIO NAPROXENO EM PLASMÍDEOS E
49
CULTURAS DE E. COLI. Publicado nos Anais da XXIII Reunião Anual da Federação de
Sociedades de Biologia Experimental, 2008, ÁGUAS DE LINDÓIA, SP.
- Fonseca, A.S.; Pereira, M J ; Frydman, J. N. G. ; Lombardi, S.S.; Pereira, M.O.;
Rocha, G.S.; Bernardo-Filho, M. AVALIAÇÃO DA INFLUENCIA DA PRIVAÇÃO DE
SONO REM E DO NADO FORÇADO NA MORFOLOGIA DE HEMÁCIAS DE RATOS
WISTAR. Publicado nos Anais do I CONGRESSO IBRO/LARC DE NEUROCIENCIAS
DA AMÉRICA LATINA, CARIBE E PENINSULA IBERICA, 2008, BUZIOS, RJ. I
IBRO/LARC CONGRESS OF NEUROSCIENCES OF LATIN AMERICA, THE
CARIBBEAN AND IBERIAN PENINSULA, 2008.
- Rocha, G.S.; Pereira, M.O.; Rocha, V.C.; Frydman, J.N.G.; Benarroz, M.O.; Lins, M.C.;
Garcia-Pinto, A.B.G.; Ribeiro, C.G.; Pereira, M J ; Fonseca, A.S.; Bernardo-Filho, M.
TRATAMENTO CRONICO COM ADOÇANTE COM SUCRALOSE NÃO ALTERA A
BIODISPONIBILIDADE DO RADIOFARMACO PERTECNETATO DE SODIO, A
MARCAÇÃO DE CONSTITUINTES SANGUINEOS COM TECNÉCIO-99M E A
MORFOLOGIA DAS HEMÁCIAS DE RATOS WISTAR. Publicado nos Anais do VI
CONGRESSO DA SOCIEDADE BRASILEIRA DE BIOCIENCIAS NUCLEARES, 2008,
CABO FRIO, RJ. MN METABOLICA, 2008. v. 10.
- Pereira, M.O.; Rocha, G.S.; Lombardi, S.S; Diniz, C.L.; Carmo, F.S.; Pereira, M.J.;
Santos-Filho, S.D.; Geller, M.; Fonseca, A.S.; Bernardo-Filho, M. AVALIAÇÃO DE
EFEITOS BIOLOGICOS DO FENOPROFENO NA MARCAÇÃO DE CONSTITUINTES
50
SANGUINEOS COM TECNECIO-99M, NA MORFOLOGIA DE HEMÁCIAS E NA
ELETROFORESE DE DNA PLASMIDIAL. Publicado nos Anais do VI CONGRESSO DA
SOCIEDADE BRASILEIRA DE BIOCIENCIAS NUCLEARES, 2008, CABO FRIO, RJ.
MN METABOLICA, 2008. v. 10.
- Benarroz, M.O.; Frydman, J.N.G.; Rocha, G.S.; Pereira, M.O.; Pereira, M.J.; Geller, M.;
Fonseca, A.S.; Bernardo-Filho, M. RADIOMARCAÇÃO DE CONSTITUINTES
SANGUINEOS E MORFOLOGIA DE HEMÁCIAS DE RATOS WISTAR TRATADOS
COM EXTRATO DE CINNAMOMUM ZEYLANICUM. Publicado nos Anais do VI
CONGRESSO DA SOCIEDADE BRASILEIRA DE BIOCIENCIAS NUCLEARES, 2008,
CABO FRIO, RJ. MN METABOLICA, 2008. v. 10.
- Lombardi, S.S.; Pereira, M.O.; Rocha, G.S.; Fonseca, A.S.; Bernardo-Filho, M.
AVALIAÇÃO DE EFEITO GENOTÓXICO E ANTIOXIDANTE DE EXTRATO AQUOSOS
DE SÁLVIA (SALVIA OFFICINALIS). Publicado nos Anais do X INTERNATIONAL
CONGRESS OF ETHNOPHARMACOLOGY E XX SIMPOSIO DE PLANTAS
MEDICINAIS DO BRASIL, 2008, SÃO PAULO.
- Diniz, C.L.; Carmo, F.S.; Lombardi, S.S.; Pereira, M.O.; Rocha, G.S.; Fonseca, A.S.;
Santos-Filho, S.D.; Bernardo-Filho, M. AVALIAÇÃO DO EFEITO CITOTÓXICO DE UM
EXTRATO AQUOSO DE CENTELLA ASIATICA EM CULTURAS BACTERIANAS DE
ESCHERICHIA COLI. Publicado nos Anais da XXIII Reunião Anual da Federação de
Sociedades de Biologia Experimental, 2008, ÁGUAS DE LINDOIA, SP.
51
- Rocha, G.S.; Pereira, M.O.; Benarroz, M.O.; Frydman, J.N.G.; Lins, M.C.; Garcia-
Pinto, A.B.G.; Ribeiro, C.G.; Bernardo, R.M.; Fonseca, A.S.; Bernardo-Filho, M.
AVALIAÇÃO DOS EFEITOS DO TRATAMENTO AGUDO E CRÔNICO DO ADOÇANTE
COMERCIAL COM SUCRALOSE NA BIODISPONIBILIDADE DO RADIOFÁRMACO
PERTECNETATO DE SÓDIO EM RATOS WISTAR. Publicado nos Anais da XXIII
Reunião Anual da Federação de Sociedades de Biologia Experimental, 2008, ÁGUAS
DE LINDOIA, SP.
52
6- Referências
1- Chahade WH, Giorgi RDN, Szajubok JCM.Nonsteroidal anti-inflammatory
drugs.Einstein 2008; 6 (Supl 1):S166-S74.
2- Costa D, Moutinho L, Lima JLF, Fernandes E. Antioxidant Activity and Inhibition of
Human Neutrophil Oxidative Burst Mediated by Arylpropionic Acid Non-steroidal Anti-
inflammatory Drugs. Biol. Pharm. Bull. 2006; 29:1659-1670.
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1. Abstract
Derivatives of propionic acid NSAIDs are irreversible inhibitors of cyclooxygenase
enzyme widely used. The aim of this study was to evaluate, through different
experimental models, biological effects of derivatives of propionic acid (fenoprofen,
naproxen, ibuprofen and ketoprofen) in cellular and molecular level. The labeling of
blood constituents with technetium-99m (99mTc) and morphological analysis of
erythrocytes of blood of rats, as well as growth, survival of cultures of Escherichia coli
(E. coli) and the assessment of bacterial plasmid electrophoretic profiles were models
used for experimental evaluation of possible biological effects of antiinflammatory drugs.
The results show that, in general, anti-inflammatory drugs evaluated were not able to
alter the labeling of blood constituents with 99mTc, the morphology of red blood cells
from blood of rats, as well as the growth of cultures of E. coli and the electrophoretic
profile of plasmid DNA. However, naproxen appears to cytotoxic effect on bacterial
cultures, plasmids and genotoxic effects in reducing the action of stannous chloride in
cultures of E. coli. The use of experimental fast performance and low cost was important
for assessment of biological effects, contributing to a better understanding of the
properties of propionic acid derivatives studied.
Keywords: anti-inflammatory, blood constituents, technetium-99m, stannous chloride,
Escherichia coli; DNA.
